Thesis-C.-Montante

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Université!de!Mons!
Faculté!des!Sciences!
Laboratoire!de!Protéomique!et!de!Microbiologie!
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Functional!Study!of!the!Plasmid9Encoded!CopB!
Protein!Involved!in!Copper!Resistance!in!
Cupriavidus*metallidurans!CH34!
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Thèse!présentée!en!vue!de!l'obtention!du!Grade!Légal!de!Docteur!en!
Sciences!Chimiques!par!Claire!MONTANTE!
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Thèse!soutenue!publiquement!le!26!septembre!2014!devant!le!jury!composé!de:!
Prof.!Ruddy!Wattiez,!UMons,!Belgium!
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Directeur!de!thèse!
Dr.!Igor!Eeckhaut,!UMons,!Belgium!
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Président!du!jury!
Dr.!David!Gillan,!UMons,!Belgium!!
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Secrétaire!
Dr.!Jacques!Covès,!IBS,!Grenoble,!France!!
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Rapporteur!externe!!
Prof.!Meta!J.!Kuehn,!Duke!University,!NC,!USA!
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Rapporteur!externe!
Prof.!Max!Mergeay,!ULB,!SCK!•!CEN,!Belgium!
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Rapporteur!externe!
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To!my!friends!and!family,!with!all!my!love.!
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Abstract(
Copper," which" is" prevalent" in" the" environment," is" an" essential" trace" element" required" by" all"
cellular" organisms." Although" this" metal" fulfills" many" biological" functions," copper" is" also" extremely"
toxic" due" to" its" ability" to" cycle" between" stable" oxidized" Cu(II)" and" unstable" reduced" Cu(I)" states,"
which" can" lead" to" cellular" damage." The" dual" role" of" copper" compels" microorganisms," such" as"
bacteria," to" develop" resistance" mechanisms" to" tightly" control" copper" homeostasis." Cupriavidus*
metallidurans" strain" CH34" is" a" βFproteobacterium" able" to" resist" and" to" survive" in" the" presence" of"
many" metal" ions" found" in" anthropogenic" biotopes." Consequently," C.* metallidurans" CH34" is" an*
important"model"for"studying"resistance"to"numerous"metals."
Copper" resistance" in" C.* metallidurans" CH34" essentially" involves" the" cop" cluster," which" is"
present" on" the" plasmid" pMOL30," copVTMKNSRABCDIJGFOLQHEW." PlasmidFencoded" Cop" proteins"
include"proteins"involved"in"periplasmic"copper"detoxification,"such"as"CopSRABCD;"proteins"involved"
in"cytoplasmic"copper"detoxification,"such"as"CopF;"and"proteins"whose"exact"functions"remain"still"
unknown." The" plasmidFencoded" CopB" protein" has" gained" attention" due" to" its" unique" NFterminal"
extremity,"which"is"rich"in"methionine"residues."The"functional"study"of"this"protein"is"the"subject"of"
this"thesis."Interestingly,"the"plasmidFencoded"CopB"protein"has"two"distinct"structural"domains,"as"
shown"experimentally:"an"unstructured"NFterminal"extremity,"wherein"a"sequential"methionineFrich"
motif" (MQGMDHSKMQGMDQGS)" is" repeated" ten" times," and" a" structural" CFterminal" domain," which"
contains" many" βFstrands" and" few" αFhelices." Studies" of" these" domains" using" synthetic" peptides" and"
recombinant" proteins" demonstrated" an" interaction" between" the" sequential" methionineFrich" motif"
and"Cu(I)"ions"and,"to"a"lesser"extent,"Cu(II)"ions."Subcellular"fractionation"and"immunogold"labeling"
of" C.* metallidurans" CH34" in" the" presence" of" copper" indicated" that" the" CopB" protein" was" outerF
membraneFassociated" and" might" be" folded" into" a" βFbarrel" conformation," as" shown" by" circular"
dichroism" and" transmembrane" protein" topology" prediction." Chemical" crossFlinking" experiments"
strengthened"this"study"and"identified"the"CopA"protein,"which"is"a"putative"multicopper"oxidase,"as"
one"of"the"CopBFinteracting"partners"involved"in"this"copper"resistance"mechanism."
Moreover," our" investigations" also" highlighted" a" vesiculation" phenomenon" that" had" not"
previously" been" described" in" C.* metallidurans" CH34" and" appears" to" be" specific" to" copper" exposure"
and"the"presence"of"metalFresistance"genes"harbored"by"plasmids"(e.g.,"pMOL28"and"pMOL30)."The"
high" abundance" of" copper" ions" complexed" by" the" periplasmic" Cop" proteins" inside" these" outer"
membrane"vesicles"suggested"that"this"vesiculation"production"may"serve"as"a"new"copper"resistance"
mechanism" in" C.* metallidurans" CH34." This" study" paves" the" way" towards" a" better" understanding" of"
the"fate"of"copper"in"this"microorganism."
I"
"
Acknowledgments(
Those"four"years"were"an"unforgettable"and"incredible"experience"for"me."I"learnt"many"things"
during"this"period,"in"particular"about"myself"and"my"ability"to"face"problems."This"thesis"is"the"result"
of"investigations,"collaborations"and"exchanges"with"great"people"and"I"would"like"dedicate"those"few"
lines"to"all"those"people"who"helped"me"to"reach"my"goals."
First" and" foremost," I" would" like" to" express" my" sincere" gratitude" to" my" supervisor," Professor"
Ruddy"Wattiez."He"placed"his"confidence"in"me"four"years"ago"and"gave"me"the"opportunity"to"join"
his" team." All" those" years," he" offered" me" his" continuous" advice" and" encouraged" me" to" think" more"
independently" about" my" experiments" and" results." I" would" like" also" to" thank" him" for" his" support"
throughout"the"tedious"process"of"postdoctoral"fellowship"research"and"his"encouragements"during"
the"most"difficult"times"when"writing"this"thesis."
This" thesis" allowed" me" to" take" part" in" many" activities," combine" chemistry" and" biology" and"
interact" with" many" people," which" constitutes" from" my" personal" point" of" view" one" of" the" greatest"
strengths"of"this"work."I"will"start"by"thanking"Professor"Patrick"Flammang"from"Laboratory"of"Biology"
of" Marine" Organisms" and" Biomimetics" who" provided" me" advice" many" times" during" experiments" of"
Scanning" and" Transmission" Electron" Microscopy." A" special" acknowledgment" goes" to" Mélanie"
Demeuldre" who" gave" me" judicious" suggestions" and" helped" me" a" thousand" times" with"
instrumentation."
My"gratitude"also"goes"to"Professor"Luce"Vander"Elst"and"Céline"Henoumont"from"Department"
of" General," Organic" and" Biomedical" Chemistry," NMR" and" Molecular" Imaging" Laboratory." Thank" you"
for"your"time"and"help"in"the"acquisition"and"interpretation"of"NMR"results."
I" would" like" to" offer" my" deepest" appreciation" to" Mathieu" Surin" and" Jenifer" Rubio" Magneto"
from" Laboratory" for" Chemistry" of" Novel" Materials." I" really" appreciated" working" with" you" both" and"
improved" my" knowledge" in" Atomic" Force" Microscopy" and" Circular" Dichroism." Thank" you" for" your"
warm"encouragements,"kindness"and"friendship!"
I"warmly"thank"the"Professor"Max"Mergeay"for"his"availability,"enthusiasm"and"kindness"as"well"
as"for"the"discussions"we"have"had"over"the"years."I"also"express"my"gratitude"to"the"members"of"the"
jury"who"accepted"to"participate"in"the"evaluation"of"this"work."
I"would"like"to"extend"my"sincere"appreciation"to"the"FRIA"fellowship"(Fonds"pour"la"formation"
à"la"Recherche"dans"l'Industrie"et"l'Agriculture)"who"provided"me"financial"support"during"those"four"
years."
II"
"
I"would"like"to"take"a"few"lines"to"sincerely"thank"my"fellow"labmates."Since"my"very"first"day,"I"
felt" like" home" within" the" lab" and" many" of" you" became" like" a" second" family." Thank" you" to" the" girls"
next"door,"Mélanie"and"Samia!"You"are"amazing,"wonderful"and"generous"friends;"I"really"appreciated"
your" help" and" the" moral" support" you" gave" me." Best" wishes" to" Jérémy" in" the" last" step" of" writing."
Thank"you"for"all"the"discussions"we"had"during"those"years,"your"advice"and"opinions"on"lab"related"
issues."A"special"acknowledgment"goes"to"my"office"mates,"Frédéric,"Quentin"and"Raphaël."There"are"
probably"no"words"to"express"the"profound"respect"and"affection"I"have"for"you."Your"unconditional"
support" through" good" and" bad" times," your" help" and" friendship" mean" a" lot" to" me!" I" also" offer" my"
special"thanks"to"my"other"office"mate,"Clotilde,"for"her"positive"outlook"and"kindness."Thank"you"to"
Baptiste"for"providing"me"many"advices"during"this"thesis,"for"his"positive"attitude"and"energy,"and"
for" cheering" me" up" when" I" needed" it." Thank" you" to" Catherine," David," Viera," Benoît," Harmonie" and"
Florian"for"your"kindness"and"support."My"sincere"thanks"also"go"to"the"people"I"met"and"who"went"
for" new" adventures:" Caroline," Vanessa," Sophie," Stéphanie," Mélissa," Aline," Sabine," Deborah" and"
Nicolas."I"spent"happy"times"with"all"of"you,"and"I"promise"that"I"won't"forget"you!"
To"my"very"best"friends,"Mélanie"and"Laure,"God"knows"how"much"I"love"you!"Thank"you"for"
your"unconditional"support,"your"kindness"and"friendship."Thank"you"to"Gil"for"his"care"and"precious"
friendship." To" my" fellow" travelers," Estelle" and" Sébastien," thank" you" for" all" the" good" moments" we"
spent"together,"for"all"your"encouraging"messages"and"your"kindness."Thank"you"to"Aurélie"for"her"
continued"friendship"through"high"school"and"after."I"wish"all"the"best"for"you"and"your"little"family."
All"of"you"my"friends,"you"are"such"amazing"people"and"I"know"that"when"we"get"old,"you"will"still"be"
there"as"supportive"and"caring"friends."
Last"but"not"least,"I"would"like"to"thank"my"parents,"my"sister"and"her"boyfriend,"and"my"grandF
parents"for"their"love,"great"patience"and"support"at"all"times."They"always"had"faith"in"me"and"my"
ability"to"reach"my"goals,"even"when"I"didn't"faith"in"myself."They"are"my"rock,"my"source"of"energy"
and"I'm"really"blessed"to"have"such"an"amazing"family."I"love"you!"
"
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"
III"
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Table(of(Contents(
Abstract........................................................................................................................................"" I(
Acknowledgments....................................................................................................................."" II"
Table(of(Contents......................................................................................................................"" IV(
List(of(Figures.............................................................................................................................."" VIII"
List(of(Tables..............................................................................................................................."" XIII(
List(of(Abbreviations................................................................................................................."" XIV(
General(Introduction................................................................................................................"" 1"
Chapter(1:( Metal(Ions(in(Biological(Systems........................................................................"" 2"
1.1."Occurrence"of"Metals"in"the"Environment"and"Definition.................................................."" 2"
1.2."Bacterial"Transport"Systems:"EffluxFMediated"Resistance.................................................."" 6"
Chapter(2:( The(Copper(Paradox............................................................................................."" 19"
2.1."General"Overview................................................................................................................"" 19"
2.2."Chemistry"and"Toxicity"of"Copper......................................................................................."" 20"
2.3."Coordination"Chemistry:"CopperFBinding"Centers.............................................................."" 22"
2.4."Models"of"Copper"Homeostasis:"Escherichia*coli"and"Enterococcus*hirae.........................."" 33"
Chapter(3:( Cupriavidus*metallidurans(CH34,(a(MetalNResistant(Bacterium..................."" 40"
3.1."General"Overview................................................................................................................"" 40"
3.2."Copper"Resistance"in"Cupriavidus*metallidurans"CH34......................................................."" 43"
3.3."Phenotypic"Analysis"of"Some"cop"Mutants........................................................................."" 49"
3.4."Thesis"Aim..........................................................................................................................."" 52"
References.................................................................................................................................."" 53(
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IV"
"
Results(and(Discussion............................................................................................................."" 61(
Chapter(4:( Functional(Study(of(the(PlasmidNEncoded(CopB(Protein(from(Cupriavidus*
metallidurans(CH34..............................................................................................." 62"
4.1."Abstract................................................................................................................................" 62"
4.2."Introduction........................................................................................................................." 63"
4.3."Results.................................................................................................................................." 66"
4.3.1."
Characterization"of"Recombinant"Proteins....................................................................."" 66"
4.3.2."
Stoichiometry"of"Metal"–"Peptide"Interactions"by"Mass"Spectrometry.............................." 68"
4.3.3."
Circular"Dichroism"(CD)"and"1D"Nuclear"Magnetic"Resonance"(NMR)"Spectroscopy..........." 70"
4.3.4."
Subcellular"Localization"of"CopB"Protein"in"C.*metallidurans"CH34...................................." 74"
4.3.5"
Identification"of"CopBFInteracting"Partners"Using"in*vivo"CrossFlinking"and"Mass"
Spectrometry.............................................................................................................."" 76"
4.4."Discussion............................................................................................................................"" 78"
4.5."Materials"and"Methods......................................................................................................."" 85"
4.5.1."
Expression"and"Purification"of"Three"Recombinant"Proteins............................................"" 85"
4.5.2."
Production"and"Purification"of"Polyclonal"Antibodies......................................................" 86"
4.5.3."
Protein"and"Antibody"Determination............................................................................." 86"
4.5.4."
Bacterial"Cells"and"Culture"Conditions............................................................................" 86"
4.5.5."
Subcellular"Fractionation"of"C.*metallidurans"CH34"and"Western"Blotting........................." 86"
4.5.6."
Immunogold"Labeling................................................................................................" 87"
4.5.7."
Synthetic"Peptides"and"Metal"Stock"Solutions.........................................................." 88"
4.5.8."
Electrospray"Mass"Spectrometry"(nESI).........................................................................." 88"
4.5.9."
Circular"Dichroism"Spectroscopy..................................................................................."" 89"
4.5.10." Nuclear"Magnetic"Resonance"(NMR)"Spectroscopy........................................................." 89"
4.5.11." Formaldehyde"CrossFlinking.........................................................................................." 90"
4.5.12." CoFImmunoprecipitation.............................................................................................."" 90"
4.5.13." LCFMS/MS"Analysis"and"Identification"of"CopBFInteracting"partners................................." 91"
4.6."References..........................................................................................................................."" 92"
V"
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4.7."Supplemental"Data.............................................................................................................."" 96"
4.8."Appendices:"Overexpression"and"Purification"of"Three"Recombinant"Proteins.................."" 97"
4.8.1." Bacterial"Transformation"and"Expression"of"Three"Recombinant"Proteins.............."" 97"
4.8.2." NonFDenaturing"Protein"Extraction.........................................................................."" 98"
4.8.3." Purification"of"the"CopB(Met)"Protein......................................................................"" 98"
4.8.4." Purification"of"the"FullFLength"CopB"Protein............................................................"" 101"
4.8.5." Purification"of"the"CopB251F495"Protein......................................................................." 103"
4.8.6." Recombinant" Plasmid" pJexpress411:56155" Encoding" the" FullFLength" CopB"
Protein"from"C.*metallidurans"CH34........................................................................." 106"
4.8.7." Recombinant" Plasmid" pJexpress411:34700" Encoding" the" Recombinant" CopB(Met)"
Protein......................................................................................................................." 108"
4.8.8." Recombinant" Plasmid" pJexpress411:56156" Encoding" the" Recombinant" CopB251F495"
Protein......................................................................................................................." 110"
4.9."Appendices:"Metal"–"Peptide"Interactions.........................................................................."" 112"
4.9.1." Mass"Spectra"of"Synthetic"Peptides"in"the"Presence"of"Cu(II)"Ions.........................."" 112"
4.9.2." Peptide"Mass"Spectra"in"the"Presence"of"Cu(I)"Ions................................................."" 116"
4.9.3." Peptide"Mass"Spectra"in"the"Presence"of"Ag(I)"Ions................................................."" 121"
4.9.4." Peptide"Mass"Spectra"in"the"Presence"of"Zn(II)"Ions................................................"" 126"
4.9.5." Determination"of"Dissociation"Constant"Values......................................................."" 128"
4.9.6."
1
HFNMR"Spectra"of"Synthetic"Peptides"Upon"Successive"Cu(I)"Additions................." 131"
4.9.7."
1
HFNMR"Spectra"of"Synthetic"Peptides"Upon"Successive"Ag(I)"Additions................." 135"
Chapter(5:( Towards( a( New( Copper( Resistance( Mechanism( in( Cupriavidus*
metallidurans(CH34,(Time(to(Vesiculate...(......................................................." 139"
5.1."Abstract................................................................................................................................" 139"
5.2."Introduction........................................................................................................................." 140"
5.3."Results.................................................................................................................................." 142"
5.3.1." Morphostructural"Alterations"of"C.*metallidurans"CH34"Induced"by"Copper(II)"Ions.............." 142"
5.3.2." External"Vesicle"Formation"Appears"to"Be"Specific"to"Copper"Exposure..............................." 144"
5.3.3." Purification"and"Characterization"of"Outer"Membrane"Vesicles.........................................." 147"
VI"
"
5.4."Discussion............................................................................................................................." 150"
5.5."Materials"and"Methods........................................................................................................" 157"
5.5.1." Bacterial"Strains"and"Culture"Conditions..........................................................................." 157"
5.5.2." Scanning"and"Transmission"Electron"Microscopy..............................................................." 157"
5.5.3." Atomic"Force"Microscopy................................................................................................" 158"
5.5.4." Isolation"and"Purification"of"OMVs"from"C.*metallidurans"CH34.........................................." 158"
5.5.5." Protein"Extraction"from"Purified"OMVs"and"Enzymatic"Digestion........................................" 158"
5.5.6." LCFMS/MS"Analysis"and"Identification"of"OMV"proteins....................................................." 159"
5.5.7." Inductively"Coupled"PlasmaFMass"Spectrometry"(ICPFMS)"analysis....................................."" 159"
5.6."References..........................................................................................................................."" 161"
5.7."Supplemental"Data.............................................................................................................."" 165"
5.8."Supplemental"Tables..........................................................................................................."" 168"
Concluding(Remarks(and(Future(Prospects......................................................................."" 181(
References..................................................................................................................................." 192"
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VII"
"
List(of(Figures(
A.(Main(Figures(
Figure"1.1:" Mendeleev's"periodic"table........................................................................................." 3"
Figure"1.2:" Transport" of" solutes" across" the" membrane," comparison" between" passive" and"
active"transport..........................................................................................................." 7"
Figure"1.3:" Model"illustrating"the"different"steps"of"ABC"exporter"mechanism..........................." 9"
Figure"1.4:" Model"of"the"CopA"P1BFtype"ATPase"of"E.*coli............................................................." 10"
Figure"1.5:" Schematic"overview"of"the"alternating"access"mechanism"used"by"PFtype"ATPase..." 11"
Figure"1.6:" Alternating"access"mechanism"for"M2+/H+"antiport"by"Cation"Diffusion"Facilitator..." 13"
Figure"1.7:" Structure"of"an"RND"efflux"pump................................................................................" 15"
Figure"1.8:" Schematic" illustration" of" the" alternating" site" functional" rotation" transport"
mechanism.................................................................................................................." 16"
Figure"1.9:" Schematic" diagram" illustrating" the" alternating" access" mechanism" for" a"
substrate/proton"symporter......................................................................................." 17"
Figure"2.1:" Ligands"predominantly"found"in"copperFbinding"sites"within"proteins......................" 23"
Figure"2.2:" Type"1"(T1)"copper"sites.............................................................................................." 25"
Figure"2.3:" Type"2"(T2)"and"type"3"(T3)"copper"sites....................................................................." 26"
Figure"2.4:" CuA,"CuB"and"CuZ"centers............................................................................................." 28"
Figure"2.5:" Copper"coordination"geometry"in"ATPases"and"chaperones......................................" 29"
Figure"2.6:" Coordination"geometry"in"copper"resistance"proteins..............................................." 32"
Figure"2.7:" Schematic" drawing" of" the" cop" operon" and" model" of" copper" homeostasis" in"
Enterococcus*hirae......................................................................................................" 34"
Figure"2.8:" Schematic"model"of"copper"homeostasis"in"Escherichia*coli......................................" 36"
Figure"2.9:" Alignment" of" similar" plasmidFborne" genes" in" Escherichia* coli" (plasmid" pRJ1004)"
and"Pseudomonas*syringae*(plasmid"pPT23D)..........................................................." 37"
Figure"2.10:" Schematic"drawing"of"the"cop"operon"and"proposed"mechanism"of"PcoFmediated"
copper"detoxification"in"E.*coli...................................................................................." 38"
Figure"3.1:" Maps"of"the"two"large"plasmids"in"C.*metallidurans"CH34........................................." 42"
Figure"3.2:" Copper"resistance"operons"in"C.*metallidurans"CH34................................................." 44"
Figure"3.3:" Schematic"illustration"of"copper"resistance"in"C.*metallidurans"CH34......................." 45"
VIII"
"
Figure"3.4:" Viability"curves"of"the"copB"mutant"in"the"presence"of"copper"ions,"in"the"context"
of"pMOL1024,"in"uninduced"preculture"conditions...................................................." 50"
Figure"3.5:" Viability"curves"of"the"copB"mutant"in"the"presence"of"copper"ions,"in"the"context"
of"pMOL30,"in"uninduced"preculture"conditions........................................................" 51"
Figure"3.6:" Viability"curves"of"the"copB"mutant"in"the"presence"of"copper"ions,"in"the"context"
of"pMOL30,"in"copperFinduced"preculture"conditions................................................" 51"
Figure"4.1:" StructureFbased" sequence" alignment" of" the" pMOL30Fencoded" CopB" protein" and"
the"chromidFencoded"CopB"protein"from*C.*metallidurans"CH34.............................." 64"
Figure"4.2:" Multiple" sequence" alignment" of" the" plasmidFencoded" CopB" protein" with"
homologous"sequences.............................................................................................." 67"
Figure"4.3:" SDSFpolyacrylamide" gel" (4F20%)" showing" the" protein" profile" of" purified"
recombinant"proteins................................................................................................." 66"
Figure"4.4:" Circular" dichroism" spectra" of" 4" µM" recombinant" proteins" in" 25" mM"
ammoniumacetate,"in"the"absence"and"presence"of"metal"ions................................" 68"
Figure"4.5:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)" spectra" of" native"
peptide"and"its"Cu(I)"adducts......................................................................................" 69"
Figure"4.6:" Circular" dichroism" spectra" of" methionineFrich" peptides" in" the" absence" and"
presence"of"Cu(II),"Cu(I),"or"Ag(I)"ions........................................................................." 71"
Figure"4.7:" Example" of" 1HFNMR" spectrum" obtained" for" apoFPep." 2" (M4" →" G)" in" 100%" D2O"
solution......................................................................................................................." 72"
Figure"4.8:" Subcellular" localization" of" the" plasmidFencoded" CopB" protein" from"
C.*metallidurans"CH34................................................................................................." 74"
Figure"4.9:" Predicted"topology"of"the"CopB251F495"recombinant"protein......................................." 75"
Figure"4.10:" Schematic"drawing"representing"the"metalFbinding"site"within"synthetic"peptides.." 81"
Figure"4.11:" Schematic"drawing"representing"the"potential"CopBFinteracting"partners................" 84"
Figure"5.1:" Morphostructural"alterations"of"C.*metallidurans"CH34"induced"by"copper(II)"ions.." 143"
Figure"5.2:" TEM"photomicrographs"of"C.*metallidurans"CH34......................................................" 144"
Figure"5.3:" Purification"and"characterization"of"outer"membrane"vesicles.................................." 147"
Figure"5.4:" Subcellular"localization"of"identified"OMV"proteins..................................................." 148"
Figure"5.5:" Schematic"representation"of"OMV"formation............................................................" 154"
Figure"6:"
Multiple" sequence" alignment" of" the" plasmidFencoded" CopB" protein" with"
homologous"sequences"from"different"strains"of"C."metallidurans............................" 185"
IX"
"
B.(Supplemental(Figures(
Figure"S4.1:" Circular" dichroism" spectra" of" methionineFrich" peptides" in" the" absence" and"
presence"of"Cu(II),"Cu(I),"or"Ag(I)"ions........................................................................." 96"
Figure"S4.2:" Circular" dichroism" spectra" of" methionineFrich" peptides" in" the" absence" and"
presence"of"Zn(II)"ions................................................................................................." 96"
Figure"S5.1:" TEM" photomicrographs" of" C.* metallidurans" CH34" treated" with" different" divalent"
metal"ions..................................................................................................................." 165"
Figure"S5.2:" TEM" photomicrographs" of" C.* metallidurans" CH34" treated" with" hydrogen"
peroxide......................................................................................................................" 165"
Figure"S5.3:" TEM"photomicrographs"of"mutants"derived"from"the"wildFtype"strain"CH34............" 166"
Figure"S5.4:" TEM"photomicrographs"of"strain"AE2214"derived"from"the"wildFtype"strain"CH34..." 167"
Figure"S5.5:" COGFbased"prediction"of"identified"OMV"proteins....................................................." 168"
C.(Appendix(Figures(
Appendix"4.1:" Overproduction"of"three"recombinant"proteins"upon"IPTG"induction"in"E.*coli...." 98"
Appendix"4.2:" Purification" of" the" recombinant" CopB(Met)" protein" by" immobilizedFmetal"
affinity"chromatography........................................................................................" 99"
Appendix"4.3:" Purification" of" the" recombinant" CopB(Met)" protein" by" anionFexchange"
chromatography...................................................................................................." 100"
Appendix"4.4:" MALDIFTOF"mass"analysis"of"the"CopB(Met)"protein............................................." 101"
Appendix"4.5:" ESIFMS"spectrum"of"the"purified"CopB(Met)"protein............................................." 101"
Appendix"4.6:" SDSFPAGE" (4F20%)" showing" the" extraction" of" the" fullFlength" CopB" protein"
based"on"two"different"treatments......................................................................." 102"
Appendix"4.7:" Purification" of" the" recombinant" fullFlength" CopB" protein" expressed" in" E.* coli"
BL21(DE3)"by"gel"filtration"chromatography........................................................." 102"
Appendix"4.8:" SDSFPAGE" (4F20%)" showing" the" extraction" of" the" CopB251F495" protein" based" on"
two"different"treatments......................................................................................." 104"
Appendix"4.9:" Purification" of" the" recombinant" CopB251F495" protein" by" affinity"
chromatography...................................................................................................." 105"
Appendix"4.10:" Map" of" the" recombinant" plasmid" pJexpress411:56155" encoding" the"
recombinant"fullFlength"CopB"protein..................................................................." 106"
Appendix"4.11:" Full" genome" sequence" of" the" recombinant" plasmid" pJexpress411:56155"
encoding"the"recombinant"fullFlength"CopB"protein............................................." 107"
X"
"
Appendix"4.12:" Map" of" the" recombinant" plasmid" pJexpress411:34700" encoding" the""
recombinant"CopB(Met)"protein..........................................................................." 108"
Appendix"4.13:" Full" genome" sequence" of" the" recombinant" plasmid" pJexpress411:34700"
encoding"the"recombinant"CopB(Met)"protein....................................................." 109"
Appendix"4.14:" Map" of" the" recombinant" plasmid" pJexpress411:56156" encoding" the"
recombinant"CopB251F495"protein............................................................................" 110"
Appendix"4.15:" Full" genome" sequence" of" the" recombinant" plasmid" pJexpress411:56156"
encoding"the"recombinant"CopB251F495"protein......................................................" 111"
Appendix"4.16:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)" spectra" of" the"
native"peptide"and"its"Cu(II)"adducts....................................................................." 112"
Appendix"4.17:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep." 1"
and"its"Cu(II)"adducts............................................................................................." 113"
Appendix"4.18:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep."2"
and"its"Cu(II)"adducts............................................................................................." 114"
Appendix"4.19:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep."3"
and"its"Cu(II)"adducts............................................................................................." 115"
Appendix"4.20:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)" spectra" of" the"
native"peptide"and"its"Cu(I)"adducts......................................................................" 116"
Appendix"4.21:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep."1"
and"its"Cu(I)"adducts.............................................................................................." 117"
Appendix"4.22:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep."2"
and"its"Cu(I)"adducts.............................................................................................." 118"
Appendix"4.23:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep."3"
and"its"Cu(I)"adducts.............................................................................................." 119"
Appendix"4.24:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)" spectra" of" the"
double"motif"peptide"and"its"Cu(I)"adducts..........................................................." 120"
Appendix"4.25:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)" spectra" of" the"
native"peptide"and"its"Ag(I)"adducts......................................................................" 121"
Appendix"4.26:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep."1"
and"its"Ag(I)"adducts.............................................................................................." 122"
Appendix"4.27:" Positive" electrospray"ionization"mass"spectrometry"(ESIFMS)"spectra"of"Pep."2"
and"its"Ag(I)"adducts.............................................................................................." 123"
Appendix"4.28:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)" spectra"of"Pep."3"
and"its"Ag(I)"adducts.............................................................................................." 124"
XI"
"
Appendix"4.29:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)" spectra" of" the"
double"motif"peptide"and"its"Ag(I)"adducts..........................................................." 125"
Appendix"4.30:" Positive" electrospray" ionization" mass" spectrometry" (ESIFMS)"spectra"of"Pep."2"
and"its"Zn(II)"adducts.............................................................................................." 126"
Appendix"4.31:" Positive" electrospray" ionization" mass" spectrometry"(ESIFMS)"spectra"of"Pep."3"
and"its"Zn(II)"adducts.............................................................................................." 127"
Appendix"4.32:" NanoFESIFMS"titration"experiments"of"20"µM"synthetic"peptide"with"increasing"
amounts"of"Cu(II)"ions............................................................................................" 128"
Appendix"4.33:" NanoFESIFMS"titration"experiments"of"20"µM"synthetic"peptide"with"increasing"
amounts"of"Cu(I)"ions............................................................................................." 129"
Appendix"4.34:" NanoFESIFMS"titration"experiments"of"20"µM"synthetic"peptide"with"increasing"
amounts"of"Ag(I)"ions............................................................................................." 130"
Appendix"4.35:" 1HFNMR" spectra" of" the" native" peptide" collected" at" 500" MHz" in" D2O" in" the"
absence"and"presence"of"various"molar"equivalents"of"Cu(I)................................" 131"
Appendix"4.36:" 1HFNMR"spectra"of"Pep."1"(H9"→"A)"collected"at"500"MHz"in"D2O"in"the"absence"
and"presence"of"various"molar"equivalents"of"Cu(I).............................................." 132"
Appendix"4.37:" 1HFNMR" spectra" of" Pep." 2" (M4" →" G)" collected" at" 500" MHz" in" D2O" in" the"
absence"and"presence"of"various"molar"equivalents"of"Cu(I)................................" 133"
Appendix"4.38:" 1HFNMR" spectra" of" Pep." 3" (M12" →" G)" collected" at" 500" MHz" in" D2O" in" the"
absence"and"presence"of"various"molar"equivalents"of"Cu(I)................................" 134"
Appendix"4.39:" 1HFNMR" spectra" of" the" native" peptide" collected" at" 500" MHz" in" D2O" in" the"
absence"and"presence"of"various"molar"equivalents"of"Ag(I)................................" 135"
Appendix"4.40:" 1HFNMR"spectra"of"Pep."1"(H9"→"A)"collected"at"500"MHz"in"D2O"in"the"absence"
and"presence"of"various"molar"equivalents"of"Ag(I).............................................." 136"
Appendix"4.41:" 1HFNMR" spectra" of" Pep." 2" (M4" →" G)" collected" at" 500" MHz" in" D2O" in" the"
absence"and"presence"of"various"molar"equivalents"of"Ag(I)................................" 137"
Appendix"4.42:" 1HFNMR" spectra" of" Pep." 3" (M12" →" G)" collected" at" 500" MHz" in" D2O" in" the"
absence"and"presence"of"various"molar"equivalents"of"Ag(I)................................" 138"
(
(
XII"
"
List(of(Tables(
A.(Main(Tables(
Table"1:"
Summary"table"of"the"different"cop/pco"genes.........................................................." 39"
Table"4.1:" Amino"acid"sequences"of"synthetic"peptides.............................................................." 69"
Table"4.2:" KD"values"determined"by"electrospray"ionization"mass"spectrometry"(ESIFMS)"for"
1:1"peptideFmetal"complexes....................................................................................." 70"
Table"4.3:" Difference" of" 1HFNMR" chemical" shifts" (in" Hz)" derived" from" the" methionineFrich"
peptides" in" the" absence" and" presence" of" 3" molar" equivalents" of" Ag(I)" or" Cu(I)"
ions.............................................................................................................................." 73"
Table"4.4:" List"of"the"potential"CopBFinteracting"partners.........................................................." 77"
Table"5.1:" OMV"production"appears"specific"to"copper"stress...................................................." 146"
Table"5.2:" List"of"the"Cop"proteins"identified"from"the"proteomic"analysis"of"OMVs.................." 150"
B.(Supplemental(Tables(
Table"S5.1:" List"of"the"vesicular"proteins"identified"using"the"ProteinPilotTM"software"(v.4.1)....." 168"
Table"S5.2:" List"of"the"bacterial"strains"used"in"the"present"study................................................" 180"
"
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"
XIII"
"
List(of(Abbreviations(
AA:"
Amino"acids"
ABC:"
ATPFbinding"cassette"
ADP:"
Adenosine"diphosphate"
AFM:"
Atomic"Force"Microscopy"
AgNO3:"
Silver"nitrate"
ATP:"
Adenosine"triphosphate"
BCA:"
Bicinchoninic"acid"
BSA:"
Bovine"serum"albumin"
CD:"
Circular"Dichroism"
CDF:"
Cation"Diffusion"Facilitator"
CO2:"
Carbon"dioxide"
COG:"
Cluster"of"Orthologous"Group"
CTD:"
Cytosolic"CFterminal"domain"
CuCl2"•"2H2O:"
Copper(II)"chloride"dihydrate"
Cu2CO3(OH)2:"
Malachite"
Cu3(CO3)2(OH)2"
Azurite"
CuFeS2:"
Chalcopyrite"
Cu(NO3)2"•"3H2O:" Copper(II)"nitrate"trihydrate"
Cu2O:"
Cuprite"
Cu2S:"
Chalcocite"
Cys:"
Cysteine"(also""C")"
D2O:"
Deuterium"oxide"(also""heavy"water")"
DNA:"
Deoxyribonucleic"Acid"
DMT"modulus:"
Derjaguin,"Muller"and"Toporov"model"
EDTA:"
Ethylenediaminetetraacetic"acid"
emPAI:"
Exponentially"modified"protein"abundance"index"
XIV"
"
EPR:"
Electron"Paramagnetic"Resonance"
ESIFMS:"
Electrospray"IonizationFMass"Spectrometry"
EXAFS:"
Extended"XFRay"Absorption"Fine"Structure"
Fe(OH)3:"
Iron(III)"hydroxide"
gtr:"
Glycosyltransferase"
H2:"
Hydrogen"
H2Asc:"
Ascorbic"acid"
H2O:"
Water"
HAE:"
Hydrophobic"and"amphiphilic"efflux"
His:"
Histidine"(also""H")"
HME:"
HeavyFmetal"efflux"
HNO3:"
Nitric"acid"
H2O2:"
Hydrogen"peroxide"
HSAB:"
Hard"and"Soft"Acids"and"Bases"
ICPFMS:"
Inductively"Coupled"Plasma"F"Mass"Spectrometry"
IgG:"
Immunoglobulin"G"
IGEPAL"CAF630:"
(Octylphenoxy)polyethoxyethanol"
IM:"
Inner"Membrane"
IMAC:"
Immobilized"metal"ion"affinity"chromatography"
IPTG:"
isopropyl"βFDF1Fthiogalactopyranoside"
KD:"
Dissociation"Constant"
LCFMS/MS:"
Liquid"Chromatography"with"Tandem"Mass"Spectrometry"Detection"
LPS:"
Lipopolysaccharide"
MALDI:"
MatrixFAssisted"Laser"Desorption/Ionization"
Met:"
Methionine"(also""M")"
MFS:"
Major"Facilitator"Superfamily"
MFP:"
Membrane"Fusion"Protein"
XV"
"
MIC:"
Minimal"Inhibitory"Concentration"
MOPS:"
4FMorpholinepropanesulfonic"acid""
MS:"
Mass"Spectrometry"
N2:"
Nitrogen"
N2O:"
Nitrous"oxide"
N2OR:"
Nitrous"oxide"reductase"
NaCl:"
Sodium"chloride"
NAD:"
Nicotinamide"adenine"dinucleotide"
NBD:"
NucleotideFbinding"domain"
NH2OH:"
Hydroxylamine"solution"
NH4HCO3:"
Ammonium"bicarbonate"
NiCl2"•"6H2O:"
Nickel(II)"chloride"hexahydrate"
NMR:"
Nuclear"Magnetic"Resonance"
NPF40:"
Nonyl"phenoxypolyethoxylethanol"
NTA:"
Nitrilotriacetic"acid"
O2:"
Oxygen"
OD600:"
Optical"density"at"600"nm"
OM:"
Outer"Membrane"
OMF:"
Outer"Membrane"Factor"
OMV:"
Outer"Membrane"Vesicle"
OsO4:"
Osmium"tetroxide"
PBS:"
Phosphate"buffered"saline"
PG:"
Peptidoglycan"
PHB:"
Polyhydroxybutyrate"
Pro:"
Proline"(also""P")"
QTOF:"
QuadrupoleFtimeFofFflight"mass"spectrometer"
Rhs:"
Rearrangement"hotspot"
XVI"
"
RND:"
ResistanceFNodulationFCell"Division"
SDSFPAGE:"
Sodium"Dodecyl"Sulfate"F"Polyacrylamide"Gel"Electrophoresis"
SEM:"
Scanning"Electron"Microscopy"
TBS:"
TrisFbuffered"saline"
TEM:"
Transmission"Electron"Microscopy"
TEMFEDX"
Transmission"Electron"Microscopy"coupled"with"Energy"Dispersive"XFray"analyzer"
TEV:"
Tobacco"etch"virus"
TIMM:"
TemperatureFInduced"Mutagenesis"and"Mortality"
TMD:"
Transmembrane"Domain"
TrisFHCl:"
Tris(hydroxymethyl)aminomethane"hydrochloride"
UHPLCFHRMS:"
"
Ultra" High" Performance" Liquid" ChromatographyFHigh" Resolution" Mass"
Spectrometer"
UV:"
Ultraviolet"
ZnCl2:"
Zinc"chloride"
"
XVII"
"
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!
General!Introduction!
___________________________________________________________________________!
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!
General!Introduction!
!
Chapter(1:( Metal(Ions(in(Biological(Systems(
1.1.(Occurrence(of(Metals(in(the(Environment(and(Definition(
The! Industrial! Revolution,! which! started! in! the! late! 18th! and! early! 19th! centuries,! had!
profound!beneficial!effects!in!many!areas.!In!addition!to!fundamental!changes!in!numerous!aspects!
of! daily! life,! anthropogenic! activities! have! been! shown! to! result! in! an! increase! in! environmental!
pollution! (Malik,! 2004).! The! release! of! toxic! industrial! contaminants! (e.g.,! organic! pollutants,!
hydrocarbons,!metals,!etc.)!in!the!air,!water!and!soil!may!be!poisonous!or!toxic!and!lead!to!harm!to!
living! organisms! in! these! polluted! biotopes! (Kabir! et! al.,! 2012;! Nagajyoti! et! al.,! 2010).! Contrary! to!
organic!components,!which!may!be!degraded!into!less!harmful!compounds!by!various!biological!or!
chemical! processes,! metals! are! nonMbiodegradable! and! persistent! environmental! contaminants!
(Kabir! et! al.,! 2012;! Mejare! and! Bulow,! 2001;! Singh! et! al.,! 2011).! Pollution! by! metals! represents! a!
problem! of! increasing! significance! due! to! their! toxic! effects! and! accumulation! into! the! food! chain,!
leading!to!ecological,!nutritional!and!environmental!issues!(Malik,!2004;!Nagajyoti!et!al.,!2010).!
The! occurrence! of! metals! in! the! environment! results! from! both! natural! and! anthropogenic!
activities!(Nagajyoti!et!al.,!2010).!The!earth's!crust!is!naturally!made!of!metallic!elements,!which!are!
largely!found!in!dispersed!form!in!rock!formations!(Nagajyoti!et!al.,!2010).!Depending!on!the!type!of!
rock! and! the! weathering! process,! the! composition! and! concentration! of! heavy! metals! in! soil! will!
fluctuate! (Nagajyoti! et! al.,! 2010).! Other! natural! phenomena! (e.g.,! wind,! rain,! snowfall,! volcanic!
eruptions,!marine!aerosols,!forest!fires,!etc.)!also!contribute!to!metal!dispersal!in!many!environments!
(Nagajyoti!et!al.,!2010).!Even!green!plants!play!a!crucial!role!in!the!biogeochemical!cycles!of!metals!in!
the! soil! via! leaching! from! leaves! and! stems! and! from! decomposition! (Nagajyoti! et! al.,! 2010).!
Urbanization! and! industrialization! have! considerably! modified! the! geochemical! cycles! and!
biochemical! balance! of! natural! metallic! constituents! (Nagajyoti! et! al.,! 2010;! Singh! et! al.,! 2011).!
Indeed,! in! addition! to! natural! sources,! an! incredible! number! of! manMmade! processes! have!
contributed! to! the! contamination! of! extensive! areas! of! the! world! (Nagajyoti! et! al.,! 2010).! This!
pollution! includes! various! sources,! such! as! the! use! of! sewage! sludge! or! municipal! wastes,!
insecticides/fungicides/pesticides! and! fertilizers! in! agriculture;! metal! residues! from! smelting! and!
mining! processes;! emission,! effluents! and! solid! discharge! from! industries! involved! in! plastics! and!
textile!processing,!wood!preservation,!etc.;!metal!emission!during!the!use!of!any!vehicles;!domestic!
effluents! and! many! more! (Nagajyoti! et! al.,! 2010;! Singh! et! al.,! 2011).! Each! of! those! contamination!
sources!has!its!own!damaging!effects.!
!
!
2!
!
General!Introduction!
!
!
3!
!
General!Introduction!
!
Metals!are!the!most!important!group!of!elements! constituting!the!periodic!table;!metals!are!
distinguished!from!other!chemical!elements!(nonmetals!and!metalloids)!by!their!physical!properties!
(Appenroth,!2010).!In!contrast!to!nonmetals,!metals!in!their!elemental!form!are!usually!solids,!strong!
and!hard!at!room!temperature,!with!the!exception!of!mercury.!These!good!conductors!of!heat!and!
electricity,! which! are! characterized! by! their! metallic! luster,! are! also! malleable! and! ductile!
(Appenroth,! 2010).! From! a! chemical! point! of! view,! the! term! "metal"! encompasses! any! of! the!
elements! able! to! readily! lose! electrons! to! form! positive! ions.! Furthermore,! each! metal! element! is!
defined! according! to! its! own! position! in! the! periodic! table.! Metals! play! an! essential! role! in! the!
nutrition! of! living! organisms;! however,! metals! are! not! accessible! to! organisms! in! their! native! form!
(i.e.,! valence! state! of! 0)! (Appenroth,! 2010).! Metals! are! only! bioavailable! to! organisms! when! in!
solution.! Consequently,! metals! must! react! with! other! elements,! called! ligands,! to! form! metal!
complexes! that! can! subsequently! be! solubilized,! thus! making! metals! available! for! use! (Appenroth,!
2010).!Notably,!during!this!chemical!transformation,!metals!completely!lose!the!physical!properties!
originally! associated! with! their! elemental! form! (Appenroth,! 2010).! By! contrast,! the! chemical!
properties! within! metal! complexes! remain! generally! related! to! their! elemental! form! and,!
consequently,!to!their!position!in!Mendeleev's!periodic!table!(Appenroth,!2010).!
Based!on!similar!arrangements!of!their!last!electron!subshell!in!the!atom!and,!consequently,!
on!similarities!in!chemical!properties,!metal!elements!can!be!subdivided!into!four!broad!categories!
(Appenroth,!2010;!Duffus,!2002).!These!subgroups!include!the!s%block!with!alkali!metals!(e.g.,!Li,!Na,!
K,! etc.)! and! alkaline! earth! metals! (e.g.,! Be,! Mg,! Ca,! etc.);! the! p%block,! which! is! a! mixed! group! of!
metals,!metalloids!and!nonmetals;!the!f%block,!also!known!as!rare!earth!metals!(i.e.,!lanthanide!and!
actinide! series);! and! the! d%block! of! transition! metals! (Fig.! 1.1)! (Appenroth,! 2010;! Duffus,! 2002).!
Transition! metals,! which! are! characterized! by! incompletely! filled! d! orbitals,! play! a! crucial! role! in!
numerous! biological! processes! (Nies,! 2007).! Because! electrons! from! unfilled! dMshells! are! loosely!
bound,! transition! metal! elements! are! able! to! exhibit! a! wide! range! of! oxidation! states.! These!
positively! charged! forms! allow! transition! metals! to! form! coordination! complexes! with! nonmetal!
ligands!containing!free!electron!pairs!(e.g.,!nitrogen,!oxygen,!or!sulfur!atoms).!The!formation!of!such!
complex! compounds! and,! particularly,! reactions! catalyzed! by! these! compounds,! highlight! an!
important!facet!of!the!role!of!transition!metals!in!biology!(Nies,!2007).!
Now! consider! the! definition! of! "heavy! metals".! This! obscure! term! has! been! commonly! used!
over! the! last! few! decades,! and! negative! connotations! of! contamination! and! toxicity! are! largely!
associated! with! this! term! in! many! people's! minds! (Duffus,! 2002).! According! to! the! authors,! many!
meanings! can! be! found! in! the! literature,! leading! to! general! confusion;! however,! no! authoritative!
definition! actually! exists! (Duffus,! 2002).! This! controversial! term! is! based! on! a! classification! of! both!
4!
!
General!Introduction!
!
the! elemental! metals! and! their! compounds! according! to! their! density! (Duffus,! 2002).! However,! no!
relation! exists! between! the! density! of! a! metal! and! its! toxicity! or! even! the! physicochemical!
characteristics!of!its!compounds!(Appenroth,!2010;!Duffus,!2002).!Consequently,!defining!this!term!in!
a!better!way,!avoiding!the!use!of!density,!is!important!because!density!is!not!a!biologically!relevant!
property! (Appenroth,! 2010;! Duffus,! 2002).! Understanding! the! bioavailability! of! metallic! elements,!
their! ions! and! their! compounds,! which! relies! on! their! physicochemical! properties! and! on!
environmental!factors!(i.e.,!pH,!temperature,!oxidationMreduction!potential!of!the!environment,!etc.),!
is! important! for! more! realistically! assessing! their! potential! toxicity! (Duffus,! 2002;! Hobman! et! al.,!
2007).! Based! on! such! considerations,! Duffus! suggested! in! his! paper! published! in! 2002! that! a!
categorization! of! metals! with! relevance! to! toxicity! must! ideally! be! based! on! both! the! chemical!
properties!of!metals!and!their!compounds!and!biological!parameters!(Duffus,!2002).!However,!thus!
far,! knowledge! regarding! the! relation! between! metal! ions! and! their! physiological! or! toxicological!
effects! on! organisms! remains! limited! (Duffus,! 2002).! Currently,! only! a! classification! of! metals! and!
their!compounds!firmly!based!on!their!chemical!properties!appears!to!be!the!most!appropriate!and!
scientifically!sound!method!for!discriminating!metals!(Duffus,!2002).!Nevertheless,!notably,!chemical!
properties! cannot! predict! the! toxic! effects! of! metal! ions! on! living! systems;! however,! chemical!
properties! remain! the! best! basis! for! explaining! the! biochemical! interactions! of! metal! ions! with!
cellular!components!(e.g.,!proteins,!water,!nucleic!acids,!etc.)!(Appenroth,!2010).!
Overall,!the!term!"heavy!metal",!which!is!commonly!used!by!the!scientific!community,!should!
clearly! be! avoided.! However,! considering! that! expecting! people! to! suddenly! abandon! this! term! is!
unrealistic,!this!term!should!be!defined!in!a!rigorous!way.!Any!definition!of!"heavy!metal"!should!be!
based! on! Mendeleev's! periodic! table,! which! describes! the! atomic! structure! of! each! element.! The!
chemical!properties!of!a!metal!ion!determine!the!possibilities!of!complex!formation.!According!to!the!
Lewis!concept,!metal!ions!are!Lewis!acids!(i.e.,!are!able!to!accept!electrons),!which!can!be!classified!
as!either!"hard",!"soft"!or!"intermediate".!Hard!metal!ions!(e.g.,!Mg2+,!Ca2+,!Na+,!etc.)!primarily!form!
ionic!complexes!with!oxygenMcontaining!ligands,!whereas!soft!Lewis!acids!(e.g.,!Ag+,!Cd2+,!Hg2+,!etc.)!
covalently! bind! to! sulfurM! and! nitrogenMcontaining! ligands.! Intermediate! metal! ions! (e.g.,! Zn2+,! Cu2+,!
etc.),!as!their!name!suggests,!form!complexes!with!oxygenM,!nitrogenM!and!sulfurMcontaining!ligands!
(Appenroth,! 2010;! Duffus,! 2002;! Hobman! et! al.,! 2007).! Based! on! this! classification,! predicting! the!
preferred! metal! ligands! and! the! general! trend! in! the! properties! of! metal! complexes! is! possible.!
However,!a!metal!is!not!toxic!per0se;!toxic!effects!induced!on!living!systems!are!always!dependent!on!
the!metal!concentration!available!to!cells.!The!threshold!toxic!concentrations!differ!for!each!metal.!
For! example,! some! metals! called! micronutrients! are! crucial! to! cells! and! are! only! required! in! trace!
5!
!
General!Introduction!
!
amounts;!however,!when!their!concentration!exceeds!a!threshold,!then!these!metals!become!toxic!
and!induce!harmful!interferences!(Appenroth,!2010).!
(
1.2.(Bacterial(Transport(Systems:(EffluxCMediated(Resistance(
Bacteria!require!metal!ions!to!perform!a!wide!variety!of!cellular!functions,!from!stabilization!of!
biological! molecules! to! structural! and! catalytic! roles! in! enzymes! and! proteins! (Bruins! et! al.,! 2000).!
Although!some!metal!ions!are!essential!for!life,!most!metals!are!nonessential!and!potentially!toxic!to!
microorganisms! (Bruins! et! al.,! 2000;! Summers,! 2009).! However,! at! high! concentrations,! both!
beneficial! and! nonessential! metal! ions! can! be! deleterious! and! lead! to! toxicity! often! through!
generating! oxygen! radicals,! which! can! damage! cellular! components! (Bruins! et! al.,! 2000;! Summers,!
2009).!Consequently,!tight!regulation!of!metal!ion!homeostasis!systems!is!important!to!avoid!metal!
ion!stress,!which!can!result!in!either!excessive!or!insufficient!amounts!of!metal!ions!in!cells!(Hobman!
et!al.,!2007).!Homeostasis!systems!ensure!an!optimal!bioavailable!concentration!of!each!metal!ion,!
allowing! the! cellular! requirements! of! metal! ions! to! be! met! while! simultaneously! minimizing! their!
potential!toxic!effects!(Bleackley!and!Macgillivray,!2011;!Ma!et!al.,!2009).!Metal!ion!homeostasis!is!
maintained!through!highly!regulated!processes!of!efficient!metal!uptake!and!intracellular!trafficking,!
storage!and!efflux!(Bleackley!and!Macgillivray,!2011;!Ma!et!al.,!2009).!Indeed,!in!each!compartment,!
a! broad! set! of! both! low! and! high! affinity! metal! ion! transporters! function! in! concert! to! provide! a!
delicate!balance!of!transport!activities!across!their!membranes!and!to!maintain!adequate!metal!ion!
concentrations.!
Selective!pressure!from!metal!ion!contamination!compelled!microorganisms!to!evolve!various!
resistance! strategies! to! respond! to! metal! ion! stress! encountered! in! their! biotopes.! Such! resistance!
mechanisms!are!mediated!by!genes!generally!on!chromosomes,!plasmids,!or!even!transposons!and!
can!be!constitutively!expressed!or!inducible!(Bruins!et!al.,!2000).!The!processes!involved!in!the!first!
line!of!defense!include!metal!exclusion!by!permeability!barrier,!intraM!and!extracellular!sequestration!
by! chelation! with! binding! proteins,! enzymatic! detoxification! converting! a! toxic! metal! ion! to! a! less!
toxic!or!less!available!form,!or!reduction!in!metal!sensitivity! by!cellular!targets!(Bruins!et!al.,!2000;!
Hobman! et! al.,! 2007).! These! basic! metal! resistance! mechanisms! are! enhanced! by! specific! defense!
systems,! which! involve! efflux! as! a! stratagem.! Metal! ion! transport! across! the! lipid! bilayer! occurs!
either! by! passive! or! active! transport! through! three! classes! of! membrane! proteins,! which! share! a!
common!feature!of!having!several!transmembrane!domains:!channels,!carriers,!and!pumps!(Fig.!1.2)!
(Alberts!et!al.,!2007).!
6!
!
General!Introduction!
!
In! passive! transport! (i.e.,! simple! and! facilitated! diffusion),! according! to! the! second! law! of!
thermodynamics,! molecules! move! from! a! compartment! of! higher! concentration! to! one! of! lower!
concentration!(Fig.!1.2A,!B!and!C).!Consequently,!the!chemiosmotic!gradient!allows!solute!diffusion!
into!the!cell!without!expending!cellular!energy!(Davidson!et!al.,!2008;!Hobman!et!al.,!2007).!Notably,!
passive! transport! is! a! high! rate! phenomenon! which! is! relatively! nonspecific! of! metal! ion! nature!
(Hobman! et! al.,! 2007).! Conversely,! active! transport! requires! the! use! of! free! energy,! such! as! ATP!
hydrolysis,!to!drive!metal!ion!uptake!(Fig.!1.2D).!This!process!is!slower!than!passive!diffusion!but!is!
much! more! specific! of! a! given! substrate! (Hobman! et! al.,! 2007).! Two! categories! of! active! transport!
systems!can!be!distinguished!according!to!the!source!of!energy!used!to!drive!the!transport!process:!
primary!active!transport!and!secondary!active!transport!(Davidson!et!al.,!2008).!
!
(
Figure( 1.2:! Transport! of! solutes! across! the! membrane,! comparison! between! passive! and! active!
transport.!(A.)!Simple!diffusion!is!the!unassisted!diffusion!of!solutes!through!the!lipid!bilayer,!no!energy!
is! required.! (B.,! C.)! Passive! transport! (facilitated! diffusion)! occurs! either! by! channelMmediated! (B.)! or!
carrierMmediated!(C.)!diffusion,!and!does!not!require!energy.!(D.)!Active!transport!is!always!mediated!by!
specific!carrier!proteins;!the!transport!of!solutes!across!the!membrane!occurs!against!a!concentration!
gradient!(E.)!and!requires!an!input!of!energy.!(Figure!modified!from!Alberts!et!al.,!2007)!
!
Primary! active! transport! is! a! process! in! which! free! energy! from! ATP! hydrolysis! is! directly!
required!to!drive!the!movement!of!ions!against!their!concentration!gradients!(Davidson!et!al.,!2008;!
Oliveira!et!al.,!2011).!Two!major!types!of!ATPMdriven!efflux!pumps!are!well!known!and!characterized;!
these! pumps! include! ATPMbinding! cassette! (ABC)! transporters! and! PMtype! ATPases.! Both! of! these!
pumps! are! known! to! undergo! conformational! changes! following! ATP! binding! and! hydrolysis,! which!
allows!the!unidirectional!pumping!of!a!bound!substrate!across!the!cellular!membrane!(Davidson!et!
al.,!2008;!Kuhlbrandt,!2004).!The!following!section!is!a!short!overview!of!the!two!aboveMmentioned!
primary!efflux!transporter!families.!
7!
!
General!Introduction!
!
ABC(systems:!Widespread!among!the!three!kingdoms!of!life!(i.e.,!bacteria,!archaea!and!eukaryotes),!
ABC! transporters! are! involved! in! numerous! physiological! processes,! from! antibiotic! and!
antifungal!resistance!in!microorganisms!to!genetic!diseases!and!anticancer!drug!resistance!in!
humans! (Davidson! et! al.,! 2008;! Higgins,! 2001;! Oliveira! et! al.,! 2011).! This! variety! of!
physiological! roles! reflects! diversity! in! substrate! specificity! (Davidson! et! al.,! 2008;! Higgins,!
2001).! Indeed,! being! relatively! specific! for! their! own! particular! substrate(s),! ABC! exporters!
are! able! to! extrude! a! myriad! of! molecules! through! the! lipid! membrane,! such! as! peptides,!
lipids,!inorganic!ions,! hydrophobic!drugs,!polysaccharides!and!toxins!(Davidson!et!al.,!2008;!
Higgins,!2001;!Oliveira!et!al.,!2011).!
Each! ABC! transporter! shares! a! common! architecture! consisting! of! four! core! domains:! two!
highly! hydrophobic! transmembrane! domains! (TMDs)! and! two! hydrophilic! ATPMbinding!
domains,!which!are!called!nucleotideMbinding!domains!(NBDs)!(Davidson!et!al.,!2008;!Higgins,!
2001;! Oliveira! et! al.,! 2011).! The! two! TMDs! span! the! membrane! via! putative! αMhelices! that!
form! a! large! pore! through! which! substrates! cross! the! membrane! (Davidson! et! al.,! 2008;!
Higgins,!2001).!The!selectivity!of!ABC!exporters!for!a!solute!is!made!through!specific!binding!
sites!within!these!TMDs!(Davidson!et!al.,!2008;!Higgins,!2001).!Unlike!ABC!importers!where!
NBDs! are! associated! with! the! cytoplasmic! surface! of! the! membrane;! the! NBDs! of! ABC!
exporters!lie!far!from!the!membrane,!separated!by!an!extramembranous!domain!(Davidson!
et!al.,!2008;!Oliveira!et!al.,!2011).!These!binding!domains!supply!energy!for!active!transport!
through!ATP!binding!and!hydrolysis!(Davidson!et!al.,!2008;!Higgins,!2001).!
Although! the! detailed! mechanisms! of! transport! remain! unclear,! currently,! the! first! step! of!
the!catalytic!cycle!is!proposed!to!be!initiated!subsequent!to!the!specific!interaction!between!
the! substrate! and! binding! domains! in! the! central! cavity! formed! by! the! TMDs! (Fig.! 1.3A)!
(Davidson!et!al.,!2008;!Higgins,!2001).!Following!this!binding,!ATPMbinding!cassette!domains!
have!an!increased!affinity!for!ATP!molecules!(Fig.!1.3B)!(Davidson!et!al.,!2008;!Higgins,!2001).!
Each! nucleotideMbinding! domain! binds! a! single! ATP! molecule,! inducing! conformational!
changes!that!lead!to!the!closure!of!the!catalyticMbinding!interface!(Fig.!1.3C)!(Davidson!et!al.,!
2008;! Oliveira! et! al.,! 2011).! ATP! hydrolysis! occurs! in! this! closed,! catalytically! active!
conformation,!inducing!structural!rearrangements!in!both!the!NBDs!and!the!TMDs!(Davidson!
et!al.,!2008;!Oliveira!et!al.,!2011).!The!energy!transduction!coupled!to!these!conformational!
modifications!allows!the!release!of!the!substrate!outside!the!membrane!(Fig.!1.3D)!(Davidson!
et! al.,! 2008;! Oliveira! et! al.,! 2011).! After! the! hydrolysis! of! ATP! molecules,! the! catalytic!
monomers! move! apart! and! release! the! postMhydrolysis! products! (i.e.,! ADP! and! inorganic!
8!
!
General!Introduction!
!
phosphate),! allowing! the! reset! of! the! transporter! for! another! catalytic! cycle! (Fig.! 1.3E)!
(Davidson!et!al.,!2008;!Oliveira!et!al.,!2011).!
(
Figure( 1.3:! Model! illustrating! the! different! steps! of! ABC! exporter! mechanism.! (A.)! A! specific! substrate! enters! in!
the! channel! which! is! open! toward! the! inside! of! the! cell,! and! binds! to! the! binding! domains! in! the! central! cavity!
formed! by! the! TMDs! (the! binding! domains! are! in! bold).! (B.)! The! substrate! binding! is! followed! by! an! increased!
affinity!for!ATP!molecules!in!the!NBDs.!(C.)!ATP!binding!induces!conformational!changes!and!the!closure!of!the!
catalyticMbinding!interface.!(D.)!Conformational!changes!are!transmitted!from!the!NBDs!with!the!hydrolysis!of!ATP!
which! allows! the! release! of! the! substrate! to! the! other! side! of! the! membrane.! (E.)! ATP! hydrolysis! resets! the!
transporter! to! its! initial! state! and! this! step! is! associated! with! the! release! of! postMhydrolysis! products.! (Figure!
modified!from!Berg!et!al.,!2012)!
!
PCtype( ATPases:! Integral! transmembrane! proteins,! which! are! members! of! the! large! PMtype! ATPase!
superfamily,! use! the! energy! stored! in! ATP! to! pump! out! specific! inorganic! cations! (e.g.,! H+,!
Na+,!K+,!Cu+,!Ag+,!Mg2+,!Ca2+,!etc.)!and!lipids!across!cellular!membranes!(Bublitz!et!al.,!2011;!
Kuhlbrandt,! 2004;! Palmgren! and! Nissen,! 2011).! Omnipresent! in! all! living! organisms! from!
bacteria! to! humans,! this! family! of! transport! proteins! functions! in! many! important! cellular!
processes!in!biology!and!medicine,!such!as!the!removal!of!toxic!ions!from!cells!(Bublitz!et!al.,!
2011;! Kuhlbrandt,! 2004).! Any! PMtype! ATPase! dysfunction! can! lead! to! severe! diseases;! one!
striking!example!is!mutations!occurring!in!the!human!genes!encoding!Cu+MATP7A!and!ATP7B,!
which! cause! Menkes! syndrome! and! Wilson's! disease,! respectively! (Bublitz! et! al.,! 2011;!
Kuhlbrandt,!2004).!Based!on!phylogenetic!analyses,!the!PMtype!ATPase!group!can!be!divided!
into!five!primary!subfamilies!(e.g.,!P1MP5),!wherein!different!subgroups!(e.g.,!A,!B,!etc.)!can!be!
distinguished!(Bublitz!et!al.,!2011;!Kuhlbrandt,!2004;!Palmgren!and!Nissen,!2011).!Each!class!
is!specific!for!particular!transported!ligands!and!differs!in!their!regulation!(Kuhlbrandt,!2004;!
Palmgren! and! Nissen,! 2011).! One! of! the! most! common! PMtype! ATPase! groups! in! bacteria! is!
the! group! of! P1BMATPase! pumps,! which! transport! transition! metals.! Members! of! this! group!
exhibit! specific! conserved! features,! such! as! CysM! or! HisMrich! metal! binding! motifs! at! the! NM
terminal! extremity! and! a! CysMProMCys! signature! sequence! in! the! sixth! transmembrane! helix!
(Osman! and! Cavet,! 2008;! Rosen,! 2002).! A! wellMcharacterized! example! of! P1BMATPases! is! the!
9!
!
General!Introduction!
!
CopA! copper! ATPase! of! Escherichia0 coli,! which! confers! resistance! to! Cu(I)! (Fig.! 1.4)! (Osman!
and!Cavet,!2008;!Rosen,!2002).!
Figure(1.4:!Model!of!the!CopA!P1BMtype!ATPase!
of! E.0 coli.! CopA! is! a! PMtype! ATPase! which!
pumps! Cu(I)! ions! from! the! cytoplasm! to! the!
periplasmic!space.!It!is!predicted!to!have!an!NM
terminal!region!with!two!cytosolic!CXXC!metal!
binding! domains! and! eight! transmembrane!
segments! (TMs).! The! actuator! domain! (AM
domain)!connects!the!TM4!and!TM5.!The!TM6!
contains! the! consensus! CPC! sequence! and! is!
connected! to! the! TM7! through! the!
phosphorylation! domain! (PMdomain),! the! ATPM
binding! domain! (NMdomain)! and! a! conserved!
sequence.! (Figure! modified! from! Rosen! et! al.,!
2002)!
!
All! PMtype! ATPases! are! multiMdomain! proteins,! which! have! common! biochemical!
characteristics.!Characterized!by!both!their!CM!and!NMtermini!in!the!cytoplasmic!space,!ATPase!
pumps!consist!of!three!wellMdefined!cytoplasmic!domains,!which!are!conserved!throughout!
the! PMtype! family,! and! are! structurally! and! functionally! distinct.! The! latter! are! called! the!
phosphorylation!(P),!nucleotideMbinding!(N),!and!actuator!(A)!domains!(Fig.!1.4)!(Bublitz!et!al.,!
2011;!Kuhlbrandt,!2004;!Palmgren!and!Nissen,!2011).!The!transmembrane!core!of!ATPases!is!
composed! of! at! least! six! hydrophobic! αMhelices,! which! surround! the! ionMbinding! sites!
embedded! in! the! membrane! domain! and! which! are! supplemented! in! most! cases! by!
additional! transmembrane! helices! (Bublitz! et! al.,! 2011;! Kuhlbrandt,! 2004;! Palmgren! and!
Nissen,!2011).!Notably,!the!PMdomain!is!the!catalytic!core!of!the!protein!due!to!the!presence!
of! a! highly! conserved! DKTGT! sequence! motif,! in! which! an! acidMstable! aspartic! residue! is!
reversibly! phosphorylated.! By! contrast,! the! dephosphorylation! process! involves! the! AM
domain,! which! contains! an! invariant! TGE! sequence! motif! (Bublitz! et! al.,! 2011;! Kuhlbrandt,!
2004;!Palmgren!and!Nissen,!2011).!
Advances!regarding!the!understanding!of!molecular!mechanisms!involved!in!ATPMdriven!ion!
transport!in!PMtype!ATPases!were!made!in!the!last!few!years,!allowing!the!establishment!of!a!
reaction! cycle! wherein! multiple! conformational! intermediates! occur.! The! following! section!
briefly!describes!the!classical!E1/E2!PostMAlbers!catalytic!cycle,!which!is!traditionally!used!to!
describe! the! PMtype! ATPase! working! mechanism.! In! this! model! the! enzyme! is! postulated! to!
alternate!between!two! primary!conformational!states,!called!E1!and!E2,!to!drive!the!active!
10!
!
General!Introduction!
!
transport! of! cations! against! an! electrochemical! gradient! (Fig.! 1.5)! (Bublitz! et! al.,! 2011;!
Kuhlbrandt,!2004;!Palmgren!and!Nissen,!2011).!The!E1!state!is!characterized!by!highMaffinity!
binding!site(s)!embedded!in!the!membrane!and!exposed!to!the!cytoplasmic!side!through!an!
access!channel.!Upon!substrate!binding,!the!protein!reacts!with!an!Mg2+MATP,!which!catalyzes!
enzyme! autoMphosphorylation,! generates! transmembrane! helix! rearrangements! occluding!
the! transported! ion(s)! in! the! membrane! domain,! and! leads! to! a! highMenergy! intermediate!
state! called! the! E1MP! state! (Bublitz! et! al.,! 2011;! Kuhlbrandt,! 2004;! Palmgren! and! Nissen,!
2011).!The!following!step,!called!the!E2MP!state,!is!a!lowMenergy!intermediate!state.!The!rateM
limiting! transition! from! the! E1MP! to! E2MP! state! is! characterized! by! conformational! changes!
that! destroy! the! highMaffinity! coordination! geometry! and! simultaneously! opens! an! ion! exit!
pathway! on! the! other! side! of! the! membrane.! In! addition! to! ion! diffusion,! the! E2MP!
conformation! binds! counterions! (i.e.,! ion! in! solution! that! associates! itself! with! an! ion! of!
opposite! charge! to! maintain! electric! neutrality)! from! the! periplasmic/extracellular! space,!
resulting!in!ion!channel!closure!(Bublitz!et!al.,!2011;!Kuhlbrandt,!2004;!Palmgren!and!Nissen,!
2011).!
Such! conformational! modifications! allow! the! hydrolysis! of! phosphorylated! Asp,! resulting! in!
the!E2!state!and!the!release!of!inorganic!phosphate.!The!return!to!a!relaxed!E1!conformation!
destroys!the!highMaffinity!counterion!binding!site(s)!in!the!transmembrane!domain!and!opens!
up!an!exit!channel!at!the!membrane!cytoplasmic!side,!which!leads!to!an!additional!substrate!
transport!cycle!(Bublitz!et!al.,!2011;!Kuhlbrandt,!2004;!Palmgren!and!Nissen,!2011).!
!
!
!
11!
!
Figure(1.5:!Schematic!overview!of!the!alternating!access!
mechanism! used! by! PMtype! ATPases! to! actively! pump!
substrates! against! an! electrochemical! gradient.! During!
each! catalytic! cycle,! the! pump! oscillates! between! four!
major!conformational!states:!E1,!E1MP,!E2MP,!and!E2.!Ions!
of!interest!(drawn!as!black!circles)!bind!to!a!highMaffinity!
site! in! the! E1! state! and! the! phosphorylation! of! the!
enzyme! by! an! ATP! nucleotide! leads! to! the!
phosphorylated! E1MP! state.! The! rateMlimiting! transition!
from! the! E1MP! to! E2MP! state! induces! conformational!
changes! generating! a! reduced! affinity! for! interest! ions!
which! escape! to! the! outside.! Then,! counterions! (drawn!
as! grey! circles)! bind! to! the! E2MP! state! resulting! in! the!
closure! of! the! ion! channel.! The! hydrolysis! of! the!
phosphorylated! Asp! induces! the! release! of! inorganic!
phosphate! leading! to! the! E2! sate.! The! last! step! of! this!
cycle! is! characterized! by! the! release! of! counterions! to!
the! interior! and! the! return! to! the! relaxed! E1! state.! The!
enzyme! is! then! ready! to! start! another! cycle.! (Figure!
modified!from!Bublitz!et!al.,!2011)!
General!Introduction!
!
Secondary! active! transport! (also! known! as! coupled! transport! or! coMtransport)! involves! two!
types!of!carrier!proteins,!symporters!and!antiporters,!which!transport!two!or!more!substrates!in!the!
same! direction! or! in! opposite! directions,! respectively.! In! contrast! to! primary! active! transport,!
secondary!active!transport!couples!the!movement!of!a!substance!against!its!concentration!gradient!
to! coMsubstrate! transport! down! its! electrochemical! gradient! (Davidson! et! al.,! 2008;! Forrest! et! al.,!
2011).!This!type!of!transport!is!designated!as!"secondary"!because!transporters!use!energy!stored!in!
coMsubstrate! electrochemical! gradients! to! drive! the! transmembrane! movement! of! interest!
substrates;!transporters!do!not!directly!consume!energy!derived!from!ATP!hydrolysis,!as!do!primary!
active!transporters!(Davidson!et!al.,!2008;!Forrest!et!al.,!2011).!The!following!section!lists!and!briefly!
describes! wellMcharacterized! secondary! transporters! that! belong! to! different! functional! families:!
cation!diffusion!facilitator!(CDF)!systems,!resistanceMnodulationMcell!division!(RND)!systems!and!major!
facilitator!superfamily!(MFS)!systems.!
(
CDF(transporters:!Ubiquitously!found!in!all!domains!of!life,!transport!proteins!belonging!to!the!cation!
diffusion! facilitator! family! (CDF,! also! known! as! the! cation! efflux! family)! are! implicated! in! a!
variety! of! functional! processes,! particularly! in! the! regulation! of! cellular! divalent! cation!
homeostasis!(Cubillas!et!al.,!2013;!Montanini!et!al.,!2007;!Zeytuni!et!al.,!2014).!In!contrast!to!
other! transporter! families,!all! CDF! substrates! known! thus! far! are! only! divalent! metals! (e.g.,!
Zn2+,!Cd2+,!Co2+,!Fe2+,!Mn2+,!or!Ni2+)!(Cubillas!et!al.,!2013;!Montanini!et!al.,!2007;!Zeytuni!et!al.,!
2014).! Those! latter! metals! are! translocated! across! cytoplasmic! and! organelles! membranes!
through! a! protonMdependent! antiport! mechanism! (Cubillas! et! al.,! 2013;! Montanini! et! al.,!
2007;! Zeytuni! et! al.,! 2014).! Originally! divided! into! three! substrateMspecific! subclasses! (i.e.,!
Zn2+MCDF,!Fe2+/Zn2+MCDF!and!Mn2+MCDF),!the!functional!classification!of!CDF!transporters!has!
been!recently!reviewed!and!extended!to!eighteen!clades!based!on!a!phylogenomic!approach!
(Cubillas! et! al.,! 2013;! Montanini! et! al.,! 2007;! Zeytuni! et! al.,! 2014).! The! majority! of! CDF!
transporters! are! involved! in! Zn2+! transport;! the! most! frequent! example! is! the! ferrousMiron!
efflux!pump!FieF!(formerly!YiiP)!from!E.0coli.!Belonging!to!the!Fe/ZnMCDF!subgroup,!FieF!is!a!
dimeric!Zn2+/H+!antiporter,!which!is!known!to!catalyze!active!efflux!of!Zn(II)!across!the!inner!
membrane!of!this!bacterium!(Coudray!et!al.,!2013;!Lu!et!al.,!2009).!
Although! high! variability! was! observed! in! both! sequence! and! size! of! CDF! proteins,! these!
proteins! have! structural! and! functional! similarities! (Lu! et! al.,! 2009;! Montanini! et! al.,! 2007;!
Zeytuni! et! al.,! 2014).! Most! CDF! transporters! are! generally! YMshaped! homodimers,! in! which!
each! protomer! shares! a! common! twoMmodular! architecture! consisting! of! a! transmembrane!
domain!(TMD)!and!a!cytosolic!CMterminal!domain!(CTD)!(Lu!et!al.,!2009;!Zeytuni!et!al.,!2014).!
12!
!
General!Introduction!
!
The!transmembrane!domain!exhibits!six!helical!segments!grouped!into!a!compact!fourMhelix!
bundle! (i.e.,! TM1,! TM2,! TM4,! and! TM5)! and! a! helix! pair! (i.e.,! TM3! and! TM6),! whereas! the!
cytosolic!CMterminal!domain!adopts!a!metallochaperoneMlike!fold,!which!allows!the!docking!of!
cytoplasmic! metal! carrier! proteins! and! metal! ion! transfer! at! the! metal! donorMacceptor!
interface!(Lu!et!al.,!2009;!Zeytuni!et!al.,!2014).!Other!structural!characteristics!specific!to!the!
CDF!family!include!a!conserved!signature!sequence!between!TM1!and!TM2,!as!well!as!some!
widely! conserved! aspartate! and! histidine! residues! within! the! transmembrane! domain! and!
likely!involved!in!cation!transport!and/or!uptake!(Haney!et!al.,!2005;!Montanini!et!al.,!2007).!
Although!numerous!details!relative!to!their!metal!transport!mechanism!remain!unclear,!CDF!
proteins! appear! to! function! as! proton/cation! antiporters! and! translocate! cations! from! the!
cytoplasmic!to!periplasmic!space!through!an!alternating!access!mechanism!(Fig.!1.6).!
!
2+
+
Figure( 1.6:! Alternating! access! mechanism! for! M /H ! antiport! by! Cation! Diffusion! Facilitator.! Bundles! of!
transmembrane! helices! (TMDs)! and! helix! pairs! are! drawn! as! grey! cylinders! and! light! grey! cylinder,!
respectively.!Cytosolic!CMterminal!domains!(CTDs)!are!drawn!as!grey!triangles.!(A.)!Upon!sufficient!cytoplasmic!
2+
metal! concentration,! CTDs! bind! divalent! metal! ions! (M ,! grey! circles)! and! undergo! conformational! changes!
toward! a! tighter! and! compact! fold.! Such! a! conformational! change! activates! the! metal! transport! through!
TMDs.! (B.)! During! metal! transport,! TMDs! undergo! alternate! conformational! changes! between! cytoplasmM
2+
facing!to!periplasmMfacing!conformation,!which!leads!to!the!release!of!M !ions!in!the!periplasm.!(C.,!D.)!Metal!
+
ion!release!is!followed!by!proton!entrance!(H ,!white!circles)!that!induces!the!reconversion!to!the!cytoplasmicM
facing!conformation.!Protons!are!then!released!in!the!cytoplasm,!which!completes!the!metal!transport!cycle.!
(Figure!modified!from!Zeytuni!et!al.,!2014)!
!
Metal! translocation! is! coupled! to! the! protonMmotive! force,! which! provides! the! energy!
required!for!the!transport!cycle.!This!process!is!used!by!many!secondary!transporter!families!
and!involves!inwardM!and!outwardMfacing!conformations!(Coudray!et!al.,!2013;!Zeytuni!et!al.,!
2014).!Recently,!Zeytuni!et0al.!improved!the!preMexisting!model!describing!the!CDF!mode!of!
action! (Zeytuni! et! al.,! 2014).! These! authors! suggested! that! the! CDF! CMterminal! domain!
undergoes!conformational!changes!upon!metal!cation!binding,!which!induce!a!more!compact!
conformation!and! which!activate!metal!transport!through!the!transmembrane!domain!(Fig.!
1.6A)!(Zeytuni!et!al.,!2014).!In!addition!to!metal!binding,!each!protomer!undergoes!a!unique!
13!
!
General!Introduction!
!
conformational! change,! subsequently! leading! to! the! scissoring! of! the! transmembrane!
domain!and!to!metal!release!on!the!periplasmic!side!(Fig.!1.6B)!(Coudray!et!al.,!2013;!Zeytuni!
et! al.,! 2014).! The! following! step! is! characterized! by! proton! entrance! to! the! binding! sites,!
which! favors! the! next! transition! from! the! periplasmicMfacing! conformation! to! the!
cytoplasmicMfacing! fold,! thus! enabling! another! transport! cycle! to! start! (Fig.! 1.6C! and! D)!
(Coudray! et! al.,! 2013;! Zeytuni! et! al.,! 2014).! Notably,! the! CDF! CMterminal! domain! does! not!
appear! to! be! affected! by! transmembrane! conformational! changes! (Coudray! et! al.,! 2013;!
Zeytuni!et!al.,!2014).!
(
RND(transporters:!ResistanceMnodulationMcell!division!(RND)!efflux!pumps!are!a!large!superfamily!of!
proteins!that!are!widely!distributed!across!all!major!kingdoms!of!life!and!that!are!involved!in!
many!important!physiological!functions!(AlvarezMOrtega!et!al.,!2013;!Kim!et!al.,!2011;!Routh!
et!al.,!2011).!Powered!by!the!protonMmotive!force,!RND!transporters!can!pump!a!wide!array!
of!substrates!directly!into!the!extracellular!space!(e.g.,!organic!substances,!transition!metals,!
polypeptides,! fatty! acids,! nodulation! factors,! etc.)! (AlvarezMOrtega! et! al.,! 2013;! Kim! et! al.,!
2011;! Routh! et! al.,! 2011).! Based! on! phylogenetic! analyses,! the! RND! superfamily! can! be!
divided! into! at! least! seven! subfamilies! (Kim! et! al.,! 2011;! Nies,! 2003).! In! GramMnegative!
bacteria,! two! important! RND! subgroups! have! been! widely! studied:! the! hydrophobic! and!
amphiphilic!efflux!family!(HAEMRND)!and!the!heavyMmetal!efflux!family!(HMEMRND)!(Kim!et!al.,!
2011;! Long! et! al.,! 2012;! Nies,! 2003).! The! best! characterized! examples! of! HAEMRND! proteins!
are! AcrB! from! E.0 coli! and! MexR! from! P.0 aeruginosa,! which! are! multidrug! efflux! pumps!
involved!in!the!detoxification!of!antibiotics,!bile!acids,!or!hydrophobic!compounds!(Kim!et!al.,!
2011;!Nies,!2003).!Unlike!HAEMRND!proteins,!HMEMRND!members!mediate!the!efflux!of!highly!
specific! transition! metal! cations! and! can! even! differentiate! monovalent! from! divalent! ions!
(Kim! et! al.,! 2011;! Long! et! al.,! 2012).! The! primary! studied! HMEMRND! transporters! are! CusA!
from! E.0 coli,! which! specifically! confers! resistance! to! Cu(I)! and! Ag(I),! and! CzcA! from! C.0
metallidurans!CH34,!which!expels!Co(II),!Zn(II)!and!Cd(II)!(Long!et!al.,!2012;!Nies,!2003).!
In! GramMnegative! bacteria,! RND! transporters! are! part! of! a! tripartite! efflux! complex,! which!
spans! the! complete! cell! wall! from! the! inner! to! the! outer! membrane! (Fig.! 1.7)! (Kim! et! al.,!
2011;!Long!et!al.,!2012).!RND!proton/substrate!antiporters!follow!a!homotrimeric!(e.g.,!CusA!
from! E.0 coli)! or! heterotrimeric! (e.g.,! ArcB! from! E.0 coli)! organization,! wherein! each! RND!
subunit! generally! consists! of! 12! transmembrane! αMhelices! along! with! two! large! hydrophilic!
loops!(Kim!et!al.,!2011;!Long!et!al.,!2012).!
14!
!
General!Introduction!
!
Figure( 1.7:! Structure! of! an! RND! efflux! pump.! The!
pump! is! a! tripartite! system! consisting! of! an! RND!
cytoplasmic! membrane! transporter! (RND),! a!
membrane! fusion! protein! (MFP)! and! an! outer!
membrane! factor! (OMF).! Such! a! complex! forms! a!
channel!spanning!the!entire!membrane!and!allows!
the!protonMdriven!transport!of!various!compounds!
(black! circles)! from! the! periplasmic! space! to! the!
extracellular!space.!When!the!compound!is!bound,!
the! pump! turns! on! and! pushes! it! out! of! the! cell.!
(Figure!modified!from!AlvarezMOrtega!et!al.,!2011)!
!
The!RND!trimeric!ringMlike!structure!is!anchored!in!the!inner!membrane!and!largely!extends!
into!the!periplasmic!space!(Kim!et!al.,!2011;!Long!et!al.,!2012).!The!periplasmic!portion!of!the!
RND! trimer! is! bridged! to! a! trimeric! outer! membrane! factor! (OMF),! which! is! located! as! a! βM
barrel!in!the!outer!membrane!and!extending!into!the!periplasm!(Kim!et!al.,!2011;!Long!et!al.,!
2012).! This! connection! is! made! possible! through! the! presence! of! periplasmic! adaptor!
proteins!(also!described!as!membrane!fusion!proteins,!MFPs),!which!form!a!hexameric!ring!
around!the!RND!and!OMF!trimers!and!which!stabilize!the!contact!(Kim!et!al.,!2011;!Long!et!
al.,! 2012).! The! continuous! channel! formed! by! these! three! components! catalyzes! the! active!
efflux! of! many! substances! directly! into! the! external! medium! (AlvarezMOrtega! et! al.,! 2013;!
Long! et! al.,! 2012;! Routh! et! al.,! 2011).! This! transport! system! is! energized! through! the!
electrochemical! proton! gradient! occurring! in! transmembrane! domains! of! the! RND! trimer!
(AlvarezMOrtega!et!al.,!2013;!Long!et!al.,!2012;!Routh!et!al.,!2011).!
The! basic! principles! of! the! functionally! rotating! mechanism,! which! is! used! by! RNDMdriven!
tripartite! protein! complexes!to! extrude! substrates,! are!relatively!well!understood,! although!
some! aspects! still! must! be! deciphered! (Murakami,! 2008;! Murakami! et! al.,! 2006;! Nikaido,!
2011).!Acting!in!synergy!with!"simple"!pumps!(e.g.,!ABC!and!MFS!transporter!families),!which!
are!known!to!export!substrates!only!into!the!periplasm,!RND!pumps!completely!rid!the!cell!of!
periplasmic!substrates!(Murakami,!2008;!Murakami!et!al.,!2006;!Nikaido,!2011).!Briefly,!the!
substrate! pumping! mechanism! is! a! threeMstep! functional! rotation,! in! which! each! RND!
protomer!adopts!successively!one!of!three!different!conformations,!which!are!called!access,!
binding!and!extrusion!states,!respectively!(Fig.!1.8)!(Murakami,!2008;!Murakami!et!al.,!2006;!
Nikaido,!2011).!
15!
!
General!Introduction!
!
!
(
Figure(1.8:!Schematic!illustration!of!the!alternating!site!functional!rotation!transport!mechanism.!Each!monomer!
is!colored!with!a!different!variation!of!grey!and!undergoes!its!own!rotating!mechanism.!The!substrate!is!illustrated!
as!diamonds.!In!the!"Access"!state,!the!substrate!cannot!access!to!the!end!of!the!binding!pocket!(represented!by!
dotted!lines)!since!the!binding!cavity!is!shrunk.!The!"Binding"!state!is!characterized!by!conformational!changes!of!
the! monomer! as! well! as! the! complete! entrance! of! the! substrate! in! the! cavity.! The! "Extrusion"! conformation!
corresponds!to!the!release!of!the!substrate!in!funnel!(white!triangle!at!the!center)!which!requires!energy!from!the!
proton!translocation!across!the!RND!transmembrane!domain.!(Figure!modified!from!Murakami!et!al.,!2006)!
!
Each! RND! protomer! has! its! own! substrateMbinding! pocket,! which! is! rich! in! aromatic! and!
hydrophobic! amino! acid! residues! (e.g.,! phenylalanine,! tyrosine,! isoleucine,! or! valine)! and!
which!is!in!their!periplasmic!domain!(Murakami,!2008;!Murakami!et!al.,!2006;!Nikaido,!2011).!
Depending!on!the!conformational!state!adopted!by!the!protomer,!the!binding!cavity!will!be!
expanded!(e.g.,!in!the!binding!state)!or!reduced!(e.g.,!in!the!access!and!extrusion!states)!to!
accommodate! and! push! out! the! substrates! into! the! top! of! the! funnel! (Murakami,! 2008;!
Murakami!et!al.,!2006;!Nikaido,!2011).!At!the!end!of!a!transport!cycle!that!corresponds!to!the!
substrate! extrusion,! the! RND! monomer! turns! back! into! the! "access"! conformation! and!
incorporates! the! next! substrate! (Murakami,! 2008;! Murakami! et! al.,! 2006;! Nikaido,! 2011).!
Conformational! changes! in! the! RND! periplasmic! domain! are! energized! by! the! proton!
translocation! across! the! RND! transmembrane! domain! (Murakami,! 2008;! Murakami! et! al.,!
2006;!Nikaido,!2011).!A!charged!amino!acid!group!(e.g.,!lysine!and!aspartic!acids),!which!can!
be!protonated!and!deprotonated,!is!in!the!transmembrane!domain!and!is!spatially!separated!
from!the!periplasmic!binding!pocket!(Murakami,!2008;!Murakami!et!al.,!2006;!Nikaido,!2011).!
The! protonation! and! deprotonation! of! these! specific! residues! induce! transmembrane!
rearrangements,!which!are!transmitted!to!the!RND!periplasmic!domain!and,!subsequently,!to!
the! membrane! fusion! protein! and! to! the! outer! membrane! channel,! allowing! substrate!
binding!and!extrusion!(Murakami,!2008;!Murakami!et!al.,!2006;!Nikaido,!2011).!
!
16!
!
General!Introduction!
!
MFS( transporters:! The! major! facilitator! superfamily! (MFS)! is! known! to! be! one! of! the! largest! and!
functionally!diverse!groups!of!secondary!active!transporters!(Reddy!et!al.,!2012;!Saier!et!al.,!
1999;!Yan,!2013).!This!ancient!protein!family!is!broadly!conserved!in!all!branches!of!life!from!
bacteria!to!mammals!and!plays!an!important!role!in!many!physiological!processes!(Reddy!et!
al.,! 2012;! Saier! et! al.,! 1999;! Yan,! 2013).! Because! its! members! function! as! uniporters,!
symporters,!or!antiporters,!the!MFS!is!also!called!the!uniporterMsymporterMantiporter!family!
(Pao!et!al.,!1998;!Reddy!et!al.,!2012;!Yan,!2013).!At!least!76!subfamilies!have!been!recognized!
within! this! superfamily! based! on! phylogenetic! data,! substrate! specificity! and! working!
mechanisms;!some!of!these!subfamilies!are!functionally!well!characterized,!whereas!almost!
half! of! these! subfamilies! still! have! unknown! or! putative! functions! (Reddy! et! al.,! 2012;! Yan,!
2013).! MFS! transporters! exhibit! specificity! for! a! wide! spectrum! of! substances,! including!
sugars,! drugs,! Krebs! cycle! metabolites,! nucleosides,! amino! acids,! peptides,! organic! and!
inorganic!ions,!etc.!(Reddy!et!al.,!2012;!Saier!et!al.,!1999;!Yan,!2013).!A!current!example!of!
this! transporter! superfamily! is! the! lactose! permease! LacY! of! E.0 coli,! which! functions! as! a!
lactose/proton!symporter!(Abramson!et!al.,!2003).!
MFS!transporters!appear!to!share!a!common!folding!pattern,!as!predicted!by!the!hydropathy!
profiles! of! their! amino! acid! sequences! (Yan,! 2013;! Zgurskaya,! 2009).! Most! MFS! carriers!
consist!of!twelve!transmembrane!αMhelices,!which!are!organized!into!two!homologous!repeat!
units!connected!by!a!large,!cytoplasmic!loop!(Fig.!1.9)!(Reddy!et!al.,!2012;!Yan,!2013).!In!the!
core! of! the! transporter,! which! is! enclosed! by! the! NM! and! CMterminal! halves,! is! localized! an!
internal! cavity,! which! is! generally! accepted! as! the! single! substrate! recognition! site! (Yan,!
2013;!Zgurskaya,!2009).!
Figure( 1.9:! Schematic! diagram!
illustrating! the! alternating!
access! mechanism! for! a!
substrate/proton!
symporter.!
The! central! cavity,! located!
approximately! halfway! into! the!
membrane! and! sandwiched! by!
the! N! and! C! domains,! is!
alternately! exposed! to! either!
side! of! the! membrane.! Such!
"rocking! bundle"! mechanism!
involves!
interconversion!
through! occluded! intermediate!
states.! (Figure! modified! from!
Yan!et!al.,!2013)!
!
17!
!
General!Introduction!
!
The! general! transport! mechanism! of! all! MSF! carriers,! which! is! also! called! the! "rocking!
bundle"! mechanism,! can! be! represented! by! an! alternating! access! model! wherein! the!
substrate! binding! site! is! alternatively! exposed! to! only! one! side! of! the! membrane! at! a! time!
(Fig.! 1.9)! (Forrest! and! Rudnick,! 2009;! Yan,! 2013).! Consequently,! to! complete! a! transport!
cycle,! transporters! are! compelled! to! undergo! rockerMtype! conformational! changes! allowing!
the! reorientation! of! the! substrateMbinding! site! from! either! an! outwardMfacing! to! inwardM
facing! conformation! or! the! opposite,! going! through! occluded! intermediate! states! (Forrest!
and!Rudnick,!2009;!Yan,!2013).!In!both!symporters!and!antiporters,!the!upload!and!release!of!
substrate(s)! across! the! lipid! bilayer! involve! the! use! of! energy! stored! in! the! transmembrane!
proton! gradient! (Forrest! and! Rudnick,! 2009;! Yan,! 2013).! Accordingly,! the! conformational!
modifications! required! for! the! substrate! transport! are! coupled! with! the! protonation! and!
deprotonation!of!specific!amino!acid!residues!in!transmembrane!domains,!such!as!glutamic!
acid,!aspartic!acid,!or!histidine!(Yan,!2013;!Zgurskaya,!2009).!
(
(
18!
!
!
!
General!Introduction!
!
Chapter(2:( The(Copper(Paradox!
2.1.(General(Overview(
Copper!is!widely!distributed!in!the!environment;!its!average!natural!abundance!in!the!Earth's!
crust! is! around! 60! mg! kgM1! (Magnani! and! Solioz,! 2007;! Oorts,! 2013).! Naturally! present! as! an!
uncompounded! mineral! (i.e.,! native! copper),! copper! also! occurs! as! different! types! of! ores,! such! as!
sulfides!(e.g.,!chalcopyrite,!CuFeS2!and!chalcocite,!Cu2S),!carbonates!(e.g.,!azurite,!Cu3(CO3)2(OH)2!and!
malachite,!Cu2CO3(OH)2)!and!oxides!(cuprite,!Cu2O)!(Magnani!and!Solioz,!2007;!Oorts,!2013).!As!the!
third!most!important!metal!used!by!humans!(Oorts,!2013),!its!price!has!fluctuated!over!the!years!due!
to! demand,! and! currently,! copper! is! valued! at! 7,055! USD/t! (5084! EUR/t).! The! world! demand! for!
copper! is! impressive! (e.g.,! around! 24! million! tons! in! 2007),! whereas! its! world! global! production! is!
barely! half! of! the! demand! (e.g.,! more! than! 15! million! tons! yearM1! in! 2008)! (Oorts,! 2013).! Under!
current! conditions,! many! people! estimate! that! the! depletion! of! existing! Cu! reserves! available! for!
mining!will!be!reached!in!the!next!25!to!60!years!(Oorts,!2013).!
The!origin!of!the!name!"copper"!(formerly!called!cyprium)!refers!to!the!island!of!Cyprus,!where!
important! extractions! of! copper! occurred! during! the! Roman! era! (Oorts,! 2013).! Discovered! in!
approximately! ⋍! 9000! B.C.,! copper! was! the! first! metal! used! by! ancient! civilizations! since! time!
immemorial! most! likely! because! native! copper! could! be! bashed! into! new! shapes! without! requiring!
any!smelting!(e.g.,!crude!knives!and!sickles)!(Grass!et!al.,!2011;!Koch!et!al.,!1997).!The!development!
of! metallurgy! with! the! invention! of! smelting! occurred! independently! in! several! parts! of! the! world!
(e.g.,!Europe,!Middle!East,!West!Africa,!Central!America,!China,!etc.)!at!different!times!(Grass!et!al.,!
2011;!Oorts,!2013).!In!Europe,!at!approximately!2500!B.C.,!the!accidental!discovery!of!bronze,!which!
is!an!alloy!of!copper!and!tin!that!is!harder!than!either!metal!on!its!own,!established!the!Bronze!Age!
(Grass!et!al.,!2011;!Oorts,!2013;!Osman!and!Cavet,!2008).!First!dedicated!to!several!daily!uses!(e.g.,!
the!creation!of!jewelry,!material!for!cooking!and!storage!vessels,!weapons,!etc.),!copper!has!rapidly!
been!exploited!for!its!antimicrobial!properties! throughout!the!ages!(Grass!et!al.,!2011;!Hodgkinson!
and! Petris,! 2012;! Koch! et! al.,! 1997;! Osman! and! Cavet,! 2008).! The! Edwin! Smith! Papyrus,! which! was!
written! between! 2600! and! 2200! B.C.,! is! most! likely! one! of! the! oldest! recorded! medical! texts!
describing!copper!as!a!sterilizing!agent!for!chest!wounds!and!for!drinking!water!(Grass!et!al.,!2011;!
Hodgkinson! and! Petris,! 2012;! Osman! and! Cavet,! 2008).! Widely! used! by! Greek,! Roman! and! Aztec!
civilizations! to! treat! many! ailments! (e.g.,! headaches,! burns,! intestinal! worms,! etc.),! its! use! as! an!
antimicrobial! agent! persisted! until! the! modern! era! of! antibiotics! in! 1932! (Grass! et! al.,! 2011;!
Hodgkinson!and!Petris,!2012).!Numerous!modernMday!applications!still!use!the!cytotoxic!properties!
19!
!
General!Introduction!
!
of! copper.! For! instance,! copper! is! commonly! exploited! as! a! fungicidal! treatment! for! plants! (e.g.,!
Bordeaux!mixture),!as!an!algaecide,!as!a!wood!preservative,!as!a!disinfectant!for!veterinary!purposes!
and! in! the! food! industry,! as! an! electrolytic! ionizer! to! counter! the! growth! of! Legionella0 in! hospital!
drinking!water,!or!as!a!selfMsanitizing!material!in!the!manufacture!of!frequently!touched!surfaces!to!
prevent!the!spread!of!nosocomial!infections!(e.g.,!door!handles!and!railings)!(Hodgkinson!and!Petris,!
2012;!Koch!et!al.,!1997;!Osman!and!Cavet,!2008).!In!addition!to!its!remarkable!antimicrobial!activity,!
copper! also! has! many! other! properties,! such! as! extremely! good! conductivity! of! both! heat! and!
electricity,! good! malleability! and! ductility,! and! high! corrosion! resistance.! All! these! properties! are!
primarily!exploited!in!electrical!applications!(65%)!(e.g.,!transformers,!motors,!and!electrical!cables),!
in!construction!(25%)!(e.g.,!plumbing,!roofing!and!cladding),!and!in!transport!(7%)!(e.g.,!trams,!trains,!
car!and!trucks)!(Oorts,!2013).!
(
2.2.(Chemistry(and(Toxicity(of(Copper(
Copper!is!an!essential!trace!element!required!by!all!living!organisms!from!bacteria!to!humans!
(Hodgkinson!and!Petris,!2012).!As!an!exception!among!the!transition!metals!from!the!first!series,!this!
micronutrient!is!characterized!by!the!following!electron!configuration:![Ar]!4s1!3d10,!in!which!the!3d!
orbital!is!filled,!corresponding!to!a!more!stable!and!lower!energy!state!(Cuillel,!2009;!Mikolay!et!al.,!
2010).! Due! to! its! basic! chemistry,! copper! can! primarily! cycle! between! two! oxidation! states:! the!
cuprous!Cu(I)!form,![Ar]!4s0!3d10,!and!the!cupric!Cu(II)!form,![Ar]!4s0!3d9!(Cuillel,!2009;!Mikolay!et!al.,!
2010;!Osman!and!Cavet,!2008).!This!ability!to!alternate!between!the!reduced!Cu(I)!and!oxidized!Cu(II)!
states! is! used! by! numerous! copperMcontaining! proteins! (e.g.,! cytochrome! oxidase,! superoxide!
dismutase,!plastocyanin,!etc.)!involved!in!a!variety!of!metabolic!processes!in!which!copper!serves!as!
a!biological!cofactor!(i.e.,!as!an!electron!donor/acceptor!or!carrier)!(Grass!et!al.,!2011;!Magnani!and!
Solioz,!2007).!
Before! the! advent! of! an! oxidizing! atmosphere! resulting! from! the! photosynthetic! activity! of!
cyanobacteria,!copper!would!likely!have!existed!primarily!in!the!reduced!Cu(I)!state,!in!the!form!of!
nonMbioavailable! and! insoluble! sulfide! salts! (i.e.! Cu2S)! (Burkhead! et! al.,! 2009;! Magnani! and! Solioz,!
2007;!Rubino!and!Franz,!2012).!Under!these!anoxic!conditions,!in!contrast!to!copper,!iron!existed!in!a!
much!more!soluble!and!bioavailable!form,!Fe(II),!and!found!application!in!a!wide!variety!of!proteins!
as!a!cofactor!of!choice!to!catalyze!redox!reactions!(Burkhead!et!al.,!2009;!MacPherson!and!Murphy,!
2007).!Some!2.7!billion!years!ago,!the!Great!Oxygenation!Event,!also!called!the!Oxygen!Catastrophe,!
had! decisive! effects! on! the! chemical! and! biological! evolution,! such! as! the! establishment! of! an! oxic!
biosphere,! the! creation! of! a! protective! stratospheric! ozone! layer,! and! the! development! of! aerobic!
20!
!
General!Introduction!
!
respiration!(Abolmaali!et!al.,!1998;!Magnani!and!Solioz,!2007;!Osman!and!Cavet,!2008;!Rubino!and!
Franz,!2012).!Concurrently,!this!revolution!in!the!environmental!conditions!also!altered!the!solubility!
and! availability! of! metal! ions,! favoring! a! change! in! their! redox! states.! Consequently,! the!
bioavailability!of!copper!and!iron!changed!dramatically!due!to!the!release!of!copper!from!insoluble!
sulfides! as! more! soluble! and! bioavailable! Cu(II)! ions! and! the! precipitation! of! iron! as! insoluble! iron!
oxides! Fe(OH)3! after! the! oxidation! of! Fe(II)! to! Fe(III)! (Cuillel,! 2009;! MacPherson! and! Murphy,! 2007;!
Magnani! and! Solioz,! 2007;! Rubino! and! Franz,! 2012).! Furthermore,! the! rise! of! atmospheric! O2!
compelled!most!living!organisms!to!adapt!their!metabolism,!inducing!an!evolution!toward!reaction!
systems!that!worked!at!higher!redox!potentials,!ranging!from!0!V!(H+/H2)!to!1.229!V!(O2/H2O),!and!
were! more! suited! to! the! oxidizing! power! of! oxygen! (Lide,! 2009).! In! this! context,! the! high! redox!
potential!of!the!Cu(II)/Cu(I)!couple!(E0!=!+!153!mV,!in!aqueous!solution!at!25°C)!fell!perfectly!within!
this! range,! allowing! copper! to! become! a! biologically! relevant! element! in! several! redox! reactions!
(Abolmaali!et!al.,!1998;!Magnani!and!Solioz,!2007;!Osman!and!Cavet,!2008;!Rubino!and!Franz,!2012).!
Although! the!redox! properties!of! copper!(i.e.,!its!ability!to!donate!or!accept!an!electron)!are!
crucial,! this! transition! metal! can! also! participate! in! the! cascade! of! free! radical! generation,! causing!
cellular! damage.! Indeed,! copper! is! able! to! catalyze! Fenton! chemistry,! in! which! the! Cu(I)! ion! reacts!
with!hydrogen!peroxide!(H2O2,!a!relatively!stable!byMproduct!of!oxidative!metabolism),!leading!to!the!
release!of!a!highly!reactive!hydroxyl!radical!(Grass!et!al.,!2011;!Hodgkinson!and!Petris,!2012;!Koch!et!
al.,! 1997;! Osman! and! Cavet,! 2008).! This! oxygen! derived! oxidant! (OH•)! produced! during! the! Fenton!
reaction,! Cu+! +! H2O2! ∏! Cu2+! +! OH–! +! OH•,! is! toxic! and! known! to! rapidly! participate! in! numerous!
reactions! detrimental! to! all! cellular! components,! such! as! sugars,! proteins,! lipids! and! nucleic! acids!
(Grass!et!al.,!2011;!Hodgkinson!and!Petris,!2012;!Koch!et!al.,!1997;!Osman!and!Cavet,!2008).!Notably,!
although!frequently!claimed,!the!Fenton!process!may!not!be!the!primary!toxic!mechanism!(Grass!et!
al.,!2011).!Actually,!to!what!extent!Fenton!chemistry!contributes!to!copper!toxicity!remains!unclear;!
moreover,!the!H2O2!concentration!appears!to!be!maintained!at!extremely!low!levels!in!cells!to!avoid!
its!powerful!oxidizing!capacity!(Grass!et!al.,!2011).!An!alternative!cytotoxic!pathway!of!copper!is!its!
ability!to!compete!with!other!metal!ions!for!important!binding!sites!in!proteins,!which!results!in!the!
displacement!of!existing!transition!metal!cations!and!alterations!to!the!structure!and/or!function!of!
proteins!(Koch!et!al.,!1997;!Osman!and!Cavet,!2008).!The!human!estrogen!receptor!(i.e.,!a!hormoneM
responsive!DNAMbinding!transcription!factor)!is!a!representative!example!of!this!disturbance,!wherein!
copper! can! substitute! for! zinc! in! zincMfinger! domains,! thus! inactivating! the! receptor,! which! is! now!
unable!to!bind!its!DNAMtarget!sequence!(Bertinato!and!L'Abbe,!2004;!Cuillel,!2009;!Koch!et!al.,!1997).!
21!
!
General!Introduction!
!
Considering!the!above!characteristics,!the!JanusMfaced!nature!of!copper!(i.e.!essential!but!also!
toxic)!compels!organisms!to!evolve!diverse!mechanisms!to!maintain!a!delicate!balance!between!the!
uptake,!distribution,!sequestration/storage!and!export!of!copper!ions!(Camakaris!et!al.,!1999;!Cuillel,!
2009;! Osman! and! Cavet,! 2008;! Rubino! and! Franz,! 2012).! From! unicellular! organisms! to! specialized!
cells! of! mammals,! the! strict! regulation! of! cellular! copper! homeostasis! involves! highly! complex!
networks! between! copper! transporters! and! copperMbinding! proteins/chaperones! to! efficiently!
acquire! and! to! appropriately! utilize! this! metal! while! preventing! the! accumulation! of! toxic! copper!
levels! (Bertinato! and! L'Abbe,! 2004;! Grass! et! al.,! 2011).! Any! copper! imbalance! can! lead! to! serious!
problems,!such!as!in!humans,!where!copper!is!known!to!be!strongly!involved!in!neurodegenerative!
diseases! (e.g.,! Alzheimer's! disease,! CreutzfeldtMJakob! disease,! familial! amyotrophic! lateral! sclerosis,!
etc.),! in! tumor! development! and! progression,! in! Menkes! syndrome! or! in! Wilson's! disease,! which!
correspond! to! disorders! of! copper! deficiency! and! accumulation,! respectively! (Koch! et! al.,! 1997;!
Krupanidhi!et!al.,!2008;!Puig!and!Thiele,!2002).!
(
2.3.(Coordination(Chemistry:(CopperCBinding(Centers(
Tight! control! of! copper! is! a! cellular! necessity;! to! meet! this! challenge,! Nature! has! developed!
sophisticated! copperMbinding! motifs! to! acquire,! transport,! sequester,! and! export! copper! in! a! safe!
way.! The! understanding! of! metalMligand! coordination! chemistry! within! cuproproteins! is! important!
because!this!coordination!plays!a!major!role!in!their!biochemical!functions!(Koch!et!al.,!1997;!Rubino!
and!Franz,!2012).!The!binding!of!copper!ions!to!ligands!is!based!on!Pearson's!hard!and!soft!acids!and!
bases! (HSAB)! theory,! which! is! a! qualitative! concept! introduced! in! the! early! 1960s! to! predict! the!
relative! stabilities! of! metal! complexes! and! the! outcomes! of! chemical! reactions! (Abolmaali! et! al.,!
1998;! Cuillel,! 2009;! Rubino! and! Franz,! 2012).! Depending! on! the! oxidation! state! of! copper,! the!
composition,! stereochemistry! and! coordination! geometry! of! binding! sites! vary! to! stabilize! and! to!
handle!this!toxic!cargo!in!a!suitable!manner!(Abolmaali!et!al.,!1998;!Cuillel,!2009).!
Because! of! its! closed! shell! 3d10! configuration,! the! Cu(I)! transition! metal! ion! is! diamagnetic,!
meaning!that!this!ion!is!silent!in!electron!paramagnetic!resonance!(EPR)!spectroscopy,!whereas!the!
unpaired! dMorbital! configuration! (i.e.,! 3d9)! makes! Cu(II)! a! paramagnetic! ion! (i.e.,! EPRMactive! form)!
(Abolmaali! et! al.,! 1998;!Koch! et! al.,! 1997;!Osman!and!Cavet,!2008).!Considering!the! HSAB! concept,!
the!highly!polarizable!Cu(I)!ion!is!categorized!as!a!soft!Lewis!acid!(i.e.,!an!electronMpair!acceptor)!and!
forms! complexes! of! different! geometries! by! covalent! binding! to! soft! Lewis! bases,! whereas! the! less!
polarizable!Cu(II)!ion!is!classified!as!borderline!(i.e.,!between!soft!and!hard!acids)!and!interacts!with!
intermediate! Lewis! bases! (Cuillel,! 2009;! Koch! et! al.,! 1997;! Rubino! and! Franz,! 2012).! Cu(I)! is! often!
22!
!
General!Introduction!
!
coordinated!by!2,!3!or!4!ligands!and!can!take!up!linear,!trigonal!planar,!or!tetrahedral!conformations,!
respectively! (Cuillel,! 2009;! Koch! et! al.,! 1997;! Rubino! and! Franz,! 2012).! In! contrast! to! the! reduced!
oxidation!state,!Cu(II)!favors!a!coordination!number!of!4,!5!or!6!and!can!adopt!square!planar,!square!
pyramidal,! or! axially! distorted! octahedral! geometries! (Cuillel,! 2009;! Koch! et! al.,! 1997;! Rubino! and!
Franz,!2012).!
With! a! few! exceptions,! the! amino! acids! predominantly! found! in! copperMbinding! sites! in!
proteins!are!representative!of!three!ligand!types,!namely,!the!side!chains!of!histidine,!cysteine,!and!
methionine!(Fig.!2.1)!(Bondarczuk!and!PiotrowskaMSeget,!2013;!Rubino!and!Franz,!2012).!Because!of!
their!chemical!properties,!these!components!provide!a!great!variety!of!possible!arrangements!within!
coordinating! motifs! (Rubino! and! Franz,! 2012).! Methionine! and! cysteine! residues! are! two! sulfurM
containing!amino!acids!(Fig.!2.1A!and!B),!whereas!histidine!is!an!amino!acid!containing!an!imidazole!
functional!group!(i.e.,!an!aromatic!heterocycle!containing!two!nitrogen!atoms)!(Fig.!2.1C).!
Figure(2.1:!Ligands!predominantly!found!in!
copperMbinding! sites! within! proteins.! (A.)!
Methionine;!(B.)!Cysteine;!(C.)!Histidine.!
!
With!its!large!aliphatic!side!chain,!methionine!is!a!nonpolar!residue!that!is!more!hydrophobic!than!
the! two! others;! this! property! can! influence! solvent! accessibility! (i.e.,! methionine! residues! are!
primarily! found! on! the! interior! of! proteins)! and! proteinMprotein! contact! (Rubino! and! Franz,! 2012).!
Unlike! the! methylthioether! side! chain! of! methionine! residue,! the! thiol! group! of! cysteine! and! the!
imidazole! ring! of! histidine! are! both! polarizable! and! ionizable.! Indeed,! at! physiological! pH,! the!
sulfhydryl! group! of! cysteine! is! potentially! acidic! (pKa! ~! 8.4),! whereas! the! heterocyclic! side! chain! of!
histidine!is!basic!(pKa!~!6.0).!Consequently,!under!such!conditions,!the!ionization!of!the!thiol!group!is!
negligible! (i.e.,! the! neutral! form! predominates),! whereas! the! imidazole! group! can! be! both! under!
neutral!and!protonated!forms!(i.e.,!the!imidazole!ring!unionized/ionized!ratio!is!10:1).!Notably,!these!
pKa! values! are! reported! for! free! amino! acids! but! can! be! altered! by! the! effects! of! the! protein!
environment! and! by! coordination! to! metal! ions.! Finally,! cysteine! residues! are! redox! active! and! can!
oxidize! to! form! disulfide! crossMlinked! cysteine;! the! resulting! unit! of! two! cysteineMlinked! is! called!
cystine.! However,! methionine! residues! are! less! prone! to! oxidation! and! do! not! form! crossMlinks;!
23!
!
General!Introduction!
!
nevertheless,! sometimes! methionines! can! be! oxidized! to! methionine! sulfoxide! and! methionine!
sulfone!under!specific!conditions.!
According! to! the! focused! review! written! by! Rubino! (2012),! cuproproteins! can! be! separated!
into! two! large! groups,! depending! on! whether! copper! is! used! as! a! cofactor! to! perform! a! specific!
function!or!as!a!cargo!to!be!transported.!
In!the!former!case,!copper!must!be!tightly!coordinated!within!highMaffinity!binding!sites!to!prevent!its!
loss!during!the!redox!cycling!(Rubino!and!Franz,!2012).!Based!on!their!structural!and!spectroscopic!
properties,! copperMbinding! sites! observed! in! cofactor! proteins! can! be! classified! into! different!
categories.!Initially,!the!subdivision!consisted!of!three! distinct!classes!of!Cu(II)!metal!centers!within!
proteins! (i.e.,! type! 1,! type! 2,! and! type! 3)! (Koch! et! al.,! 1997).! However,! over! the! years,! the! copper!
center!classification!has!been!further!extended!to!the!acquisition!of!new!crystal!structures!of!copper!
proteins.! The! current! categorization! is! based! on! six! different! classes,! as! briefly! summarized! below!
(Rubino!and!Franz,!2012).!
(
Type(1:!The!type!1!(T1)!copper!sites!are!most!likely!the!most!intensively!studied!group.!These!sites!
are! found! in! copper! proteins! that! perform! single! electron! transfer! in! a! wide! variety! of!
biochemical!processes,!such!as!photosynthesis!and!metabolism!(Dennison,!2008;!Lucas!and!
Karlin,!2009;!MacPherson!and!Murphy,!2007).!Structurally,!the!Cu!coordination!sphere!of!a!
typical! type! 1! center! is! formed! by! three! strong! equatorial! ligands,! which! are! provided! by! a!
cysteine!and!two!histidine!residues,!in!a!trigonal!planar!arrangement!(Dennison,!2008;!Lucas!
and! Karlin,! 2009;! MacPherson! and! Murphy,! 2007).! Usually,! one! or! two! additional! axial!
ligands!(e.g.,!methionine,!glycine,!or!glutamine!residue)!complement!these!primary!ligands,!
resulting! in! a! distorted! stereochemistry,! as! in! plastocyanin! (i.e.,! distorted! tetrahedral!
conformation)! and! in! azurin! (i.e.,! distorted! bipyramidal! geometry)! (Fig.! 2.2A! and! B,!
respectively)!(Dennison,!2008;!Lucas!and!Karlin,!2009;!MacPherson!and!Murphy,!2007).!
Mononuclear!T1!copper!centers!are!characterized!by!an!intense!absorption!band!at!600!nm!
in! the! UVMvisible! spectrum,! which! has! been! assigned! to! a! ligandMtoMmetal! charge! transfer!
transition!(i.e.,!SCys!π→!Cu(II)!dx2My2)!(Dennison,!2008;!Lucas!and!Karlin,!2009;!MacPherson!and!
Murphy,! 2007).! This! spectral! feature! generates! a! bright! blue! color,! conferring! the! name! of!
"blue! copper! proteins"! to! cuproproteins! containing! these! T1! copper! sites! (Dennison,! 2008;!
Lucas! and! Karlin,! 2009;! MacPherson! and! Murphy,! 2007).! These! sites! also! display! unusually!
narrow! hyperfine! splitting! in! EPR! spectroscopy,! which! has! been! attributed! to! the! highly!
covalent!nature!of!the!sulfur!(Cys)MtoMcopper!bond!(Dennison,!2008;!Lucas!and!Karlin,!2009;!
MacPherson!and!Murphy,!2007).!Last,!another!important!feature!of!these!T1!copper!sites!is!
24!
!
General!Introduction!
!
their!high!redox!potential,!which!is!greater!than!that!of!Cu(II)/Cu(I)!aqua!couple!(i.e.,!ranging!
from! +! 180! to! +! 800! mV),! explaining! their! presence! in! redox! enzymes,! such! as! nitrite!
reductase,!and!in!multicopper!oxidases!(Dennison,!2008;!Lucas!and!Karlin,!2009;!MacPherson!
and!Murphy,!2007).!
!
!
Figure(2.2:!Type!1!(T1)!copper!sites.!(A.)!Plastocyanin!harbors!a!distorted!tetrahedral!conformation!formed!by!two!
histidine,!one!cysteine!and!one!methionine!residues.!(B.)!Azurin!harbors!a!distorted!bipyramidal!geometry!formed!
by!two!histidine,!one!cysteine,!one!methionine!and!one!glycine!residues.!
(
Type(2:!The!type!2!(T2)!copper!centers!are!less!studied!than!blue!ones!because!of!their!inability!to!be!
spectrophotometrically! studied.! Consequently,! in! contrast! to! the! T1! copper! sites,! the! T2!
copper! centers! are! usually! called! "nonMblue! copper! centers"! (Abolmaali! et! al.,! 1998;!
MacPherson! and! Murphy,! 2007;! Rubino! and! Franz,! 2012).! The! stereochemistry! of!
mononuclear!T2!centers!is!neither!uniform!in!terms!of!amino!acid!ligands!nor!in!coordination!
number.! Most! type! 2! Cu! sites! predominantly! exhibit! a! distorted! square! planar! geometry,!
wherein!the!imidazole!side!chain!of!histidines!are!primarily!involved!(Abolmaali!et!al.,!1998;!
Koch!et!al.,!1997;!MacPherson!and!Murphy,!2007;!Rubino!and!Franz,!2012).!However,!other!
amino!acids,!such!as!methionine,!glutamate,!glutamine,!or!tyrosine!residues,!can!be!found!in!
the! Cu(II)! coordination! sphere! (Abolmaali! et! al.,! 1998;! MacPherson! and! Murphy,! 2007;!
Rubino!and!Franz,!2012).!Notably,!some!coordination!positions!in!T2!copper!centers!can!be!
either! vacant! or! occupied! by! exogenous! ligands! (e.g.,! O2,! H2O,! etc.).! This! special! feature!
justifies!why!these!T2!copper!sites!appear!in!oxidases,!oxygenases,!nitrite!reductases,!and!in!
Cu,ZnMsuperoxide! dismutases! (Fig.! 2.3A)! (Abolmaali! et! al.,! 1998;! MacPherson! and! Murphy,!
2007;! Rubino! and! Franz,! 2012).! Indeed,! the! catalytic! activity! of! an! enzyme! directly! results!
from!the!binding!of!its!substrate!within!the!active!T2!copper!site.!
(
25!
!
General!Introduction!
!
(
Figure( 2.3:! Type! 2! (T2)! and! type! 3! (T3)! copper! sites.! (A.)! Cu,ZnMsuperoxide! dismutase! harbors! a! T2! copper! site!
which!is!formed!by!four!histidine!residues.!(B.)!Hemocyanin!harbors!a!T3!copper!site!wherein!two!Cu(II)!ions!are!
both!coordinated!by!three!histidine!ligands!and!reversibly!bind!oxygen.!
(
Type( 3:! The! type! 3! (T3)! copper! centers! are! involved! in! a! variety! of! biological! processes,! such! as!
pigment! formation,! innate! immunity,! and! dioxygen! transport.! For! instance,! these! sites! are!
present! in! some! oxidases! (e.g.,! catechol! oxidase! and! tyrosinase)! and! oxygenMtransport!
proteins!(e.g.,!hemocyanin)!(Fig.!2.3B)!(Aguilera!et!al.,!2013;!Maria!et!al.,!2011;!Rubino!and!
Franz,!2012).!All!type!3!copper!proteins!possess!a!binuclear!active!site!consisting!of!two!Cu(II)!
ions,!which!are!both!coordinated!by!three!conserved!histidine!residues,!and!reversibly!bind!
dioxygen!(Aguilera!et!al.,!2013;!Maria!et!al.,!2011;!Rubino!and!Franz,!2012).!In!contrast!to!the!
T1!and!T2!copper!centers,!which!possess!paramagnetic!properties,!the!type!3!copper!site!is!
diamagnetic!(Aguilera!et!al.,!2013;!Maria!et!al.,!2011;!Rubino!and!Franz,!2012).!Consequently,!
the!T3!Cu!center!is!silent!in!EPR!spectroscopy!due!to!antiferromagnetic!coupling!between!the!
two!Cu(II)!metal!ions,!which!are!bridged!by!molecular!oxygen!(Aguilera!et!al.,!2013;!Maria!et!
al.,!2011;!Rubino!and!Franz,!2012).!
(
CuA(center:!Primarily!identified!in!cytochrome!c!oxidases!and!nitrous!oxide!reductases!where!these!
centers! are! involved! in! longMrange! electron! transfers! to! the! catalytic! site,! CuAMtype! centers!
are! highly! unusual! bimetallic! sites! (Chacon! and! Blackburn,! 2012;! Gamelin! et! al.,! 1998;!
Gennari!et!al.,!2011;!Tsai!et!al.,!2013).!The!highly!covalent!core!of!these!purple!CuA!centers!
consists! of! two! copper! ions,! which! are! bridged! by! two! thiolate! sulfur! atoms! of! cysteine!
residues!in!a!nearly!planar!geometry!and!which!are!directly!bonded!with!an!extremely!short!
distance! (i.e.,! 2.4! –! 2.5! Å)! (Fig.! 2.4A)! (Chacon! and! Blackburn,! 2012;! Gamelin! et! al.,! 1998;!
Gennari! et! al.,! 2011;! Tsai! et! al.,! 2013).! Each! copper! ion! is! further! coordinated! by! one!
equatorial!histidine!residue!and!by!a!weak!axial!ligand,!which!is!a!methionine!residue!in!the!
26!
!
General!Introduction!
!
first! copper! center! and! in! the! carbonyl! oxygen! of! a! glutamate! residue! in! the! second! one!
(Chacon!and!Blackburn,!2012;!Gamelin!et!al.,!1998;!Gennari!et!al.,!2011;!Tsai!et!al.,!2013).!In!
its! oxidized! state,! the! CuAMtype! site! is! a! mixedMvalence! system! (i.e.,! Cu2+! and! Cu+),! which! is!
formally! denoted! as! Cu1.5+MCu1.5+,! wherein! the! single! unpaired! electron! is! completely!
delocalized! over! both! copper! nuclei! (Chacon! and! Blackburn,! 2012;! Gamelin! et! al.,! 1998;!
Gennari! et! al.,! 2011;! Tsai! et! al.,! 2013).! This! mixedMvalence! oxidation! state! generates! a!
characteristic! hyperfine! splitting! pattern! in! EPR! spectroscopy! (Chacon! and! Blackburn,! 2012;!
Gamelin!et!al.,!1998;!Gennari!et!al.,!2011;!Tsai!et!al.,!2013).!
(
CuB(center:!The!CuB!centers!are!found!in!cytochrome!c!oxidases!in!the!immediate!vicinity!of!a!heme!
a3! group,! resulting! in! a! heme! a3MCuB! cluster! (Colbran! and! PaddonMRow,! 2003;! Du! and!
Noodleman,! 2013;! Kaila! et! al.,! 2009).! Buried! in! subunit! I! of! cytochrome! c! oxidases,! this!
heteronuclear!Fea3MCuB!cluster!constitutes!the!active!site!where!molecular!oxygen!is!fixed!and!
reduced! to! two! water! molecules! in! a! fourMelectron! pathway! according! to! the! following!
equation:! 8H+in! +! O2! +! 4eM! →! 4H+out! +! 2H2O! (Colbran! and! PaddonMRow,! 2003;! Du! and!
Noodleman,!2013;!Kaila!et!al.,!2009).!The!mononuclear!copper!center!is!coordinated!by!three!
histidine! residues! (Fig.! 2.4B)! and! is! usually! spectroscopically! invisible! due! to! the!
antiferromagnetic! coupling! between! CuB! and! Fea3! (Colbran! and! PaddonMRow,! 2003;! Du! and!
Noodleman,! 2013;! Kaila! et! al.,! 2009).! Notably,! a! tyrosine! residue! is! postMtranslationally!
bonded!to!one!of!the!histidine!residues,!and!one!electron!together!with!a!proton!necessary!
for!the!oxygen!reduction!reaction!are!thought!to!be!derived!from!this!covalently!linked!HisM
Tyr!ligand!(Colbran!and!PaddonMRow,!2003;!Du!and!Noodleman,!2013;!Kaila!et!al.,!2009).!
(
CuZ(center:!Recently!discovered!and!characterized,!CuZ!centers!are!much!more!complex!than!simple!
binuclear!type!3!copper!centers!(Dell'Acqua!et!al.,!2011;!Rubino!and!Franz,!2012).!Found!in!
nitrous! oxide! reductases! (N2OR),! where! these! centers! constitute! the! catalytic! center!
responsible! for! the! reduction! of! nitrous! oxide! (N2O)! to! nitrogen! and! water,! these! unique!
structures! consist! of! four! copper! ions! (labeled! as! CuI,! CuII,! CuIII,! and! CuIV)! coordinated! by!
seven!wellMconserved!histidine!residues!in!a!distorted!tetrahedron!(Fig.!2.4C)! (Dell'Acqua!et!
al.,! 2011;! Rubino! and! Franz,! 2012).! Inside! this! unusual! tetranuclear! copper! center,! notably,!
one!of!these!copper!ions!(i.e.,!CuIV)!is!only!ligated!by!one!histidine!residue!(Dell'Acqua!et!al.,!
2011;! Rubino! and! Franz,! 2012).! Consequently,! the! pocket! between! CuI! and! CuIV! of! the! CuZ!
center! is! thought! to! be! the! enzyme! substrate! binding! site! and! can! accommodate! oxygen!
species,! such! as! a! water! molecule! or! a! hydroxyl! group! (Dell'Acqua! et! al.,! 2011;! Rubino! and!
Franz,! 2012).! Another! feature! of! CuZ! centers! is! the! bridging! of! the! four! copper! ions! by! an!
27!
!
General!Introduction!
!
inorganic! sulfur! atom,! whose! role! in! the! enzyme! catalytic! mechanism! remains! unclear!
(Dell'Acqua!et!al.,!2011;!Rubino!and!Franz,!2012).!
!
Figure(2.4:!CuA,!CuB!and!CuZ!centers.!(A.)!Cytochrome!c!oxidase!harbors!CuAMtype!center!consisting!of!two!copper!
ions!bridged!by!two!cysteine!residues.!Each!Cu!ion!is!further!coordinated!by!one!equatorial!histidine!and!a!weak!
axial!ligand!(i.e.!glutamate!or!methionine!residue).!(B.)!CuBMtype!centers!are!found!in!cytochrome!c!oxidases!near!
to! a! heme! a3! group.! Such! sites! contain! a! mononuclear! copper! center! which! is! coordinated! by! three! histidine!
residues!(C.)!Nitrous!oxide!reductase!harbors!a!complex!CuZ!center!which!contains!four!copper!ions!coordinated!
by! seven! histidine! residues.! Each! copper! is! ligated! by! two! histidine! residues,! with! the! exception! of! the! CuIV!
coordinated!only!by!one!histidine!residue.!Consequently,!the!cavity!generated!between!CuI!and!CuIV!is!proposed!
to!accommodate!the!enzyme!substrate!(i.e.!oxygen!species).!
!
In! contrast! to! canonical! cuproenzymes,! metalMtrafficking! proteins! use! copper! as! a! cargo! that!
must!be!protected!and!delivered!to!a!specific!target.!For!this!purpose,!copper!must!be!bound!tightly!
enough!to!prevent!adventitious!reactions!or!its!release;!however,!copper!must!also!be!coordinated!in!
a! way! allowing! its! easy! transfer! to! another! copper! protein! partner.! Consequently,! the! copper!
coordination! chemistry! of! such! proteins! is! atypical! and! quite! distinct! from! the! coordination!
geometries! seen! above! (Finney! and! O'Halloran,! 2003;! Rubino! and! Franz,! 2012).! CopperMtrafficking!
proteins! constitute! a! large! and! varied! family,! including,! in! particular,! integral! transmembrane! P1BM
28!
!
(
General!Introduction!
!
type!ATPases,!intracellular!copper!chaperones,!resistance!proteins!and!copper!binding!transcription!
factors! (Finney! and! O'Halloran,! 2003;! Rubino! and! Franz,! 2012).! Hereafter,! we! briefly! describe! the!
copper!chemistry!of!some!extensively!reviewed!copper!transport!proteins.!
(
P1BCtype( ATPases:! Copper! transporting! PMtype! ATPases! belong! to! a! subclass! of! the! PMtype! ATPases,!
which! are! involved! in! heavy! metal! pumping! across! biological! membranes! and! which! are!
classified! as! P1BMtype! ATPases! or! CPxMtype! ATPases! (Lubben! et! al.,! 2009;! Rubino! and! Franz,!
2012;! Solioz! and! Vulpe,! 1996).! All! CPxMtype! ATPases! share! the! defining! motifs! of! PMtype!
ATPases!(i.e.,!a!phosphatase!domain!TGE,!a!phosphorylation!domain!DKTGT,!an!ATP!binding!
domain,! etc.);! however,! these! ATPases! also! exhibit! additional! features.! As! their! name!
suggests,! CPxMtype! ATPases! have! a! conserved! intramembranous! cysteineMprolineMcysteine!
(CPC)! or! cysteineMprolineMhistidine! (CPH)! motif! required! for! metal! transport.! These! ATPases!
also! contain! various! numbers! of! metalMbinding! motifs,! which! are! arranged! as! CXXC,! in! the!
aminoMterminal!region.!Such!conserved!and!flexible!motifs!play!an!important!role!in!copper!
transport! by! coordinating! metal! in! a! near! linear! geometry! (Fig.! 2.5)! (Lubben! et! al.,! 2009;!
Rubino!and!Franz,!2012;!Solioz!and!Vulpe,!1996).!CopA!from!E.0coli!is!a!good!representative!
of! CPxMATPases,! which! possesses! two! putative! cytoplasmic! CXXC! motifs! in! the! NMterminal!
region.!The!protein!CopA!transports!Cu(I)!from!the!cytoplasm!into!the!periplasm,!utilizing!the!
energy! derived! from! ATP! hydrolysis! (Bondarczuk! and! PiotrowskaMSeget,! 2013;! Osman! and!
Cavet,!2008;!Rosen,!2002).!
Figure( 2.5:! Copper! coordination!
geometry!in!ATPases!and!chaperones.!
!
Copper( chaperones:! Copper! chaperones! are! small,! soluble! and! intracellular! proteins! that! are!
involved! in! copper! transport! and! delivery! into! the! active! sites! of! specific! copper! partners!
(Harrison! et! al.,! 2000;! Rubino! and! Franz,! 2012;! Urvoas! et! al.,! 2003).! In! addition! to! ferrying!
copper! to! the! desired! location,! these! proteins! also! protect! the! cell! from! the! deleterious!
effects! of! free! copper! by! preventing! inappropriate! copper! interactions! with! cellular!
components!(Harrison!et!al.,!2000;!Rubino!and!Franz,!2012).!Prevalent!from!prokaryotes!to!
humans,! these! proteins! belong! to! a! family! of! highly! homologous! metalMbinding! proteins.!
Members!of!this!homologous!family!share!a!consensus!metalMbinding!motif,!MXCXXC,!which!
is!involved!in!Cu(I)!chelation.!The!copper!binding!occurs!essentially!via!the!two!cysteines!in!a!
29!
!
General!Introduction!
!
lowMcoordinationMnumber!environment,!which!allows!extremely!tight!binding!(Fig.!2.5).!Such!
chaperones! also! display! extremely! similar! folding,! which! consists! of! four! antiparallel! βM
strands! and! two! αMhelices! (βαββαβ),! known! as! "openMfaced! β! sandwich"! (Finney! and!
O'Halloran,!2003;!Harrison!et!al.,!2000;!Urvoas!et!al.,!2003).!CopZ!from!Enterococcus0hirae!is!
a!good!example!of!a!copper!chaperone.!This!protein!appears!to!load!intracellular!Cu(I)!from!
CopA! ATPase! and! delivers! the! metal! to! CopY! according! to! a! transitory! interaction.! After!
copper! delivery,! the! chaperone! returns! to! its! apoMform! and! starts! a! new! copperMdelivery!
cycle,! thereby! fulfilling! its! vital! role! of! copper! transport! to! specific! targets! (Harrison! et! al.,!
2000).!
(
CopperCbinding(transcription(factors:!Transcription!factors!are!proteins!that!function!in!the!reducing!
environment! of! the! cytosol! and! that! regulate! gene! expression! (Magnani! and! Solioz,! 2007;!
Rademacher! and! Masepohl,! 2012;! Rubino! and! Franz,! 2012).! Transcription! factors! are!
predicted! to! interact! with! DNA! sequences! in! a! specific! manner! and! either! upM! or!
downregulate!gene!transcription.!The!binding!of!small!ligands,!such!as!copper,!can!modulate!
the!DNAMbinding!affinity!of!transcription!factors!by!inducing!structural!rearrangements!that!
either! promote! or! suppress! proteinMDNA! interactions! (Rademacher! and! Masepohl,! 2012;!
Rubino!and!Franz,!2012).!CueR!from!E.0coli!is!a!wellMknown!copperMresponsive!transcription!
factor! that! activates! the! expression! of! copA! and! cueO! genes! at! lowMtoMmoderate! copper!
concentrations! (Osman! and! Cavet,! 2008;! Rademacher! and! Masepohl,! 2012;! Rubino! and!
Franz,! 2012).! Crystal! structure! analyses! reveal! that! CueR! is! a! dimer! consisting! of! three!
functional!domains:!an!NMterminal!helixMturnMhelix!DNA!binding!region,!a!central!dimerization!
domain! and! a! CMterminal! copperMbinding! domain! (Magnani! and! Solioz,! 2007;! Rademacher!
and!Masepohl,!2012).!In!this!protein,!copper!binding!occurs!in!a!buried,!solventMinaccessible!
site! at! the! dimer! interface! and! involves! two! conserved! cysteine! ligands,! CXXC.! This! site!
contributes! to! the! selectivity! toward! copper! monovalent! ions! which! are! coordinated! in! a!
nearly! linear! geometry,! S–Cu+–S! (Magnani! and! Solioz,! 2007;! Osman! and! Cavet,! 2008;!
Rademacher!and!Masepohl,!2012;!Rubino!and!Franz,!2012).!Notably,!even!if!both!apoM!and!
holoMregulators! bind! the! target! promoter,! only! the! CueR! holoMregulator! is! able! to! activate!
gene!transcription!(Rademacher!and!Masepohl,!2012).!
(
Copper( resistance( proteins:! Copper! sensing! and! trafficking! proteins! displaying! lowMcoordinationM
number! Cys2! sites! that! favor! Cu(I)! binding! have! been! well! documented.! Such! cysteineMrich!
motifs! are! primarily! found! in! cytosolic! proteins! where! these! cysteine! residues! are! reduced!
and! available! for! Cu(I)! coordination.! Nevertheless,! numerous! soluble! periplasmic! proteins!
30!
!
General!Introduction!
!
associated! with! copper! resistance! systems! in! GramMnegative! bacteria! are! known! to! adopt!
new! types! of! copper! coordination! chemistry! involving! methionineMrich! motifs! (Davis! and!
O'Halloran,!2008;!Finney!and!O'Halloran,!2003).!This!Cu(I)Mthioether!coordination!geometry!is!
generally!featured!by!a!higher!coordination!number!(3!or!4)!and!by!a!low!affinity,!in!contrast!
to! a! Cu(I)Mdithiolate! ligation! (Davis! and! O'Halloran,! 2008;! Rubino! and! Franz,! 2012).! For!
example,! XMray! crystallography! studies! have! shown! that! CusA! (i.e.,! an! inner! membrane!
protein)! and! CusB! (i.e.,! a! membraneManchored! periplasmic! protein)! from! E.0 coli! coordinate!
monovalent! copper! ions! in! a! threeMcoordination! methionine! environment! (Fig.! 2.6A)! (Davis!
and!O'Halloran,!2008;!Rubino!and!Franz,!2012).!Unusual!copper!coordination!chemistry!has!
also! been! described! for! the! periplasmic! CusF! protein! from! E.0 coli! (Fig.! 2.6B).! The! latter!
coordinates! a! Cu(I)! ion! through! a! site! consisting! of! a! Met2His! motif,! and! a! strong! Cu(I)Mπ!
interaction!with!the!aromatic!ring!of!a!nearby!tryptophan!residue!also!participates!in!copper!
recognition!(Davis!and!O'Halloran,!2008;!Rubino!and!Franz,!2012).!A!trigonal!Met2His!site!was!
also! revealed! in! PcoC! from! E.0 coli! by! spectroscopic! and! crystallographic! analysis;! this! motif!
was!found!in!a!solventMexposed!loop!on!the!protein!surface!(Fig.!2.6C)!(Davis!and!O'Halloran,!
2008;!Finney!and!O'Halloran,!2003;!Rubino!and!Franz,!2012).!However,!such!Cu(I)!binding!is!
slightly!more!ambiguous!in!the!closely!related!CopC!protein!from!Pseudomonas0syringae.!In!
fact,! NMR! and! EXAFS! analyses! suggest! a! tetrahedral! geometry! involving! three! methionine!
and! one! histidine! residues! (Fig.! 2.6D),! whereas! the! crystal! structure! shows! a! coordination!
geometry! with! only! methionine! residues,! Cu(I)Met4! (Fig.! 2.6E)! (Davis! and! O'Halloran,! 2008;!
Rubino!and!Franz,!2012).!
!
!
!
31!
!
General!Introduction!
!
!
!
Figure( 2.6:! Coordination! geometry! in! copper! resistance! proteins.! (A.)! CusA! and! CusB! from! E.0 coli! coordinate!
monovalent!copper!ions!in!a!threeMcoordination!methionine!environment.!(B.)!CusF!from!E.0coli!coordinates!Cu(I)!
ions! through! a! Met2His! motif! and! also! involves! a! tryptophan! residue.! (C.)! PcoC! from! E.0 coli! contains! a! trigonal!
Met2His! site! able! to! bind! Cu(I)! ions,! while! its! homologue! CopC! from! P.0 syringae! harbors! a! tetragonal! geometry!
either!through!Met3His!(D.)!or!Met4!(E.).!
(
(
32!
!
General!Introduction!
!
2.4.(Models(of(Copper(Homeostasis:(Escherichia)coli(and(Enterococcus)hirae)
Considering! the! above! descriptions,! the! high! chemical! reactivity! of! copper! clearly! compels!
microorganisms!to!tightly!control!copper!transport!and!traffic!through!different!compartments.!Such!
regulation!of!copper!availability!is!achieved!by!the!concerted!activity!of!numerous!protein!networks!
that! manage! copper! ions! properly! and! efficiently.! However,! the! complete! understanding! of!
mechanisms! involved! in! copper! trafficking! and! homeostasis! in! microorganisms! requires! further!
investigation.! In! the! following! section,! we! briefly! described! the! two! best! characterized! bacterial!
copperMresistance! systems,! namely,! copper! homeostasis! in! E.0 coli! and! E.0 hirae0 (Rensing! and! Grass,!
2003).!
Copper! homeostasis! in! the! GramMpositive! bacterium! E.0 hirae! is! one! of! the! simplest! and! best!
understood! systems! (Fig.! 2.7)! (Harrison! Krick! and! Dameron,! 2013;! Lu! et! al.,! 2003;! Magnani! and!
Solioz,!2005;!Solioz!and!Stoyanov,!2003).!Copper!resistance!within!this!bacterium!is!accomplished!by!
the! chromosomal! copYZAB! operon,! which! is! induced! by! copper! and! which! is! regulated! at! the!
transcriptional!level!by!the!copperMresponsive!repressor!CopY.!In!addition!to!the!CopY!repressor,!the!
cop! operon! encodes! two! copperMtranslocating! PMtype! ATPases,! CopA! and! CopB,! as! well! as! a!
metallochaperone,! CopZ.! The! following! is! an! overview! of! the! current! model! of! copper! circulation.!
Both! localized! in! the! cytoplasmic! membrane,! CopA! appears! to! be! responsible! for! copper(I)! uptake!
under! copperMlimiting! conditions,! whereas! CopB! appears! to! export! copper! out! of! the! cell! when!
intracellular!Cu(I)!ions!reach!toxic!concentrations!(Harrison!Krick!and!Dameron,!2013;!Lu!et!al.,!2003;!
Magnani! and! Solioz,! 2005;! Solioz! and! Stoyanov,! 2003).! The! chaperone! CopZ! serves! in! intracellular!
copper!routing!by!specifically!delivering!copper!to!CopY.!CopZ!also!interacts!with!CopA,!which!may!
correspond! to! the! mechanism! by! which! CopZ! loads! the! metal.! An! extracellular! copper! reductase,!
which!has!been!tentatively!called!CorA,!may!also!be!involved!in!the!copper!homeostatic!process!by!
reducing!extracellular!Cu(II)!ions!to!Cu(I)!ions,!which!are!then!taken!up!by!CopA!(Magnani!and!Solioz,!
2005;!Solioz!and!Stoyanov,!2003).!Under!limited!copper!concentrations,!the!CopY!repressor!binds!to!
the!cop!promoter!region!as!a!homodimer!containing!Zn(II)!ions,![Zn(II)CopY]2.!In!this!manner,!CopY!
represses! its! own! transcription,! as! well! as! that! of! the! three! other! cop! genes.! When! copper!
concentration!increases!in!the!cytoplasm,!the!DNAMbound!repressor!protein!leaves!the!cop!promoter!
region,! which! allows! the! transcription! of! downstream! genes.! CopY! dissociation! proceeds! by! the!
binding!of!Cu(I)!ions,!which!are!delivered!by!the!copper!chaperone!CopZ,!and!by!the!displacement!of!
bound! Zn(II)! ions! (Magnani! and! Solioz,! 2005;! Solioz! and! Stoyanov,! 2003).! However,! notably,! high!
Cu(I)CopZ! concentrations! are! toxic! to! cell.! Consequently,! CopZ! is! degraded! by! a! copperMactivated!
proteolytic! activity! under! high! copper! concentrations.! This! controlled! proteolysis! is! extremely!
33!
!
General!Introduction!
!
important! and! illustrates! an! additional! safeguarding! mechanism! for! copper! homeostasis! in! E.0 hirae!
(Lu!et!al.,!2003;!Magnani!and!Solioz,!2005;!Solioz!and!Stoyanov,!2003).!
!
Figure(2.7:!Schematic!drawing!of!the!cop!operon!and!model!of!copper!homeostasis!in!Enterococcus0hirae.!At!physiological!
copper! levels! (copperMlimiting! conditions),! [Zn(II)CopY]2! is! bound! to! the! promoter! and! transcription! of! the! cop! operon! is!
turned!down.!Under!high!copper!conditions,!Cu(I)!ions!would!enter!cells!by!the!ATPase!CopA!and!would!be!picked!up!by!
CopZ,! a! copper! metallochaperone.! Then,! CopZ! can! deliver! safety! Cu(I)! ions! to! the! CopY! repressor! which! loses! the! bound!
zinc! and! dissociates! from! the! DNA! inducing! the! cop! operon! transcription.! If! intracellular! copper! is! excessive! (drawn! as!
dotted!line),!CopZ!may!also!deliver!copper!ions!to!the!ATPase!CopB!for!expulsion,!and!excess!of!Cu(I)CopZ!is!degraded!by!a!
copperMactivated!protease.!CorA!would!be!the!extracellular!copper!reductase!that!reduces!Cu(II)!ions!to!Cu(I)!ions!before!
their!uptake!by!the!CopA!ATPase.!(Figure!modified!from!Solioz!et!al.,!2003)!
!
In!contrast,!copper!resistance!in!the!GramMnegative!bacterium!E.0coli!is!a!multiMlayered!process!
that!is!primarily!conferred!by!two!chromosomallyMencoded!systems:!the!CuMsensing!(cus)!locus!and!
the! CuMefflux! (cue)! regulon! (Fig.! 2.8)! (Bondarczuk! and! PiotrowskaMSeget,! 2013;! Osman! and! Cavet,!
2008;! Rensing! and! Grass,! 2003).! The! cue! system! is! the! primary! copper! resistance! mechanism! in! E.0
coli,!which!is!expressed!under!moderate!to!high!copper!concentrations,!as!well!as!under!both!aerobic!
and!anaerobic!conditions.!This!system!is!regulated!by!CueR,!a!copperMresponsive!regulator.!
CueR! (for! copper! export! regulator)! belongs! to! the! MerRMfamily! of! metal! responsive!
transcriptional! activators,! which! occurs! in! a! range! of! bacterial! genera! and! which! modulates!
transcription! in! response! to! different! environmental! stimuli,! including! in! particular! metal! ions!
(Humbert! et! al.,! 2013;! Osman! and! Cavet,! 2008).! Other! members! of! the! MerRMfamily! include! zincM
sensing!ZntR,!mercuryMsensing!MerR,!cadmiumMsensing!CadR,!leadMsensing!PbrR,!and!cobaltMsensing!
CoaR!(Osman!and!Cavet,!2008).!These!regulators!bind!to!the!promoters!of!their!target!genes!in!the!
presence!and!absence!of!their!effector!metals.!Depending!on!their!metalMbinding!states,!the!MerRM
family!metalloregulators!change!their!interactions!with!DNA!to!suppress!or!activate!transcription.!In!
34!
!
General!Introduction!
!
the! absence! of! metal,! the! regulator! is! bound! to! DNA! in! the! repressing! conformation! maintaining!
repression! of! the! promoter.! The! binding! of! metal! ions! induces! a! conformational! change! of! the!
regulator,! which! adopts! an! activating! conformation.! The! activation! of! the! regulator! results! in! a!
realignment!of!the!promoter!elements!that!allows!a!productive!interaction!between!RNA!polymerase!
and!DNA!and!initiates!the!transcription!(Andoy!et!al.,!2009;!Humbert!et!al.,!2013;!Osman!and!Cavet,!
2008).!
In!E.0coli,!the!cytoplasmic!copper!sensor!CueR!mediates!the!transcription!of!at!least!two!genes:!
copA!and!cueO,!which!are!not!organized!in!an!operon.!These!target!genes!are!expressed!in!response!
to! either! Cu(I),! Ag(I),! or! Au(I)! ions! (Humbert! et! al.,! 2013;! Osman! and! Cavet,! 2008).! Notably,! the!
extraordinary!high!sensitivity!of!the!E.0coli!copper!sensor!CueR!to!free!Cu(I),!which!was!determined!to!
be! 10–21! M! (zeptomolar)! and! which! was! translated! to! less! than! one! free! copper! atom! in! the!
cytoplasm,! indicates! that! there! is! essentially! no! free! cytosolic! copper! ions! (Hodgkinson! and! Petris,!
2012;!Osman!and!Cavet,!2008).!
The!copA!gene!under!the!control!of!CueR!regulator!encodes!the!CopA!protein,!a!copper!efflux!
PMtype!ATPase!that!expels!monovalent!copper!ions!from!the!cytoplasm!into!the!periplasm,!whereas!
the! cueO! gene! also! under! the! CueR! control! encodes! the! CueO! protein,! a! periplasmic! multicopper!
oxidase!that!oxidizes!toxic!Cu(I)!ions!to!less!toxic!Cu(II)!ions!(Fig.!2.8).!Under!aerobic!conditions,!the!
cus!system!is!an!auxiliary!system!that!is!only!required!at!extremely!high!copper!concentrations!when!
the!cue!system!is!overwhelmed.!This!system!is!particularly!important!under!anaerobic!conditions!and!
appears! to! safeguard! the! periplasmic! compartment! from! copperMinduced! stress! because! the!
multicopper! oxidase! CueO! does! not! function! under! oxygen! limitation! (Bondarczuk! and! PiotrowskaM
Seget,!2013;!Osman!and!Cavet,!2008;!Rensing!and!Grass,!2003).!The!chromosomal0cus!determinant!
consists! of! two! operons:! cusRS! genes,! which! encode! a! twoMcomponent! regulatory! system! that!
responds!to!an!increase!in!the!periplasmic!copper!concentration,!and!cusCFBA!genes,!which!encode!a!
proton/cation!antiporter!complex.!
In!bacteria,!environmental!signals!(e.g.,!stressors,!growth!conditions,!etc.)!are!transduced!into!
the!cell!predominantly!by!twoMcomponent!systems.!The!class!of!twoMcomponent!regulatory!systems!
is!one!of!the!most!widespread!means!by!which!bacteria!sense,!respond,!and!adapt!to!environmental!
stresses! (Laub! and! Goulian,! 2007;! Mitrophanov! and! Groisman,! 2008;! Sheng! et! al.,! 2012).! In! the!
prototypical! twoMcomponent! system,! the! sensor! histidine! kinase! is! spatially! decoupled! from! the!
response!regulator,!which!has!a!major!advantage!over!cytoplasmically!located!regulators!(Hobman!et!
al.,! 2007).! Typically! located! in! the! inner! membrane,! the! sensor! can! sense! and! transduce! a! signal!
across! the! inner! membrane! to! a! cytoplasmic! response! regulator,! which! acts! on! the! expression! of!
genes!required!in!response!to!the!environmental!signals!(Laub!and!Goulian,!2007;!Mitrophanov!and!
35!
!
General!Introduction!
!
Groisman,! 2008;! Sheng! et! al.,! 2012).! The! signal! transduction! occurs! via! phosphorylation.! Basically,!
the!activation!of!a!sensor!kinase!leads!to!autophosphorylation!on!a!conserved!histidine!residue!using!
ATP.! Subsequently,! the! phosphoryl! group! is! transferred! to! a! conserved! aspartate! residue! on! the!
cognate!response!regulator.!Phosphorylation!of!the!response!regulator!affects!its!affinity!for!target!
promoters,!controlling!gene!expression!(Hobman!et!al.,!2007;!Laub!and!Goulian,!2007;!Mitrophanov!
and!Groisman,!2008;!Sheng!et!al.,!2012).!
The!expression!of!the!cusCFBA!genes!is!regulated!by!the!CusRS!system.!The!CusCBA!transport!
complex!forms!a!channel!that!spans!both!membranes!and!the!periplasmic!space,!allowing!the!efflux!
of!Cu(I)!into!the!extracellular!milieu,!whereas!the!CusF!protein!is!a!small!periplasmic!metal!chaperone!
that!sequesters!and!transfers!excess!Cu(I)!to!the!tripartite!transenvelope!protein!complex!(Fig.!2.8).!
Although! copper! export! mechanisms! in! E.0 coli! have! been! elucidated,! no! protein! responsible! for!
copper! uptake! across! the! cytoplasmic! membrane! has! conclusively! been! identified! thus! far!
(Bondarczuk!and!PiotrowskaMSeget,!2013;!Osman!and!Cavet,!2008;!Rensing!and!Grass,!2003).!
!
!
Figure(2.8:!Schematic!model!of!copper!homeostasis!in!Escherichia0coli.!Under!physiological!conditions!(i.e.!in!the!presence!
of!O2),!the!Cus!system!is!not!induced!and!copper!homeostasis!is!achieved!by!CopA!and!CueO.!CopA!is!a!Cu(I)!translocating!
PMtype!ATPase!which!expels!copper!from!the!cytoplasm!into!the!periplasm.!Once!in!the!periplasmic!space,!Cu(I)!ions!can!be!
oxidized! by! the! multicopper! oxidase! CueO! to! less! toxic! Cu(II)! ions.! Under! oxygenMdepleted! conditions,! CopA! still! exports!
Cu(I)!ions!from!the!cytosol!to!the!periplasm!but!CueO!is!inactivated.!A!second!system!is!then!activated,!the!multicomponent!
copper!efflux!pump!CusCFBA!(i.e.,!a!RND!transporter).!CusCBA!is!a!tripartite!pump!which!allows!the!efflux!of!Cu(I)!ions!into!
the! extracellular! space,! whereas! CusF! is! a! soluble! copper! chaperone! which! sequesters! excess! Cu(I)! ions! and! gives! it! to!
CusCBA.! The! mechanism! by! which! copper! enters! the! periplasmic! space! is! still! unknown.! Abbreviations:! OM! outer!
membrane,!P!periplasm!and!IM!inner!membrane.!(Figure!modified!from!Bondarczuk!et!al.,!2013)!
!
!
!
36!
!
General!Introduction!
!
In! addition! to! these! extensively! studied! copper! resistance! mechanisms,! additional! plasmidM
encoded! copper! resistance! determinants! have! also! been! identified,! inter0 alia,! in! some! strains! of! E.0
coli! and! P.0 syringae! (Bondarczuk! and! PiotrowskaMSeget,! 2013;! Clayton! et! al.,! 2011;! Magnani! and!
Solioz,!2007;!Rensing!and!Grass,!2003).!These!extrachromosomal!genetic!elements!confer!resistance!
to!copper!above!the!innate!chromosomal!copper!homeostatic!system,!allowing!bacterial!survival!in!
copperMrich!environments.!Copper!resistance!from!the!P.0syringae!pathovar!tomato!plasmid!pPT23D!
is!conferred!by!the!copABCDRS!operon,!which!is!homologous!to!the!pco!gene!cluster!from!the!E.0coli!
pRJ1004! plasmid! pcoABCDRS! (Fig.! 2.9).! The! Pco/Cop! systems! of! E.0 coli! and! P.0 syringae! share! four!
structural! genes,! pco/copABCD,! as! well! as! two! other! genes,! pco/copRS,! which! encode! a! twoM
component! regulatory! system! required! for! the! copperMinducible! expression! of! copper! resistance.!
Notably,! farther! downstream! of! pcoRS,! an! additional! gene! pcoE! has! also! been! identified! in! the!
pRJ1004! plasmid.! This! pcoE! gene! is! transcribed! from! its! own! copperMinducible! promoter,! which! is!
under!the!control!of!CusRS,!unlike!the!pcoABCD!operon,!which!is!primarily!regulated!by!PcoRS.!Thus!
far,!no!P.0syringae!homolog!has!been!described!(Bondarczuk!and!PiotrowskaMSeget,!2013;!Clayton!et!
al.,!2011;!Magnani!and!Solioz,!2007;!Rensing!and!Grass,!2003).!Although!the!E.0coli!pco!determinant!
and!the!cop!operon!in!P.0syringae!have!considerable!similarity,!the!mechanism!of!copper!resistance!
conferred!by!those!determinants!appears!to!function!differently,!and!such!differences!have!not!yet!
been!explained!(Brown!et!al.,!1995;!Lu!and!Solioz,!2002).!In!E.0coli,!the!pco!system!enhances!copper!
efflux,!which!results!in!reduced!cellular!copper!accumulation,!whereas!the!reverse!response!occurs!in!
P.0 syringae,! where! the! cop! system! appears! to! result! in! increased! copper! uptake,! leading! to! copper!
bioaccumulation!by!bacterial!cells!(Brown!et!al.,!1995;!Lu!and!Solioz,!2002).!
!
Figure( 2.9:! Alignment! of! similar! plasmidMborne! genes! in! Escherichia0 coli! (plasmid! pRJ1004)! and! Pseudomonas0
syringae! (plasmid! pPT23D).! The! plasmidMborne! pcoE! gene! from! the! plasmid! pRJ1004! has! no! homolog! in! other!
bacterial!cop!operon.!
!
Despite!the!molecular!mechanism!of! copper!resistance!by!the!pco!system!and!the!related! P.0
syringae0cop!system!remain!unclear,!PcoA/CopA!may!be!a!periplasmic!multicopper!oxidases!similar!
to!CueO!of!E.0coli,!oxidizing!Cu(I)!to!Cu(II)!ions.!PcoB/CopB!and!PcoD/CopD!are!copper!pumps!in!the!
outer! and! inner! membranes,! respectively.! Both! of! these! pumps! participate! in! copper! transport;!
however,!their!exact!function!remains!elusive.!PcoC/CopC!are!soluble!periplasmic!copper!chaperones!
that! bind! Cu(I)! and! Cu(II)! in! two! separate! binding! sites! with! specific! affinities.! These! proteins! may!
37!
!
General!Introduction!
!
potentially! shuttle! copper! to! PcoA/CopA! or! PcoB/CopB! to! detoxify! excess! copper! or! to! deliver!
essential! copper! to! PcoD/CopD! (Bondarczuk! and! PiotrowskaMSeget,! 2013;! Clayton! et! al.,! 2011;!
Magnani! and! Solioz,! 2007;! Rensing! and! Grass,! 2003).! PcoE! also! appears! as! a! periplasmic! copper!
chaperone,! which! does! not! seem! to! be! strictly! required! for! copper! resistance! in! E.0 coli0 (Fig.! 2.10).!
However,! this! chaperone! may! provide! a! rapid! response! by! acting! as! a! primary! defense! factor! that!
sequesters!copper!in!the!periplasm!before!the!complete!induction!of!the!pco!system!is!achieved!and!
that!transports!Cu(I)!ions!to!PcoC,!which,!in!turn,!will!pass!the!sequestered!Cu(I)!ions!to!PcoA!or!PcoD!
(Bondarczuk!and!PiotrowskaMSeget,!2013;!Rensing!and!Grass,!2003;!Zimmermann!et!al.,!2012).!
!
!
Figure( 2.10:! Schematic! drawing! of! the! cop! operon! and!
proposed! mechanism! of! PcoMmediated! copper!
detoxification!in!E.0coli.!The!four!structural!genes!pcoABCD!
are! under! the! control! of! pcoRS,! while! pcoE! is! transcribed!
from! its! own! promoter! and! is! under! the! control! of! cusRS.!
Copper! enters! the! periplasm! by! an! unknown! mechanism,!
possibly! through! porins.! In! the! periplasm,! the! multicopper!
oxidase!PcoA!can!oxidize!Cu(I)!ions!from!PcoC!to!less!toxic!
Cu(II)! ions.! PcoC! is! predicted! as! a! periplasmic! copper!
chaperone! which! can! bind! Cu(I)! and! Cu(II)! ions! in! two!
distinct! binding! sites.! This! protein! can! potentially! deliver!
copper!ions!to!PcoA!and/or!to!the!outer!membrane!protein!
PcoB!to!detoxify!copper!excess,!or!shuttle!essential!copper!
to! PcoD! located! in! the! inner! membrane.! PcoE! is! a!
periplasmic! chaperone! which! can! act! as! a! copper! storage!
element! by! binding! both! Cu(I)! and! Cu(II)! ions.! It! was!
proposed! that! PcoE! may! deliver! sequestered! Cu(I)! ions! to!
PcoC!which,!in!turn,!would!pass!ions!to!PcoA!for!oxidation.!
Abbreviations:! OM! outer! membrane! and! IM! inner!
membrane.!(Figure!modified!from!Rensing!et!al!2003)!
!
In!the!last!decade,!several!bacterial!strains,!such!as!Cupriavidus0metallidurans!CH34,!Ralstonia0
solanacearum!and!Pseudomonas0aeruginosa!PA01,!have!been!shown!to!be!resistant!to!copper!and!to!
encode! all! or! part! of! these! operons.! Genes! homologous! to! pco/cop! have! been! identified! on! their!
plasmids! and! chromosomes,! and! the! plasmidMencoded! pco/cop! operon! may! have! evolved! from! its!
chromosomal!counterpart!(Bondarczuk!and!PiotrowskaMSeget,!2013).!
Notably,!metal!resistance!in!bacteria!is!a!very!wide!area,!which!is!still!poorly!investigated.!Few!
systems! are! really! well! known! and! numerous! systems! or! interactions! still! remain! unanswered.!
Moreover,!no!classification!of!metal!resistance!systems!exists.!The!absence!of!any!classification!is!in!
conjunction!with!the!fact!that!many!proteins!have!the!same!name!in!different!bacteria,!but!they!do!
not!have!the!same!function!(Table!1).!
(
(
38!
!
General!Introduction!
!
Table(1:!Summary!table!of!the!different!cop/pco!genes!and!their!putative!functions!in!each!strain!mentioned!in!
this!introduction.!
Strain(
Gene(
Putative(protein(function(
E.)coli)
!
!
!
!
!
!
!
!
0
copA0
pcoA0
pcoB0
pcoC0
pcoD0
pcoS0
pcoR0
pcoE0
!
copper!ATPase!
multicopper!oxidase!
copper!pump!
copper!chaperone!
copper!pump!
sensor!kinase!
response!regulator!
copper!chaperone!
P.)syringae)pv.)tomato)
!
!
!
!
!
!
0
copA0
copB0
copC0
copD0
copS0
copR0
E.)hirae)
!
!
!
!
0
copY0
copZ0
copA0
copB0
C.)metallidurans(CH34(
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
0
copW0
copE0
copH0
copQ0
copL0
copO0
copF0
copG0
copJ0
copI0
copD10
copC10
copB10
copA10
copR10
copS10
copN0
copK0
copM0
copT0
copV0
!
!
multicopper!oxidase!
copper!pump!
copper!chaperone!
copper!pump!
sensor!kinase!
response!regulator!
!
!
copperMresponsive!repressor!
metallochaperone!
copper!ATPase!
copper!ATPase!
!
(
39!
!
!
unknown!
unknown!
Cu(II)!binding!as!a!Cu(II)!sensor!
unknown!
unknown!
chaperone!
PMtype!ATPase!involved!in!Cu(I)!efflux!
putative!cupredoxin!
putative!cytochrome!c!protein!
oxidoreductase!
canal!involved!in!Cu(II)/Cu(I)!uptake!
copperMbinding!protein!
copperMbinding!protein!
multicopper!oxidase!
response!regulator!
sensor!kinase!
unknown!
copper!chaperone!
unknown!
putative!cytochrome!
unknown!
!
!
General!Introduction!
!
Chapter(3:(Cupriavidus)metallidurans(CH34,(a(MetalCResistant(Bacterium(
3.1.(General(Overview(
Naturally!soilMdwelling,!C.0metallidurans!CH34!is!a!GramMnegative!bacterium!belonging!to!the!
βMproteobacterial!family!Burkholderiaceae!of!the!order!Burkholderiales0(von!Rozycki!and!Nies,!2009).!
Formerly! assigned! to! genera! Wausteria,! Alcaligenes! and! Ralstonia,! this! metalMresistant! bacterium!
was! isolated! in! the! late! 1970s! from! the! sludge! of! a! nonMferrous! metallurgical! plant! near! Liège,! in!
Belgium,!which!was!contaminated!with!high!concentrations!of!zinc,!cadmium!and!cobalt!(Mergeay!et!
al.,!2009;!Taghavi!et!al.,!1997;!von!Rozycki!and!Nies,!2009).!C.0metallidurans!CH34!and!related!strains,!
which! are! particularly! well! adapted! to! colonize! and! to! adapt! to! harsh! industrial! environments! and!
which! are! affected! by! the! presence! of! wastes! rich! in! toxic! metals! and! often! mixed! with! organic!
recalcitrant! compounds! and! hydrocarbons,! are! prevalent! all! around! the! world! (e.g.,! Belgium,!
Germany,! Zaire,! New! Zealand,! Japan,! etc.)! (Diels! et! al.,! 2009;! von! Rozycki! and! Nies,! 2009).! Their!
ability!to!grow!in!the!presence!of!high!metal!concentrations!(i.e.,!millimolar!concentrations),!such!as!
Cu(II),! Zn(II),! Cd(II),! Co(II),! Pb(II),! Ni(II),! etc.,! makes! this! bestMstudied! Cupriavidus! representative! an!
interesting! model! system! to! investigate! microbial! responses! to! numerous! metals! (Monchy! et! al.,!
2006;!Monsieurs!et!al.,!2011).!
As!a!facultative!chemolithotrophic!bacterium!possessing!two!hydrogenases!(i.e.,!a!cytoplasmic,!
NADMreducing! hydrogenase! and! a! membraneMbound! hydrogenase),! C.0 metallidurans! strain! CH34! is!
able! to! grow! aerobically! in! the! presence! of! H2! (used! as! energy! source)! and! CO2! (used! as! carbon!
source)!(Mergeay!et!al.,!1985;!Taghavi!et!al.,!1997).!Also!characterized!by!an!oxidative!metabolism,!
this! bacterium! can! degrade! a! wide! range! of! substrates,! which! are! subsequently! used! as! carbon!
sources!(with!the!exception!of!sugars,!which!are!not!assimilated)!(Taghavi!et!al.,!1997;!Vaneechoutte!
et! al.,! 2004;! von! Rozycki! and! Nies,! 2009).! These! nonMspore! forming! and! peritrichously! flagellated!
bacteria,!which!are!described!as!unicellular!short!rods!with!a!diameter!of!up!to!0.8!µm!and!a!length!
of!up!to!2!µm!occur!singly,!in!pairs!or!in!short!chains;!these!bacteria!form!round,!opaque!and!slightly!
pink!colonies!on!agar!plates!(Goris!et!al.,!2001;!Guine!et!al.,!2006;!Taghavi!et!al.,!1997;!Vaneechoutte!
et! al.,! 2004).! This! mesophilic! bacterium! has! an! optimal! growth! temperature! of! 30°C,! and! a!
phenomenon! described! as! temperatureMinduced! mutagenesis! and! mortality! (i.e.,! TIMM)! appears!
beyond!37°C!(Taghavi!et!al.,!1997).!
!
!
40!
!
General!Introduction!
!
The! complex! genome! of! C.0 metallidurans! CH34! was! completely! sequenced,! thoroughly!
analyzed!and!annotated!by!the!Joint!Genome!Institute!(JGI)!in!Walnut!Creek,!CA,!USA!(Janssen!et!al.,!
2010).! The! genome! consists! of! four! circular! replicons:! a! chromosome! (3.9! Mb),! a! chromid! (i.e.,! a!
"second!chromosome",!2.6!Mb)!as!well!as!two!megaplasmids!pMOL28!(171!kb)!and!pMOL30!(234!kb)!
(Janssen!et!al.,!2010;!Van!Houdt!and!Mergeay,!2012).!The!chromosome!shelters!most!of!the!essential!
"housekeeping"! genes! (e.g.,! genes! required! for! DNA! replication! and! repair,! cell! division,!
transcription,! translation,! etc.),! whereas! the! chromid! carries! essential! genes! involved! in! specialized!
metabolic! and! biosynthetic! activities! and! in! adaptive! responses! (e.g.,! genes! involved! in! acetone!
utilization! and! tolerance,! sulfite! oxidation,! biofilm! formation,! exopolysaccharide! synthesis,! etc.)!
(Janssen! et! al.,! 2010).! Although! some! metal! resistance! genes! have! been! discovered! on! the!
bacterium's! chromosome! and! chromid,! plasmid! curing! and! genetic! transfer! experiments!
demonstrated! that! both! megaplasmids! carry! the! primary! key! genes! encoding! for! transition! metal!
resistance! systems! which! allow! growth! in! metalMcontaminated! niches! (Monchy! et! al.,! 2007;! von!
Rozycki!and!Nies,!2009).!
Cloning!and!sequencing!experiments!of!numerous!fragments!from!plasmid!pMOL28!revealed!
many!determinants!associated!with!resistance!to!nickel!and!cobalt!(i.e.,!cnr!operon),!chromate!(i.e.,!
chr!operon)!and!mercury! (i.e.,!mer!operon)!(Fig.!3.1A)!(Mergeay!et!al.,!2009;!Monchy!et!al.,!2007).!
The!annotation!of!plasmid!pMOL28!indicates!that!this!plasmid!shares!a!backbone!of!common!genes!
with! two! other! plasmids,! namely,! pRALTA,! from! Cupriavidus0 taiwanensis,! and! pHG1,! from!
Cupriavidus0eutrophus!H16.!These!genes!contribute!to!plasmid!replication!and!maintenance,!as!well!
as!to!conjugative!transfer!(Janssen!et!al.,!2010;!Mergeay!et!al.,!2009;!Monchy!et!al.,!2007).!Backbone!
genes!are!clearly!distinguishable!from!the!three!genomic!islands!defined!in!pMOL28.!The!first!one!is!
the! island! CMGIM28a,! which! shelters! the! metal! resistance! determinants! mentioned! above! (i.e.,! cnr,!
chr! and! mer! operons)! (Mergeay! et! al.,! 2009;! Van! Houdt! et! al.,! 2009).! The! second! one! is! the! Rhs!
island,!which!is!called!CMGIM28b.!Rearrangement!hotspot!(rhs)!elements!were!first!identified!as!sites!
that!promote!recombination!in!C.0metallidurans!CH34.!These!genes!encode!proteins!rich!in!tyrosineM
aspartate!(YD)!motifs,!whose!functions!are!not!well!understood!(Mergeay!et!al.,!2009;!Van!Houdt!et!
al.,!2009).!Finally,!the!last!one!is!the!putative!island!CMGIM28c,!which!contains!primarily!hypothetical!
genes.! Because! of! the! proximity! between! CMGIM28a! and! CMGIM28c,! these! genomic! islands! may! be!
merged!into!one!(Mergeay!et!al.,!2009;!Van!Houdt!et!al.,!2009).!
In! contrast! to! pMOL28,! a! direct! relation! between! the! plasmid! pMOL30! and! other!
megaplasmids! is! less! evident.! Nevertheless,! the! annotation! of! pMOL30! revealed! some! backbone!
genes!that!are!related!to!those!genes!from!the!megaplasmid!pBVIE01!of!Burkholderia0vietnamiensis!!
41!
!
General!Introduction!
!
(
A.(
!
!
!
B.(
!
Figure(3.1:!Maps!of!the!two!large!plasmids!in!C.0metallidurans!CH34.!Genomic!islands!are!highlighted!in!grey.!(A.)!pMOL28!
plasmid;! the! plasmid! backbone! in! synteny! with! pHG1! from! C.0 eutrophus! H16! is! marked! in! dotted! lines.! (B.)! pMOL30!
plasmid;!the!plasmid!backbone!in!synteny!with! pBVIE01!from!B.0vietnamiensis!G4!is!marked!in!dotted!lines.!(Figure!from!
Mergeay!et!al!2009)!
!
!
42!
!
General!Introduction!
!
G4!(Janssen!et!al.,!2010;!Mergeay!et!al.,!2009;!Monchy!et!al.,!2007).!Similar!to!pMOL28,!two!genomic!
islands! are! distinguishable! from! the! plasmid! backbone! (Fig.! 3.1B).! The! CMGIM30a! island! of! pMOL30!
harbors!a!variety!of!genes!conferring!resistance!to!cobalt,!zinc!and!cadmium!(i.e.,!czc!operon),!lead!
(i.e.,!pbr!operon)!and!mercury!(i.e.,!mer!genes),!whereas!the!genomic!island!CMGIM30b!is!associated!
with!resistance!to!silver!(i.e.,!sil!operon)!and!copper!(i.e.,!cop!operon)!(Mergeay!et!al.,!2009;!Monchy!
et!al.,!2007;!Van!Houdt!et!al.,!2009).!Some!nre/ncc!genes!were!also!identified!in!CMGIM30b;!however,!
these! genes! seem! to! be! partially! deleted! or! damaged! and,! consequently,! do! not! appear! to! be!
functional!because!no!nickel!resistance!phenotype!was!linked!to!these!genes!(Mergeay!et!al.,!2009).!
In! addition! to! determinants! for! metal! resistance! found! in! both! genomic! islands,! notably,! some!
putative! glycosyltransferase! (gtr)! genes! were! also! identified! and! overexpressed! in! the! presence! of!
transition!metal!ions.!These!genes,!which!are!situated!between!some!metal!resistance!loci,!play!an!
important! role! in! maintaining! the! integrity! of! the! cell! wall! (Mergeay! et! al.,! 2009;! Monchy! et! al.,!
2007).!
Transcriptomic! data! obtained! after! various! challenges! by! transition! metal! ions! highlighted!
multiple! crossMresponses! exhibited! by! most! of! the! metal! resistance! operons! (Mergeay! et! al.,! 2009;!
Monchy! et! al.,! 2007).! These! multipleMmetal! responses! result! from! gene! induction! by! not! only! their!
respective! substrate! but! also! other! metals.! For! example,! mer! genes! are! overexpressed! in! the!
presence!of!not!only!Hg(II)!but!also!Cd(II)!and!Pb(II)!ions,!whereas!the!expression!of!the!cop!genes!is!
activated! in! response! to! a! wide! range! of! metals! (e.g.,! Cu,! Zn,! Cd,! Co! and! Ni).! Additionally,! the! cnr!
genes! of! pMOL28! are! induced! in! response! to! Ni(II),! Cu(II)! and! Cd(II)! ions! (Mergeay! et! al.,! 2009;!
Monchy!et!al.,!2007).!Of!all!the!transition!metals!tested,!the!most!upregulated!genes!were!localized!
on! both! megaplasmids;! however,! many! of! these! genes! were! also! found! on! the! two! chromosomes!
(Monchy! et! al.,! 2007).! Consequently,! the! cellular! defense! mechanism! of! C.0 metallidurans! CH34!
against!transition!metal!stress!most!likely!has!multiple!stages,!suggesting!a!more!general!response!to!
various!signals!first,!which!is!then!followed!by!a!more!metalMspecific!response!(Monchy!et!al.,!2007).!
Understanding! molecular! mechanisms! underlying! defenses! against! transition! metal! ions! and! their!
regulation! is! crucial.! Such! detailed! knowledge! will! help! to! improve! industrial! applications! using!
metalMresistant!bacteria,!such!as!biomonitoring!(via!biosensors),!waste!water!treatment!and!polluted!
soils!recycling!(i.e.,!bioaugmentation!and!phytoremediation)!(Mergeay!et!al.,!2009).!
(
3.2.(Copper(Resistance(in(Cupriavidus)metallidurans(CH34(
As! mentioned! earlier,! several! examples! of! chromosomal! and! plasmid! copperMresistance!
systems!have!been!reported!in!bacteria!(e.g.,!E.0hirae,!E.0coli,!P.0syringae!pv.!tomato,!etc.).!However,!
43!
!
General!Introduction!
!
copper!resistance!in!C.0metallidurans!CH34!is!interesting!because!of!particular!features!that!are!not!
present! in! homologous! systems.! Understanding! copper! resistance! in! C.0 metallidurans! CH34! is!
essential!and!required!for!an!inMdepth!understanding!of!copper!handling!by!bacteria!and!of!copper!
fate! in! these! cells.! Copper! resistance! in! C.0 metallidurans! CH34! is! fairly! well! documented,! although!
numerous!aspects!remain!unclear.!These!bacteria!are!particularly!well!adapted!to!resist!high!copper!
concentrations,! particularly! due! to! plasmidMencoded! Cop! proteins! allowing! copper! detoxification! in!
both!the!cytoplasmic!space!and!periplasmic!space!(Bersch!et!al.,!2008;!Mergeay!et!al.,!2003;!Monchy!
et!al.,!2007).!
In!fact,!a!microarray!analysis!highlighted!two!cop!clusters!induced!by!copper!found!on!both!the!
chromid! copSRABCD! and! the! plasmid! pMOL30! copVTMKNSRABCDIJGFOLQHEW! (Fig.! 3.2).! Although!
both! clusters! are! induced! after! a! challenge! with! elevated! Cu(II)! concentrations,! only! the! pMOL30M
borne!cop!cluster!displayed!high!and!clear!transcriptional!activation!compared!with!that!of!chromid!
cop! genes! (Monchy! et! al.,! 2006;! Monsieurs! et! al.,! 2011).! Notably,! the! plasmid! cop! cluster! is! also!
upregulated! by! various! metal! ions! (e.g.,! Cu,! Cd,! Ni,! Zn,! and! Co),! whereas! almost! no! transcriptional!
activation!of!the!chromid!cop!cluster!occurs!for!those!same!metals.!Consequently,!the!plasmid!cop!
genes! might! arguably! act! on! a! broader! copper! concentration! range! and! handle! other! metal! ions!
(Monsieurs!et!al.,!2011).!
!
!
Figure( 3.2:! Copper! resistance! operons! in! C.0 metallidurans! CH34.! The! organization! of! the! plasmidMborne! cop! cluster! is!
presented! on! the! top! of! the! schematic! drawing.! Similar! copSRABCD! genes! are! found! on! the! chromid! cop! operon! of! C.0
metallidurans!CH34,!as!observed!at!the!bottom!of!the!figure.!
!
Notably,! both! cop! gene! clusters! share! the! basic! copSRABCD! operon,! which! is! homologous! to!
the! previouslyMdiscussed! plasmidMborne! pco/cop! systems! and! which! encodes! proteins! expected! to!
participate!in!basic!mechanisms!for!periplasmic!copper!removal!(Mergeay!et!al.,!2003).!In!contrast!to!
the!copRS!genes!from!the!P.0syringae!pPT23D!plasmid!or!the!pcoRS!genes!from!the!E.0coli!pRJ1004!
plasmid,!which!are!downstream!of!the!cop/pcoABCD!operon!and!which!are!transcribed!in!the!same!
direction,!the!copRS!genes!on!pMOL30!are!directly!upstream!of!copABCD!and!are!transcribed!in!the!
opposite! direction! (Mergeay! et! al.,! 2003;! Monchy! et! al.,! 2006).! These! regulatory! genes! encode!
44!
!
General!Introduction!
!
proteins! that! are! members! of! the! twoMcomponent! family! of! sensor! regulators.! In! the! presence! of!
external!stimuli,!the!sensor!kinase!CopS,!which!is!in!the!inner!membrane,!autophosphorylates!at!an!
internal!histidine!and!then!transfers!the!high!energy!phosphate!group!to!an!aspartyl!residue!on!the!
response! regulator! CopR! (Mergeay! et! al.,! 2003).! However,! although! they! are! related,! the! plasmid!
copSRABCD! operon! encloses! special! features! that! differ! from! its! chromid! counterpart,! such! as! the!
pMOL30! copB! gene,! which! encodes! a! protein! containing! an! amplified! methionineMrich! motif!
(Mergeay! et! al.,! 2009;! Monsieurs! et! al.,! 2011).! Another! difference! is! that! the! copSRABCD! operon!
found!on!the!pMOL30!plasmid!is!included!in!a!larger!group!of!genes!that!appear!to!be!unique!to!C.0
metallidurans!CH34!or!even!to!the!Cupriavidus!genus!or!to!related!βMproteobacteria!(Mergeay!et!al.,!
2009;!Monchy!et!al.,!2006).!The!function!of!these!additional!Cop!proteins!remains!unclear;!however,!
all!plasmidMencoded!Cop!proteins!are!thought!to!be!required!to!ensure!an!optimal!resistance!to!high!
copper!concentrations!found!in!anthropogenic!metalMrich!biotopes!(Bersch!et!al.,!2008;!Sarret!et!al.,!
2010).!
Let! us! focus! on! some! general! details! regarding! plasmidMencoded! Cop! proteins.! The! large! cop!
cluster! on! pMOL30! is! composed! of! at! least! twentyMone! genes,! copVTMKNSRABCDIJGFOLQHEW,!
which!can!be!classified!into!three!categories!according!to!Monchy!et!al.!(Monchy!et!al.,!2006).!The!
first! category! encompasses! genes! encoding! proteins! involved! in! periplasmic! copper! detoxification,!
the! second! corresponds! to! genes! that! may! encode! proteins! involved! in! cytoplasmic! copper!
detoxification,!and!the!last!category!includes!genes!for!which!no!putative!function!could!be!assigned!
to! the! gene! product! (Monchy! et! al.,! 2006).! The! characterization! of! some! members! of! C.0
metallidurans'! cop! cluster! has! begun! to! improve! insight! into! their! involvement! in! the! copper!
resistance!mechanism!of!C.0metallidurans!CH34!(Fig.!3.3).!
!
Figure(3.3:!Schematic!illustration!of!copper!resistance!in!C.0metallidurans!CH34.!Cu(I)!and!Cu(II)!ions!are!drawn!in!the!shape!
of!white!and!black!balls,!respectively.!
45!
!
General!Introduction!
!
Proteins(likely(to(be(involved(in(periplasmic(copper(detoxification(
The!CopA(protein!shares!homology!with!multicopper!oxidases!CopA!from!P.0syringae!pv.!tomato!and!
PcoA!from!E.0coli!plasmid!pRJ1004!(Monchy!et!al.,!2006;!Rensing!and!Grass,!2003).!Found!in!both!the!
cytoplasmic!and!periplasmic!space,!this!putative!multicopper!oxidase!contains!a!MGGM!motif!that!is!
repeated!five!times!in!its!sequence.!Similar!methionineMrich!regions!were!also!identified!in!CopA!in!P.0
syringae!and!in!PcoA!in!E.0coli!(Bondarczuk!and!PiotrowskaMSeget,!2013).!
The!CopB(protein!is!predicted!to!be!an!outer!membraneMbound!protein!(Mergeay!et!al.,!2003).!This!
protein! contains! a! peculiar! methionineMrich! NMterminal! extremity! (i.e.,! 46! methionine! residues)!
wherein!the!same!motif,!MQGMDHSKMQGMDQGS,!is!repeated!ten!times.!By!contrast,!only!one!copy!
of!this!characteristic!motif!exists!in!the!chromid!CopB!counterpart.!This!motif!is!also!present!once!or!
few!times!in!other!CopB!proteins,!such!as!CopB!in!P.0syringae!or!CopB!in!Ralstonia0solanacearum,!and!
may!be!involved!in!Cu(I)!fixation!(Monchy!et!al.,!2006).!Nevertheless,!similar!amplified!methionineM
rich! motifs! have! also! been! found! in! the! NMterminal! extremity! of! copper! resistance! B! precursors! in!
Ralstonia0pickettii!12J!and!12D,!for!which!no!putative!function!has!yet!been!assigned.!
The! CopC( protein! is! a! soluble! periplasmic! protein! that! contains! two! predicted! copperMbinding! sites!
with! specific! affinities! for! Cu(I)! and! Cu(II)! (Monchy! et! al.,! 2006).! As! observed! in! numerous! CopC!
homologs! (such! CopC! in! P.0 syringae! pv.! tomato),! the! Cu(I)Mbinding! site! includes! a! methionineMrich!
motif,! MTGMPGMADHSPM,! similar! to! methionineMrich! motifs! found! on! the! CopB! protein!
(Bondarczuk!and!PiotrowskaMSeget,!2013;!Monchy!et!al.,!2006).!Based!on!this!specific!feature,!a!close!
interaction! may! occur! between! CopC! and! CopB! proteins.! In! contrast,! the! Cu(II)Mbinding! center! is! a!
type!2!copper!center!composed!of!nitrogen!and!oxygen!ligands!(such!as!histidine,!glutamic!acid!and!
aspartic! acid)! and! conserved! in! other! CopC! proteins! (Bondarczuk! and! PiotrowskaMSeget,! 2013;!
Monchy!et!al.,!2006).!Consequently,!these!two!copperMbinding!sites!may!provide!a!dual!function!to!
the!CopC!protein.!
The! CopD( protein! is! composed! of! eight! transmembrane! αMhelices! that! embed! CopD! in! the! inner!
membrane.!This!protein!most!likely!forms!a!channel!allowing!the!import!of!essential!copper!into!the!
cytoplasm!(Bondarczuk!and!PiotrowskaMSeget,!2013).!
The!CopI(protein!is!a!small!periplasmic!protein!that!shares!homology!with!blue!copper!proteins!and!
that!may!function!as!an!oxidoreductase!(Monchy!et!al.,!2006).!The!CopI!protein!is!also!characterized!
by!the!presence!of!a!methionineMrich!motif,!MEHEIM,!which!is!similar!to!the!motif!MXHXXM!found!
on!the!CopB!protein!(Monchy!et!al.,!2006).!
46!
!
General!Introduction!
!
The!CopK(protein!is!a!small!soluble!protein!that!accumulates!to!high!levels!in!the!periplasm!during!
challenge! with! high! copper! concentrations! (Monchy! et! al.,! 2006).! This! protein! is! unique! to! this!
system!and!does!not!have!any!homolog!in!the!Pco/Cop!systems!(Chong!et!al.,!2009).!ApoMCopK!can!
bind!both!Cu(I)!and!Cu(II)!ions!in!two!distinct!and!specific!metal!sites!with!an!unprecedented!binding!
cooperativity! (Chong! et! al.,! 2009;! Sarret! et! al.,! 2010).! This! protein! can! bind! a! Cu(I)! ion! with! high!
affinity!(KD!~!2!x!10M11!M)!in!a!tetrathioether!environment!made!of!methionine!residues,!whereas!this!
protein! binds! a! Cu2+! ion! with! low! affinity! (KD! >! 10M6! M)! (Chong! et! al.,! 2009;! Sarret! et! al.,! 2010).!
Notably,!the!binding!of!Cu(I)!enhances!the!affinity!of!the!protein!for!Cu(II)!(by!a!factor!at!least!106)!
whereas,!in!turn,!Cu(II)!binding!increases!KD(Cu+)!by!a!factor!of!~!102!(Chong!et!al.,!2009).!The!exact!
role! of! CopK! remains! unknown;! however,! this! protein! may! likely! function! as! a! periplasmic! copperM
sequestering!protein!and/or!a!copperMtransport!protein!(Chong!et!al.,!2009).!
Little!information!exists!regarding!the!small!CopG(protein.!Containing!a!short!cysteineMrich!motif!in!
its! sequence! (i.e.,! CXCC),! this! protein! appears! to! be! a! metalMbinding! periplasmic! protein!that! might!
function! as! a! putative! cupredoxin! containing! a! type! 1! copper! center! (Bondarczuk! and! PiotrowskaM
Seget,! 2013;! Monchy! et! al.,! 2006).! Although! the! detailed! mechanism! of! copper! handling! by! the!
protein!CopG!remains!unclear,!its!function!in!the!periplasm!is!assumed!to!be!closely!related!to!the!
CopA! ATPase! from! Vibrio0 cholerae,! which! is! homologous! to! the! CopA! ATPase! found! in! E.0 coli!
(Bondarczuk!and!PiotrowskaMSeget,!2013).!
The! CopH( protein! is! a! dimeric! protein! in! the! periplasmic! space! that! shares! identity! with! the! CzcE!
protein,! which! is! involved! in! cadmium/zinc/cobalt! resistance! in! C.0 metallidurans! CH34! (Mergeay! et!
al.,!2003).!Although!its!function!remains!unclear,!CopH!may!be!involved!in!the!late!response!phase!of!
copper!resistance!mechanism!(Monchy!et!al.,!2006).!In!fact,!a!microarray!analysis!indicated!that!the!
copH!gene!was!maximally!transcribed!after!only!one!hour!of!copper!exposure,!whereas!the!other!cop!
genes!were!optimally!induced!after!30!minutes!(Monchy!et!al.,!2006).!Characterized!by!the!absence!
of! cysteine! or! methionine! residues! in! it! sequence,! nevertheless,! this! protein! displays! two! histidine!
residues! that! contribute! to! the! formation! of! two! copperMbinding! sites.! Indeed,! CopH! can! bind!
specifically! two! Cu(II)! ions! per! dimer! with! high! affinity! (KD! ~36! nM,! evaluated! from! isothermal!
titration!calorimetry!experiments)!in!type!2!copper!sites!made!of!nitrogen!and!oxygen!ligands.!Once!
the! highMaffinity! sites! are! occupied,! the! CopH! protein! remains! able! to! bind! additional! copper! ions!
with!a!lower!affinity!(KD!~2.5!µM)!(Sendra!et!al.,!2009).!
The!CopT(protein!is!predicted!to!be!a!periplasmic!protein!similar!to!the!cytochrome!domain!of!PbrT,!
which!is!seemingly!an!importer!of!lead.!This!putative!cytochrome!protein!is!also!characterized!by!the!
presence!of!a!short!cysteineMrich!motif,!CXXCH!(Mergeay!et!al.,!2009;!Monchy!et!al.,!2006).!
47!
!
General!Introduction!
!
The!CopJ(protein!is!a!small!protein!predicted!to!be!in!the!periplasm.!Characterized!by!a!CXXCH!motif,!
this!protein!shows!similarity!with!cytochrome!c!proteins!(Mergeay!et!al.,!2009;!Monchy!et!al.,!2006).!
Proteins(likely(to(be(involved(in(cytoplasmic(copper(detoxification(
The!CopF(protein!is!a!transmembrane!P1Mtype!ATPase!that!harbors!an!NMterminal!histidineMrich!type!
of! metal! binding! motif! and! an! NMterminal! TRASH! domain! (i.e.,! a! wellMconserved,! extended! cysteine!
motif)! anticipated! to! be! involved! in! metal! coordination! (Ettema! et! al.,! 2003;! Mergeay! et! al.,! 2003;!
Monchy! et! al.,! 2006).! This! protein! may! be! in! the! inner! membrane! and! may! form! a! cation! channel!
with! its! transmembrane! αMhelices.! The! CopF! protein! may! mediate! the! efflux! of! Cu(I)! ions! from! the!
cytoplasm!to!the!periplasmic!space!(Mergeay!et!al.,!2003;!Monchy!et!al.,!2006).!
Proteins(for(which(no(putative(function(could(be(assigned(
The!CopQ(protein!is!an!extremely!small!protein!encoded!by!the!copQ!gene,!which!belongs!to!a!group!
of!nineteen!homologous!genes!encoding!putative!small!stress!response!proteins.!These!genes!appear!
to! be! restricted! to! the! genera! Cupriavidus! and! Ralstonia! (Janssen! et! al.,! 2010).! Predicted! as! being!
localized!in!the!periplasmic!space,!the!CopQ!protein!is!highly!induced!in!the!presence!of!many!metals!
(e.g.,!Cu,!Cd,!Co,!Zn,!Pb!and!Ni)!(Mergeay!et!al.,!2009;!Monchy!et!al.,!2007).!
The!CopL(protein!may!be!a!putative!membrane!protein!that!contains!a!cysteineMrich!motif,!HXCXCC.!
Its! precise! role! remains! unknown;! however,! the! CopL! protein! may! be! involved! in! regulating! CopF!
ATPase!(Mergeay!et!al.,!2003;!Monchy!et!al.,!2006).!
The!CopO(protein!is!a!very!small!protein!with!a!predicted!length!of!63!amino!acids.!The!sequence!of!
this! protein! is! characterized! by! a! CMterminal! extremity! rich! in! histidine! residues.! Because! the! copO!
gene! is! associated! with! the! copF! gene! that! encodes! a! PMtype! ATPase,! the! CopO! protein! may! be! a!
putative! conserved! chaperone! for! PMtype! ATPases! in! the! inner! membrane! (van! Aelst,! 2008).! The!
CopE,(CopN(and(CopV(proteins!are!predicted!to!be!in!the!cytoplasm,!whereas!the!CopM(and!CopW(
proteins!may!be!in!the!periplasmic!space.!All!these!proteins!do!not!have!any!equivalent!in!current!
databases,!and!no!putative!function!can!be!assigned!to!them!(Mergeay!et!al.,!2009;!Monchy!et!al.,!
2006).!
The!complexity!of!the!protein!assembly!induced!by!copper!is!obvious,!and!we!remain!far!from!
understanding!the!complete!function!of!this!protein!network.!In!fact,!in!addition!to!the!plasmid!cop!
genes,! other! genes! harbored! by! the! plasmid! pMOL30! may! also! be! required! for! full! resistance! to!
copper! ions! (Mergeay! et! al.,! 2009).! Some! chromosomal! genes! may! also! be! involved! in! copper!
48!
!
General!Introduction!
!
resistance!and!homeostasis,!such!as!the!RNDMencoding!cusDCBAF!cluster!on!the!chromid!or!the!cup!
cluster!on!the!chromosome!encoding!a!PMtype!ATPase!named!CupA!(Mergeay!et!al.,!2003;!Monchy!et!
al.,! 2006).! However,! a! recent! microarray! analysis! demonstrated! that! these! chromosomal! genes!
previously!marked!as!copperMresponsive!were!not!upregulated!after!copper!exposure!(Monsieurs!et!
al.,!2011).!Nevertheless,!the!neighboring!sil!genes!on!the!genomic!island!CMGIM30b!of!pMOL30!may!
play!a!role!in!copper!detoxification!based!on!RNDMmediated!efflux!(Fig.!3.3).!Notably,!almost!all!the!
genes!between!the!sil!and!cop!clusters!are!overexpressed!under!copper!exposure,!which!most!likely!
indicates!that!all!of!these!genes!are!required!for!maximal!expression!of!copper!resistance!(Mergeay!
et!al.,!2009).!To!provide!insight!regarding!this!copper!resistance!mechanism,!we!focused!our!studies!
on!the!plasmidMencoded!CopB!protein,!which!is!particularly!appealing!due!to!its!peculiar!methionineM
rich!extremity,!and!for!which!numerous!questions!remain!unanswered.!
!
3.3.(Phenotypic(Analysis(of(Some(cop(Mutants(
The! construction! of! mutants! is! a! common! approach! in! biology! to! understand! the! role! of! a!
protein!and!its!involvement!in!resistance!mechanisms.!Insertional!mutants!have!been!constructed!for!
14! cop! genes! in! C.0 metallidurans! CH34! on! pMOL30! plasmid! and! on! pMOL1024! (i.e.,! a! derivative! of!
the!cosmid!pLAFR3!wherein!only!the!cop!genes!from!copV!to!copH!were!cloned).!They!were!made!
isogenic! due! to! homologous! recombination! between! pMOL1024! and! pMOL30! (van! Aelst,! 2008).!
These! mutants! were! investigated! in! a! previous! study! carried! out! by! Sébastien! van! Aelst! using! two!
preculture! conditions! (i.e.,! uninduced! and! copperMinduced)! (van! Aelst,! 2008).! Although! our!
understanding! of! copper! resistance! is! still! far! from! being! complete,! phenotypic! analysis! of! these!
mutants!have!shown!interesting!facts!that!are!relevant!for!our!study!as!illustrated!by!the!copS,!the!
copR!and!the!copB!mutants!discussed!below.!
The!phenotypic!analysis!was!carried!out!by!looking!at!the!viable!counts!of!the!mutants!and!the!
reference!strains!exposed!to!various!Cu(II)!concentrations!(from!0.3!mM!to!3!mM).!As!shown!in!Fig.!
3.4! to! 3.6,! reference! strains! included! the! wildMtype! strain,! its! plasmidMfree! derivative! AE104! and! a!
derivative!of!AE104!carrying!the!recombinant!plasmid!pMOL1024!(i.e.,!pLAFR3!where!the!genes!copV!
to! copH! were! cloned).! As! far! as! the! reference! strains! are! concerned,! Fig.! 3.4,! 3.5! and! 3.6! revealed!
that!the!wildMtype!resistance!to!copper!is!expressed!in!a!narrow!concentration!domain!if!compared!
to!the!resistance!domain!of!the!plasmidMfree!derivative!(i.e.,!AE104).!This!is!in!strong!contrast!with!
the!resistance!observed!for!other!metals!especially!zinc!and!cobalt!(Mergeay,!1995).!Surely,!it!does!
not! help! to! understand! why! so! many! genes! seem! to! be! involved! in! the! response! to! copper! and!
49!
!
General!Introduction!
!
especially,!genes!that!look!unique!for!the!species!C.0metallidurans!and!its!mobile!genetic!elements.!
Yet,!(i)!the!response!to!the!other!metals!validate!the!use!of!viable!counts!to!describe!the!response!to!
heavy!metals,!and!(ii)!it!should!be!recalled!that!the!chromid!of!C.0metallidurans!also!carries!genes!for!
resistance! to! copper! and! that! these! genes! (e.g.,! copABCD! and! cus...)! are! functional! according! to!
transcriptomic! studies! (Monsieurs! et! al.,! 2011)! and! may! display! a! resistance! concentration! domain!
that!recovers!a!substantial!part!of!the!wildMtype!resistance!domain.!On!the!other!hand,!pMOL30!also!
seems!to!carry!additional!functions!contributing!to!the!resistance!to!copper,!as!suggested!in!Fig.!3.5!
(i.e.,!the!wildMtype!strain!is!indeed!somewhat!more!resistant!to!Cu(II)!than!the!strain!AE1744).!
Figure( 3.4:! Viability! curves! of! the! copB! mutant! in! the!
presence!of!copper!ions,!in!the!context!of!pMOL1024,!in!
uninduced!preculture!conditions.!
CH34! (pMOL30! and! pMOL28),! AE104! (no! plasmid),!
AE1744!(plasmid!pMOL1024,!which!is!a!derivative!of!the!
cosmid! pLAFR3! wherein! the! cop! genes! were! cloned),!
–
pMOL1024copB (copB! mutant! in! the! context! of!
pMOL1024).!Figure!from!S.!van!Aelst,!2008.!
!
As!shown!in!Fig.!3.6,!the!wildMtype!strain!displays!two!kinds!of!responses!in!the!tested!domain!
of!Cu(II)!concentrations:!one!up!to!~2.1!mM!roughly!describes!the!resistance!domain!and!the!second!
one!that!lies!beyond!the!minimal!inhibitory!concentration!(MIC)!shows!the!presence!of!survivors!at!a!
rather! constant! percentage! (~0.3%).! These! survivors! are! likely! not! resistant! mutants,! but! look! like!
"persister"! (Harrison! et! al.,! 2005;! Lewis,! 2008;! van! Aelst,! 2008).! From! the! phenotypic! analysis! in!
copper! induced! conditions! of! the! available! cop! mutants,! it! seems! that! (i)! some! genes! are! mainly!
affected!in!the!resistance!copper!concentration!domain!(i.e.,!copS!and!copD)!as!expected!but!provide!
a! wildMtype! amount! of! survivors,! (ii)! other! genes! are! mainly! affected! in! the! concentration! domain!
where!survivors!are!observed!(i.e.,!copV,!copT!and!copG),!and!(iii)!copR!is!affected!in!both!domains.!It!
is!very!important!because!it!seems!that!some!cop!genes!(especially!those!that!look!to!be!unique!to!C.0
metallidurans! species)! are! involved! in! the! survival! or! the! general! response! to! Cu(II)! concentrations!
that! incompatible! with! active! growth! as! it! is! typically! the! case! in! industrial! wastes! where! many! C.0
metallidurans!strains!were!found!(Diels!and!Mergeay,!1990)!
In!this!respect,!the!copB!mutant!displays!some!remarkable!features:!in!the!context!of!plasmid!
pMOL1024! and! in! NONMinduced! conditions,! this! mutant! is! hypersensitive! to! copper! (Fig.! 3.4,! green!
curve).! This! phenotype! is! absolutely! unique! to! this! mutant! and! is! not! observed! in! the! pMOL30!
genetic!context!suggesting!that!in!the!pMOL30!genetic!context,!some!phenotypic!complementation!
50!
!
General!Introduction!
!
may!exist!and!that!in!the!pMOL1024,!there!is!no!observed!phenotypic!complementation!by!chromid!
based!functions!(likely!cop!or!cus!chromid!genes).!
Figure( 3.5:! Viability! curves! of! the! copB! mutant! in! the!
presence! of! copper! ions,! in! the! context! of! pMOL30,! in!
uninduced!preculture!conditions.!
CH34! (pMOL30! and! pMOL28),! AE104! (no! plasmid),!
–
pMOL30copB (copB!mutant!in!the!context!of!pMOL30).!
Figure!from!S.!van!Aelst,!2008.!
!
In! the! pMOL30! genetic! context,! in! uninduced! preculture! conditions,! the! copB! mutant! has! a!
wildMtyp!phenotype!for!most!of!the!tested!concentration!domain.!This!is!surprising!and!suggests!that!
its! high! content! in! methionineMrich! motifs! is! mainly! useful! at! concentrations! above! the! MIC.! In! the!
pMOL30!context,!in!copperMinduced!preculture!conditions,!a!hyperresistant!phenotype!was!observed!
for!the!copB!mutant!(Fig.!3.6,!green!curve).!This!phenotypic!observation!might!suggest!that!the!copB!
gene!is!not!necessary!in!induced!copper!conditions,!at!least!for!the!concentration!range!investigated.!
However,!a!drastic!drop!in!viability!is!nevertheless!observed!after!2.1!mM!of!Cu(II),!meaning!that!the!
copB!gene!is!likely!essential!at!high!copper!concentrations.!
Figure( 3.6:! Viability! curves! of! the! copB! mutant! in! the!
presence! of! copper! ions,! in! the! context! of! pMOL30,! in!
copperMinduced!preculture!conditions.!
CH34! (pMOL30! and! pMOL28),! AE104! (no! plasmid),!
AE1744!(plasmid!pMOL1024,!which!is!a!derivative!of!the!
cosmid! pLAFR3! wherein! the! cop! genes! were! cloned),!
–
pMOL30copB (copB!mutant!in!the!context!of!pMOL30).!
Figure!from!S.!van!Aelst,!2008.!
!
The! work! of! Sébastien! van! Aelst! clearly! demonstrated! that! the! individual! cop! genes! may! be!
compensated! by! other! genes! (e.g.,! other! cop! genes! and/or! other! genes! such! as! sil! genes).! In!
summary,!the!cop!genes!may!be!classified!in!three!categories:!(i)!cop0genes!which!have!an!influence!
on!copper!resistance!but!not!on!the!survival!at!high!copper!concentrations!such!as!the!copS!gene,!(ii)!
cop! genes! that! influence! copper! resistance! and! survival! at! high! copper! concentrations! such! as! the0
copR!gene,!and!(iii)!cop!genes!that!influence!only!the!survival!at!high!copper!concentrations!such!as!
the!copB!gene.!
51!
!
General!Introduction!
!
3.4.(Thesis(Aim(
This! work! focused! on! copper! resistance! in! C.0 metallidurans! CH34.! The! copper! regulation!
mechanism! of! C.0 metallidurans! CH34! is! known! to! essentially! involve! the! pMOL30Mencoded! cop!
cluster,!copVTMKNSRABCDIJGFOLQHEW,!which!is!highly!upregulated!by!Cu(II)!and!by!a!wide!range!of!
other! metals.! Although! this! copper! resistance! has! been! extensively! studied,! numerous! questions!
remain!unanswered!thus!far.!During!this!work,!we!attempted!to!provide!insight!regarding!a!few!of!
these!questions!to!decipher!the!complex!response!of!C.0metallidurans!CH34!to!copper!on!a!molecular!
level.!Providing!answers!regarding!the!fate!of!copper!in!C.0metallidurans!CH34!and!characterizing!the!
plasmidMencoded!CopB!protein!are!the!primary!objectives!of!this!research.!
The!following!"Results!and!Discussion"!section!is!organized!into!two!parts.!The!first!part!(i.e.,!
Chapter! 4)! details! the! characterization! and! metalMbinding! properties! of! the! plasmidMencoded! CopB!
protein!in!C.0metallidurans!CH34,!which!is!characterized!by!an!amplified!sequential!methionineMrich!
motif! (MQGMDHSKMQGMDQGS).! Using! complementary! techniques! (MS,! NMR,! CD),! synthetic!
peptides! that! deviated! from! this! methionineMrich! domain! were! shown! to! significantly! interact! with!
Cu(I)! ions! and,! to! a! lesser! extent,! with! Cu(II)! ions.! Chemical! crossMlinking! experiments! also! allowed!
the! identification! of! CopA,! which! is! a! putative! multicopper! oxidase,! as! one! of! the! CopBMinteracting!
partners!involved!in!the!copper!resistance!mechanism!of!C.0metallidurans!CH34.!
The!second!part!(i.e.,!Chapter!5)!highlights!a!vesiculation!phenomenon!upon!copper!exposure,!
which!has!never!been!described!thus!far!in!C.0metallidurans!CH34.!The!study!of!mutants!derived!from!
the! wildMtype! strain! CH34,! the! presence! of! copper! and! several! Cop! proteins! inside! vesicles! suggest!
that!this!vesiculation!process!might!provide!a!defensive!role!in!C.0metallidurans!CH34.!
(
(
52!
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General!Introduction!
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Results!and!Discussion!
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Results'and'Discussion'
'
Chapter(4:( Functional(Study(of(the(Plasmid9Encoded(CopB(Protein(from(
Cupriavidus*metallidurans(CH34(
(
4.1.(Abstract*
Copper' is' a' paradoxical' element,' both' essential' and' extremely' toxic,' which' compels'
microorganisms'to'develop'resistance'mechanisms'to'tightly'control'its'homeostasis.'In'Cupriavidus*
metallidurans' CH34,' copper' resistance' essentially* involves' the* cop' cluster' of' genes' on' the' plasmid'
pMOL30.'Among'the'Cop'protein'products'expressed'from'this'cluster,'CopB'is'of'particular'interest'
due' to' the' presence' of' ten' identical' methionineIrich' motifs' (MQGMDHSKMQGMDQGS)' at' its'
extreme'NIterminus.'Based'on'secondary'structure'predictions,'this'protein'is'thought'to'have'two'
different' structural' domains:' an' unstructured' NIterminal' extremity' and' a' structural' CIterminal'
subdomain'containing'numerous'βIstrands'and'few'αIhelices.'In'order'to'characterize'molecular'and'
functional' properties' of' the' CopB' protein,' recombinant' proteins' and' synthetic' peptides' were'
investigated' by' mass' spectrometry,' circular' dichroism' and' 1HInuclear' magnetic' resonance'
spectroscopies'in'the'absence'and'presence'of'Cu(II),'Cu(I)'or'Ag(I)'ions.'Upon'additions'of'copper'or'
silver,'the'results'established'that'the'native'peptide'containing'four'methionines'and'one'histidine'
residue'was'able'to'bind'one'copper'ion'[Cu(I)'or'Cu(II)]'or'one'silver'ion.'However,'it'also'appeared'
that'the'native'peptide'preferentially'bound'Cu(I)'with'a'moderate'affinity'(KD'~49'µM)'over'either'
Cu(II)' or' Ag(I)' ions' (KD~71.9' and' 70.4' µM,' respectively)' in' a' relatively' unordered' binding' site.' In'
agreement' with' this' result,' the' native' double' motif' peptide' bound' two' Cu(I)' ions,' suggesting' that'
CopB'might'bind'at'least'ten'Cu(I)'ions.'This'study'was'strengthened'by'subcellular'fractionation'and'
immunogold' labeling' of' copperIexposed' C.* metallidurans.' These' data' furthermore' demonstrated'
that' CopB' was' an' outer' membraneIassociated' protein' that' was' most' likely' folded' into' a' βIbarrel'
conformation,' as' showed' by' circular' dichroism' and' transmembrane' protein' topology' prediction.'
Chemical' crossIlinking' experiments' were' also' performed' and' highlighted' the' periplasmic' CopA'
protein' as' a' potential' CopBIinteracting' partner.' These' findings' are' likely' to' improve' our'
understanding'of'copper'resistance'mechanism'in'C.*metallidurans.'
(
(
62'
'
Results'and'Discussion'
'
4.2.(Introduction(
Copper' is' an' essential' micronutrient' that' is' required' for' all' living' organisms' and' serves' as' a'
cofactor' for' many' enzymes' (e.g.,' superoxide' dismutase,' cytochrome' c' oxidase,' etc.),' carrying' out'
fundamental'cellular'functions'(Bondarczuk'and'PiotrowskaISeget,'2013).'Although'copper'is'crucial'
for'cell'metabolism,'an'excess'of'copper'is'harmful'to'cells'and'leads'to'cellular'damage.'Therefore,'a'
strong' regulation' of' cellular' copper' levels' is' required' in' all' organisms' to' preclude' toxic' effects'
(Bondarczuk'and'PiotrowskaISeget,'2013).'
Initially' isolated' from' the' sediment' of' a' nonIferrous' metallurgical' plant' in' Belgium,' the' βI
proteobacterium' Cupriavidus* metallidurans' strain' CH34' is' a' microorganism' that' is' characteristic' of'
metalIcontaminated'biotopes'(Mergeay'et'al.,'2003).'Particularly'wellIadapted'to'tolerate'and'resist'
the' presence' of' millimolar' concentrations' of' numerous' metals,' this' bacterium' represents' an'
important' model' for' studying' metal' resistances' (Bersch' et' al.,' 2008).' The' complex' genome' of' C.*
metallidurans'CH34'consists'of'a'main'chromosome,'a'chromid'and'two'large'plasmids,'pMOL28'and'
pMOL30,'carrying'the'primary'key'gene'clusters'encoding'machinery'for'resistance'to'various'metals'
(Monchy'et'al.,'2007;'Van'Houdt'and'Mergeay,'2012;'von'Rozycki'and'Nies,'2009).'
Genetic' determinants' for' copper' resistance' in' C.* metallidurans' CH34' essentially' include' the'
large' cop' cluster' on' the' plasmid' pMOL30,' copVTMKNSRABCDIJGFOLQHEW,' which' is' highly'
upregulated' by' Cu(II)' and' a' few' other' metals,' and' appears' to' be' required' for' maximal' resistance'
(Monsieurs' et' al.,' 2011).' This' set' of' plasmidIencoded' cellular' components' participate' both' in'
cytoplasmic'and'periplasmic'copper'efflux'(Mergeay'et'al.,'2003;'Monchy'et'al.,'2007).'Genes'such'as'
copK,'copH,'copM,'copV,'copN'of'the'cop'cluster'appear'to'be'unique'to'C.*metallidurans'CH34,'or'
even'to'the'Cupriavidus'genus'or'related'βIproteobacteria'and'the'exact'role'of'such'gene'products'
remains'elusive'(Mergeay'et'al.,'2009;'Monchy'et'al.,'2006).'On'the'other'hand,'the'cop'cluster'also'
includes'the'basic'copSRABCD'operon,'which'is'homologous'to'the'pcoABCDRSE'and'copABCDRS'loci'
found' in' some' strains' of' Escherichia* coli' and' Pseudomonas* syringae' pathovar' tomato,' respectively'
(Bondarczuk' and' PiotrowskaISeget,' 2013;' Sendra' et' al.,' 2006).' A' particular' feature' of' C.*
metallidurans'CH34'is'the'presence'of'the'copSRABCD'cluster,'duplicated'on'both'the'chromid'and'
plasmid' pMOL30.' The' copSRABCD' cluster' is' expected' to' be' involved' in' basic' mechanisms,' allowing'
for' the' efflux' of' cellular' copper' from' the' periplasmic' space'(Monchy' et' al.,' 2006;' Monsieurs' et' al.,'
2011).' Although' they' are' closely' related,' the' copSRABCD' operon' on' the' plasmid' contains' unique'
features'that'differ'from'the'complementary'operon'on'the'chromid,'as'observed'with'the'pMOL30I
encoded'CopB'protein'(Mergeay'et'al.,'2009;'Monsieurs'et'al.,'2011).'
63'
'
Results'and'Discussion'
'
The'plasmidIencoded'CopB'protein,'which'is'highly'induced'in'the'presence'of'copper'salts,'is'
interesting' by' virtue' of' its' amplified' methionineIrich' motif,' absent' in' its' chromidIencoded' CopB'
counterpart.' Its' NIterminal' extremity' contains' 46' methionine' residues,' thought' to' be' involved' in'
Cu(I)' binding,' and' are' mainly' arranged' as' ten' identical' sequential' motifs' with' the' consensus'
sequence' MXXMXHXXMXXMX' (Monchy' et' al.,' 2006).' Interestingly,' based' on' secondary' structure'
predictions,'it'appears'that'the'pMOL30Iencoded'CopB'protein'has'two'distinct'structural'domains,'
as' shown' in' Figure' 4.1:' an' unstructured' NIterminal' extremity' and' a' structured' CIterminal' domain'
that'contains'numerous'βIstrands'and'few'αIhelices.'This'structural'domain'is'identical'to'the'one'of'
the'chromidIencoded'CopB'counterpart.'Predicted'as'an'outer'membraneIbound'protein'(Mergeay'
et'al.,'2003),' it' was' hypothesized' that' the' plasmidIencoded'CopB'protein'might'be'a'copper'pump'
allowing'for'the'efflux'of'metal'from'the'cell,'as'predicted'for'its'PcoB'homologs'in'some'strains'of'E.*
coli' (Chaturvedi' and' Henderson,' 2014).' However,' to' the' best' of' our' knowledge,' the' exact' function'
and'copperIbinding'abilities'of'this'outer'membrane'protein'have'yet'to'be'demonstrated.'
'
Figure(4.1:'StructureIbased'sequence'alignment'of'the'pMOL30Iencoded'CopB'protein'and'the'chromidIencoded'CopB'
protein' from* C.* metallidurans' CH34,' colorIcoded' for' secondary' structure' type' (red' for' helices' and' blue' for' strands).'
Amino' acid' sequence' alignment' of' CopB' proteins' was' performed' using' the' PRALINE' software' tool'
(http://www.ibi.vu.nl/programs/)' with' the' BLOSUM62' weights' matrix.' Secondary' structure' prediction' was' performed'
using'DSSP'(Kabsch'and'Sander,'1983)'and'PSIPRED'(Jones,'1999).'
64'
'
Results'and'Discussion'
'
To'characterize'the'molecular'and'functional'properties'of'the'plasmidIencoded'CopB'protein,'
synthetic' peptides' and' recombinant' proteins' were' investigated' by' mass' spectrometry,' circular'
dichroism'and' 1HInuclear'magnetic'resonance'spectroscopies'in'the'absence'and'presence'of'Cu(II),'
Cu(I),'or'Ag(I)'ions.'Upon'additions'of'copper'or'silver,'model'methionineIrich'peptides'were'able'to'
bind'one'copper'ion'[Cu(I)'or'Cu(II)]'or'one'Ag(I)'ion.'Moreover,'it'appeared'that'they'preferentially'
bound' Cu(I)' over' Cu(II)' and' Ag(I)' ions.' Dissociation' constant' (KD)' values' were' also' determined' by'
mass' spectrometry' and' ranged' from' 9.9' to' 59.5' µM' in' the' presence' of' Cu(I)' ions.' Subcellular'
fractionation' and' immunogold' labeling' of' copperIexposed' C.* metallidurans' also' showed' that' the'
CopB' protein' was' outer' membraneIassociated' and' was' most' likely' folded' into' a' βIbarrel'
conformation,' as' shown' by' circular' dichroism' and' transmembrane' protein' topology' predictions.'
CrossIlinking' experiments' reinforced' these' results' and' allowed' for' the' identification' of' the' CopA'
protein,' a' putative' multicopper' oxidase,' as' being' one' of' the' CopBIinteracting' partners' involved' in'
copper'resistance'mechanisms.'Taken'together,'these'results'promote'a'better'understanding'of'the'
role'of'CopB'and'open'up'interesting'horizons'for'the'comprehension'of'copper'resistance'molecular'
mechanisms'in'C.*metallidurans'CH34.'
(
(
65'
'
Results'and'Discussion'
'
4.3.(Results(
4.3.1.(( Characterization(of(Recombinant(Proteins(
The' methionineIrich' NIterminal' extremity' of' CopB' is' unusual' among' related' proteins,' as'
shown' in' Figure' 4.2.' Multiple' amino' acid' sequence' alignment' of' the' different' CopB' proteins'
present'in'the'databank'revealed'that'the'CIterminal'extremity'was'highly'conserved'among'all'of'
these' proteins.' However,' the' methionineIrich' sequence' is' arranged' as' multiple' repeating' motifs'
and'appeared'to'be'specific'to'the'plasmidIencoded'CopB'protein'from'C.*metallidurans'CH34.'This'
methionineIrich' extremity' was' partially' identified' in' the' CopB' homolog' from' Ralstonia* Pickettii'
12D,' but' no' equivalent' sequence' was' observed' in' the' C.* metallidurans' CH34' chromidIencoded'
counterpart.'
In'this'study,'three'recombinant'proteins'were'overexpressed'and'purified,'as'shown'in'Figure'
4.3.' Such' recombinant' proteins' corresponded' to' (i)' the' fullIlength'plasmidIencoded'CopB'protein'
(Fig.' 4.3A);' (ii)' its' methionineIrich' NIterminal' extremity,' hereafter' referred' to' as' CopB(Met)' (Fig.'
4.3B);'and'(iii)'its'CIterminal'extremity,'predicted'to'be'structured,'CopB251I495'(Fig.'4.3C).'Polyclonal'
antisera' were' also' raised' against' the' fullIlength' CopB' protein' and' clearly' recognized' the' three'
different'recombinant'proteins'(data'not'shown).'
'
Figure( 4.3:' SDSIpolyacrylamide' gel' (4I20%)' showing' the'
protein' profile' of' purified' recombinant' proteins.' (A.)' FullI
length'plasmidIencoded'CopB'protein'from'C.*metallidurans'
CH34;' (B.)' CopB(Met)' protein' corresponding' to' the'
methionineIrich' NIterminal' extremity' of' the' CopB' protein;'
(C.)' CopB251I495' corresponding' to' the' CIterminal' subdomain'
of'the'CopB'protein.'
'
'
In'an'attempt'to'characterize'the'three'purified'recombinant'proteins,'circular'dichroism'(CD)'
experiments'were'performed'in'25'mM'ammonium'acetate'in'the'absence'and'presence'of'metal'
ions'(i.e.,'Cu2+'and'Ag+).'As'expected,'in'the'absence'of'metal,'the'CD'spectrum'associated'with'the'
CopB(Met)'protein'showed'spectral'features'typical'of'randomIcoil'proteins'(Fig.'4.4A,'blue'curve).'
Estimations'of'the'fractional'percentages'(67%'unordered,'13%'turns,'18%'strands'and'2%'helices)'
of'secondary'structure'for'CopB(Met)'were'in'line'with'experimental'data.'
66'
'
Results'and'Discussion'
'
'
67'
'
Results'and'Discussion'
'
By'contrast,'the'CD'spectrum'of'the'fullIlength'CopB'protein'appeared'different'when'compared'to'
data' for' CopB(Met)' (Fig.' 4.4A,' black' curve).' This' difference' was' reflected' in' the' percentage' (26%'
unordered,'28%'turns,'41%'strands'and'5%'helices)'of'secondary'structures'and'was'in'accordance'
with' the' secondary' structure' prediction' shown' in' Figure' 4.1.' However,' the' spectroscopic'
investigation'of'CopB251I495'protein'was'unsuccessful'due'to'instability'of'the'protein'in'the'absence'
of'6'M'urea,'which'is'required'for'its'extraction'from'inclusion'bodies.'Upon'additions'of'metal,'the'
fullIlength' CopB' protein' was' completely' unstable,' resulting' in' our' inability' to' characterize' this'
protein.' By' contrast,' freshly' purified' CopB(Met)' protein' was' studied' by' circular' dichroism' upon'
additions' of' Cu(II)' or' Ag(I)' (Fig.' 4.4B' and' C,' respectively).' However,' with' the' exception' of' a'
significant' positive' shift' in' the' negative' 197' nm' band,' no' major' changes' in' the' spectra' were'
observed,'suggesting'that'the'CopB(Met)'protein'remained'largely'unfolded.'
'
Figure(4.4:'Circular'dichroism'spectra'of'4'µM'recombinant'proteins'in'25'mM'ammonium'acetate,'in'the'absence'and'
presence'of'metal'ions.'(A.)'FullIlength'plasmidIencoded'CopB'protein'(black'line)'and'CopB(Met)'protein'(blue'line),'in'
the' absence' of' metal' ions.' (B.)' CopB(Met)' protein' in' the' absence' and' presence' of' CuCl2' •' 2H2O.' The' apoIprotein' is'
represented'by'a'solid'black'line;'additions'of'2'molar'Eq,'10'molar'Eq,'and'14'molar'Eq'of'CuCl2'•'2H2O'are'indicated'by'
the'dashed'blue,'green,'and'grey'lines,'respectively.'(C.)'CopB(Met)'protein'in'the'absence'and'presence'of'AgNO3.'The'
apoIprotein' is' represented' by' a' solid' black' line;' additions' of' 2' molar' Eq,' 10' molar' Eq,' and' 14' molar' Eq' of' AgNO3' are'
indicated' by' the' dashed' blue,' green,' and' grey' lines,' respectively.' Each' curve' represents' an' average' of' three' scans' and'
was'baselineIcorrected'by'subtraction'of'the'background'for'the'spectrum'obtained'with'buffer'alone.'
'
4.3.2.(( Stoichiometry(of(Metal(—(Peptide(Interactions(by(Mass(Spectrometry(
The'difficulties'associated'with'recombinant'proteins'prompted'us'to'study'the'metal'binding'
properties' of' the' protein' of' interest' using' synthetic' peptides.' Five' model' peptides' were'
investigated' in' this' study' and' are' listed' in' Table' 4.1.' They' were' studied' by' ESIIMS' in' an' effort' to'
elucidate'the'binding'stoichiometry'with'copper'[Cu(II)'and'Cu(I)]'as'well'as'silver,'a'surrogate'for'
Cu(I)' ions.' These' peptides' corresponded,' respectively,' to' the' native' MetIrich' motif' from' the'
plasmidIencoded'CopB'protein'(hereafter'referred'to'as'"nPep."),'the'native'double'motif'(named'
"dPep.")'and'three'analogs'of'the'native'peptide:'one'with'His9'replaced'by'Ala'("Pep.'1"),'another'
with'Met4'replaced'by'Gly'("Pep.'2"),'and'one'with'Met12'replaced'by'Gly'("Pep.'3").'These'analogs'
were' chosen' to' determine' whether' the' presence' of' histidine' at' position' 9' or' methionines' at'
68'
'
Results'and'Discussion'
'
positions'4'and'12'contributes'to'the'metalIbinding'geometry'of'the'peptide.'Methionine'residues'
were'replaced'by'glycine'residues'to'confer'a'greater'flexibility'on'peptides.'Such'flexibility'would'
presumably'allow'a'more'favored'binding'coordination'as'well'as'a'greater'binding'affinity.'
'
Table(4.1:'Amino'acid'sequences'of'the'synthetic'peptides'
Native'peptide'(nPep.)'
Double'peptide'(dPep.)'
Pep.'1'(H9→A)'
Pep.'2'(M4→G)'
Pep.'3'(M12→G)'
AcIQGSMQGMDHSKMQGMD'
AcIQGSMQGMDHSKMQGMDQGSMQGMDHSKMQGMD'
AcIQGSMQGMDASKMQGMD'
AcIQGSGQGMDHSKMQGMD'
AcIQGSMQGMDHSKGQGMD'
'
'
Mass' spectra' were' recorded' for' each' peptide' sample' at' a' concentration' of' 20' µM' in' the'
absence' and' presence' of' various' molar' equivalents' of' Cu(II),' Cu(I),' and' Ag(I).' The' Cu(I)' ions' were'
obtained'by'reducing'Cu(II)'ions'with'5'mM'H2Asc,'as'referenced'in'Rubino'et'al.'(2010)'and'Jiang'et'
al.'(2005)'(Jiang'et'al.,'2005;'Rubino'et'al.,'2010).'For'all'apoIpeptides'with'the'exception'of'dPep.,'
the' doubly' charged' ion' was' the' most' intense' signal,' as' shown' by' the' strong' [M' +' 2H]2+' peak' in'
Figure'4.5.'
'
'
Figure( 4.5:' Positive' electrospray' ionization' mass' spectrometry' (ESIIMS)' spectra' of' the' native' peptide' (AcI
QGSMQGMDHSKMQGMD)' and' its' Cu(I)' adducts.' Only' the' doublyIcharged' state' distribution' is' presented' on' the'
2+
spectra.'In'the'absence'of'copper'(0'Eq.),'the'major'peak'corresponds'to'[M'+'2H] ,'with'an'm/z'of'905.24.'With'
successive'additions'of'CuCl2'•'2H2O'in'the'presence'of'5'µM'ascorbic'acid,'a'peak'corresponding'to'a'single'Cu(I)'
adduct'emerges'with'an'm/z'of'936.75.'
'
69'
'
Results'and'Discussion'
'
Upon' successive' additions' of' Cu(I)' or' Ag(I)' ions,' the' apoIpeptide' signal' for' [M' +' 2H]2+' of' each'
peptide'decreased,'whereas'a'new'signal'corresponding'to'the'binding'of'Cu(I)'or'Ag(I)'emerged'at'
higher'm/z'ratios.'Such'emerging'signals'intensified'at'higher'metal:peptide'ratios'at'the'expense'of'
apoIpeptide'signals.'Consequently,'all'studied'peptides'were'able'to'effectively'bind'one'Cu(I)'ion'
or' one' Ag(I)' ion.' In' contrast,' at' high' Cu(II)' concentrations,' the' apoIpeptide' signal' remained'
important' regardless' of' the' peptide' considered' and' only' a' small' peptide' portion' was' associated'
with' a' single' Cu(II)' ion.' Another' divalent' metal' ion' (Zn2+)' was' also' investigated,' but' only' a' weak'
holoIpeptide' signal' was' recorded' at' very' high' Zn(II)' concentrations' (Appendices' 4.30' and' 4.31).'
Investigations' of' the' dPep.' showed' that' the' triply' charged' ion' [M' +' 3H]3+' was' the' most' intense'
signal'observed'in'the'absence'of'metal.'This'signal'decreased'upon'successive'additions'of'Cu(I)'or'
Ag(I)'ions'and'new'signals'corresponding'to'Cu(I)'or'Ag(I)'adducts'emerged'at'higher'm/z'ratios.'In'
agreement' with' the' results' obtained' above' for' the' nPep.,' the' double' motif' peptide' efficiently'
bound' two' Cu(I)' ions' or' two' Ag(I)' ions.' These' results' were' strengthened' by' the' determination' of'
dissociation'constant'(KD)'values.'
Dissociation'constants'determined'for'each'peptide'upon'metal'titration'are'listed'in'Table'4.2.'
They' were' calculated' as' described' in' the' Materials' and' Methods.' It' was' clear' that' all' peptides'
examined'had'less'affinity'for'Cu(II)'ions'over'Cu(I)'and'Ag(I)'ions.'The'high'KD'value'obtained'for'
Pep.'1'(H9'→'A)'as'opposed'to'that'of'other'peptides'underscored'a'plausible'involvement'of'the'
histidine'residue'in'Cu(II)Ibinding'geometry.'The'replacement'of'histidine'and'methionine'residues'
had'a'noteworthy'impact'on'calculated'dissociation'constants'in'the'presence'of'Cu(I)'or'Ag(I)'ions.'
The' low' KD' values' obtained' in' comparison' with' the' native' peptide' might' be' due' to' structural'
changes'which'favor'the'interaction'with'metal'ions.'
'
Table(4.2:'KD'values'determined'by'electrospray'ionization'mass'spectrometry'(ESIIMS)'for'1:1'peptideImetal'
complexes'
'
Native'Peptide'
Pep.'1'(H9→A)'
Pep.'2'(M4→G)'
Pep.'3'(M12→G)'
'
Cu(II)'KD'(µM)'
71.9'
270.3'
121.9'
102'
'
Cu(I)'KD'(µM)'
49'
10.9'
59.5'
9.9'
'
Ag(I)'KD'(µM)'
70.4'
29.2'
17.4'
21.1'
'
(
4.3.3.(( Circular(Dichroism((CD)(and(1D(Nuclear(Magnetic(Resonance((NMR)(Spectroscopy(
CD' and' 1D' NMR' spectroscopy' were' used' to' monitor' any' structural' changes' occurring' in' the'
synthetic'peptides'upon'metal'ligation'and'provide'complementary'information'to'that'obtained'by'
ESIIMS.'The'farIUV'CD'spectra'obtained'for'all'small'peptides'in'the'absence'of'any'metal'showed'a'
70'
'
Results'and'Discussion'
'
strong'negative'band'at'197'nm,'suggesting'a'high'content'of'random'coil'conformations'(Fig.'4.6'
and' Fig.' S4.1)' (Rubino' et' al.,' 2010).' Upon' successive' additions' of' AgNO3,' spectral' changes' were'
observed'for'all'peptides'(Fig.'4.6C,'F'and'Fig.'S4.1C,'F).'These'marked'modifications'can'be'divided'
into'two'groups.'The'first'group,'represented'by'peptides'containing'only'three'Met'residues'(Pep.'
2' and' Pep.' 3),' was' characterized' by' a' significant' positive' shift' of' the' negative' 197' nm' band' in'
conjunction'with'a'significant'increase'in'the'negative'ellipticity'near'225'nm,'suggesting'a'βItype'
turn'conformation'(Fig.'4.6F'and'Fig.'S4.1F)'(Liu'et'al.,'2005;'Rubino'et'al.,'2010).'The'second'group,'
represented' by' peptides' with' four' Met' residues' (nPep.' and' Pep.' 1),' was' also' characterized' by' a'
positive' shift' of' the' negative' 197' nm' band,' but' no' emergence' of' a' negative' peak' at' 225' nm' was'
observed' (Fig.' 4.6C' and' Fig.' S4.1C).' However,' it' should' be' noted' that' Pep.1' (H9' →' A)' was' also'
characterized' by' the' emergence' of' a' positive' peak' at' approximately' 215' nm,' which' was' also'
associated'with'a'βItype'turn'conformation'(Fig.'S4.1C)'(Ananthanarayanan'et'al.,'1985;'Rubino'et'
al.,'2010).'Similar'spectral'changes'were'observed'upon'the'addition'of'Cu(I)'in'the'presence'of'10'
molar'equivalents'NH2OH'(Fig.'4.6B,'E'and'Fig.'S4.1B,'E).'In'contrast,'CD'spectra'relating'to'Cu(II)I
peptides' were' only' slightly' altered' upon' the' addition' of' Cu(II)' ions,' in' comparison' to' changes'
observed' with' the' additions' Ag(I)' or' Cu(I)' ions' (Fig.' 4.6A,' D' and' Fig.' S4.1A,' D).' Another' divalent'
cation'(Zn2+)'was'also'studied'to'determine'whether'it'induced'changes'in'secondary'structure,'but'
no'significant'spectral'changes'were'observed'(Fig'S4.2A,'B).'
'
'
Figure( 4.6:' Circular' dichroism' spectra' of' methionineIrich' peptides' in' the' absence' and' presence' of' Cu(II),' Cu(I),' or' Ag(I)'
ions.'Panels'above'(A.,'B.,'and'C.)'and'below'(D.,'E.,'and'F.)'correspond'to'the'native'peptide'(nPep.)'and'Pep.'2'(M4'→'G),'
respectively.'CD'spectra'of'20'µM'peptide'were'recorded'in'5.3'mM'ammonium'acetate,'pH'6.8.'Each'curve'represents'an'
average' of' three' scans' and' was' baselineIcorrected' by' subtraction' of' the' background' for' the' spectrum' obtained' with'
buffer'alone.'ApoIpeptides'are'shown'in'solid'black'lines.'(A.'and'D.)'Additions'of'2'(dashed'blue'line)'and'5'(dashed'red'
line)'molar'equivalents'of'Cu(II).'(B.'and'E.)'Additions'of'2'(dashed'blue'line)'and'5'(dashed'red'line)'molar'equivalents'of'
Cu(I),'in'the'presence'of'10'molar'equivalents'NH2OH.'(C.'and'F.)'Additions'of'2'(dashed'blue'line)'and'5'(dashed'red'line)'
molar'equivalents'of'Ag(I).'
71'
'
Results'and'Discussion'
'
1
HINMR' spectroscopy' was' used' to' further' characterize' Cu(I)' and' Ag(I)' coordination' by'
methionineIrich'peptides.'OneIdimensional' 1H'NMR'spectra'were'collected'for'each'small'peptide'
in'the'absence'and'presence'of'monovalent'cations,'at'500'MHz'in'D2O'at'room'temperature.'In'the'
absence' of' any' metal,' all' synthetic' peptides' were' largely' unstructured' and' characterized' by' 1D'
NMR'spectra'which'lacked'signal'dispersion'and'resembled'spectra'of'free'amino'acid'mixtures'(Fig.'
4.7)'(Zerbe'and'Jurt,'2013).'These'results'are'consistent'with'CD'studies,'showing'that'all'peptides'
were' basically' unfolded' in' the' absence' of' metal.' Upon' successive' additions' of' AgNO3,' similar'
spectral'features'were'observed'for'peptides'containing'only'three'methionine'residues'(Pep.'2'and'
Pep.' 3).' Peaks' corresponding' to' methyl' protons' of' each' methionine' residue' moved' significantly'
downfield' (Δδ' 130' –' 155' Hz),' while' peaks' associated' with' histidine' protons' shifted' more' slightly'
upfield' (Table' 4.3,' A.).' These' results' suggested' a' strong' AgIpeptide' interaction' involving' a'
methionineIthioether' coordination' environment' wherein' the' histidine' residue' has' some' minimal'
interaction.' When' histidine' is' replaced' by' alanine' (in' Pep.' 1),' methyl' protons' of' the' four'
methionine'residues'shifted'downfield'in'a'similar'way'as'observed'for'Pep.'2'and'Pep.'3.'This'result'
suggests' an' analogous' interaction' with' silver,' in' agreement' with' KD' values' obtained' by' mass'
spectrometry.' With' regard' to' the' native' peptide,' only' one' methyl' group' was' significantly' shifted'
downfield'while'the'three'others'moved'more'slightly'downfield'(Table'4.3,'A.).'In'contrast'to'Pep.'
2'and'Pep.'3,'peaks'associated'with'histidine'protons'shifted'significantly'downfield,'suggesting'the'
involvement'of'the'histidine'residue'in'the'metal'coordination'geometry.'
'
1
Figure(4.7:'Example'of( HINMR'spectrum'obtained'for'apoIPep.'2'(M4'→'G)'in'100%'D2O'solution.'The'arrow'indicates'
residual'water'signal.'
'
NMR'experiments'with'Cu(I)'ions'were'investigated'in'the'presence'of'excess'ascorbic'acid'in'
order'to'efficiently'reduce'Cu(II)'ions'to'Cu(I).'Pep.'1'(H9'→'A)'in'the'presence'of'Cu(I)'ions'showed'
similar' results' to' those' ones' obtained' in' the' presence' of' Ag(I)' ions' (Table' 4.3,' B.).' Indeed,' peaks'
72'
'
Results'and'Discussion'
'
corresponding'to'methyl'protons'of'each'methionine'residue'moved'significantly'downfield'(Δδ'104'
–' 115' Hz),' suggesting' a' strong' Cu(I)Ipeptide' interaction' wherein' methionine' residues' would'
interact'in'similar'ways.'Pep.'3'(M12'→'G)'also'showed'methyl'protons'for'two'methionine'residues'
which'were'significantly'shifted'downfield'(Δδ'115'–'129'Hz),'while'the'methyl'group'of'the'third'
methionine'residue'moved'only'slightly'downfield'(Δδ'60'Hz)'(Table'4.3,'B.).'It'should'be'noted'that'
the' less' deshielded' histidine' proton' was' shifted' significantly' upfield' (Δδ' I100' Hz),' an' event' not'
observed'in'the'presence'of'Ag(I)'ions.'Methyl'proton'chemical'shift'differences'observed'for'Pep.'2'
(M4'→'G)'appeared'weaker'(Δδ'25'–'40'Hz)'than'those'observed'for'Ag(I),'while'peaks'associated'
with'histidine'protons'moved'significantly'downfield'(Table'4.3,'B.).'These'NMR'data'suggested'that'
Pep.'2'(M4'→'G)'would'interact'differently'with'Cu(I)'than'with'Ag(I),'in'agreement'with'KD'values'
determined'by'mass'spectrometry.'By'contrast,'methyl'protons'of'the'native'peptide'moved'much'
more' downfield' upon' Cu(I)' titration' in' comparison' with' Ag(I)' titration' (Table' 4.3,' B.).' The' same'
spectral'feature'applied'for'histidine'protons,'suggesting'that'histidine'would'be'more'involved'in'
the'Cu(I)Ibinding'site'than'in'the'Ag(I)Ibinding'site.'Based'on'these'results,'it'was'argued'that'the'
native' peptide' would' interact' much' more' with' Cu(I)' ions' than' with' Ag(I)' ions,' as' highlighted' by'
dissociation'constant'values'determined'by'mass'spectrometry.'
'
1
Table( 4.3:' Difference' of' HINMR' chemical' shifts' (in' Hz)' derived' from' the' methionineIrich' peptides' in' the'
absence'and'presence'of'3'molar'equivalents'of'Ag(I)'(A.)'or'Cu(I)'(B.).'
A.(
'
'
'
'
'
'
Ag(I)'
nPep.'
Pep.'1'
(H9→A)'
Pep.'2'
(M4→G)'
Pep.'3'
(M12→G)'
B.(
'
'
'
'
'
'
Cu(I)'
nPep.'
Pep.'1'
(H9→A)'
Pep.'2'
(M4→G)'
Pep.'3'
(M12→G)'
Difference'of'chemical'shift'at'3'molar'equivalents'(Δδ,'Hz)'
Methyl'protons'of'methionine'residues'
Protons'of'histidine'residue'
Hα*'
Hβ*'
108.24'
74.35'
72.72'
65.68'
56.53'
35.05'
134.25'
131.53'
131.41'
120.86'
—'
—'
154.73'
151.20'
130.40'
—'
I14.20'
I40.99'
159.25'
153.24'
139.70'
—'
I2.30'
I18.34'
'
Difference'of'chemical'shift'at'3'molar'equivalents'(Δδ,'Hz)'
Methyl'protons'of'methionine'residues'
Protons'of'histidine'residue'
Hα*'
Hβ*'
109.99'
109.99'
87.73'
87.73'
98.25'
69.46'
114.99'
114.95'
111.08'
104.49'
—'
—'
39.30'
29.86'
24.55'
—'
68.95'
45.74'
128.59'
115.61'
60.58'
—'
I100.85'
10.96'
*' Hα' corresponds' to' the' less' deshielded' proton' located' between' the' nitrogen' and' carbon' atoms' in' the'
imidazole'ring;'Hβ'corresponds'to'the'much'more'deshielded'proton'located'between'the'two'nitrogen'atoms'in'
the'imidazole'ring.'
73'
'
Results'and'Discussion'
'
4.3.4.(( Subcellular(Localization(of(CopB(Protein(in(C.*metallidurans(CH34(
Knowledge'about'a'protein's'subcellular'localization'often'carries'important'information'and'is'
critical' to' a' full' understanding' of' its' function.' Based' on' its' signal' sequence'
(MYKYTRIAVAALLTAGMSAAWAQ)' and' results' obtained' from' the' bacterial' localization' prediction'
tool'(PSORTb'v3.0.2),'the'plasmidIencoded'CopB'protein'was'predicted'to'be'localized'to'the'outer'
membrane.' To' date,' however,' this' has' never' been' experimentally' demonstrated.' To' this' end,'
subcellular' fractionation' of' copperItreated' C.* metallidurans' CH34' was' performed' and' membrane'
proteins' (inner' membrane/outer' membrane)' were' separated' from' soluble' proteins'
(cytosolic/periplasmic),'as'described'in'the'Materials'and'Methods.'Further'saline'treatments'of'the'
purified' membrane' fraction' with' 500' mM' NaCl' resulted' in' disruptions' to' the' electrostatic'
interactions' between' peripheral' and' integral' membrane' proteins,' leading' to' the' extraction' of'
peripheral' membrane' proteins.' Purified' polyclonal' antibodies' raised' against' the' fullIlength' CopB'
protein' were' used' to' demonstrate' the' subcellular' localization' of' this' protein' in* C.* metallidurans'
CH34.'As'shown'in'Figure'4.8A,'the'protein'of'interest'was'detected'in'the'membrane'fraction.'The'
signal'obtained'from'the'C.*metallidurans'CH34'membrane'fraction'treated'with'0.8'mM'Cu(II)'was'
strongly'enhanced'when'compared'to'fractions'isolated'from'untreated'control'bacteria.'This'result'
suggested'that'the'CopB'protein'was'associated'with'the'membrane'fraction.'
'
'
Figure(4.8:'Subcellular'localization'of'the'plasmidIencoded'CopB'protein'from'C.*metallidurans'CH34.'(A.)'Western'blots'
of'soluble'proteins'(SPs),'membrane'proteins'(MPs)'and'peripheral'membrane'proteins'(PPs)'from'untreated'(gluconate'
control)' and' copperItreated' C.* metallidurans' CH34' probed' with' antiICopB' antibody' raised' against' the' fullIlength' CopB'
protein.' Briefly,' supernatants' corresponding' to' soluble' proteins' and' crude' membrane' pellets' of' untreated' and' copperI
treated'bacteria'were'extracted'as'described'in'the'Materials'and'Methods.'Crude'membrane'pellets'were'subsequently'
treated'with'highIsalt'extraction'buffer'(100'mM'TrisIHCl,'pH'7.3,'500'mM'NaCl),'which'results'in'pellets'enriched'in'outer'
and'inner'membrane'proteins'(MP)'as'well'as'supernatants'containing'peripheral'membrane'proteins'(PPs).'(B.'and'C.)'
Transmission' electron' micrographs' illustrating' immunogold' localization' of' CopB' protein' in' copperItreated' C.*
metallidurans'CH34'(B.)'and'untreated'bacteria'(C.).'Ultrathin'sections'were'labeled'with'antiICopB'antibody,'followed'by'
gold'conjugated'secondary'antibodies.'Gold'particles'appear'in'or'near'the'outer'membrane'of'copperItreated'bacteria.'
Untreated'cells'showed'a'widely'similar'distribution'of'gold'granules,'with'a'significantly'reduced'amount'of'labeling.'
74'
'
Results'and'Discussion'
'
Immunogold' labeling' was' further' used' to' strengthen' the' previous' result' and' to' specify' the'
subcellular' localization' of' CopB' protein' within' C.* metallidurans' CH34.' Ultrathin' sections' of'
untreated' and' 0.8' mM' Cu(II)Itreated' bacteria' were' processed' as' described' in' the' Materials' and'
Methods'and'observed'by'transmission'electron'microscopy.'Cells'treated'with'copper'and'probed'
for'CopB'clearly'exhibited'gold'particles'in'or'near'the'outer'membrane,'raising'the'possibility'that'
CopB' might' be' associated' with' the' outer' membrane' (Fig.' 4.8B).' Untreated' cells' showed' a' widely'
similar' distribution' of' gold' granules,' but' the' amount' of' labeling' was' significantly' reduced' (Fig.'
4.8C).' As' controls,' ultrathin' sections' were' treated' according' to' the' same' procedure' with'
preimmune'serum'and'another'antibody.'The'controls'showed'nonspecific'staining,'indicating'that'
the'CopB'localization'to'the'outer'membrane'was'not'the'result'of'an'artifactual'labeling'process'
(data'not'shown).'Taken'together,'immunolocalization'data'support'conclusions'derived'from'cellI
fractionation' experiments' and' showed' that' the' CopB' protein' was' largely' outer' membraneI
associated'in'C.*metallidurans'CH34.'
These' results' were' supported' by' transmembrane' protein' topology' predictions.' Indeed,'
structural' predictions' made' using' the' program' PREDITMBB' indicated' that' the' CopB251I495' protein'
might'form'a'12Istranded'βIbarrel'in'the'outer'membrane'(Fig.'4.9).''
'
Figure( 4.9:' Predicted' topology' of' the' CopB251I495'
recombinant' protein' using' the' program' PREDI
TMBB.'Aspartic'acids'(D)'and'glutamic'acids'(E)'are'
shown'in'red.'
'
'
This' prediction' is' consistent' with' results' obtained' by' circular' dichroism' for' the' fullIlength' CopB'
protein,' which' showed' a' significant' content' of' strands' (i.e.,' 41%).' It' is' noteworthy' that' no'
methionine' or' cysteine' residues,' known' to' bind' copper' ions,' were' found' inside' the' βIbarrel.'
However,' a' significant' number' (18' residues)' of' glutamic' acids' were' present' within' the' βIbarrel.'
Notably,' this' topology' prediction' does' not' correspond' to' previously' reported' categories' of'
transporters' such' as' OmpC' (16' strands)' and' maltoporin' (18' strands).' On' the' basis' of' these'
75'
'
Results'and'Discussion'
'
observations,' we' attempted' to' demonstrate' the' probable' function' of' this' protein' as' a' cargo'
transporter.' Electrophysiological' experiments' were' done' to' probe' the' putative' channel' activity.'
The'fullIlength'CopB'protein'was'reconstituted'in'planar'lipid'bilayers'formed'from'soy'lipid'extract.'
An' electric' potential' difference' was' then' applied' across' the' membrane' to' detect' a' change' in'
conductance'through'the'lipid'bilayer.'No'change'in'conductance'was'observed,'preventing'us'from'
concluding'that'the'CopB'protein'might'transport'ions'out'of'the'cell.'
'
4.3.5.(( Identification( of( CopB9Interacting( Partners( Using( in* vivo( Cross9linking( and( Mass(
( Spectrometry(
The' identification' of' CopBIinteracting' partners' was' achieved' by' coIimmunoprecipitation'
combined' with' nanoLCIMS/MS' analysis.' In' our' experimental' strategy,' control' and' copperItreated'
C.* metallidurans' CH34' bacteria' were' chemically' crossIlinked' using' low' concentrations' of'
formaldehyde'(1.2%)'during'a'short'reaction'time'(10'minutes).'These'chemical'treatments'allowed'
for' the' covalent' binding' of' interacting' proteins' spatially' located' in' close' proximity' (2.3I2.7' Å).'
Purified' antiICopB' antibodies' were' used' to' specifically' target' the' protein' complexes' of' interest,'
resulting' in' their' coIimmunoprecipitation' out' of' the' cell' lysates.' Eluted' crossIlinked' complexes'
were'digested'inIsolution'and'the'resulting'peptides'were'analyzed'by'tandem'mass'spectrometry.'
In'theory,'chemical'crossIlinking'coupled'with'mass'spectrometry'allows'the'identification'of'direct'
interacting' partners' within' a' protein' complex' and' can' pinpoint' binding' interfaces' (Yang' et' al.,'
2012).'However,'conventional'database'search'algorithms'(such'as'those'ones'used'by'ProteinPilot)'
often' do' not' allow' the' identification' of' monoIlinked,' loopIlinked' or' interlinked' peptides,' which'
result' in' more' complex' fragmentation' spectra' (Yang' et' al.,' 2012).' Consequently,' only' regular'
peptides' were' identified' in' the' digest' with' ProteinPilot.' With' regard' to' the' crossIlinked' copperI
treated'cell'lysate,'the'CopB'bait'protein'was'detected'at'the'top'of'the'identified'protein'list'with'
87'peptides'and'sequence'coverage'of'83.23%'with'95%'confidence.'This'protein'was'also'detected'
with' a' similarly' high' number' of' peptides' in' the' crossIlinked' gluconate' control' as' well' as' in' nonI
crossIlinked'controls,'highlighting'the'specificity'of'the'antibodyIantigen'recognition.'
To'focus'the'study'on'a'few'high'quality'candidates,'an'arbitrary'cutIoff'point'was'chosen'and'
only' proteins' observed' with' twelve' or' more' identified' peptides' were' considered' for' further'
analysis,'as'listed'in'Table'4.4.'Among'the'relevant'candidates,'the'periplasmic'CopA'protein'drew'
our'attention.'Identified'with'46'peptides'(sequence'coverage'of'68.40%'at'95%'confidence),'this'
protein' appeared' as' a' valuable' protein' partner' for' the' outer' membraneIlocalized' CopB' protein.'
Indeed,'this'protein'was'identified'with'only'2'peptides'(sequence'coverage'of'2.93%'at'95%'confiI'
76'
'
Results'and'Discussion'
'
'
77'
'
Results'and'Discussion'
'
dence)'in'the'crossIlinked'gluconate'control'and'was'not'found'in'nonIcrossIlinked'controls'nor'in'
experiments' conducted' with' preimmune' serum' (Table'4.4).' Interestingly,' four' additional' proteins'
belonging' to' the' ABC' transporter' superfamily' were' also' identified,' Rmet_0399,' Rmet_2480,'
Rmet_3518,' and' Rmet_1211.' Such' ABC' proteins' are' of' considerable' interest' owing' to' their'
significant'number'of'peptides'and'high'sequence'coverage'(>'43%)'at'95%'confidence.'Once'again,'
ABC' proteins' appeared' as' being' specific' to' the' crossIlinked' copperItreated' cell' lysates' and' were'
not' detected' in' nonIcrossIlinked' controls' nor' in' experiments' performed' with' preimmune' serum'
(Table'4.4).'A'few'other'proteins'were'also'observed'but'did'not'appear'to'be'specific'to'the'crossI
linked'copperItreated'cell'lysate'and'were'therefore'considered'to'be'background'contaminants.'
(
4.4.(Discussion(
C.* metallidurans' strain' CH34' is' a' model' metalIresistant' bacterium,' able' to' resist' high'
concentrations' of' numerous' metal' ions' (in' particular' copper' ions)' that' are' typically' found' in'
metallurgical'tailings'and'industrial'biotopes'(Mergeay'et'al.,'2009;'Monsieurs'et'al.,'2011).'Copper'is'
a' metallic' trace' element' essential' for' bacterial' life,' of' which' the' cellular' concentration' is' precisely'
controlled' to' avoid' toxic' effects' generated' by' elevated' copper' levels' (Cuillel,' 2009).' Numerous'
mechanisms'are'used'by'bacteria'to'regulate'copper'homeostasis,'such'as'sequestration,'enzymatic'
transformation,'or'active'trafficking'(Cha'and'Cooksey,'1991;'Rensing'and'Grass,'2003;'Rubino'et'al.,'
2010).'Copper'resistance'in'C.*metallidurans'CH34'involves'an'intricate'network'of'molecular'actors'
which' are' mainly' encoded' by' the' cop' cluster' on' the' plasmid' pMOL30' and' are' responsible' for'
periplasmic' and' cytoplasmic' copper' detoxification' (Monchy' et' al.,' 2006).' Among' the' twentyIone'
identified' cop' genes' copVTMKNSRABCDIJGFOLQHEW,' the' basic' operon' copSRABCD' is' found.' This'
operon'is'homologous'to'the'cop/pco'basic'cluster'found,'in'particular,'in'the'P.*syringae'pathovar'
tomato'plasmid'pPT23D'and'in'the'E.*coli'plasmid'pRJ1004'(Bondarczuk'and'PiotrowskaISeget,'2013).'
Duplicates'of'these'basic'genes'are'found'in'C.*metallidurans'CH34,'both'on'the'plasmid'pMOL30'and'
on' the' chromid.' Nevertheless,' these' basic' cop* clusters' are' not' identical,' as' demonstrated' by' the'
subject'of'this'study,'the'plasmidIencoded'CopB'protein.'
Although' plasmidIencoded' CopB' proteins' are' found' in' most' copperIresistant' bacteria,' few'
information'is'known'at'both'molecular'and'structural'levels.'The'plasmidIencoded'CopB'protein'in'
C.*metallidurans'CH34'has'two'distinct'domains,'as'shown'by'the'secondary'structure'prediction'and'
the'experimental'demonstration:'an'unstructured'NIterminal'extremity'and'a'structured'CIterminal'
subdomain.'The'structured'CIterminal'domain'is'highly'conserved'among'the'different'known'CopB'
proteins,' in' particular' the' plasmidI' and' chromidIencoded' CopB' proteins' in' C.* metallidurans' CH34.'
78'
'
Results'and'Discussion'
'
The'NIterminal'extremity'is'extremely'rich'in'methionine'residues'and'appears'to'be'unique'among'
the'Cop/PcoB'homologues.'This'domain'contains'46'methionine'and'13'histidine'residues,'which'are'
mainly' arranged' as' ten' identical' sequential' motifs' with' a' consensus' sequence' of'
MQGMDHSKMQGMDQGS.' Notably,' this' methionineIrich' sequence' is' considerably' smaller' in' the' C.*
metallidurans' CH34' chromidIencoded' counterpart.' MethionineIrich' motifs' are' found' in' several'
proteins' involved' in' bacterial' copper' detoxification' systems,' such' as' CueO' in' Escherichia* coli,' PcoA'
and'PcoC'from'the'E.*coli'plasmid'pRJ1004,'CopA'and'CopC'from'P.*syringae'pv.'tomato,'and'CusB'
from'E.*coli'(Bagai'et'al.,'2007;'Bondarczuk'and'PiotrowskaISeget,'2013;'Jiang'et'al.,'2005;'Singh'et'
al.,'2011).'These'methionineIrich'motifs'are'widely'known'to'bind'Cu(I)'ions'and'Ag(I)'ions,'which'are'
considered'as'a'surrogate'for'Cu(I)'ions'(Bagai'et'al.,'2007;'Loftin'et'al.,'2007;'Rubino'et'al.,'2010).'
The' NIterminal' domain' of' the' plasmidIencoded' CopB' protein' in' C.* metallidurans' CH34' is'
connected'to'the'CIterminal'domain'through'a'conserved'sequential'motif,'GS,'which'is'also'present'
in' the' chromidIencoded' counterpart.' The' presence' of' residues' such' as' glycine' and' serine' offer'
rotational'freedom'of'the'protein'backbone,'so'that'the'adjacent'protein'domains'are'free'to'move'
relative'to'one'another'and'thus'suggests'that'the'methionineIrich'extremity'might'be'a'flexible'and'
unfolded' loop' (Reddy' Chichili' et' al.,' 2013).' Although' bacterial' copper' resistance' mechanisms' are'
quite' well' studied,' it' is' surprising' that' little' chemical' evidence' exists' to' clarify' the' molecular'
interactions' between' methionineIrich' motifs' and' copper' or' silver' in' the' CopB' protein,' and' little' is'
known'regarding'its'physiological'role'in'copper'resistance.'
In' an' attempt' to' characterize' molecular' and' functional' properties' of' the' plasmidIencoded'
CopB' protein,' three' recombinant' proteins' were' first' overexpressed' and' purified' during' our'
investigation.' These' proteins' corresponded' to' (i)' the' fullIlength' CopB' protein,' (ii)' its' NIterminal,'
methionineIrich' extremity,' referred' as' CopB(Met),' and' (iii)' its' structured' CIterminal' subdomain'
named'CopB251I495.'Characterization'of'apoIstructures'by'circular'dichroism'confirmed'the'theoretical'
predictions' of' protein' secondary' structure' showing' that' the' fullIlength' protein' contained' 41%'
strands,' 5%' helices,' 28%' turns' and' 26%' unordered' structures,' while' CopB(Met)' was' mainly'
unordered' (67%' unordered).' Notably,' in' our' experimental' conditions,' these' recombinant' proteins'
have'problems'of'longIterm'stability'(i.e.,'freeze/thaw'cycles'or'storage'at'4°C'even'under'a'nitrogen'
atmosphere'lead'to'the'precipitation'of'the'samples).'Moreover,'the'CIterminal'subdomain'CopB251I
495'could'not'be'characterized'by'circular'dichroism'due'to'instability'of'the'protein'in'the'absence'of'
urea,' which' is' required' for' extraction' of' the' protein' from' inclusion' bodies' and' absorbs' at' low'
wavelength.'
79'
'
Results'and'Discussion'
'
Characterization'of'structural'changes'upon'copper'or'silver'addition'appeared'quite'intricate.'
In' fact,' any' copper' or' silver' addition' induced' the' bioprecipitation' of' the' fullIlength' CopB' protein,'
making'its'study'impossible.'In'contrast,'the'freshly'purified'CopB(Met)'protein'was'investigated'by'
circular' dichroism' in' the' presence' of' Cu(II)' ions,' as' well' as' Ag(I)' ions.' However,' regardless' of' the'
metal'studied,'no'significant'structural'change'was'observed'in'the'CD'spectra'and'the'loss'of'the'CD'
signal'might'suggest'a'likely'aggregation/precipitation'of'the'protein.'It'can'be'concluded'that'(i)'the'
CopB(Met)'protein'remains'primarily'unfolded'in'the'presence'of'metal'ions'and'(ii)'the'addition'of'
metal'ions'appears'to'increase'the'hydrophobicity'of'the'proteins'in*vitro.'
As' a' result' of' aggregation/solubility' problems,' investigation' of' metal' binding' by' mass'
spectrometry' to' determine' the' number' of' metal' ions' bound' by' the' recombinant' proteins' was'
impossible.' Synthetic' peptides' were' then' produced' to' circumvent' problems' encountered' with'
recombinant'proteins'in'the'presence'of'metal'ions'and'to'clarify'the'molecular'interactions'between'
methionine' motifs' and' copper' in' another' way.' Synthetic' peptides' offer' a' good' model' for' binding'
studies' and' folding' events' because' they' enable' the' detailed' characterization' of' binding' sites.' Five'
model' peptides' were' investigated' using' three' complementary' techniques:' MS,' NMR,' and' CD.' The'
native' peptide,' AcIQGSMQGMDHSKMQGMD,' and' the' double' motif' peptide' were' derived' from' the'
methionineIrich' NIterminal' extremity' of' CopB,' while' the' three' others' were' designed' by' replacing'
specific'amino'acids'to'investigate'their'contribution'to'the'binding'site:'Pep.'1'(H9'→'A),'Pep.'2'(M4'
→' G),' and' Pep.' 3' (M12' →' G).' Unsurprisingly,' in' the' absence' of' any' metal' ions,' the' four' small'
peptides' remained' largely' unfolded' and' lacked' any' stable' secondary' structure,' as' observed' by' CD'
and' NMR' spectroscopy.' This' result' is' in' agreement' with' the' large' conformational' flexibility'
associated' with' small' linear' peptides,' which' cannot' provide' the' formation' of' stable' secondary'
structures'due'to'their'low'number'of'amino'acids'(Chitta'and'Gross,'2004).'
Following'the'addition'of'copper'or'silver'ions,'it'was'established'that'the'native'peptide'might'
bind'Cu(I)'or'Ag(I)'ions'with'a'linear'coordination'involving'the'single'histidine'and'four'methionine'
residues.'Indeed,'1HINMR'experiments'with'Cu(I)'or'Ag(I)'ions'showed'a'significant'downfield'shift'of'
the' methyl' group' relating' to' each' methionine' residue,' as' well' as' a' downfield' shift' of' histidine'
protons.'Moreover,'no'significant'changes'were'observed'in'CD'signatures'induced'by'titration'with'
Cu(I)'or'Ag(I),'suggesting'a'small'degree'of'ordering'(Fig.'4.10A'and'B).'Similarly,'it'was'argued'that'
Pep.'1'(H9'→'A)'might'bind'Cu(I)'or'Ag(I)'ions'in'a'binding'geometry'involving'the'four'methionine'
residues' (Fig.' 4.10C' and' D).' NMR' experiments' showed' that' the' methyl' group' of' each' methionine'
residue'was'significantly'and'similarly'moved'downfield.'This'result'suggests'a'folding'of'the'peptide'
back' upon' itself,' so' that' each' methionine' residue' can' contribute' similarly' to' the' metalIbinding'
80'
'
Results'and'Discussion'
'
coordination.' The' folding' was' confirmed' by' spectral' changes' observed' in' circular' dichroism,' which'
were'assigned'to'a'βItype'turn'conformation'(Ananthanarayanan'et'al.,'1985).'
'
Figure( 4.10:' Schematic' drawing' representing' the'
metalIbinding' site' within' the' synthetic' peptides'
studied'by'mass'spectrometry,'circular'dichroism'and'
1
H' nuclear' magnetic' resonance.' Cu(I)' and' Ag(I)' ions'
are' shown' as' black' and' white' circles,' respectively.'
When' the' residues' appear' to' be' more' influenced' by'
the'presence'of'metal'ions,'the'dotted'lines'are'much'
more'tight.'(A.'and'B.)'Native'peptide;'(C.'and'D.)'Pep.'
1' (H9' →' A);' (E.' and' F.)' Pep.' 2' (M4' →' G);' (G.' and' H.)'
Pep.' 3' (M12' →' G).' This' schematic' drawing' does' not'
reflect' the' accurate' distances' between' the' residues'
and'the'metal'ion.'
'
'
By' contrast,' some' differences' were' observed' in' the' copperI' and' silverIbinding' geometries'
relating'to'the'two'other'peptides.'Upon'the'addition'of'Ag(I)'ions,'it'appears'that'Pep.'2'(M4'→'G)'
and' Pep.' 3' (M12' →' G)' are' likely' to' bind' a' Ag(I)' ion' in' a' trigonal' coordination' (Fig.' 4.10F' and' H,'
respectively).'In'fact,'NMR'experiments'showed'a'significant'and'similar'downfield'shift'of'the'methyl'
group'of'two'methionine'residues'(Δδ'~150'Hz)'and'a'slightly'less'significant'downfield'shift'of'the'
methyl' group' of' the' third' methionine' residue' (Δδ' ~130' Hz),' suggesting' that' the' three' methionines'
might' similarly' contribute' to' the' binding' geometry.' This' result' suggests' a' folding' of' the' peptide;'
indeed,' this' was' confirmed' by' the' identification' of' significant' changes' in' CD' signatures.' Spectral'
changes' observed' upon' the' addition' of' Ag(I)' might' be' assigned' to' the' formation' of' a' βItype' turn,'
suggesting'the'folding'of'the'peptide'back'upon'itself'and'strengthening'NMR'experiments'(Liu'et'al.,'
2005;'Rubino'et'al.,'2010).'
Although' the' chemical' properties' of' Cu(I)' and' Ag(I)' are' quite' similar,' it' appears' that' Cu(I)'
binding' geometries' within' Pep.' 2' and' Pep.' 3' are' slightly' different' than' those' identified' in' the'
presence'of'Ag(I)'ions.'As'observed'by'NMR'spectroscopy,'Pep.'2'(M4'→'G)'might'bind'a'Cu(I)'ion'in'a'
coordination' involving' the' three' methionine' and' histidine' residues' (Fig.' 4.10E).' The' three'
methionines' appear' to' contribute' equally' within' the' binding' site' since' the' methyl' groups' were'
similarly'shifted'downfield'(Δδ'25'—'40'Hz).'However,'their'chemical'shift'differences'were'weaker'in'
81'
'
Results'and'Discussion'
'
comparison'to'other'investigated'peptides,'suggesting'that'these'methionine'residues'are'less'likely'
to' be' involved' in' the' binding' geometry.' Moreover,' histidine' protons' were' also' significantly' shifted'
downfield,' underscoring' the' involvement' of' histidine' in' the' binding' site.' As' mentioned' above,' the'
same' contribution' of' methionine' residues' suggests' a' folding' of' the' peptide' back' upon' itself.' This'
result'was'confirmed'by'spectral'changes'observed'by'CD'spectroscopy,'which'were'assigned'to'the'
formation'of'a'βItype'turn'(Liu'et'al.,'2005;'Rubino'et'al.,'2010).'By'contrast,'Pep.'3'(M12'→'G)'might'
bind'a'Cu(I)'ion,'likely'in'a'linear'coordination'involving'the'histidine'and'three'methionine'residues'
(Fig.' 4.10G).' Indeed,' no' significant' spectral' change' was' observed' by' circular' dichroism,' suggesting'
that'the'peptide'would'remain'unfolded'upon'the'addition'of'Cu(I).'Moreover,'in'NMR'experiments,'
only'two'methyl'groups'and'the'less'deshielded'histidine'proton'were'significantly'shifted'downfield'
and'upfield,'respectively;'while'the'third'methyl'group'was'slightly'less'shifted'downfield.'
Structural' changes' and' coordination' environments' accompanying' metal' binding' were'
supplemented' by' the' calculation' of' effective' dissociation' constants' (KDs)' using' mass' spectrometry.'
Upon'additions'of'copper'or'silver'ions,'it'was'demonstrated'that'the'native'peptide'was'able'to'bind'
one'copper'ion'or'one'silver'ion.'However,'it'preferentially'bound'Cu(I)'ions'with'a'moderate'affinity'
(KD~5'x'10–5'M)'over'either'Cu(II)'and'Ag(I)'ions'(KD'~'7'x'10–5'M).'This'moderateIaffinity'binding'was'
in'contrast'to'that'observed'in'other'methionineIrich'copper'trafficking'proteins,'such'as'CopK'(KD'~'2'
x'10–11'M),'CopC'(KD'values'of'10–7'to'10–13'M)'or'CusF'(KD'of'5'x'10–7'M)'(Chong'et'al.,'2009;'Rubino'et'
al.,' 2010;' Sarret' et' al.,' 2010).' The' moderate' affinity' suggests' that' (i)' the' CopB' protein' is' not' a'
metallochaperone' and' (ii)' high' copper' concentrations' are' required' to' saturate' the' protein.'
Moreover,'it'appeared'that'the'replacement'of'histidine'residue'decreases'the'affinity'for'Cu(II)'ions,'
illustrating'the'importance'of'the'histidine'residue'in'Cu(II)'binding.'The'replacement'of'histidine'and'
methionine' residues' had' an' impact' on' dissociation' constant' values' determined' in' the' presence' of'
Cu(I)'and'Ag(I)'ions.'The'low'KD'values'determined'for'the'peptides'derived'from'the'native'peptide'
are'correlated'with'structural'changes'observed'by'circular'dichroism'and'NMR,'which'favor'a'higher'
affinity'for'Cu(I)'and'Ag(I)'ions'in'comparison'with'the'unfolded'native'peptide.'Investigations'using'
the'double'motif'peptide'showed'that'it'was'able'to'efficiently'bind'two'Cu(I)'ions,'in'agreement'with'
results' obtained' for' the' native' peptide.' Although' the' role' of' CopB' in' mechanisms' of' copper'
resistance'remains'unclear,'the'present'study'suggests'that'CopB'might'bind'ten'copper'or'silver'ions'
(i.e.,' one' ion' per' methionineIrich' motif)' with' moderate' affinity' in' relatively' unstructured' binding'
sites.' However,' it' cannot' be' excluded' that' some' methionineIrich' motifs' might' partially' be'
reorganized' to' coordinate' metal' ions' (i.e.,' several' methionineIrich' motifs' might' be' involved' in' the'
coordination'of'one'metal'ion).'
82'
'
Results'and'Discussion'
'
These'methionineIrich'motifs'might'easily'promote'transfer'of'metal'ions'either'out'of'the'cell'
(if'the'CIterminal'subdomain'of'CopB'acts'as'a'cargo'transporter)'or'to'other'copper'binding'motifs'
harbored'by'CopBIinteracting'partners.'The'study'of'proteinIprotein'interactions'is'very'important'to'
the' understanding' of' underlying' molecular' networks' of' numerous' biological' processes' such' as'
copper' homeostasis' in' C.* metallidurans' CH34.' Our' preliminary' results,' obtained' by' using' a' crossI
linking' approach,' demonstrated' that' the' CopA' protein' is' a' likely' biological' partner' of' the' CopB'
protein.'This'result'is'not'surprising'and'appears'consistent'with'previous'reports.'Indeed,'copA'and'
copB' genes' are' contiguous' in' the' plasmid' and' chromid' of' C.* metallidurans' CH34,' as' well' as' in' the'
genome' of' numerous' copperIresistant' organisms' (HernándezIMontes' et' al.,' 2012).' Moreover,' the'
expression'of'copA*is'highly'upregulated'by'copper'salts,'similar'to'copB'(Monchy'et'al.,'2006).'The'
CopA' protein' from' C.* metallidurans' CH34' is' homologous' to' the' P.* syringae' CopA' (56%' sequence'
identity)'and'E.*coli'PcoA'(58%'sequence'identity)'proteins,'which'suggests'that'they'most'likely'have'
a'similar'function'(Lee'et'al.,'2002).'Predicted'to'be'a'putative'multiIcopper'oxidase,'CopA'converts'
Cu(I)'ions'to'the'less'toxic'Cu(II)'form'(Monchy'et'al.,'2006).'The'presence'of'a'twinIarginine'motif'in'
its'leader'sequence'indicates'that'this'protein'is'most'likely'exported'across'the'inner'membrane'to'
the'periplasm'by'the'twinIarginine'translocation'(TAT)'pathway'with'copper'bound'in'its'binding'sites'
(Monchy'et'al.,'2006;'Rensing'and'Grass,'2003).'Indeed,'this'protein'contains'36'methionine'residues'
(most' of' which' are' localized' to' five' MGGM' motifs' in' the' extreme' CIterminus)' and' 21' histidine'
residues.'
It'is'tempting'to'speculate'that'the'MetIrich'extremity'of'CopA'might'dock'by'direct'interaction'
with'the'MetIrich'Cu(I)'sites'of'CopB,'allowing'for'the'export'of'metal'ions'out'of'the'cell'in'the'event'
that'the'CIterminal'extremity'of'CopB'protein'functions'as'a'cargo'transporter'(Fig.'4.11).'However,'it'
is'also'possible'that'CopB'might'donate'its'Cu(I)'ions'to'CopA,'which'could'oxidize'Cu(I)'to'Cu(II)'and'
subsequently'conduct'copper'to'other'protein'partners'such'as'Sil'or'Cus'RNDIdriven'efflux'systems'
(Fig.'4.11).'Surprisingly,'periplasmicIbinding'components'of'an'ABC'superfamily'were'also'identified'
as' potential' CopBIinteracting' partners,' with' a' significant' number' of' peptides' identified' by' mass'
spectrometry' with' good' sequence' coverage.' Such' periplasmic' metal' transporters' are' essential' for'
maintaining'many'cellular'processes,'such'as'homeostasis,'in'several'pathogenic'and'nonIpathogenic'
bacteria' (Chandra' et' al.,' 2007).' They' are' known' to' recognize' a' wide' variety' of' substrates' (i.e.,'
metallic' cations,' amino' acids,' proteins,' and' lipids)' and' deliver' their' cargo' to' the' import' complex'
formed'by'the'cytoplasmic'membrane'protein'and'nucleotideIbinding'protein,'both'members'of'the'
ABCItype'family'(Chandra'et'al.,'2007).'It'cannot'be'excluded'that'such'proteins'might'be'background'
contaminants,'even'though'they'are'not'detected'in'the'control'samples.'However,'it'is'also'possible'
that' the' formaldehyde' crossIlinking' stabilizes' interactions' between' proteins' that' do' not' directly'
83'
'
Results'and'Discussion'
'
interact,' such' as' CopB' protein' and' periplasmicIbinding' components' of' ABC' superfamily,' but' which'
have'a'binding'partner'in'common'(i.e.,'CopA)'(Klockenbusch'and'Kast,'2010).'
Figure( 4.11:' Schematic' drawing'
representing' the' potential' CopBI
interacting' partners.' CopA' is' a'
putative' multicopper' oxidase,'
while'SilABC'and'CusABC'are'RNDI
type'tripartite'efflux'systems.'
'
'
With' regard' to' the' structured' CIterminal' domain,' conserved' among' the' different' CopB'
proteins' as' well' as' among' the' plasmidIencoded' and' chromidIencoded' CopB' proteins' in' C.*
metallidurans'CH34,'it'might'legitimately'be'wondered'if'this'extremity'might'form'a'transmembrane'
channel.' This' study' experimentally' demonstrated' that' the' plasmidIencoded' CopB' protein' was'
associated' to' the' outer' membrane,' in' agreement' with' the' predicted' localization' of' CopB' found' in'
previous' studies' (Bondarczuk' and' PiotrowskaISeget,' 2013;' Mergeay' et' al.,' 2009;' Mergeay' et' al.,'
2003).' (i)' The' electron' microscopy' investigations' (i.e.,' immunogold' labeling' experiments)' showed'
that' the' CopB' protein' was' localized' in' the' outer' membrane.' (ii)' The' subcellular' fractionation'
experiments'strengthened'this'localization.'Indeed,'the'CopB'protein'was'not'found'in'the'peripheral'
membrane'fraction,'which'was'isolated'after'the'highIsalt'extraction'treatment'(i.e.,'0.5'M'NaCl'or'
even'at'higher'saline'concentrations,'1'M'NaCl)'of'the'purified'membrane'fraction.'The'presence'of'
the'CopB'protein'in'the'integral'membrane'fraction'after'detergent'treatment'(i.e.,'sodium'dodecyl'
sulfate)' suggests' that' the' protein' of' interest' might' be' associated' to' the' hydrophobic' core' of' the'
membrane.'
The' structured' CIterminal' extremity' of' the' CopB' protein' was' predicted' as' forming' a' 12I
stranded'betaIbarrel'in'the'outer'membrane'using'the'program'PREDITMBB.'However,'this'topology'
prediction' (i.e.,' 12' strands)' does' not' correspond' to' previously' reported' categories' of' transporters.'
Notably,' this' prediction' appears' consistent' with' results' obtained' by' circular' dichroism' for' the' fullI
length' CopB' protein,' which' showed' a' significant' content' of' strands' (i.e.,' 41%).' Electrophysiological'
experiments,' based' on' electrical' current' measurements' across' an' artificial' lipid' membrane,' were'
done' to' demonstrate' the' putative' channel' activity' of' this' protein.' No' conclusive' results' were'
84'
'
Results'and'Discussion'
'
obtained' suggesting' that' (i)' the' fullIlength' recombinant' protein' might' be' misfolded,' (ii)' the' likely'
folding'of'the'flexible'NIterminal'extremity'inside'the'barrel'might'hamper'the'metal'ion'transfer,'or'
it' cannot' be' excluded' that' (iii)' the' fullIlength' CopB' protein' might' not' form' a' channel' through' the'
membrane.' Consequently,' it' might' be' concluded' that' the' plasmidIencoded' CopB' protein' in' C.*
metallidurans' CH34' is' associated' to' the' hydrophobic' core' of' the' outer' membrane,' but' it' might' be'
possible'that'this'protein'does'not'form'a'channel'in'the'membrane.'This'hypothesis'is'strengthened'
by'the'absence'of'any'histidine'and'methionine'residues'inside'the'betaIbarrel,'which'are'known'to'
be'used'by'efflux'system'in'order'to'bind'and'transport'copper'ions'(Long'et'al.,'2012).'However,'a'
significant' number' of' glutamic' acids' were' found' inside' the' betaIbarrel.' Further' experimental'
investigations' regarding' the' structure' of' the' CIterminal' extremity' are' required' (e.g.,' protein'
reconstitution' into' liposomes' and' enzymatic' treatment)' to' improve' the' understanding' of' its'
membrane'topology.'
In' summary,' the' plasmidIencoded' CopB' protein' involved' in' copper' resistance' in' C.*
metallidurans'CH34'is'associated'to'the'hydrophobic'core'of'the'outer'membrane.'This'protein'has'a'
structured'CIterminal'domain'containing'a'high'percentage'of'betaIstrands'and'an'unstructured'NI
terminal' domain,' which' is' rich' in' methionine' residues.' The' methionineIrich' extremity' is' able' to'
interact' with' copper' and' silver' with' a' moderate' affinity' and' might' form' a' flexible' loop' in' the'
periplasmic'space.'This'moderate'binding'affinity'involves'that'the'CopB'protein'requires'high'metal'
concentrations' to' be' saturated' and' consequently,' it' suggests' that' this' protein' might' act' in' a' high'
metal'concentration'range.'
(
4.5.(Materials(and(Methods(
4.5.1.( Expression(and(Purification(of(Three(Recombinant(Proteins(
Three' recombinant' proteins' were' overexpressed' and' purified' during' this' study.' These'
correspond'to'(i)'the'plasmidIencoded'fullIlength'CopB'protein'from'C.*metallidurans'CH34,'(ii)'its'
methionineIrich' NIterminal' extremity' (from' AA' 51' to' 250),' hereafter' referred' to' as' "CopB(Met)",'
and' (iii)' its' CIterminal' subdomain' named' CopB251I495.' The' overexpression' and' purification' of' each'
recombinant' protein' were' detailed' in' Appendices' 4.8.' The' CopB251I495' topology' prediction' was'
performed' based' on' a' Hidden' Markov' Method' using' the' program' PREDITMBB' available' on' the'
World'Wide'Web'at'http://bioinformatics.biol.uoa.gr/PREDITMBB/'(Bagos'et'al.,'2004).'
(
(
85'
'
Results'and'Discussion'
'
4.5.2.( Production(and(Purification(of(Polyclonal(Antibodies(
AntiICopB' antibodies' were' produced' by' the' CERIGroupe' Marloie' (Belgium)' and' generated' in'
rabbits.'Polyclonal'antibody'purification'was'achieved'by'gradually'adding'solid'ammonium'sulfate'
(VWR'BDH'Prolabo)'to'the'serum'to'35%'saturation'with'gentle'stirring'at'4°C.'After'the'complete'
addition' of' ammonium' sulfate,' the' sample' was' constantly' mixed' for' 1' h' at' 4°C' to' allow' a'
precipitate'to'form;'this'step'was'followed'by'a'centrifugation'at'11,000'rpm'for'20'min'at'4°C.'The'
pellet' was' subsequently' dissolved' in' a' minimal' volume' of' PBS' (BupH™' PhosphateIBuffered' Saline'
Packs,'Thermo'Scientific)'and'dialyzed'at'4°C'against'PBS.'The'preimmune'serum'from'rabbits'was'
purified'in'the'same'way.'
4.5.3.( Protein(and(Antibody(Determination(
Protein' and' antibody' concentrations' were' determined' by' the' Bradford' method' (Bradford,'
1976)'using'bovine'serum'albumin'(Thermo'Scientific)'as'a'standard.'
4.5.4.( Bacterial(Cells(and(Culture(Conditions(
C.*metallidurans'strain'CH34'was'grown'at'30°C'under'aerobic'conditions'(rotary'shaker,'160'
rpm)'in'MOPSIsalt'minimal'medium'with'2'g'LI1'gluconate'as'a'unique'carbon'source'(adapted'from'
(Mergeay'et'al.,'1985)).'Minimal'medium'for'copperItreated'bacteria'was'supplemented'with'0.8'
mM'Cu(NO3)2'•'3H2O.'Growth'was'monitored'by'optical'density'at'600'nm'(OD600)'and'harvested'by'
centrifugation'during'the'midIexponential'phase'(OD600'of'~'0.6).'
4.5.5.( Subcellular(Fractionation(of(C.*metallidurans(CH34(and(Western(Blotting(
Untreated' and' copperItreated' bacteria' from' a' 250ImL' culture' were' harvested' by'
centrifugation'(6,000'rpm,'20'min,'at'4°C).'Each'pellet'was'subsequently'resuspended'in'100'mM'
TrisIHCl,'pH'7.3,'containing'a'protease'inhibitor'cocktail'(Complete,'EDTAIfree,'Roche)'and'passed'
through'a'French'press'(Mini'CELL'FAI003,'Thermo'Electron'Corporation)'at'10,000'psi.'They'were'
then' supplemented' with' Benzonase®' Nuclease' (Purity' >99%,' 10' KUN,' Novagen®)' and' incubated'
with'mild'agitation'for'15'min'at'room'temperature.'Cell'lysates'were'subsequently'clarified'by'lowI
speed'centrifugation'(6,000'rpm'for'15'min'at'4°C)'and'subjected'to'ultracentrifugation'(120,000'x'
g'for'1'h'20'min'at'4°C)'using'a'Beckman'Type'50.2'Ti'Ultracentrifuge'rotor.'Crude'membranes'were'
recovered' as' pellets' after' highIspeed' centrifugation.' Supernatants' corresponding' to' soluble'
proteins' were' subjected' to' another' round' of' ultracentrifugation' to' remove' membrane'
contaminants.' Crude' membranes' were' further' resuspended' in' a' highIsalt' extraction' buffer' (100'
86'
'
Results'and'Discussion'
'
mM' TrisIHCl,' pH' 7.3,' 500' mM' NaCl),' incubated' with' mild' agitation' for' 15' min' at' 4°C' and'
ultracentrifuged'again'(120,000'x'g'for'1'h'20'at'4°C).'As'a'result'of'this'highIsalt'extraction'step,'
pellets' enriched' in' outer' and' inner' membrane' proteins' were' isolated'in' addition' to' supernatants'
containing'peripheral'membrane'proteins.'Enriched'membrane'pellets'were'resuspended'in'300'µL'
Laemmli'sample'buffer.'
For'immunoblotting,'membrane'proteins'were'diluted'500Ifold'in'Laemmli'sample'buffer.'0.1'
µg' of' soluble' (i.e.,' cytoplasmic' and' periplasmic' proteins)' and' peripheral' membrane' proteins'
prepared'in'the'same'buffer'were'separated'on'a'4I20%'MiniIProtean®'precast'gel'(BioIRad).'The'
proteins'from'the'gel'were'transferred'onto'a'nitrocellulose'membrane'(Amersham™'Hybond™IECL,'
pore'size'0.45'µm,'GE'Healthcare)'using'a'wet'tank'blotting'system'(BioIRad).'The'membrane'was'
blocked' in' TBS' containing' 1%' Tween' 20' and' 5%' nonfat' dry' milk.' All' subsequent' washes' were'
carried'out'at'room'temperature'in'TBS'containing'1%'Tween'20.'The'nitrocellulose'membrane'was'
then'incubated'with'antiICopB'antibody'at'1:100,000'overnight'at'4°C.'The'membrane'was'washed'
as' before' and' then' incubated' with' secondary' antibody' (ECL™' antiIrabbit' IgG,' horseradish'
peroxidaseIlinked' whole' antibody' (from' donkey),' GE' Healthcare)' at' 1:8,000' for' 1' h' at' room'
temperature.' After' washing,' the' membrane' was' developed' using' the' peroxide' and' the' luminol'
enhancer'solutions'from'the'Promega'kit'(ECL'Western'Blotting'Substrate,'Promega).'
4.5.6.( Immunogold(Labeling(
For' postIembedding' labeling' of' ultrathin' sections' of' bacteria,' cell' pellets' were' prepared'
following'a'method'adapted'from'Chikwamba'et'al.,'2003'(Chikwamba'et'al.,'2003).'Untreated'and'
copperItreated' bacteria' were' washed' with' 8' mM' NaCl' and' 0.05%' (w/v)' ruthenium' red' in' 0.1' M'
cacodylate'buffer'(pH'7.8).'Samples'were'immediately'fixed'in'2%'(v/v)'paraformaldehyde'and'0.5%'
(v/v)'glutaraldehyde'in'the'same'buffer'for'2'h'at'4°C.'The'chemical'fixation'was'followed'by'rinses'
with'four'10Imin'changes'of'cacodylate'buffer.'Samples'were'subsequently'dehydrated'in'a'graded'
ethanol' series' up' to' 70%,' and' embedded' in' LR' White' resin' (62662' LR' White' kit,' SigmaIAldrich).'
Ultrathin'sections'(70I90'µm)'were'cut'with'a'Leica'Ultracut'UCT'ultramicrotome'equipped'with'a'
diamond'knife.'Immunogold'labeling'was'then'carried'out'by'the'protocol'of'Flammang'et'al.,'1998'
(Flammang'et'al.,'1998).'Briefly,'thin'sections'on'gold'grids'were'blocked'on'drops'of'10%'normal'
goat'serum'(SouthernBiotech)'in'PBS'for'30'min'at'room'temperature.'Grids'were'then'incubated'
overnight'at'4°C'on'drops'of'antiICopB'antibody'diluted'1:250'in'PBS,'1%'Tween'20,'and'3%'BSA.'
Grids' were' washed' four' times' in' PBS' for' 2' min' at' room' temperature.' Ultrathin' sections' were'
subsequently'incubated'for'1'h'at'room'temperature'on'drops'of'goldIconjugated'goat'antiIrabbit'
IgG'(15'nm;'BBInternational)'diluted'1:100'in'PBS,'1%'Tween'20,'and'3%'BSA.'Lastly,'the'grids'were'
87'
'
Results'and'Discussion'
'
washed'four'times'in'PBS'and'one'time'in'ultrapure'water'for'2'min'each'at'room'temperature,'and'
allowed' to' dry.' The' immunoIlabeled' sections' were' further' counterstained' with' aqueous' uranyl'
acetate' and' lead' citrate' and' observed' using' a' Zeiss' LEO' 906E' transmission' electron' microscope'
operated' at' 60' kV.' TEM' images' were' acquired' with' the' program' analySIS' (Soft' Imaging' System,'
Switzerland).'
4.5.7.( Synthetic(Peptides(and(Metal(Stock(Solutions(
Synthetic'peptides'corresponding'to'the'repetitive'methionineIrich'motif'of'the'CopB'protein'
were' purchased' from' GenScript' Corporation' (Table' 6.1).' All' peptides' contain' an' NIterminal'
acetylation'modification.'Peptide'stock'solutions'were'prepared'by'solubilizing'lyophilized'peptides'
in'50'mM'ammonium'acetate,'pH'6.8'and'stored'under'N2.'Peptide'concentration'was'determined'
by'quantitative'amino'acid'analysis'(PICK''n'POST™,'Alphalyse,'Denmark).'CuCl2'•'2H2O,'AgNO3'and'
ZnCl2'stock'solutions'were'prepared'in'reagentIgrade'H2O.'L(+)IAscorbic'acid'stock'solution'(H2Asc,'
UCB'Pharma)'was'used'as'a'biologically'relevant'reductant'to'convert'Cu(II)'to'Cu(I)'ions'and'was'
freshly'prepared'in'reagentIgrade'H2O'daily'(Jiang'et'al.,'2005).'
4.5.8.( Electrospray(Mass(Spectrometry((nESI)(
All' mass' spectrometry' experiments' were' performed' on' a' Waters' (Manchester,' UK)' QToF2'
mass' spectrometer' equipped' with' a' ZIspray' source.' Ionization' was' achieved' in' the' positive' ion'
mode'by'application'of'+2'kV'at'the'capillary'entrance;'the'cone'voltage'and'source'temperature'
were'20'V'and'150°C,'respectively.'Argon'was'used'as'the'collision'gas.'Spectra'were'recorded'in'
the' mass/charge' (m/z)' range' of' 100' —' 2,000.' Data' were' acquired' in' continuum' mode' until'
acceptable'averaged'data'were'obtained,'and'analyzed'using'the'MassLynx'v4.1'software.'
MetalIbinding'experiments'were'performed'in'50'mM'ammonium'acetate,'pH'6.8,'by'titrating'
a' constant' concentration' of' peptide' together' with' increasing' amounts' of' metal.' Assuming' a' 1:1'
peptideImetal' interaction,' Peptide' +' Metal' ∏' PeptideIMetal,' the' expression' for' the' dissociation'
constant' (KD)' was' described' by' the' following' equation:' KD' =' [Peptidefree]' *' [Metalfree]' /' [PeptideI
Metal].'For'KD'calculation,'we'assumed'that'(i)'the'total'signal'intensity'for'each'individual'species'
(i.e.,' free' peptide' and' proteinImetal' complex)' is' proportional' to' their' concentration' in' the' gas'
phase,'and'consequently,'reflects'their'concentration'in'solution;'and'(ii)'the'ionization'efficiency'is'
the' same' for' the' complex' and' free' peptide.' These' assumptions' allowed' us' to' be' free' from'
concentration' dependence,' so' that' we' can' use' the' intensity' ratios' of' unbound' peptide' over'
complex'instead'of'their'concentrations'in'solution'(Jecklin'et'al.,'2009).'Consequently,'the'complex'
88'
'
Results'and'Discussion'
'
formation'of'the'peptide'with'the'metal'can'be'described'by'the'following'equations'(Yin'and'Loo,'
2009):'
(a)'[PeptideIMetal]'='[Peptidetotal]*Icomplex'/'(Ipeptide'free'+'Icomplex)''
(b)'[Peptidefree]'='[Peptidetotal]I[PeptideIMetal]'
(c)'[Metalfree]'='[Metaltotal]I[PeptideIMetal]'
For'each'titration'experiment,'the'ratio'of'complex'over'unbound'peptide'was'plotted'against'the'
concentration'of'unbound'metal,'which'gave'a'linear'relationship'with'a'slope'equivalent'to'1/KD.'
4.5.9.( Circular(Dichroism(Spectroscopy(
Circular' dichroism' (CD)' measurements' were' performed' with' a' ChirascanTMIplus' CD'
Spectrometer' (Applied' Photophysics),' using' a' 1.0' mm' pathIlength' suprasil' quartz' cell' (Hellma'
Analytics)'at'room'temperature.'Circular'dichroism'data'were'collected'from'190'to'280'nm'with'a'
spectral' bandwidth' set' to' 1' nm' and' a' current' time' per' point' of' 1' s.' For' each' spectrum,' the' CD'
signal'corresponds'to'an'average'of'three'scans'and'was'baselineIcorrected'by'subtraction'of'the'
background'for'the'spectrum'obtained'with'buffer'alone.'Experiments'with'recombinant'proteins'
were' performed' in' 25' mM' ammonium' acetate' buffer' containing' 4' µM' of' protein.' Estimations' of'
the' fractional' percentage' of' secondary' structures' were' carried' out' using' the' CDSSTR' algorithm'
(DICROWEB' website,' http://dichroweb.cryst.bbk.ac.uk)' (Sreerama' and' Woody,' 2000;' Whitmore'
and' Wallace,' 2008).' CD' spectra' of' the' CopB(Met)' protein' were' recorded' in' the' absence' and'
presence'of'2,'10'and'14'molar'equivalents'of'Cu(II)'or'Ag(I).'Experiments'with'synthetic'peptides'
were'performed'in'5.3'mM'ammonium'acetate'buffer,'containing'20'µM'of'peptide.'CD'spectra'of'
20'µM'each'peptide'were'recorded'in'the'absence'and'presence'of'2'and'5'molar'equivalents'of'
Cu(II),' Cu(I)' and' Ag(I),' respectively.' Hydroxylamine' solution' (NH2OH,' SigmaIAldrich)' was' used' to'
reduce'Cu(II)'to'Cu(I)'ions.'The'observed'ellipticity'is'reported'in'millidegrees.'
4.5.10.(( Nuclear(Magnetic(Resonance((NMR)(Spectroscopy(
1D'proton'NMR'experiments'were'conducted'on'an'Avance'II'500'spectrometer'equipped'with'
an' 11.75' T' superconducting' magnet' (Bruker,' Karlsruhe,' Germany).' Synthetic' peptides' were'
dissolved'in'degassed'100%'D2O'solution,'typically'at'a'final'concentration'of'2'mM.'As'for'of'AgNO3'
and' CuCl2' •' 2H2O,' stock' solution' of' L(+)Iascorbic' acid,' which' was' used' in' excess' (15' molar'
equivalents)'to'efficiently'reduce'Cu(II)'to'Cu(I)'ions,'was'also'prepared'in'D2O'solution.'All' 1HINMR'
spectra'were'acquired'using'the'same'acquisition'parameters'(20°C,'64'scans),'and'collected'before'
and' after' successive' additions' of' metal' (from' 0.25' to' 3' molar' equivalents).' Chemical' shits' were'
reported'in'Hz.'
89'
'
Results'and'Discussion'
'
4.5.11.(( Formaldehyde(Cross9Linking(
Formaldehyde'crossIlinking'experiments'were'carried'out'following'a'procedure'adapted'from'
Klockenbusch'et'al.,'2010'(Klockenbusch'and'Kast,'2010).'A'formaldehyde'solution'was'prepared'by'
dissolving' 1.2%' paraformaldehyde' (Carl' Roth)' (w/v)' in' PBS' (BupH™' PhosphateIBuffered' Saline'
Packs,' Thermo' Scientific).' Preparation' of' depolymerized' paraformaldehyde' solution' requires'
heating' to' ~60°C' (~30' min)' followed' by' the' dropwise' addition' of' concentrated' sodium' hydroxide'
solution' until' the' solution' of' paraformaldehyde' clears' (Ellis,' 2009).' The' solution' was' filtered'
through' cellulose' acetate' membranes' with' 0.2' µmIsized' pores' (16534K,' Minisart®' NML' syringe'
filters,'Sartorius'AG)'and'stored'in'the'dark'at'room'temperature'for'a'maximum'of'four'weeks.'
For' crossIlinking' experiments,' untreated' and' copperItreated' bacteria' from' a' 20ImL' culture'
were' harvested' by' centrifugation' (6,000' rpm,' 10' min,' at' room' temperature).' Pelleted' cells' were'
resuspended' in' 10' mL' PBS' and' centrifuged' again.' Each' bacterial' pellet' was' incubated' with' mild'
agitation'in'4ImL'formaldehyde'solution'for'7'min'at'room'temperature.'The'crossIlinked'cells'were'
subsequently'centrifuged'at'2,000'x'g'for'3'min'at'room'temperature.'Bacterial'cells'are'therefore'
exposed' to' formaldehyde' for' a' maximum' of' 10' min.' The' supernatants' were' then' discarded,' and'
the' reaction' is' quenched' by' adding' 2' mL' iceIcold' 1.25' M' glycine/PBS.' Resuspended' cells' were'
incubated' with' mild' agitation' for' 5' min' at' room' temperature,' centrifuged' (2,000' x' g,' 15' min,' at'
4°C),' washed' once' in' 5ImL' PBS,' and' centrifuged' again.' Control' cells' were' prepared' in' the' same'
manner,'with'the'exception'of'formaldehyde'treatment.'
Pelleted' cells' were' lysed' in' 1.5' mL' RIPA' buffer' [50' mM' TrisIHCl,' pH' 8.0,' 150' mM' NaCl,' 1%'
IGEPAL®CAI630,' 0.5%' sodium' deoxycholate,' 0.1%' SDS,' 1' mM' EDTA,' protease' inhibitor' cocktail'
(Complete,'EDTAIfree,'Roche)]'for'60'minutes'on'ice.'Cell'lysates'were'subsequently'passed'three'
times' through' a' French' press' (Mini' CELL' FAI003,' Thermo' Electron' Corporation)' at' 10,000' psi.'
Soluble' proteins' were' separated' from' insoluble' debris' by' centrifugation' for' 30' min' at' 20,000' x' g'
and'4°C.'Soluble'protein'concentrations'were'determined'using'a'BCA™'Protein'Assay'Kit'(Thermo'
Scientific).'
4.5.12.(( Co9Immunoprecipitation(
CoIimmunoprecipitation'experiments'were'achieved'using'the'Pierce'CoIImmunoprecipitation'
Kit' (CoIIP' Kit' no.' 26149,' Thermo' Scientific).' Briefly,' purified' antiICopB' or' preimmune' antibodies'
(100'µg)'were'directly'and'covalently'coupled'onto'an'amineIreactive'resin'(AminoLink'Plus'Resin)'
for' 2' hours' under' gentle' rotation' at' room' temperature.' Covalent' antibody' immobilizations' were'
followed'by'washes'according'to'the'manufacturer's'instructions.'All'coIIP'steps'were'subsequently'
90'
'
Results'and'Discussion'
'
performed'at'4°C.'Cell'lysates'(250'µg)'were'incubated'with'agarose'beads'for'2'hours'under'gentle'
rotation'at'4°C.'After'a'series'of'washing'steps'(ten'washes'of'2'min'each)'with'IP'Lysis/Wash'Buffer'
(25'mM'Tris,'pH'7.4,'150'mM'NaCl,'1'mM'EDTA,'1%'NPI40,'5%'glycerol),'bound'material'was'eluted'
with'a'low'pH'solution'(Elution'Buffer,'pH'2.8)'and'immediately'rebuffered'with'1'M'Tris,'pH'9.5'for'
downstream'enzymatic'assays.'
4.5.13.(( LC9MS/MS(Analysis(and(Identification(of(CopB9Interacting(Partners(
The' entire' eluates' were' reduced' and' alkylated' according' to' the' manufacturer's' instructions'
(ICPL' kit,' Serva).' Eluted' proteins' were' recovered' through' acetone' precipitation' and' digested' inI
solution' with' trypsin' (Promega' V51' 11)' at' a' 1:50' (enzyme/substrate)' ratio' in' 50' mM' NH4HCO3'
overnight'at'37°C.'Trypsic'digestions'were'stopped'by'addition'of'formic'acid'(0.1%,'v/v,'final).'
The' identification' of' proteolytic' peptides' was' performed' using' a' labelIfree' strategy' on' an'
UHPLCIHRMS'platform'combining'a'NanoLCIUltra'system'(Eksigent)'with'a'TripleTOF®'5600'System'
(AB'SCIEX).'Peptides'were'separated'on'a'25'cm'C18'reverseIphase'column'(Acclaim'PepMap'100,'3'
µm,' Dionex)' by' a' linear' acetonitrile' gradient' (4—35%,' v/v,' flow' rate' of' 300' nL' minI1,' 20' min)' in'
water' containing' 0.1%' (v/v)' formic' acid.' The' TOF' analyzer' was' regularly' and' automatically'
calibrated' with' trypsic' peptides' of' βIgalactosidase' from' E.* coli,' which' maintains' an' average' mass'
error'below'10'ppm'across'all'injections.'Mass'spectra'(MS)'were'acquired'across'400—1500'm/z'
with'an'accumulation'time'of'0.5'sec.'A'maximum'number'of'50'precursors'per'cycle'were'selected'
according' to' an' intensity' threshold' of' 200' counts/sec.' Each' selected' precursor' was' accumulated'
during'50'ms'and'submitted'to'a'fragmentation'with'N2' as'the'collision'gas.'MS/MS'spectra'were'
acquired'across'100—1800'm/z'and'an'exclusion'time'of'30'seconds'was'applied.'
Acquired' data' were' analyzed' using' ProteinPilotTM' Software' v.' 4.1' (AB' SCIEX' MA,' USA)' and'
searched'against'the'UniProt'TrEMBL'database'(10/26/2011'version),'restricted'to'C.*metallidurans'
CH34.' The' search' parameters' included' differential' amino' acid' mass' shifts' for' carbamidomethyl'
cysteine,'all'biological'modifications'and'amino'acid'substitutions,'and'missed'trypsin'cleavage.'
(
(
91'
'
Results'and'Discussion'
'
4.6.(References(
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cooperativity' between' Cu(I)' and' Cu(II)' in' the' copper' resistance' protein' CopK' from'
Cupriavidus* metallidurans' CH34:' implications' from' structural' studies' by' NMR'
spectroscopy'and'XIray'crystallography.'J'Am'Chem'Soc'131,'3549I3564.'
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Ellis,' E.A.,' 2009.' A' fast,' simple,' and' safe' way' to' prepare' paraformaldehyde' solutions.' Microscopy'
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Flammang,' P.,' Michel,' A.,' Cauwenberge,' A.V.,' Alexandre,' H.,' Jangoux,' M.,' 1998.' A' study' of' the'
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HernándezIMontes,' G.,' Argüello,' J.M.,' Valderrama,' B.,' 2012.' Evolution' and' diversity' of' periplasmic'
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Jecklin,'M.C.,'Schauer,'S.,'Dumelin,'C.E.,'Zenobi,'R.,'2009.'LabelIfree'determination'of'proteinIligand'
binding' constants' using' mass' spectrometry' and' validation' using' surface' plasmon'
resonance'and'isothermal'titration'calorimetry.'Journal'of'molecular'recognition':'JMR'22,'
319I329.'
Jiang,' J.,' Nadas,' I.A.,' Kim,' M.A.,' Franz,' K.J.,' 2005.' A' Mets' motif' peptide' found' in' copper' transport'
proteins'selectively'binds'Cu(I)'with'methionineIonly'coordination.'Inorg'Chem'44,'9787I
9794.'
Jones,'D.T.,'1999.'Protein'secondary'structure'prediction'based'on'positionIspecific'scoring'matrices.'
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Kabsch,' W.,' Sander,' C.,' 1983.' Dictionary' of' protein' secondary' structure:' pattern' recognition' of'
hydrogenIbonded'and'geometrical'features.'Biopolymers'22,'2577I2637.'
Klockenbusch,' C.,' Kast,' J.,' 2010.' Optimization' of' formaldehyde' crossIlinking' for' protein' interaction'
analysis'of'nonItagged'integrin'beta1.'J'Biomed'Biotechnol'2010,'927585.'
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Lee,'S.M.,'Grass,'G.,'Rensing,'C.,'Barrett,'S.R.,'Yates,'C.J.,'Stoyanov,'J.V.,'Brown,'N.L.,'2002.'The'Pco'
proteins'are'involved'in'periplasmic'copper'handling'in'Escherichia*coli.'Biochem'Biophys'
Res'Commun'295,'616I620.'
Liu,'M.,'Tang,'Y.C.,'Fan,'K.Q.,'Jiang,'X.,'Lai,'L.H.,'Ye,'Y.H.,'2005.'Cyclization'of'several'linear'pentaI'and'
heptapeptides' with' different' metal' ions' studied' by' CD' spectroscopy.' The' journal' of'
peptide'research':'official'journal'of'the'American'Peptide'Society'65,'55I64.'
Loftin,' I.R.,' Franke,' S.,' Blackburn,' N.J.,' McEvoy,' M.M.,' 2007.' Unusual' Cu(I)/Ag(I)' coordination' of'
Escherichia* coli' CusF' as' revealed' by' atomic' resolution' crystallography' and' XIray'
absorption'spectroscopy.'Protein'Sci'16,'2287I2293.'
Long,' F.,' Su,' C.C.,' Lei,' H.T.,' Bolla,' J.R.,' Do,' S.V.,' Yu,' E.W.,' 2012.' Structure' and' mechanism' of' the'
tripartite' CusCBA' heavyImetal' efflux' complex.' Philos' Trans' R' Soc' Lond' B' Biol' Sci' 367,'
1047I1058.'
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Mergeay,' M.,' Monchy,' S.,' Janssen,' P.,' Van' Houdt,' R.,' Leys,' N.,' 2009.' Megaplasmids' in' Cupriavidus'
genus' and' metal' resistance,' In:' Schwartz,' E.' (Ed.),' Microbial' Megaplasmids,' Springer' ed,'
Münster,'Germany,'pp.'209I238.'
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adapted'to'toxic'metals:'towards'a'catalogue'of'metalIresponsive'genes.'FEMS'Microbiol'
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eutrophus'CH34'is'a'facultative'chemolithotroph'with'plasmidIbound'resistance'to'heavy'
metals.'J'Bacteriol'162,'328I334.'
Monchy,' S.,' Benotmane,' M.A.,' Janssen,' P.,' Vallaeys,' T.,' Taghavi,' S.,' van' der' Lelie,' D.,' Mergeay,' M.,'
2007.'Plasmids'pMOL28'and'pMOL30'of'Cupriavidus*metallidurans'are'specialized'in'the'
maximal'viable'response'to'heavy'metals.'J'Bacteriol'189,'7417I7425.'
Monchy,' S.,' Benotmane,' M.A.,' Wattiez,' R.,' van' Aelst,' S.,' Auquier,' V.,' Borremans,' B.,' Mergeay,' M.,'
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Sarret,'G.,'Favier,'A.,'Coves,'J.,'Hazemann,'J.L.,'Mergeay,'M.,'Bersch,'B.,'2010.'CopK'from'Cupriavidus*
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Yang,'B.,'Wu,'Y.J.,'Zhu,'M.,'Fan,'S.B.,'Lin,'J.,'Zhang,'K.,'Li,'S.,'Chi,'H.,'Li,'Y.X.,'Chen,'H.F.,'Luo,'S.K.,'Ding,'
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'
(
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Results'and'Discussion'
'
4.7.(Supplemental(Data(
'
'
Figure( S4.1:' Circular' dichroism' spectra' of' methionineIrich' peptides' in' the' absence' and' presence' of' Cu(II),' Cu(I),' or' Ag(I)'
ions.' Panels' above' (A.,' B.,' and' C.)' and' below' (D.,' E.,' and' F.)' correspond' to' Pep.' 1' (H9' →' A)' and' Pep.' 3' (M12' →' G),'
respectively.'CD'spectra'of'20'µM'peptide'were'recorded'in'5.3'mM'ammonium'acetate,'pH'6.8.'Each'curve'represents'an'
average'of'three'scans'and'was'baselineIcorrected'by'subtraction'of'the'background'for'the'spectrum'obtained'with'buffer'
alone.' ApoIpeptides' are' shown' in' solid' black' lines.' (A.' and' D.)' Additions' of' 2' (dashed' blue' line)' and' 5' (dashed' red' line)'
molar'equivalents'of'Cu(II).'(B.'and'E.)'Additions'of'2'(dashed'blue'line)'and'5'(dashed'red'line)'molar'equivalents'of'Cu(I),'in'
the'presence'of'10'molar'equivalents'of'NH2OH.'(C.'and'F.)'Additions'of'2'(dashed'blue'line)'and'5'(dashed'red'line)'molar'
equivalents'of'Ag(I).'
'
'
'
'
Figure( S4.2:' Circular' dichroism' spectra' of' methionineIrich' peptides' in' the' absence' and' presence' of' Zn(II)' ions.' The' panel'
(A.)'corresponds'to'Pep.'2'(M4'→'G),'while'panel'(B.)'corresponds'to'Pep.'3'(M12'→'G).'CD'spectra'of'20'µM'peptide'were'
recorded' in' 5.3' mM' ammonium' acetate,' pH' 6.8.' Each' curve' represents' an' average' of' three' scans' and' was' baselineI
corrected'by'subtraction'of'the'background'for'the'spectrum'obtained'with'buffer'alone.'ApoIpeptides'are'shown'in'solid'
black'lines;'additions'of'2'and'5'molar'equivalents'of'Zn(II)'are'in'dashed'blue'and'red'lines,'respectively.'
'
96'
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Results'and'Discussion'
'
4.8.(Appendices:(Overexpression(and(Purification(of(Three(Recombinant(Proteins(
During' this' thesis,' three' recombinant' proteins' were' overexpressed' and' purified:' (i)' the'
plasmidIencoded' fullIlength' CopB' protein' from' Cupriavidus* metallidurans' CH34;' (ii)' the'
recombinant' protein' corresponding' to' its' NIterminal' fragment' (from' AA' 51' to' 250),' hereinafter'
called'"CopB(Met)";'and'(iii)'the'protein'corresponding'to'its'CIterminal'extremity'(from'AA'251'to'
495),'which'has'a'predicted'structure'and'which'is'named'the'CopB251I495'protein.'This'chapter'is'
dedicated'to'the'overexpression'and'purification'of'these'three'recombinant'proteins.'
4.8.1.( Bacterial(Transformations(and(Expression(of(Three(Recombinant(Proteins(
The'recombinant'plasmids'pJexpress411:56155,'pJexpress411:34700'and'pJexpress411:56156'
were' purchased' from' DNA' 2.0' (Menlo' Park,' CA' 94025,' USA)' and' used' according' to' the'
manufacturer's' instructions.' These' plasmids' pJexpress411:56155,' pJexpress411:34700' and'
pJexpress411:56156' encoded' (i)' the' plasmidIencoded' fullIlength' CopB' protein,' (ii)' the' CopB(Met)'
protein'and'(iii)'the'CopB251I495'protein,'respectively.'All'recombinant'plasmids'also'carried'a'gene'
coding' for' resistance' to' kanamycin.' These' three' expression' vectors' were' transformed'
independently'into'chemically'competent'BL21(DE3)'Escherichia*coli'(One'shot®'BL21'Star™'(DE3),'
Invitrogen)' according' to' the' manufacturer's' instructions.' Transformed' bacteria' were' grown' using'
LB'broth'supplemented'with'kanamycin'sulfate'(100X'kanamycin'sulfate,'Gibco®).'Bacterial'growth'
was' monitored' by' optical' density' at' 600' nm' (OD600).' Once' cultures' reached' an' OD600' of' ~0.6,'
isopropyl'βIDI1Ithiogalactopyranoside'(IPTG,'CalbioChem®)'was'added'to'a'final'concentration'of'1'
mM,' and' cultures' were' incubated' for' an' additional' 3' h' at' 140' rpm,' 37°C.' Then,' bacteria' were'
harvested'by'centrifugation'at'6,000'x'g'for'15'min'at'4°C.'
The' recombinant' protein' expression' was' evaluated' using' sodium' dodecyl' sulfate'
polyacrylamide' gel' electrophoresis' (SDSIPAGE).' Recovered' bacterial' pellets' before' and' after' IPTG'
induction' were' dissolved' in' an' appropriate' volume' of' Laemmli' sample' buffer,' and' proteins' were'
subsequently'separated'on'a'4I20%'MiniIProtean®'precast'gel'(BioIRad)'(App.'4.1).'
'
'
97'
'
Results'and'Discussion'
'
'
Appendix(4.1:'Overproduction'of'three'recombinant'proteins'upon'IPTG'induction'in'E.*coli.'Lysates'of'nonIinduced'cells'
(U)'and'IPTGIinduced'cells'(I)'were'separated'by'SDSIPAGE'(4I20%)'and'CoomassieIstained.'(A.)'The'plasmidIencoded'fullI
length'CopB'protein'from'C.*metallidurans'CH34.'(B.)'The'recombinant'protein'corresponding'to'the'methionineIrich'NI
terminal'extremity'of'CopB'protein,'called'the'CopB(Met)'protein.'(C.)'The'recombinant'protein'corresponding'to'the'CI
terminal'subdomain'of'CopB'protein'and'which'is'named'the'CopB251I495'protein.'
'
4.8.2.( Non9Denaturing(Protein(Extraction(
All'induced'bacterial'pellets'were'resuspended'in'50'mM'TrisIHCl,'pH'7.8,'containing'protease'
inhibitor' cocktail' (Complete,' EDTAIfree,' Roche)' and' rLysozymeTM' solution' (1200' KU,' Novagen®).'
Crude'lysates'were'passed'through'a'French'press'(Mini'CELL'FAI003,'Thermo'Electron'Corporation)'
at'10,000'psi'and'then'supplemented'with'Benzonase®'Nuclease'(Purity'>99%,'10'KUN,'Novagen®)'
and'incubated'with'mild'agitation'for'15'min'at'room'temperature.'Cell'lysates'were'subsequently'
clarified'by'centrifugation'at'6,000'rpm'for'15'min'at'4°C.'
'
4.8.3.( Purification(of(the(CopB(Met)(Protein(
Unlike' the' two' other' recombinant' proteins,' which' were' found' in' inclusion' bodies,' the'
CopB(Met)' recombinant' protein' is' a' soluble' protein' found' in' the' supernatant' after' the' nonI
denaturing' extraction.' The' CopB(Met)' protein' purification' occurred' in' two' steps:' first,' by'
immobilized' metal' ion' affinity' chromatography' (IMAC)' and' then,' by' anionIexchange'
chromatography.'
The'protein'sample'was'passed'through'an'iminodiacetic'acidIagarose'column'(I4758,'SigmaI
Aldrich)' previously' loaded' with' Cu2+' ions.' Then,' the' column' was' thoroughly' washed' with' 50' mM'
TrisIHCl,' pH' 7.8,' and' elution' was' performed' with' 50' mM' TrisIHCl,' pH' 7.8,' containing' 20' mM'
imidazole' (flow' rate' =' 1' mL/min).' The' chromatogram' relating' to' the' CopB(Met)' purification' by'
IMAC'and'SDSIPAGE'(4I20%)'protein'profiles'of'collected'fractions'are'shown'in'Appendix'4.2A'and'
B,' respectively.' The' CopB(Met)Icontaining' fractions' were' pooled' (App.' 4.2A,' grey' strip' and' 4.2B,'
arrows),'supplemented'with'2'mM'EDTA'and'dialyzed'against'50'mM'TrisIHCl,'pH'7.8.''
98'
'
Results'and'Discussion'
'
'
Appendix( 4.2:' Purification' of' the' recombinant' CopB(Met)' protein' expressed' in' E.* coli' BL21(DE3)' by' immobilizedImetal'
2+
affinity'chromatography,'IMAC.'(A.)'Chromatogram'showing'the'purification'of'the'CopB(Met)'protein'via'Cu IIMAC.'(a.)'
The'protein'sample'is'loaded'onto'an'IDAISepharose'6B'column,'running'at'a'flow'rate'of'1'mL/min.'(b.)'Then,'the'column'
is'thoroughly'washed'with'50'mM'TrisIHCl,'pH'7.8.'(c.)'The'elution'is'carried'out'with'50'mM'TrisIHCl,'pH'7.8,'containing'
20' mM' imidazole.' The' grey' area' corresponds' to' the' fractions' which' were' pooled.' (B.)' SDSIPAGE' (4I20%)' stained' with'
2+
Coomassie'blue'of'the'crude'extract'and'some'fractions'collected'from'Cu IIMAC.'The'arrows'indicate'the'CopB(Met)I
containing'fractions'which'were'pooled.'
'
The' second' chromatographic' step' was' anionIexchange' chromatography' (SOURCE™' 15Q,'
Amersham'Biosciences).'After'injection'of'the'pooled'sample,'the'column'was'equilibrated'with'50'
mM'TrisIHCl,'pH'7.8.'Then,'a'NaCl'gradient'(from'0'to'500'mM)'was'applied'to'separate'the'various'
proteins' from' the' CopB(Met)' recombinant' protein' (flow' rate' =' 1' mL/min).' Then,' the' column' was'
eluted'with'50'mM'TrisIHCl,'pH'7.8,'1'M'NaCl'and'subsequently'equilibrated'with'50'mM'TrisIHCl,'
pH' 7.8.' The' chromatogram' relating' to' the' CopB(Met)' purification' by' anionIexchange'
chromatography'is'shown'in'Appendix'4.3A.'As'previously'mentioned'for'IMAC'purification,'eluted'
fractions'were'monitored'for'protein'content'by'SDSIPAGE,'and'only'the'fractions'of'interest'were'
pooled'(App.'4.3A,'grey'strip'and'4.3B,'arrows).'
'
99'
'
Results'and'Discussion'
'
'
Appendix( 4.3:' Purification' of' the' recombinant' CopB(Met)' protein' by' anionIexchange' chromatography.' (A.)'
Chromatographic'profile'obtained'for'the'purification'of'the'CopB(Met)'protein'by'anionIexchange'chromatography.'(a.)'
TM
The' protein' sample' is' loaded' onto' a' column' packed' with' SOURCE ' 15Q,' running' at' a' flow' rate' of' 1' mL/min.' (b.)' The'
column'is'thoroughly'washed'with'50'mM'TrisIHCl,'pH'7.8.'(c.)'The'elution'is'performed'by'a'continuous'sodium'chloride'
gradient'from'0'to'0.5'M'(drawn'as'dotted'line)'with'flow'rate'of'1'mL/min.'(d.)'The'column'is'eluted'with'50'mM'TrisI
HCl,' pH' 7.8,' 1' M' NaCl.' (e.)' Equilibration' of' the' column' with' 50' mM' TrisIHCl,' pH' 7.8.' The' grey' area' corresponds' to' the'
fractions'which'were'pooled.'(B.)'SDSIPAGE'(4I20%)'stained'with'Coomassie'blue'of'the'crude'extract'and'some'fractions'
collected' from' anionIexchange' chromatography.' The' arrows' indicate' the' fractions' containing' the' purified' CopB(Met)'
protein'which'were'pooled.'
'
The'purity'of'CopB(Met)'recombinant'protein'was'evaluated'by'MALDIITOF'mass'spectrometry'
in' the' linear' mode' and' estimated' at' >95%,' as' observed' in' Appendix' 4.4.' The' experimental' molar'
mass' of' the' apoICopB(Met)' protein' determined' by' QToF' mass' spectrometry' under' denaturing'
conditions' was' 21,466.13' Da' (App.' 4.5).' This' value' was' consistent' with' the' predicted' value' of'
21,469' Da.' The' CopB(Met)' protein' was' stored' at' 4°C' under' a' nitrogen' atmosphere' to' avoid'
methionine' oxidation.' However,' this' recombinant' protein' appeared' unstable' in' a' relatively' short'
period.'Consequently,'all'analyses'were'conducted'with'freshly'purified'CopB(Met)'protein.'
'
100'
'
Results'and'Discussion'
'
'
Appendix( 4.4:' MALDIITOF' mass' analysis' of' the' CopB(Met)' protein.' Mass' spectrum' of' the' purified' CopB(Met)'
TM
recombinant'protein'was'obtained'using'the'linear'mode'on'a'Micromass'M@ldi 'spectrometer'(Waters)'equipped'with'
a' 337Inm' nitrogen' laser.' The' peak' at' m/z' 21475' corresponds' to' singly' charged' state,' while' the' peak' at' m/z' 10738.8'
corresponds'to'the'doubly'charged'ion'and'the'peak'at'm/z'7159.8'corresponds'to'the'triply'charged'state.'
'
'
Appendix(4.5:'ESIIMS'spectrum'of'the'purified'CopB(Met)'protein'tenIfold'diluted'in'50%'acetonitrile/0.1%'formic'acid.'
Mass' spectrum' of' the' purified' CopB(Met)' recombinant' protein' was' obtained' in' the' positive' mode' on' a' QToF2' mass'
spectrometer'(Waters)'equipped'with'a'ZIspray'source.'The'experimental'molar'mass'(21,466.13'Da)'is'consistent'with'
that'calculated'from'the'protein'sequence'(21,469'Da).'The'spectrum'consists'of'ions'of'different'charge'(+8'to'+29).'
'
4.8.4.( Purification(of(the(Full9Length(CopB(Protein(
The'fullIlength'CopB'protein'was'found'in'the'pellet'obtained'after'centrifuging'the'cell'lysate,'
which' followed' the' nonIdenaturing' extraction.' To' efficiently' extract' the' protein' of' interest,' two'
different' treatments' were' tested.' In' the' one' treatment,' the' recovered' pellet' was' dissolved' in' an'
appropriate'volume'of'50'mM'TrisIHCl,'pH'7.8,'8'M'urea,'and'in'the'other'treatment,'dissolving'the'
protein'in'50'mM'TrisIHCl,'pH'7.8,'0.5%'Triton'XI100,'was'attempted.'The'protein'suspensions'were'
incubated'under'mild'stirring'for'30'min'at'room'temperature'and'then'centrifuged'at'10,000'x'g'
for'15'min'at'4°C.'The'recovered'pellets'and'supernatants'for'each'treatment'were'dissolved'in'an'
appropriate'volume'of'Laemmli'sample'buffer,'and'the'proteins'were'subsequently'separated'on'a'
4I20%' MiniIProtean®' precast' gel' (BioIRad),' as' shown' in' Appendix' 4.6.' The' denaturing' buffer'
101'
'
Results'and'Discussion'
'
containing'8'M'urea'appeared'to'be'much'more'efficient'at'extracting'the'fullIlength'CopB'protein'
from'the'pellet'compared'with'the'nonIdenaturing'buffer'containing'0.5%'Triton'XI100.'This'result'
suggested'that'the'fullIlength'CopB'protein'was'overexpressed'as'inclusion'bodies.'
'
'
Appendix(4.6:'SDSIPAGE'(4I20%)'showing'the'extraction'
of' the' fullIlength' CopB' protein' based' on' two' different'
treatments.' The' fullIlength' CopB' protein' was' found' in'
the' pellet' after' centrifuging' the' cell' lysate,' which'
followed' the' nonIdenaturing' extraction.' In' one'
treatment,' the' pellet' was' dissolved' in' 50' mM' TrisIHCl,'
pH'7.8,'and'8'M'urea,'and'in'the'other'treatment,'it'was'
resuspended'in'50'mM'TrisIHCl,'pH'7.8,'and'0.5%'Triton'
XI100.' The' protein' suspensions' were' incubated' for' 30'
min' at' room' temperature' and' then' centrifuged' at'
10,000' x' g' for' 15' min' at' 4°C.' The' recovered' pellets' (P)'
and' supernatants' (S)' were' diluted' in' Laemmli' sample'
buffer' and' proteins' were' separated' by' SDSIPAGE' (4I
20%).'
'
Consequently,'the'denaturing'treatment'(i.e.,'50'mM'TrisIHCl,'pH'7.8,'8'M'urea)'was'used'to'
efficiently' extract' the' fullIlength' CopB' protein.' Following' the' centrifugation' step' (10,000' x' g,' 15'
min,'4°C),'the'supernatant'was'thoroughly'dialyzed'against'50'mM'TrisIHCl,'pH'7.8,'0.5'M'NaCl'and'
purified'by'gel'filtration'chromatography.'The'protein'sample'(200'µL)'was'applied'onto'a'TSKIgel'
G3000SW' column' (7.5' mm' ID' x' 30.0' cm' L,' 10' µm' particle' size,' Tosoh' Bioscience),' which' is'
previously'equilibrated'and'run'in'50'mM'TrisIHCl,'pH'7.8,'0.5'M'NaCl'(flow'rate'='1'mL/min).'The'
chromatogram' relating' to' the' fullIlength' CopB' purification' by' gel' filtration' chromatography' is'
shown'in'Appendix'4.7A.'
'
'
Appendix( 4.7:' Purification' of' the' recombinant' fullIlength' CopB' protein' expressed' in' E.* coli' BL21(DE3)' by' gel' filtration'
chromatography.'(A.)'Chromatographic'profile'obtained'for'the'purification'of'the'recombinant'fullIlength'CopB'protein'
by'gel'filtration'chromatography.'The'protein'sample'was'applied'onto'a'TSKIgel'G3000SW'column'with'a'flow'rate'of'1'
mL/min.' The' column' was' previously' equilibrated' and' run' in' 50' mM' TrisIHCl,' pH' 7.8,' 0.5' M' NaCl.' The' grey' area'
corresponds'to'the'fractions'which'were'pooled.'(B.)'SDSIPAGE'(4I20%)'stained'with'Coomassie'blue'of'the'crude'extract'
and' some' fractions' collected' from' gel' filtration' chromatography.' The' arrows' indicated' the' fullIlength' CopB' proteinI
containing'fractions'which'were'pooled.'
102'
'
Results'and'Discussion'
'
Protein' detection' was' performed' at' 595' nm' using' Coomassie' blue' GI250,' and' eluted' fractions' of'
interest'were'then'electrophoresed'on'a'4I20%'MiniIProtean®'precast'gel'(BioIRad)'subsequently'
stained' with' Coomassie' blue' RI250' (App.' 4.7B).' Fractions' containing' only' the' protein' of' interest'
were'pooled,'as'shown'in'Appendix'4.7A'(grey'strip)'and'B'(arrows).'Based'on'this'chromatogram'
and'the'corresponding'SDSIPAGE,'the'purity'of'the'sample'was'estimated'at'higher'than'95%.'
Similar'to'the'CopB(Met)'recombinant'protein,'the'fullIlength'CopB'protein'was'stored'at'4°C'
under'a'nitrogen'atmosphere'to'avoid'methionine'oxidation.'However,'identical'to'that'observed'
for' the' CopB(Met)' protein,' the' fullIlength' CopB' protein' appeared' unstable' in' a' relatively' short'
period.'Consequently,'all'analyses'were'conducted'with'freshly'purified'CopB'protein.'
'
4.8.5.( Purification(of(the(CopB2519495(Protein(
Identical'to'the'fullIlength'CopB'protein,'the'recombinant'CopB251I495'protein'was'found'in'the'
pellet' after' the' centrifugation' step,' which' followed' the' nonIdenaturing' extraction.' Similar' to' the'
fullIlength' CopB' protein,' two' different' treatments' were' tested' to' efficiently' extract' the' protein'
from'the'pellet.'Therefore,'the'recovered'pellet'was'dissolved'in'either'50'mM'TrisIHCl,'pH'7.8,'8'M'
urea' or' 50' mM' TrisIHCl,' pH' 7.8,' 0.5%' Triton' XI100.' These' protein' suspensions' were' incubated'
under'mild'agitation'for'30'min'at'room'temperature'and'then'centrifuged'at'10,000'x'g'for'15'min'
at' 4°C.' The' recovered' pellets' and' supernatants' relating' to' each' treatment' were' dissolved' in' an'
appropriate'volume'of'Laemmli'sample'buffer,'and'proteins'were'subsequently'separated'on'a'4I
20%'MiniIProtean®'precast'gel'(BioIRad),'as'shown'in'Appendix'4.8.'identical'to'that'observed'with'
the' fullIlength' CopB' protein,' the' denaturing' buffer' containing' 8' M' urea' appeared' much' more'
efficient' for' extracting' the' CopB251I495' protein' from' the' pellet' than' the' nonIdenaturing' buffer'
containing' 0.5%' Triton' XI100.' This' result' suggested' that' the' CopB251I495' protein' was' also'
overexpressed'as'inclusion'bodies.'
Consequently,'the'denaturing'lysis'buffer'(i.e.,'50'mM'TrisIHCl,'pH'7.8,'and'8'M'urea)'was'used'
to' efficiently' extract' the' recombinant' CopB251I495' protein' from' the' pellet.' This' protein' was'
subsequently' purified' based' on' the' presence' of' 6xHisITag' at' the' NIterminus' through' a' nickelI
nitrilotriacetic' acid' (NiINTA)' affinity' column' (HISISelect®' High' Flow' Cartridge,' H7788I5EA,' SigmaI
Aldrich)'under'denaturing'conditions.'After'loading'the'lysate'on'the'NiINTA'column'and'extensive'
washing'steps,'the'recombinant'protein'was'eluted'with'elution'buffer'(i.e.,'50'mM'TrisIHCl,'pH'7.8,'
8'M'urea,'and'300'mM'imidazole).'The'chromatogram'relating'to'the'purification'of'the'CopB251I495'
protein' by' NiINTA' affinity' column' is' shown' in' Appendix' 4.9A.' Eluted' fractions' of' interest' were'
electrophoresed'on'a'4I20%'precast'gel'and'subsequently'stained'with'Coomassie'blue'RI250'(App.'
103'
'
Results'and'Discussion'
'
4.9B).'Only'the'fractions'containing'the'protein'of'interest'were'pooled,'as'shown'in'Appendix'4.9A'
(grey' strip)' and' B' (arrows).' After' the' purification,' the' protein' was' dialyzed' to' remove' urea' to'
enzymatically'digest'the'HisITag'using'TEV'protease.'However,'the'protein'was'completely'unstable'
after'removing'urea.'Consequently,'the'HisITag'could'not'be'removed,'and'the'protein'could'not'be'
characterized.'
To' circumvent' this' precipitation' problem,' another' treatment' was' tested.' Removed' inclusion'
bodies'were'dissolved'in'100'mM'TrisIHCl,'50'mM'NaCl,'2'M'urea,'and'5'mM'EDTA,'pH'12.0,'and'
incubated' for' 30' min' at' room' temperature' under' mild' agitation.' This' treatment' efficiently'
solubilized'the'inclusion'bodies'because'the'protein'of'interest'was'found'in'the'supernatant'after'
the' centrifugation' step' (8,000' rpm,' 10' min,' 4°C).' The' supernatant' was' subsequently' dialyzed' to'
remove' urea' while' maintaining' a' basic' pH.' The' removal' of' urea' was' a' success.' However,' protein'
precipitation'was'also'observed'when'returned'to'physiological'pH,'once'again'preventing'the'use'
of'TEV'protease'and'the'protein'characterization.'
'
'
'
'
'
'
'
'
104'
'
Appendix( 4.8:' SDSIPAGE' (4I20%)' showing' the'
extraction' of' the' CopB251I495' protein' based' on'
two' different' treatments.' The' recombinant'
protein' was' found' in' the' pellet' after'
centrifuging'the'cell'lysate,'which'followed'the'
nonIdenaturing' extraction.' In' one' treatment,'
the'pellet'was'dissolved'in'50'mM'TrisIHCl,'pH'
7.8,'and'8'M'urea,'and'in'the'other'treatment,'
it'was'resuspended'in'50'mM'TrisIHCl,'pH'7.8,'
and' 0.5%' Triton' XI100.' The' protein'
suspensions' were' incubated' for' 30' min' at'
room' temperature' and' then' centrifuged' at'
10,000' x' g' for' 15' min' at' 4°C.' The' recovered'
pellets'(P)'and'supernatants'(S)'were'diluted'in'
Laemmli' sample' buffer' and' proteins' were'
separated'by'SDSIPAGE'(4I20%).'
Results'and'Discussion'
'
'
Appendix(4.9:'Purification'of'the'recombinant'CopB251I495'protein'by'affinity'chromatography'using'NiINTA'resin,'under'
denaturing'conditions.'(A.)'Chromatographic'profile'obtained'for'the'purification'of'the'recombinant'CopB251I495'protein'
by' affinity' chromatography.' (a.)' The' protein' sample' was' loaded' onto' a' NiINTA' affinity' column' with' a' flow' rate' of' 1'
mL/min.' (b.)'The'column'is'thoroughly'washed'with'50'mM'TrisIHCl,'pH'7.8,'and'8'M'urea.'(c.)'Elution'was'performed'
using' 50' mM' TrisIHCl,' pH' 7.8,' 8' M' urea,' 300' mM' imidazole.' The' grey' area' corresponds' to' the' fractions' which' were'
pooled.' (B.)' SDSIPAGE' (4I20%)' stained' with' Coomassie' blue' of' the' crude' extract' and' some' fractions' collected' from'
affinity'chromatography.'The'arrows'indicate'the'fractions'containing'the'purified'CopB251I495'protein'which'were'pooled.'
'
'
'
105'
'
Results'and'Discussion'
'
4.8.6.( Recombinant(Plasmid(pJexpress411:56155(Encoding(the(Full9Length(CopB(Protein(
from(C.*metallidurans(CH34(
(
'
Appendix( 4.10:' Map' of' the' recombinant' plasmid' pJexpress411:56155' encoding' the' recombinant' fullIlength' CopB'
protein' from' C.* metallidurans' CH34.' The' gene' encoding' the' protein' of' interest' is' drawn' in' red,' whereas' the' gene'
encoding'kanamycin'resistance'is'drawn'in'green.'
'
106'
'
Results'and'Discussion'
'
'
Appendix(4.11:'Full'genome'sequence'of'the'recombinant'plasmid'pJexpress411:56155'encoding'the'recombinant'fullI
length'CopB'protein'from'C.*metallidurans'CH34.'The'sequence'encoding'the'protein'of'interest'is'drawn'in'red.'
107'
'
Results'and'Discussion'
'
4.8.7.( Recombinant(Plasmid(pJexpress411:34700(Encoding(the(Recombinant(CopB(Met)(
Protein(
(
'
Appendix( 4.12:' Map' of' the' recombinant' plasmid' pJexpress411:34700' encoding' the' recombinant' CopB(Met)' protein.'
The'gene'encoding'the'protein'of'interest'is'drawn'in'red,'whereas'the'gene'encoding'kanamycin'resistance'is'drawn'in'
green.'
108'
'
Results'and'Discussion'
'
'
Appendix( 4.13:' Full' genome' sequence' of' the' recombinant' plasmid' pJexpress411:34700' encoding' the' recombinant'
CopB(Met)'protein.'The'sequence'encoding'the'protein'of'interest'is'drawn'in'red.'
'
'
'
109'
'
Results'and'Discussion'
'
4.8.8.( Recombinant( Plasmid( pJexpress411:56156( Encoding( the( Recombinant( CopB2519495(
Protein(
(
'
Appendix( 4.14:' Map' of' the' recombinant' plasmid' pJexpress411:56156' encoding' the' recombinant' CopB251I495' protein.'
The'gene'encoding'the'protein'of'interest'is'drawn'in'red,'whereas'the'gene'encoding'kanamycin'resistance'is'drawn'in'
green.'
'
'
110'
'
Results'and'Discussion'
'
'
Appendix( 4.15:' Full' genome' sequence' of' the' recombinant' plasmid' pJexpress411:56156' encoding' the' CopB251I495'
protein.'The'sequence'encoding'the'protein'of'interest'is'drawn'in'red.'
'
(
111'
'
Results'and'Discussion'
'
4.9.(Appendices:(Metal(—(Peptide(Interactions(
4.9.1.( Mass(Spectra(of(Synthetic(Peptides(in(the(Presence(of(Cu(II)(Ions(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
Appendix( 4.16:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' the' native' peptide' and' its' Cu(II)'
adducts.'In'the'absence'of'copper'(0'Eq.),'the'
2+
major'peak'corresponds'to'[M'+'2H] 'with'an'
m/z' of' 905.24.' With' successive' additions' of'
CuCl2'•'2H2O,'a'peak'corresponding'to'a'first'
Cu(II)'adduct'emerges'with'an'm/z'of'935.73'
and'a'second'one'appears'at'm/z'of'966.21.'
(
(
'
112'
'
Results'and'Discussion'
'
'
'
'
'
Appendix( 4.17:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 1' (H9' →' A)' and' its' Cu(II)'
adducts.'In'the'absence'of'copper'(0'Eq.),'the'
2+
major'peak'corresponds'to'[M'+'2H] 'with'an'
m/z' of' 872.33.' With' successive' additions' of'
CuCl2' •' 2H2O,' a' peak' corresponding' to' a'
single' Cu(II)' adduct' emerges' with' an' m/z' of'
902.79.'
'
'
'
'
'
'
'
113'
'
'
Results'and'Discussion'
'
'
'
'
'
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
Appendix( 4.18:( Positive' electrospray'
ionization'mass'spectrometry'(ESIIMS)'spectra'
of' Pep.' 2' (M4' →' G)' and' its' Cu(II)' adducts.' In'
the'absence'of'copper'(0'Eq.),'the'major'peak'
2+
corresponds' to' [M' +' 2H] ' with' an' m/z' of'
868.34.' With' successive' additions' of' CuCl2' •'
2H2O,' a' peak' corresponding' to' a' first' Cu(II)'
adduct' emerges' with' an' m/z' of' 898.79' and' a'
second' one' appears' at' 5' molar' equivalents'
Cu(II)'at'm/z'of'929.25.'
'
'
'
114'
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Results'and'Discussion'
'
'
'
'
'
Appendix( 4.19( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 3' (M12' →' G)' and' its' Cu(II)'
adducts.'In'the'absence'of'copper'(0'Eq.),'the'
2+
major' peak' corresponds' to' [M' +' 2H] ' with'
an' m/z' of' 868.33.' With' successive' additions'
of' CuCl2' •' 2H2O,' a' peak' corresponding' to' a'
first' Cu(II)' adduct' emerges' with' an' m/z' of'
898.79' and' a' second' one' appears' at' m/z' of'
929.26.'
'
'
'
'
115'
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Results'and'Discussion'
'
4.9.2.( Peptide(Mass(Spectra(in(the(Presence(of(Cu(I)(Ions(
(
Appendix( 4.20:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' the' native' peptide' and' its' Cu(I)'
adducts.'In'the'absence'of'copper'(0'Eq.),'the'
2+
major'peak'corresponds'to'[M'+'2H] 'with'an'
m/z' of' 905.25.' With' successive' additions' of'
CuCl2' •' 2H2O' in' the' presence' of' 5' µM'
ascorbic' acid,' a' peak' corresponding' to' a'
single' Cu(I)' adduct' emerges' with' an' m/z' of'
936.24.'
(
(
(
'
116'
'
Results'and'Discussion'
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'
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'
(
(
(
(
(
(
(
(
(
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(
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(
Appendix( 4.21:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 1' (H9' →' A)' and' its' Cu(I)'
adducts.'In'the'absence'of'copper'(0'Eq.),'the'
2+
major' peak' corresponds' to' [M' +' 2H] ' with'
an' m/z' of' 872.37.' With' successive' additions'
of' CuCl2' •' 2H2O' in' the' presence' of' 5' µM'
ascorbic' acid,' a' peak' corresponding' to' a'
single' Cu(I)' adduct' emerges' with' an' m/z' of'
903.34.'
'
'
117'
'
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Results'and'Discussion'
'
'
'
Appendix( 4.22:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 2' (M4' →' G)' and' its' Cu(I)'
adducts.'In'the'absence'of'copper'(0'Eq.),'the'
2+
major' peak' corresponds' to' [M' +' 2H] ' with'
an' m/z' of' 868.34.' With' successive' additions'
of' CuCl2' •' 2H2O' in' the' presence' of' 5' µM'
ascorbic' acid,' a' peak' corresponding' to' a'
single' Cu(I)' adduct' emerges' with' an' m/z' of'
899.31.'
'
'
'
'
'
'
'
'
'
118'
'
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Results'and'Discussion'
'
'
'
(
(
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(
(
(
Appendix( 4.23:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 3' (M12' →' G)' and' its' Cu(I)'
adducts.'In'the'absence'of'copper'(0'Eq.),'the'
2+
major' peak' corresponds' to' [M' +' 2H] ' with'
an' m/z' of' 868.36.' With' successive' additions'
of' CuCl2' •' 2H2O' in' the' presence' of' 5' µM'
ascorbic' acid,' a' peak' corresponding' to' a'
single' Cu(I)' adduct' emerges' with' an' m/z' of'
899.34.'
'
'
'
119'
'
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Results'and'Discussion'
'
'
'
'
Appendix( 4.24:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' the' double' motif' peptide' and' its'
Cu(I)' adducts.' In' the' absence' of' copper' (0'
Eq.),' the' major' peak' corresponds' to' [M' +'
3+
3H] ' with' an' m/z' of' 1186.77.' With'
successive' additions' of' CuCl2' •' 2H2O' in' the'
presence' of' 5' µM' ascorbic' acid,' a' peak'
corresponding'to'a'first'Cu(I)'adduct'emerges'
with'an'm/z'of'1207.42'and'a'second'one'at'
m/z'of'1228.06.'
'
'
'
'
'
'
'
'
120'
'
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Results'and'Discussion'
'
4.9.3.( Peptide(Mass(Spectra(in(the(Presence(of(Ag(I)(Ions(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
Appendix( 4.25:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' the' native' peptide' and' its' Ag(I)'
adducts.' In' the' absence' of' silver' (0' Eq.),' the'
2+
major'peak'corresponds'to'[M'+'2H] 'with'an'
m/z' of' 905.25.' With' successive' additions' of'
AgNO3,'a'peak'corresponding'to'a'single'Ag(I)'
adduct'emerges'with'an'm/z'of'958.24.'
(
(
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121'
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Results'and'Discussion'
'
'
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(
(
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(
(
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(
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(
(
(
Appendix( 4.26:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 1' (H9' →' A)' and' its' Ag(I)'
adducts.'In'the'absence'of'silver'(0'Eq.),'the'
2+
major' peak' corresponds' to' [M' +' 2H] ' with'
an' m/z' of' 872.36.' With' successive' additions'
of' AgNO3,' a' peak' corresponding' to' a' first'
Ag(I)'adduct'emerges'with'an'm/z'of'925.33'
and'a'second'one'at'm/z'of'979.29.'
'
'
'
'
122'
'
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Results'and'Discussion'
'
'
'
Appendix( 4.27:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 2' (M4' →' G)' and' its' Ag(I)'
adducts.'In'the'absence'of'silver'(0'Eq.),'the'
2+
major' peak' corresponds' to' [M' +' 2H] ' with'
an' m/z' of' 868.33.' With' successive' additions'
of' AgNO3,' a' peak' corresponding' to' a' single'
Ag(I)'adduct'emerges'with'an'm/z'of'921.29.'
'
'
'
'
'
'
123'
'
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Results'and'Discussion'
'
'
'
'
(
(
(
(
(
(
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(
(
(
Appendix( 4.28:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 3' (M12' →' G)' and' its' Ag(I)'
adducts.' In' the' absence' of' silver' (0' Eq.),' the'
2+
major'peak'corresponds'to'[M'+'2H] 'with'an'
m/z' of' 868.37.' With' successive' additions' of'
AgNO3,'a'peak'corresponding'to'a'single'Ag(I)'
adduct'emerges'with'an'm/z'of'921.34.'
'
'
124'
'
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Results'and'Discussion'
'
'
'
'
Appendix( 4.29:( Positive' electrospray'
ionization'mass'spectrometry'(ESIIMS)'spectra'
of' the' double' motif' peptide' and' its' Ag(I)'
adducts.' In' the' absence' of' silver' (0' Eq.),' the'
3+
major'peak'corresponds'to'[M'+'3H] 'with'an'
m/z' of' 1186.78.' With' successive' additions' of'
AgNO3,' a' peak' corresponding' to' a' first' Ag(I)'
adduct'emerges'with'an'm/z'of'1222.07'and'a'
second'one'at'm/z'of'1257.38.'
'
'
'
'
'
'
'
125'
'
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Results'and'Discussion'
'
4.9.4.( Peptide(Mass(Spectra(in(the(Presence(of(Zn(II)(Ions(
(
Appendix( 4.30:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 2' (M4' →' G)' and' its' Zn(II)'
adducts.' In' the' absence' of' zinc' (0' Eq.),' the'
2+
major'peak'corresponds'to'[M'+'2H] 'with'an'
m/z' of' 868.36.' With' successive' additions' of'
ZnCl2,'a'peak'corresponding'to'a'single'Zn(II)'
adduct'emerges'with'an'm/z'of'899.35.'
(
(
126'
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Results'and'Discussion'
'
'
'
'
Appendix( 4.31:( Positive' electrospray'
ionization' mass' spectrometry' (ESIIMS)'
spectra' of' Pep.' 3' (M12' →' G)' and' its' Zn(II)'
adducts.' In' the' absence' of' zinc' (0' Eq.),' the'
2+
major'peak'corresponds'to'[M'+'2H] 'with'an'
m/z' of' 868.38.' With' successive' additions' of'
ZnCl2,'a'peak'corresponding'to'a'single'Zn(II)'
adduct'emerges'with'an'm/z'of'899.36.'
'
127'
'
'
Results'and'Discussion'
'
4.9.5.( Determination(of(Dissociation(Constant(Values(
(
'
Appendix(4.32:'NanoIESIIMS'titration'experiments'of'20'µM'synthetic'peptide'with'increasing'amounts'of'Cu(II)'ions.'The'
ratio'of'complex'signal'over'free'protein'signal'([complex]/[peptidefree])'is'plotted'against'the'free'ligand'concentration'
and' the' data' were' fitted' with' linear' regression.' From' the' slope' of' the' line,' the' solution' KD' value' of' the' proteinIligand'
interaction'was'determined'as'being'1/slope.'The'titration'with'the'native'peptide'is'represented'in'the'top'left,'with'Pep.'
1'(H9'→'A)'in'the'top'right,'with'Pep.'2'(M4'→'G)'in'the'bottom'left,'and'with'Pep.'3'(M12'→'G)'in'the'bottom'right,'
respectively.'
'
'
'
'
'
'
'
'
'
128'
'
Results'and'Discussion'
'
'
'
'
Appendix( 4.33:' NanoIESIIMS' titration' experiments' of' 20' µM' synthetic' peptide' with' increasing' amounts' of' Cu(I)' ions.'
Copper(I)'ions'were'obtained'by'reducing'CuCl2'•'2H2O'in'the'presence'of'5'µM'ascorbic'acid.'The'ratio'of'complex'signal'
over'free'protein'signal'([complex]/[peptidefree])'is'plotted'against'the'free'ligand'concentration'and'the'data'were'fitted'
with'linear'regression.'From'the'slope'of'the'line,'the'solution'KD'value'of'the'proteinIligand'interaction'was'determined'
as'being'1/slope.'The'titration'with'the'native'peptide'is'represented'in'the'top'left,'with'Pep.'1'(H9'→'A)'in'the'top'right,'
with'Pep.'2'(M4'→'G)'in'the'bottom'left,'and'with'Pep.'3'(M12'→'G)'in'the'bottom'right,'respectively.'
'
'
'
'
'
'
'
'
'
129'
'
Results'and'Discussion'
'
'
'
'
Appendix(4.34:'NanoIESIIMS'titration'experiments'of'20'µM'synthetic'peptide'with'increasing'amounts'of'Ag(I)'ions.'The'
ratio'of'complex'signal'over'free'protein'signal'([complex]/[peptidefree])'is'plotted'against'the'free'ligand'concentration'
and' the' data' were' fitted' with' linear' regression.' From' the' slope' of' the' line,' the' solution' KD' value' of' the' proteinIligand'
interaction'was'determined'as'being'1/slope.'The'titration'with'the'native'peptide'is'represented'in'the'top'left,'with'Pep.'
1'(H9'→'A)'in'the'top'right,'with'Pep.'2'(M4'→'G)'in'the'bottom'left,'and'with'Pep.'3'(M12'→'G)'in'the'bottom'right,'
respectively.'
'
'
130'
'
Results'and'Discussion'
'
4.9.6.( 1H9NMR(Spectra(of(Synthetic(Peptides(Upon(Successive(Cu(I)(Additions(
(
'
1
Appendix(4.35:' HINMR'spectra'of'the'native'peptide'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Cu(I).'Copper(I)'ions'were'obtained'by'reducing'CuCl2'•'2H2O'in'the'presence'of'15'molar'equivalents'
ascorbic'acid.'(A.)'Evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'the'addition'of'Cu(I)'ions.'
Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'(B.)'Evolution'of'chemical'shift'of'histidine'
protons'upon'the'addition'of'Cu(I)'ions.'Hα'corresponds'to'the'less'deshielded'proton'located'between'the'nitrogen'and'
carbon' atoms' in' the' imidazole' ring.' Hβ' corresponds' to' the' much' more' deshielded' proton' located' between' the' two'
nitrogen'atoms'in'the'imidazole'ring.'
'
'
'
131'
'
Results'and'Discussion'
'
'
'
'
'
1
Appendix(4.36:' HINMR'spectra'of'Pep.'1'(H9'→'A)'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Cu(I).'Spectra'show'the'evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'
the'addition'of'Cu(I)'ions.'Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'Copper(I)'ions'
were'obtained'by'reducing'CuCl2'•'2H2O'in'the'presence'of'15'molar'equivalents'ascorbic'acid.'
'
'
'
'
'
'
'
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'
'
'
'
'
132'
'
Results'and'Discussion'
'
'
'
'
'
1
Appendix(4.37:' HINMR'spectra'of'Pep.'2'(M4'→'G)'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Cu(I).'Copper(I)'ions'were'obtained'by'reducing'CuCl2'•'2H2O'in'the'presence'of'15'molar'equivalents'
ascorbic'acid.'(A.)'Evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'the'addition'of'Cu(I)'ions.'
Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'(B.)'Evolution'of'chemical'shift'of'histidine'
protons'upon'the'addition'of'Cu(I)'ions.'Hα'corresponds'to'the'less'deshielded'proton'located'between'the'nitrogen'and'
carbon' atoms' in' the' imidazole' ring.' Hβ' corresponds' to' the' much' more' deshielded' proton' located' between' the' two'
nitrogen'atoms'in'the'imidazole'ring.'
'
'
'
133'
'
Results'and'Discussion'
'
'
'
'
'
1
Appendix(4.38:' HINMR'spectra'of'Pep.'3'(M12'→'G)'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Cu(I).'Copper(I)'ions'were'obtained'by'reducing'CuCl2'•'2H2O'in'the'presence'of'15'molar'equivalents'
ascorbic'acid.'(A.)'Evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'the'addition'of'Cu(I)'ions.'
Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'(B.)'Evolution'of'chemical'shift'of'histidine'
protons'upon'the'addition'of'Cu(I)'ions.'Hα'corresponds'to'the'less'deshielded'proton'located'between'the'nitrogen'and'
carbon' atoms' in' the' imidazole' ring.' Hβ' corresponds' to' the' much' more' deshielded' proton' located' between' the' two'
nitrogen'atoms'in'the'imidazole'ring.'
'
'
'
134'
'
Results'and'Discussion'
'
4.9.7.( 1H9NMR(Spectra(of(Synthetic(Peptides(Upon(Successive(Ag(I)(Additions(
(
'
1
Appendix(4.39:' HINMR'spectra'of'the'native'peptide'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Ag(I).'(A.)'Evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'the'addition'
of'Ag(I)'ions.'Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'(B.)'Evolution'of'chemical'shift'
of'histidine'protons'upon'the'addition'of'Ag(I)'ions.'Hα'corresponds'to'the'less'deshielded'proton'located'between'the'
nitrogen'and'carbon'atoms'in'the'imidazole'ring.'Hβ'corresponds'to'the'much'more'deshielded'proton'located'between'
the'two'nitrogen'atoms'in'the'imidazole'ring.'
'
'
'
'
'
'
'
'
135'
'
Results'and'Discussion'
'
'
'
'
'
1
Appendix(4.40:' HINMR'spectra'of'Pep.'1'(H9'→'A)'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Ag(I).'Spectra'show'the'evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'
the'addition'of'Ag(I)'ions.'Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'
'
'
'
'
'
'
'
'
'
'
'
'
'
136'
'
Results'and'Discussion'
'
'
'
'
'
1
Appendix(4.41:' HINMR'spectra'of'Pep.'2'(M4'→'G)'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Ag(I).'(A.)'Evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'the'addition'
of'Ag(I)'ions.'Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'(B.)'Evolution'of'chemical'shift'
of'histidine'protons'upon'the'addition'of'Ag(I)'ions.'Hα'corresponds'to'the'less'deshielded'proton'located'between'the'
nitrogen'and'carbon'atoms'in'the'imidazole'ring.'Hβ'corresponds'to'the'much'more'deshielded'proton'located'between'
the'two'nitrogen'atoms'in'the'imidazole'ring.'
'
'
'
'
'
137'
'
Results'and'Discussion'
'
'
'
'
'
1
Appendix(4.42:' HINMR'spectra'of'Pep.'3'(M12'→'G)'collected'at'500'MHz'in'D2O'in'the'absence'and'presence'of'various'
molar'equivalents'of'Ag(I).'(A.)'Evolution'of'chemical'shift'of'methyl'protons'from'methionine'residues'upon'the'addition'
of'Ag(I)'ions.'Peaks'of'interest'are'circled'in'black'on'the'spectrum'in'the'absence'of'metal.'(B.)'Evolution'of'chemical'shift'
of'histidine'protons'upon'the'addition'of'Ag(I)'ions.'Hα'corresponds'to'the'less'deshielded'proton'located'between'the'
nitrogen'and'carbon'atoms'in'the'imidazole'ring.'Hβ'corresponds'to'the'much'more'deshielded'proton'located'between'
the'two'nitrogen'atoms'in'the'imidazole'ring.'
'
'
138'
'
!
!
Results'and'Discussion'
'
Chapter(5:( Towards(a(New(Copper(Resistance(Mechanism(in(Cupriavidus*
metallidurans(CH34,(Time(to(Vesiculate...(
(
5.1.(Abstract*
Copper'is'an'essential'trace'element'that'provides'a'challenge'to'biological'systems'due'to'its'
ability' to' cycle' between' a' stable' oxidized' state,' Cu(II),' and' an' unstable' reduced' state,' Cu(I),' which'
can' lead' to' oxidative' stress.' Multidisciplinary' studies' of' Cupriavidus* metallidurans' CH34' and' its'
mutant'derivatives'exposed'to'copper'stress'have'shown'an'indisputable'impact'on'its'morphology'
and' ultrastructural' organization.' More' importantly,' a' vesiculation' phenomenon' never' previously'
described' was' highlighted' upon' copper' exposure.' These' vesicles' appear' to' be' specific' to' the'
presence'of'copper'in'the'culture'medium'and'were'not'observed'with'redoxHinert'metal'ions.'It'also'
appears' that' this' phenomenon' is' associated' with' the' presence' of' genetic' elements' carried' by'
plasmids' pMOL28' and' pMOL30,' particularly' the' cop' genes' harbored' by' the' pMOL30' plasmid.'
Proteomic' analysis' showed' that' outer' membrane' vesicles' (OMVs)' are' significantly' enriched' in'
periplasmic,' outer' membrane' and' extracellular' proteins.' Numerous' periplasmic' and' outer'
membrane'Cop'proteins'involved'in'copper'resistance'in'C.*metallidurans'CH34'were'also'identified'
in' the' OMVs' such' as' CopB1,' A1,' C1,' H,' I,' K' and' G.' Moreover,' the' OMVs' contained' a' higher'
concentration' of' copper' [2.98' ±' 0.46' ng/µg' protein]' than' that' found' in' the' cell' membrane.' This'
vesiculation' phenomenon' appears' to' serve' as' a' new' copper' resistance' mechanism' in' C.*
metallidurans' CH34' that' involves' the' secretion' of' toxic' copper' compounds' complexed' to'
overexpressed' proteins' and/or' damaged' lipids' under' copper' stress.' These' findings' pave' the' way'
towards'a'better'understanding'of'the'fate'of'copper'in'C.*metallidurans'CH34.'
(
(
139'
'
Results'and'Discussion'
'
5.2.(Introduction(
Metal'ions'play'important'roles'in'microbial'nutrition.'Some'metals,'such'as'Cu,'Ni,'Co,'Zn'and'
Mn,' are' essential' micronutrients' for' cells' but' are' required' only' in' trace' amounts' due' to' their'
potential'toxicity,'which'can'cause'cellular'stress'(Hobman'et'al.,'2007;'Ivanov'et'al.,'1996).'At'high'
levels,' both' essential' and' nonHessential' metal' ions' become' toxic' to' organisms' and' can' induce'
biochemical'damage'and'deficiencies'in'bacterial'cells.'The'cell'membrane,'corresponding'to'the'first'
surface' by' which' bacteria' contact' their' surrounding' environment,' is' the' main' target' (MashburnH
Warren' et' al.,' 2008)' and' especially' the' outer' membrane' because' it' acts' as' a' permeability' barrier'
preventing'the'entry'of'toxic'molecules'(Silhavy'et'al.,'2010).'An'excessive'concentration'of'essential'
metal' ions' can' also' alter' enzyme' specificity,' disrupt' cellular' functions' and' damage' the' structure' of'
DNA'(Markowicz'et'al.,'2010;'Monsieurs'et'al.,'2011;'Rathnayake'et'al.,'2010).'Copper'is'an'abundant'
environmental'element'fulfilling'many'common'functions'within'bacteria,'particularly'taking'part'in'
redox'reactions,'but'it'is'also'known'to'exert'toxic'effects'on'cell'membranes'(Cuillel,'2009).'This'dual'
nature' of' copper,' essential' but' also' extremely' dangerous,' compels' all' living' organisms' to' develop'
mechanisms'to'tightly'control'its'homeostasis'(Cuillel,'2009).'
The' βHproteobacterium* Cupriavidus* metallidurans' strain' CH34' is' a' good' representative' for'
various'related'strains'found'in'natural'and'anthropogenic'biotopes'contaminated'with'moderate'to'
high' concentrations' of' many' metals' (Monsieurs' et' al.,' 2011).' This' soil' bacterium' is' an' interesting'
model'organism'for'studying'microbial'responses'to'several'metals'such'as'Cd2+,'Co2+,'Cu2+,'Ni2+,'Zn2+,'
and' Pb2+' (Monsieurs' et' al.,' 2011).' The' resistance' and' management' of' these' metal' ions' are' made'
possible' by' at' least' twentyHfour' gene' clusters' distributed' over' the' bacterium's' four' replicons:' the'
chromosome,' the' chromid' (Van' Houdt' and' Mergeay,' 2012)' and' two' megaplasmids' (pMOL28' and'
pMOL30)' (Janssen' et' al.,' 2010;' Mergeay' et' al.,' 2003).' Copper' resistance' in' C.* metallidurans' CH34'
essentially'involves'the'cop'cluster'present'on'the'bacterial'plasmid'pMOL30' (Monchy'et'al.,'2007;'
Monchy' et' al.,' 2006;' Monsieurs' et' al.,' 2011).' The' large' cop' cluster' consisting' of' twentyHone' genes'
(copVTMKNSRABCDIJGFOLQHEW)'is'highly'upregulated'by'Cu(II)'but'also'by'a'wide'range'of'metals,'
such'as'Ni(II),'Zn(II)'and'Cd(II)'(Monchy'et'al.,'2007;'Monsieurs'et'al.,'2011).'Most'of'these'cop'genes'
appear' to' be' specific' to' the' Cupriavidus' genus' except' for' 1°)' the' copSRABCD' genes' involved' in'
periplasmic'detoxification'and'for'which'high'identity'paralogs'are'present'on'the'chromid,'and'2°)'
the' copF' gene' encoding' a' PHtype' copperHefflux' ATPase' essential' for' cytoplasmic' detoxification'
(Monchy'et'al.,'2007;'Monchy'et'al.,'2006).'
Among' the' unique' cop' genes' harbored' by' the' cop' cluster' is' the' copK' gene' encoding' a'
periplasmic' protein' that' cooperatively' binds' Cu(I)' and' Cu(II)' (Chong' et' al.,' 2009).' This' protein' is'
140'
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Results'and'Discussion'
'
known'to'strongly'chelate'periplasmic'copper'ions'(Chong'et'al.,'2009),'and'it'can'be'considered'as'
an' additional' copper' detoxification' system' in' the' presence' of' high' copper' levels.' By' contrast,' the'
functions'of'the'other'cop'genes'appear'to'be'less'understood'or'completely'unknown.'Four'genes'
involved'in'membraneHrelated'functions'and'which'are'upregulated'by'Cu(II)'ions'are'also'found'in'
the' immediate' vicinity' of' the' cop' cluster:' ompP2' (Rmet_6127),* gtrB2' (Rmet_6128),' gtrA2'
(Rmet_6129),' and' gtrM2' (Rmet_6130)' (Monchy' et' al.,' 2007).' The' region' of' interest' encoding' for'
copper' resistance/response' is' completed' by' the' ubiE' gene' (Rmet_6131),' which' is' upregulated' by'
Cu(II)' ions' and' encodes' a' putative' methyl' transferase;' and' the* silDCBA' cluster' (Rmet_6133H6136),'
which' is' mainly' induced' by' Ag(I)' but' also' by' Cu(II)' ions' and' encodes' a' RNDHdriven' efflux' system'
(Monchy' et' al.,' 2007).' All' together,' these' gene' products' are' involved' in' active' efflux' mechanisms'
allowing'both'cytoplasmic'and'periplasmic'copper'detoxification'(Monchy'et'al.,'2006).'
Often,' effluxHbased' mechanisms' are' linked' to' physiological' changes' that' alter' the'
microenvironment' of' the' cell' creating' favorable' conditions' for' metal' ion' precipitation' as' metal'
sulfides,'carbonates,'hydroxides'or'phosphates'(Diels'et'al.,'2009;'Mahvi'and'Diels,'2004;'Mergeay,'
1997).'However,'this'does'not'look'to'be'the'case'for'copper'in'C.*metallidurans'CH34.'In'addition'to'
the' efflux' system,' bacteria' can' use' metallochaperones' to' sequester' intracellular' copper' (Mergeay,'
1997).' Copper' accumulation' within' the' cell's' periplasm' occurs' in' particular' in' E.* coli' harboring' the'
PcoHplasmid' pRJ1004' and' P.* syringae' carrying' the' plasmid' pT23D' with' the' copABCDRS' system.' In'
fact,' E.* coli' strains' form' brown' colonies' in' the' presence' of' copper' salts,' whereas' P.* syringae' turn'
bright'blue'upon'copper'exposure'(Magnani'and'Solioz,'2007).'Once'again,'such'intracellular'copper'
sequestration' has' never' been' reported' in' C.* metallidurans' CH34.' Consequently,' although' copper'
resistance' mechanisms' are' fairly' well' studied' at' the' molecular' level' in' C.* metallidurans' CH34,'
numerous'questions'are'still'unanswered,'in'particular'the'fate'of'copper.'
This'study'sheds'some'light'on'the'fate'of'copper'in'C.*metallidurans'CH34'with'evidence'of'a'
previously' undescribed' specific' vesiculation' phenomenon' under' copper' challenge.' The' outer'
membrane'vesicles'(OMVs)'were'highlighted'further'via'highHresolution'investigations'on'the'surface'
and'morphological'alterations'induced'in'C.*metallidurans'CH34'after'copper'exposure.'The'presence'
of'periplasmic'Cop'proteins'associated'with'these'OMVs,'the'detection'of'high'copper'content'[2.98'
(±'0.46)'ng/µg'protein]'within'these'vesicles,'as'well'as'the'connection'between'OMV'production'and'
the' presence' of' genetic' elements' carried' by' plasmids' all' suggest' that' outer' membrane' vesicles'
constitute'an'active'mechanism'allowing'periplasmic'copper'detoxification.'
(
(
(
141'
'
Results'and'Discussion'
'
5.3.(Results(
5.3.1.(Morphostructural(Alterations(of(C.*metallidurans(CH34(Induced(by(Copper(II)(Ions(
C.* metallidurans' CH34' treated' with' different' concentrations' of' copper(II)' underwent'
morphological' alterations' in' comparison' to' control' cells' when' observed' by' a' scanning' electron'
microscope'as'shown'in'Figure'5.1A'and'B.'Control'C.*metallidurans'CH34'cells'appeared'intact'and'
had'a'typical'rodHshaped'morphology'with'smooth'and'regular'membrane'surfaces'(Fig.'5.1A).'They'
were'approximately'1'µm'long'and'0.4'µm'wide,'the'size'and'shape'being'in'accordance'with'the'
dimensions'of'C.*metallidurans'CH34'cited'in'the'literature'(Goris'et'al.,'2001;'Guine'et'al.,'2006;'
Taghavi'et'al.,'1997;'Vaneechoutte'et'al.,'2004).'
However,'upon'exposure'to'copper(II)'ions,'the'treated'cells'showed'membrane'invaginations'
and'irregularities'on'their'surface'(Fig.'5.1B).'Detailed'surface'features'of'untreated'and'treated'C.*
metallidurans' CH34' bacteria' were' visualized' at' the' nanometerHscale' resolution' by' atomic' force'
microscopy.'The'AFM'images'showed'evidence'of'alterations'in'morphology'due'to'copper'stress'
and' corroborated' the' SEM' observations.' The' average' thickness' of' untreated' bacteria' on' a' mica'
surface' was' determined' to' be' 275' ±' 10' nm' (Fig.' 5.1C).' The' analysis' of' membrane' topography'
revealed'the'presence'of'grooves'perpendicular'to'the'long'axes'of'the'bacteria,'as'particularly'well'
observed' in' the' DMT' modulus' images,' which' maps' nanomechanical' properties' at' the' nanoscale'
(Fig.' 5.1D)' (Heu' et' al.;' Pittenger' et' al.).' These' furrows' in' the' membrane' presented' a' certain'
periodicity,'with'an'average'distance'between'the'furrows'of'75'(±'20)'nm.'These'grooves'were'not'
observed' on' the' surface' of' bacterial' cells' treated' with' copper' (Fig.' 5.1E' and' F).' However,'
membrane' alterations' appeared' upon' Cu(II)' challenge.' Small' bulges' were' identified' at' numerous'
places'on'the'external'membrane'as'shown'in'the'3D'topography'(Fig.'5.1E,'arrows),'and'residues'
were'also'recorded'around'some'of'the'bacteria'(Fig.'5.1F).'
Further'evidence'of'toxic'effects'induced'by'copper'on'the'C.*metallidurans'CH34'membrane'
was'obtained'by'transmission'electron'microscopy.'Untreated'C.*metallidurans'CH34'were'used'as'
control'samples'to'ensure'that'the'observed'characteristics'of'the'copperHtreated'cells'were'due'to'
the'copper'exposure'and'not'to'the'preparation'method'itself.'TEM'photomicrographs'of'untreated'
C.*metallidurans'CH34'showed'a'relatively'uniform'wavy'bacterial'wall'consisting'of'clearly'defined'
outer' and' inner' membranes' (Fig.' 5.2A' and' B).' As' observed' in' Figure' 5.2A,' large' bright' inclusion'
bodies'were'also'present'in'most'bacteria'most'likely'corresponding'to'polyhydroxybutyrate'(PHB)'
granules' (Janssen' et' al.,' 2010).' Two' distinct' areas' were' visualized' in' the' ultrathin' sections' of'
untreated'cells:'a'darker'peripheral'zone'most'likely'corresponding'to'a'ribosomeHrich'area'and'a''
142'
'
Results'and'Discussion'
'
(
Figure( 5.1:' Morphostructural' alterations' of' C.* metallidurans' CH34' induced' by' copper(II)' ions.' SEM' micrographs' of' C.*
metallidurans' CH34' on' cellulose' membrane' filters' (0.22Hµm' pore' size):' untreated' bacteria' (A.)' and' 0.2' mM' copperH
treated'bacteria'(B.).'AFM'images'of'C.*metallidurans'CH34'deposited'on'a'freshlyHcleaved'mica'surface'acquired'in'air'
at'room'temperature'using'the'peak'force'tapping'mode:'topographic'image'of'untreated'bacteria'(C.),'DMT'modulus'
picture'of'untreated'bacteria'(D.).'3D'Topography'of'a'single'cell'treated'with'0.2'mM'Cu(II);'the'arrows'indicate'small'
bulges'on'the'external'membrane'(E.).'DMT'modulus'picture'of'bacteria'treated'with'0.2'mM'Cu(II)'(F.).'
143'
'
Results'and'Discussion'
'
lighter' central' region' related' to' the' nucleoid' containing' genetic' material' (Fig.' 5.2A' and' B).'
Compared' with' the' control' samples,' some' morphostructural' alterations' appeared' upon' copper'
exposure.' A' bulging' out' of' the' cell' wall' was' observed' in' almost' all' bacteria' at' different' localized'
places,' and' various' stages' of' bulging' were' recorded' (Fig.' 5.2C).' It' is' important' to' note' that' each'
outer'membrane'vesicle'consisted'of'a'portion'of'the'bacterium's'periplasmic'space,'surrounded'by'
a'single'membrane'bilayer'(Fig.'5.2C'and'D,'inserts).'In'addition'to'these'vesicles,'numerous'vesicle'
strings' were' recorded' in' many' copperHtreated' bacteria' (Fig.' 5.2D).' Intracytoplasmic' changes'
attributable'to'the'copper'treatment'included'a'more'compact'electronHlucent'central'region,'the'
nucleoid' concentrated' in' the' center' of' bacterium' and' filled' with' filaments' of' chromatin,' and' the'
peripheral' space' appearing' to' be' a' thicker' and' more' electronHdense' area' (Fig.' 5.2C' and' D).'
Moreover,' different' stages' of' cell' death' were' also' recorded' upon' copper' challenge' as' some' cell'
walls' appeared' distorted' (e.g.,' alteration' and' enlargement' of' the' periplasmic' space' at' the' apical'
ends),'damaged'or'broken'(data'not'shown).'
'
Figure(5.2:'TEM'photomicrographs'of'C.*metallidurans'CH34.'Longitudinal'ultrathin'section'of'an'untreated'bacterium'
(A.),'transverse'ultrathin'section'of'an'untreated'bacterium'(B.).'In'(A.)'and'(B.),'arrows'indicate'the'outer'membrane'
(OM)' and' inner' membrane' (IM)' as' well' as' polyhydroxybutyrate' granules' (PHB).' Longitudinal' ultrathin' sections' of' C.*
metallidurans' CH34' treated' with' 0.2' mM' Cu(II)' (C.' and' D.).' Zoom' of' the' bacterial' membrane' (C.,' insert).' Transverse'
ultrathin'section'of'C.*metallidurans'CH34'treated'with'0.2'mM'Cu(II)'(D.,'insert).'
(
5.3.2.(External(Vesicle(Formation(Appears(to(Be(Specific(to(Copper(Exposure(
The' specificity' of' the' vesiculation' phenomenon' was' investigated' upon' exposure' to' different'
divalent' metal' ions' (i.e.,' Cu,' Ni' and' Zn)' by' TEM' study' (Table' 5.1,' A.).' A' noticeable' difference' was'
visualized' between' bacteria' treated' with' copper,' nickel' or' zinc.' Although' numerous' outer'
membrane' vesicles' were' observed' among' copperHtreated' bacteria' (Fig.' S5.1A),' no' vesicles' were'
recorded'upon'nickel'or'zinc'exposure'(Fig.'S5.1B'and'C).'The'TEM'photomicrographs'showed'that'
144'
'
Results'and'Discussion'
'
nickelH'and'zincHtreated'bacteria'had'a'cell'wall'that'was'perfectly'unaffected'by'metal'treatment'in'
comparison'to'copperHtreated'cells;'neither'membrane'fragments'nor'cell'damage'were'visualized,'
and'cellular'integrity'was'apparently'preserved'(Fig.'S5.1B'and'C).'The'response'of'C.*metallidurans'
CH34'to'shortHterm'oxidative'stress'from'hydrogen'peroxide'was'also'investigated'(Table'5.1,'A.).'
Indeed,' some' bacteria' such' as' Pseudomonas* aeruginosa' are' known' to' produce' OMVs' under'
oxidative'stress'(Macdonald'and'Kuehn,'2013).'However,'in'our'experimental'conditions,'this'stress'
treatment'did'not'induce'OMV'production'in'C.*metallidurans'CH34'(Fig.'S5.2).'
Some' clues' relating' to' the' OMV' biogenesis' were' highlighted' by' studying' derivatives' of' C.*
metallidurans' CH34' that' differ' by' their' plasmid' content' and' treating' them' with' 0.2' mM' Cu(II)'
(Table'5.1,'A.).'TEM'observations'enabled'an'apparent'connection'between'the'presence'of'metal'
resistance' genes' harbored' by' some' mobile' genetic' elements' and' the' vesiculation' process' to' be'
established.'Untreated'derivatives'showed'morphostructural'features'and'a'size'distribution'similar'
to' those' observed' for' the' untreated' wildHtype' strain' (Fig.' S5.3A,' C,' E' and' G,' and' Fig.' S5.4A).'
However,' upon' copper' treatment,' ultrastructural' changes' were' visualized' for' each' mutant.'
Similarly' to' copperHtreated' wildHtype' C.* metallidurans,' each' mutant' showed' a' more' compact'
electronHlucent' central' region' filled' with' filaments' of' chromatin' and' surrounded' by' a' darker'
external'zone'(Fig.'S5.3B,'D,'F'and'H,'and'Fig.'S5.4B).'It'should'also'be'noted'that'for'each'copperH
treated'sample,'a'few'bacterial'cells'were'identified'with'a'cell'wall'that'was'distorted,'damaged'or'
destroyed'resulting'in'their'death'(data'not'shown).'Surprisingly,'it'was'recorded'that'only'strains'
carrying'metal'resistance'genes'on'their'plasmid'─'AE126'(pMOL28),'AE128'(pMOL30)'and'AE1744'
(pMOL1024)' ─' showed' OMVs' under' copper' stress' exposure' (Fig.' S5.3D,' F' and' H).' In' contrast,' no'
vesicles' were' observed' for' the' plasmidHfree' derivative' AE104' (Fig.' S5.3B)' or' the' strain' AE2214'
carrying'the'vector'pLAFR3'(Fig.'S5.4B).'This'observation'underlies'the'possible'implication'of'metal'
resistance'genes'harbored'by'mobile'genetic'elements'in'the'vesiculation'phenomenon.'
'
'
145'
'
Results'and'Discussion'
'
Table(5.1:'OMV'production'appears'specific'to'copper'stress.(
A.'OMV'phenotype'of'C.*metallidurans'strain'CH34'and'its'mutant'derivatives'observed'by'TEM'upon'a'stress'
treatment.'The'symbol'"+"'corresponds'to'the'presence'of'OMVs,'"++"'means'that'the'OMVs'were'abundant,'
while'"—"'corresponds'to'the'absence'of'OMVs.'
Strains'
WildHtype'
WildHtype'
WildHtype'
WildHtype'
WildHtype'
WildHtype'
AE104'(no'plasmid)'
AE104'(no'plasmid)'
AE126'(pMOL28)'
AE126'(pMOL28)'
AE128'(pMOL30)'
AE128'(pMOL30)'
AE1744'(pMOL1024)'
AE1744'(pMOL1024)'
AE2214'(pLAFR3)'
AE2214'(pLAFR3)'
Stress'Treatments'
Gluconate'
0.2'mM'Cu(II)'
0.8'mM'Cu(II)'
1'mM'Zn(II)*'
0.42'mM'Ni(II)*'
6'mM'H2O2*'
Gluconate*'
0.2'mM'Cu(II)*'
Gluconate*'
0.2'mM'Cu(II)*'
Gluconate*'
0.2'mM'Cu(II)*'
Gluconate*'
0.2'mM'Cu(II)*'
Gluconate*'
0.2'mM'Cu(II)*'
Phenotype'(OMVs)'
—'
+'+'
+'+''
—'
—'
—'
—'
—'
—'
+'
—'
+'
—'
+'
—'
—'
H1
All'strains'were'grown'at'30°C'under'aerobic'conditions'in'MOPSHsalt'minimal'medium'with'2'g'L 'gluconate'as'
a'unique'carbon'source.'Minimal'medium'was'supplemented'with'low'or'high'copper'concentrations'based'on'
the'copper'minimal'inhibitory'concentration:'0.2'mM'or'0.8'mM'Cu(NO3)2'•'3H2O,'respectively.'In'addition,'two'
other' metals' were' investigated' as' controls' at' low' concentrations' based' on' their' minimal' inhibitory'
concentrations:'0.42'mM'NiCl6'•'6H2O'and'1'mM'ZnCl2.'Another'type'of'oxidative'stress'was'also'investigated'
by'adding'6'mM'hydrogen'peroxide'(H2O2)'to'the'growth'medium.'This'stress'treatment'was'applied'for'2'hours'
when'the'optical'density'at'600'nm'(OD600)'was'~'0.4.'The'mutant'derivatives'were'only'treated'with'0.2'mM'
Cu(II)'because'numerous'vesicles'were'observed'for'the'wildHtype'strain'at'this'concentration.'All'bacterial'cells'
were' harvested' by' centrifugation' (6,000' rpm,' 10' min,' at' 4' °C)' during' midHexponential' phase' (OD600' of' ~' 0.6).'
Bacteria'were'prepared'as'described'in'the'Materials'and'Methods,'and'the'presence'of'OMV'was'observed'by'
TEM.'
*TEM'photomicrographs'are'shown'in'supplemental'data.'
(
B.'Copper'content'(in'ng/µg'protein)'determined'by'ICPHMS'in'the'purified'OMVs'and'C.*metallidurans'CH34'
cells.'
Sample'
OMVs'
C.*metallidurans'CH34'
Gluconate'
—'
b'
<'0.008'(±'0.001)
0.8'mM'Cu(II)'
a'
2.98'(±'0.46)
b'
0.234'(±'0.007)
Pure'OMVs'were'prepared'as'described'in'the'Materials'and'Methods.'Bacteria'were'grown'in'the'absence'and'
presence'of'0.8'mM'Cu(II),'they'were'harvested'by'centrifugation'during'the'midHexponential'phase'(OD600nm'of'
~0.6).' The' OMV' and' bacteria' samples' were' rinsed' with' ultrapure' water' and' mineralized' using' HNO3.'
Afterwards,'ultrapure'water'was'added'to'the'solution'and'the'titration'was'carried'out'by'ICPHMS.'ICPHMS'gave'
TM
[copper]' in' µg/L.' Protein' concentration' was' determined' using' a' Micro' BCA ' Protein' Assay' Kit' (Thermo'
Scientific).'The'final'copper'concentration'was'expressed'as'ng'metal'per'µg'protein.'
In'parentheses'is'the'standard'error'of'the'mean'(SEM).'
a
From'n=3'biological'replicates.'
b
From'n=3'technical'replicates'
146'
'
Results'and'Discussion'
'
5.3.3.(Purification(and(Characterization(of(Outer(Membrane(Vesicles(
Preparation'of'pure'OMVs'from'bacterial'supernatant'is'important'when'studying'the'protein'
content' of' OMVs.' Consequently,' avoiding' cell' lysis' is' crucial' for' producing' highHquality' OMV'
fractions.' The' purity' of' OMV' preparations' was' evaluated' by' transmission' electron' microscopy.'
Figure' 5.3A' shows' TEM' photomicrograph' of' OMVs' purified' from' C.* metallidurans' grown' in' the'
presence'of'0.8'mM'Cu(II).'Examination'of'the'purified'OMVs'revealed'the'presence'of'both'large'
and'small'closed'vesicles'as'well'as'numerous'strings'of'vesicles,'as'previously'described'in'copperH
treated' bacteria.' Observed' as' being' spherical' to' eggHshaped' and' relatively' electronHlucent,' the'
OMVs' varied' widely' in' size' with' an' estimated' diameter' ranging' from' 20H180' nm.' No' cell' debris'
contamination'was'observed'by'TEM,'suggesting'that'our'OMV'preparations'were'very'pure.'
A' profile' of' vesicular' protein' extract' resolved' on' SDSHPAGE' is' shown' in' Figure' 5.3B.' This'
approach'highlighted'some'proteins'appearing'as'thick'bands'in'the'gel'(Fig.'5.3B,'arrows).'Analysis'
of' those' bands' of' interest' by' mass' spectrometry' (nano' LCHMS/MS)' enabled' us' to' associate'
identified'proteins'with'their'apparent'molecular'mass'as'assessed'by'SDSHPAGE.'As'a'result,'it'was'
demonstrated'that'the'most'intense'band,'approximately'45'kDa,'mostly'corresponds'to'the'FliC2'
flagellar'filament'structural'protein'(flagellin)'(Rmet_5252)'of'C.*metallidurans,'which'may'play'an'
important'role'in'the'function'of'the'OMVs'as'reported'in'Escherichia*coli'W3110'by'Manabe'et'al.,'
2013'(Manabe'et'al.,'2013).'
'
'
Figure(5.3:'Purification'and'characterization'of'outer'membrane'vesicles.'(A.)'TEM'photomicrograph'of'purified'outer'
membrane'vesicles.'OMVs'were'isolated'and'purified'from'the'supernatant'of'C.*metallidurans'CH34'cultures'treated'
with'0.8'mM'Cu(II)'as'described'in'the'Materials'and'Methods.'(B.)'SDSHpolyacrylamide'gel'(4H20%)'showing'the'protein'
profile'of'pure'OMVs'isolated'from'a'culture'of'C.*metallidurans'CH34'treated'with'0.8'mM'Cu(II).'(C.)'Venn'diagram'of'
proteins'overlapping'between'the'three'independent'assays'and'the'number'of'proteins'identified'in'each'experiment.'
For'each'trial,'only'proteins'with'two'or'more'identified'peptides'were'taken'into'consideration.'
'
147'
'
Results'and'Discussion'
'
This'result'was'strengthened'by'a'proteomic'analysis'of'the'total'protein'content'of'OMVs'that'
was'performed'on'three'biological'replicates.'OMV'proteins'were'extracted'through'6'M'guanidine'
chloride,'digested'with'trypsin'and'analyzed'by'nano'LCHMS/MS.'By'setting'a'cutHoff'of'two'or'more'
identified'peptides,'295'unique'vesicular'proteins'were'identified'with'high'confidence'(95%)'from'
the'three'independent'experiments'(Table'S5.1);'210,'183'and'201'proteins'were'identified'in'the'
first,' second' and' third' assays,' respectively.' Notably,' the' Venn' diagram' in' Figure' 5.3C' shows' that'
130'proteins'overlap'between'the'three'independent'trials.'This'high'rate'of'protein'identification'
in' separate' experiments' suggests' good' protein' identification' reproducibility' and' emphasizes' the'
reliability'of'the'results.'
The' identified' OMV' proteins' were' analyzed' based' on' their' subcellular' localization' and' the'
resulting'prediction'is'shown'in'Figure'5.4.'This'reveals'that'the'vesicular'proteins'identified'in'this'
study'derive'from'the'extracellular'space'(15'proteins,'5%),'the'outer'membrane'(24'proteins,'8%),'
the' periplasmic' space' (56' proteins,' 19%),' the' inner' membrane' (11' proteins,' 3.7%),' and' the'
cytoplasmic' space' (101' proteins,' 34.3%).' It' should' also' be' mentioned' that' almost' half' of' the'
cytoplasmic' proteins' (48.5%)' identified' were' ribosomal' proteins.' Moreover,' it' may' be' observed'
that'no'localization'was'attributed'to'88'proteins'(30%).'However,'a'predicted'signal'peptide'was'
assigned'to'58'of'them,'which'means'that'those'proteins'may'potentially'be'translocated'across'the'
inner'membrane.'Compared'with'the'complete'theoretical'proteome'of'C.*metallidurans'CH34,'it'is'
obvious'that'extracellular,'outer'membrane'and'periplasmic'proteins'are'highly'enriched'in'OMVs'
produced'in'the'presence'of'0.8'mM'Cu(II).'By'contrast,'it'appears'that'inner'membrane'proteins'
are' excluded' from' OMVs' because' only' 11' inner' membrane' proteins' were' identified' out' of' a'
theoretical' total' of' 1371' in' C.* metallidurans' CH34.' Consequently,' this' global' proteomic' profile' of'
native'outer'membrane'vesicles'derived'from'C.*metallidurans'CH34'provides'a'true'and'fair'view'
of'the'periplasmic'space'and'the'outer'membrane.'
Figure( 5.4:' Subcellular' localization' of'
the' identified' OMV' proteins.(
Comparison' of' the' identified' OMV'
proteome' with' the' theoretical'
proteome' of' C.* metallidurans' CH34'
based' on' subcellular' localization'
predicted' using' the' PSORTb' algorithm'
(v.3.0.2'package).'
'
'
148'
'
Results'and'Discussion'
'
The'identified'OMV'proteins'were'also'classified'based'on'their'functions'predicted'using'the'
C.* metallidurans' CH34' COG' protein' database' available' on' GenoScope's' MaGe' system.' A' total' of'
15.6%'of'the'identified'proteins'are'poorly'characterized'and'13.6%'of'them'are'not'classified'in'a'
COG'group.'The'major'identified'proteins'are'involved'in'metabolism'(25.1%),'information'storage'
and'processing'(23%),'and'cell'processes'and'signaling'(22.7%).'Each'COG'group'is'subdivided'into'
functional' categories' designated' by' oneHletter' abbreviations.' The' distribution' of' the' functional'
categories'associated'with'the'identified'OMV'proteins'is'shown'in'Figure'S5.5.'Compared'with'the'
theoretical' proteome' of' C.* metallidurans' CH34,' it' may' be' observed' that' many' vesicular' proteins'
identified'in'this'study' are'likely'to'be'involved'in'translation,'ribosomal'structure'and'biogenesis'
([J],' 23.5%);' in' cell' wall/membrane/envelope' biogenesis' ([M],' 11.8%);' in' posttranslational'
modification,' protein' turnover' and' chaperones' ([O],' 7.1%);' and' in' cell' motility' ([N],' 5.1%).' In'
contrast,'it'is'clear'that'some'functions'are'less'represented'in'this'study'in'comparison'to'the'C.*
metallidurans' CH34' proteome.' These' latter' are' especially' involved' in' replication,' recombination'
and' repair' ([L],' 0.8%);' transcription' ([K],' 2.4%);' signal' transduction' mechanisms' ([T],' 0.8%);' lipid'
transport'and'metabolism'([I],'1.6%);'and'coenzyme'transport'and'metabolism'([H],'0.4%).'
Unsurprisingly,' the' FliC2' flagellar' filament' structural' protein' (flagellin)' of' C.* metallidurans'
CH34'was'the'most'frequently'identified'protein'with'341'peptides'(82%'sequence'coverage'at'95%'
confidence).'In'addition'to'flagellar'hook'proteins'(e.g.,'FlgE,'FliD3,'FlgK,'FlgL,'etc.),'the'OMV'protein'
profile' contains' outer' membrane' proteins' (e.g.,' OmpA,' OmpW,' OmpP2,' etc.),' ABCHtransporters,'
lipoproteins'and'transcription'factors.'A'number'of'ribosomal'proteins'(e.g.,'RpsA,'RpsB,'RplA,'RplB,'
etc.),' chaperones,' murein' hydrolases' (e.g.,' MltA)' and' elongation' factors' were' also' identified.' The'
substrateHbinding' chaperonin' GroEL' was' the' second' most' frequently' identified' protein' with' 64'
peptides'giving'78.4%'sequence'coverage'at'95%'confidence.'Interestingly,'numerous'Cop'proteins'
involved' in' copper' resistance' in' C.* metallidurans' CH34' were' also' present' in' the' OMV' proteome'
such'as'CopB1'(Rmet_6113),'CopA1'(Rmet_6112),'CopC1'(Rmet_6114),'CopH,'CopI,'CopK'and'CopG'
(Table' 5.2).' It' should' be' noted' that' the' CopB1' protein' was' the' third' most' frequently' identified'
protein'with'43'peptides'giving'71%'sequence'coverage'at'95%'confidence.'
'
'
'
'
'
149'
'
Results'and'Discussion'
'
Table(5.2:'List'of'the'Cop'proteins'identified'from'the'proteomic'analysis'of'OMVs.'The'"%Cov(95)"'corresponds'
to'the'sequence'coverage'at'95%'confidence.'The'number'of'peptides'was'identified'at'95%'confidence.'The'
emPAI'is'the'exponentially'modified'protein'abundance'index.'
Name'
CopB1'
CopA1'
CopC1'
CopA2'
CopH'
CopI'
CopG'
CopK'
b
CopC2 '
b
CopN '
b
CopM '
Accession'
Rmet_6113'
Rmet_6112'
Rmet_6114'
Rmet_5671'
Rmet_6122'
Rmet_6116'
Rmet_6118'
Rmet_6108'
Rmet_5669'
Rmet_6109'
Rmet_6107'
%Cov(95)'
71,11'
39,09'
60,61'
15,21'
62,09'
46,20'
32,85'
26,60'
7,03'
7,93'
13,97'
a
Peptides'(95%)'
43'
21'
9'
7'
7'
7'
5'
4'
1'
1'
1'
a
emPAI '
12,54'
2,00'
4,62'
0,45'
9,00'
1,93'
2,59'
1,03'
0,23'
0,18'
0,26'
PAI
emPAI' is' the' exponentially' modified' protein' abundance' index' which' is' equal' to' 10 ' H' 1,'
where' PAI' =' (Nobserved' /' Nobservable);' Nobserved' is' the' number' of' experimentally' observed'
peptides'and'Nobservable'is'the'number'of'observable'peptides'when'the'protein'sequence'is'
digested'in*silico'by'trypsin.'
b
Proteins'identified'with'only'one'unique'peptide'
'
ICPHMS'analysis'of'the'total'copper'content'within'the'purified'OMVs'revealed'an'appreciable'
amount' of' copper' estimated' at' 2.98' (±' 0.46)' ng' per' µg' protein' in' comparison' with' the' total' cellH
associated'copper'content'of'C.*metallidurans'CH34'estimated'at'0.234'(±'0.007)' ng'per'µg'protein'
(Table'5.1,'B.).'
(
5.4.(Discussion(
Copper'is'an'essential'trace'element'that'occurs'widely'in'the'environment'and'is'required'by'
all'cell'types.'This'study'investigated'the'effects'of'copper'on'the'morphology'and'ultrastructure'of'C.*
metallidurans' CH34' bacteria' by' a' multidisciplinary' approach' coupling' atomic' force' microscopy' and'
electron' microscopy' (SEM' and' TEM)' to' understand' the' fate' of' copper' within' these' bacteria.' Many'
wellHknown'studies'have'used'AFM'in'air'to'investigate'morphological'alterations'after'exposure'to'
various' chemicals' (Kasas' et' al.,' 1994;' Peng' et' al.,' 2004;' Rossetto' et' al.,' 2007).' This' powerful'
technique'complements'structural'information'obtained'by'SEM'and'TEM,'and'it'gives'details'about'
the'topography'of'bacterial'surfaces'with'unparalleled'lateral'resolution'(Braga'and'Ricci,'1998).'
It' is' indisputable' that' copper' has' an' impact' on' the' morphology' and' ultrastructural'
organization'of'C.*metallidurans'CH34.'Many'membrane'surface'invaginations'and'irregularities'were'
observed'by'SEM'similar'to'those'observed'in'copperHresistant'Ralstonia'isolates'from'lake'sediments'
contaminated'with'high'concentrations'of'copper'(Konstantinidis'et'al.,'2003).'Interestingly,'the'highH
150'
'
Results'and'Discussion'
'
resolution' AFM' imaging' highlighted' the' presence' of' bulges' on' the' cellular' envelope' of' copperH
treated' cells.' This' observation' correlates' with' the' outer' membrane' vesicles' observed' during'
ultrastructural' analysis' of' copperHtreated' bacteria.' The' bulges' are' pinched' off' from' the' outer'
membrane' in' a' way' that' leads' to' the' inclusion' of' periplasmic' material' and' released' into' the'
surrounding' environment.' It' has' been' reported' that' the' vesiculation' process' is' not' the' result' of'
bacterial'lysis'or'disintegration'of'the'bacterial'membrane,'and'no'correlation'has'been'established'
between'this'phenomenon'and'membrane'instability'(Chatterjee'and'Chaudhuri,'2012;'McBroom'et'
al.,'2006;'Schwechheimer'et'al.,'2013).'
Although'copper'resistance'within'C.*metallidurans'CH34'has'been'relatively'well'studied,'the'
fate' of' copper' in' the' cells' has' remained' unknown.' Until' now,' this' vesiculation' phenomenon' in' C.*
metallidurans' wildHtype' strain' CH34' in' response' to' copper' stress' has' never' been' described.' The'
vesiculation'phenomenon'is'a'ubiquitous'physiological'process'for'most'gramHnegative'bacteria,'both'
pathogenic'and'nonpathogenic'species'such'as'Escherichia*coli'and'Pseudomonas*aeruginosa'(Kuehn'
and'Kesty,'2005;'MashburnHWarren'et'al.,'2008;'McBroom'and'Kuehn,'2007;'Schwechheimer'et'al.).'
The' production' of' vesicles' by' bacteria' is' influenced' by' a' variety' of' environmental' factors'
(temperature,' nutrient' availability,' exposure' to' toxic' agents,' etc.)' and' contributes' to' their' growth'
and'survival'under'challenging'circumstances'(Chatterjee'and'Chaudhuri,'2012;'McBroom'and'Kuehn,'
2007).' Diverse' functional' roles' have' been' assigned' to' outer' membrane' vesicles' depending' on' the'
organism' from' which' they' originate.' They' notably' function' in' aggressive' roles' carrying' virulence'
factors' from' pathogenic' bacteria,' they' play' a' role' in' bacterial' cooperation' by' sharing' antibiotic'
resistance' enzymes' and' exogenous' DNA,' and' they' also' perform' a' defensive' role' in' promoting' the'
survival' of' nonpathogenic' bacteria' by' reducing' levels' of' harmful' compounds' such' as' toluene'
(Berleman'and'Auer,'2013;'Kuehn'and'Kesty,'2005;'MashburnHWarren'et'al.,'2008;'Schwechheimer'
et' al.,' 2013;' Tashiro' et' al.,' 2012).' The' mechanisms' allowing' the' production' of' outer' membrane'
vesicles'are'still'poorly'known,'nonetheless'it'would'appear'that'this'vesiculation'process'constitutes'
a'new'envelope'stress'response'independent'and'distinct'from'all'those'previously'known'(McBroom'
and' Kuehn,' 2007).' Consequently,' it' may' be' that' the' outer' membrane' vesicles' observed' in' C.*
metallidurans'CH34'upon'copper'exposure'constitute'an'effective'protective'mechanism'for'survival'
in'the'presence'of'toxic'copper'compounds.'
In' an' attempt' to' explore' the' molecular' machinery' involved' in' OMV' biogenesis,' a' global'
proteomic'view'of'OMVs'derived'from'C.*metallidurans'CH34'upon'copper'treatment'was'generated'
in' the' present' study.' Vesicles' isolated' by' filtration' of' bacterial' supernatant' followed' by'
ultracentrifugation' were' examined' by' transmission' electron' microscopy.' Observation' of' TEM'
photomicrographs' essentially' revealed' numerous' ovoidHtoHroundHshaped' OMVs' with' an' average'
151'
'
Results'and'Discussion'
'
diameter' of' 20H180' nm' in' accordance' with' the' diameter' range' referred' to' previous' papers'
(Bonnington'and'Kuehn,'2014;'Kuehn'and'Kesty,'2005;'Li'et'al.,'1998;'Schwechheimer'et'al.,'2013).'
Moreover,' the' electron' microscopy' appears' to' show' no' significant' contamination' by' bacteria' or'
membrane' fragments.' Nano' LCHMS/MS' analysis' of' OMV' proteins' identified' 295' unique' vesicular'
proteins'with'high'confidence'from'three'independent'experiments.'Notably,'the'proteomic'analysis'
appears' to' show' very' little' contamination' with' cytoplasmic/inner' membrane' proteins.' The' major'
component' of' OMVs' derived' from' copperHtreated' C.* metallidurans* CH34' is' FliC2' (Rmet_5252),' a'
flagellar'filament'structural'protein'also'known'as'flagellin.'Numerous'flagellar'proteins'such'as'FlgE,'
FliD3,' FlgK,' FlgL,' FlgG,' FlgM,' FlgF' and' FlgB' were' also' identified' in' our' experiments' with' a' relatively'
low'abundance'in'comparison'with'the'FliC2'protein.'All'these'proteins'are'required'for'the'structure'
and' assembly' of' the' external' portions' of' the' bacterial' flagellum,' the' external' structures' being'
defined' as' any' structures' that' are' outside' of' the' inner' membrane' (Chevance' and' Hughes,' 2008).'
However,' no' flagellar' proteins' associated' to' the' cytoplasmic' membrane' were' detected' in' our'
samples.'
The' FliC' (flagellin)' protein' is' the' major' subunit' protein' of' bacterial' flagella' and' it' has' been'
observed' in' numerous' OMV' proteomes' from' various' gramHnegative' bacteria' (Altindis' et' al.,' 2014;'
Bauman' and' Kuehn,' 2006;' Lee' et' al.,' 2007;' Park' et' al.,' 2011).' However,' there' is' a' debate' about'
flagellar' proteins,' which' are' considered' as' contaminants' in' some' papers.' Indeed,' some' authors'
supplement' the' purification' of' OMVs' by' sucroseHdensity' gradient' centrifugation' to' remove' these'
flagellar' contaminants' (Bauman' and' Kuehn,' 2006;' Kulp' and' Kuehn,' 2010;' Lee' et' al.,' 2007).'
Nevertheless,' Manabe' et' al.' (2013)' fractionated' their' OMV' preparations' by' density' gradient'
centrifugation' and' demonstrated' that' FliC' is' still' associated' with' membranes' and' might' be' a'
component'of'the'OMVs'but'not'a'contaminant.'Moreover,'these'authors'suggest'that'the'ability'to'
form'flagella'affects'the'production'of'OMVs'in*E.*coli'W3110'(Manabe'et'al.,'2013).'
The' OMV' proteome' annotated' according' to' subcellular' distribution' using' the' PSORTb'
algorithm'showed'a'strong'significant'enrichment'in'periplasmic,'outer'membrane'and'extracellular'
proteins' whereas' inner' membrane' proteins' were' almost' excluded.' This' distribution' is' in' complete'
agreement'with'previous'reported'OMV'proteomes'(AvilaHCalderon'et'al.,'2012;'Kulkarni'et'al.,'2014;'
Lee'et'al.,'2007;'Lee'et'al.,'2008).'Indeed,'besides'the'flagellar'proteins,'the'most'abundant'proteins'
identified'with'a'high'number'of'peptides'and'a'good'sequence'coverage'are'periplasmic'and'outer'
membrane'proteins'such'as'Ca2+'sensor'(periplasmic'protein,'27'peptides),'mucD'TrypsinHlike'serine'
protease' (periplasmic' protein,' 25' peptides),' gltl' glutamate' and' aspartate' transporter' subunit'
(periplasmic' protein,' 24' peptides),' CopB1' outer' membrane' protein' (43' peptides),' yfgL' outer'
membrane' protein' assembly' complex' subunit' YfgL' (22' peptides),' porin' Rmet_3234' (21' peptides),'
152'
'
Results'and'Discussion'
'
etc.' Regarding' the' inner' membrane' proteins,' they' were' identified' with' a' few' number' of' peptides'
(e.g.,' TatA' TatABCE' protein' translocation' system' subunit' (2' peptides),' IorB' Isoquinoline' 1H
oxidoreductase' subunit' beta' (2' peptides),' putative' substrateHbinding' region' of' ABCHtype' glycine'
betaine' transport' system:' QAT' family' (3' peptides),' etc.)' and' might' be' considered' as' trace'
contaminants'due'to'the'fact'that'mass'spectrometry'is'an'extremely'sensitive'analytical'method,'in'
particular'with'the'TripleTOF®'instrument'used'in'this'study.'
We' cannot' exclude' that' the' intriguing' presence' of' numerous' cytosolic' proteins' in' OMVs,' in'
particular'ribosomal'proteins,'resulted'from'contamination'of'our'OMV'preparation.'The'enzymes'for'
glycolysis'are'known'as'being'the'most'abundant'and'stable'proteins'found'in'the'cytosol.'However,'
only'three'glycolytic'enzymes'were'identified'with'a'few'number'of'peptides'(4H8'peptides)'such'as'
enolase,' cbbG1' glyceraldehydeH3Hphosphate' dehydrogenase' A' and' cbbG2' glyceraldehydeH3H
phosphate' dehydrogenase' A,' suggesting' that' the' observed' contamination' is' really' weak.' Notably,'
the' presence' of' cytosolic' proteins,' in' particular' ribosomal' proteins,' in' OMVs' has' consistently' been'
documented,'even'if'it'is'still'debated'(Lee'et'al.,'2007;'Lee'et'al.,'2008;'Maredia'et'al.,'2012;'Park'et'
al.,'2011).'Nevertheless,'in'our'analysis,'ribosomal'proteins'appear'to'be'overrepresented'in'term'of'
concentration' (i.e.,' proteins' identified' with' a' high' number' of' peptides' and' with' a' good' sequence'
coverage),' but' not' in' term' of' number' in' comparison' with' the' global' proteome' of' C.* metallidurans'
CH34.'The'small'amount'of'contamination'in'our'sample'might'be'circumvented'by'sucroseHdensity'
gradient'centrifugation'(Bauman'and'Kuehn,'2006;'Kulp'and'Kuehn,'2010;'Lee'et'al.,'2007).'
Numerous' Cop' proteins' involved' in' copper' resistance' in' C.* metallidurans' CH34' were' also'
identified' in' the' OMV' proteome' such' as' CopA1,' CopB1,' CopC1,' CopA2,' CopK,' CopH,' CopI,' CopG,'
CopC2,'CopN'and'CopM.'These'proteins'are'known'to'be'localized'in'the'periplasmic'space'and'to'be'
able' to' bind' Cu(I)' and/or' Cu(II)' ions' (Monchy' et' al.,' 2006)' (Fig.' 5.5).' Notably,' among' these' Cop'
proteins,' CopC1,' CopA1' and' in' particular' CopB1' are' the' most' abundant' proteins' with' an'
exponentially'modified'protein'abundance'index'(emPAI)'of'9,'21'and'43,'respectively.'Moreover,'the'
results'of'the'proteomic'analysis'suggest'specific'mechanisms'for'sorting'proteins'into'OMVs.'Indeed,'
the' well' known' periplasmic' proteins' are' not' all' identified' in' the' proteome' of' OMVs.' The' CopB1'
protein'appears'to'be'overrepresented'in'the'OMV'proteome'in'comparison'with'the'tripartite'efflux'
systems' (i.e.,' SilABC' and' CusABC' systems),' for' which' the' periplasmic' adaptor' proteins' known' as'
being'characteristic'of'the'periplasmic'proteome'analysis'were'not'identified'in'our'analysis'(Derock'
et'al.,'in'preparation)'(Fig.'5.5).'The'hypothesis'of'specific'mechanisms'for'sorting'proteins'into'OMVS'
is' suggested' in' numerous' papers,' but' it' is' still' far' from' being' understood' (Bonnington' and' Kuehn,'
2014;'Kulkarni'et'al.,'2014).'
153'
'
Results'and'Discussion'
'
Figure(5.5:'Schematic'representation'
of'OMV'formation.'Outer'membrane'
vesicles'consist'of'OM'phospholipids,'
LPS,' porins,' OM' proteins' and'
periplasmic' proteins' such' as' Cop'
proteins' identified' by' mass'
spectrometry'and'involved'in'copper'
resistance.' (LPS)' lipopolysaccharide;'
(Pp)' periplasm;' (OM)' outer'
membrane;'(PG)'peptidoglycan;'(IM)'
inner' membrane;' (Cyt)' cytoplasm.'
Cu(I)'and'Cu(II)'ions'are'represented'
by' white' and' black' balls,'
respectively.'
'
'
As'aforementioned,'all'identified'Cop'proteins'are'known'to'bind'Cu(I)'and/or'Cu(II)'ions'and'
an' appreciable' copper' content' [2.98' (±' 0.46)' ng/µg' protein]' was' detected' associated' to' these'
vesicles' by' ICPHMS,' which' is' tenfold' higher' than' the' total' cellHassociated' copper' content' in' C.*
metallidurans'CH34.'Notably,'transmission'electron'microscope'coupled'with'energy'dispersive'XHray'
analyzer'(TEMHEDX)'was'also'used'to'determine'whether'copper'was'associated'to'the'membrane'or'
present' inside' the' vesicles.' No' conclusive' results' were' obtained,' preventing' to' accurately' localize'
copper.' It' might' be' hypothesized' that' the' overabundance' of' the' Cop' proteins,' which' coordinate'
copper'ions'in'the'periplasmic'space,'might'be'one'of'the'key'elements'underlying'the'vesiculation'
phenomenon.' This' hypothesis' is' strengthened' by' the' fact' that' some' Cop' proteins' in' OMVs,' in'
particular' the' abundant' CopB' protein,' appear' to' be' less' soluble' when' metal' ions' bound.' The'
vesiculation' phenomenon' is' known' as' an' insoluble' secretion' pathway,' which' is' regulated' by'
overexpression'of'periplasmic'proteins'(Berleman'and'Auer,'2013;'Kulp'and'Kuehn,'2010).'
The' specificity' of' the' vesicle' formation' was' also' investigated' under' exposure' to' different'
divalent' metals' such' as' copper,' nickel' and' zinc.' At' first' glance,' these' three' metals' are' structurally'
similar;'they'have'similar'atomic'radii'and'carry'a'double'positive'charge.'Monsieurs'et'al.'(Monsieurs'
et' al.,' 2011)' showed' that,' regardless' of' any' physicochemical' explanation,' these' divalent' cations'
belong'to'the'same'group'of'metals'giving'rise'to'a'similar'transcriptomic'response'in'strain'CH34.'
Nevertheless,' no' outer' membrane' vesicles' were' observed' in' the' wildHtype' strain' CH34' exposed' to'
Ni(II)'or'Zn(II)'ions.'A'plausible'explanation'for'this'phenomenon'could'involve'the'toxicity'of'copper.'
Indeed' based' on' their' respective' minimal' inhibitory' concentrations' (MICs),' strain' CH34' shows' a'
pattern' of' metal' tolerance' in' the' order' Zn2+' >' Ni2+' >' Cu2+;' consequently,' C.* metallidurans' CH34' is'
154'
'
Results'and'Discussion'
'
highly'sensitive'to'Cu2+'ions.'Because'the'redox'potential'of'the'Cu2+/Cu+'couple'(H268'mV'at'30°C,'pH'
7.0)' ranges' between' those' of' the' H+/H2' couple' (H421' mV)' and' the' O2/H2O' couple' (+808' mV),'
copper(II)'ions'may'be'reduced'by'the'cell.'By'contrast,'Zn2+'(H1180'mV)'and'Ni2+'(H678'mV)'ions'are'
outside'of'this'redox'potential'range'and'may'not'be'reduced'(Nies,'1999).'Consequently,'Zn(II)'and'
Ni(II)' ions' are' redoxHinert' while' copper' ions' can' lead' to' oxidative' stress' and' destructive' lipid'
peroxidation'damage'(Galhardi'et'al.,'2004).'Indeed,'the'reduction'products'of'copper'are'more'toxic'
than' the' starting' ions.' Free' Cu(I)' ions' are' among' the' most' toxic' trace' elements' because' they' can'
induce'FentonHlike'reactions,'generating'highly'reactive'hydroxyl'radicals'OH•'that'can'react'with'all'
cellular'components'and'inhibit'the'cellular'machinery'(Cuillel,'2009).'Therefore,'it'is'crucial'to'export'
the' reduced' products' out' of' the' cell,' and' it' is' likely' that' the' observed' OMVs' might' constitute' an'
excellent'way'to'trap'and'excrete'monovalent'copper'ions.'
However,'even'if'these'three'metal'ions'give'rise'to'a'similar'transcriptomic'response'for'the'
cop'genes'in'strain'CH34,'the'proteomic'analysis'of'the'periplasmic'space'showed'that'the'relative'
abundance'of'Cop'proteins'upon' copper'exposure'appears'much'more'important'than'upon'nickel'
exposure' (Derock' et' al.,' in' preparation).' Moreover,' the' large' majority' of' Cop' proteins' (e.g.,' CopB,'
CopK,'CopI,'etc.)'are'not'able'to'bind'zinc'or'nickel'ions;'consequently,'it'might'be'possible'that'these'
proteins'do'not'undergo'changes'in'their'physicochemical'properties'upon'nickel'and'zinc'additions.'
Notably,' cadmium' also' belongs' to' the' same' group' than' copper,' nickel' and' zinc' and' gives' rise' to' a'
similar' transcriptomic' response' for' the' cop' genes' (Monsieurs' et' al.,' 2011).' Nevertheless,' the'
proteomic'analysis'of'the'periplasm'also'showed'that'the'relative'abundance'of'Cop'proteins'upon'
cadmium' exposure' is' more' important' than' upon' copper' treatment' (Derock,' in' preparation).'
Consequently,' it' would' be' interesting' to' investigate' if' the' cadmium' exposure' leads' to' the' same'
vesiculation'phenomenon'than'observed'upon'copper'exposure.'
The'vesiculation'phenomenon'in'C.*metallidurans'CH34'obviously'appears'to'be'connected'to'
the'presence'of'copper'in'the'growth'culture.'Nevertheless,'the'study'of'mutants'derived'from'the'
wildHtype' strain' CH34' and' treated' with' copper' highlighted' another' interesting' point.' Although' the'
copper' treatment' induced' ultrastructural' modifications' in' each' mutant,' only' the' derivative' strains'
harboring'metal'resistance'genes'on'their'plasmid'appeared'to'produce'OMVs'in'various'proportions'
during'copper'stress'challenge.'Indeed,'observations'of'ultrathin'sections'relating'to'the'plasmidHfree'
derivative'AE104'of'C.*metallidurans'CH34'and'strain'AE2214'carrying'the'vector'pLAFR3'devoid'of'
metal' resistance' genes' showed' no' outer' membrane' vesicles' in' the' presence' of' copper;' whereas'
various'outer'membrane'vesicles'were'produced'upon'copper'exposure'by'all'derivative'strains'of'C.*
metallidurans'harboring'metal'resistance'genes'on'their'plasmid:'AE126'(pMOL28),'AE128'(pMOL30)'
and'AE1744'(pMOL1024).'
155'
'
Results'and'Discussion'
'
The'identification'of'genes'carried'by'plasmids'and'involved'in'vesicle'formation'turns'out'to'
be' extremely' complex.' Both' plasmids' pMOL28' (171' kb)' and' pMOL30' (234' kb)' carry' many' genetic'
determinants'for'high'resistance'to'metals.'They'have'been'completely'sequenced'and'annotated'in'
previous'studies'(Monchy'et'al.,'2007;'Monsieurs'et'al.,'2011)'and'also'studied'at'the'transcriptomic'
level'after'various'metal'challenges'such'as'Cd(II),'Co(II),'Cu(II),'Ni(II),'and'Zn(II)'(Monchy'et'al.,'2007).'
It'was'shown'by'Monchy'et'al.'(2007)'that'many'genes'are'induced'on'both'plasmids'after'copper'
treatment' (Monchy' et' al.,' 2007).' Among' the' copperHinduced' genes,' it' should' be' noted' that' Cu(II)'
causes'the'entire'cnr'cluster'on'pMOL28'to'be'overexpressed.'Interestingly,'these'genes'are'involved'
only'in'nickel'and'cobalt'resistance'in'C.*metallidurans'CH34'and'not'in'copper'resistance.'Another'
cluster'highly'induced'by'Cu(II)'is'the'cop'cluster'on'pMOL30,'which'is'involved'in'copper'resistance'
in'strain'CH34.'In'addition'to'those'metal'resistance'determinants,'other'plasmidHborne'genes'were'
found' to' be' strongly' expressed' after' copper' challenge.' Some' of' them' are' hypothetical' genes'
whereas' others' are' involved' in' pilus' biosynthesis' (such' as' pilQ' on' pMOL28' encoding' a' hexameric'
ATPase' required' for' pilus' assembly),' in' membrane' biogenesis' and' maintenance' (such' as' putative'
glycosyltransferase' genes' on' pMOL30),' and' in' conjugative' transfer' and' transposition' (such' as' trbN'
lytic* murein* transglycosylase' (Rmet_6298)' on' pMOL28)' (Monchy' et' al.,' 2007).' Based' on' current'
knowledge'regarding'copper'resistance'in'C.*metallidurans'CH34,'we'are'not'able'to'determine'which'
genes'underlie'the'vesiculation'phenomenon.'Although'further'study'of'this'issue'is'warranted,'our'
findings'provide'evidence'that'vesiculation'occurs'upon'copper'exposure'and'most'likely'requires'the'
presence'of'certain'genes'carried'by'mobile'genetic'elements'(e.g.,'cop'genes'and/or'genes'involved'
in'membrane'maintenance'and'restoration).'Nevertheless,'the'releasing'of'OMVs'by'the'strain'AE126'
(pMOL28)' raises' many' questions,' even' if' the' entire' cnr' cluster' on' pMOL28' is' overexpressed' upon'
copper' exposure.' Further' investigations' will' be' required' to' improve' the' biogenesis' of' OMVs;' in'
particular,' it' would' be' interesting' to' study' the' proteome' of' OMVs' produced' by' the' different'
derivative'strains'of'C.'metallidurans'CH34,'which'differ'by'their'plasmid'content.'
Taken' together,' these' results' support' the' aforementioned' hypothesis' that' outer' membrane'
vesicles' might' be' an' excellent' way' to' transport' toxic' copper' ions.' This' phenomenon' might' be' a'
putative' clustering' mechanism' similar' to' the' bioprecipitation/biomineralization.' The' putative'
clustering'of'toxic'compounds'offers'a'significant'benefit'to'the'bacteria'in'comparison'to'the'efflux'
mechanisms,'which'are'energetically'not'favorable.'
(
(
156'
'
Results'and'Discussion'
'
5.5.(Materials(and(Methods(
5.5.1.(Bacterial(Strains(and(Culture(Conditions(
C.* metallidurans' strain' CH34' and' its' mutant' derivatives' (listed' in' Table' S5.2)' were' kindly'
provided'by'Natalie'Leys'and'Max'Mergeay'from'SCK•CEN,'Belgium'and'Françoise'Van'Vliet'from'
ULB,'Belgium.'All'strains'were'grown'at'30°C'under'aerobic'conditions'(rotary'shaker,'160'rpm)'in'
MOPS' (4HMorpholinepropanesulfonic' acid)Hsalt' minimal' medium' with' 2' g' LH1' gluconate' as' unique'
carbon'source'(adapted'from'(Mergeay'et'al.,'1985)).'Minimal'medium'was'supplemented'with'low'
or' high' copper' concentrations' based' on' the' copper' minimal' inhibitory' concentration:' 0.2' mM' or'
0.8' mM' Cu(NO3)2' •' 3H2O,' respectively' (Monchy' et' al.,' 2006).' In' addition,' two' other' metals' were'
investigated' as' controls' at' low' concentrations' based' on' their' minimal' inhibitory' concentrations:'
0.42' mM' NiCl2' •' 6H2O' (Mergeay' et' al.,' 2003)' and' 1' mM' ZnCl2' (Monchy,' 2007).' Another' type' of'
oxidative'stress'was'also'investigated'by'adding'either'3'mM'or'6'mM'hydrogen'peroxide'(H2O2)'to'
the'growth'medium.'This'stress'treatment'was'applied'for'2'hours'when'the'optical'density'at'600'
nm' (OD600)' was' ~' 0.4.' The' mutant' derivatives' were' only' treated' with' 0.2' mM' Cu(II).' All' bacterial'
cells' were' harvested' by' centrifugation' (6,000' rpm,' 10' min,' at' 4°C)' during' the' midHexponential'
phase'(OD600'of'~'0.6).'
5.5.2.(Scanning(and(Transmission(Electron(Microscopy(
Pelleted'cells'were'washed'with'8'mM'NaCl,'0.05%'(w/v)'red'ruthenium'in'0.1'M'cacodylate'
buffer'(pH'7.8)'and'immediately'fixed'for'1'h'45'at'4°C'by'using'3%'(v/v)'glutaraldehyde'in'the'same'
buffer.'The'chemical'fixation'was'followed'by'three'10Hmin'rinses'with'changes'of'cacodylate'buffer'
and'a'postfixation'with'1%'OsO4'(w/v)'in'the'same'buffer'(1'h'45,'at'room'temperature).'For'SEM'
experiments,'the'samples'were'carefully'applied'to'cellulose'membrane'filters'with'a'0.22Hµm'pore'
size' (GSWP,' MFHMillipore).' They' were' subsequently' dehydrated' in' a' graded' ethanol' series' up' to'
100%' and' dried' by' the' criticalHpoint' method.' Desiccated' samples' were' mounted' on' aluminum'
stubs,'coated'with'gold'in'a'sputterHcoater'(JEOL'JFCH1100E'ion'sputter)'and'observed'with'a'JEOL'
JSMH6100'scanning'electron'microscope.'For'TEM'investigations,'dehydrated'cells'were'embedded'
in'Spurr'resin'(TAAB'Laboratories'Equipment,'Berks,'England).'Ultrathin'sections'(70H90'µm)'were'
cut' with' a' Leica' Ultracut' UCT' ultramicrotome' equipped' with' a' diamond' knife.' Then,' they' were'
stained' with' uranyl' acetate' and' lead' citrate' and' observed' with' a' Zeiss' LEO' 906E' transmission'
electron'microscope'operated'at'60'kV.'TEM'images'were'acquired'with'the'program'analySIS'(Soft'
Imaging'System,'Switzerland).'
157'
'
Results'and'Discussion'
'
5.5.3.(Atomic(Force(Microscopy(
Samples' for' AFM' experiments' were' prepared' with' a' procedure' similar' to' that' described' in'
Gillis'et'al.,'2012'(Gillis'et'al.,'2012).'Droplets'of'100'µL'of'bacterial'cell'suspension'were'deposited'
on' a' freshly' cleaved' muscovite' mica' surface' and' were' incubated' for' 2' h' at' room' temperature.'
Then,' the' surface' was' gently' rinsed' three' times' with' ultrapure' water' (MilliHQ)' and' dried' at' room'
temperature.' AFM' images' were' acquired' in' air' at' room' temperature' using' an' ICON' AFM' model'
(Bruker)'operating'in'the'Peak'Force'Tapping'mode.'The'Peak'Force'QNMTM'is'a'new'investigation'
technique,'very'suitable'to'soft'samples'such'as'bacteria,'allowing'the'simultaneous'mapping'of'the'
topography'and'nanomechanical'properties'at'a'speed'similar'to'the'tapping'mode'and'with'high'
resolution' (Heu' et' al.;' Pittenger' et' al.).' The' AFM' images' were' acquired' using' silicon' nitride' tips'
(Model'SNL,'f0'='16H28'kHz,'k'='0.12N/m,'Bruker)'at'several'locations'on'each'sample.'The'images'
were'analyzed'and'processed'using'the'Nanoscope'image'analysis'program.'
5.5.4.(Isolation(and(Purification(of(OMVs(from(C.*metallidurans(CH34(
Isolation'and'purification'of'OMVs'from'C.*metallidurans'CH34'grown'aerobically'at'30°C'in'the'
presence' of' 0.8' mM' Cu(NO3)2' •' 3H2O' were' performed' according' to' a' procedure' adapted' from'
Kobayashi' et' al.' and' AvilaHCalderon' et' al.' (AvilaHCalderon' et' al.,' 2012;' Kobayashi' et' al.,' 2000).'
Briefly,' cells' from' a' 500HmL' culture' were' harvested' by' centrifugation' (10,000' x' g,' 15' min,' at' 4°C)'
during' midHlog' phase' (OD600' of' ~' 0.6).' The' supernatant' was' collected' and' sequentially' filtered'
through' 0.45Hµm' (16555K,' Minisart®' NML' syringe' filters,' Sartorius' AG)' and' 0.2Hµm' (16534K,'
Minisart®'NML'syringe'filters,'Sartorius'AG)'pore'cellulose'acetate'membranes'to'remove'residual'
cells.'The'resulting'cellHfree'filtrate'was'subjected'to'ultracentrifugation'at'100,000'x'g'for'3'h'at'4°C'
using' a' Beckman' Type' 50.2' Ti' Ultracentrifuge' rotor.' Purified' preparations' were' confirmed' to' be'
cellHfree'by'transmission'electron'microscopy'following'the'procedure'described'above.'
5.5.5.(Protein(Extraction(from(Purified(OMVs(and(Enzymatic(Digestion(
On' the' one' hand,' a' purified' OMV' pellet' (~' 125' mL' cellHfree' supernatant)' was' mixed' with'
Laemmli'sample'buffer'and'heated'for'5'min'at'95°C.'The'sample'was'electrophoresed'on'a'4H20%'
MiniHProtean®'precast'gel'(BioHRad)'and'subsequently'stained'with'Coomassie'blue'RH250.'Only'the'
more' intense' protein' bands' were' excised.' Each' slice' was' subsequently' reduced' with' 50' mM'
dithiothreitol,'alkylated'with'50'mM'iodoacetamide'and'then'inHgel'digested'with'trypsin'(Promega'
V51' 11)' overnight' at' 37°C.' On' the' other' hand,' a' second' purified' OMV' pellet' (~' 125' mL' cellHfree'
supernatant)'was'resuspended'in'50'µL'6'M'guanidine'chloride'pH'8.5'(lysis'buffer'from'the'ICPL'
kit,'Serva).'The'cells'were'sonicated'on'ice'using'a'U50'IKA'Technik'(20%'amplitude,'3'cycles'of'1's,'
158'
'
Results'and'Discussion'
'
1' pulse' rate).' The' supernatant' containing' soluble' proteins' was' recovered' by' centrifugation' at'
13,000' rpm' for' 15' min' at' 4°C.' Due' to' its' very' low' concentration,' the' whole' supernatant' was'
reduced'and'alkylated'according'to'the'manufacturer's'instructions'(ICPL'kit,'Serva).'OMV'proteins'
were'recovered'through'acetone'precipitation'and'inHsolution'digested'with'trypsin'(Promega'V51'
11)' at' a' ratio' of' 1:50' (enzyme/substrate)' in' 50' mM' NH4HCO3' overnight' at' 37°C.' Both' trypsic'
digestions'were'stopped'by'the'addition'of'formic'acid'(0.1%,'v/v,'final).'
5.5.6.(LCTMS/MS(Analysis(and(Identification(of(OMV(proteins(
The'identification'of'proteolytic'peptides'resulting'from'both'inHgel'and'inHsolution'digestions'
was' performed' using' a' labelHfree' strategy' on' a' UHPLCHHRMS' platform' combining' a' NanoLCHUltra'
system'(Eksigent)'with'a'TripleTOF®'5600'System'(AB'SCIEX).'Peptides'were'separated'in'a'25'cm'
C18'reverseHphase'column'(Acclaim'PepMap'100,'3'µm,'Dionex)'by'a'linear'acetonitrile'gradient'(4'
to'35%'v/v,'flow'rate'of'300'nL'minH1,'20'min)'in'water'containing'0.1%'(v/v)'formic'acid.'The'timeH
ofHflight' (TOF)' analyzer' was' regularly' and' automatically' calibrated' with' trypsic' peptides' of' βH
galactosidase'from'Escherichia*coli'which'maintains'an'average'mass'error'below'10'ppm'across'all'
injections.'Mass'spectra'(MS)'were'acquired'across'400H1500'm/z'with'0.5'sec'accumulation'time.'A'
maximum' number' of' 50' precursors' per' cycle' was' selected' according' to' an' intensity' threshold' of'
200' counts' per' sec.' Each' selected' precursor' was' accumulated' for' 50' ms' and' submitted' to'
fragmentation'with'N2' as'the'collision'gas.'MS/MS'spectra'were'acquired'across'100H1800'm/z'and'
an'exclusion'time'of'30'seconds'was'applied.'
The'acquired'data'were'analyzed'using'ProteinPilotTM'Software'v.'4.1'(AB'SCIEX'MA,'USA)'and'
searched'against'the'UniProt'TrEMBL'database'(10/26/2011'version)'restricted'to'C.*metallidurans'
CH34.' The' search' parameters' included' differential' amino' acid' mass' shifts' for' carbamidomethyl'
cysteine,' all' biological' modifications' and' amino' acid' substitutions,' and' missed' trypsin' cleavage.' A'
computational' analysis' was' performed' for' each' identified' protein' by' the' PSORTb' algorithm' to'
predict'its'subcellular'localization'(PSORTb'v.3.0.2'package).'The'SignalP'algorithm'(v.4.1)'was'used'
to'assign'a'predicted'signal'peptide'to'identified'proteins'with'an'unknown'predicted'localization.'
Classification'of'the'identified'OMV'proteins'based'on'their'predicted'functions'was'achieved'using'
the'COG'protein'database'of'C.*metallidurans'CH34'available'on'GenoScope's'MaGe'system.'
5.5.7.(Inductively(Coupled(PlasmaTMass(Spectrometry((ICPTMS)(analysis(
Purified' OMVs' and' both' untreated' and' 0.8HmM' copperHtreated' C.* metallidurans' CH34' were'
prepared'as'previously'described.'The'samples'were'thoroughly'rinsed'several'times'with'ultrapure'
water'and'mineralized'by'using'200'µL'of'HNO3'(Merck,'suprapur)'for'48'h'at'30°C.'Afterwards,'5'
159'
'
Results'and'Discussion'
'
mL'of'ultrapure'water'was'added'to'the'solution'and'titration'was'carried'out'by'ICPHMS'(Thermo'
Elemental'X'series)'after'an'additional'dilution'by'a'factor'10.'ICPHMS'gave'[copper]'in'µg/L.'Protein'
extractions'were'achieved'as'previously'described'using'6'M'guanidine'chloride'pH'8.5'(lysis'buffer'
from' the' ICPL' kit,' Serva),' performing' sonication' on' ice' and' centrifugation' at' 13,000' rpm.' Protein'
concentrations' were' determined' using' a' Micro' BCATM' Protein' Assay' Kit' (Thermo' Scientific).' Final'
copper'concentrations'were'expressed'as'ng'metal'per'µg'protein.'
(
(
160'
'
Results'and'Discussion'
'
5.6.(References(
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164'
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5.7.(Supplemental(Data(
'
Figure( S5.1:' TEM' photomicrographs' of' C.*
metallidurans' CH34' treated' with' different'
divalent' metal' ions.' (A.)' Bacteria' treated'
with' 0.2' mM' Cu(II),' the' arrows' indicate'
vesicles.' (B.)' Bacteria' treated' with' 0.42'
mM'Ni(II).'(C.)'Bacteria'treated'with'1'mM'
Zn(II).' Each' bacterial' sample' was' fixed' in'
3%'(v/v)'glutaraldehyde,'postfixed'with'1%'
OsO4' (w/v),' dehydrated' in' a' graded'
ethanol' series' and' embedded' in' Spurr'
resin.'
(
'
'
Figure(S5.2:'TEM'photomicrographs'of'C.*metallidurans'CH34'treated'with'hydrogen'peroxide.'(A.)'
Bacteria'treated'with'3'mM'H2O2.'(B.)'Bacteria'treated'with'6'mM'H2O2.'Each'bacterial'sample'was'
fixed' in' 3%' (v/v)' glutaraldehyde,' postfixed' with' 1%' OsO4' (w/v),' dehydrated' in' a' graded' ethanol'
series'and'embedded'in'Spurr'resin.'
165'
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'
Figure( S5.3:' TEM' photomicrographs' of' mutants' derived' from' the' wildHtype' strain' CH34.' (A.)' Untreated' bacterial'
strain'AE104'(carrying'no'plasmid).'(B.)'Bacterial'strain'AE104'treated'with'0.2'mM'Cu(II).'(C.)'Untreated'bacterial'
strain' AE128' (carrying' only' the' plasmid' pMOL30).' (D.)' Bacterial' strain' AE128' treated' with' 0.2' mM' Cu(II).' (E.)'
Untreated' bacterial' strain' AE126' (carrying' only' the' plasmid' pMOL28).' (F.)' Bacterial' strain' AE126' treated' with' 0.2'
mM'Cu(II).'(G.)'Untreated'bacterial'strain'AE1744'(carrying'only'the'cosmid'pMOL1024).'(H.)'Bacterial'strain'AE1744'
treated' with' 0.2' mM' Cu(II).' Each' bacterial' sample' was' fixed' in' 3%' (v/v)' glutaraldehyde,' postfixed' with' 1%' OsO4'
(w/v),'dehydrated'in'a'graded'ethanol'series'and'embedded'in'Spurr'resin.'
166'
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'
'
'
'
Figure( S5.4:( TEM' photomicrographs' of' strain' AE2214' derived' from' the' wildHtype' strain' CH34.' (A.)'
Untreated'bacterial'strain'AE2214'(carrying'vector'pLAFR3'without'any'metal'resistance'genes).'(B.)'
Bacterial' strain' AE2214' treated' with' 0.2' mM' Cu(II).' Each' bacterial' sample' was' fixed' in' 3%' (v/v)'
glutaraldehyde,'postfixed'with'1%'OsO4'(w/v),'dehydrated'in'a'graded'ethanol'series'and'embedded'
in'Spurr'resin.'
(
'
'
'
Figure( S5.5:' COGHbased' prediction' of' identified' OMV' proteins.' Comparison' of' the' identified' OMV' proteome' with'
the'theoretical'proteome'of'C.*metallidurans'CH34'based'on'functions'predicted'using'the'COG'protein'database'of'
C.* metallidurans' CH34' available' on' GenoScope's' MaGe' system.' Each' COG' group' is' subdivided' into' functional'
categories'designated'by'oneHletter'abbreviations:'[C]'energy'production'and'conversion;'[D]'cell'cycle'control,'cell'
division,' chromosome' partitioning;' [E]' amino' acid' transport' and' metabolism;' [F]' nucleotide' transport' and'
metabolism;' [G]' carbohydrate' transport' and' metabolism;' [H]' coenzyme' transport' and' metabolism;' [I]' Lipid'
transport' and' metabolism;' [P]' Inorganic' ion' transport' and' metabolism;' [Q]' secondary' metabolites' biosynthesis,'
transport' and' catabolism;' [M]' Cell' wall/membrane/envelope' biogenesis;' [N]' cell' motility;' [O]' posttranslational'
modification,' protein' turnover,' chaperones;' [T]' signal' transduction' mechanisms;' [U]' intracellular' trafficking,'
secretion,' and' vesicular' transport;' [J]' translation,' ribosomal' structure' and' biogenesis;' [K]' transcription;' [L]'
replication,'recombination'and'repair;'[R]'general'function'prediction'only;'[S]'function'unknown.'
167'
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5.8.(Supplemental(Tables(
'
168'
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5.8.(Supplemental(Tables(
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169'
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5.8.(Supplemental(Tables(
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170'
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5.8.(Supplemental(Tables(
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171'
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5.8.(Supplemental(Tables(
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172'
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5.8.(Supplemental(Tables(
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173'
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Results'and'Discussion'
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5.8.(Supplemental(Tables(
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174'
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5.8.(Supplemental(Tables(
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175'
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5.8.(Supplemental(Tables(
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176'
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5.8.(Supplemental(Tables(
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177'
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5.8.(Supplemental(Tables(
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178'
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5.8.(Supplemental(Tables(
'
179'
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Results'and'Discussion'
'
Table(S5.2:(List'of'the'bacterial'strains'used'in'the'present'study.'
Strains'
Cupriavidus*metallidurans'CH34'
AE104'
AE126'
AE128'
'
AE1744'
AE2214'
Mobile'Genetic'Elements'
Plasmids'pMOL28'and'pMOL30'
No'plasmid'
Plasmid'pMOL28'
Plasmid'pMOL30'
Cosmid' pMOL1024' which' is' build' from' the' vector'
pLAFR3'and'carries'almost'all'cop'genes'of'the'plasmid'
pMOL30,'copVTMKNSRABCDIJGFOLQH''
Vector'pLAFR3'without'the'cop'genes'
'
'
'
'
180'
'
Provided'by'
SCK'•'CEN'
SCK'•'CEN'
SCK'•'CEN'
SCK'•'CEN'
'
ULB'
SCK'•'CEN'
!
!
!
!
!
!
!
!
Concluding!Remarks!and!!
Future!Prospects!
___________________________________________________________________________!
!
!
!
!
!
!
!
!
!
!
!
!
Concluding*Remarks*and*Future*Prospects*
*
Copper*presents*a*great*paradox*in*biological*systems*given*that*it*is*both*essential*for*life*and*
highly*reactive*even*at*low*concentrations.*Consequently,*elaborate*mechanisms*are*required*in*all*
organisms* to* tightly* control* intracellular* copper* availability* (Cuillel,* 2009).* This* nonHbiodegradable*
metal*accumulates*in*the*environment,*in*particular*in*anthropogenic*biotopes,*and*can*often*reach*
hazardous*concentration*levels*adversely*affecting*the*quality*of*life.*One*of*the*main*challenges*of*
modern*times*consists*of* environmental*decontamination* of*polluted* sites.* In* many* cases,* copperH
polluted*environments*are*contaminated*with*other*metal*ions*(e.g.,*zinc,*iron,*lead,*etc.)*(Kabir*et*
al.,*2012).*In*this*context,*multipleHmetalHresistant*bacteria*are*required*for*bioremediation*purposes*
because*of*their*unique*properties*(Shamim*and*Rehman,*2013).*To*survive*in*stressful*environments*
and* cope* with* metal* toxicity,* metalHresistant* microorganisms* have* developed* numerous* defense*
mechanisms,*such*as*efflux*systems,*metal*absorption,*bioaccumulation,*and*detoxification*(Mejare*
and*Bulow,*2001;*Shamim*and*Rehman,*2013).*The*understanding*of*such*systems*at*the*molecular*
level* is* an* extremely* important* challenge* since* they* can* be* used* to* develop* genetically* modified*
bacteria* and* plants* for* the* bioremediation* of* polluted* waters* and* soils* (Mejare* and* Bulow,* 2001).*
For* example,* genetic* modifications* in* the* biosynthesis* of* metalHbinding* proteins* have* been* widely*
exploited* to* increase* the* metal* binding* capacity,* accumulation,* or* tolerance* of* bacteria* and* plants*
(Mejare* and* Bulow,* 2001;* Pattanayak* et* al.,* 2014;* Valls* et* al.,* 2000).* Knowledge* on* the* regulatory*
mechanisms* of* bacterial* metalHregulated* operons* has* also* resulted* in* the* development* of* other*
applications,*such*as*wholeHcell*metal*biosensors*based*on*gene*fusions*between*a*metalHinducible*
operon*and*the*promotorless*luciferase*operon*luxCDABE*of)Vibrio)fischeri*(Diels*et*al.,*2009).*These*
bioluminescent*bacterial*sensors*are*widely*used*for*detecting*and*quantifying*bioavailable*metals*in*
contaminated*environments*(e.g.,*soils,*wastes,*solids*or*minerals)*due*to*their*ability*to*produce*a*
detectable*response*upon*activation*(Diels*et*al.,*2009).*
Isolated* from* polluted* environments* contaminated* with* numerous* metals,* Cupriavidus)
metallidurans*strain*CH34*is*an*important*metalHresistant*model*for*bioremediation*purposes*as*well*
as* for* studying* metal* resistances,* in* particular* copper* resistance* (Bersch* et* al.,* 2008).* In* C.)
metallidurans*CH34,*resistance*to*high*copper*concentrations*in*the*millimolar*range*mainly*involves*
the*large*cop*cluster*harbored*by*the*plasmid*pMOL30,*copVTMKNSRABCDIJGFOLQHEW*(Mergeay*et*
al.,*2009;*Sarret*et*al.,*2010).*Most*of*the*cop*genes*appear*to*be*specific*to*the*Cupriavidus*genus,*
with*the*exception*of*1°)*the*copSRABCD*genes,*which*are*involved*in*periplasmic*detoxification*and*
for* which* high* identity* paralogs* are* also* present* on* the* chromid;* and* 2°)* the* copF* gene,* which*
encodes*for*a*PHtype*ATPase*required*for*efficient*copper*detoxification*from*the*cytoplasmic*space*
(Monchy* et* al.,* 2007;* Monchy* et* al.,* 2006).* Although* the* copSRABCD* and* copF* genes* are* well*
documented,* the* function* of* most* other* cop* gene* products* remains* unknown* or* not* fully*
182*
*
Concluding*Remarks*and*Future*Prospects*
*
understood.* It* may* be* hypothesized* that* such* unique* and* specific* genes* are* specialized* in* the*
response*to*intense*copper*stress.*This*assumption*is*based*on*the*study*of*CopK,*a*small*periplasmic*
protein*able*to*cooperatively*bind*Cu(I)*and*Cu(II)*(Chong*et*al.,*2009).*In*the*presence*of*high*copper*
levels,* such* a* protein* strongly* coordinates* the* periplasmic* copper* fraction* and,* as* a* consequence,*
CopK* might* be* considered* as* an* additional* copper* detoxification* system* in* C.) metallidurans* CH34.*
Other*genes*appear*to*be*involved*in*copper*resistance*such*as*the*silDCBA*cluster,*which*is*mainly*
induced* by* Ag(I)* ions* but* also* Cu(II)* ions.* Such* a* cluster* encodes* an* RNDHdriven* efflux* system* that*
assists*the*CopF*protein*in*cytoplasmic*copper*detoxification.*Although*proteomic*(NoelHGeoris*et*al.,*
2004)*and*transcriptomic*(Monchy*et*al.,*2006)*studies*supplemented*with*phenotypic*analyses*(van*
Aelst,*2008)*of*various*mutants*derived*from*the*wildHtype*strain*have*provided*a*good*indication*of*
proteins* involved* in* copper* resistance,* no* clear* mechanism* has* been* developed* thus* far.* Indeed,*
there*are*still*a*number*of*unknowns*such*as*the*role*of*some*Cop*proteins*and*the*fate*of*copper*in*
the*cell.*Moreover,*most*of*the*cop*genes*can*also*be*induced*by*other*metal*ions*(i.e.,*Ni2+,*Zn2+*and*
Cd2+),* which* increases* the* mechanistic* complexity* of* established* systems.* Such* a* synergy* between*
partially*common*mechanisms*to*different*metals*is*perfectly*understandable*since*such*a*strain*was*
found* in* biotopes* contaminated* by* multiple* metals.* This* work* attempted* to* shed* light* on* some*
unanswered*questions,*including*the*fate*of*copper*in*the*cell*and*the*molecular*characterization*of*
the*plasmidHencoded*CopB*protein,*which*contains*a*peculiar*methionineHrich*NHterminal*extremity.*
The*plasmidHencoded*CopB*protein*is*of*particular*interest.*Overexpressed*in*the*presence*of*
copper* ions,* this* protein* possesses* unique* features* absent* from* or* only* partially* found* in* other*
Cop/PcoB* homologues,* in* particular* the* CopB* protein* from* Ralstonia) Pickettii* 12D.* Multiple*
sequence* alignment* of* the* plasmidHencoded* CopB* protein* with* homologous* proteins* found* in* the*
databank* revealed* the* presence* of* a* highlyHconserved* CHterminal* domain* amongst* the* different*
Cop/PcoB*proteins,*which*contains*numerous*βHstrands*and*few*αHhelices.*This*CHterminal*extremity*
appears* to* be* conserved* amongst* different* species* and* strains,* but* also* amongst* the* plasmidH
encoded* and* chromidHencoded* proteins.* Moreover,* this* structured* domain* is* connected* through* a*
conserved* sequential* motif* (i.e.,* GS)* to* a* second* domain,* which* is* an* unstructured* and* flexible* NH
terminal* extremity.* This* NHterminal* domain* is* extremely* rich* in* methionine* residues,* primarily*
arranged* as* ten* identical* sequential* motifs* (MXXMXHXXMXXMX),* and* is* thought* to* be* involved* in*
Cu(I)*binding*(Monchy*et*al.,*2006).*Notably,*the*NHterminal*methionine*content*is*variable*amongst*
species*and*strains,*as*well*as*amongst*the*plasmidHencoded*and*chromidHencoded*proteins.*Indeed,*
the*chromidHencoded*paralog*from*C.)metallidurans*CH34*does*not*have*a*similarly*high*methionine*
content*in*comparison*with*the*plasmidHencoded*CopB*protein.*
183*
*
Concluding*Remarks*and*Future*Prospects*
*
Interestingly,*the*comparison*between*the*methionineHrich*sequence*of*the*plasmidHencoded*
CopB* protein* from*C.) metallidurans* CH34* and* the* sequence* of* the* CopB* proteins* from* different*C.)
metallidurans* strains* reveals* a* difference* in* the* methionine* content* (Fig.* 6).* This* difference* in* the*
methionine* content* might* be* connected* to* higher* metal* concentrations* that* reflect* the* biotope*
wherein* the* different* strains* were* found.* Our* sequence* alignment* showed* that* the* methionine*
content* is* higher* in* the* plasmidHencoded* CopB* proteins* of* C.) metallidurans* CH34* and* C.)
metallidurans* H1130* (isolated* from* decantation* tank* of* Belgian* zinc* factory* and* from* an* invasive*
human* infection,* respectively)* than* in* the* CopB* proteins* of* C) metallidurans* strains* isolated* from*
spaceHrelated*environments*such*as*C.)metallidurans*NA1,*C.)metallidurans*NA4*and*C.)metallidurans*
NE12* (Mijnendonckx* et* al.,* 2013;* Monsieurs* et* al.,* 2013).* However,* further* analyses* of* the* CopB*
protein* sequences* from* strains* isolated* from* extreme* biotopes* will* be* required* to* confirm* this*
relationship* between* the* methionine* content* and* the* biotope* wherein* the* strains* were* found.* As*
suggested*by*the*results*obtained*by*S.*van*Aelst*regarding*the*copB*mutants,*it*might*be*possible*
that*the*plasmidHencoded*CopB*protein*from*C.)metallidurans*CH34*may*be*involved*in*the*bacterial*
survival*at*high*copper*concentrations.*
In* an* attempt* to* improve* our* knowledge* of* the* physiological* role* of* CopB* and* to* clarify* its*
copperHbinding* abilities,* three* recombinant* proteins* were* overexpressed* and* purified* during* this*
work.* Respectively,* they* corresponded* to* (i)* the* fullHlength* plasmidHencoded* CopB* protein* from* C.)
metallidurans* CH34,* (ii)* its* methionineHrich* NHterminal* extremity* named* CopB(Met),* and* (iii)* its* CH
terminal*domain,*which*is*named*CopB251H495.*
Aggregation/solubility* problems* were* encountered* with* these* recombinant* proteins,* forcing*
us* to* work* with* freshlyHpurified* proteins.* In* the* absence* of* metal* ions,* recombinant* protein*
characterization* by* circular* dichroism* was* in* accordance* with* the* secondary* structure* predictions.*
Indeed,*the*CopB(Met)*protein*was*mainly*found*to*possess*an*unordered*conformation,*while*the*
fullHlength*CopB*protein*contained*41%*strands,*5%*helices,*28%*turns*and*26%*unordered*structure.*
It*was*impossible*to*characterize*the*CopB251H495*recombinant*protein*due*to*aggregation*problems*in*
the* absence* of* urea.* Overexpressed* in* E.) coli* as* inclusion* bodies,* the* extraction* of* CopB251H495*
required* denaturation* with* 8* M* urea* treatment* because* the* treatment* with* detergent* was*
unsuccessful.*Such*a*drastic*treatment*did*not*allow*us*to*enzymatically*remove*the*polyhistidineHtag*
required*for*purifying*the*protein*nor*characterize*the*protein*by*spectroscopic*techniques*because*
urea* absorbs* at* low* wavelengths.* All* attempts* to* remove* or* decrease* the* concentration* of* urea*
resulted*in*the*precipitation*of*the*protein.*Moreover,*in*the*presence*of*copper*or*silver*ions,*the*
fullHlength* CopB* protein* was* completely* unstable,* leading* to* aggregation/precipitation* of* the*
recombinant*protein*and*preventing*its*characterization.*The*CopB(Met)*characterization*by*circular*
184*
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Concluding*Remarks*and*Future*Prospects*
*
resp* metal* ions* and* (ii)* the* addition* of* metal* ions* appears* to* increase* the* hydrophobicity* of* the*
different*recombinant*proteins*in)vitro.*
*
*
185*
*
Concluding*Remarks*and*Future*Prospects*
*
*
*
186*
*
Concluding*Remarks*and*Future*Prospects*
*
dichroism* did* not* show* any* significant* spectral* change* irrespective* of* the* metal* tested,* and* the*
observed* loss* of* the* CD* signal* might* suggest* a* likely* aggregation/precipitation* of* the* protein.*
Consequently,* it* can* be* concluded* that* (i)* the* CopB(Met)* recombinant* protein* remains* largely*
unfolded* in* the* presence* of* metal* ions* and* (ii)* the* addition* of* metal* ions* appears* to* increase* the*
hydrophobicity*of*the*different*recombinant*proteins*in)vitro.*
Synthetic*peptides*derived*from*the*methionineHrich*NHterminal*extremity*of*CopB*were*then*
produced*and*allowed*us*to*circumvent*problems*encountered*with*recombinant*proteins*and*clarify*
molecular*interactions*between*methionineHrich* motifs* and* metal* ions.*Therefore,*through*the*use*
of* complementary* techniques* (MS,* CD* and* RMN)* it* was* shown* that,* upon* additions* of* copper* or*
silver*ions,*the*native*peptide*(AcHQGSMQGMDHSKMQGMD)*was*able*to*bind*one*copper*ion*[Cu(I)*
or*Cu(II)]*or*one*silver*ion.*This*methionineHrich*motif*preferentially*bound*Cu(I)*ions*with*a*moderate*
affinity* (KD* ~5* x* 10–5* M)* over* either* Cu(II)* or* Ag(I)* (KD* ~7* x* 10–5* M,* respectively)* in* a* relatively*
unfolded*binding*site*involving*the*four*methionines*and*one*histidine*residue,*as*shown*by*circular*
dichroism* and* resonance* magnetic* nuclear* spectroscopy.* This* moderateHaffinity* binding* suggests*
that*(i)*the*CopB*protein*is*not*a*metallochaperone*such*as*the*CopK*protein*(KD*~*2*x*10–11*M)*and*(ii)*
high* copper* concentrations* are* required* to* saturate* the* protein.* Moreover,* preliminary* results*
obtained* by* mass* spectrometry* showed* that* the* histidine* residue* appears* to* be* more* involved* in*
Cu(II)Hbinding.*However,*further*investigation*is*required*to*more*precisely*define*the*nature*of*the*
copperHbinding*site.*It*could*be*useful*and*interesting*to*study*the*same*peptides*labeled*with*15N*or*
a* combination* of* 15N* and* 13C,* which* can* help* to* reduce* the* complexity* of* spectra* and* obtain* new*
correlations*between*atoms*for*more*complete*structural*information*(e.g.,*backbone*torsion*angles,*
or*distances*between*nuclei*in*the*peptide,*etc.).*
Investigations* by* mass* spectrometry* of* the* double* motif* peptide* showed* that* it* was* able* to*
bind*two*Cu(I)*ions*(one*metal*ion*per*methionineHrich*motif),*in*agreement*with*results*mentioned*
above.* Consequently,* in* our* experimental* conditions,* several* lines* of* evidence* suggest* that* CopB*
methionineHrich* motifs* might* bind* ten* copper* or* silver* ions* (one* ion* per* motif)* with* moderate*
affinity* in* relatively* unstructured* binding* sites.* However,* it* cannot* be* excluded* that* some*
methionineHrich*motifs*might*partially*be*reorganized*to*coordinate*metal*ions.*
With* regard* to* the* conserved* CHterminal* domain,* predicted* as* being* structured,* it* might*
legitimately* be* wondered* if* this* extremity* might* form* a* channel* across* the* membrane.* It* was*
experimentally*demonstrated*by*immunogold*labelling*that*the*plasmidHencoded*CopB*protein*was*
an*outer*membraneHassociated*protein,*in*agreement*with*the*previous*predictions*of*its*localization*
(Bondarczuk* and* PiotrowskaHSeget,* 2013;* Mergeay* et* al.,* 2009;* Mergeay* et* al.,* 2003).* Moreover,*
187*
*
Concluding*Remarks*and*Future*Prospects*
*
subcellular* fractionation* experiments* coupled* with* highHsalt/detergent* extraction* treatments*
suggest*that*the*protein*of*interest*might*be*associated*to*the*hydrophobic*core*of*the*membrane.*
The* transmembrane* protein* topology* predictions* using* the* program* PREDHTMBB* showed* that* the*
CopB251H495* protein* might* form* a* 12Hstranded* βHbarrel* in* the* outer* membrane.* Although* this*
prediction* is* in* accordance* with* the* results* obtained* by* circular* dichroism* (i.e.,* 41%* beta* strands*
predicted*for*the*fullHlength*recombinant*protein),*it*is*possible*that*this*topology*prediction*might*
be* incorrect.* Indeed,* this* prediction* does* not* correspond* to* any* previously* reported* category* of*
transporters* such* as* OmpC* (16* stands)* and* maltoporin* (18* strands).* Moreover,* conducted*
electrophysiological*experiments*do*not*allow*us*to*conclude*to*a*putative*channel*activity*for*this*
protein,* presumably* due* to* (i)* the* misfolding* of* the* fullHlength* recombinant* protein,* (ii)* the* likely*
folding* of* the* flexible* NHterminal* extremity* inside* the* barrel,* which* might* hamper* the* metal* ion*
transfer,*or*(iii)*the*CopB*protein*might*not*form*a*channel*though*the*membrane.*In*the*future,*it*
will* be* very* interesting* to* combine* the* direct* refolding* of* the* fullHlength* recombinant* protein* into*
liposomes* with* proteolytic* digestion* in* order* to* specifically* characterize* the* transmembrane*
topology* (Ge* et* al.,* 2014).* Moreover,* due* to* the* likely* folding* of* the* flexible* NHterminal* extremity*
which* might* hamper* the* metal* ion* transfer,* investigations* of* the* CopB251H495* recombinant* protein*
(i.e.,* without* the* flexible* NHterminal* extremity* from* AA* 1* to* 250)* will* be* achieved.* The* likely*
spontaneous*refolding*of*CopB251H495*into*artificial*lipid*membranes*may*help*us*circumvent*problems*
encountered*with*the*fullHlength*CopB*protein*and*evaluate*the*potential*formation*of*a*βHbarrel*to*
potentiate*efflux*out*of*the*cell.*
Another*aspect*of*this*work*was*dedicated*to*the*identification*of*potential*CopBHinteracting*
partners* using* a* chemical* crossHlinking* approach* combined* with* immunoprecipitation* and* mass*
spectrometry* analysis.* Chemical* crossHlinking* gives* a* snapshot* of* a* molecule's* environment* and*
combined* with* mass* spectrometry,* constitutes* a* very* promising* and* relevant* method* to* map*
proteinHprotein* interactions.* This* technique* allowed* us* to* highlight* the* CopA* protein,* a* putative*
multicopper* oxidase,* as* a* significant* partner* of* the* CopB* protein.* This* result* is* not* surprising* and*
appears* consistent* as* copA* and* copB* genes* are* contiguous* in* the* plasmid* and* chromid* of* C.)
metallidurans* CH34,* as* well* as* in* the* genome* of* numerous* copperHresistant* organisms* such* as* P.)
syringae*pathovar*tomato*and*some*E.)coli*strains*(HernándezHMontes*et*al.,*2012).*Moreover,*the*
CopA* protein* contains* 36* methionine* residues* mainly* arranged* as* five* CHterminal* MGGM* motifs*
together* with* 21* histidine* residues.* Consequently,* it* is* tempting* to* speculate* that* methionineHrich*
extremity* of* CopA* might* dock* by* direct* interaction* with* the* methionineHrich* sites* of* CopB* protein,*
which*might*transport*copper*out*of*the*cell*in*the*event*that*CopB*acts*as*a*cargo*transport.*But,*it*
can*also*be*hypothesized*that*CopB*might*give*its*copper*ions*to*CopA,*potentially*converting*Cu(I)*
188*
*
Concluding*Remarks*and*Future*Prospects*
*
ions*to*the*less*toxic*Cu(II)*form*and*subsequently*conducting*copper*to*other*protein*partners*(e.g.,*
Sil*or*Cus*tripartite*efflux*systems).*However,*further*investigations*are*required*to*identify*the*metal*
transfer*direction,*notably,*by*determining*the*KD*value*of*the*CopA*protein*in*the*presence*of*Cu(I)*
ions.*It*would*be*also*interesting*to*identify,*understand,*and*characterize*the*potential*binding*sites*
at*the*interface*of*the*two*proteins.*These*experiments*will*require*the*use*of*tailored*software*able*
to* interpret* complex* MS/MS* spectra* obtained* following* chemical* crossHlinking.* Moreover,*
considering* the* promising* results* obtained* with* chemical* crossHlinking* experiments,* this* technique*
could* also* be* applied* to* the* different* Cop* proteins* overexpressed* and* purified* to* date* (e.g.,* CopI,*
CopT,* CopK,* CopM,* etc.)* to* generate* a* global* map* of* CopHinteracting* partners* involved* in* copper*
resistance*in*C.)metallidurans*CH34,*together*with*detailed*mapping*of*the*interaction*interfaces.*
Although* numerous* questions* remain* unanswered,* the* results* obtained* during* this* study*
demonstrated* four* evidences.* First,* the* CopB* protein* is* associated* to* the* hydrophobic* core* of* the*
outer*membrane*in*C.)metallidurans*CH34.*Second,*the*CopB*protein*does*not*function*as*a*copper*
pump*in*our*experimental*conditions,*even*if*this*hypothesis*cannot*be*completely*excluded.*Third,*
the*role*of*CopB*appears*to*be*connected*to*the*one*of*the*CopA*protein.*And*finally,*the*saturation*
state* of* CopB* occurs* at* high* copper* concentrations,* suggesting* that* it* might* be* involved* in* the*
bacterial*survival*at*high*copper*amounts.*Interestingly,*the*plasmidHencoded*CopB*protein*was*also*
the*major*protein*identified*in*the*OMV*proteomic*content.*
Indeed,*the*second*part*of*this*thesis*was*dedicated*to*the*understanding*of*the*copper*fate*in*
the* cell.* Often,* effluxHbased* mechanisms* developed* by* microorganisms* to* resist* high* metal*
concentrations*are*linked*to*physiological*changes*that*lead*to*bioprecipitation*and*sequestration*of*
metals* around* the* bacterial* cell* (Diels* et* al.,* 2009;* Mahvi* and* Diels,* 2004;* Mergeay,* 1997).* Some*
copperHresistant*bacteria*harboring*plasmidHborne*copper*resistance*genes*are*known*to*sequester*
this* metal* within* the* cell's* periplasm* (e.g.,* E.) coli* harboring* the* PcoHplasmid* pRJ1004* form* brown*
colonies*when*grown*in*the*presence*of*CuSO4*and*P.)syringae*carrying*the*plasmid*pT23D*with*the*
copABCDRSHsystem* turn* bright* blue* in* the* presence* of* copper* salt)* (Magnani* and* Solioz,* 2007)* or*
cytoplasm* (e.g.,* formation* of* black* copper* sulfide* precipitate* in* Mycobacterium) scrofulaceum)*
(Bruins*et*al.,*2000;*Mergeay,*1991).*Intracellular*accumulation*is*a*very*useful*strategy*that*prevents*
exposure*to*essential*cellular*components,*avoiding*the*reentry*of*toxic*metals*and*the*consequent*
energetic*exhaustion*of* bacteria.*Nevertheless,*such*a*sequestration*has*never*been*reported*in*C.)
metallidurans*CH34*in*the*presence*of*copper.*Our*multidisciplinary*investigations*have*definitively*
shown* that* copper* has* an* impact* on* the* morphology* and* ultrastructural* organization* of* C.)
metallidurans*CH34*and*its*mutant*derivatives*that*differ*in*their*plasmid*content.*More*importantly,*
189*
*
Concluding*Remarks*and*Future*Prospects*
*
our* results* have* allowed* us* to* highlight* a* vesiculation* phenomenon* upon* copper* exposure* never*
described*in*C.)metallidurans*CH34*to*date.*
The* vesiculation* phenomenon* is* common* in* gramHnegative* bacteria* (Kulp* and* Kuehn,* 2010).*
By*virtue*of*the*metabolic*cost*implicit*in*replacing*the*lipids*and*proteins,*it*is*difficult*to*imagine*
that* OMV* formation* would* be* purposeless* (Kulp* and* Kuehn,* 2010).* Indeed,* depending* on* the*
organisms*from*which*they*originated,*OMVs*may*have*a*myriad*of*biological*roles*in*the*growth*and*
survival* of* bacteria* (Kulp* and* Kuehn,* 2010).* The* protective* role* of* OMVs,* as* observed* in* C.)
metallidurans* CH34* upon* copper* exposure,* has* already* been* identified* in* other* gramHnegative*
bacteria* (e.g.,* Pseudomonas) aeruginosa* (MashburnHWarren* et* al.,* 2008)* or* solventHtolerant*
Pseudomonas) putida* strains* (Kobayashi* et* al.,* 2000))* under* conditions* of* environmental* stress*
affecting* the* cellular* envelope.* Mechanisms* by* which* bacteria* produce* OMVs* are* not* fully*
understood,* but* it* is* likely* that* multiple* factors* may* collectively* contribute* to* their* biogenesis.*
Previous*studies*of*OMVs*have*established*that*such*a*phenomenon*is*regulated*by*environmental*
factors* (e.g.,* temperature,* nutrient* availability,* exposure* to* toxic* agents,* etc.),* induction* of* some*
stress*response*pathways,*and*overexpression*of*periplasmic*proteins.*It*appears*that*production*of*
OMVs*is*also*regulated*at*the*genetic*level*(Chatterjee*and*Chaudhuri,*2012;*Kulp*and*Kuehn,*2010;*
McBroom*and*Kuehn,*2007).*
Global*proteome*analysis*of*OMVs*released*upon*copper*exposure*showed*a*strong*significant*
enrichment*in*periplasmic,*outer*membrane*and*extracellular*proteins.*Interestingly,*numerous*Cop*
proteins* (i.e.,* CopB1,* A1,* C1,* H,* I,* K* and* G)* involved* in* copper* resistance* in* C.) metallidurans* CH34*
were*identified*in*the*OMV*proteome.*The*Cop*proteins*are*known*to*be*localized*in*the*periplasmic*
space*and*to*be*able*to*bind*Cu(I)*and/or*Cu(II)*ions.*Moreover,*an*appreciable*copper*content*was*
also*detected*associated*to*these*vesicles*in*comparison*to*the*total*cellHassociated*copper*content*
in*C.)metallidurans*CH34.*This*vesiculation*event*was*only*observed*in*the*presence*of*copper*ions*
since*no*vesicles*were*observed*with*redoxHinert*metal*ions*in*our*experimental*conditions*(i.e.,*Ni(II)*
and* Zn(II)* ions).* Although* the* transcriptomic* profiles* of* those* three* metals* are* similar,* proteomic*
evidences* showed* that* the* relative* abundance* of* the* Cop* proteins* upon* copper* exposure* appears*
much*more*important*than*upon*nickel*exposure*(Derock*et*al.,*in*preparation).*Moreover,*only*few*
periplasmic* Cop* proteins* are* known* to* bind* zinc* and* nickel* ions.* Consequently,* it* might* be*
hypothesized* that* the* copper* binding* to* major* periplasmic* Cop* proteins* changes* their*
physicochemical* properties* and* that* this* event* might* be* one* of* the* key* elements* underlying* the*
vesiculation*phenomenon*(i.e.,*in*particular*the*CopB*protein).*
190*
*
Concluding*Remarks*and*Future*Prospects*
*
In*an*attempt*to*better*understand*the*mechanisms*underlying*the*vesiculation*process*in*C.)
metallidurans*CH34,*a*number*of*mutants*derived*from*the*wildHtype*strain*were*investigated*upon*
copper* exposure* by* microscopy.* Although* it* appeared* that* some* genetic* elements* carried* by*
plasmids* pMOL28* and* pMOL30* might* be* associated* with* this* vesiculation* phenomenon* (i.e.,* cop*
genes* on* the* pMOL30* plasmid),* preliminary* results* obtained* did* not* allow* us* to* identify* which*
specific* genes* were* involved* in* this* mechanism.* Further* investigations* will* be* required* to* fully*
understand*the*mechanism*of*OMV*biogenesis*in*C.)metallidurans)CH34*upon*copper*challenge.*As*a*
first* step* toward* this* goal,* it* would* be* interesting* to* use* mass* spectrometry* to* examine* the*
proteome* of* OMVs* produced* by* the* different* mutant* derivatives,* which* differ* by* their* plasmid*
content,* in* order* to* draw* a* global* map* of* proteins* involved* in* the* mechanism* of* vesiculation* and*
identify* conserved* components.* Another* interesting* prospect* would* be* to* quantitatively* compare*
the*protein*contents*of*pure*OMVs*and*OMs*using*metabolic*labeling*with* 15N;*this*would*allow*for*
the* precise* determination* of* the* protein* compositions* of* OMV* and* OM* fractions,* allowing* for* the*
observation* of* a* possible* enrichment* or* depletion* of* proteins* and* lipids* in* OMVs.* It* would* also* be*
important*to*determine*the*quantity*of*copper*in*purified*OMVs*isolated*from*the*different*mutant*
derivatives*in*order*to*correlate*potential*genetic*elements*with*an*increase*or*decrease*in*the*ability*
to* accumulate* copper* in* OMVs.* Lastly,* the* study* of* the* cellular* lipid* profile* in* the* presence* and*
absence* of* copper* would* also* be* very* useful* to* highlight* a* likely* lipid* peroxidation* phenomenon,*
potentially*relevant*to*the*understanding*of*vesicle*biogenesis.*
Altogether,* the* results* obtained* during* our* investigations* suggest* that* these* vesicles* play* a*
defensive*role*allowing*for*bacterial*survival.*It*is*tempting*to*hypothesize*that*OMVs*might*be*a*way*
to*secrete*harmful*copper*compounds*most*likely*bound*to*Cop*proteins.*This*phenomenon*might*be*
a* putative* clustering* mechanism* offering* a* significant* benefit* to* the* bacteria* in* comparison* to* the*
efflux*mechanisms,*which*are*energetically*less*favorable.*
In* conclusion,* this* thesis*reveals* new* insights* into* the* fate* of* copper* within* C.) metallidurans*
CH34* and* promotes* a* better* understanding* of* the* role* of* CopB* in* copper* resistance.* Numerous*
questions*remain*unanswered,*but*the*results*presented*within*this*thesis*have*revealed*interesting*
horizons*and*paved*the*way*for*promising*experiments*in*the*future.*
"
"
"
191*
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Concluding*Remarks*and*Future*Prospects*
*
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