UNIVERSIDADE!DE!LISBOA!
FACULDADE!DE!FARMÁCIA!
DEPARTAMENTO!DE!MICROBIOLOGIA!E!IMUNOLOGIA!
!
THE!LSR!SYSTEM:!A!SWITCHER!FOR!BACTERIAL!
BEHAVIOURS?!
!
!
Jorge!André!Matias!Pereira!
!
DISSERTAÇÃO!
MESTRADO!EM!CIÊNCIAS!BIOFARMACÊUTICAS!
2013!
UNIVERSIDADE DE LISBOA
FACULDADE!DE!FARMÁCIA!
DEPARTAMENTO!DE!MICROBIOLOGIA!E!IMUNOLOGIA!
!
THE!LSR!SYSTEM:!A!SWITCHER!FOR!BACTERIAL!
BEHAVIOURS?!
Dissertação!orientada!por:! Doutora!Karina!Xavier!(IGC)!e!Professora!Doutora!Madalena!Pimentel!(FFUL)!!
Jorge!André!Matias!Pereira!
MESTRADO!EM!CIÊNCIAS!BIOFARMACÊUTICAS!
2013!
!
2013
!
THE!LSR!SYSTEM:!A!SWITCHER!FOR!BACTERIAL!
BEHAVIORS?
!
Jorge!André!Matias!Pereira!
MASTER’S!THESIS!
2013!
This! thesis! was! fully! performed! at! the! Bacterial! Signalling! Laboratory! of! Instituto! Gulbenkian! de! Ciência! (Oeiras,! Portugal)! under! the! direct! supervision! of! Dr.! Karina! Xavier.! Professor! Madalena! Pimentel! was! the! internal! designated! supervisor! in! the! scope!of!the!Master!in!Biopharmaceutical!Sciences!of!the!Faculty!of!Pharmacy!of!the! University!of!Lisbon.!
ACKNOWLEDGEMENTS..
First"and"foremost,"I"would"like"to"thank"my"supervisor,"Karina"Xavier,"for"her" guidance"and"support"throughout"this"thesis"with"her"knowledge"and"friendship."Her" persistence" and" passion" for" Science" has" inspired" and" fed" my" wish" of" pursuing" a" scientific" career." I" would" also" like" to" express" my" gratitude" towards" my" internal" supervisor,"Professor"Madalena"Pimentel,"for"all"her"encouragement"and"advice"during" this"stage."" " " " " " " " " "
" A" warm" and" very" special" “thank" you”" goes" to" each" of" my" colleagues" in" the" Bacterial"Signalling"Lab"at"IGC:""
Rita" –" for" setting" me" up" in" the" lab" and" for" all" the" help," guidance," encouragement" and" extreme" generosity." I" really" appreciate" everything" I’ve" learned"with"you"over"this"time."
Oz"–" Dostluğun,"desteğin"ve"tüm"güzel"anılarımız"için"teşekkür"ederim.""
Ana" Rita" –" for" sharing" with" me" the" preVadventures" of" the" PhD," for" the" friendship"and"the"advice"about"writing"a"good"thesis."I"wish"you"all"the"best"in" this"next"stage." Pol"–"" for"rising"up"the"good"mood"in"the"lab"even"more"and"for"helping"me" when"I"needed"the"most." Jess"–"for"the"comforting"“Good"morning”"every"day,"all"the"nice"conversations," the"good"advices"and"constructive"critiques"that"made"me"improve"my"work." Filipe" –" for" your" good" sense" of" humor" that" contributed" to" an" even" better" ambiance"in"the"lab."
Catarina" Pereira" –" for" all" the" help" you" gave" me" to" start" my" project" and" for" showing"me"the"wonders"of"Quorum"Sensing."
Patrícia"V"for"all"your"interest"in"my"work"and"your"advice."
I" would" also" like" to" thank" the" Evolutionary" Biology" Lab" for" the" scientific" discussions"and"for"making"our"“chinhos”"even"funnier."" Last,"but"not"least,"I"would"like"also"to"acknowledge"my"family"and"friends:" Ao"meu"pai"e"à"Lídia"por"terem"estado"sempre"presentes,"por"toda"a"paciência," apoio"e"carinho"que"tanto"me"reconfortaram." À"minha"mãe,"ao"Gustavo"e"ao"Guga"pelo"por"todo"o"apoio"e"positivismo"que" me"levantaram"o"ânimo"mesmo"nas"alturas"mais"difíceis."
Aos" meus" amigos" por" toda" a" amizade" e" pelas" fortes" gargalhadas" que" me" relembram"sempre"o"quão"a"Vida"é"bela.""
" Thank"you"all"for"your"friendship"and"for"everything"you"have"taught"me."These" have"been"amazing"times!"
ABSTRACT
.
Quorum" sensing" is" a" cellVtoVcell" communication" mechanism" that" enables" bacteria" to" monitor" population" density" and" synchronize" behaviors" on" a" communityV wide"scale"by"collectively"regulating"their"gene"expression."This"process"is"mediated"by" the"production,"release"and"detection"of"signaling"molecules"called"autoinducers."The" bestVstudied"signal"that"mediates"interVspecies"communication"is"autoinducerV2"(AIV2)." This" molecule" has" been" shown" to" regulate" important" bacterial" behaviors" such" as" virulence,"biofilm"formation"and"antibiotic"resistance."In"enteric"bacteria,"this"signal"is" imported"and"processed"by"a"system"called"Lsr."The"biological"function"of"Lsr"is"still"a" matter"of"debate."It"was"proposed"that"through"this"system"AIV2"regulates"phenotypes" other" than" only" its" incorporation" and" degradation." However," further" studies" are" needed"to"fully"prove"this"hypothesis."" " " " " "" The"aim"of"this"research"work"is"to"elucidate"the"role"of"Lsr"in"the"regulation"of" AIV2Vdependent"behaviors,"by"constructing"and"testing"phenotypically"a"set"of"mutants" on" the" AIV2" signalling" system" in" two" different" model" organisms:" Escherichia) coli) and" Photorhabdus) luminescens." E.) coli" mutants" were" tested" for" their" ability" to" form" biofilms."Results"from"this"study"indicate"that"AIV2"signalling"through"the"Lsr"system"is" not" involved" in" the" regulation" of" biofilm" formation" in" this" bacterium." However," this" work" reveals" for" the" first" time" a" phenotype" for" the" lsrF) mutant," which" raises" the" hypothesis" that" LsrF," an" enzyme" involved" in" AIV2" processing," is" important" for" the" detoxification" of" the" AIV2" degradation" subproducts." Furthermore," mutants" of" the" Lsr" system" in" P.) luminescens" were" constructed" and" the" function" of" this" system" in" AIV2" internalization"was"determined."The"constructed"mutants"are"currently"being"analyzed" for"their"ability"to"form"biofilms."In"future"work"these"mutants"can"be"used"to"test"the" role" of" Lsr" in" symbiosis" and" pathogenesis" using" in) vivo" models" for" P.) luminescens." " Understanding" how" the" ability" to" communicate" and" regulate" bacterial" behaviors" is" dependent" on" the" Lsr" system" could" aid" the" development" of" novel" strategies"to"manipulate"bacterial"controlled"phenotypes"to"our"profit.""
Keywords:
.
Quorum" Sensing;" autoinducerV2;" Lsr" system;" cellVtoVcell" communication;"biofilm"formation;"bacterialVhost"interactions"
RESUMO.
A"detecção"de"quorum"é"um"processo"usado"por"bactérias"para"determinarem" a" sua" densidade" populacional," bem" como" coordenarem" os" seus" comportamentos" a" nível" comunitário." Este" mecanismo" é" caracterizado" pela" produção," libertação" e" detecção" de" moléculas" sinalizadoras" denominadas" autoindutores." O" sinal" melhor" estudado"que"medeia"a"comunicação"entre"diferentes"espécies"é"o"autoindutorV2"(AIV 2)." Esta" molécula" regula" comportamentos" bacterianos" importantes," tais" como" a" virulência,"a"formação"de"biofilmes"e"a"resistência"a"antibióticos."Nas"enterobactérias" este" sinal" é" importado" e" processado" pelo" sistema" Lsr." A" função" fisiológica" de" Lsr" é" ainda"controversa."Existe"a"possibilidade"do"AIV2"regular"outros"fenótipos"através"deste" sistema," que" não" apenas" a" sua" incorporação" e" degradação." Contudo," estudos" adicionais"são"necessários"para"provar"esta"hipótese."
O"objectivo"deste"trabalho"de"investigação"é"elucidar"o"papel"do"sistema"Lsr"na" regulação" dos" comportamentos" bacterianos" dependentes" de" AIV2," através" da" construção"e"teste"fenotípico"de"um"conjunto"de"mutantes"no"sistema"de"sinalização" do" AIV2" em" dois" organismosVmodelo" diferentes:" Escherichia) coli) and" Photorhabdus) luminescens."O"mutantes"de"E.)coli"foram"testados"para"a"sua"capacidade"de"formar" biofilmes." Os" resultados" deste" estudo" indicam" que" a" sinalização" mediada" por" AIV2" através"do"sistema"Lsr"não"está"envolvida"na"regulação"da"formação"de"biofilmes"nesta" espécie." Contudo," este" trabalho" apresenta," pela" primeira" vez," um" fenótipo" para" o" mutante" lsrF," que" levanta" a" hipótese" de" que" LsrF," uma" enzima" envolvida" no" processamento" do" AIV2," é" importante" para" a" destoxificação" dos" subprodutos" de" degradação"deste"sinal."Adicionalmente,"os"mutantes"no"sistema"Lsr"em"P.)luminescens) foram"construídos"e"foi"determinada"a"função"deste"sistema"na"internalização"do"AIV2." As" estirpes" construídas" estão" atualmente" a" ser" testadas" para" a" sua" capacidade" de" formar" biofilmes." Para" estudos" futuros," estes" mutantes" poderão" ser" utilizados" para" testar"o"papel"do"sistema"Lsr"na"simbiose"e"patogénese,"usando"modelos"in)vivo"para" P.)luminescens."
Compreender" se" a" capacidade" de" comunicação" e" de" regulação" dos" comportamentos" bacterianos" é" dependente" do" sistema" Lsr," poderá" potenciar" o"
desenvolvimento" de" novas" estratégias" para" manipular" os" fenótipos" bacterianos" para" proveito"do"ser"humano.""
Palavras>chave:" Detecção" de" quórum;" autoindutorV2;" sistema" Lsr;" comunicação"
célulaVaVcélula;"formação"de"biofilmes;"interações"bactériaVhospedeiro."
Contents'
ACKNOWLEDGEMENTS...i' ABSTRACT...iii' RESUMO...iv' INTRODUCTION' 1.'Bacterial+language:+Quorum+sensing...1+ 2'–+Broadcast+without+frontiers:+bacterial+signaling+within+and+across+ species...3+ 2.1.+Autoinducer>2>based+communication...3+ 2.2.+AI>2+in+signaling+and+metabolism………...+3+ 2.3.+Tuning+into+the+right+frequency:+listening+to+AI>2...+4+ 2.4.+AI>2+sensing+and+response+in+enteric+bacteria...+7+ 2.5.+Assembling+the+puzzle:+the+biological+significance+of+the+Lsr+system.10+ 3.+AI>2+mediated+regulation+of+biofilm+formation...11+4.' Photorhabdus+ luminescens:+ insights+ into+ AI>2>regulated+ phenotypes+ in+ entomopathogenic+bacteria………...+13+ 5.'Objectives...15+ ' CHAPTER'I'A'The$role$of$AI+2$signalling$through$Lsr$in$biofilm$formation$in!E.!coli$ 1.'Materials'and'methods...16'' 1.1.+Bacterial+strains+and+growth+conditions...16++ 1.2.+Genetic+and+molecular+techniques...17+ 3.1.+Preparation+of+P1+bacteriophage+lysates...19+ 3.2.+P1+phage+transduction...20+ 1.4.+Electrocompetent+cells+and+electroporation...20+ 1.5.+AI>2+activity+assay...+21+ 1.6.+Quantification+of+Crystal+Violet>stained+attached+cells...21+ 1.7.+Growth+of+bacterial+macrocolonies……….22+ 1.8.+Statistical+analysis...+22+ 2.'Results'and'discussion...22+ 2.1.$Construction$of$AI12$signalling$system$mutants$on$E.#coli#W3110...+23+ 2.2.$AI12$production$and$internalization$by$E.#coli$W3110$$ and$mutants$on$AI12$signalling$system...26$
2.3.$The$mutants$on$AI12$system$are$not$affected$on$biofilm$attachment$in$LB$ medium$...28$ 2.4.$Glucose$inhibits$biofilm$attachment$in$E.#coli$W3110...31$ 2.5.$The$lsrF$mutant$is$affected$in$biofilm$attachment$in$$ M9C$Cit$+$100$μM$FeSO4...34$ $ CHAPTER'II'−'The$role$of$Lsr$in$AI+2$uptake$in$P.!luminescens' 1.'Materials'and'methods...44'' 1.+Bacterial+strains+and+growth+conditions...44++ 1.2.$AI12$activity$in$P.#luminescens#cultures...44$ 1.3.$Genetic$and$molecular$techniques...45$ 1.4.$Construction$of$deletion$mutants$in$P.#luminescens$using$a$$ suicide$vector1based$approach...48+ 1.5.$Quantification$of$Crystal$Violet1stained$attached$cells...49$ 2.'Results'and'discussion...49+ 2.1.$Construction$of$AI12$signalling$mutants$on$Photorhabdus#luminescens...50+$ 2.2.$The$Lsr$system$of$Photorhabdus#luminescens$has$a$similar$functionality$$ to$that$of$Escherichia#coli$...54$ CONCLUDING$REMARKS$ ' ' ' ' ' ' ''''''''60' REFERENCES$ $ $ $ $ $ $ $ $ $$$$$$$$62$ APPENDICES' ' ' ' ' ' ' ' ' '''''''''''I' + $ $
INTRODUCTION.
1..Bacterial.language:.Quorum.sensing.
In" nature," microorganisms" face" everVchanging" environmental" conditions" for" which"they"have"to"constantly"adapt"to"in"order"to"maintain"homeostasis."
Quorum"sensing"(QS)"is"a"cellVtoVcell"signaling"mechanism"that"enables"bacteria" to"act"in"group,"by"synchronizing"behaviors,"such"as"in"multicellular"tissues."Typically," such"group"behaviors"are"only"productive"at"high"cellular"densities"and"are"performed" by"collectively"regulating"gene"expression"of"the"bacterial"population"(Lazdunski"2004," Reading" 2006," Bassler" 2006)." QS" involves" the" production," secretion" and" detection" of" diffusible"small"signaling"molecules"named"autoinducers"(AIs),"that"accumulate"in"the" extracellular" environment" as" bacteria" divide" (Bassler" 2006," Ng" WL" 2009)." When" a" threshold" concentration" is" reached" in" the" extracellular" milieu," gene" expression" is" coordinately" shifted" towards" the" development" of" group" behavior" such" as" the" production"of"virulence"factors,"toxins"and"secondary"metabolites"(such"as"pigments"or" antibiotics),"the"formation"of"biofilms,"conjugation,"competence,"bioluminescence"and" sporulation" (Williams" 2007," Ng" WL" 2009," Boyer" 2009)." Since" many" of" these" QSV regulated" phenotypes" are" important" for" the" establishment" and" maintenance" of" bacterialVhost"interactions,"interference"with"QS"processes"–"Quorum"Quenching"V"has" been"pointed"to"be"an"attractable"alternative"therapeutic"approach"to"antibiotics"for" treating"bacterial"infections,"since"it"offers"lower"selective"pressure"for"the"acquisition" of" resistance." " Therefore," understanding" the" molecular" mechanisms" underlying" QS" regulation" might" aid" the" design" of" promising" new" therapeutics" that" fight" bacterial" disease," or" to" take" advantage" of" beneficial" properties" of" microbes" to" develop" new" probiotics"(Reviewed"in"Pereira"2013a"and"Zhu"2013)."
QS" systems" are" diverse" but" their" molecular" mechanisms" can" be" distinguished" according" to" their" ability" to" promote" intraV" and" interVspecies" communication."The" paradigm"of"QS"regulation"for"GramVnegative"bacteria"is"exemplified"by"the"regulation" of" bioluminescence" production" in" Vibrio) fischeri) (Ng" WL" 2009)," where" QS" was" first" described" (Nealson" 1970)." The" AIs" produced" by" this" bacterium" are" acylVhomoserine" lactones"that"are"detected"by"LuxRVtype"response"regulators"(Figure"1A)."GramVpositive"
bacteria" use" mainly" modified" small" peptides" (AIPs)" as" their" QS" signals" and" their" detection" mechanism" relies" on" twoVcomponent" systems" (Figure" 1B)." Both" AHLs" and" AIPs"share"highly"specificity"since"they"foster"communication"within"the"same"species," or"in"the"later"case,"even"between"bacteria"belonging"to"the"same"strain." " When"it"comes"to"communication"between"different"bacterial"species,"the"best" well"studied"signal"is"AutoinducerV2"(AIV2)."This"molecule"is"produced"by"many"bacteria," including"GramVnegative"and"GramVpositive"species"(Chen"2002,"Federle"2003,"Xavier" 2003)."More"than"one"of"the"described"QS"circuits"can"coexist"in"one"bacterial"species" to"assure"a"fineVtune"regulation"of"behaviors"and"optimal"adaptation"to"its"ecological" niche"(Ng"WL"2009)." " Microbes"use"a"surprisingly"diverse"chemical"lexicon"to"communicate"and"there" is" an" increasing" collection" of" molecules" shown" to" act" as" AI," with" many" more" novel" “languages”"waiting"to"be"deciphered."In"this"work"focus"will"be"laid"on"AIV2Vmediated" communication"in"bacteria.""
Figure.1>.The.two.canonical.species>specific.quorum.sensing.circuits..(A)"The"LuxI/LuxR"paradigm"for"GramV
negative"bacteria."Red"pentagons"represent"acylVhomoserine"lactones."LuxI"is"the"autoinducer"synthase"and" LuxR" is" the" response" regulator" that" binds" intracellular" AHL" and" activates" transcription" of" quorum" sensing" controlledVgenes." (B)" TwoVcomponent" system" model" for" GramVpositive" bacteria." Yellow" tailedVpentagons" represent"autoinducing"peptides"after"processing"or"modification."When"the"histidine"kinase,"HK,"detects"the" autoinducer,"undergoes"autophosphorilation"and"transfers"the"phosphate"group"to"the"response"regulator," RR,"that,in"turn,"can"bind"DNA"and"regulate"quorum"sensing"target"genes."Adapted"from:"A"–"Waterfields"&" Bassler"2005;"B"–"Novick"&"Geisinger"2008.""
.
B " A"2..Broadcast.without.frontiers:.bacterial.signaling.within.and.across. species.
! 2.1..Autoinducer>2>based.communication.
AIV2"is"a"lowVweight"molecule"that"is"produced"and"detected"by"a"wide"variety" of"bacteria"and"constitutes"the"first"identified"interspecies"cellVtoVcell"communication" signal" (Waters" 2005," Vendeville" 2005," Hardie" 2008)." The" first" evidence" for" “communication"without"frontiers”"was"supported"by"the"work"of"Greenberg"in"which" the" addition" of" cellVfree" supernatant" from" other" bacterial" species" to" Vibrio) harveyi) cultures"led"to"the"production"of"light"(Greenberg"1979,"Bassler"1997,"Barnard"2007)" and" later" on," the" wide" distribution" of" its" synthase" gene," luxS," amongst" the" bacterial" kingdom,"having"been"identified"in"more"than"half"of"the"sequenced"genomes"(Surette" 1999,"Xavier"2003)."The"term"AIV2"was"first"coined"by"Bassler"and"colleagues"to"identify" the" second" QS" autoinducer" found" in" this" organism) for" the" regulation" of" bioluminescence" (Bassler" 1993)." This" signal" has" been" shown" to" regulate" many" other" important"bacterial"behaviors"(Table.1),"as"discussed"later."
2.2..AI>2.in.signalling.and.metabolism.
The" AIV2" synthase," LuxS," uses" as" substrate" SVribosylmethionine" (SRH)," an"
intermediate"of"the"Activated"Methyl"Cycle"(Figure.2),"an"important"pathway"involved" in"central"metabolism."For"this"reason"the"strict"role"of"AIV2"as"a"signaling"molecule"has" been"questioned."The"phenotypes"attributed"to"luxS"disruption"could"be"viewed"as"a" direct" consequence" of" metabolic" disturbance" of" the" Activated" Methyl" Cycle."This" important" metabolic" pathway" recycles" SVadenosylmethionine" (SAM)," the" major" methyl" donor" used" by" celular" methyltransferases" to" carry" out" vital" celular" processes" such" as" nucleic"acid"and"protein"methylation"(Shauder"2001"and"Grillo"2008)."The"LuxS"enzyme"is" involved"in"the"last"step"of"the"cycle"by"regenerating"Homocysteine,"with"production"of" DPD# (4,5(dihydroxy(2,3(pentanedione),# the" linear" form" of" AIV2." " In" solution"DPD" cyclizes" and" spontaneously" rearranges" into" chemically" distinct" molecular" forms" (Chen" 2002," Vendeville" 2005)" that" are" recognized" by" different" bacterial" species," and" are" generically"designated"AIV2"(Xavier"2003"and"Shauder"2001)"(Figure.2)."
Experimentally,"complementation"of"luxS"mutants"with"synthetic"AIV2"can"prove" the"role"of"this"molecule"as"a"signal"by"discriminating"between"metabolic"and"signaling" effects."This"approach"has"been"successfully"performed"in"many"studies"showing,"that" in"some"species"AIV2"has"evolved"to"become"a"QS"signal"with"the"capacity"of"integrating" information"of"the"metabolic"status"of"the"cell"to"the"regulation"of"gene"expression."In" fact,"other"QS"signalling"molecules,"such"as"AHLs"and"cholerae"autoinducerV1,"SAM"is" also"the"precursor"for"its"synthesis." " Figure.2.–.The.Activated.Methyl.Cycle.and.the.synthesis.of.AI>2.in.LuxS>containing.bacteria..Transfer"of"
the" methyl" group" from" SAM" to" an" aceptor" molecule" generates" a" toxic" compound," SV adenosylhomocysteine"(SAH)."In"bacteria"harboring"the"LuxS"enzyme,"SAH"is"processed"in"two"steps:""Pfs" uses"SAH"as"substrate"to"first"generate"adenine"and"SVribosylhomocysteine"(SRH)."SRH"is"then"converted" into" homocysteine" and" DPD," the" linear" formo" f" AIV2." The" regenation" of" Homocysteine" enables" the" reclycling"of"SAM"via"the"de)novo"synthesis"of"methionine"(MET)."Unstable"DPD"undergoes"spontanious" cyclization"rearrangements"into"chemically"distinct"molecular"forms"collectively"designated"AIV2."(2R,4S)V 2VmethylV2,3,3,4Vtetrahydroxytetrahydrofuran" (RVTHMF)" is" recognized" by" LsrBVtype" receptors" from" enteric" bacteria," while" furanosyl" borate" diester" (SVTHMFVborate)" is" detected" by" LuxPVtype" receptors" form"Vibrio)spp.""Modified"from"Hardie"&"Heurlier"2008.""
#
2.3..Tuning.in.to.the.right.frequency:.listening.to.AI>2..
(Chen" 2002," Miller" 2004):" LuxPVtype" proteins," which" are" found" in" Vibrio" spp." and" recognize"a"borated"form"of"AIV2,"and"the"LsrB"family"in"enteric"bacteria,"first"described" in"Salmonella)enterica)serovar" Typhimurium" and) Escherichia) coli," but" also" present" in" members"of"the"Rhizobiaceae"and"Bacillaceae"families"(Pereira"2009)."LsrB"receptors" bind" a" different" conformation" that" does" not" require" an" atom" of" boron," the" (2RV4S)< methylV2,3,3,4Vtetrahydroxytetrahydrofuran"(RVTHMF)"(Figure.2)."A"RbsBVlike"protein"in) Aggregatibacter)actinomycetemcomitans,"sharing"high"homology"with"this"periplasmic" binding"component"of"an"ABCVtype"transporter"of"E.)coli,"has"been"proposed"as"AIV2" receptor"in"addition"to"LsrB"(James"2006;"Shao"2007a,b)."However,"in"this"case,"further" structural" studies" in" complex" with" ligand" are" needed" to" clarify" whether" this" protein" represents"a"third"class"of"AIV2"receptors."More"AIV2"detection"systems"are"expected"to" exist" in" nature" since" different" bacterial" species" have" been" shown" to" respond" to" this" signal"although"they"lack"homologous"receptors"to"the"ones"already"described"(Table.
1).""
Focus"will"be"laid"on"QS"regulation"through"LsrB–type"receptors"as"the"aim"of" the"present"work"is"to"study"the"role"of"a"system"that"uses"LsrB"as"the"mechanism"for" signal"recognition."
Table.1.–.Functions.regulated.by.AI>2.signal.a"(modified"from"Pereira"2013b)" . . . a"Whole"genome"expression"profiles"such"as"microarrays"analysis"were"not"included"in"this"table" " " " " """"""""""""""""""" b"DPD"supplemented"to"wild"type"strain"cultures" " " " " " " " " """""""""""""….." c"This"receptor"is"present"in"this"bacteria"but"the"connection"to"the"regulated"phenotypes"remains"to"be"demonstrated." "
2.4..AI>2.sensing.and.response.in.enteric.bacteria.
In"the"model"Escherichia)coli,"and"also"in"S."Tiphymurium,"AIV2"is"produced"and" exported" to" the" extracellular" environment" where" it" accumulates" as" cells" divide." However,"unlike"typical"AIs,"a"peak"of"AIV2"concentration"is"achieved)during"the"mid"to" late" exponential" phase" and" then" it" declines" abruptly" during" the" entry" to" stationary" phase" of" growth." This" rapid" removal" of" the" extracellular" AIV2" is" due" to" its" fast" incorporation" by" Lsr" (for" “LuxS" regulated”)," an" ABCVtype" transport" system," which" allows" its" processing" inside" the" cell" (Taga" 2001," Taga" 2003," Xavier" 2005a)." The" apparatus"of"the"Lsr"transport"system"is"encoded"in"the"lsr"operon"(Figure.3),"which"is" regulated"by"two"proteins,"LsrK"and"LsrR,"and"also"by"catabolite"repression"trough"the" cAMPVCRP"complex"(Taga"2001,"Wang"2005a,"Xavier"2005a).""
When"the"extracellular"concentration"of"AIV2"increases,"as"bacteria"divide,"it"is" first" internalized" by" a" low" affinity" Phosphotransferase" system" (PTS)" Vdependent" transport" (Pereira" 2012b)." Basal" levels" of" LsrR" repressor" are" expressed" since" lsrR) promoter" expression" is" always" higher" than" that" of" lsrACDBFG," preventing" the" premature"induction"of"the"Lsr"system"(Pereira"2012b)."LsrR"also"represses"the"lsrRK" operon,"transcribed"in"the"opposite"direction"of"lsr,"thus"regulating"its"own"expression" and"that"of"LsrK"(Wang"2005b,"This"2010)."AIV2"fast"internalization"(Figure.3)"starts"with" its"binding"to"the"ABC"transporter"by"interacting"with"the"periplasmic"receptor"LsrB"and" its" transport" through" the" two" transmembrane" domains," LsrC" and" LsrD," into" the" bacterial"cell."This"process"requires"energy"from"ATP"hydrolysis"catalyzed"by"LsrA,"the" ATPase"of"the"system"(Taga"2001,"Taga"2003,"Xavier"2005a)."The"imported"AIV2"is"then" phosphorylated"by"the"cytoplasmic"LsrK"kinase,"producing"AIV2VPhosphate"(AIV2VP),"the" inducer" of" the" lsr" operon" expression:" AIV2VP" binds" to" LsrR" for" relieving" the" transcriptional" repression" of" the" lsr" operon" (Taga" 2003," Xavier" 2007)." As" a" consequence,"the"expression"of"the"genes"encoding"the"Lsr"apparatus"is"induced"and" upregulated,"and"this"positive"feedback"loop"is"responsible"for"the"abrupt"depletion"of" AIV2"from"the"extracellular"medium"before"reaching"the"stationary"phase"(Taga"2001," Taga" 2003," Xavier" 2005a)." " Cytoplasmic" AIV2VP" is" further" processed" by" two" enzymes" codified" downstream" in" the" lsr" operon" (Figures. 3)," LsrG" and" LsrF" (Taga" 2003," Xavier" 2007,"Diaz"2009,"Marques"2011)."LsrG"is"an"isomerase"that"catalyses"the"isomerization"
of" AIV2VP" to" 3,4,4VtrihydroxyV2VpentanoneV5Vphosphate" (PVTPO)" (Marques" 2011)." Recently," João" Marques" in" the" laboratory" of" K." Xavier" showed" that" LsrF" processes" thisisomer" into" PhosphoVHidroxyacetone" and" AcetilVCoA," which" are" intermediates" of" glycolysis" and" the" Krebs" Cycle," respectively" (unpublished)." Therefore," through" the" action"of"these"two"enzymes,"transcription"termination"of"lsrACDB"is"achieved"and"the" signaling"cycle"ends"(Xavier"2007)."An"extra"protein,"LsrE,"whose"function"has"not"yet" been" identified," is" encoded" downstream" of" this" AIV2" processing" enzymes" in" the" lsr" operon"of"S.)Typhimurium,"but"not"in"E.)coli"(Taga"2003)."In"summary,"in"E.)coli,"AIV2"is" internalized,"processed"and"degraded"in"a"selfVinduced"way"via"the"Lsr"system."
!
Figure'3'–'AI,2'incorporation'and'processing'by'the'Lsr'transport'system'in'Escherichia)coli.' (A)'During'early'bacterial'growth,'extracellular'levels'of'AI92'are'low'and'the'expression'of'the'
Lsr' system' is' inhibited' due' to' the' action' of' the' LsrR' repressor.' (B)' As' cells' divide' AI92' accumulates' 'extracellularly' and' its'first' internalized' by' a'PTS9dependent' permease(s).' Once' inside'the'cell,'AI92'is'phosphorylated'by'the'LsrK'kinase.'The'activated'molecule'binds'the'LsrR' regulator'and'relieves'the'transcriptional'repression'of'the'lsr'operon.'(C)'The'Lsr9dependent' transport'starts.'The'expression'of'the'system'is'up9regulated'leading'to'a'rapid'depletion'of' the' extracellular' AI92' at' the' entry' to' stationary' phase.'AI929P'is' then' processed' by'LsrG'and' LsrF'to'terminate'the'signaling'cycle.'Pentagons'represent'AI92'molecule;'P'–'PTS9dependent' permease.'Adapted'from'Pereira'2013b.' ! lo g! (O D 60 0 ) lo g! (O D60 0 ) lo g! (O D 60 0 ) 0! 0! 1! 10! 1! 5! 50! 0! 2! 4! 6! 8! ! [A I/ 2] !( μM) Time!(h) 0! 0! 1! 10! 1! 5! 50! 0! 2! 4! 6! 8! ! [A I/ 2] !( μM) Time!(h) 0! 0! 1! 10! 1! 5! 50! 0! 2! 4! 6! 8! ! [A I/ 2] !( μM) Time!(h) A B C
2.5$–$Assembling$the$puzzle:$the$biological$significance$of$the$Lsr$system$
The! physiological! function! of! Lsr! system! is! not! yet! clear:! why! some! bacteria! produce!and!export!AI>2!to!then!uptake!and!degrade!it!is!still!a!controversial!issue.!As! the!Lsr!system!imports!self!and!non>self!AI>2,!it!has!been!suggested!that!it!may!be!a! mechanism! of! interfering! with! AI>2>based! processes! of! other! bacterial! species! that! inhabit! the! same! environment! and! rely! upon! this! molecule! for! signaling! purposes! (Xavier!2005b).!This!is!supported!by!the!observation!that,!not!only!when!E.#coli!is!co> cultured! with! V.# harveyi,# but! also! when! it! shares! the! same! environment! with! the! human!pathogenic!species#Vibrio#cholerae,!it!uses!the!Lsr!apparatus!to!internalize!AI>2! produced! by! the! two! species.! Consequently,! the! extracellular! levels! of! this! molecule! rapidly!decrease.!This!uptake!prevents!AI>2!regulated!behaviors!in!both!V.#harveyi!and! V.# cholerae.! Moreover,! Sinorhizobium# meliloti,# a! bacterium! that! lacks! the! luxS! gene! and,! therefore,! does! not! produce! AI>2,! is! also! capable! of! eliminating! the! signal! produced!by!other!bacteria!from!the!environment,!using!its!own!Lsr!system!(Pereira! 2008).!Studies!using!co>colonization!in#vivo!are!required!to!evaluate!the!relevance!of! this! phenomenon! of! interference! in! natural! contexts.! Interference! with! AI>2! is! proposed! to! confer! competitive! advantages! upon! Lsr>containing! bacteria! in! multispecies!consortia!through!the!disruption!of!signaling!in!nearby!bacteria!(Pereira! 2008).!However,! according! to! Social! Evolution! theories! it! is! unlikely! that! the! unique! purpose!of!the!Lsr!system!is!to!interfere!with!other!species.!It!was!also!proposed!that! Lsr>mediated!uptake!of!AI>2!could!confer!a!metabolic!benefic.!However,!AI>2!does!not! appear!to!be!a!carbon!source,!since!neither!S.#Typhimurium!nor!S.#meliloti!are!able!to! grow! when! AI>2! is! used! as! the! sole! carbon! source! (Taga! 2001,! Pereira! 2008).! Additionally,!S.#meliloti!shows!no!increase!in!growth!in!the!presence!of!synthetic!AI>2,! compared!to!media!without!this!supplement!(Pereira!2008).!
!Bentley!and!colleagues!propose!a!canonical!QS!function!for!the!AI>2!and!the!Lsr! system:!in!organisms!with!the!Lsr!system,!the!interspecies!signal!regulates!other!genes! than! only! those! necessary! for! its! uptake! and! degradation! by! Lsr! (DeLisa! 2001,! Ren! 2004,!Li!2007).!Supporting!this!hypothesis,!Bentley!and!co>workers!reported!that!in!E.# coli,!where!AI>2!induces!Lsr!expression,!AI>2!regulates!biofilm!formation!and!motility! (Reviewed!in!Pereira!2013b).!!
Choi# et.# al! showed! that! S.# Typhimurium! luxS! mutant! phenotypes,! such! as! defects! in! the! induction! of! virulence! genes! and! invasion! ofepithelial! cells,! were! restored!by!supplying!AI>2!to!the!cultures!and!rescued!in!a!luxS#lsrR!double!mutation! (Choi! 2007,! Choi! 2012),! tough! analysis! of! more! mutants! in! the! components! of! Lsr! needs!to!be!accomplished.!!
Nevertheless,!new!data!start!to!shed!light!on!this!issue!(Table$1).!E.#coli!K;12! RP437!seems!to!alter!its!chemotactic!behavior!in!response!to!AI>2!through!both!LsrB! and!the!L>serine!receptor!(Hegde!2011).!Chemotaxis!was!not!affected!in!lsrC!mutant,! indicating! that! incorporation! of! AI>2! is! unnecessary! for! this! process! (Hegde! 2011).! Moreover,!AI>2!signaling!through!both!LsrB!and!an!extra!receptor,!the!RbsB,!has!been! implicated! in! the! formation! of! biofilms! in! Aggregatibacter! actinomycetemcomitans! (Shao!2007a,!Shao!2007b).!Biofilm!growth!was!reduced!in!a!lsrB!mutant,!and!in!wild> type!(WT)!supplemented!with!ribose,!a!competitor!with!AI>2!for!binding!to!RbsB.!lsrB# rbsB!double!mutant!was!impaired!in!this!behavior!(Shao!2007b).!Moreover,!AI>2!and! functional!lsrK!and!lsrR!homologs!seem!to!be!required!for!optimal!biofilm!formation!in! this! species! (Torres>Escobar! 2013,! Novak! 2010,! Shao! 2007A,! Shao! 2007B).! However,! the!functionality!of!Lsr!in!A.!actinomycetemcomitans!remains!largely!elusive,!and!may! be!different!from!the!well>studied!E.#coli.$
Clearly!more!information!is!needed!to!fully!understand!the!role!of!Lsr!and!its! potencial!function!as!a!typical!QS!signal!system.!In!E.#coli!MG1655!our!laboratory!has! not! been! able! so! far! to! identify! AI>2>regulated! genes! other! then! the! lsr! genes.! This! could!be!a!strain!related!difference!and!this!will!be!addressed!in!this!work.!!
3.$AI@2$mediated$regulation$of$biofilm$formation$
Biofilms! are! single! or! multi>species! multicellular! communities! attached! to! a! surface!and!cohesively!embedded!in!a!self>produced!extracellular!matrix!(López!2010).! These! living! structures! impact! humans! in! many! ways! as! they! can! form! in! natural,! medical,!and!industrial!settings!(López!2010).!For!instance,!shortening!of!the!lifetimes! modules! in! fermenters! due! to! bacterial! biofilms! is! a! major! problem! in! Bioindustry! (Sung! 2006),! with! impacted! productivity! and! economical! consequences.! Moreover,! pathological! conditions! in! some! organs! and! tissues,! such! as! those! observed! on! oral! cavity,! skin,! urinary! tract,! in! ears! and! lungs,! have! been! associated! with! biofilm!
formation! (Duan! 2003,!López! 2010,!Thornton! 2011).! Biofilm! assembly! on! medical! devices!used!in!clinical!practice,!such!as!catheters!or!implants!is!an!open!door!for!the! establishment! of! often! difficult>to>treat! chronic! infections! (Qu! 2009,! López! 2010,! Francolini! 2010).! The! treatment! outcome! of! such! conditions! is! often! compromised! since!this!architecturally!complex!organization!confers!resistance!to!many!antibiotics! to! an! extent! of! up! to! 1000>fold! more! than! observed! in! planktonic! cells! (Mah! 2003,!
Lopéz!2010,!Beaudoin!2012).!!
It!is!well!established!that!in!many!bacteria!QS!is!involved!in!regulating!biofilm!
formation! (Henke! 2004a,! Ng! WL! 2009,! Boyer! 2009).!Specifically,! several! studies!
indicate!that!luxS!gene!and!AI>2!signaling!are!important!for!proper!regulation!of!this! behavior! in! many! different! species,! including! important! human! pathogens,! such! as,! Streptococcus# pneumonia! (Vidal! 2011),! Vibrio# cholerae,! Moraxella# catarrhalis! (Armbruster!2010)!and!Haemophilus#influenza!(Armbruster!2011)!(Table$1).!Probably! the! most! well>documented! role! of! AI>2! in! biofilm! development! is! in! the! oral! cavity! environment!where!it!has!also!been!shown!to!control!biofilm!formation!both!in!single! and!multi>species!set>ups!that!include!commensal!and!pathogenic!bacteria!(reviewed! in!Pereira!2013b!and!Kolenbrander!2010).!Importantly,!AI>2!has!been!shown!to!control! biofilm! formation! between! oral! cavity! pioneer! colonizing! bacteria! (Cuadra>Saenz! 2012),!which!suggests!that!it!might!affect!all!the!ecological!succession!that!shapes!the! development! of! the! oral! ecosystem.! Although! several! oral! bacteria! respond! to! AI>2,! only! the! pathogen! Aggregatibacter# actinomycetemcomitans! harbours! known! AI>2! receptors,!a!LsrB!and!a!RbsB!homologs!(Kolenbrander!2010),!but!the!function!of!these! receptors!in!AI>2>dependent!regulation!of!biofilm!formation!has!not!been!established.$ In! fact! except! for! V.# cholerae,! which! uses! the! LuxP>AI>2! receptor,! the! mechanisms! behind! the! detection! of! AI>2! and! the! signal! transduction! that! regulates! biofilm! formation!in!most!bacteria!remain!unknown.$
More!specifically,!the!role!of!AI>2!signaling!in!biofilm!formation!through!the!Lsr! system! has! not! been! shown! yet.! Bentley! and! co>workers! determined! that! biofilm! formation!in!E.#coli!K12!is!up>regulated!by!AI>2!in!a!LsrK!dependent!fashion!(Gonzalez! 2006,!reviewed!in!Beloin!2008).!However,!the!conclusions!from!this!work!are!not!clear! since! their! studies! are! based! on! non>isogenic! strains.! Importantly,! microarray! experiments!in!lsrR!and!lsrK!mutants!indicate!that!Lsr!is!implicated!in!biofilm!formation!
and!motility!(Li!2007),!though!additional!experiments,!such!as!testing!the!phenotypes! of! luxS# mutant,! which! is! impaired! on! the! production! of! the! signal,! and! lsrB! mutant,! which!is!unable!to!detect!the!signal,!are!required!to!fully!confirm!these!conclusions.! ! Advances! in! the! elucidation! of! the! regulation! mechanisms! behind! these! behaviors! are! of! great! importance! since! manipulation! of! the! Lsr! system! could! be! a! potentially! attractive! alternative! in! the! fight! against! bacterial! diseases.! Because! therapies!that!interfere!with!QS!are!based!on!the!manipulation!of!bacterial!behavior,! virulence!can!be!stopped!without!killing!bacteria,!creating!a!lower!selective!pressure! for!acquiring!resistance!(Rasko!2010,!Marques!2011).Concretely,!AI>2>based!strategies! are!of!particular!economical!interest,!as!a!single!therapeutic!could!be!developed!with! broad! effects! upon! a! multitude! of! species! or! diseases.! This! would! be! particularly! important!for!the!treatment!of!infections!caused!by!multi>species!biofilms!that!might! rely!on!interspecies!signals!for!persistence,!which!are!frequently!a!major!problem!in! chronic!diseases!such!as!that!associated!with!cystic!fibrosis!or!otitis!media!(Duan!2003,! Armbruster!2010,!Thornton!2011).!For!these!cases,!it!is!predicted!that!targeting!inter> species!signaling!is!likely!to!be!more!effective!than!targeting!species>specific!signals.!! 4.$Photorhabdus$luminescens:$insights$into$the$role$of$Lsr$in$AI@2@regulated$ phenotypes$in$entomopathogenic$bacteria$ ! AI>2>dependent!phenotypes!have!also!been!studied!in!other!bacterial!species! harboring!LsrB>like!receptors,!such!as!Photorhabdus#luminescens.!!P.# luminescens! is! a! Gram>negative! bioluminescent! entomopathogenic! bacterium.!Subspecies! laumondii! strain!TT01!contains!orthologues!(plu3141>plu3148)!to!all!the!genes!of!the#lsr!operon! of! E.# coli# (Pereira! 2009),! indicating! that! it! has! the! ability! to! import! and! process! AI>2! (Xavier!2005a).!Its!genome!has!already!been!sequenced!(Duchaud!2003).!
! This!bacterium!possesses!a!peculiar!life!cycle!with!two!host>associated!stages:!a! symbiotic!stage,!in!which!the!microorganism!lives!in!the!intestinal!tract!of!a!nematode;! and!a!pathogenic!stage,!in!which!bacteria,!in!association!with!the!nematode,!infect!and! kills!a!wide!variety!of!insect!larvae!(Figure$4)!(Goodrich>Blair!2007,!Eleftherianos!2010).! Photorhabdus! establishes! an! exclusive! symbiotic! relationship! with! nematodes! from! the!genus$Heterorhabditis!which!usually!require!their!specific!cognate!bacterial!strains! for! growth! (Gaudriault! 2006,! Waterfield! 2009),! suggesting! a! metabolic! dependency!
(Waterfield! 2009,! Lango! 2010).! However,! development! is! also! dependent! on! factors! present!in!the!insect!(Waterfield!2009).! # ! ! ! ! ! ! ! ! ! ! ! ! ! !
The! switch! between! the! two! lifestyles! can! be! distinguished! by! the! bacterial! growth! phase:! the! exponential! growth! is! associated! with! the! pathogenicity! and! the! post>exponential! phase! of! growth! with! the! mutualistic! interaction! (ffrench>Constant! 2003).!Therefore,!it!is!possible!that!a!detection!system!for!cell>density,!such!as!QS,!is! important!for!the!regulation!of!the!transition!between!these!two!different!lifestyles.!
Studies!in!this!organism!showed!that!LuxS!plays!a!role!in!the!regulation!of!carbapenem! production,! a! broad>spectrum! antibiotic! thought! to! prevent! contamination! of! the! insect! carcass! by! other! microorganisms! (Derzelle! 2002).! Furthermore,! a! global! expression!profile!study,!comparing!the!WT!to!a!luxS!mutant!grown!in!the!presence!of! enzymatically! synthesized! AI>2,! showed! that! this! signal! regulates! more! than! 100! targets!in!P.#luminescens,!including!the!lsr!operon#(Krin!2006).!The!expression!of!other! genes!that!encode!outer!membrane>associated!proteins,!such!as!flagella!and!pili,!that!
Figure$ 4$ @$ Life$ cycle$ of$ Photorhabdus! luminescens.! The! life! cycle! of! Photorhabdus!
begins!with!the!colonization!of!the!gut!of!a!soil>dwelling,!motile,!non>feeding!stage!of! the! heterorhabditid! nematode,! known! as! the! infective! juvenile! (IJ),! which! is! developmentally! arrested! and! resistant! to! environmental! stresses.! After! locating! the! insect,!IJs!enter!the!host!and!release!their!bacterial!symbionts!into!the!haemolymph.! Within! the! insect’s! blood,! bacteria! reproduce! and! kill! the! insect! in! 48>72h! while! converting! its! carcass! into! nutrients! that! can! be! readily! used! by! the! nematode! for! growth! and! development.! After! 2>3! rounds! of! reproduction,! nematodes! enter! an! alternative!developmental!pathway!that!leads!to!a!new!generation!of!IJs,!which!can!be! colonized! by! bacteria.! These! IJs! emerge! from! the! insect! cadaver! to! search! for! new! insects!and!initiate!another!cycle.!Source:!Goodrich>Blair!2007!
mediate! virulence! and! that! promote! resistance! to! oxidative! stress,! was! also! AI>2! sensitive! (Krin! 2006).! Phenotypic! assays! indicated! that! AI>2! is! implicated! in! biofilm! formation,!motility,!resistance!to!ROS!and!virulence,!though!were!performed!without! signal! complementation! (Krin! 2006).! UvrY,! another! regulatory! QS! component! in! P.# luminescens,!was!more!recently!shown!to!regulate!bioluminescence!and!resistance!to! oxidative!stress,!as!well!as!23!genes/proteins!previously!reported!to!be!regulated!by! AI>2!(Krin!2008).!However,!it!is!important!to!determine!whether!AI>2!regulates!these! phenotypes! through! the! LsrB! receptor.! Importantly,! most! of! these! AI>2! responsive! phenotypes!are!important!for!symbiotic!and!pathogenic!associations!but!the!role!of!AI> 2! signaling! in! P.# luminescens! interactions! with! its! hosts! remains! to! be! explored.! Therefore,!such!studies!may!shed!a!light!on!the!importance!of!AI>2!signaling!in!both! symbiosis! and! pathogenesis! in! P.# luminescens.! Research! into! Photorhabdus–host! interactions!is!also!expected!to!give!clues!about!important!bacterial>host!interactions! in! humans,! including! pathogenicity! and! mammal! responses! to! microbes,! since! many! traits! and! molecular! pathways! seem! to! be! evolutionary! conserved! (Casadevall! 2005,! Eleftherianos! 2010).! This! idea! is! further! supported! if! we! consider! the! evolution! of! Photorhabdus#asymbiotica,!which!is!both!an!insect!and!human!pathogen!(Waterfield! 2009).! Specifically,! since! Photorhabdus# luminescens! is! a! member! of! the! Enterobacteriaceae!family,!it!is!likely!that!this!research!will!also!contribute!to!similar! studies! of! important! mammalian! pathogens! such! as! E.# coli,! Salmonella# spp.! and! Yersinia# spp.! (Eleftherianos! 2010,! Easom! 2010).! Thus,! the! Photorhabdus>nematode> insect! tripartite! system! provides! a! useful! model! in! which! these! questions! can! be! directly!addressed,!being!an!ideal!well>established!model!for!this!type!of!studies.!
5.$Objectives$
The!main!goal!of!this!research!work!was!to!elucidate!the!physiological!role!of! the!Lsr!system!by!evaluating!the!hypothesis!of!Bentley!and!colleagues!(Li!2007)!in!the! context! of! biofilm! formation! in! E.# coli! and! in! bacterial>host! interactions! in! the! P.# luminescens>nematode>insect! system.! This! work! can! lead! to! develop! strategies! to! explore!the!possible!use!of!the!Lsr!system!to!manipulate!bacterial!behaviors!a!possibly! contribute! to! design! new! clinical! and! biotechnological! strategies! towards! the! modulation!of!relevant!bacterial!behaviors!such!as!virulence.!
CHAPTER(I(*(
The(role(of(AI*2(signalling(through(Lsr(in(biofilm(formation(
in!E.!coli(
(
1.(Materials(and(Methods(
1.1.(Bacterial(strains(and(growth(conditions(!
The! strains! used! in! this! chapter! are! listed! in! Table( 1.! Escherichia) coli) K212! W3110!was!used!as!the!parental!strain!for!all!the!subsequent!genetic!manipulations.! Unless!otherwise!mentioned,!the!strains!were!grown!with!aeration!at!37ºC,!in!Luria2 Bertani! (LB)! or! LB! medium! supplemented! with! antibiotics! at! the! following! final! concentrations:! Chloramphenicol! (cm),! 25! mg.L21;! Kanamycin! (kan),! 50! mg.L21! and! Ampicillin!(amp),!100!mg.L21.!!
!
Table(1(–(E.!coli(strains(used(and(constructed(in(this(chapter.!Only!odd!number!strains!are!
shown!for!each!mutant!from!JMP17–JMP51!since!the!others!have!the!same!genotype.( Strain( Relevant(genotype( Plasmid( Marker(s)( Donor(strain( Parent(strain( construction(Source(or(
W3110!Wild!type!2!Lab!stock!(PS2444)! 2! 2! 2! 2! E.)coli!Genetic!Stock!Center! (CGSC4474)! MG1655!Wild!type!2!Lab!stock! 2! 2! 2! 2! Lab!stock!
AB3073!1(K43N)ΔgalK::(cat!ΔlacIZYA::Frt!rpsL2 Plac::cfp)!
2! cmR;!strpR! 2! MG1655!
(seq)!
Lab!of!Alex! Boehm! AB1006! rpoS2! 2! kanR;!strpR! 2! KX1001! Lab!of!Bonnie!
Bassler! JMP56! 2! pKD46! ampR,!TempS! 2! W3110! Electroporation!
of!pKD46! JMP17! ΔluxS::cm! 2! cmR! mutant!in!JMP56! obtained!by!Red2 swap!method! W3110! This!study! JMP19! ΔluxS! 2! 2! 2! JMP17! This!study! JMP21! ΔlsrK::cm! 2! cmR! mutant!in!JMP56!obtained!by!Red2 swap!method! W3110! This!study! JMP23! ΔlsrK! 2! 2! 2! JMP21! This!study! JMP25! ΔlsrR::cm! 2! cmR! mutant!in!JMP56!obtained!by!Red2 swap!method! W3110! This!study! JMP27! ΔlsrR! 2! 2! 2! JMP25! This!study! JMP29! ΔlsrACDB::cm! 2! cmR! mutant!in!JMP56! obtained!by!Red2 swap!method! W3110! This!study!
Legend(–(ampR:!amp!resistance!gene;!cmR:!cm!resistance!gene;!kanR:!kan!resistance!gene;!TempS:! Temperature!sensitive!origin!of!replication.!
!
!1.2.(Genetic(and(molecular(techniques(
Deletion! mutants! on! AI22! system! were! constructed! using! the! Red2swap! method!(Protocol(1,(see(Appendices)!described!by!Datsenko!and!Wanner!(Datsenko! &! Wanner! 2000)! in! E.) coli! W3110.! Using! this! method! the! gene! of! interest! was! replaced! with! a! cm! resistance! cassette.! The! plasmid! pKD3! (cmR)! was! used! as! template! for! the! amplification! of! a! linear! DNA! fragment! by! polymerase! chain! reaction! (PCR)! with! the! primers! listed! in! Table( 2.! Each! of! these! primers! contains! homology!extensions!at!the!5’!end!of!approximately!50!nt!to!the!flanking!regions!of! the!gene!to!be!replaced!2!H1!for!the!fw!(forward)!or!H2!for!the!rv!(reverse)!primers!2! and!20!or!21!nt!priming!sequences!(P1!for!the!fw,!or!P2!for!the!rv!primers)!at!the!3’! end!to!amplify!the!antibiotic!cassette!from!pKD3.!( ( Table(2!–!Primers!used!for!Red2swap!method.!P1!and!P2!sequences!are!highlighted!for!each! primer.!! Primer(
number( Primer(name( Oligonucleotide(sequence((5'*3')(
40( lsrR1! GTGAAGAATGAATTATGACAATCAACGATTCGGCAATTTCAGAACAGG GAGTGTAGGCTGGAGCTGCTTC! 552( lsrK2fw! AGAACGTATAGAGGAACAGACAGAACGCATAAGCCGAGGATAATCTA ATGGTGTAGGCTGGAGCTGCTTC! 553( lsrK2rv! CGGTCATCATGATGTGGCTGTCAATGAAACTATAACCCAGGCGCTTTCC ACATATGAATATCCTCCTTAGT! JMP31! ΔlsrACBD! 2! 2! 2! JMP29! This!study! JMP33! ΔlsrB::cm! 2! cmR! mutant!in!JMP56!obtained!by!Red2 swap!method! W3110! This!study! JMP35! ΔlsrB! 2! 2! 2! JMP33! This!study! JMP37! ΔlsrF::cm! 2! cmR! mutant!in!JMP56! obtained!by!Red2 swap!method! W3110! This!study! JMP39! ΔlsrF! 2! 2! 2! JMP37! This!study! JMP41! ΔlsrG::cm! 2! cmR! mutant!in!JMP56! obtained!by!Red2 swap!method! W3110! This!study! JMP43! ΔlsrG! 2! 2! 2! JMP41! This!study! JMP49! ΔfliC::kan! 2! kanR! JW1908!(Keio!
collection)! W3110! This!study! JMP51! ΔfliC! 2! 2! 2! JMP49! This!study! JMP57! ΔluxS)lsrF! 2! 2! JMP17! JMP39! This!study!
555( lsrR2rv! ATCATTCCCGGTAATAAGGTCATGCAAATTTAACTACGTAAAATCGCCG CCATATGAATATCCTCCTTAGT! 556( lsrACDB2fw! GAAATACATTTGTTCAAAACTCACCTGCAAAACTGAACGGGGGAAATA TGGTGTAGGCTGGAGCTGCTTC! 557( lsrACDB2rv! CATAACTTACTCCGGTTAAGTAATGCACATCAGAAATCGTATTTGCCGA TCATATGAATATCCTCCTTAGT! 558( luxS2fw! AAGAAAGAGTTCAGAAAATTTTTAAAAAAATTACCGGAGGTGGCTAAA TGGTGTAGGCTGGAGCTGCTTC! 559( luxS2rv! TTCAATTAATTGTGAAGATAGTTTACTGACTAGATGTGCAGTTCCTGCA ACATATGAATATCCTCCTTAGT! 560( lsrF2fw! ATCGGCAAATACGATTTCTGATGTGCATTACTTAACCGGAGTAAGTTAT GGTGTAGGCTGGAGCTGCTTC! 561( lsrF2rv! GTGCATAATTCTTCTCCTTAGATCCGCAGTTACTGTTTTTCACTCAGATA CATATGAATATCCTCCTTAGT! 562( lsrG2fw! CTCTATCTGAGTGAAAAACAGTAACTGCGGATCTAAGGAGAAGAATTA TGGTGTAGGCTGGAGCTGCTTC! 563( lsrG2rv! CAATTGTAGATAAAATTGATAAATTCGCCTCACGGCATCAAACCATTGA ACATATGAATATCCTCCTTAGT! 779( lsrB2fw! TGGCTGGCGCGTCGGGCCAATAACCCATTGCCATAAAGGATATCTTCG TGTAGGCTGGAGCTGCTTC! !
!fliC! mutant! and! luxS) lsrF) double! mutant! were! constructed! by! P1! bacteriophage! transduction! of! chromosomal! deletions! carrying! the! specified! antibiotic!resistance!markers!to!E.)coli!W3110.!In!the!case!of!fliC::kan,!the!respective! mutant! from! the! Keio! Collection! of! single! deletion! mutants! (Baba! 2006),! obtained! from!the!National!BioResource!Project!(Japan),!was!used!as!donor!strain.!For!the!luxS) lsrF! mutant! the! ΔluxS::cm! marker! from! JMP17! strain! was! transduced! into! the! ΔlsrF::cm!JMP37!strain.!The!kanR!and!cmR!cassettes!were!eliminated!by!introducing! the!plasmid!pCP20!expressing!the!FLP!recombinase!into!the!strains.!To!confirm!the! deletions! of! the! mutants! obtained! by! the! Red2swap! method! and! the! P1! phage! transductions,! all! the! mutants! were! confirmed! by! Colony! PCR! (Protocol( 2,(
Appendices)!using!primers!that!have!homology!to!the!flanking!regions!of!the!genes!
of!interested!(Table(3).!Agarose!gel!electrophoresis!was!performed!to!evaluate!PCR! product!size.!To!determine!PCR!product!size!the!following!DNA!ladders!were!used:! 100!bp!DNA!ladder!and!1!Kb!DNA!ladder!(New!England!Biolabs).!Purification!of!PCR! products! was! achieved! using! QIAquick! PCR! Purification! Kit! (Qiagen)! following! the! manufacturer! protocol.! All! plasmids! were! purified! using! QIAprep! Spin! Miniprep! (QIAGEN)!kit!following!the!manufacturer!protocol.!Nucleic!acid!concentrations!were! determined! using! Nanodrop! (Thermo! Scientific)! and! Nanodrop! 1000! Spectrophotometer!3.7.1!software.!PCR!was!performed!using!Taq!DNA!polymerase!
(New! England! Biolabs)! in! a! T100! thermal! cycler! (Bio2rad).! All! primers! used! in! this! study! were! designed! using! Vector! NTI! Advance! 11.0! (Invitrogen).! All! the! plasmids! used!in!this!chapter!for!mutant!construction!are!shown!on!Table(4.!!
( Table( 3( –( Primers! used! for! PCR! confirmation! of! the! deletion! mutants! constructed! in! this!
chapter!
Primer(number( Primer(name( Oligonucleotide(sequence(((5'*3')( 8( K2! CGGTGCCCTGAATGAACTGC! 23( lsrBu! CCCATTGCCATAAAGGATATC! 49( LuxS_P1! TTAAGCTTCGGTGCGCACTAAGTACAAC! 50( LuxS_P2! TGCTAGCGATGATAACAAATGCGCGTCT! 332( lsrR2RACE2seq! CCGAATCGTTGATTGTCATAATTCA! 428( lsrBconfirm2R! TCCGCGATAAGCGTGACTGCA! 443( lsrK!fw! GCAGGGGGAGAAAATAAAGC! 444( lsrK!rv! AAAATAAGGGTTGTGCTTAATGCT! 316( lsrK2GSP1! AAACAATGCCTTCACGCA! 28( fklsrR1! AGGTGGACATTGCACATATTTCCGAC! 603( lsr2gfp!Cm!2! ATCACAAACGGCATGATGAA! 228( Ec2lsrG2P12seq! CAGTAACTGCGGATCTAAGGAG! 230( EC2lsrF2P22seq! ACCAGTGTGACGTGCATAATTC! 231( Ec2lsrF2P12seq! ACCGGAGCGCGTGATATTCAAC! 25( lsrGD! GCCAAGATCGGCAACATATTCGAC! 799( fliC!P1!confirm!fw! ATTGAGCCGACGGGTGGAAACC! 800( fliC!P1!confirm!rv! GGTTACAGGTAAATTCCAGGCAGAA! ( ! Table(4*(Plasmids!used!in!this!chapter.(
Plasmid( Marker(s)( Relevant(genotype(or(property( Source(
pKD3! cmR,!ampR! cmR!!cassete!flanked!by!FRT!
sites! Datsenko!&!Wanner!2000! pKD4! kanR,!ampR! kanR!cassete!flanked!by!FRT!
sites! Datsenko!&!Wanner!2000! pKD46! ampR,!TempS! oriR101!w/repA101ts;!araC,!
ParaB::gam,)exo,)bet)
Datsenko!&!Wanner!2000! pCP20! cmR,!ampR,!
TempS! ts2rep(ts),!bla,!cat,!FLP! Datsenko!&!Wanner!2000!
Legend(–(ampR:!amp!resistance!gene;!cmR:!cm!resistance!gene;!kanR:!kan!resistance!gene;!TempS:!
Temperature!sensitive!origin!of!replication.(
(
1.3.1.(Preparation(of(P1(bacteriophage(lysates!
P1!lysate!stock!grown!in!E.)coli!MG1655!(Protocol(3,(Appendices).!The!donor!strain! was!infected!with!the!phage!in!LB!+!0.2%!glucose!+!5!mM!CaCl2!for!2!hours,!until!the! mixture! became! translucent.! Remaining! cells! were! lysed! with! chloroform! and! the! supernatant!of!the!mixture!(phage!lysate)!was!carefully!recovered!after!spin!down! and!stored!at!4ºC!until!use.!
1.3.2.(P1(bacteriophage(transduction!
The!insertion!of!genes!into!E.)coli!W3110!by!P1!bacteriophage!transduction! was! carried! out! following! Protocol( 4( (Appendices).! Cm! resistance! cassettes! that! resulted!from!the!deletion!of!the!luxS!and!the!lsr!genes!by!the!Red2swap!protocol! were!inserted!back!into!the!W3110!WT!strain!by!infection!with!lysates!from!each!of! the!respective!mutants.!Transduction!events!were!selected!by!platting!in!LB!medium! supplemented! with! cm! 25! mg.L21.! fliC! was! constructed! by! infection! with! lysates! of! the!respective!mutant!from!the!Keio!Collection,!whose!chromosomal!deletions!carry! the! kanR! marker.! The! ΔfliC::kan! deletion! was! transduce! directly! into! the! W3110) strain.!Transduction!events!were!selected!in!LB!medium!supplemented!with!kan!50! mg.L21.! All! the! mutants! resulting! from! P1! bacteriophage! transduction! were! confirmed!by!PCR!using!primers!that!flank!the!transduced!deleted!gene!(Table(3).!! !
1.4.(Electrocompetent(cells(and(electroporation(
The! plasmids! pKD46! and! pCP20! were! transformed! into! E.) coli! W3110! (PS2444)!or!the!derived!mutants!by!electroporation!at!2.2!Kvolts!after!the!cells!were! made!electrocompetent!by!consecutive!washings!with!sucrose!300!mM!(Protocol(5,(
Appendices).! For! electroporation! of! Red2swap! method! linear! DNA! (Protocol( 1,( Appendices)!cells!were!made!electrocompetent!through!consecutive!washings!with!
10!%!glycerol.!Electroporation!was!achieved!using!EC2!program!(200!Ω,!25!μF!and! 2.5! Kvolts).! All! electroporations! were! performed! in! a! MicroPulser! (Bio2rad)! equipment.((
(
1.5.(AI*2(activity(assay(!
To!measure!AI22!extracellular!activity!in!E.)coli)supernatants,!overnight!(ON)! cultures!were!diluted!to!a!final!optical!density!at!600!nm!(OD600)!of!0.05!into!fresh!LB! medium! supplemented! with! 100! mM! MOPS! buffer! pH7! (LB! MOPS)! in! Erlenmeyer! flasks!and!grown!with!aeration!at!37ºC!in!a!water!bath!shaker!for!the!indicated!time.! Aliquots!were!collected!at!the!times!indicated!and!used!to!analyze!the!OD600!and!to! prepare!cell2free!culture!fluids.!The!AI22!detection!and!quantification!in!the!cell2free! culture! fluids! was! measured! using! a! LuxP2fluorescence! resonance! energy! transfer! (FRET)!assay!as!previously!described!(Taga!&!Xavier!2011)!(Protocol(6,(Appendices).! Cell2free!culture!fluids!were!prepared!by!filtration!of!liquid!cultures!through!0.22!μm! pore! size! 962well! filtration! plates! (Millipore).! Fluorescence! was! measured! using! Victor3! multilabel! counter! (Wallac).! Each! sample! was! assessed! in! duplicates.! This! experiment!was!performed!twice!in!independent!days.!!
1.6.(Quantification(of(Crystal(Violet*stained(attached(cells(
Attached! cells! were! quantified! as! described! previously! (O'Toole! &! Kolter! 1998)!with!a!few!modifications!(Protocol(7,!Appendices).!For!these!assays!different! culture! media! were! tested:! LB,! LB! +! 0.2%! (wt/vol)! glucose! (LB! glu),! M9! minimal! media! +! Casamino! acids! 0.4%! (wt/vol)! +! 0.4%! (wt/vol)! glucose! (M9C! glu)! or! M9! minimal!media!+!Casamino!acids!0.4%!(wt/vol)!+!100!μM!FeSO4!(M9C!Cit!+!Fe).!ON! were!cultured!at!30ºC!with!agitation!and!normalized!to!an!OD600=!0.05!and!100!μL! were! dispensed! in! each! well! of! a! Costar! Clear! Polystyrene! 962Well! Plate! (Fisher! Scientific)!and!incubated!at!25ºC!without!agitation.!After!24h!(for!rich!media)!or!48h! (for!minimal!media)!of!incubation,!the!wells!were!washed!twice!with!PBS!1x,!fixed! with!200!μl!of!methanol!and!stained!with!200!μl!of!0.1%!Crystal!Violet!(CV),!rinsed( and!thoroughly!dried.!The!CV!was!solubilized!by!the!addition!of!200!μl!of!Acetic!Acid! 33%,! and! OD600! was! determined! using! Victor3! multilabel! counter! (Wallac).! fliC! mutant!was!included!as!a!negative!control.!These!experiments!were!performed!at! least,!in!three!independent!days!with!8!technical!replicates!per!strain!tested.!(
1.7.(Growth(of(bacterial(macrocolonies(
Bacterial! macrocolonies! were! grown! as! described! previously! (Serra! 2013)! (Protocol(8,!Appendices).!Cells!were!grown!ON!in!LB!with!agitation!at!37°C.!A!total!of! 5!µl!of!the!ON!cultures!was!spotted!on!salt2free!LB!1.8%!agar!plates!supplemented! with! Congo! red! (40!µg.ml−1)! and! Coomassie! brilliant! blue! (20!µg.ml−1).! In! order! to! grow!all!strains!to!be!compared!in!parallel!on!a!single!agar!plate,!1402mm2diameter! petri!dishes!(VWR)!were!used.!Plates!with!macrocolonies!were!incubated!at!28°C!for! up! to! 6!days! and! were! visualized! at! ×10! magnification! with! a! Leica! MZ75! stereomicroscope.!Digital!images!were!captured!with!a!Leica!DFC420!C!Camera,!and! a!Canon!PowerShot!S5!IS!digital!camera!using!the!SuperMacro!tool.(
(
1.8.(Statistical(analysis(
Data! was! plotted! and! analyzed! by! performing! a! Mann–Whitney–Wilcoxon! test!using!Prism!6!software!(GraphPad),!with!a!Holm2Bonferroni!correction!method! to! adjust! P2values! for! multiple! comparisons,! estimated! in! R! program,! by! using! the! following!command:! p!<2!c(P1,!P2,…,!Pn)! p.adjust!(p,!"holm")!! Where!P!represents!each!P2value!in!n!comparisons.! Error!bars!correspond!to!the!standard!deviation!calculated!as!SD!=!√!(σ12!+!σ22!+….! σn2),!taking!into!account!the!standard!deviations!(σ)!of!the!average!of!the!technical! replicates!in!each!independent!n!experiments.!*!=!P!<!0.05,!**!=!P!<!0.01,!***!=!P!<! 0.001,!****!=!P!<!0.0001.!( (
Results(and(discussion
(By! the! time! this! study! had! started! there! was! a! lot! of! controversy! in! the! literature!regarding!a!possible!role!of!AI22!and!the!Lsr!system!on!biofilm!formation!in! E.)coli.!The!main!goal!of!this!chapter!was!to!clarify!if!this!regulation!indeed!occurs!in! this!bacterium!by!using!the!most!appropriate!experimental!approaches.!
!The!Lsr!system!in!E.)coli!is!well!characterized!(Xavier!2005a,!Pereira!2012b).! However,! until! now! our! laboratory! has! been! unable! to! identify! other! phenotypes!
regulated!by!AI22!besides!its!incorporation!and!processing!in!our!model!E.)coli)strain,! the!domesticated!MG1655.!Because!this!strain!is!highly!adapted!to!the!laboratory!it! might!be!affected!in!the!ability!to!perform!many!group!behaviors!typically!regulated! by! QS,! such! as! biofilm! formation.! In! fact,! we! have! shown! that! the! MG1655! strain! stock!that!we!were!using!previously!doesn’t!have!an!insertion!sequence!(IS)!element! in!the!regulatory!region!of!the!flhDC!promoter!(Pereira!2012),!which!was!shown!to! confer!increase!motility!(Barker!2004).!As!motility!is!crucial!for!biofilm!development! (Pratt!&!Kolter!1998),!this!might!explain!why!this!strain!forms!poor!biofilm.! (In!this!study,!E.)coli!W3110!was!chosen!as!model!strain!since!it!is!flhDC+,!exhibits!WT! motility!(Barker!2004),!and!has!been!shown!to!form!biofilms!(Pesavento!2008,!Clarke! 2009).!The!laboratory!stock!of!E.)coli!W3110!was!confirmed!by!PCR,!as!described!by! Barker! et) al.,! to! have! an! intact! flhDC) operon! and! an! IS! element! upstream! of! this! region!(Pereira!2012).!!
!
2.1.(Construction(of(AI*2(signalling(system(mutants(on(E.!coli!W3110(
To!study!the!function!of!AI22!signalling!on!biofilm!formation!in!E.)coli!W3110,! a!set!of!isogenic!deletion!mutants!on!the!AI22!synthase!and!all!the!genes!of!the!lsr) system! were! constructed:! luxS,! lsrK,! lsrR,! lsrACDB,! lsrB,! lsrF! and! lsrG! mutants.! The! construction!of!all!these!strains!was!achieved!trough!three!main!steps((Figure(1)!by!a! method!for!gene!deletion!in!E.)coli!known!as!Red2swap!(Datsenko!&!Wanner!2000).! The!mutants!can!be!readily!selected!since!the!gene!to!be!disrupted!is!replaced!by!an! antibiotic! resistance! cassette.! The! selective! markers! are! flanked! by! FTR! (flipase! recognition!target)!sequences!to!enable!its!elimantion!upon!induction!of!a!flipase.! Marker! elimination! can! be! very! useful! when! constructing! multiple! mutants! in! the! same!strain.!
! !
!
