SequencingSalmonella,hilA,HilA,A,A,A...

AsequenceanalysisoftwomutanthilAgenes

SlaterSharpandBeccaLensing
CornellCollege
9/22/2014

INTRODUCTION
Salmonella,afacultativeintracellularparasite,isoneofthemostcommoncausesoffoodborne
bacterialdiseaseintheworld.Itbecomesincorporatedintothehumangastrointestinalsystemprimarily
throughtheingestionofinfectedfoodproducinganimals(RychlikandBarrow,2005).Virulence,the
capacityofSalmonellatoinvadeandinfecthostepithelialcells,isdependentonbothenvironmentaland
geneticfactors.Genesencodingpathwaysforintracellularpathogenesisarelocatedonpathogenicity
islandsinthebacterialchromosome(Bumleretal.,2000).Althoughtwelvepathogenicityislandshave
beenidentified,therolesofspecificgenesinvirulencearenotwellunderstood(Hensel,2004).
Understandingthegeneticcontrolsandmechanismsresponsiblefortheinvasivepropertiesof
Salmonellamayenablethepreventionofhealthendemics,economicsuffering,andproductivitylosses
causedbythepathogen.ThehilAgeneencodesanimportanttranscriptionalregulatorofSalmonella
pathogenicityisland1(SPI1).MutationsinhilAcanleadtothereducedexpressionofSPI1genes,
resultinginavirulence(LostrohandLee,2001).InourstudythehilAgenesoftwoavirulentSalmonella
strains,mutatedinvitro,weresequencedandcomparedtothatofwildtypeSalmonella.Basepair
mutationsconservedacrossthetwoavirulentstrainsindicatedcrucialsitesforSalmonella
pathogenicity.Throughthisretrospectiveapproach,wewereabletoprovideinsightintothenatureof
Salmonellavirulencebydrawingconclusionsaboutthegeneticcausesofavirulence.
TheprimaryroleofhilAregulatedgenesonSPI1istheexpressionofthetypeIIIsecretion
system1(T3SS1),aknowndeterminantofSalmonellavirulence.T3SS1isorganizedintoa
supramolecularstructureknownastheneedlecomplex",responsibleforthetransferofbacterial
proteinsintoeukaryoticepithelialhostcells(LostrohandLee,2001).DiscreteT3SS1substructures
includeaneedlelikeprojectionthatextendsoutwardsfromthebacterialenvelopeandabasethatspans
thewidthofthedoublemembrane(Kuborietal.,2000).Thebaseiscomposedoftwopairsofrings
joinedbyahollowcylindricalstructure.StructuralproteinInvGcomprisestheouterringswhilePrgH
andPrgKformtheinnerringsandcylinder.Theneedleismadeupofasingleprotein,PrgI,stabilizedby
PrgJ(Kuborietal.,2000).InvGisencodedontheinvFoperonatoneendofSPI1,whilePrgH,PrgK,
PrgI,andPrgJareencodedontheprgHoperonattheoppositeendofthe40Kbppathogenicityisland
(LostrohandLee,2001).
TranscriptionofT3SS1structuralgenesinvFandprgHisactivatedbyHilA,atranscription
factorcodedforbythegenehilA.HilArecognizesspecific17nucleotidelongsequencesintheinvF
andprgHpromotersonSPI1.Eachofthese17nucleotidesequencescontainstwocopiesofthe
hexamericrepeatTTXYAT,whereXrepresentsanadenineorthyminebaseandYrepresentsan
adenine,thymine,orcytosinebase.ThisrepeatedmotifmakesuptheHilAproteinbindingsite,orthe
HilAbox.BindingofHilAtotheHilAboxdirectlyactivatesthepromotersP
invF
andP
prgH
,whichin
turnactivatetheinvF1andprgHgenes,producingtheproteinsrequiredforT3SS1biosynthesis
(LostrohandLee,2001).
SalmonellavirulencerequiresaHilAdependentcascadeoftranscriptionalactivation,and
throughtheexpressionofinvFandprgH,initiatestheconstructionoftheT3SS1needlecomplex
(LostrohandLee,2001).MutationsinthehilAgenestructurallyaltertheproteinHilA,potentially
affectingitsabilitytobindtotheHilAboxandactivatetheinvFandprgHpromoters,compromisingthe
cascade.Thebacteria,unabletoassembleT3SS1,isrenderedavirulent.
HerewereportthehilAsequenceanalysisoftwoavirulentSalmonellastrains(A5andB3)and
characterizethemutantsequences.Selectmutationswereconservedacrossthestrains,indicatingcrucial
basepairsequencesnecessaryforthetranslationoffunctionalHilAprotein.Originsofavirulencewere
derivedatthenucleotidelevel.

METHODSANDMATERIALS
PlasmidIsolation
C.PhoebeLostrohintroducedrandommutationsintopHilAandisolatedmutantplasmids
unabletoactivatetheinvFandprgHgenes.E.colicontainingpHilAstrainsA5andB3weregrown
overnightinLBBrothcontaining100g/mLampicillin.PlasmidDNAwasisolatedusingtheIBIFast
IonPlasmidIsolationKit.Cellswerepelletedat7,000rpmfor15minutes.Thebacterialpelletswere
resuspendedinbufferPM1(50mMTrisCl,pH8.0,10mMEDTA,100g/mLRNaseA)and
incubatedatroomtemperatureforfiveminutesinbufferPM2(100mMNaOH,1%w/vSDS).Buffer
PM3(3.0Mpotassiumacetate,pH5.5)wasadded,andthelysatewascentrifugedat11,000rpmfor
20minutes.Thesupernatantswerespunat11,000rpmfor15minutesandtransferredintoIBI
anionexchangecolumnsequilibratedwithPEQbuffer(750mMNaCl,50mMMOPSpH7.0,15%
v/visopropanol,0.15%TritonX100).ThecolumnswerewashedwithPWbuffer(1MNaCl,50mM
MOPSpH7.0,15%v/visopropanol)andtheplasmidswereelutedwithPELbuffer(50mMTrisCl
pH8.5,1.25MNaOH,15%v/visopropanol).PlasmidDNAwasprecipitatedwith0.75volumesof
roomtemperatureisopropanolandcentrifugedat12,000rpmfor30minutesat4C.TheDNAwas
ethanol(75%)precipitated,centrifugedat12,000rpmfor10minutes,andresuspendedinsteriledH
2
0.
PlasmidmasswasdeterminedbyscanningtheDNAfrom230350nmonaNanodrop
spectrophotometerandrecordingpeakabsorbanceat260nm.

PlasmidCharacterization
PlasmidspHilA(A5)andpHilA(B3)(0.5g)weredigestedwithNcoIandXbaItoremove
thehilAinsert,XbaItolinearizeplasmidtheconstruct,orleftundigestedtofunctionasnegative
controls.Lambda(0.5g)digestedwithHindIIIandEcoRIanda100b.p.ladderservedasmolecular
weightmarkers.Samplesweredigestedovernightat37C.Digestedsamplescontainingtrackingdye
(50%v/vglycerol,0.4%w/vbromophenolblue)ranona0.7%(w/v)agarosegelmadeinTrisacetate
buffer(0.04MTrisacetate,0.35Mglacialaceticacid,0.01MEDTA,pH8.0)containing0.5LMidori
GreenAdvanceDNAStain,anonmutagenicalternativetoEthidiumbromide.Sampleswererunat65
voltsinTrisacetatebuffer(0.04MTrisacetate,0.35Mglacialaceticacid,0.01MEDTA,pH8.0)and
werephotographedunderultravioletlight.

PCRAmplificationandSequencing
PlasmidspHilA(A5)andpHilAB3weresequencedusingdideoxyDNAsequencing.The
PCRreactioncontainedpHilAplasmidtemplate(1,2,3g),USBThermoSequenaseReactionBuffer
(11.76%v/v),USBThermoSequenaseDNAPolymerase(11.76%v/v),M13Forwardprimer
ATGCCATAGCATTTTTATCC(88nM),M13ReverseprimerCCTGATACAGATTAAATC
(88nM),anddNTPnucleotidemix(5.88%v/v).Thefinalmastermixvolumewas17L.4Lofthe
mixwasaliquotedintothinwalledPCRtubesand4Ldideoxynucleosidetriphosphate(A,T,G,orC)
wasadded.Thetubeswereruninathermocyclerat92Cfortwominutes,thencycledat92Cfor30
seconds,47Cfor30seconds,and70Cfor1minuteforatotalof30cycles.Thereactionswere
denaturedat92Cfor3minutesandrunon5.5%(w/v)denaturingpolyacrylamidegels(7.0Murea)in
0.8XTBE(0.072MTris,0.072Mboricacid,0.0016MEDTA)at2,000voltsat45Candreadunder
infraredbyaLICOR4300DNAAnalyzer.ComputerprogramLICOReSeqtranslatedthe
resultingbandingpatternintoabasepairsequence.

RESULTSANDDISCUSSION
GelElectrophoresisofhilA
Priortoourinvestigation,twoinvitromutated1,600basepair(bp)hilAsequenceswereinserted
into4,100bppBADplasmids.TheresultingplasmidpHilA,measuringaround5,700bp,wasinsertedinto
anE.colihosttoallowforitsreplication.Followingplasmidisolation,pHilA(A5)andpHilA(B3)were
characterizedusingrestrictionenzymesNcoIandXbaIandrunonanagarosegel(Figure1)inorder
toconfirmtheidentityofthepHilAplasmidsandtheisolatedhilAsequence.Undigestedplasmid
functionedasanegativecontrolinordertoconfirmthatthelinearizedplasmidswereproperlydigested.
Lambda()/EcoRIpresented21,226bp,7,421bp,5,8045,645bpand4,878bpmolecular
weightmarkersandLambda()/HindIIIdisplayedweightmarkersatthe23,130bp,9,416bp,6,557
bp,2,322bp,and2,027bpregions.Afaint4,361bpmarkerwasalsopresent.The1,500bpand500
bpmarkerswerethemostdistinguishedbandsofthe100bpladder.` Prominentbandsofundigested
plasmidpresentednearthe7,421bpmarkerof/EcoRI.PlasmidslinearizedwithonlyXbaIexpressed
multiplebandingpatterns,mostofwhichwereconservedacrossthelinearizedpHilA(A5)and
pHilA(B3).Thebandofinterestappearedatthe5,8045,643bpmarkerof/EcoRI.Otherbands
presentedatthe20,000bp,18,500bp,14,000bp,and7,500bpregions,approximately.Plasmids
digestedwithNcoIandXbaIexhibitedthreeconsistentbandsnearthe5,8045,643bpandslightly
belowthe4,878bpmarkersof/EcoRIandjustabovethe1,500bpmarkerof100bpladder.
Bothundigestedplasmidsappearednearthe7,421bpmarkerof/EcoRI,despitetheir5.7
Kbplength.Thisismostlikelyduetotheplasmidssupercoiledconformations,alteringtheirabilitiesto
travelthroughthegel.TheundigestedplasmidsandXbaIlinearizedpHilAexhibiteddissimilarbanding
patterns,confirmingthatthecutplasmidswereproperlydigested.The5,8045,643bplocalizedbands
oftheXbaIlinearizedpHilAindicatedthelengthofthelinearizedplasmidstobeapproximately5.7
Kbplong.ThismodelisconsistentwithpHilA.TheextraneousbandsofXbaIlinearizedpHilAwere
unaccountedfor,andwerelikelytheresultofsamplecontamination.NcoIandXbaIdigestedplasmids
eachexhibitedthreelinearizedfragments5.7Kbp,4.1Kbp,and1.6Kbpinlength.The4,100bp
fragmentswerecharacteristicoflinearizedpBAD,andthe1,600bpfragmentswerepresumptivelythe
linearizedhilAinserts.The5.7KbpfragmentsarelikelypartiallydigestedpHilAplasmidsthathave
beenlinearizedbyasinglerestrictionenzyme.Thisassumptionissupportedbytheirbandingsimilarity
withtheXbaIlinearizedpHilAfragments.Thesefinalresultsstronglysupporttheconclusionthatthe
plasmidswereindeedpHilA,andthatbothplasmidsincludedthehilAinsert.

DideoxyDNASequencingofhilA
hilAtemplateDNAwasamplifiedthroughpolymerasechainreaction(PCR)using
pBADspecificM13forward(ATGCCATAGCATTTTTATCC)andreverse
(CCTGATACAGATTAAATC)primers.Elongationofthetemplateswasterminatedin1bp
incrementsbytheadditionofaspecificdideoxynucleosidetriphosphate(ddNTP).Thefourreactions
(ddATP,ddCTP,ddGTP,andddTCP)wererunseparatelyona5.5%denaturingpolyacrylamidegel
wherevariationsinchainlengthassmallasonenucleotidecouldbedifferentiated.Eachbandonthegel
markedaspecificterminationpointinthedaughterstrandtherefore,byrunningthefourreactionsin
parallel,thesequenceofthehilAgenecouldbereadoffthegelbytheLICOReSeqcomputer
program.Multiplefourreactionsamplesofdifferentvolumes(1.0L,0.75L,and0.5L)wererun
simultaneouslyforeachofthepHilA(A5)andpHilA(B3)plasmids.
TheresultingsequencesweretestedagainstthefullSalmonellagenomeofSalmonella
entericasubsp.entericaserovarEnteritidisstrainSEJ,asreleasedbytheLosAlamosNational
Laboratory(BishopLillyet.al,2014),andtheNationalCenterforBiotechnologyInformation.Inorder
toverifythattheconservednonhomologybetweentheavirulentstrainswasnotduetonatural
variationsbetweentheSEJsubspeciesstrainandtheoriginalstrainmutatedbyLostroh,theconserved
regionswerecomparedtoSalmonellaentericasubsp.entericaserovarEnteritidisstrains
OLFSE60021916(SE6),OLFSE110191(SE1),andOLFSE511042(SE5),alsofromthe
NationalCenterforBiotechnologyInformation.
MutatedhilA(A5)wassequenced720bpintheforwarddirectionand562bpinthereverse
directionwith99%homology(714/720and559/562).These1,282nucleotidescomprise77.4%ofthe
1,656bphilAgene.Theunsequencedmiddleregionmeasures374bpinlength.MutatedhilA(B3)was
sequenced511bpintheforwarddirectionand312bpinthereversedirectionwith99%homology
(505/511and310/312).These820nucleotidescomprise49.5%ofthehilAgene.Theunsequenced
middleregionmeasures836bpinlength.Sequencedregionsdisplayinghigherratesofambiguitywere
disregardedtheprobabilityofanonhomologousbaseintheregionsbeingtheresultofamutationwas
decidedlyhigher.However,thesedissimilaritiesbetweentheSEJhilAsequenceandthemutatedhilA
sequencesmayhavebeentheresultofexperimentalerrorandnotrandommutation.
ComparisonofhilA(A5)totheSalmonellaentericaSEJstraingenomerevealedtwopoint
mutations,twoinsertions,andfivedeletions.ComparisonofhilA(B3)totheSalmonellaentericaSEJ
straingenomerevealedtwopointmutations,oneinsertion,andfivedeletions.Ofthese,twopoint
mutations(CtoGandAtoT)andtwodeletions(CandT)ontheforwardstrandsandonedeletion
(A)onthereversestrandareconservedbetweenthemutatedhilA(A5)andhilA(B3)sequences.When
hilA(A5)andhilA(B3)werecomparedtotheSE6,SE1,andSE5strains,thesemutationsremained
conserved.
OnepossibleexplanationfortheavirulenceofSalmonellastrainsA5andB3isthattheinduced
mutationsinhilAmodifiedtheaminoacidsequenceofproteinHilA.Thesechangesmayhaverendered
theproteinsactivesiteinert,byalteringeitherproteinfoldingortheactivesiteitselfconsequently,the
abilityofproteinHilAtobindtotheHilAboxmayhavebeencompromised.Withoutafunctional
transcriptionfactorforinvFandprgH,theT3SS1structuralproteinswerenotexpressed.These
Salmonellabacteriawereunabletophysicallypierce,andthereforeinfect,eukaryoticepithelialhost
cells.ConservationofthesemutationsbetweentheavirulentSalmonellastrainsA5andB3suggests
thatSalmonellaentericasubsp.entericaserovarEnteritidisstrainSEJbasepairs1201733,
1201736,1201809,1201813,and1203340maybenecessaryforproperhilAfunction,andby
extensionSalmonellavirulence.

APPENDIX
Figure1:Agarosegelelectrophoresis
Table1:Gelelectrophoresisloadswithrestrictionenzymes
Lane# Sample Enzyme
1 pHilA(A5)
2 pHilA(A5) XBAI
3 /EcoRI
4 pHilA(B3)
5 pHilA(B3) XBAI
6 /HindIII
7 pHilA(A5) NCOI+XBAI
8 100bpLadder
9 pHilA(B3) NCOI+XBAI
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