Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio ...

14 Pages·2008·2.73 MB·English

JOURNALOFBACTERIOLOGY,Oct.2008,p.6646–6659 Vol.190,No.20
0021-9193/08/$08.00(cid:1)0 doi:10.1128/JB.00466-08
Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved.
Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and
(cid:1)
Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation †
Jiunn C. N. Fong and Fitnat H. Yildiz*
DepartmentofMicrobiologyandEnvironmentalToxicology,UniversityofCalifornia,SantaCruz,SantaCruz,California95064
Received4April2008/Accepted4August2008
Vibriocholeraeisafacultativehumanpathogen.TheabilityofV.choleraetoformbiofilmsiscrucialforits
survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during
epidemics.Formationofmaturebiofilmsrequirestheproductionofextracellularmatrixcomponents,includ-
ing Vibrio polysaccharide (VPS) and matrix proteins. Biofilm formation is positively controlled by the tran- D
scriptional regulators VpsR and VpsT and is negatively regulated by the quorum-sensing transcriptional o
regulator HapR, as well as the cyclic AMP (cAMP)-cAMP receptor protein (CRP) regulatory complex. w
n
Transcriptome analysis of cyaA (encoding adenylate cyclase) and crp (encoding cAMP receptor protein) lo
deletionmutantsrevealedthatcAMP-CRPnegativelyregulatestranscriptionofbothVPSbiosynthesisgenes a
andgenesencodingbiofilmmatrixproteins.FurthermutationalandexpressionanalysisrevealedthatcAMP- d
e
CRP negatively regulates transcription of vps genes indirectly through its action on vpsR transcription. d
However,negativeregulationofthegenesencodingbiofilmmatrixproteinsbycAMP-CRPcanalsooccurindepen- f
r
dent of VpsR. Transcriptome analysis also revealed that cAMP-CRP regulates the expression of a set of genes o
m
encodingdiguanylatecyclases(DGCs)andphosphodiesterases.Mutationalandphenotypicanalysisofthediffer-
entiallyregulatedDGCsrevealedthataDGC,CdgA,isresponsiblefortheincreaseinbiofilmformationinthe(cid:1)crp h
t
mutant,showingtheconnectionbetweenofcyclicdi-GMPandcAMPsignalinginV.cholerae. tp
:
/
/
jb
Vibrio cholerae, the causative agent of cholera (26), is a vps-II cluster consists of vpsL to vpsQ (VC0934 to VC0939). .
a
natural inhabitant of aquatic environments (14). Seasonal Recently,weidentifiedproteincomponentsofthebiofilmma- s
m
choleraoutbreaksoccurwherethediseaseisendemicandcan trix of V. cholerae and showed that the RbmA, RbmC, and
.
spread worldwide (14, 34). The ability of V. cholerae to cause Bap1 proteins are also required for the formation of a wild- or
g
epidemics is linked to its ability to survive in natural aquatic typebiofilm.Mutantsthatarenotabletoproducethesematrix /

ecosystems. One important factor for environmental survival proteinsformbiofilmsthatarestructurallyunstable(15,16). o
n
and transmission of V. cholerae is its ability to form biofilms TheregulationofbiofilmformationinV.choleraeiscomplex
J
(14, 60, 63). Biofilms are surface-attached microbial commu- and involves several transcriptional regulators. Two proteins a
n
nities composed of microorganisms and the extrapolymeric thatpositivelyregulateVPSproductionandbiofilmformation u
substancesthattheyproduce(9).Thebiofilmmodeofgrowth have been identified, VpsR and VpsT. Disruption of vpsR ar
isthepreferredlifestyleinthemicrobialworld,asitenhances prevents expression of the vps genes and production of VPS, y
3
survival in natural settings. In addition, biofilms protect the and it eliminates formation of typical 3D biofilm structures ,
constituent microbes from predators, such as protozoa and 2
(61).AvpsTmutantexhibitsreducedvpsgeneexpressionand 0
viruses, and from toxic compounds, such as antimicrobial 1
biofilm-forming capacity (7). A population-density-dependent
9
agents(4,10,13,36,41,63). regulatorysystem,knownasthequorum-sensingsystem,neg- b
The process of biofilm development in V. cholerae can be ativelyregulatesbiofilmformationinV.cholerae.HapRisthe y

divided into distinct stages: transport and attachment of bac- g
masterregulatorofthequorum-sensingregulatorysystem,and u
teria to the surface, colonization of the attached surface, for- ahapRmutanthasincreasedbiofilm-formingcapacity(19,62, e
mation of a monolayer of cells, and synthesis of the extracel- s
64). Consistent with this observation, expression of the vps t
lular matrix, leading to formation of a mature biofilm with a
genes, including vpsR and vpsT, is increased in the hapR mu-
characteristicthree-dimensional(3D)architecture.TheVibrio
tant (62). In addition, a second messenger, cyclic di-GMP
polysaccharide (VPS), encoded by the vps genes, is essential
(c-di-GMP), which is produced by diguanylate cyclases
for the development of 3D biofilm structures (63). The vps
(DGCs) containing a GGDEF amino acid motif and is de-
genesareclusteredintworegionsonthelargechromosomeof
gradedbyphosphodiesterases(PDEs)thathaveEALorHD-
V. cholerae O1 El Tor; the vps-I cluster consists of vpsU
GYP domains (8, 46, 49), positively regulates biofilm forma-
(VC0916) and vpsA to vpsK (VC0917 to VC0927), and the
tion in V. cholerae (3, 31, 56). We recently determined that
cdgA(encodingaDGC),whosetranscriptionispositivelyreg-
ulated by VpsR and negatively regulated by HapR, positively
*Correspondingauthor.Mailingaddress:DepartmentofMicrobi-
ologyandEnvironmentalToxicology,UniversityofCalifornia,Santa regulatesbiofilmformationinV.cholerae(2).
Cruz,SantaCruz,CA95064.Phone:[phone].Fax:(831)459- The cyclic AMP (cAMP)-cAMP receptor protein (CRP)
3524.E-mail:[email protected].
regulatorycomplexwasrecentlyidentifiedasanegativeregu-
†Supplemental material for this article may be found at http://jb
latorofbiofilmformationinV.cholerae(29,30).Thesestudies
.asm.org/.
(cid:1)Publishedaheadofprinton15August2008. showed that cAMP-CRP negatively regulates vpsL and vpsT
6646
VOL.190,2008 REGULATION OF BIOFILM MATRIX PRODUCTION BY cAMP-CRP 6647
expressionandpositivelyregulatesvpsRexpression.Additional (cid:2)-Galactosidaseassays.(cid:5)-Galactosidaseassayswerecarriedoutbyusinga
study showed that growth in the presence of glucose, which protocolsimilartothatdescribedbyMiller(38).Briefly,overnightcultureswere
diluted200-foldinLBmediumsupplementedwithampicillinandincubatedat
leads to a decrease in cellular cAMP levels, induces biofilm
30°Cfor10hwithshaking(200rpm).Theopticaldensitiesat600nm(OD )
formation,whileadditionofcAMPtoagrowthmediumleads 600
of the stationary-phase cultures were determined, and 1-ml portions of the
toadecreaseinbiofilmformationinwild-typeV.cholerae(23). cultures were harvested and washed with 1 ml of buffer Z (16.1 g/liter
TofurtherevaluatethemechanismbywhichcAMP-CRPneg- NaHPO (cid:1)7HO, 5.5 g/liter NaHPO (cid:1)HO, 0.75 g/liter KCl, 0.246 g/liter
2 4 2 2 4 2
atively regulates biofilm formation in V. cholerae, we deter- MgSO4(cid:1)7H2O;pH7.0).Cellswerelysedbyresuspendingacellpelletin1mlof
bufferZcontaining0.69%(cid:5)-mercaptoethanol,0.02%cetyltrimethylammonium
mined whole-genome expression profiles of cyaA (encoding
bromide,and0.01%deoxycholicacid(sodiumsalt),followedbyincubationat
adenylate cyclase) and crp (encoding CRP) deletion mutants. roomtemperaturefor5min.Celllysates(100(cid:4)l)ofdifferentdilutionswere
Our analysis revealed that cAMP-CRP negatively regulates pipetted into flat-bottom 96-well microtiter plates, and 20-(cid:4)l portions of an
transcription of VPS biosynthesis genes and genes encoding o-nitrophenyl-(cid:5)-D-galactopyranosidesolution(4mg/ml)wereadded,followedby
incubationat30°Cuntilsufficientcolordevelopmentwasobserved.Thereactions
biofilmmatrixproteins.cAMP-CRPnegativelyregulatestran-
werestoppedbyadding50(cid:4)lof1MNaCO,andthecolorintensitieswere
scription of both vps genes and the genes encoding biofilm 2 3
measuredatOD andOD .Thedurationofcolordevelopmentwasnoted,
420 550
matrixproteinsindirectly,throughitsactiononvpsRtranscrip- and the (cid:5)-galactosidase activity (expressed in Miller units) was calculated as
tion.Inaddition,cAMP-CRPcanalsonegativelyregulatetran- previouslydescribed(15,38).Theassayswererepeatedwithatleasttwodifferent D
scription of the genes encoding biofilm matrix proteins inde- biologicalreplicatesandeighttechnicalreplicates. ow
Generation of gfp-tagged strains and confocal laser scanning microscopy
pendent of VpsR. We also determined that cAMP-CRP n
regulatestheexpressionofasetofgenesencodingDGCsand (CLSM).V.choleraestrainswerechromosomallytaggedwiththegeneencoding lo
greenfluorescentprotein(gfp),usingapreviouslydescribedprocedure(2,15). a
PDEs. Through mutational and phenotypic analysis, we Non-flow-cellexperimentswerecarriedoutusingLab-TekIIchamberedcover- d
showed that CdgA is largely responsible for the increased glass systems (Nalge Nunc) and a previously described protocol (2). Briefly, e
d
transcriptionofvpsandbiofilmmatrixproteingenes,aswellas overnightculturesweredilutedtoobtainanOD600of0.2,and3-mlportionsof f
enhanced biofilm formation in a (cid:2)crp mutant, revealing the thedilutedcultureswereplacedintochambersandincubatedat30°Cfor8h. ro
Thechamberswerethenwashedtwicewith1mlofLBmedium.Biofilmsformed m
connectionbetweenc-di-GMPandcAMP-CRPsignalinginV. bynon-gfp-taggedstrainswerestainedfor15minatroomtemperatureinthe
cholerae. dark with 1 ml of 5 (cid:4)M SYTO9 (Molecular Probes). Images of the biofilms ht
t
formed in the chambers were acquired using a Zeiss Axiovert 200 M laser p
scanning microscope. 3D images of the biofilms were reconstructed using :/
MATERIALSANDMETHODS IMARISsoftware(Bitplane)andwerequantifiedusingtheCOMSTATprogram /jb
Bacterialstrains,plasmids,andcultureconditions.Bacterialstrainsandplas- (22). Non-flow-cell experiments were carried out with at least two different .a
midsusedinthisstudyarelistedinTable1.AllV.choleraeandEscherichiacoli biologicalreplicates. s
strains were routinely grown aerobically in Luria-Bertani (LB) medium (1% Biofilmformationassays.Biofilmswereformedin96-wellpolyvinylchloride m
tryptone,0.5%yeastextract,1%NaCl;pH7.5)at30and37°C,respectively, microtiterplatesbyusing100-(cid:4)lportionsofovernightculturesdilutedtoobtain .o
unlessotherwisenoted.TheE.coliDH10BandCC118(cid:3)pirstrainswereusedfor anOD600of0.2.Themicrotiterplateswereincubatedat30°Cfor8h.Crystal rg
DNAmanipulation,whiletheE.coliS17-1(cid:3)pirstrainwasusedforconjugation violetstainingandethanolsolubilizationwerecarriedoutaspreviouslydescribed /

with V. cholerae A1552. Conjugation with other V. cholerae strains (C6706, (15,63).Theassayswererepeatedwithtwodifferentbiologicalreplicatesand o
n
N16961,andMO10)wascarriedoutusingtheE.coliSM10(cid:3)pirstrain.Agar eighttechnicalreplicates.
mediumcontained1.5%granulatedagar(Difco),unlessotherwisenoted.Am- RNAisolation.TotalRNAwasisolatedfromV.choleraestrainsinstationary Ja
picillin,rifampin,andstreptomycinwereusedataconcentrationof100(cid:4)g/ml, growthphasebyusingapreviouslydescribedprotocol(62).Briefly,overnight n
whilegentamicinwasusedataconcentrationof50(cid:4)g/ml.Weobservedthat culturesofV.choleraegrowninLBmediumat30°Cwithshaking(200rpm)were u
a
(cid:2)cyaAand(cid:2)crpmutantshadincreaseddoublingtimesinLBmediumat30°C diluted200-foldinLBmediumandincubatedat30°Cfor10h.Aliquots(2ml) r
comparedtothewildtype(datanotshown).Thisfindingissimilartothegrowth ofthecultureswerecollectedandcentrifugedfor2minatroomtemperature. y
defectreportedforacrpmutantgrowninLBmediumat37°C(52).Tominimize Thecellpelletswereimmediatelyresuspendedin1mlofTRIzol(Invitrogen) 3
theeffectofreducedgrowthratesinourexperiments,expressionprofilingand andstoredat(cid:7)80°C.TotalRNAwasisolatedaccordingtothemanufacturer’s , 2
(cid:5)-galactosidaseassayswerecarriedoutwithculturesgrowntostationaryphase. instructions. To remove contaminating DNA, total RNA was incubated with 0
RecombinantDNAtechniques.DNAmanipulationswerecarriedoutbyusing RNase-freeDNaseI(Ambion),andanRNeasyminikit(Qiagen)wasusedto 1
9
standardmoleculartechniques(47).RestrictionandDNAmodificationenzymes cleanupRNAafterDNasedigestion.
b
werepurchasedfromNewEnglandBiolabs.PCRswerecarriedoutusingprim- Whole-genomeexpressionprofiling.Whole-genomeexpressionprofilingwas y
erspurchasedfromOperonTechnologies(Table2)andahigh-fidelityPCRkit performedbyusingapreviouslydescribedprocedure(3).Acommonreference g
(Roche).DNAsequencingwascarriedoutbytheUCBerkeleyDNASequencing RNAwasused,whichcontainedequalamountsoftotalRNAisolatedfromV. u
Facility. choleraecellsgrowntostationaryphaseinLBmedium.Normalizedsignalratios e
s
Generation of in-frame deletion mutants. Deletion mutants of V. cholerae wereobtainedwithLOWESSprinttipnormalizationusingtheBioconductor t
strains were generated by using a previously described protocol (16). The packages (http://www.bioconductor.org) in the R environment (18). Differen-
DNAsequencesoftheconstructeddeletionplasmidswereverifiedbyDNA tiallyregulatedgenesweredetermined(withthreebiologicalandtwotechnical
sequencing. Primers used in the construction of the deletion plasmids are replicatesforeachdatapoint)usingtheSignificanceAnalysisofMicroarrays
showninTable2. (SAM)software(58)witha(cid:1)2-folddifferenceingeneexpressionandafalse
Pellicleformationandmotilityassays.Pellicleformationexperimentswere discoveryrate(FDR)of(cid:2)1%ascutoffvalues,unlessotherwisenoted.
carriedoutusingglassculturetubes(18by150mm)containing5mlofmedium. qPCR.QuantitativePCR(qPCR)wascarriedoutbyfirstsynthesizingcDNA
Themediumwasinoculatedwithovernightcultures,resultingin200-folddilu- from1(cid:4)gofatotalRNAsampleusinganiScriptcDNAsynthesiskit(Bio-Rad).
tion.Thetubeswereincubatedat30°Cwithoutshakingfor2days.LBsoftagar ThecDNAproductwasthendiluted1:4withwater,and4(cid:4)lwasusedasa
plates(0.3%agar)wereusedtodeterminethemotilityofthebacterialstrains. templatewith12pmolofeachqPCRprimer(Table2)inaPCRperformedwith
The diameter of each migration zone (in cm) was measured after 18 h of the Expand high-fidelity PCR system (Roche). The PCR conditions were as
incubationat30°C.Assayswererepeatedwithatleasttwobiologicalreplicates. follows:94°Cfor2minandthen25cyclesof94°Cfor30s,55°Cfor30s,and72°C
GenerationoflacZtranscriptionalfusionconstructs.lacZtranscriptionalfu- for30sandafinalincubationat72°Cfor2min.Theamplifiedproductswere
sionswithpromotersofrbmCandbap1wereconstructedbycloningthePCR- analyzedona2%agarosegelandwerequantifiedusingtheImageQuant5.2
amplified(cid:6)300-bppromoterregionsimmediatelyupstreamofthestartcodons software(MolecularDynamics).TheintensityofeachDNAbandwasnormal-
ofrbmCandbap1intopRS415(51)asdescribedpreviously(15).Theresulting izedtothatofthecorrespondingrecAbandamplifiedwithprimersRecA578and
transcriptionalfusionplasmidsweresequenced.Theplasmidswereelectropo- RecA863(29).Thedatapresentedbelowarefromthreebiologicalreplicates,
ratedintoV.choleraestrainscontainingalacZin-framedeletion.Theprimers andreactionmixturescontainingnotemplateorreversetranscriptasewereused
usedforamplificationofthepromoterregionsareshowninTable2. asnegativecontrols.
6648 FONG AND YILDIZ J.BACTERIOL.
TABLE 1. Bacterialstrainsandplasmidsusedinthisstudy
Strainorplasmid Relevantgenotypeandphenotype Sourceorreference
E.colistrains
DH10B F(cid:7)mcrA(cid:2)(mrr-hsdRMS-mcrBC)(cid:8)80lacZ(cid:2)M15(cid:2)lacX74recA1endA1ara(cid:2)139(cid:2)(araleu) Invitrogen
7697galUgalK(cid:3)-rpsL(Smr)nupG
CC118(cid:3)pir (cid:2)(ara-leu)araD(cid:2)lacX74galEgalKphoA20thi-1rpsErpoBargE(Am)recA1(cid:3)pir 21
S17-1(cid:3)pir TprSmrrecAthipror (cid:7)m (cid:1)RP4::2-Tc::MuKmTn7(cid:3)pir 11
SM10(cid:3)pir thithrleutonAlacYsuKpEreKcA(RP4-2-Tc::Mu)(cid:3)pirR6KKmr(cid:9)(cid:1) 54
V.choleraestrains
FY_Vc_1 V.choleraeO1ElTorA1552,wild-typevariant,Rifr 63
C6706 V.choleraeO1ElTorC6706,wild-typevariant,Smr 35
N16961 V.choleraeO1ElTorN16961,wild-typevariant,Smr 20
MO10 V.choleraeO139MO10,wild-typevariant,Smr P.Watnick
FY_Vc_2322 (cid:2)cyaA,Rifr Thisstudy
FY_Vc_2326 (cid:2)crp,Rifr Thisstudy
D
FY_Vc_237 FY_Vc_1mTn7-gfp,RifrGmr 3
o
FY_Vc_2448 (cid:2)cyaAmTn7-gfp,RifrGmr Thisstudy w
FY_Vc_2451 (cid:2)crpmTn7-gfp,RifrGmr Thisstudy n
FY_Vc_3 FY_Vc_1(cid:2)lacZ,Rifr 7 lo
FY_Vc_3756 C6706(cid:2)lacZ Thisstudy a
FY_Vc_3748 N16961(cid:2)lacZ Thisstudy d
e
FY_Vc_3763 MO10(cid:2)lacZ Thisstudy d
FY_Vc_2456 FY_Vc_1(cid:2)cyaA(cid:2)lacZ,Rifr Thisstudy f
FY_Vc_2459 FY_Vc_1(cid:2)crp(cid:2)lacZ,Rifr Thisstudy ro
FY_Vc_3759 C6706(cid:2)crp(cid:2)lacZ Thisstudy m
FY_Vc_3751 N16961(cid:2)crp(cid:2)lacZ Thisstudy h
FY_Vc_3766 MO10(cid:2)crp(cid:2)lacZ Thisstudy t
t
FY_Vc_2919 FY_Vc_1(cid:2)hapR(cid:2)lacZ,Rifr Thisstudy p
FY_Vc_2922 FY_Vc_1(cid:2)vpsT(cid:2)lacZ,Rifr Thisstudy ://
FY_Vc_2874 FY_Vc_1(cid:2)vpsR(cid:2)lacZ,Rifr Thisstudy jb
FY_Vc_344 FY_Vc_1(cid:2)cdgA,Rifr 31 .a
FY_Vc_360 FY_Vc_1(cid:2)cdgAmTn7-gfp,RifrGmr 31 s
m
FY_Vc_3296 FY_Vc_1(cid:2)cdgA(cid:2)lacZ,Rifr Thisstudy
FY_Vc_350 FY_Vc_1(cid:2)cdgB,Rifr 31 .o
FY_Vc_987 FY_Vc_1(cid:2)cdgF,Rifr 3 rg
FY_Vc_1592 FY_Vc_1(cid:2)cdgH,Rifr Beyhanet.al., /
o
submitted n
FY_Vc_956 FY_Vc_1(cid:2)cdgI,Rifr Thisstudy
J
FY_Vc_354 FY_Vc_1(cid:2)rocS,Rifr 31 a
FY_Vc_152 FY_Vc_1(cid:2)VC0072,Rifr Thisstudy n
FY_Vc_869 FY_Vc_1(cid:2)VC1376,Rifr Thisstudy ua
FY_Vc_158 FY_Vc_1(cid:2)VC2750,Rifr Thisstudy r
FY_Vc_154 FY_Vc_1(cid:2)VCA0217,Rifr Thisstudy y
FY_Vc_2779 FY_Vc_1(cid:2)crp(cid:2)hapR(cid:2)lacZ,Rifr Thisstudy 3,
FY_Vc_2781 FY_Vc_1(cid:2)crp(cid:2)vpsT(cid:2)lacZ,Rifr Thisstudy 2
FY_Vc_2916 FY_Vc_1(cid:2)crp(cid:2)vpsR(cid:2)lacZ,Rifr Thisstudy 0
FY_Vc_3299 FY_Vc_1(cid:2)crp(cid:2)cdgA(cid:2)lacZ,Rifr Thisstudy 19
FY_Vc_3712 FY_Vc_1(cid:2)crp(cid:2)cdgA(cid:2)lacZmTn7-gfp,RifrGmr Thisstudy
b
FY_Vc_3311 FY_Vc_1(cid:2)crp(cid:2)cdgB(cid:2)lacZ Thisstudy y
FY_Vc_3323 FY_Vc_1(cid:2)crp(cid:2)cdgF(cid:2)lacZ Thisstudy g
FY_Vc_3317 FY_Vc_1(cid:2)crp(cid:2)cdgH(cid:2)lacZ Thisstudy u
FY_Vc_3308 FY_Vc_1(cid:2)crp(cid:2)cdgI(cid:2)lacZ Thisstudy e
s
FY_Vc_3314 FY_Vc_1(cid:2)crp(cid:2)rocS(cid:2)lacZ Thisstudy t
FY_Vc_3305 FY_Vc_1(cid:2)crp(cid:2)VC0072(cid:2)lacZ Thisstudy
FY_Vc_3318 FY_Vc_1(cid:2)crp(cid:2)VC1376(cid:2)lacZ Thisstudy
FY_Vc_3320 FY_Vc_1(cid:2)crp(cid:2)VC2750(cid:2)lacZ Thisstudy
FY_Vc_3322 FY_Vc_1(cid:2)crp(cid:2)VCA0217(cid:2)lacZ Thisstudy
FY_Vc_3302 FY_Vc_1(cid:2)hapR(cid:2)cdgA(cid:2)lacZ,Rifr Thisstudy
FY_Vc_337 FY_Vc_1(cid:2)flaA,Rifr Beyhanet.al.,
submitted
FY_Vc_231 FY_Vc_1(cid:2)vps-I,Rifr 3
FY_Vc_3787 FY_Vc_1(cid:2)crp(cid:2)vps-I,Rifr Thisstudy
FY_Vc_3411 FY-Vc_1(cid:2)vps-I(cid:2)vps-II,Rifr Thisstudy
FY_Vc_3788 FY-Vc_1(cid:2)crp(cid:2)vps-I(cid:2)vps-II,Rifr Thisstudy
FY_Vc_102 FY_Vc_1(cid:2)rbmA,Rifr 15
FY_Vc_3789 FY_Vc_1(cid:2)crp(cid:2)rbmA,Rifr Thisstudy
FY_Vc_3790 FY_Vc_1(cid:2)bap1,Rifr Thisstudy
FY_Vc_3791 FY_Vc_1(cid:2)crp(cid:2)bap1,Rifr Thisstudy
Continuedonfollowingpage
VOL.190,2008 REGULATION OF BIOFILM MATRIX PRODUCTION BY cAMP-CRP 6649
TABLE 1—Continued
Strainorplasmid Relevantgenotypeandphenotype Sourceorreference
Plasmids
pGP704-sacB28 pGP704derivative,mob/oriTsacB,Apr G.Schoolnik
pFY-659 pGP704-sacB28::(cid:2)vps-IIoperon,Apr Thisstudy
pFY-308 pGP704-sacB28::(cid:2)cyaA,Apr Thisstudy
pFY-333 pGP704-sacB28::(cid:2)crp,Apr Thisstudy
pFY-149 pGP704-sacB28::(cid:2)cdgA,Apr 31
pFY-447 pGP704-sacB28::(cid:2)cdgI,Apr Thisstudy
pCC27 pGP704-sacB28::(cid:2)vpsR,Apr 7
pCC2 pGP704-sacB28::(cid:2)lacZ,Apr 7
pFY-252 pGP704-sacB28::(cid:2)VC0072,Apr Thisstudy
pFY-384 pGP704-sacB28::(cid:2)VC1376,Apr Thisstudy
pFY-237 pGP704-sacB28::(cid:2)VC2750,Apr Thisstudy
pFY-250 pGP704-sacB28::(cid:2)VCA0217,Apr Thisstudy
pRS415 PromoterlesslacZcloningvectorfortranscriptionalfusionstudies,Apr 51
D
pCC12 pRS415vpsLpromoter,Apr 7
o
pCC25 pRS415vpsTpromoter,Apr 7 w
pCC10 pRS415vpsRpromoter,Apr 7 n
pFY-169 pRS415rbmApromoter,Apr 15 lo
pFY-578 pRS415rbmCpromoter,Apr Thisstudy a
pFY-581 pRS415bap1promoter,Apr Thisstudy d
e
pFY-150 pACYC177::cdgAoperon(includesVCA0074andVCA0075),Apr 31 d
pMCM11 pGP704::mTn7-gfp,GmrApr M.MillerandG. f
r
Schoolnik o
pUX-BF13 oriR6Khelperplasmid,mob/oriT,providestheTn7transpositionfunctionintrans,Apr 1 m

h
t
t
p
:
/
RESULTS genes and vpsT were higher in both the (cid:2)cyaA and (cid:2)crp mu- /jb
Identificationofgenesdifferentiallyregulatedin(cid:1)cyaAand tantsthaninthewildtype(Table3).Geneexpressionprofiling .as
(cid:1)crpmutants.cAMP-CRPnegativelyregulatesbiofilmforma- also revealed that the expression of hapR was decreased in m
both the (cid:2)cyaA and (cid:2)crp mutants. This finding is consistent .
tion in V. cholerae. To further understand how cAMP-CRP o
withpreviouslydescribedresults(29,50).Inadditiontothese r
regulates biofilm formation, we generated in-frame cyaA g
(VC0122)andcrp(VC2614)deletionmutantsofourprototype genes, we observed that the levels of expression of the genes o/
encoding the biofilm matrix proteins, rbmA, rbmC, and bap1, n
V. cholerae O1 El Tor A1552 strain and performed whole-
were higher in both the (cid:2)cyaA and (cid:2)crp mutants than in the J
genome expression profiling of these mutants. The gene ex- a
pression data were analyzed by using the SAM software and wildtype.Toverifythisfinding,wemonitoredtranscriptionof n
the genes encoding biofilm matrix proteins by using rbmA- u
the following criteria to define significantly regulated genes, a
unless otherwise indicated: an FDR of (cid:2)1% and a (cid:1)2-fold lacZ,rbmC-lacZ,andbap1-lacZfusionconstructsandmeasur- ry
transcriptabundancedifferencebetweensamples.Thisanalysis ing (cid:5)-galactosidase activities. In parallel, we also monitored 3
transcriptionofthegenesinvolvedinVPSbiosynthesisusinga ,
revealedthatcAMP-CRPdifferentiallyregulatestranscription 2
vpsL-lacZ fusion construct. As expected, vpsL transcription 0
opfroafilleasrgoeftsheet(cid:2)ocfygaeAnaensdan(cid:2)dcrpthmatuttahnetsoavereraslilmgielanre(seexeprFeisgs.ioSn1 was increased in both the (cid:2)cyaA mutant (205-fold) and the 19
inthesupplementalmaterial).Altogether,889genes(22.9%of (cid:2)crp mutant (142-fold) compared to the wild type (Fig. 1A). b
y
thegenome)and822genes(21.2%ofthegenome)arediffer- Transcription of the genes encoding biofilm matrix proteins g
entiallyregulatedinthe(cid:2)cyaAand(cid:2)crpmutantscomparedto was also increased in the (cid:2)cyaA mutant (15-fold for rbmA, u
the wild type, respectively. Of the 889 differentially regulated 36-fold for rbmC, and 32-fold for bap1) and the (cid:2)crp mutant es
genes in the (cid:2)cyaA mutant, 431 are upregulated and 458 are (13-fold for rbmA, 27-fold for rbmC, and 15-fold for bap1) t
downregulated, whereas of the 822 differentially regulated compared to the wild type (Fig. 1B to D). This indicates that
genes in the (cid:2)crp mutant, 386 are upregulated and 436 are cAMP-CRP negatively regulates transcription of genes re-
downregulated. All the differentially regulated genes are quired for the production of both VPS and biofilm matrix
showninTablesS1andS2inthesupplementalmaterial. proteins.
In this study, our main objective was to determine how cAMP-CRP negatively regulates transcription of vpsT and
cAMP-CRP negatively regulates biofilm formation. Thus, for vpsR in V. cholerae O1 El Tor A1552. To understand how
the genes that are differentially regulated by cAMP-CRP, we cAMP-CRPnegativelyregulatestranscriptionofvpsgenesand
focused on two sets of genes: the genes required for biofilm biofilmmatrixproteingenes,weanalyzedhowtranscriptionof
matrixproductionanditsregulationandthegenespredictedto vpsR and vpsT is altered in (cid:2)cyaA and (cid:2)crp mutants. Using
beinvolvedintheproductionanddegradationofc-di-GMP,as vpsT-lacZandvpsR-lacZfusionconstructs,wedeterminedthat
thissecondmessengerregulatesbiofilmformationinV.cholerae. vpsTandvpsRtranscriptionwasincreasedinthe(cid:2)cyaAmutant
cAMP-CRP negatively regulates transcription of the vps, (178-fold for vpsT and 4-fold for vpsR) and the (cid:2)crp mutant
rbmA, rbmC, and bap1 genes. As expected for a negative reg- (144-fold for vpsT and 4-fold for vpsR) compared to the wild
ulator of biofilm formation, the levels of expression of vps type,indicatingthatcAMP-CRPalsonegativelyregulatestran-
6650 FONG AND YILDIZ J.BACTERIOL.
TABLE 2. Sequencesofoligonucleotidesusedinthisstudy scription of vpsT and vpsR (Fig. 2A and B). Interestingly,
Primer Sequence(5(cid:10)–3(cid:10)) transcription of vpsR was shown to be positively regulated by
cAMP-CRP in the V. cholerae O1 El Tor C7258 strain (30).
vps-II_del_A..............CATGCCATGGCATGCGGCTGGTCTATGT Differences in the regulation of vpsR by cAMP-CRP in our
GGCTTG
prototypestrain,V.choleraeO1ElTorA1552,andV.cholerae
vps-II_del_B..............CGAGCATAGTCCCTAGCAAGGCAACC
GAAA O1 El Tor C7258 prompted us to look at vpsR regulation in
vps-II_del_C..............GTCTTGCTAGGGACTATGCTCGCGGGTT othercommonlyusedV.choleraestrains.Tothisend,wegen-
TACTGC erated in-frame crp deletion mutants of V. cholerae strains
vps-II_del_D.............CGAGCTCGCTCGATCTTTGCCGATCACC
N16961,C6706,andMO10andintroducedareporterplasmid
cyaA_del_A...............GATCCCATGGGTTTTCCCGCTTGATT
harboring a vpsR-lacZ fusion construct into these strains. We
GTGT
cyaA_del_B...............CGCGGTTGGCCTGCTGGATAGGATTGG thenmonitoredtranscriptionofvpsRbymeasuring(cid:5)-galacto-
CTTC sidase activity (Fig. 2C). In our prototype A1552(cid:2)crp strain,
cyaA_del_C...............TATCCAGCAGGCCAACCGCGTTGAAGT vpsR-lacZtranscriptionwasincreased3.9-foldcomparedtothe
CTAT wild-type transcription. N16961(cid:2)crp also exhibited a similar
cyaA_del_D...............GATCTCTAGACAAATCGATTGATGGC
GAAT increase in transcription of vpsR-lacZ (3.4-fold), while D
crp_del_A..................GATCTCTAGATGAGTTTTGCGATGGA MO10(cid:2)crp exhibited a slight increase (1.3-fold) in vpsR-lacZ ow
TTTG transcriptioncomparedtothecorrespondingwild-typestrains. n
crp_del_B..................TGATCTTGATCGCAACTGAACCTTTTACG Ontheotherhand,inC6706andtranscriptionofvpsR-lacZin lo
crp_del_C..................TTCAGTTGCGATCAAGATCACTCGCCA a
AGAG C6706(cid:2)crp did not differ significantly. These results indicate d
e
crp_del_D..................GATCGAGCTCCCAACATGGCTTTAGC thatwhilecAMP-CRPnegativelyregulatesvpsRtranscription d
ATCA intheA1552,N16961,andMO10(albeitslightly)strainsofV. f
cdgI_del_A................CTAGCCATGGCCTCTTCGTGTCCCGGAG cholerae, there is no such regulation in strain C6706. Hence, ro
TATC m
theresultsareconsistentwiththeideathatvpsRregulationby
cdgI_del_B.................CAACGGGTAAGGCCAAAGAATGAATC
h
TTGC cAMP-CRP varies in strains of V. cholerae. Together, our re- t
t
cdgI_del_C................TTCTTTGGCCTTACCCGTTGCGACAAA sults indicate that in our prototype strain, cAMP-CRP re- p
:
CAGT pressesbiofilmformationinV.choleraebynegativelyregulat- //
cdgI_del_D................CTAGTCTAGACTCATCAAGGAATCGC jb
ATCA ing transcription of the genes required for VPS biosynthesis .a
VC0072_del_A.........GATCTCTAGACTAGGCTAATCGAACGCT and matrix protein production, as well as the genes encoding s
m
CATTCC thepositivetranscriptionalregulatorsVpsTandVpsR.
.
VC0072_del_B..........GCTGCAAAGCTAACGGTTGGCTCGAT crp is epistatic to hapR in the regulation of vpsT and vpsR o
AAGG transcription. Biofilm formation in V. cholerae is negatively rg
VC0072_del_C..........CCTAGATCGCGTTAGCTTTGCAGCAGATTGG regulatedbybothHapR(19,62,64)andcAMP-CRP(29,30). o/
n
VC0072_del_D.........GGAGCTCGAGACTGATGCGCTCACTGAC Since cAMP-CRP positively regulates hapR transcription, re-
J
VC1376_del_A.........CGAGCTCCAGCCCAGCATGGAGCATATC pression of biofilm formation by cAMP-CRP could be medi- a
VC1376_del_B..........ACCTTTCGATACCAGCAAGGGCACAA atedthroughHapR.Thus,tobetterevaluatethemechanismby n
TCAC u
which cAMP-CRP and HapR negatively regulate biofilm for- a
VC1376_del_C..........CCTTGCTGGTATCGAAAGGTCGAAATC r
mation,weanalyzedvpsTandvpsRtranscriptioninwild-type, y
GTGT
VC1376_del_D.........CATGCCATGGCATGCATTCACCAGCCAA (cid:2)crp,(cid:2)hapR,and(cid:2)crp(cid:2)hapRstrainsharboringvpsT-lacZand 3
,
CAGACG vpsR-lacZ fusion plasmids (Fig. 2D and E). We observed 27- 2
VC2750_del_A.........CATGCCATGGCAGCCAAAGAGCTC and 3.2-fold increases in (cid:5)-galactosidase activities in the 01
GGAG (cid:2)hapR mutants harboring vpsT-lacZ and vpsR-lacZ fusion 9
VC2750_del_B..........GCAACCGACAAACGGCAGTATGATGGC
VC2750_del_C..........TTTGTCGGTTGCCCACGCGGGCAAGGC plasmids, respectively, compared to the wild type. These re- by
VC2750_del_D.........CGATTCTAGAGTACCAAAGGTGCGGCTC sults are congruent with the results of our previous studies g
VCA0217_del_A......GATCTCTAGACTAGTGCGCCATGTAACC showingthatHapRnegativelyregulatestheexpressionofvpsT u
AATAGA e
and vpsR in strain A1552 (2, 62). It is noteworthy that in V. s
VCA0217_del_B.......GCTTTTACCGATGCGCTATTGGGTTC t
choleraestrainsC6706andC7258negativeregulationofvpsR
AACT
VCA0217_del_C.......AATAGCGCATCGGTAAAAGCAGGAGA by HapR has not been observed (19, 30, 59), indicating that,
GTGA similar to vpsR repression by cAMP-CRP, vpsR repression by
VCA0217_del_D......GGAGCTCGGTCTTATTGATGCGGGAGCA HapRalsovariesinstrainsofV.cholerae.Interestingly,similar
rbmC_proF..............GATCGAATTCCTAGAAAATGCTTCTTGA
increases in the transcriptional levels of vpsT (210-fold in the
rbmC_proR..............GATCGGATCCTTGTAAGACTCCCTTT
ACCT (cid:2)crpmutantand231-foldinthe(cid:2)crp(cid:2)hapRmutant)andvpsR
bap1_proF................GAGAGAATTCCGCCGCGTTGCTGAG (6.2-fold in the (cid:2)crp and mutant 6.0-fold in the (cid:2)crp (cid:2)hapR
bap1_proR...............GAGAGGATCCGGCTTGACCTTCATCT mutant)wereobservedforthe(cid:2)crpand(cid:2)crp(cid:2)hapRmutants,
RecA578....................GTGCTGTGGATGTCATCGTTGTTG
indicating that crp is epistatic to hapR in regulating vpsT and
RecA863....................CCACCACTTCTTCGCCTTCTTTGA
cdgA_rtF...................CAAGCGATCTGGTTCTTATTCC vpsRtranscription.
cdgA_rtR..................AAAACGGCTCCAAGTCAGC cAMP-CRP regulates rbmC and bap1 expression both
cdgI_rtF....................GATGTGGAAAGGCAAAGAGC through and independent of VpsR. Formation of mature bio-
cdgI_rtR...................CTGTGCTATGCGAGTTTTGC filmsinV.choleraerequiresproductionofVPSandthematrix
rocS_rtF....................CAGGTTGCACCTCTTTACTCG
proteinsRbmA,RbmC,andBap1.Asdiscussedabove,cAMP-
rocS_rtR...................ACCCGTGTCGGTTATACAGC
CRPnegativelyregulatestheexpressionofbothvpsgenesand
VOL.190,2008 REGULATION OF BIOFILM MATRIX PRODUCTION BY cAMP-CRP 6651
TABLE 3. Differentiallyexpressedgenesinvolvedinbiofilmmatrix
productionandc-di-GMPsignalingin(cid:2)cyaAand(cid:2)crpmutants
comparedtothewildtypea
Change(fold)
Geneno. Designation (cid:2)cyaA/wild (cid:2)crp/wild
type type
Biofilmmatrixproduction
genes
VC0916 vpsU 3.76 3.35
VC0917 vpsA 1.51
VC0918 vpsB 2.93 2.16
VC0919 vpsC 2.36 2.37
VC0922 vpsF 2.04
VC0928 rbmA 5.89 5.45
VC0929 rbmB 2.83 3.57
VC0930 rbmC 21.04 8.01 D
VC0931 rbmD 2.30 o
VC0932 rbmE 7.46 4.27 w
VC0933 rbmF 5.89 5.99 n
VC0935 vpsM 12.75 8.06 lo
VC0936 vpsN 2.63 2.32 FIG. 1. cAMP-CRP negatively regulates expression of genes in- a
volved in VPS biosynthesis and biofilm matrix protein production. d
VC0937 vpsO 1.61 1.99 (cid:5)-Galactosidaseassaysofwild-type,(cid:2)cyaA,and(cid:2)crpstrainsharboring e
VC0939 vpsQ 2.73 2.84 d
VC0583 hapR 0.11 0.48 (A) vpsL-lacZ, (B) rbmA-lacZ, (C) rbmC-lacZ, and (D) bap1-lacZ f
VC1888 bap1 7.84 6.28 fusion constructs were performed. The data are representative of at ro
VCA0952 vpsT 74.62 48.70 leasttwoindependentexperiments.Theerrorbarsindicatestandard m
deviations.
h
c-di-GMPsignalinggenes: t
GGDEFandEAL tp
VVCC00067523 rocS 1.91 21..5831 genes encoding matrix proteins. However, we have a limited ://jb
VC0658 cdgI 2.95 3.34 understanding of the mechanism by which cAMP-CRP regu- .
VC0703 mbaA 1.78 1.77 lates transcription of these genes and do not know whether as
VC1934 0.42 0.44 m
vps and matrix protein genes are regulated differently by
VC2750 1.70
.
VCA0785 cdgC 1.53 cAMP-CRP. o
r
WepreviouslyreportedthatVpsRisthemostdownstream g
c-di-GMPsignalinggenes: regulatorofvpsgenetranscriptionandgenesencodingmatrix o/
onlyGGDEF
n
VC1029 cdgB 1.88 2.17 proteinsintheVpsT,VpsR,andHapRregulatorycircuitry(2).
J
VC1067 cdgH 1.58 We wanted to determine how cAMP-CRP contributes to this a
VC1216 0.56 0.45 regulatorycircuitry.Tothisend,wemonitoredtranscriptionof n
VC1353 0.53 u
VC1367 cdgE 0.64 vpsL, rbmC, and bap1 using lacZ transcriptional fusion con- a
VC1370 0.37 structs in the wild-type strain and (cid:2)crp, (cid:2)vpsT, (cid:2)vpsR, (cid:2)crp ry
VC1376 1.99 (cid:2)vpsT,and(cid:2)crp(cid:2)vpsRmutants(Fig.3).Asdiscussedabove,in 3
VC1599 0.44 0.40 ,
VCA0049 0.58 the(cid:2)crpstrainharboringthefusionconstructvpsL-lacZ,tran- 2
0
VCA0074 cdgA 1.61 scriptionofvpsLwasmarkedlyincreasedcomparedtothatin 1
VCA0165 0.56 thewildtype.TranscriptionofvpsL-lacZwas1.7-and4.6-fold 9
VCA0217 1.94
VCA0697 cdgD 0.09 0.11 lowerinthe(cid:2)vpsTand(cid:2)vpsRmutants,respectively,thaninthe by
VCA0956 cgdF 1.90 1.87 wildtype(Fig.3A).Thisfindingisconsistentwithourprevious g
VCA0965 0.31 0.50 reportthatthemagnitudeofregulationofvpsgeneexpression u
e
c-di-GMPsignalinggenes: by VpsR is greater than that by VpsT (2). In the (cid:2)crp (cid:2)vpsT s
t
onlyEAL double-deletion mutant, a 2.1-fold increase in vpsL transcrip-
VC0137 1.69 1.93 tion was observed compared to the wild type. This slight in-
VC1086 0.38 0.40
VC1211 1.58 crease in vpsL transcription was likely due to decreased ex-
VC1641 1.66 2.56 pressionofhapRinthe(cid:2)crpgeneticbackground.cAMP-CRP
VC1710 0.21 0.29 positively regulates hapR expression, and a decrease in hapR
message abundance, in turn, leads to an increase in vpsL and
c-di-GMPsignalinggenes:
HD-GYP vpsRexpression(2,62).Unlikethefindingsforthe(cid:2)crp(cid:2)vpsT
VC1087 0.43 0.55 mutant, the vpsL transcription in the (cid:2)crp (cid:2)vpsR mutant was
VC1348 0.62
3.3-fold lower than that in the wild type (similar to the
VC2340 2.57
VC2497 0.54 0.47 (cid:2)vpsR mutant), indicating that VpsR acts downstream of
VCA0210 1.76 cAMP-CRP.
VCA0895 0.46 0.61 Transcription of rbmC and bap1 was increased in the (cid:2)crp
VCA0931 0.48
mutantcomparedtothewildtype.AlthoughdeletionofvpsR
aDifferentiallyexpressedgenesweredeterminedusingtheSAMsoftwarewith resulted in decreases in transcription of rbmC (7.6-fold) and
a(cid:1)1.5-foldchangeingeneexpressionandanFDRof(cid:2)3%asthecriteria.
bap1 (6.8-fold), deletion of vpsT did not significantly alter
6652 FONG AND YILDIZ J.BACTERIOL.
encoding matrix proteins are regulated differently by cAMP-
CRP, we wanted to determine the contribution of VPS and
biofilmmatrixproteinstobiofilmformationinthe(cid:2)crpgenetic
background.Tothisend,wegenerated(cid:2)crp(cid:2)vps-I(vps-Iclus-
terdeletion),(cid:2)crp(cid:2)vps-I(cid:2)vps-II(vps-Iandvps-IIclusterde-
letion), (cid:2)crp (cid:2)rbmA, and (cid:2)crp (cid:2)bap1 mutants and compared
their biofilm-forming capacities to those of the wild type and
(cid:2)crp,(cid:2)vps-I,(cid:2)vps-I(cid:2)vps-II,(cid:2)rbmA,and(cid:2)bap1singlemutants.
Deletionofthevps-Iclusterorthevps-Iandvps-IIclustersin
the(cid:2)crpgeneticbackgroundeliminatedformationofapellicle
(biofilm formed at the air-liquid interface) (data not shown)
and drastically reduced biofilm formation in both 96-well mi-
crotiter plate and non-flow-cell systems (Fig. 3D and E). De-
letion of rbmA or bap1 in the (cid:2)crp genetic background de-
creased,butdidnoteliminate,biofilmformation.Comparedto D
the (cid:2)crp mutant biofilm, the biofilms formed by the (cid:2)crp ow
(cid:2)rbmA and (cid:2)crp (cid:2)bap1 mutants were less structured, and n
therewerefewermaturepillarsinthebiofilmsformedbythe lo
a
double-deletion mutants (Fig. 3E). Although the total bio- d
e
masses and average biofilm thicknesses were not significantly
d
different for the (cid:2)crp, (cid:2)crp (cid:2)rbmA, and (cid:2)crp (cid:2)bap1 mutants, f
r
the maximum biofilm thickness was greater for the (cid:2)crp mu- o
m
tant than for the (cid:2)crp (cid:2)rbmA and (cid:2)crp (cid:2)bap1 mutants (data

h
not shown). Together, these results indicate that under the
t
t
experimental conditions that we utilized, VPS production is p
:
essential for biofilm formation in a (cid:2)crp genetic background, //
jb
whilebiofilmmatrixproteinshaveanaccessoryrole.Wedonot .
a
know yet how biofilm matrix proteins function, whether they s
m
bind VPS carbohydrates or mediate cell-cell or cell-surface
FIG. 2. cAMP-CRPnegativelyregulatesvpsTandvpsRexpression.
.
(AandB)(cid:5)-Galactosidaseassaysofwild-typestrainA1552and(cid:2)cyaA interactions. It is possible that the relative contributions of o
and(cid:2)crpmutantsharboring(A)vpsT-lacZand(B)vpsR-lacZfusion VPSandbiofilmmatrixproteinstobiofilmformationaredif- rg
constructs.(C)(cid:5)-GalactosidaseassaysofdifferentV.choleraestrains ferentwhentheorganismsaretestedunderdifferentenviron- o/
(A1552,N16961,C6706,andMO10)and(cid:2)crpdeletionstrainsharbor- n
ingthevpsR-lacZfusionconstruct.(DandE)(cid:5)-Galactosidaseassays mentalconditionsusingdifferentsurfaces. J
ofwild-type,(cid:2)crp,(cid:2)hapR,and(cid:2)crp(cid:2)hapRstrainsharboring(D)vpsT- cAMP-CRPdifferentiallyregulatestheexpressionofasetof a
lacZand(E)vpsR-lacZfusionconstructs.Thedataarerepresentative genes encoding proteins harboring GGDEF, EAL, and HD- n
u
ofatleasttwoindependentexperiments.Theerrorbarsindicatestan- GYP domains. Biofilm formation in V. cholerae is positively a
darddeviations. r
regulatedbyc-di-GMP(3,31,32,56).SincecAMP-CRPneg- y

ativelyregulatesbiofilmformationinV.cholerae,wewantedto 3
,

investigateifthereisaconnectionbetweencAMP-CRPregu- 2
0
rbmC and bap1 transcription compared to the wild type (Fig. latorycircuitryandc-di-GMPsignalinginregulationofbiofilm 1
3BandC).Thisresultsuggeststhat,likethefindingsforvpsL formation. We hypothesized that c-di-GMP levels may be in- 9

transcription, the magnitude of transcriptional regulation by creased in (cid:2)cyaA and (cid:2)crp mutants due to increased expres- b
y
VpsRisgreaterthanthemagnitudeoftranscriptionalregula- sion of a gene(s) encoding a key DGC or due to decreased g
tion by VpsT for rbmC and bap1. Intriguingly, unlike the re- expression of a gene(s) encoding a key PDE. Indeed, whole- u
e
sults for vpsL transcription, deletion of crp in both the (cid:2)vpsT genome expression profiling of (cid:2)cyaA and (cid:2)crp mutants re- s
and (cid:2)vpsR genetic backgrounds resulted in increased tran- vealedthatgenesencodingproteinspredictedtoexhibitDGC t
scription of rbmC (16.4-fold in the (cid:2)crp (cid:2)vpsT mutant and and PDE activities were differentially regulated in (cid:2)cyaA
3.0-fold in the (cid:2)crp (cid:2)vpsR mutant) and bap1 (8.1-fold in the and/or(cid:2)crpmutantscomparedtothewildtype(Table3).
(cid:2)crp (cid:2)vpsT mutant and 2.8-fold in the (cid:2)crp (cid:2)vpsR mutant) Inthissetofdifferentiallyregulatedgenes,wefocusedon10
compared to the wild type. Together, these findings indicate genesthatencodeproteinswithaconservedGGDEFdomain
that cAMP-CRP negatively regulates the transcription of vps whichispredictedtoactasaDGC:VC0072,VC0653(rocS),
genes and genes encoding matrix proteins in a different man- VC0658, VC1029 (cdgB), VC1067 (cdgH), VC1376, VC2750,
ner. While VpsR is the most downstream positive transcrip- VCA0074 (cdgA), VCA0217, and VCA0956 (cdgF) (referred
tionalregulatorofvpsgeneexpression,cAMP-CRPnegatively tobelowastheGGDEFgenes).Toexaminetheinvolvement
regulates transcription of the genes encoding matrix proteins oftheGGDEFgenesintheregulationofbiofilmformationin
boththroughandindependentofVpsR.Whethertheactionof V.cholerae,wegeneratedmutantswithin-framedeletionmu-
cAMP-CRP is mediated by direct binding to the rbmC and tationsinthesegenesinthewild-typebackground,aswellasin
bap1 promoter regions or indirectly through another regula- the (cid:2)crp genetic background. We then analyzed the biofilm-
toryprotein(s)remainsunknown. forming capacity of each of these mutants using pellicle for-
Since we observed that VPS biosynthesis genes and genes mationinglassculturetubesandbiofilmformationin96-well
VOL.190,2008 REGULATION OF BIOFILM MATRIX PRODUCTION BY cAMP-CRP 6653
D
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f
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:
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.
a
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.
o
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o
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3
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2
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9

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FIG. 3. AnalysisofthecAMP-CRPcontributiontoVpsRregulationofrbmCandbap1expression.(AtoC)(cid:5)-Galactosidaseassaysofwild-type,
(cid:2)crp,(cid:2)vpsT,(cid:2)crp(cid:2)vpsT,(cid:2)vpsR,and(cid:2)crp(cid:2)vpsRstrainsharboring(A)vpsL-lacZ,(B)rbmC-lacZ,and(C)bap1-lacZfusionconstructs.Thedata
arerepresentativeofatleasttwoindependentexperiments.Theerrorbarsindicatestandarddeviations.(D)Quantitativecomparisonofbiofilm
formationbywild-type,(cid:2)crp,(cid:2)vps-I,(cid:2)crp(cid:2)vps-I,(cid:2)vps-I(cid:2)vps-II,(cid:2)crp(cid:2)vps-I(cid:2)vps-II,(cid:2)rbmA,(cid:2)crp(cid:2)rbmA,(cid:2)bap1,and(cid:2)crp(cid:2)bap1strains.The
dataarerepresentativeoftwoindependentexperiments.Theerrorbarsindicatestandarddeviations.(E)Biofilmsformedafter8hofincubation
at30°Cinanon-flow-cellsystembythewild-type,(cid:2)crp,(cid:2)vps-I,(cid:2)crp(cid:2)vps-I,(cid:2)vps-I(cid:2)vps-II,(cid:2)crp(cid:2)vps-I(cid:2)vps-II,(cid:2)rbmA,(cid:2)crp(cid:2)rbmA,(cid:2)bap1,and
(cid:2)crp(cid:2)bap1strains.BiofilmswerestainedwithSYTO9,andimageswereacquiredbyCLSM.Thelargeimagesareimagesoftheuppersurfaces
ofbiofilms,andtheimagesbelowandtorightofthelargeimagesareorthogonalviews.Bars(cid:11)40(cid:4)m.
6654 FONG AND YILDIZ J.BACTERIOL.
D
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FIG. 4. PhenotypiccharacterizationofGGDEFdeletionmutantsandGGDEFcrpdouble-deletionmutants.(A)Pellicleformation,(B)quan- e
titativecomparisonofbiofilmformation,and(C)motilityassaysforthewildtype,for(cid:2)crpandGGDEFsingle-deletionmutants,andformutants s
t
withGGDEFdeletionsgeneratedinthe(cid:2)crpgeneticbackground.Thedataarerepresentativeoftwoindependentexperiments.Theerrorbars
indicatestandarddeviations.
microtiterplateswithacrystalvioletstainingassay.Asshown violet staining assays also revealed that deletion of rocS and
in Fig. 4A and B, deletion of GGDEF genes in the wild type VC0658 (designated cdgI [cyclic di-guanylate I]) in the (cid:2)crp
did not alter the pellicle and biofilm formation phenotypes. geneticbackgroundfurtherenhancedbiofilmformation.Both
However, when the genes were deleted in the enhanced bio- RocS and CdgI contain conserved GGDEF-EAL domains.
film-forming (cid:2)crp genetic background, only the (cid:2)crp (cid:2)cdgA SuchproteinscanfunctionaseitherDGCsorPDEs,andthe
double-deletionmutantfailedtoformpelliclesandexhibiteda enzymaticfunctionsarecommonlyregulatedbyenvironmental
reduced biofilm-forming capacity. This finding indicates that stimuli. Although the enzymatic activities of these proteins
theincreasedpellicle-andbiofilm-formingcapacitiesofa(cid:2)crp havenotbeendeterminedyet,basedonbiofilm-formingphe-
mutantareduetoincreasedexpressionofcdgA.Interestingly, notypes, RocS and CdgI appear to function as a PDE under
werecentlyreportedthatcdgApositivelyregulatescolonyrug- theexperimentalconditionsthatweutilized.
osity and biofilm formation in V. cholerae (2, 31). The crystal Flagellarmotilityisnegativelyregulatedbyc-di-GMPinV.
VOL.190,2008 REGULATION OF BIOFILM MATRIX PRODUCTION BY cAMP-CRP 6655
givingrisetotheincreasedbiofilm-formingcapacitiesobserved
forthe(cid:2)cyaAand(cid:2)crpmutants.Regulationoftheexpression
ofthesegenesbycAMP-CRPinV.choleraeiscurrentlyunder
investigation.
CdgA is required for the enhanced biofilm-forming pheno-
type in the (cid:1)crp mutant and negative regulation of vpsL and
rbmCexpressionbycAMP-CRP.Intheexperimentsdescribed
above we showed that while the (cid:2)crp single-deletion mutant
exhibits enhanced pellicle- and biofilm-forming capacities (as
FIG. 5. qPCRanalysisofcdgA,cdgI,androcSmessagelevelsinthe
(cid:2)crp mutant: quantification of relative repression of (A) cdgA, determined by the crystal violet staining assay), the crp and
(B)cdgI,and(C)rocSinthewildtypeandthe(cid:2)crpmutant,normal- cdgAdouble-deletionmutant((cid:2)crp(cid:2)cdgAmutant)exhibiteda
ized using recA. The results are from three independent biological decrease in biofilm formation. We confirmed this finding by
replicates.Theerrorbarsindicatestandarddeviations.Pvalues(two- carrying out a CLSM analysis of biofilms formed by the wild
tailedttest)areindicatedatthetop. type and the (cid:2)crp, (cid:2)cdgA, and (cid:2)crp (cid:2)cdgA mutants in a non-
flow-cell system (Fig. 6A). Afte r 8 h ofbiofilm development, D
the(cid:2)crpmutantformedathickerandmore-structuredbiofilm, ow
cholerae. Therefore, we sought to understand the effect of and the total biomass, average and maximum biofilm thick- n
deletion of GGDEF genes on motility. To this end, we mea- nesses, and substratum coverage were greater for the (cid:2)crp lo
a
sured the migration zone formed by each single-deletion mu- mutant than for the wild type (Table 4). The (cid:2)cdgA mutant d
e
tant,aswellasthemigrationzonesformedbymutantsgener- formed biofilms with reduced total biomass, average biofilm
d
atedinthe(cid:2)crpgeneticbackground,whentheorganismswere thickness,andsubstratumcoveragecomparedtothewild-type f
r
grownonLBsoftagarplates(Fig.4C).Wealsoused(cid:2)flaAas biofilms.AlthoughdeletionofcdgAinthe(cid:2)crpmutantsignif- o
m
a control. Of the GGDEF single-deletion mutants tested, the icantlyreducedbiofilmformation,the(cid:2)crp(cid:2)cdgAdoublemu-

(cid:2)rocS mutant exhibited a decrease in motility and the (cid:2)cdgH tant formed biofilms whose total biomass, average biofilm h
t
t
mutantexhibitedanincreaseinmotilitycomparedtothewild thickness,andsubstratumcoverageweregreaterthanthoseof p
:
type.Theseresultsareconsistentwithourpreviousreport(31) thebiofilmsformedbythe(cid:2)cdgAmutant(Fig.6AandTable //
jb
andrecentfindingsofBeyhanetal.(S.Beyhanetal.,submit- 4). These results indicate that the negative regulation of bio- .
a
ted for publication). Deletion of crp led to a decrease in mo- film formation by cAMP-CRP is largely mediated by CdgA, s
m
tility, consistent with the expression profiling data showing but additional factors may also contribute to an increase in
downregulationofgenesinvolvedinflagellarbiosynthesisand biofilmformationinthe(cid:2)crpmutant. .o
r
chemotaxis in (cid:2)cyaA and (cid:2)crp mutants compared to the wild To determine if the altered pellicle- and biofilm-forming g
/
type(seeFig.S1inthesupplementalmaterial).Deletionofcrp phenotypes of the (cid:2)crp (cid:2)cdgA double-deletion mutant were o
n
in individual GGDEF deletion mutants also led to further due to a reduction in the transcription of genes involved in

decreasesinmotilitycomparedtothecorrespondingGGDEF biofilm matrix production, we carried out (cid:5)-galactosidase as- Ja
single-deletion mutants. One of the mutants tested, the (cid:2)crp says with the wild-type, (cid:2)crp, (cid:2)cdgA, and (cid:2)crp (cid:2)cdgA strains n
u
(cid:2)rocSdouble-deletionmutant,exhibitedafurtherreductionin harboringvpsL-lacZandrbmC-lacZtranscriptionalfusioncon- a
motilitycomparedtoboththe(cid:2)crpand(cid:2)rocSsingle-deletion structs (Fig. 6B and C). While the (cid:2)crp mutant exhibited in- ry

mutants, suggesting that cAMP-CRP and RocS have additive creased transcription of vpsL-lacZ (154-fold) and rbmC-lacZ 3
,
effectsonmotilityregulation. (37-fold), the (cid:2)cdgA mutant exhibited decreases in the tran- 2
0
In summary, for the 10 GGDEF genes tested, deletion of scription of vpsL-lacZ (1.8-fold) and rbmC-lacZ (1.3-fold) 1
cdgA, rocS, and cdgI in the (cid:2)crp genetic background altered comparedtothewildtype,consistentwithourpreviousfinding 9

biofilm-formingphenotypes.Sincewechosethesegenesbased (obtainedusinggeneexpressionprofiling)thatCdgApositively b
y
ontheirincreasedexpressioninthe(cid:2)cyaAand/or(cid:2)crpmutant regulates vps and rbm gene expression (2). The (cid:2)crp (cid:2)cdgA g
inwhole-genomeexpressionprofilingexperiments,wefurther double-deletion mutant exhibited a decrease in the transcrip- u
e
confirmed their message abundance using qPCR. In agree- tionofvpsL-lacZ(79-fold)andrbmC-lacZ(7.1-fold)compared s
ment with microarray data, the message levels of cdgA, rocS, tothe(cid:2)crpsingle-deletionmutant.Theseresultsindicatethat t
andcdgIweregreaterinthe(cid:2)crpmutantthaninthewildtype the decreased biofilm-forming capacities of the (cid:2)crp (cid:2)cdgA
(Fig.5). double mutant compared to the (cid:2)crp mutant were due to
Expression of VC1934, which encodes a protein containing decreased expression of genes involved in VPS biosynthesis
anEALdomainandanonconservedGGDEFdomain,expres- andmatrixproteinproduction.Interestingly,inthe(cid:2)crp(cid:2)cdgA
sion of VC1086 and VC1710, which encode proteins contain- double-deletionmutantthelevelsoftranscriptionofvpsL-lacZ
ing an EAL domain, and expression of VC1087, VC1348, and rbmC-lacZ were higher than those in the wild type (1.9-
VC2497, VCA0895, and VCA0931, which encode proteins fold higher for vpsL-lacZ and 5.1-fold higher for rbmC-lacZ)
containing a HD-GYP domain, were downregulated, with and in the (cid:2)cdgA mutant (3.5-fold higher for vpsL-lacZ and
(cid:1)1.5-foldchangesandFDRof(cid:2)3%inthe(cid:2)cyaAand/or(cid:2)crp 6.5-fold higher for rbmC-lacZ). These findings suggest that
mutant(Table3). besides CdgA, other factors and processes also contribute to
Itispossiblethatadecreaseintheexpressionofthesegenes anincreaseinbiofilmformationinthe(cid:2)crpmutant.
encodingproteinspredictedtohavePDEactivity(leadingtoa Wealsocarriedoutacomplementationassaybyintroducing
decrease in the cellular c-di-GMP level) (45, 49) may also cdgA,inwhichtranscriptionisdrivenfromitsownpromoter,in
contribute to an overall increase in the c-di-GMP level, thus a multicopy number plasmid into the wild type and into the