Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato ...

19 Pages·2008·0.7 MB·English

MolecularMicrobiology(2008)68(4),871–889 (cid:2) doi:10.1111/j.1365-2958.2008.06209.x
Firstpublishedonline8April2008
Characterization of the PvdS-regulated promoter motif
in Pseudomonassyringae pv. tomato DC3000 reveals
regulon members and insights regarding PvdS function
in other pseudomonads
BryanSwingle,1,2*DeeptiThete,2MonicaMoll,1 were used to query genomes of DC3000 and other
ChristopherR.Myers,3DavidJ.Schneider1,2and fluorescent pseudomonads for similar motifs. We
SamuelCartinhour1,2 conclude that the role of PvdS as a regulator of
1UnitedStatesDepartmentofAgriculture-Agricultural pyoverdine synthesis is conserved among the fluo-
ResearchService,Ithaca,NY14853,USA. rescent pseudomonads, but the promoters recog-
2DepartmentofPlantPathologyandPlant-Microbe nized by PvdS orthologues may differ subtly from
Biology,and3ComputationalBiologyServiceUnit, speciestospecies.
CenterforAdvancedComputing,CornellUniversity,
Ithaca,NY14853,USA.
Introduction
Bacterial survival depends on co-ordinated and system-
Summary
atic gene regulation in response to changing conditions.
Bacteria that survive under variable conditions Thenatureandvarietyofthechangesarereflectedinthe
possessanassortmentofgeneticregulatorstomeet genome, with species that are free living in the environ-
these challenges. The group IV or extracytoplasmic ment tending to encode proportionally more transcrip-
function (ECF) sigma factors regulate gene expres- tionalregulators(Stoveretal.,2000).Promoterselection
sioninresponsetospecificenvironmentalsignalsby byalternativeformsofRNApolymeraseholoenzymecon-
altering the promoter specificity of RNApolymerase. tainingdifferentsigmafactorsubunitsisaprimarymecha-
We have undertaken a study of PvdS, a group IV nism for regulating transcription of collections of genes
sigma factor encoded by Pseudomonas syringae pv. (Gruber and Gross, 2003). The sigma subunit confers
tomato DC3000 (DC3000), a plant pathogen that is thesequence-specificDNA-bindingdeterminantstoRNA
likely to encounter variations in nutrient availability polymerase allowing it to recognize and correctly bind to
as well as plant host defences. The gene encoding promotersequences(Murakamietal.,2002).
PvdS was previously identified by sequence similar- Most bacteria contain multiple members of the s70
ity to the Pseudomonas aeruginosa orthologue, familyofsigmafactors.Theprimarys70-typesigmafactor
which directs transcription of genes encoding the is responsible for directing expression of housekeeping
biosynthesisofpyoverdine,asiderophoreinvolvedin functions during exponential growth. The most diverse
ironacquisition,andisresponsibleforthecharacter- group of s70-type sigma factors is the group IV or extra-
istic fluorescence of the pseudomonads. We identi- cytoplasmicfunction(ECF)sigmafactors,whicharegen-
fied 15 promoters regulated by PvdS in DC3000 and erally involved in regulating transcription in response
characterized the promoter motif using computa- to environmental signals (Helmann, 2002; Potvin etal.,
tional analysis. Mutagenesis of conserved nucle- 2008).ThemodelplantpathogenPseudomonassyringae
otides within the motif interfered with promoter pv. tomato DC3000 (DC3000) (Buell etal., 2003) is
function and the degree of the effect was different predicted to encode 10 group IV sigma factors (Oguiza
dependingonwhichregionofthemotifwasmutated. etal., 2005). Based on sequence similarity, five appear
Hidden Markov models constructed from alignments to be dedicated to regulating the expression of genes
ofsequencemotifsextractedfromDC3000andPAO1 whose products are involved with iron acquisition and
homeostasis.
Pseudomonas syringae and other fluorescent
Accepted 7 March, 2008. *For correspondence. E-mail Bryan.
pseudomonads are characterized by their ability to
[email protected]; Tel. [phone]; Fax [phone]
4471. secrete pyoverdine in low-iron conditions (Meyer, 2000).
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Pyoverdinesarechemicallydiverse,yellow-greenfluores- necessaryforpromoterfunctionandthatthetwodomains
cent, high-affinity siderophores capable of chelating differintheirsensitivitytosingle-nucleotidesubstitutions.
Fe(III) as a mechanism for scavenging iron from the Three hidden Markov models (HMM) were constructed
environment.Pyoverdinesaretheprimaryironacquisition usingeither(i)theexperimentallyverifiedPvdS-regulated
system in pseudomonads and give these organisms a promoter regions, (ii) a set of 13 IS-box promoters from
competitive advantage in the environment and in their P.aeruginosa PAO1 (PAO1) based on data summarized
associationwithplantoranimalhosts(PooleandMcKay, in Ravel and Cornelis (2003), or (iii) the combination of
2003; Sarkar etal., 2006). In addition to their role in iron sequences from both (i) and (ii) to yield a combined
acquisition,pyoverdinesalsofunctionasextracellularsig- DC3000/PAO1-basedmodel.Thesemodelswereusedto
nallingmoleculesimportantforinducingtheproductionof scanthegenomesof10fluorescentpseudomonads,each
virulence factors in Pseudomonas aeruginosa (Lamont encoding PvdS orthologues sharing greater than 80%
etal.,2002).Presumably,thelowironconcentrationinthe identitywiththeDC3000PvdSandinvariantwithrespect
hostsignalstherequirementforpyoverdinesynthesisas totheresiduesimportantforpromoterbindinginthePAO1
well as other factors required to mount a successful orthologue (Wilson and Lamont, 2006). As expected,
infection. manyofthegeneslinkedwiththemotifwerepyoverdine-
The genes coding for the biosynthesis of pyoverdine related but in every species some genes were detected
are organized in genomic clusters in all sequenced that were not obviously involved with pyoverdine or iron
pseudomonad genomes that have been analysed metabolism. The results of the HMM scans varied from
(Buell etal., 2003; Ravel and Cornelis, 2003; Feil etal., species to species, suggesting the possibility that the
2005; Joardar etal., 2005; Paulsen etal., 2005). In PvdS-boxmaynotbestrictlyconservedwithinthegenus.
P.aeruginosatheexpressionofthesegenesisregulated
by the group IV sigma factor PvdS (Leoni etal., 2000;
Wilson and Lamont, 2000). The gene encoding PvdS in Results
P.aeruginosaisregulatedinresponsetoironconcentra-
Genome-widescreenidentifies
tioninthegrowthmediaviatheferricuptakeregulator,Fur
PvdS-regulatedpromoters
(Cunliffe etal., 1995; Leoni etal., 1996). It has been
hypothesized that pyoverdine synthesis is regulated by Aconventional promoter trap library screen was used to
similar mechanisms in other pseudomonads (Cunliffe isolatePvdS-regulatedpromotersencodedintheDC3000
etal.,1995;Rombeletal.,1995;Leonietal.,2000;Ravel genome.Thelibrarywasconstructedusingsize-selected,
and Cornelis, 2003). Lamont and co-workers identified a partially digested DC3000 genomic DNA that was sub-
conserved TAAAT nucleotide motif known as the IS-box cloned in a vector (pBS29) upstream of a promoterless
upstream of PvdS-regulated genes. This motif has been ′lacZa gene. In this construct, the ′lacZa gene is
proposedtofunctionaspartofthe-35elementofPvdS- expressed only when the genomic insert encodes an
regulated promoters (Rombel etal., 1995; Wilson etal., activepromoter.ThescreeningwascarriedoutinEscheri-
2001). A conserved CGT nucleotide triplet positioned chia coli where previous studies have shown that PvdS-
16bp downstream of the IS-box has also been noted in regulatedpromotersfromP.aeruginosaareactiveonlyin
some PvdS-regulated promoters (Ochsner etal., 2002; the presence of PvdS expressed in trans (Cunliffe etal.,
Visca etal., 2002).The location, spacing and function of 1995).Thishelpedfocusthescreentoidentifypromoters
these motifs are consistent with the hypothesis that they that were directly regulated by PvdS and to minimize
are part of the -35 and -10 elements of PvdS-regulated complications due to secondary regulatory effects or
promotersinP.aeruginosa. induction from other iron-regulated sigma factors that
The orthologue of PvdS (PSPTO_2133) was identified couldoccurinthenativeDC3000background.
in DC3000 by sequence analysis (Ravel and Cornelis, ScreeningforPvdS-regulatedpromoterswasperformed
2003;Oguizaetal.,2005)butthefunctionofthegenehas in two steps. First, E.coli cells transformed with the pro-
not been demonstrated. To characterize the DC3000 motertraplibraryandaDC3000PvdSexpressionvector
PvdSregulon,PvdS-responsivepromoterswereobtained werescreenedforalac+phenotypeon5-bromo-4-chloro-
by screening a DC3000 genomic DNA promoter trap 3-indolyl-b-D-galactopyranoside (X-gal) indicator media,
libraryandfurtheranalysedusingtranscriptionalreporter which identified clones containing active promoters.
fusions, quantitative real-time (qRT)-PCR and transcrip- Second,thelac+cloneswereevaluatedtoidentifyisolates
tionalstartsitemapping.ThePvdS-boxmotifwasidenti- where reporter expression was dependent on the pres-
fiedusingGibbssamplingandwasanalysedbyscanning ence of the PvdS expression construct. From 116000
mutagenesis to identify conserved nucleotides whose initialtransformants,2992wereisolatedinsteponeofthe
presenceisrequiredforfullpromoterfunction.Theresults screen. Of these, 97 clones were confirmed to exhibit
indicatethattheputative-10and-35elementsareboth PvdS-dependent expression in step two, and the inserts
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PvdSregulationinPseudomonassyringae 873
weresequencedandmappedontotheDC3000genome. A
The 25 unique sequences from this set that contained Fold
Description Relative Luminescence
Difference
intergenic regions (seeTableS1) were used to construct
vector cntl - PvdS 2
preliminary hidden Markov models (data not shown) for P0753::lux + PvdS 23
scanningthegenometoidentifycandidatepromotersand P2137::lux 173
toguidesubsequentexperimentationasdescribedbelow. P2145::lux 467
P2146::lux 138
P2147::lux 593
P2149::lux 72
Multiplemethodsvalidatecandidatepromoters P2152::lux 111
P2156::lux 39
The candidate promoters suggested by the process
P5624::lux 676
abovewerechosenforfunctionalvalidationusingreporter P2175::lux 42
100 1,000 10,000 100,000 1,000,000
fusions, qRT-PCR and transcription start site mapping.
B
Thisstrategyreflectsourgoalofbothassociatingfunction Fold
Description Relative Induction
with particular upstream sequence fragments and con- Difference
- cntl 1.5
firmingPvdS-regulatedexpressionofdownstreamgenes. 0753 6
The lux reporter fusion assay was used to associate 2134 263
2136 229
promoteractivitywithsmallDNAfragmentsandtoisolate 2137 204
2145 83
promoter activity from potentially confounding effects 2146 291
suchasreadthroughfromtranscriptsinitiatedupstreamin 2147 174
2149 258
thenativegenomiccontext.Eachregionwasclonedasa 2151 5
transcriptionalfusionwitha′luxCDABEoperon.Promoter 22115526 13376
activity of these constructs was assessed in E.coli cells 5624 329
2160 124
transformed with either a PvdS expression vector or the 2161 237
2982 32
empty vector control. Ten regions showed strong PvdS- 3290 10
dependent expression, as displayed in Fig.1A. Expres- 3556 4
4923 6
sionvariedfrom23-to676-fold,comparedwithatwofold 1 10 100 1,000 10,000
change in the promoterless reporter control, confirming
promoteractivityinthesefragments. Fig.1. PvdS-dependentpromoteractivity.Genomicregions
assessedforPvdS-dependentpromoteractivitywereidentifiedby
AqRT-PCRassaywasusedtoassessPvdS-dependent
promotertrappingorbyscanningwithpreliminarybioinformatics
transcriptionofgenesdownstreamofcandidatepromoters modelsconstructedusingsequencesidentifiedinthepromoter
in their native context in DC3000 (see Fig.1B). Relative trappingexperiments.PromoterandgenenumbersrefertoPSPTO
numbersintheannotatedDC3000genomesequenceNC_004578
expressionfor18genomiclociwasassessedbycompar-
(Buelletal.,2003)hereandthroughoutthereport,exceptwhere
ingtranscriptlevelsinDC3000cellstransformedwiththe noted.Errorbarsshowstandarddeviation.
PvdSexpressionvectorversusDC3000cellstransformed A.RelativeluminescenceexpressedfromE.colicells(sixbiological
replicas)transformedwiththepromoter::luxfusionconstructand
with the empty vector. This confirmed PvdS-dependent
eithertheemptyexpressionvectorcontrol(lightbars)orthe
differential expression of all 18 genes, 15 of which are PvdS-expressingconstruct(darkbars).Thevectorcontrol(vector
directlydownstreamofcandidatepromoters,twoofwhich cntl)wasthepromotertrapvectorwithoutapromoterinsert.
B.qRT-PCRanalysisshowingPvdS-dependentdifferential
(PSPTO_2136/daT and PSPTO_2151) are potentially
expressioninDC3000(threebiologicalreplicas).Relativeinduction
withinoperonsprecededbycandidatepromoters,andone istheratioofnormalizedtranscriptlevelinDC3000cells
of which, PSPTO_3556 (glcE), is in an unconventional transformedwiththePvdSexpressionvectorversusDC3000cells
transformedwiththeemptyvectorcontrol.Transcriptlevelsfor
orientationrelativetothecandidatepromoter(seeFig.1B
eachgenewerenormalizedtogap1.ThegyrAgene,whichisnot
andbelow).Overall,differentialexpressioninthisexperi- predictedtobePvdS-regulated,wasusedasanegativecontrol
ment ranged from 5- to 329-fold. The results from both (-cntl).AllgenesweredirectlydownstreamfromthePvdS-box
exceptfor2136and2151,whicharepotentiallypartof
transcriptional fusion assays and qRT-PCR assays were
multicistronicoperonsprecededbyPvdS-boxmotifs,and3556,for
consistent with these regions containing cis-acting ele- whichthePvdS-boxispositionedwithintheindicatedgeneinthe
ments that function as PvdS-regulated promoters. antisenseorientation.For2175,differentialexpressionwasnot
detected(datanotshown).
However, in one case (PSPTO_2175, leuB), PvdS pro-
moteractivitywasobservedusingthepromoterfusionbut
couldnotbedetectedusingqRT-PCRatthenativelocation Thepointatwhichtranscriptioninitiatesisconstrainedby
(datanotshown).Itispossiblethatexpressionatthislocus thestructureoftheRNApolymeraseholoenzymeboundat
inDC3000requiresotherregulators[e.g.LrpasinE.coli thepromoter,andisgenerallynotmorethan10or11bp
(NewmanandLin,1995)]. downstreamofthe-10elementfors70-dependentpromot-
A 5′ RACE strategy was used to map the location of ers(Murakamietal.,2002;LewisandAdhya,2004).The
transcriptionstartsitesforseveralPvdS-regulatedgenes. transcription start points for alternative sigma factors are
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874 B.Swingleetal. (cid:2)
Fig.2. 5′RACEanalysisforseven
AAGTCATGCTGAAAAATTTTGAATTAATTCTCCCGTCCGTCGTTCTCTTTCAGGTAAGCCGGT PSPTO_0753 PvdS-dependenttranscripts.TheDC3000
genomicsequencecorrespondingtothe
CGCGGCGGGCATTCACGCCTAATTATTTGCAGACGCCATCCGTTCTCATTGGTGACAGGCCCG PSPTO_2134
regionupstreamoftheindicatedORFis
GGAAATGGCGTAAGGCGGCTAATTTTTCAGCCGCGAAGTACGTTCAATCTGCACATGGGTGTT PSPTO_2146 shownwiththepromoterelementsofthe
PvdS-boxshaded.Thedirectionof
GGTTTTGTGAAAAATTCATTAAATAATCGTTAATCGCTTTCGTTCTCATTAACAGCGAACTGC PSPTO_2152 transcriptionisindicatedbyanarrow;a
trianglebeneaththearrowindicatesarange
TCCAAACGGTGATCGGCGCTAAATTCCGGCCTGCATTTCTCGTTTAACTGGCAGCTCTGTTTG PSPTO_2160 ofpossiblestartsitesduetoambiguitiesin
thesequencechromatogramand/orthe
ACCGTTTGGACGAATCAGCTAAAAATTTTCGCCCGCGCTTCGTTTAACGGGGGGCGGCCAATT PSPTO_2161 additionofhomopolymertailsduringcDNA
synthesis.Theunderlinedregionsshowthe
CGGTTTTTCTACTAGTCTCTCAAGAAATCAACCACTTGAACGTTTTACCTTTATCGGCAGTGC PSPTO_2982 49nucleotidesusedforGibbssampling.
expected to be similar to the prototypical s70-regulated werefound.Thesequencelogosuggeststhepresenceof
promoter elements (Nonaka etal., 2006; Rhodius etal., two conserved regions, separated by a less conserved
2006). Therefore the location of the start site was deter- spacer. This structure resembles promoters recognized
minedinordertofocusoursearchforthepromotermotifin by s70-type sigma factors (Gross etal., 1998), where
the smallest possible region. We analysed seven tran- these conserved regions are referred to as the -10 and
scripts(Fig.2);fiveloci(PSPTO_2134,2146,2152,2160 -35 elements. Note that the IS-box TAAAT-(N16)-CGT
and 2161) are within the pyoverdine cluster, and two foundupstreamofiron-responsivegenesinP.aeruginosa
(PSPTO_0753and2982)areindifferentregionswithno PAO1(Wilsonetal.,2001;Ochsneretal.,2002)occupies
apparent role in pyoverdine metabolism. Transcription positions2–25inthelogo.Giventhedegreeofsequence
start sites were mapped by 5′ RACE using RNA from conservation,weadoptedthefollowingconventiontorep-
DC3000cellstransformedwiththePvdSexpressioncon- resent the consensus sequence for the DC3000 PvdS-
struct.Forallofthegenestested,thetranscriptionstartsite box(whichdiffersslightlyfromthatforthePAO1IS-box):
wasdeterminedwithamaximaluncertaintyoffivenucle- TAAAT[A/T]-(N15)-CGTT[C/T][T/A]. For cases where
otides. The relation of the transcription start site to the mapped transcription start sites are available, the 5′ end
promotermotifisdiscussedbelow. of the transcript was located between 3 and 9bp down-
stream of the 3′ end of the conserved CGTT[C/T][T/A]
domainofthemotif(Fig.2).Thesourceofthisuncertainty
IdentificationofthePvdS-boxmotifbyGibbssampling
isduetothetechnicallimitationsofthetranscriptmapping
Thecombinedevidencefromthepromotertraplibrary,lux method(seeExperimentalprocedures).
fusions, qRT-PCR and transcription start site data was Thesedataareconsistentwiththeconservedfeatures
usedtodefineasequenceinputsetforGibbssamplingto of this motif functioning as the -10 and -35 promoter
identifyanalignedpromotermotifasdescribedpreviously elementsrecognizedbyPvdS,althoughcloseinspection
(Ferreiraetal.,2006).Whereapplicable,sequenceswere reveals that the geometry of the motif is slightly more
trimmedasfollows:(i)overlappinglibraryisolatesandlux compact than the canonical s70 promoters in E.coli
fusion sequences were trimmed to the common subse- (Shultzabergeretal.,2007).Wehypothesizethatthecon-
quence, (ii) sequences were trimmed at the 3′ end to served nucleotides in Fig.3 correspond to the promoter
remove overlap with annotated coding regions, (iii) sequence recognized by PvdS in DC3000, and hence-
sequences were limited to extend no further than 300 forthrefertothismotifasthePvdS-box.
nucleotidesupstreamoftheannotatedcodingregion,and
(iv) fragments for which transcription start site data were
MutagenesisinthePvdS-boxreducespromoteractivity
available were trimmed to 49 nucleotides as shown in
Fig.2. The final set of 16 sequences used for Gibbs Typically, s70-type promoters contain nucleotides within
sampling is summarized in TableS2 with experimental the-10and-35promoterelementsthatarenecessaryfor
evidencevalidatingtheirPvdS-dependentregulation. specific recognition by the sigma factor (McClure etal.,
Gibbs sampling was conducted for a range of motif 1983; Gross etal., 1998; Murakami etal., 2002); there-
window sizes. One motif was consistently found in 15 of fore, some nucleotide substitutions within the conserved
theinputsequencesandhadastructureconsistentwitha elementsareexpectedtoalterpromoterfunction.
promoterforanECFsigmafactor.Theresultsofatypical TheregionupstreamofPSPTO_2152,aputativeTonB-
sampling run with a window size of 29 nucleotides are dependentreceptorinthepyoverdinelocus,waschosen
showninFig.3alongwiththeassociatedsequencelogo; as an example of a promoter containing consensus
we find this same basic structure preserved in larger sequences in the -10 and -35 elements (Fig.3).Atotal
window sizes as well. Each input sequence yielded one of 23 mutant derivatives were constructed from the wild-
motif except for PSPTO_2136 (daT), for which no motifs type promoter sequence using transversion mutations
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PvdSregulationinPseudomonassyringae 875
A
2 -35 -10
Bits 1
0
B 5 10 15 20 25
Motif co-ordinates Downstream ORF
5‘ 3‘ ID Distance Evidence
5573286..5573258 T T A A T T A C T G G C C C T C G C T A A T C G T T C T T 4923 167
2322443..2322471 T T A A T T T T T C G A C G T A G C G A T A C G T T C A A 2137 56
2330474..2330502 T C T A A A T A T T T C C C C G C C A A T T C G T T C T T 2147 146
2306852..2306880 C T A A T T A T T T G C A G A C G C C A T C C G T T C T C 2134 128
3717248..3717276 A T A A A A A C A T G C C A G A C C G A G A C G T T T A T 3290 27
2374231..2374259 C T A A A A A T T T T C G C C C G C G C T T C G T T T A A 2161 50
2367722..2367694 C T A A A T T T T T C G C C C A T C C G C T C G T T C T C 2156 56
2367750..2367778 C T A A A T T T G T C A G C C G G T T T T A C G T T C T A 5624 39
2329291..2329319 C T A A T T T T T C A G C C G C G A A G T A C G T T C A A 2146 66
800053..800081 T T G A A T T A A T T C T C C C G T C C G T C G T T C T C 0753 146
2329218..2329190 C A A A A T A T T C C T A C A G G A A T C T C G T T C T C 2145 96
2362184..2362156 T T A A A T A A T C G T T A A T C G C T T T C G T T C T C 2152 152
2374204..2374176 C T A A A T T C C G G C C T G C A T T T C T C G T T T A A 2160 69
2341777..2341805 T A A A T C A C T G G A G G C A C T C A A A C G T T T A T 2149 46
3353645..3353617 C T C A A G A A A T C A A C C A C T T G A A C G T T T T A 2982 65
Consensus N T A A A T NTA N T N N N N C N N N N N N N N C G T T C T N Average = 87
Fig.3. PvdS-boxsequencemotif.ThisalignmentwasproducedbyGibbssamplinganalysisofsequencesconfirmedtocontainapromoter
regulatedbyPvdS.Asummaryofthepropensityofeachnucleotidetooccupyeachpositionisrepresentedabovethealignmentasa
sequencelogo.
A.The-35motif(TAAAT[A/T])isontheleft-handsideandisseparatedmostoftenbya15bpspacerfromthe-10motifwhichcontainsthe
invariantCGTTnucleotides.
B.Individualsequencemotifsalignedandshownwithrelevantpositioninformation.ThedistanceisfromtheinvariantTatposition26andthe
firstnucleotideoftheannotatedstartcodon.TheevidencecolumnsummarizestheexperimentalevidencesupportingPvdS-dependent
regulation:L,promotertraplibraryisolate;F,luxfusion;R,confirmedbyqRT-PCR;T,transcriptstartsitelocated.
(A↔C and T↔G) to test specific bases for a functional G163T, T164G and T165G in Table1). As expected,
roleinPvdS-dependentexpression(Table1).Twenty-one similar levels were observed when the entire -10 region
contained single-base substitutions and two contained wasmutated.Onepositionwithinthespacerregionadja-
multiple substitutions in either the -10 or -35 regions. cent to the -10 element that was not identified as being
Plasmids containing transcriptional fusions of the wild- conserved among the promoters was associated with
type promoter region for PSPTO_2152 or the mutant modest reductions in expression (T160G), suggesting
derivatives with the ′luxCDABE operon were introduced a role for flanking nucleotides. In contrast to the -10
into E.coli along with the PvdS expression construct element, single-base-pair mutations in the -35 element
(pBS49).The effect of the mutations on reporter expres- did not have a statistically significant effect on transcrip-
sion was assessed and compared with the wild-type tion,exceptinonecase(T141G;65%reduction).Multiple
controltoinfertranscriptionalactivity. substitutions disrupting the -35 element eliminated pro-
Severalmutationsseverelyreducedtranscription,while moter activity, confirming the essential role of this
none of the mutant promoters was significantly more element.
active than wild type at P(cid:2)0.05. The single-nucleotide These results indicate that both the -10 and -35 ele-
mutations with the greatest impact on transcription were ments are required for promoter function and that an
allinthe-10region.Substitutionofanyofthefourinvari- important difference exists between the two domains of
ant nucleotides in the -10 element reduced transcription this motif in terms of their sensitivity to single-nucleotide
86% or more compared with wild type (see C162A, substitutions. The scanning mutagenesis of the con-
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876 B.Swingleetal. (cid:2)
Table1. MutagenesisofPvdS-boxmotif.
Plasmid Promotersequence LogRLU Promoteractivity
pBS102(WT) TTCATTAAATAATCGTTAATCGCTTTCGTTCTCAT 5.70((cid:3)0.45) 1.00
pBS102(A139C) TTCCTTAAATAATCGTTAATCGCTTTCGTTCTCAT 5.73((cid:3)0.02) 1.06(0.87)
pBS102(T140G) TTCAGTAAATAATCGTTAATCGCTTTCGTTCTCAT 5.72((cid:3)0.04) 1.05(0.91)
pBS102(T141G) TTCATGAAATAATCGTTAATCGCTTTCGTTCTCAT 5.25((cid:3)0.04) 0.35(0.03)
pBS102(A142C) TTCATTCAATAATCGTTAATCGCTTTCGTTCTCAT 5.42((cid:3)0.40) 0.52(0.21)
pBS102(A143C) TTCATTACATAATCGTTAATCGCTTTCGTTCTCAT 5.45((cid:3)0.38) 0.57(0.26)
pBS102(A144C) TTCATTAACTAATCGTTAATCGCTTTCGTTCTCAT 5.62((cid:3)0.40) 0.84(0.72)
pBS102(T145G) TTCATTAAAGAATCGTTAATCGCTTTCGTTCTCAT 5.46((cid:3)0.06) 0.58(0.18)
pBS102(A146C) TTCATTAAATCATCGTTAATCGCTTTCGTTCTCAT 5.78((cid:3)0.41) 1.21(0.70)
pBS102(A147C) TTCATTAAATACTCGTTAATCGCTTTCGTTCTCAT 5.64((cid:3)0.05) 0.88(0.73)
pBS102(T148G) TTCATTAAATAAGCGTTAATCGCTTTCGTTCTCAT 5.66((cid:3)0.06) 0.91(0.81)
pBS102(C149A) TTCATTAAATAATAGTTAATCGCTTTCGTTCTCAT 5.57((cid:3)0.35) 0.77(0.52)
pBS102(G150T) TTCATTAAATAATCTTTAATCGCTTTCGTTCTCAT 5.78((cid:3)0.03) 1.21(0.62)
pBS102(141–145) TTCATGCCCGAATCGTTAATCGCTTTCGTTCTCAT 3.74((cid:3)0.02) 0.01(6E-6)
pBS102(T160G) TTCATTAAATAATCGTTAATCGCTGTCGTTCTCAT 5.22((cid:3)0.04) 0.33(0.02)
pBS102(T161G) TTCATTAAATAATCGTTAATCGCTTGCGTTCTCAT 5.43((cid:3)0.49) 0.54(0.27)
pBS102(C162A) TTCATTAAATAATCGTTAATCGCTTTAGTTCTCAT 4.28((cid:3)0.07) 0.04(4E-5)
pBS102(G163T) TTCATTAAATAATCGTTAATCGCTTTCTTTCTCAT 3.58((cid:3)0.08) 0.01(2E-6)
pBS102(T164G) TTCATTAAATAATCGTTAATCGCTTTCGGTCTCAT 4.08((cid:3)0.24) 0.02(3E-6)
pBS102(T165G) TTCATTAAATAATCGTTAATCGCTTTCGTGCTCAT 4.83((cid:3)0.35) 0.14(9E-4)
pBS102(C166A) TTCATTAAATAATCGTTAATCGCTTTCGTTATCAT 5.64((cid:3)0.34) 0.88(0.78)
pBS102(T167G) TTCATTAAATAATCGTTAATCGCTTTCGTTCGCAT 5.39((cid:3)0.36) 0.49(0.15)
pBS102(C168A) TTCATTAAATAATCGTTAATCGCTTTCGTTCTAAT 5.39((cid:3)0.04) 0.49(0.09)
pBS102(162–165) TTCATTAAATAATCGTTAATCGCTTTATGGCTCAT 3.80((cid:3)0.04) 0.01(6E-6)
Theactivityofpromoterswithsingle-andmultiple-nucleotidesubstitutionswithinandflankingconservedpositionsofthePvdS-boxmotifwas
assessedinvivobycomparingtherelativeluminescenceproducedfromwildtype(WT=PSPTO_2152)andmutatedPvdS-boxderivatives.Log
RLUshowstheaverage(n=6)ofthelog10relativelightunitsandstandarddeviation.Promoteractivityistheratioofexpressionfromtheindicated
promoterrelativetowildtypewiththeP-valueshowninparentheses.The-35and-10promoterelementsareindicatedbyunderscoringandthe
mutatednucleotidesarehighlighted.
servedregionsofthePvdS-box,alongwiththetranscrip- scores greater than or equal to 11.2. Of these, 11 were
tionstartmapping,providesextremelystrongevidencefor located within the pyoverdine cluster, while four mapped
theassociationofpromoteractivitywiththismotif. to other regions of the genome (Fig.6). This HMM
scan identified one additional PvdS-box that is plausibly
positioned to function as a promoter, upstream of
ConstructionanduseofPvdS-box
PSPTO_3172, which is predicted to encode a NorM-like
hiddenMarkovmodels
memberoftheMATEfamilyofeffluxtransporters.The11
The 15 DC3000 promoter sequences that were experi- implausible motifs were either positioned within genes,
mentallyvalidated(Fig.3)anddetectedbyGibbssampling were located or oriented in ways that are not typically
were manually realigned to optimize conservation of associated with promoter function based on existing
the -10 and -35 elements, and are summarized in the annotation,andgenerallyhadlowerscores((cid:2)11.2).We
sequence logo shown in Fig.4A. HMM model A was cannot,however,ruleoutthepossibilitythatoneormore
trainedonthisrealignedsetandusedtoscanthegenomes are functional. For example, the PvdS-box embedded in
of10fullysequencedfluorescentpseudomonads,includ- PSPTO_3556(glcE)exhibitsmodestbutsignificantdiffer-
inghumanpathogens(P.aeruginosa),aninsectpathogen entialexpression(seeFig.1B)althoughitispositionedto
(P.entomophila), saprophytes (P.fluorescens), bioreme- generate an antisense transcript within this gene, and
diation agents (P.putida) and plant pathogens (P.syrin- in fact evidence for transcriptional activity generated
gae).Theresultsofthegenomescansaresummarizedin from promoters in unconventional orientations has been
Fig.5A, where we characterize the number of ‘plausible’ observed in at least one other system (Eiamphungporn
and‘implausible’matchestomodelAforeachgenomeat and Helmann, 2008). In general the scanning results in
twodifferentHMMscorevaluecut-offs. DC3000 suggest that model A provides a reasonably
ScanningtheDC3000genomeidentified27matchesto completedescriptionofthePvdSreguloninDC3000.
the motif (16 plausible and 11 implausible) at a HMM The other pseudomonads appear to group into two
score cut-off of 10.0 (Table2 and Fig.5A). All 15 classes based on the tendency for sequences similar to
sequencesinthemodelAtrainingsetwererecoveredwith the DC3000 PvdS-box to be located upstream of anno-
Journalcompilation©2008BlackwellPublishingLtd,MolecularMicrobiology,68,871–889
NoclaimtooriginalUSgovernmentworks
PvdSregulationinPseudomonassyringae 877
A Fig.4. ComparisonofPvdS-regulated
promotermotifs.Sequencelogoshavebeen
2.0 scaledtoreflectaG/Cnucleotidebiasof
61%,theapproximateaverageG/Ccontentin
the10genomesanalysed.ModelA(A),
DC3000-based;modelB(B),PAO1-based;
Bits1.0 modelC(C),DC3000andPAO1combined.
Alignedsequencesrepresentedinthese
sequencelogosandusedtotrainHMMs
0.0 (modelsA,BandC)areavailableinClustal
5 10 15 20 25 30 formatinSupplementarymaterialat
http://www.blackwell-synergy.com/doi/abs/
B
10.1111/j.1365-2958.2008.06209.x
2.0
Bits1.0
0.0
5 10 15 20 25
C
2.0
Bits1.0
0.0
5 10 15 20 25 30
Fig.5. PvdS-boxmatchesin10pseudomonadgenomes.HMMswereusedtoscangenomesattwodifferentcut-offscores(10.0and11.0).
Matcheswereconsideredplausibleiftheywereorientedcorrectlywithrespecttoadownstreamgene,andiftheir5′endswerelocatedinthe
intervalbeginning300ntupstreamofanannotatedgenestartand50ntdownstreamofthisstart.Weincluded50ntoftheN-terminusto
accountforthepossibilityofmisannotatedstartcodons.Bothplausible(blue)andimplausible(yellow)matchesareshownforeachgenome.
Theresultsfromscansusingacut-offof11.0areshownusingthelightercolours.Themorerelaxedcut-off(10.0,withdarkercolours)was
includedtodemonstratethegeneraldegradationofthemodelasthecut-offwasreduced.(A)modelA;(B)modelB;(C)modelC.
Journalcompilation©2008BlackwellPublishingLtd,MolecularMicrobiology,68,871–889
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878 B.Swingleetal. (cid:2)
Table2. PvdS-boxHMMscanresultsinDC3000.
Co-ordinates
HMM
5′ 3′ PvdS-boxsequence G score Orientation
2306853..2306880 TAATTATTTGCAGACGCCATCCGTTCTC • 16.9 U/S 2134
2322444..2322471 TAATTTTTCGACGTAGCGATACGTTCAA • 16.4 U/S 2137
2374232..2374259 TAAAAATTTTCGCCCGCGCTTCGTTTAA • 16.1 U/S 2161
5573285..5573258 TAATTACTGGCCCTCGCTAATCGTTCTT • 16.0 U/S 4923
2367751..2367778 TAAATTTGTCAGCCGGTTTTACGTTCTA • 15.8 U/S 5624
2329292..2329319 TAATTTTTCAGCCGCGAAGTACGTTCAA • 15.4 U/S 2146
2330476..2330502 TAAATATTTCCCCGCCAATTCGTTCTT • 15.4 U/S 2147
3717249..3717276 TAAAAACATGCCAGACCGAGACGTTTAT • 15.2 U/S 3290
2367721..2367694 TAAATTTTTCGCCCATCCGCTCGTTCTC • 15.0 U/S 2156
4013974..4014001 AAAAGAACGGCAGACGCTGTTCGTTCAA 13.2 E/A 3556
2374203..2374176 TAAATTCCGGCCTGCATTTCTCGTTTAA • 12.4 U/S 2160
3565679..3565652 AAAATATAGCCACGCGACAGACGATCAT 12.3 U/S 3172
800054..800081 TGAATTAATTCTCCCGTCCGTCGTTCTC • 12.1 U/S 0753
2329217..2329190 AAAATATTCCTACAGGAATCTCGTTCTC • 12.1 U/S 2145
3200665..3200692 TAAATATTTCATACTGCACTCCGTTCTT 11.8 I/A 2850
5055286..5055259 TGAAGTTTTCCGAGCACAGTTCGTTTTA 11.6 E/S 4488
2341777..2341805 TAAATCACTGGAGGCACTCAAACGTTTAT • 11.3 U/S 2149
2362183..2362156 TAAATAATCGTTAATCGCTTTCGTTCTC • 11.3 U/S 2152
3353644..3353617 TCAAGAAATCAACCACTTGAACGTTTTA • 11.2 U/S 2982
646696..646723 TCGATATTCCAGCCAGCAAGACGTTCAA 11.1 E/S 0587
1209925..1209952 TAAAAAATGCTCCACAAGATACGATCAC 11.1 I/A 1098
5758588..5758561 TAAAGAAAAGCACACGCTAATCATTCTT 11.1 E/S 5056
225903..225876 TAATTAGCGGAACCACTGTTACGTTTTT 10.8 E/S 0202
3717077..3717050 TAAAAAAAGCGCCCCGAAGGACGCTTTA 10.7 I/A 3289
3244256..3244283 AAAAAACCCGCCGAAGCGGGTCGTTTTT 10.6 I/A 2881
312912..312939 TAAGATTTTGAACGCATTACACGCTCAA 10.3 U/S 0287
6224284..6224257 TGAATTTTGCGCCGAGCGATTCGATCTG 10.3 E/A 5463
Allregionsofthegenomewithsignificantmatches(score(cid:4)10.0)totheDC3000-basedPvdS-boxmodelAareshownwiththelocationindicated
bygenomeco-ordinatesandarelistedindescendingorderbyHMMscore.Shadedentriesindicatedassociationwithpyoverdine.The-10and
-35elementsareunderlined.AdotintheGibbscolumn(G)indicatesmembershipinboththeGibbsinputandoutput(seeTableS2andFig.3).
TheHMMscorerepresentsthedegreeofsimilaritybetweenthegenomicsequenceandtheHMM.Theorientationcolumndescribesthepositioning
ofthepromoterrelativetoannotatedgenes:U/S(upstream/sense),upstreamandpositionedtofunctionasapromoterinthesenseorientationfor
theindicatedgene.Theremainingthreecategoriesdescribematchesthatareunlikelytofunctionasconventionalpromoters:E/S(embedded/
sense),embeddedinageneandorientedtotranscribeinthesensedirection.I/A(intergenic/antisense),intergenicbutorientedtogeneratean
antisensetranscriptfortheindicatedgene.E/A(embedded/antisense),embeddedinageneandorientedtogenerateanantisensetranscript.
tated reading frames (see Fig.5A). In the first class Experimental procedures). Model C is a consistently
(P.syringae B728a, P.syringae 1448A, P.fluorescens aligned combination of the DC3000-based model A and
Pf-5andP.fluorescensPfO-1),wefindlargernumbersof thePAO1-basedmodelB.Sequencelogosofthetraining
plausible matches and a higher ratio of plausible-to- sets for modelsA, B and C are shown in Fig.4. Table3
implausible promoters; between 67% (12/18) and 88% lists the plausible promoters resulting from scanning
(15/17)ofthematchestotheDC3000-basedmodelAwith pseudomonadgenomeswithmodelsA,BandC.
scores(cid:4)11.0arelocatedinpositionsconsistentwiththeir Using the PAO1-based model B to scan the 10
functionaspromoters.Inthefiveremaininggenomesthat pseudomonad genomes revealed that this model identi-
comprisethesecondclass,significantlysmallernumbers fiedplausiblepromotersinthetwoP.aeruginosastrains,
ofplausiblematcheswereidentified,andlowerplausible- and also identified other plausible promoters associated
to-implausible ratios, suggesting the possibility that the with pyoverdine in several species not found using the
PvdS-regulated promoters of these organisms are mea- DC3000-based model A. However, Fig.5 reveals that
surablydifferentfromthosefoundinDC3000. there are systematic differences in the performance of
To understand the differences in specificity of the modelsAand B, suggesting that neither model is trans-
DC3000-based model described above, we constructed ferable across the genus. This observation provided our
two additional HMMs using sequences conferring PvdS- motivation for constructing the combined model C.
dependentexpressioninPAO1.ModelBwasconstructed Figure5C indicates that model C generally performs
using 13 sequences from PAO1 containing IS-boxes as better across the genus than either A or B separately,
describedinRavelandCornelis(2003)(seeFig.S1and identifying greater numbers of plausible promoters while
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PvdSregulationinPseudomonassyringae 879
Product/FunctionMFS transportePrvdS; SigmPav fdaGc; tTohrioPesvtseAr/aPsvedL; DNaTR; PASminoMtrbatnHs-lfiekrea speroteiAnBC transporters Hypothetical MpreotmebirnasnTae uprirnote eidnePoxvydIgenasePvdJ PvdK PvdD TonB-siddeepreonpdheornte rPevcdeEp; tAorBsC tPrvadnsO;p oHrytperoPtvhdetNi; cAalminPovtdraM;n sfDierpaePspvetiddP; asTeAT-siRgNnDa l efpfleuptxi tAdrBeaCn sefpflorutx etrRraNnD sepffolrtuxe rtPrvadnQs; pAocrtylearseTannase/ferulPooyrl ien,s tOerprasD efaAzmiluryin
Chromophore Membrane proteins Peptide NRPS Rcptr. Efflux
0753 2133 2134 2135 21362137 2138-2145 2146 2147-2150 2151 2152 2153 2154 2155 2156 5624 2158 21592160 2161 2982 3290 4923
P. syringae DC3000
0657 1943 19441945 1946 1947 1948-1955 1956 1957-1960 1961 1962 1963 1964 1965 1966 1967 1968-1970 1971 2863 4807 0591
P. syringae B728a
4640 1909 19101911 1912 1913 1914-1921 1922 1923-1926 1927 1928 1929 1930 1931 1932 1933 1934-1936 1937 2381 0586
P. syringae 1448A
0713 4190 4189 4179 4178 4176-4169 4095-4093 4092 4091 4089 4088 4087 4086 4083-4081 2902 0592
P. fluorescens Pf-5
0664 3941 3940 3932 3931 3930-3923 1845-1847 1848 1849 1850 1851 1852 1853 1854 1855-1857 2563 3618 0546
P. fluorescens PfO-1
3573 24262425 2424 2413 2412 2410-2403 2402-2399 2398 2397 2395 2394 2393 2392 2391-2389 2385 2505 4922
P. aeruginosa PAO1
1809033260 3327033280 3350033510 33530-33600 33610-33650 3368033690 337103372033730 33740 33750-33770 33820 32270 65000
P. aeruginosa PA14
0628 1623 1624 1640 1642 1643 1644 1674-1677 1678 1679 1680 1681 1683-1685 1961 1965-1972 2790 0060 4748
P. putida F1
0589 4244 4243 4223 4222 4221-4219 4217 4216 4215 4214 4213 4212 4211-4209 3808 3804-3797 2901 0046 4870
P. putida KT2440
0672 18141815 3220 3221 3222 3224 3225 3229-3234 3235-3242 3329 3664-3666 3667 3669 2083 2340 4921
P. entomophila L48
Fig.6. TheDC3000PvdSregulonwithpredictedorthologuesinninepseudomonadgenomes.GenesinDC3000downstreamof
experimentallyvalidatedPvdS-boxes(reddots)andpredictedoperonmembers(shaded)areshownintheirgenomiccontext.TheDC3000
pyoverdineclusterislocatedinasinglelocusthatspans70.5kb(from2133to2161).Intheotherpseudomonads,matches(scores(cid:4)10.0)
toanyofthethreemodelsdetailedinTable3areshownwithgreendots.Numbersaboveeachgenerefertoannotationintherespective
genomes.
retaining an acceptable ratio of plausible-to-implausible results confirm the role of PvdS in the expression of
matches. In addition, model C identifies PvdS-boxes pyoverdine-related genes and suggest that PvdS inte-
upstreamofgenesrelatedtopyoverdinemetabolismthat grates iron status with other functions beyond pyover-
were missed by the single-species models A and B. dine metabolism.
Finally,othergenesareidentifiedthatappeartovaryfrom These experiments establish two important aspects
organism to organism (see Table3), and the union of pertaining to the details of regulation by PvdS. First, we
resultsfromscansA,BandCprovidesnovelhypotheses report a de novo identification of a PvdS-regulated pro-
regarding species-specific PvdS regulation across the motermotifusingsequencesthatexhibitPvdS-dependent
fluorescentpseudomonads. activityinDC3000.Theobservedsequenceconservation
intheconfirmedpromoters,alongwithmutagenesisdata,
suggestsimportantanddistinctrolesforthe-10and-35
Discussion
elements in the interactions of PvdS with cognate
Genome-wide approaches are well suited for investigat- promoters. Second, we have developed computational
ing the role and scope of individual sigma factors, both models that can be used to predict regulation by PvdS.
because of the essential role in transcription of these WehaveappliedthesemodelsbothtoDC3000,confirm-
regulators and because of their potential for ing that our inventory of PvdS-dependent promoters in
co-ordinating global organismal responses. We used a DC3000isnearlycomplete,andtootherfullysequenced
combination of computational and molecular genetic pseudomonads to generate predictions about PvdS-
approaches to identify the DC3000 promoter motif rec- dependent regulation in those organisms. The DC3000
ognized by PvdS, a group IV/ECF sigma factor, PvdS-box refines and extends the IS-box previously
members of which regulate transcription in response to described in P.aeruginosa (Rombel etal., 1995; Wilson
specific environmental signals.This work is an important etal.,2001;Ochsneretal.,2002;Viscaetal.,2002)and
step towards understanding the regulation of pyoverdine supports the prediction that pyoverdine genes are regu-
metabolism, which makes vital contributions to lated by similar mechanisms among the fluorescent
pseudomonad fitness in environmental and host interac- pseudomonads(Rombeletal.,1995;RavelandCornelis,
tions (Meyer etal., 1996; Poole and McKay, 2003). The 2003).
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880 B.Swingleetal. (cid:2)
Table3. GenesdownstreamofHMMmatchesinDC3000andnineothersequencedpseudomonadgenomes.
Co-ordinates HMM
5′ 3′ Sequence A B C ORF Product
P.syringaepv.tomatoDC3000 NC_004578
2306853..2306880 TAATTATTTGCAGACGCCATCCGTTCTC 16.9 14.6 PSPTO_2134 PVDchromophorethioesterase
2322444..2322471 TAATTTTTCGACGTAGCGATACGTTCAA 16.4 15.3 PSPTO_2137 MbtH-likeprotein
2374232..2374259 TAAAAATTTTCGCCCGCGCTTCGTTTAA 16.1 15.5 PSPTO_2161 PvdQ;acylase
5573285..5573258 TAATTACTGGCCCTCGCTAATCGTTCTT 16.0 14.1 PSPTO_4923 Azurin
2367751..2367778 TAAATTTGTCAGCCGGTTTTACGTTCTA 15.8 12.5 16.4 PSPTO_5624 PvdP:TATsignal
2329292..2329319 TAATTTTTCAGCCGCGAAGTACGTTCAA 15.4 10.6 15.4 PSPTO_2146 Taurinedeoxygenase
2330476..2330502 TAAATATTTCCCCGCCAATTCGTTCTT 15.4 14.2 PSPTO_2147 PVDpeptideNRPS
3717249..3717276 TAAAAACATGCCAGACCGAGACGTTTAT 15.2 11.3 PSPTO_3290 Porin;OprDfamily
2367721..2367694 TAAATTTTTCGCCCATCCGCTCGTTCTC 15.0 15.3 PSPTO_2156 Dipeptidase
2374203..2374176 TAAATTCCGGCCTGCATTTCTCGTTTAA 12.4 11.8 14.2 PSPTO_2160 RNDeffluxtransporter,porin
3565679..3565652 AAAATATAGCCACGCGACAGACGATCAT 12.3 PSPTO_3172 Effluxtransporter
800054..800081 TGAATTAATTCTCCCGTCCGTCGTTCTC 12.1 10.7 PSPTO_0753 Drugresistancetransporter
2329217..2329190 AAAATATTCCTACAGGAATCTCGTTCTC 12.1 PSPTO_2145 Membraneprotein
2362183..2362156 TAAATAATCGTTAATCGCTTTCGTTCTC 11.3 11.1 PSPTO_2152 TonB-dependentreceptor
2341777..2341805 TAAATCACTGGAGGCACTCAAACGTTTAT 11.3 PSPTO_2149 PVDpeptideNRPS
3353644..3353617 TCAAGAAATCAACCACTTGAACGTTTTA 11.2 PSPTO_2982 Tannase/feruloylesterase
4725717..4725744 TGAATTCGACTGTCGGTGATTCGTTTCA 10.8 PSPTO_4196 Glucosedehydrogenase
4378372..4378345 TGAGTTTCAGGCGGCCAGTTACGTTCAT 10.7 10.8 PSPTO_3864 PsyI;N-acylhomoserinelactonesynthase
4131225..4131252 TAAAGCCTACCGCGCCGCCATCGTTCAC 10.0 11.3 PSPTO_3663 Ganinedeaminase
4357542..4357569 TAAATGCCTGCGCCAGAACATCGTTTCA 12.3 PSPTO_3849 RhselementVgrprotein
1925512..1925539 AAAAATTCAGGCCGAGTTGATCGTTCCC 11.1 PSPTO_1754 UDP-glucose4-epimerase
693151..693178 TAAAGTTCTGCTTGGCCCGCACGTTTCC 11.0 PSPTO_0628 50SribosomalproteinL23
2394871..2394898 TAAATTTTTTGCCGTATTCAGCGTTATA 10.5 PSPTO_2175 LeuB;3-isopropylmalatedehydrogenase
P.syringaepv.syringaeB728a NC_007005
2216773..2216800 TAATTATTTGCAGACGCCATCCGTTCTC 16.9 14.5 Psyr_1944 PVDchromophorethioesterase
2232356..2232383 TAATTTTTCGACGTAGTGATACGTTCAA 16.4 15.6 Psyr_1947 MbtH-likeprotein
2239795..2239822 TAATTTTTCAGCGACGCAGTACGTTCAA 14.8 14.4 Psyr_1956 Taurinedeoxygenase
3435473..3435446 TGAATAATCGCCAGGCTTAAACGTTTTA 14.8 13.6 Psyr_2863 Tannase/feruloylesterase
2285734..2285707 TAAATTCCTGCCGGCACGCCTCGTTTAA 14.5 14.0 17.0 Psyr_1970 RNDeffluxtransporter,porin
2279024..2278997 TAAATTTATCGGCGATCCGCTCGTTCTC 13.9 10.6 15.2 Psyr_1966 Dipeptidase
2241308..2241334 TAAATATTTCCTCGCCAATTCGTTCTT 13.9 12.4 Psyr_1957 PVDpeptideNRPS
679878..679905 TAATTACCGGGTCCCGTGAATCGTTCTT 13.8 10.4 14.4 Psyr_0591 Azurin
2279054..2279081 TAAATTTGTCTGCCGGGTTTACGTTCTA 13.7 11.6 15.1 Psyr_1967 PvdP:TATsignal
744682..744709 TGAATTAATTCCCCCGTTCGTCGTTCTC 13.0 11.6 Psyr_0657 Drugresistancetransporter
2252732..2252760 TAAATCACTGGAGGCACTCAAACGTTTAT 11.4 Psyr_1959 PVDpeptideNRPS
849265..849238 GAAATCCTTCAAGACCCAAGTCGTTCAA 11.3 Psyr_0746 IS66Orf2like
3187125..3187098 TAAATACTGGGGCCAGGTGCTCTTTCTA 11.2 11.2 Psyr_2626 Hypotheticalprotein
2605313..2605340 TAAAGCTTTGGTAGCGACAGACGTTAAC 11.1 Psyr_2241 Chemotaxissensorytransducer
5703584..5703611 TAAAAAAAGCGCCCCGAAGGACGCTTAA 11.1 Psyr_4808 Hypotheticalprotein
1648899..1648926 TAAATTCAGAGCCGTGGCGCTCGTTTTT 10.3 11.8 13.3 Psyr_1462 GCN5-relatedN-acetyltransferase
1417924..1417951 TCAATAACTGGCTGGATGCGTCGTTCAC 10.1 10.9 Psyr_1261 GuaB
5017688..5017661 TGAATTTGCCCGACGGCTGCGCGTTTCA 10.7 10.0 Psyr_4211 ABCtransporter
1085896..1085923 TGAAGTTCTGGCGGGCGGGGTCGATGCA 10.5 Psyr_0950 HemK;methyltransferase
4713662..4713689 TAAAAAACAGGCGCCCGGATGCGTCTGA 10.1 Psyr_3966 Membranetransport
2086497..2086470 TAAAGCCTACCGCGCCGCCATCGTTCAC 10.0 11.3 Psyr_1812 Guaninedeaminase
1012883..1012856 TGAAGTCCTGCCTGCGCCTTGCGTTTCA 10.0 10.5 Psyr_0891 ChrR;transcriptionactivator
5401838..5401811 TAAAGTTCTGCTTGGCCCGCACGTTTCC 10.9 Psyr_4546 50SribosomalproteinL23
2285763..2285790 TAAACATCAGCGTCCGTGTTCCGTTTAA 10.3 Psyr_1971 PvdQ;acylase
2489035..2489008 TAAATTGTCGGCCCGACGGATCGTTTCG 10.3 Psyr_2129 MFStransporter
4025673..4025700 TGATTGTCGAAGGCCGCGGAACGTTCAA 10.2 Psyr_3367 IronpermeaseFTR1
2273489..2273462 TAAATAAACGTTATTCGCTTTCGTTCTC 10.1 Psyr_1962 TonB-dependentreceptor
P.syringaepv.phaseolicola1448A NC_005773
2215579..2215606 TAATTATTTGCAGACGCCATCCGTTCTC 17.0 14.6 PSPPH_1910 PVDchromophorethioesterase
2231162..2231189 TAATTTTTCGACGTAGCGATACGTTCAA 16.4 15.3 PSPPH_1913 MbtH-likeprotein
2239548..2239574 TAAATATTTCCCCGCCAATTCGTTCTT 15.4 14.3 PSPPH_1923 PVDpeptideNRPS
2276764..2276737 TAAATTTTTCGCCCATCCGCTCGTTCTC 15.1 15.4 PSPPH_1932 Dipeptidase
2762672..2762699 TGAATAATTGCCTCGCTTAAACGTTTTA 14.2 12.6 PSPPH_2381 Tannase/feruloylesterase
2238138..2238165 TAATTTTCTCGTGGCGAAGTACGTTCAA 14.0 10.7 14.6 PSPPH_1922 Taurinedeoxygenase
2276794..2276821 TAAATTTGTCTGCCGGGTTTACGTTCTA 13.7 11.6 15.1 PSPPH_1933 PvdP;TATsignal
693174..693201 TAATTACCTGCTCCCGTGAATCGTTATT 12.7 12.9 PSPPH_0586 Azurin
1689357..1689330 TAAATTCATCCACACTGGATACGATCAA 11.7 11.0 PSPPH_1448 Hypotheticalprotein
5279635..5279608 TGAATTAATTACCCTGTCCTTCGTTCTC 11.6 12.0 PSPPH_4640 Drugresistancetransporter
2283545..2283518 TAAATTCCCGTCCTCATCCCTCGTTTAA 11.3 12.0 14.0 PSPPH_1936 RNDeffluxtransporter,porin
2250912..2250940 TAAATCACTGGAGGCACTCAAACGTTTAT 11.2 PSPPH_1925 PVDpeptideNRPS
2300785..2300812 TAAATTTTTCGCCGCTGCGAGCGTTATA 10.3 13.0 13.9 PSPPH_1954 LeuB;3-isopropylmalatedehydrogenase
1850987..1851014 GAAATTCCAGCGGGCGCAAGTCGTTCAT 10.2 10.6 PSPPH_1597 ISPsy18,transposase
1543459..1543486 TCAATAACTGGCTGGATGCGTCGTTCAC 10.0 10.8 PSPPH_1333 GuaB
4485522..4485549 TGAATTCGACCGTCGGTGATTCGTTTCA 12.5 11.1 PSPPH_3927 Glucosedehydrogenase
1191967..1191994 TGAAGTTCTGGCGGGCGGGGTCGATGCA 10.1 PSPPH_0998 HemK;methyltransferase
2283574..2283600 TAAATTTTGCGTCTGCGCTTCGTTTAA 11.3 PSPPH_1937 PvdQ;acylase
5225800..5225773 TAAAGTTCTGCTTGGCCCGCACGTTTCC 11.0 PSPPH_4590 50SribosomalproteinL23
593310..593337 TGATTACCCGTGCCCGCCAGACGTTCCA 10.9 PSPPH_0509 GlnE
2201685..2201658 TCAATGTCTGCGGGGGTATCTCGTTTTT 10.4 PSPPH_1897 Integrase
2271222..2271195 TAAATAAACGTTATTCGCTTTCGTTCTC 10.2 PSPPH_1928 TonB-dependentreceptor
184783..184810 TAAATCCCCGGCATACGGGACCGTTCAT 10.0 PSPPH_0157 Hypotheticalprotein
P.fluorescensPf-5 NC_004129
4876034..4876007 TAATTATTTCAAGACGTCATCCGTTCTC 15.1 13.2 PFL_4189 PvdL;PVDchromophoreNRPS
4589567..4589540 TAAATTCCTCACGGGCTGTTACGTTTTA 13.8 13.5 16.2 PFL_3974 Hypotheticalprotein
4738601..4738573 TAAATCTTTGGAGGCCCTCAAACGTTTAT 13.6 11.4 PFL_4093 PVDpeptideNRPS
1032891..1032918 TAAATTGAGCATAGAGGAAAACGTTTTC 12.4 10.7 PFL_0889 AuxinEffluxCarrier
Journalcompilation©2008BlackwellPublishingLtd,MolecularMicrobiology,68,871–889
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