An SOS-regulated operon involved in damage-inducible

12 Pages·2005·1.24 MB·English

Published online May 10, 2005
Nucleic Acids Research, 2005, Vol. 33, No. 8 2603–2614
doi:10.1093/nar/gki551
An SOS-regulated operon involved in
damage-inducible mutagenesis in
Caulobacter crescentus
Rodrigo S. Galhardo, Raquel P. Rocha, Marilis V. Marques and Carlos F. M. Menck*
DepartmentofMicrobiology, Institute ofBiomedicalSciences,University of Sa˜o Paulo,Sa˜o Paulo,
SP 05508-900,Brazil
D
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ReceivedFebruary25,2005;RevisedandAcceptedApril15,2005 n
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ABSTRACT replication forks (1), although it can bypass some types of fro
DNA damage (2). dinB (also called dinP) encodesa member m
DNApolymerasesoftheY-family,suchasEscherichia oftheY-familyofDNApolymerases,DNAPolIV(3,4).This h
coli UmuC and DinB, are specialized enzymes ttp
enzyme is responsible for untargeted SOS mutagenesis of s
inducedbytheSOSresponse,whichbypasslesions phage l, and induces frameshift mutations when overex- ://a
c
allowingthecontinuationofDNAreplication.umuDC pressed (5). More recently, dinB has been associated with ad
e
orthologs are absent in Caulobacter crescentus and thephenomenonofmutationunderstressconditions,oradapt- m
other bacteria, raising the question about the exist- ive mutagenesis [reviewed in (6)]. ic.o
ence ofSOS mutagenesisin these organisms.Here, In E.coli, ultraviolet (UV) and chemical mutagenesis is up
wereportthattheC.crescentusdinBorthologisnot heavily dependent on the SOS-regulated umuDC operon .co
m
involvedindamage-inducedmutagenesis.However, (7,8). The UmuC protein has been identified as a DNA /n
anoperoncomposedoftwohypotheticalgenesand polymerase (Pol V), a distributive enzyme able to perform ar/a
dnaE2,encodingasecondcopyofthecatalyticsub- TLS and highly inaccurate even when using undamaged rtic
templates (9,10), which interacts with the processed form of le
unitofPolIII,isdamageinducibleinarecA-dependent the co-expressed protein UmuD. Both in vivo and in vitro -ab
manner, and is responsible for most ultraviolet (UV) s
experiments have shown that efficient TLS requires the tra
aTnhdemreitsoumltyscidneCm-oinndsutrcaetdemthuatatttiohnestihnrCee.crgeesnceesntaurse. UDmNAuDP02oCl IIcIomenpcloexd,edRbeycAth,eSdsbn,aEthgeencea,taalnydticthseubbunciltamopf ct/33
/8
required for the error-prone processing of DNA [reviewed in (11)]. /2
6
lesions. The two hypothetical genes were named DNApolymeraseIIIisamultiproteincomplexresponsible 0
3
imuA and imuB, after inducible mutagenesis. ImuB for the replication of the bacterial chromosome. Although /2
4
issimilartoproteinsoftheY-familyofpolymerases, the Y-family DNA polymerases, specialized in TLS, seem 01
5
and possibly cooperates with DnaE2 in lesion to be ubiquitous throughout nature, it has been shown that 21
theasubunitofPolIII,whichbearsthecatalyticpolymerase b
bypass. The mutations arising as a consequence y
activity, is also able to perform TLS. In Bacillus subtilis, the g
oftheactivityoftheimuABdnaE2operonarerather u
dnaE gene is SOS regulated and the purified enzyme can e
s
unusual for UV irradiation, including G:C to C:G perform TLS in vitro, an activity also shown for the t o
transversions. Streptococcus pyogenes protein (12,13). Depletion of n 0
4
B.subtilis DnaE inhibits UV-induced mutagenesis, although A
p
INTRODUCTION ittheisnedeifdficfourltthtoisaesnczeyrtmaientowheexttheenrdtthhies breyqpuaisrsemreeancttiorenflpecetrs- ril 20
1
DNA damage-induced mutagenesis is, in a major extent, an formedbyY-familyDNApolymerases,orresultsfromactual 9
activeprocessthatrequiresspecializedDNApolymerasesable lack of lesion bypass (13).
to perform translesion synthesis (TLS). In Escherichia coli, It has recently been shown that a second copy of the
three SOS-regulated DNA polymerases are involved in dnaE gene mediates SOS mutagenesis in Mycobacterium
damage tolerance: Pol II, encoded by the polB gene, Pol IV tuberculosis (14). The expression of this gene is regulated
and Pol V. The major role of Pol II is the rescue of stalled by the SOS response in this organism, being induced by
*TowhomcorrespondenceshouldbeaddressedatDepartmentofMicrobiology,InstituteofBiomedicalSciences,UniversityofSa˜oPaulo,AvenueProfessor
LineuPrestes,1374,Sa˜oPaulo,SP05508-900,Brazil.Tel:[phone];Fax:[phone];Email:[email protected]
ªTheAuthor2005.PublishedbyOxfordUniversityPress.Allrightsreserved.
Theonlineversionofthisarticlehasbeenpublishedunderanopenaccessmodel.Usersareentitledtouse,reproduce,disseminate,ordisplaytheopenaccess
versionofthisarticlefornon-commercialpurposesprovidedthat:theoriginalauthorshipisproperlyandfullyattributed;theJournalandOxfordUniversityPress
areattributedastheoriginalplaceofpublicationwiththecorrectcitationdetailsgiven;ifanarticleissubsequentlyreproducedordisseminatednotinitsentiretybut
onlyinpartorasaderivativeworkthismustbeclearlyindicated.Forcommercialre-use,[email protected]
2604 Nucleic Acids Research, 2005, Vol. 33, No. 8
DNA damaging agents in a recA-dependent manner (15,16). at30(cid:3)Cwithconstantshaking.Plasmidswereintroducedinto
The dnaE gene is presented in duplicate in several bacterial C.crescentus by conjugation with E.coli S17-1 strain. When
genomes, most frequently in a potential operon with two appropriated, the culture medium was supplemented with
hypothetical genes, one of which is a dinB-related one. This kanamycin(50mg/ml),nalidixicacid(25mg/ml),spectinomy-
widespread operon has been shown to be regulated by cin(50mg/ml)ortetracycline(1mg/ml).E.colistrainDH10B
LexA in Pseudomonas putida, indicating that it is involved (Invitrogen, CA) was used for cloning purposes. E.coli was
in DNA damage response (17). This operon is present in grown at 37(cid:3)C in Luria–Bertani medium supplemented with
several a-proteobacteria genomes completely sequenced to ampicillin(100mg/ml),kanamycin(50mg/ml)ortetracycline
date, including C.crescentus (18). (15 mg/ml) when necessary.
In C.crescentus, a recA-dependent inducible repair system Genetic disruptions were achieved using the pNPTS138
was demonstrated through the existence of Weigle reactiva- vector, and selecting for two consecutive recombination
tionofirradiatedphage(19).Thisorganismisalsomutableby events. First, Kanr conjugants of C.crescentus were selected
D
UV radiation, suggesting the existence of error-prone path- inthescreeningforthefirstrecombinationevent.Lossofthe ow
ways,althoughumuDCorthologsareabsentfromitsgenome plasmid after the second recombination was selected in PYE nlo
a
(18,19). In this work, we investigated the involvement of the mediacontaining3%sucrose.Whenappropriated,aconcom- d
e
dnaE2-containing gene cassette and the dinB ortholog in itantSpcrselectionwasalsoapplied.Strainsgeneratedinthis d
damage-induced mutagenesis, detected in rifampicin resist- way were analyzed by Southern blot to confirm gene disrup- fro
m
anceassays.TheresultsshowthatthednaE2operonisupregu- tions. The construction of gene targeting plasmids was per- h
lated in response to DNA damage in a recA-dependent formed withPCR products amplifiedwith theprimersshown ttp
s
manner, and demonstrate that all three genes that compose inSupplementaryTable1S.Theseproductswereclonedinthe ://a
this expression unit are involved in UV and mitomycin pGEM-Teasyvectorandsequencedtoassuresequenceinteg- ca
d
C-inducedmutagenesis.Ontheotherhand,dinBisnotneces- rity.FordisruptionofrecA,fragmentsamplifiedwithprimers e
m
sary for mutagenic repair. We propose to name the genes recaA · recaB, and recaC · recaD were fused in the ic
.o
co-expressed with dnaE2, after inducible mutagenesis, as pNPTS138 vector using the restriction sites introduced, gen- u
p
imuA and imuB, and show that the imuAB/dnaE2-dependent eratinga1.8kbfragmentcontainingthefirst81bpfusedtothe .c
o
mutational spectra of UV-induced lesions is different from last75bpofrecAplusflankingregions,leadingtoanin-frame m
/n
the prototypical E.coli one, including a high proportion of deletion of recA. In-frame deletions were constructed in the a
G:C ! C:G transversions. same way for imuA and imuB using, respectively, primers r/a
CC3213A toDand CC3212AtoD. imuA disruption resulted rtic
le
MATERIALS AND METHODS inadeletioncontainingonlythefirst36andthelast54bpof -a
b
thegene,andimuBdisruptionresultedinadeletioncontaining s
Bacterial strains and plasmids only the first 63 and the last 81 bp of the gene. dnaE2 dis- trac
Bacterialstrainsandplasmidsusedinthisstudyareshownin ruptionwasperformedbytheinsertionoftheVspecrcassette t/3
3
Table1.C.crescentusstrainsweregrowninPYEmedium(20) (21) in the natural EcoRI site of a 1629 bp fragment of this /8
/2
6
0
3
/2
Table1.Bacterialstrainsandplasmidsusedinthisstudy 40
1
5
Description Source 21
b
y
Strain g
NA1000 Parentalstrain,C.crescentusCB15derivative (49) ue
GGMM1200 NNAA11000000,,((DDrimecuAA)) TThhiissssttuuddyy st on
GM30 NA1000,(DimuB) Thisstudy 0
4
GM40 NA1000,(dnaE2::V) Thisstudy A
GM50 NA1000,(dinB::V) Thisstudy p
GM34 NA1000,(DimuB,dnaE2::V) Thisstudy ril 2
GM35 NA1000,(DimuB,dinB::V) Thisstudy 01
S17.1 E.colistrainforplasmidmobilization (50) 9
DH10B E.colistrainforcloningpurposes Invitrogen
Plasmid
pGEM-Teasy Cloningvector Promega
pNPTS138 pNPTS129derivative,oriTsacBKanr (51)
pMR20 Broad-host-rangelowcopyvector,Tetr (52)
pHP45V Vcassette(Spcr)containingvector (21)
pRKlacZ290 pRK2derivedvectorwithapromoterlesslacZgene,Tetr (53)
pPUVRA uvrApromoterclonedinthepLACZ290vector Thisstudy
pP3213 CC3213promoterclonedinthepLACZ290vector Thisstudy
pRECDEL In-framedeletionofrecAgeneandflankingregionsclonedinpNPTS138 Thisstudy
pDINDEL dinBinterruptedwiththeVcassetteintheinternalEcoRVsite,clonedinpNTPS138 Thisstudy
pDNAE2DEL dnaE2interruptedwiththeVcassetteintheinternalEcoRIsite,clonedinpNTPS138 Thisstudy
p3212DEL In-framedeletionofCC3212geneandflankingregionsclonedinpNPTS138 Thisstudy
p3213DEL In-framedeletionofCC3213geneandflankingregionsclonedinpNPTS138 Thisstudy
pMR3213 CC3213geneclonedinthepMR20vector Thisstudy
Cosmid3A4 pLAFR5containingnt3469882–3497288oftheC.crescentusgenome (54)
Nucleic Acids Research, 2005, Vol. 33, No. 8 2605
gene, amplified with the dnaE2re and dnaE2fo primers. The primers 1112A and 1112B were used to check imuB/dnaE2
insertionintheEcoRIsiteresultedinapredictedtruncationat co-expression, and primers 1213A and 1213B were used to
position 598 of the protein, leading to loss of the last 485 check imuA/imuB co-expression. RNA from exponentially
amino acids. dinB knock-out was constructed by insertion growingcellswasextractedwithTrizol1reagent(Invitrogen),
oftheVspecrcassetteintheEcoRVsiteofa1.2kbfragment, and treated with DNAseI to eliminate contaminant DNA.
amplified with primers dinbfo and dinbre. This insertion res- RT–PCR experiments were carried out with 300 ng of RNA
ultedinapredictedtruncationatposition167ofthe419amino usingtheSuperscriptOneStepkit(Invitrogen),inthefollow-
acid DinB protein. ing cycling conditions: 50(cid:3)C for 20 min; 94(cid:3)C for 2 min;
PCRswerealsocarriedouttoamplifypromoterregionsof 40 cycles of 95(cid:3)C for 1 min; 61(cid:3)C for 30 s; 72(cid:3)C for 50 s;
uvrAandimuAgenes.Theprimerspuvrafoandpuvrarewere followedbyincubationat72(cid:3)Cfor7minandcoolingto4(cid:3)C.
usedtoobtainafragmentrangingfrom–492to+54ofthestart A control for DNA contamination of samples was performed
codonofuvrA [open readingframe(ORF)CC2590].Primers with PCR lacking reverse transcriptase.
D
p3213foandp3213reamplifiedafragmentrangingfrom(cid:4)513 Measurements of promoter activity with lacZ transcrip- ow
to+74oftheimuAstartcodon.Bothfragmentswereclonedin tionalfusionswereperformedafterUVirradiation,inthesame nlo
a
the pRKlacZ290 vector using the restriction sites introduced conditions used for survival experiments (see above), allow- d
e
in the primers. ing cells to recover in PYE medium for 90 min after treat- d
Inordertopromotecomplementationofthephenotypesof ment. After recovery, cells were assayed for b-galactosidase fro
m
the imuA strain, a full-length gene including the promoter activity as described previously (22). h
region was amplified with the 3213A and 3213D oligonuc- ttp
s
leotides, and cloned in the low copy number pMR20 vector, Real-time analysis ofgeneexpression ://a
using the restriction sites introduced in the primers. ca
RelativeexpressionofimuA,imuB,dnaE2anddinBafterUV d
e
irradiation was determined with quantitative PCR experi- m
Survivaland mutagenesis experiments ic
ments. An aliquot of 2 mg of RNA pre-treated with DNAse I .o
Caulobacter crescentus strains were grown until mid-log were used as template for total cDNA synthesis in 20 ml up
phase (5 · 108 cells/ml), and then submitted to the different reactions with random hexamers using the Superscript First- .co
m
treatments.A5mlbatchofculturewasUV-irradiatedwitha Strand Synthesis System for RT–PCR (Invitrogen). For real- /n
PhilipsTUV15W/G15T8germicidallamp(mainly254nm)in timePCR,anamountofcDNAcorrespondingto25ngofinput ar/a
Petri dishes. Aliquots were removed before and after irradi- RNA was used in each reaction. Reactions were performed rtic
ation for serial dilutions and plating in PYE medium, for the withtheSYBRGreenPCRMasterMix(AppliedBiosystems), le
determination of viable cell counts after 48 h incubation at and analyzed in the ABI 7500 Real-Time System. Relative -ab
30(cid:3)C. For mutagenesis experiments, a 200 ml aliquot of irra- expressionlevelswerecalculatedasdescribedpreviously(23), stra
diatedcellswasinoculatedin1mlofPYEliquidmediumand usingtherpoDgeneastheendogenouscontrol.Primersused ct/3
cultivated overnight, to allow mutation fixation. Then, the in this analysis are listed in Supplementary Table 1S. 3/8
cultures were submitted to serial dilutions and plating on /2
6
PYE medium for viable cell counts. The cultures were also Phylogenetic analyses 03
plated in PYE medium containing 100 mg/ml rifampicin to /2
score Rifr cells. Mutation frequencies were calculated by Protein sequences of genes CC3211, CC3212 and CC3213 40
1
were obtained from the National Center of Biotechnology 5
dividingthenumbersofmutantsbytheestimatedtotalnumber 2
of cells analyzed in the experiments. Mitomycin C (mit C) Information GenBank database (www.ncbi.nlm.nih.gov/ 1 b
survival and mutagenesis experiments were conducted in a Entrez/). The accession numbers are respectively y g
similar manner, with drug treatments performed in 2 ml of NP_422005, NP_422006 and NP_422007. Orthologous pro- ue
s
culture for 1 h at 30(cid:3)C. For mit C mutagenesis, cells were tein sequences from complete genomes were obtained by t o
centrifuged and resuspended in PYE before inoculating for BLAST analyses (24). The lack of orthologs in several gen- n 0
overnight incubation, to remove the drug from the medium. omes was ascertained by exhaustive BLAST analysis. 4 A
For phylogenetic analysis, the protein sequences were p
DeterminationofthemutationspectraintherpoBgene aligned using the ClustalX multiple sequence alignment pro- ril 2
0
gram (25) with manual adjustment using Genedoc v2.6.001 1
9
Rifampicin-resistant clones, obtained after UV irradiation in
(26) (www.psc.edu/biomed/genedoc). Only unambiguously
theexperimentsdescribedabove,werecultivatedovernightfor
aligned positions (excluding poorly conserved and gap
theextractionofchromosomalDNA.TheprimersrpoB2foand
regions) were used. Phylogenetic trees were generated for
rpoB2re(SupplementaryTable1S)wereusedinPCRsdesigned
each group of protein homologs from sequence alignments
fortheamplificationoftheregionIIofrifampicinresistance.
usingthePHYLIPprogramversion3.5(27).Distanceanalyses
PCRproductswerepurifiedandsequencedinbothstrandsusing
were performed using the neighbor-joining method in
rpoB2fo and rpoB2re primers with the Big Dye Terminator
PHYLIP,withthedistancePAMmatrixmodel(28).Bootstrap
Cycle Sequencing kit and analyzed in the ABI Prism 3100
support (resampled 1000 times) was calculated, and strict
GeneticAnalyzer(AppliedBiosystems,CA).Onlyindepend-
consensus trees constructed. Only bootstrap values >50%
entmutationsfromeachexperimentwerescored.
areshown.Theconsensustreesobtainedwereviewedthrough
TreeView software (29).The same set ofprokaryotic species
RT–PCRexperiments and b-galactosidase assays
was used in all analyses, and includes CC3211 (dnaE2) and
The operon organization of genes CC3211, CC3212 and CC3212(imuB)orthologsfromfullysequencedgenomesand
CC3213 was assayed through RT–PCR experiments. The E.coli proteins.
2606 Nucleic Acids Research, 2005, Vol. 33, No. 8
RESULTS such as several Actinomycetales and the planctomycete
Rhodopirellula baltica, also possess a dnaE2 gene, although
Genomic contextand phylogenetic analyses
indifferentgenomiccontexts(Figure1).Atotalofsixdiffer-
The CC3211 gene of C.crescentus encodes a second copy of ent gene arrangements were found in bacterial genomes con-
the dnaE gene, hereafter referred to as dnaE2. dnaE2 is in a taining dnaE2. Remarkably, the large majority of organisms
conservedorganizationwithtwogenesencodinghypothetical presentingdnaE2alsopossessanimuBortholog,althoughnot
proteins,CC3212(imuB)andCC3213(imuA),similartoother necessarilyinanoperonorganization.InR.baltica,imuAand
proteobacteria (Figure 1). This operon organization is even- imuBformapotentialoperon,whichdoesnotincludednaE2,
tually associated with a duplication of lexA, such as in located apart in the genome. The M.tuberculosis imuB gene,
P.putida. However, this is not the case for C.crescentus and encodedbytheRv3394cORF,isinanoperonwithRv3395c,a
relatedbacteria,whichhaveonlyonecopythelexAgene(30). putativeRecA-likeprotein.Mostinterestingly,bothRv3395c
ItwaspreviouslyshownthattheoperonorganizationofimuAB and Rv3394c are damage inducible in M.tuberculosis, and
D
and dnaE2 is conserved in all genomes of proteobacteria thepromoterofRv3395chasbeenshowntocontainaMyco- ow
containingthesegenes(17).However,otherbacterialspecies, bacterial SOSbox(14,16).Asimilarpicturecanbeobserved nlo
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Figure1.PrototypicalgenomicorganizationsofdnaE2andaccompanyingimuAandimuBgenes.ThesameorganizationfoundinC.crescentusisalsopresentin
a-proteobacterialgenomes,Pseudomonasaeruginosa,Vibriovulnificus,Vibrioparahaemolyticus,Ralstoniasolanacearum,BordetellabronchisepticaandBor-
detellaparapertussis.AduplicationoflexAprecedingtheoperon,asfoundinP.putida,isalsopresentinMethylococcuscapsulatus,Pseudomonassyringae,
XanthomonascampestrisandXanthomonasaxonopodis.TheM.tuberculosisgenearrangementisconservedinallspeciesofthegeneraMycobacteriumand
Corynebacterium,andinNocardiafarcinica.SmallboxesrepresentknownSOSoperators,andinterruptedlinesindicatethatthegenesarenotcontiguousinthe
genome.Locinumbersasprovidedbythegenomeannotationareindicatedabove,thearrowsrepresentinggenes,andourannotationisgiveninsidethearrows.
Differentoriginalgeneannotationsareindicatedinparentheses.
Nucleic Acids Research, 2005, Vol. 33, No. 8 2607
in the genomes of other Actinomycetales, except Propioni- majority of fully sequenced genomes from a-proteobacteria,
bacteriumacnes,wheretheRv3395corthologformsaputative butalsofoundinotherproteobacteriaandinActinomycetales
operon with both imuB and dnaE2. Although proteins with (Figure2).ThebranchofDnaEincludestheE.coliproteinand
significant similarity to ImuA cannot be found in Actinomy- theproductoftheCC1926gene,whichseemstobeinvolved
cetales,thegenomiccontextanalysissuggeststhattheRecA- in DNA replication in C.crescentus (31). DnaE2 orthologs
likeproteinencodedbyM.tuberculosisRv3395mayrepresent clearly form a distinct branch, including the DnaE2 from
a functional counterpart of ImuA in these organisms. It is C.crescentus. The prototypical member of this branch is the
evidentthatseveralrearrangementsofthegenomicorganiza- M.tuberculosisproteinRv3370c.Thisproteinhasbeenfound
tionofdnaE2andimuBhavetakenplaceduringevolution,but to be damage inducible, and implicated in cell survival and
notably these genes always co-exist in bacterial genomes. mutagenesis after UV treatment (14).
These data indicate that the imuB and dnaE2 genes compose The CC3213 ortholog from P.putida, PP3117, was annot-
ahighlyconservedco-evolvinggeneticsystem,whichislikely ated as sulA2 (32). However, PSI-BLAST analyses of the
D
to be involved in DNA damage response. protein encoded by CC3213 shows only very low levels of ow
PhylogeneticanalysesoftheDnaEproteinsofthemicroor- similarity to SulA (24), and also some similarity to RecA/ nlo
a
ganismspresentingsuchduplicationindicatethatthisismost RadA recombinases (data not shown). A phylogenetic tree d
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probablyaveryancestralduplicationevent,presentinthevast of this protein and its orthologs has been presented d
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Figure2.PhylogeneticrelationshipsbetweenDnaEandDnaE2.PhylogeneticanalysesincludedDnaEandDnaE2proteinsofallfullysequencedgenomeswhere
suchduplicationcouldbefound.OnlyM.tuberculosisandMycobacteriumboviswereincludedforsimplification,althoughtheduplicationcanbefoundinseveral
otherMycobacterialgenomes.ThenamesoftheORFsandspeciesnameabbreviationsshownarethesameadoptedinthegenomeannotationofeachorganism.
Abbreviationsoforganismnamesareasfollows:AGR,A.tumefaciensstr.C58;b,E.coliK12;BB,B.bronchisepticaRB50;bll,BradyrhizobiumjaponicumUSDA
110;BMA,BurkholderiamalleiATCC23344;BMEI,Brucellamelitensis16M;BPP,B.parapertussis12822;BPSL,BurkholderiapseudomalleiK96243;BR,
Brucellasuis1330;CC,C.crescentusCB15;CE,CorynebacteriumefficiensYS-314;DIP,CorynebacteriumdiphtheriaeNCTC13129;Mb,M.bovisAF2122/97;
MCA,M.capsulatusstr.Bath;mllandmlr,MesorhizobiumlotiMAFF303099;NCgl,CorynebacteriumglutamicumATCC13032;Nfa,N.farcinicaIFM10152;PA,
PseudomonasaeruginosaPAO1;PP,P.putidaKT2440;PPA,P.acnesKPA171202;PSPTO,P.syringaepv.tomatostr.DC3000;RB,R.balticaSH1;RPA,
RhodopseudomonaspalustrisCGA009;RscandRsp,R.solanacearumGMI1000;Rv,M.tuberculosisH37Rv;SAV,S.avermitilisMA-4680;SmcandSma,S.meliloti
1021;VP,V.parahaemolyticusRIMD2210633;VV,V.vulnificusYJ016;XAC,X.axonopodispv.citristr.306;andXCC,Xanthomonascampestrispv.campestris
str.ATCC33913.Thenumbersindicatethebootstrapvalues>50%.
2608 Nucleic Acids Research, 2005, Vol. 33, No. 8
previously (17),butthe levelsofproteinsimilaritiesbetween ofthesameorganisms,indicatingthatthisisadivergentDinB/
CC3213 orthologs and known proteins, such as SulA and UmuC homolog (Figure 3). The only possible exception is
RecA, are too low to precisely ascribe any protein function. theSAV6556proteinofStreptomycesavermitilis,whichcan-
We tested the filamentaion in both the wild type and the notbesurelyassignedasImuBthroughphylogeneticanalyses,
derivative CC3213 deletion strain in several conditions by although it is present in a putative operon with dnaE2
microscopic examination. C.crescentus shows a filamentous (Figure1).Proteinsimilaritycomparisonamongtheseproteins
morphology afterprolonged exposure tomit C, and the dele- reinforcesthedifferencebetweenImuBandDinB,e.g.E.coli
tionofCC3213didnotaffectthiscellularresponse,indicating DinB and UmuC proteins share 45% identity, while
that this gene is not involved in cell division suppression C.crescentus DinB and the ImuB proteins share only 35%
(Supplementary Figure 1S). Therefore, we propose to name identity.Therefore,weconcludedthattheCC3212geneprod-
theCC3213geneimuA,afterinduciblemutagenesis,giventhe uctisamemberofadistinctbranchintheUmuCsuperfamily
functional characterization conducted (see below). of proteins, and not a DinB ortholog. We named this gene
D
Similarly,CC3212orthologsidentifiedbyBLASTanalysis imuB, in accordance with the nomenclature adopted for ow
present diverse annotations: the P.putida gene PP3118 CC3213. nlo
a
was annotated as dinP (32), and the PPA1651 gene of d
e
P.acneswasannotatedasumuC(33),whilemostotherortho- d
logs (including CC3212 itself) were annotated as genes with Characterization ofthe imuAB/dnaE2 expression fro
m
unknown function. In this case, phylogenetic analysis was The genomic organization of the CC3213 (imuA), CC3212 h
conducted including all fully sequenced genomes where (imuB) and CC3211 (dnaE2) genes suggests that they ttp
s
CC3212orthologswerefound,forcomparisonbetweenthese may constitute an operon (Figure 1). In fact, in P.putida, ://a
proteinsandDinB,theclosestrelatedproteinofknownfunc- Sinorhizobium meliloti and Agrobacterium tumefaciens, the ca
d
tion.AllorganismswithaCC3212orthologalsohaveoneor orthologs of these genes are co-expressed in a single RNA e
m
more typical dinB genes. The product of CC3212 and its (17). In order to evaluate the co-expression of these genes in ic
orthologsformabranchthatisdistinctfromtheDinBproteins C.crescentus, we designed RT–PCR experiments with the .ou
p
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Figure3.PhylogeneticrelationshipsbetweenImuBandDinB.PhylogeneticanalysesincludedImuBandDinBproteinsofallfullysequencedgenomeswherethe
imuBgenecouldbefound.OnlyM.tuberculosisandM.boviswereincludedforsimplification,althoughimuBcanbefoundinseveralotherMycobacterialgenomes.
ThenamesoftheORFsandspeciesnameabbreviationsshownarethesameadoptedinthegenomeannotationofeachorganism.Abbreviationsoforganismnamesare
indicatedinthelegendofFigure2.Thenumbersindicatethebootstrapvalues>50%.
Nucleic Acids Research, 2005, Vol. 33, No. 8 2609
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Figure4.ExpressionoftheimuABdnaE2operon.(A)Schematicdiagramforthisoperonindicatingthepositionoftheoligonucleotides(smallarrows)usedinthe ://a
c
RT–PCRstrategytodetecttheco-expressionofthethreegenes.TemplateRNAwasextractedfromexponentiallygrowingcells,bothfromnon-irradiatedcultures a
d
andfromculturesirradiatedwith60J/m2ofUVandrecoveredafter45min.TheRT–PCRproductswerevisualizedafterelectrophoresisin1%agarosegelstained e
m
withethidiumbromide.(B)ExpressionofuvrAandimuApromotersintranscriptionalfusionswiththelacZgene.b-Galactosidaseactivitywasmeasured90minafter ic
irradiationwithUVlight,bothinthewild-typeandrecAstrains. .o
u
p
.c
o
oligonucleotides represented in Figure 4A. The results con- investigated by quantitative RT–PCR experiments. In addi- m
firmed that the three genes are expressed as a single operon, tion, the expression of the dinB gene (encoded by the /na
and the polycistronic RNA can be detected both in UV- CC2466 ORF), which does not present an SOS box in its r/a
irradiated and non-irradiated exponentially growing cells. putative promoter region, was also tested. The results rtic
le
In a-proteobacteria, the LexA-binding site has been shown in Figure 5 demonstrate that imuA, imuB and dnaE2 -a
b
characterized for many species. The typical sequence expression levels are increased similarly in response to UV s
GexTaTmCinNe7dGTtoTCdaitsea(p3p4a,r3e5n)t.lyTwheellopcoernosnervceodntianinainllgspimecuiAes, dgaemneasgeis. aFbuortlhisehremdorien, tthheerdeacmAasgteraiinn,duincdtiiocnatinogf tthheattthhreeye tract/3
3
imuBanddnaE2presentsthistypicalLexA-bindingsequence are coordinately upregulated by the SOS response. The /8
/2
66bpupstreamfromtheimuAstartcodon,indicatingthatthis SOS box-containing promoter upstream of imuA presumably 6
0
operon is SOS regulated. In order to examine whether the mediates this coordinated expression. In contrast, the expres- 3/2
expression of this operon is regulated in response to DNA sionofdinBremainsunaffectedbyDNAdamage,suggesting 40
1
damage, we cloned the promoter of imuA upstream the that this gene is not involved in damage tolerance in 5
2
reportergenelacZinthepRKlacZ290vector.Forcomparison, C.crescentus. 1
b
the promoter of the uvrA gene, which also bears a putative y g
a-proteobacteria type SOS box 78 bp upstream the start ue
Geneticcharacterizationof damage-inducible s
cbo-gdaolna,ctwosaisdacselonaecdtiviintythweassapmerefovrmecetodr.boDthetefromrinthaetiownildo-f mutagenesis inC.crescentus t on 0
typeNA1000strain,andforaderivativerecAdeletionmutant, SeveralstrainsdisruptedintheimuA,imuBanddnaE2genes, 4 A
constructed in this work. Both promoters were strongly aswellasinthedinBgene,wereconstructed(Table1).These p
induced 90 min after UV irradiation in a dose-responsive gene disruptions had no effect upon cell growth (data not ril 2
0
fashion in the wild-type strain (Figure 4B), although the shown). Compared with the wild type, all the single mutant 1
9
expression of lacZ under control of the imuAB/dnaE2 pro- strainsdeficientinthegenesoftheoperonwereslightlysens-
moter is much higher than the one observed for the uvrA itive to UV (Figure 6A). In contrast, the dinB strain showed
promoter. wild-typelevelsofUVresistance,indicatingthatDinBisnot
In our conditions, optimal promoter expression was involved in UV damage tolerance. The double mutant imuB
achieved 90–120 min after irradiation (data not shown), dnaE2showednoincreasedsensitivitytoUV,indicatingthat
whichisingoodagreementwiththepreviousreportsofWeigle theproductsofthesegenesactinthesametolerancepathway.
reactivation time course experiments in this organism (19). AdinBimuBdoublemutantwasalsoconstructed,toevaluate
Ontheotherhand,damage-inducedactivityofbothpromoters if DinB could be acting in DNA damage tolerance in an
iscompletelyabolishedinarecA-deletedstrain,indicatingthat imuAB-deficientbackground.ThedinBmutationhadnoeffect
bothareregulatedinaRecA-dependentmanner.Theseresults ontheresistancetoUV,furtherindicatingthatthisgeneisnot
confirm the prediction that both uvrA and the imuAB/dnaE2 involved in UV damage resistance.
operon are controlled by the SOS regulon in C.crescentus. The mutagenesis of these strains was determined by the
The increased activity of the imuA promoter resulting in rifampicinresistanceassay.Thisassaydetectspointmutations
the induction of all three genes in the operon was further in the rpoB gene, which encodes the b-subunit of bacterial
2610 Nucleic Acids Research, 2005, Vol. 33, No. 8
RNA polymerase, and is very useful due to the high conser-
vation of the target gene, allowing its utilization in a wide
variety of bacterial species (36,37). Figure 6B shows the
mutagenesis induced by UV light in all the strains. It is
clearthatdinBdepletiondidnotaffectUV-inducedmutagen-
esis, in contrast to the disruption of imuA, imuB and dnaE2.
The UV-induced mutations are almost equally diminished in
these three strains, and reduced to approximately the same
extentintheimuBdnaE2doublemutant.Altogether,theres-
ults shown in Figure 6 demonstrate that the imuAB dnaE2
operon is involved in mutagenic repair of UV lesions, and
suggest that the proteins encoded by this operon cooperate
D
inthesame pathway,asevidencedbythe similarphenotypes ow
ofthe singlemutants,andbytheepistaticeffectofimuBand nlo
a
dnaE2 mutations. The dinB imuB strain presents the same d
e
levelsofUV-inducedmutagenesisastheimuBsinglemutant, d
confirmingthatDinBisnotimplicatedinerror-pronerepairin fro
m
the conditions tested. h
Similar experiments were performed to investigate the ttp
s
effects of the UV mimetic drug mit C in C.crescentus. The ://a
effectsofmitConthesurvivalofthedifferentstrainsaremuch ca
d
more pronounced than those of UV light (Figure 7A). The e
m
singlemutantsimuA,imuBanddnaE2mutantsareextremely ic
.o
sensitive to this condition, revealing that this operon plays a u
p
majorroleinthetolerancetothemitC-inducedDNAlesions. .c
o
The imuB dnaE2 double mutant does not exhibit an exacer- m
/n
bated phenotype, indicating again that these genes cooperate a
inthesamepathwayofdamagetolerance.Ontheotherhand, r/a
the dinB mutant is as resistant as the wild type, and the dinB rtic
le
imuB double mutant shows no further sensitivity enhance- -a
b
ment,demonstratingthatDinBisnotinvolvedinthetolerance s
to mit C-induced DNA lesions. Both the UV and the mit C trac
Figure 5. Quantitative RT–PCR analysis of UV-induced gene expression. sensitivity phenotypes of imuA could be complemented by t/3
RNAs were extracted from exponentially growing recA and wild-type 3
cellsbeforeand45minafterirradiationwith60J/m2UVC.Relativelevels vector pMR3213, containing the wild-type imuA allele. Sim- /8/2
ofexpressionshownrepresenttheaverageoftwoindependentexperiments ilarly,theimuBstraincouldbecomplementedbycosmid3A4, 6
0
doneintriplicate. which contains integral imuA and imuB genes, but lacking 3/2
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Figure 6. Effects of imuAB, dnaE2 and dinB mutations on UV resistance and mutagenesis in C.crescentus. (A) Survival curves. (B) Frequency of
RifrmutationsinducedbyUVintherpoBgene.Theresultsshownarethemeanofatleastfiveindependentexperimentsdoneintriplicate.Errorbarsindicate
theSD.
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Figure 7. Effects of imuAB, dnaE2 and dinB mutations on mit C resistance and mutagenesis in C.crescentus. (A) Survival curves. (B) Frequency of Rifr ://a
mutationsinducedbymitCintherpoBgene.Theresultsshownarethemeanofatleastfourindependentexperimentsdoneintriplicate.Errorbarsindicate ca
theSD. de
m
ic
dnaE2(datanotshown).Theseresultsconfirmthatthepheno- mutagenicspectrumofUVlight inE.coli(37,38).These res- .ou
p
typesofimuAandimuBstrainsarenotduetonegativeeffects ultsindicatethatimuBanddnaE2cooperateinalesionbypass .c
o
on the expression of downstream genes in the operon. pathway,whichhasadifferentspecificitythanthatinvolving m
The mutagenesis induced by mit C was also investigated umuDC in E.coli. /na
using the rifampicin assay (Figure 7B). The results show r/a
a dramatic decrease in mit C mutagenesis in all strains rtic
le
devoid of genes belonging to the damage-inducible operon -a
DISCUSSION b
ofC.crescentus.Ontheotherhand,dinBisclearlynotrequired s
at all for mit C mutagenesis. These data confirm that the In the present work, we demonstrate that a highly conserved trac
imuAB/dnaE2 operon accounts for most of the mutagenic gene expression unit consisting of the genes imuAB and t/3
3
DNA repair in C.crescentus cells exposed to UV light and dnaE2, controlled by the SOS regulon, is responsible for /8
/2
mit C. most of the damage-inducible mutagenesis in C.crescentus. 6
0
ThegenesimuAandimuBencodepreviouslyuncharacterized 3/2
proteins,whichareshownhereforthefirsttimetobeinvolved 40
Mutational specificityofUV lightin C.crescentus 1
inDNAdamagetolerance.Thesegenesarepresentinseveral 5
2
ThenatureofimuAB/dnaE2-dependentandindependentmuta- genomeswhereumuDCorthologscannotbefound,suchasthe 1 b
tions caused by UV light was analyzed in the rpoB gene. a-proteobacteria branch, and it is intuitive to argue that they y g
Rifampicin-resistant mutants obtained after UV radiation of may functionally replace these proteins in damage-inducible ue
s
thewild-type,dnaE2andimuBstrainswererandomlyselected mutagenesis in a wide variety of bacterial species. It is very t o
for sequencing of the rpoB cluster II of rifampicin resistance interesting to note that, independently of being organized as n 0
(37), which contained the Rifr mutations in all mutants singletranscriptionalunitsorasindependentgenes,imuBand 4 A
analyzed. The mutation spectra observed are shown in dnaE2alwaysco-existinbacterialgenomes,stronglysuggest- p
Figure 8. The mutation signature of UV light is remarkably ing that their activities are interconnected. ril 2
0
similarindnaE2andimuBstrains.G:C!A:Ttransitionsare The data obtained here clearly indicate that this operon 1
9
by far the most common type of substitution, constituting plays a major role in damage-induced mutagenesis in
(cid:5)90% of the base changes in both strains (Table 2). On C.crescentus, especially after mit C exposure. The lack of
the other hand, UV mutagenesis in the parental strain is mitCmutagenesisinimuAB/dnaE2-deficientstrainscorrelates
significantly modified to a high proportion of G:C ! C:G well with the extreme sensitivity they show to this agent.
transversions and tandem substitutions concentrated in a hot- Although mit C mutagenesis is mostly dependent on these
spot consisting of a run of three Cs, which gives rise to only genes, at least one-third of the UV-induced mutations are
G:C ! A:T mutations indnaE2 andimuB strains (Figure8). still generated in the imu- or dnaE2-deficient genetic back-
Thisincreaseintransversionsandtandemsubstitutionsinthe grounds. These mutations are not caused by the action of the
parentalstrainisaccompaniedbyconcomitantreductioninthe dinBgene,asevidencedbythephenotypesofdinBandimuB
fractionofG:C!A:Ttransitions,asshowninTable2.These dinB mutant strains. The nature of this mutagenic process
othertypesofmutationsareprobablyanoutcomeofthemuta- remains to be elucidated. In E.coli, it has been shown that
genic activity of the imuAB/dnaE2 operon of C.crescentus, umu-independent UV mutagenesis may occur, depending on
since they are not observed in the dnaE2 and imuB mutants. theexperimentalsystemused(39).Also,inB.subtilis,residual
G:C ! C:G transversions are not a significant part of the UV mutagenesis can be detected in varying levels, even in
2612 Nucleic Acids Research, 2005, Vol. 33, No. 8
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Figure8.UVmutationspectraintherpoBgene.Nucleotides1557–1625,comprisingtheclusterIIofrifampicinresistance,areshowninthisfigure.Allmutants -a
b
analyzedhadmutationsinthisregionoftherpoBgene.Mutationsinshadedbackgroundcorrespondtorepetitionsofthesamebasesubstitution,andtandemmutations s
areunderlined.Thetotalnumberofindependentmutationssequencedisindicatedforeachstrain. tra
c
t/3
3
Table2.FrequenciesofbasesubstitutionsintherpoBgeneinducedbyUV while wild-type cells also have a high proportion of tandem /8/2
light substitutionsandG:C!C:Gtransversions.Thisnovelmuta- 6
0
tionalsignature,dependentontheactivityoftheimuAB/dnaE2 3/2
Typeofmutation Wt(%) dnaE2(%) imuB(%) operon, probably reflects a difference in polymerase prefer- 40
1
G:C!A:T 42.9 88.6 93.1 ence for nucleotide incorporation during TLS. On the other 52
G:C!C:G 28.5 — — hand,themutationsinducedbyUVlightinM.tuberculosisina 1 b
AATa::TTnd!!emGCs::uGCbstitutions 11204...943 —1—1.4 —6—.9 dCn.carEe2sc-deenptuesn,dernestemmabnlninegr atrheediUffVeremntuftarogmenitchesopbecsetrruvmed oinf y gues
E.coli (14). More detailed studies of both systems are still t o
needed to clarify this discrepancy, although it is clear that n 0
there are fundamental differences between these organisms, 4 A
cellsdevoidofbothY-familyDNApolymerases(40,41).Itis since imuA orthologs are not present in M.tuberculosis and p
interestingtonotethatnootherproteinbelongingtotheUmuC relatedbacteria.Inaddition,theuseoftherpoBgeneastarget ril 2
0
superfamily can be found in the C.crescentus genome, and for mutational analysis has intrinsic limitations, such as the 1
9
neither there are polB orthologs. Thus, imuAB/dnaE2- underestimation of frameshifts and the limited number of
independent UV mutagenesis might occur via a still unchar- mutations detectable as gain of function alleles for Rifr.
acterized pathway. Recently, it has been suggested that the The development of more sophisticated genetic tools for the
dnaE gene of C.crescentus may also be a part of the SOS studyofmutagenesisismandatoryforadeeperunderstanding
regulon (42). Overexpression of the replicative polymerase ofthebasicmechanismsofthisaspectofDNAmetabolismin
encoded by dnaE may account for the imuAB/dnaE2- C.crescentus.
independentUVmutagenesis.Ontheotherhand,mitCmuta- The DNA polymerase III holoenzyme is responsible for
genesis shows an absolute dependence on the activity of the chromosomalreplication.Thea-subunitofDNApolymerase
imuAB dnaE2 operon, indicating that DnaE and the ImuAB III, encoded by the dnaE gene, is essential in E.coli (43). In
DnaE2machinerymayhavedifferentlesionbypasscapacities. C.crescentus, the involvement of the dnaE gene (ORF
ThemutationspectraofUV-inducedlesionsinC.crescentus CC1926) in DNA replication has also been demonstrated
showadramaticqualitativedifferencebetweenwild-typeand previously (31). However, in M.tuberculosis, the deletion of
imuBordnaE2strains.G:C!A:Ttransitions,ahallmarkof dnaE2 causes no growth defect (14), similar to what was
UVmutagenesisinE.coli,areobservedinthemutantstrains, observed for C.crescentus. It was demonstrated years ago