The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach

The functions of DNA methylation by CcrM inCaulobacter crescentus a global approachDiego Gonzalez1 Jennifer B Kozdon23 Harley H McAdams2 Lucy Shapiro2 and

Justine Collier1

1Department of Fundamental Microbiology University of Lausanne Lausanne CH 1015 Switzerland2Department of Developmental Biology Stanford University CA 94305 USA and 3Department of ChemistryStanford University CA 94305 USA

Received October 7 2013 Revised December 3 2013 Accepted December 5 2013

ABSTRACT

DNA methylation is involved in a diversity ofprocesses in bacteria including maintenanceof genome integrity and regulation of geneexpression Here using Caulobacter crescentus asa model we exploit genome-wide experimentalmethods to uncover the functions of CcrM aDNA methyltransferase conserved in mostAlphaproteobacteria Using single moleculesequencing we provide evidence that most CcrMtarget motifs (GANTC) switch from a fullymethylated to a hemi-methylated state when theyare replicated and back to a fully methylated stateat the onset of cell division We show that DNAmethylation by CcrM is not required for the controlof the initiation of chromosome replication or forDNA mismatch repair By contrast our transcrip-tome analysis shows that gt10 of the genes aremisexpressed in cells lacking or constitutivelyover-expressing CcrM Strikingly GANTC methyla-tion is needed for the efficient transcription ofdozens of genes that are essential for cell cycleprogression in particular for DNA metabolism andcell division Many of them are controlled by pro-moters methylated by CcrM and co-regulated byother global cell cycle regulators demonstratingan extensive cross talk between DNA methylationand the complex regulatory network that controlsthe cell cycle of C crescentus and presumably ofmany other Alphaproteobacteria

INTRODUCTION

Methylated bases can be found in the genomes of organ-isms from all three domains of life as well as in the genome

of viruses (1ndash4) Aberrant cytosine methylation isassociated with a variety of human diseases includingneurospychiatric disorders and cancers exemplifying thecritical functions of cytosine methylation largely throughits effects on the regulation of gene expression in highermetazoans (5) The functions of DNA methylation havebeen poorly investigated for most of the bacterial kingdom(6) Bacterial DNA methyltransferases are mostlyassociated with endonucleases in restriction-modificationsystems which are generally considered as a defensemechanism that bacteria use to identify and destroy dif-ferentially methylated foreign DNA (7ndash9) A number oflsquoorphanrsquo DNA methyltransferases that are not associatedwith a cognate endonuclease have also been identified(710ndash12) The best studied examples are the DNAadenine methyltransferases Dam and CcrM Dam methy-lates 50-GATC-30 (hereafter called GATC) motifs inthe genomes of a subset of Gammaproteobacteria (13)whereas CcrM methylates 50-GANTC-30 (hereaftercalled GANTC) motifs in the genomes of manyAlphaproteobacteria

Dam has pleiotropic functions in Gammaproteobacteriait is involved in the control of the initiation of chromo-some replication (14ndash16) in the DNA mismatch repair(MMR) process (1718) and in the regulation of gene ex-pression (710) DNA microarrays were used to compareglobal transcription patterns in wild type dam andDam-over-expressing Escherichia coli cells (19ndash22) Thesestudies revealed that hundreds of genes were misregulatedin these two mutant strains Similar effects were observedin Salmonella enterica (23) In addition detailed studies inE coli and S enterica have led to the identification of anumber of genes that show a Dam-dependent transcrip-tional phase variation creating heterogeneity of expres-sion rates within the bacterial population (1124) Suchmechanisms of regulation often involve transcriptionfactors that compete with Dam for DNA binding andwhose binding affinity is sensitive to the methylation

To whom correspondence should be addressed Tel +41 21 692 56 10 Fax +41 21 692 56 05 Email justinecollierunilch

3720ndash3735 Nucleic Acids Research 2014 Vol 42 No 6 Published online 7 January 2014doi101093nargkt1352

The Author(s) 2014 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby30) whichpermits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited

state of the DNA at or near their binding site In somecases the presence of one or more under-methylatedGATC motifs in a promoter is an indication that it isregulated by such a bistable epigenetic switch althoughthere also exists under-methylated GATC motifs that arenot part of epigenetic mechanisms regulating gene expres-sion (2526) Dam is to date the only DNA adeninemethyltransferase whose global effects on gene expressionhave been studied in bacteria

The cell cyclendashregulated methyltransferase CcrMwas first identified in Caulobacter crescentus anAlphaproteobacterium that divides asymmetrically intotwo morphologically different progeny cells thedaughter stalked cell that immediately initiates DNA rep-lication and cell division and the daughter swarmer cellthat first differentiates into a stalked cell before replicationand cell division (Figure 1) (2728) This bacterium initi-ates the replication of its chromosome only once per cellcycle and specifically in the stalked progeny or during theswarmer-to-stalked cell transition (2930) The expressionof CcrM is restricted to the late predivisional stage nearthe end of chromosome replication which implies thatGANTC motifs on the chromosome are supposedlyfully methylated (methylated on both DNA strands) inswarmer cells and then become hemi-methylated(methylated on one strand only) on DNA replication instalked cells and stay so until the end of the replication ofthe chromosome in pre-divisional cells (Figure 1) (30ndash32)Maintaining the chromosome constitutively fullymethylated by over-expressing CcrM leads to morpho-logical and cell cyclendashrelated defects in C crescentus sug-gesting that the temporal regulation of the transition fromthe fully methylated to the hemi-methylated state has bio-logical importance (3133) In C crescentus the methyla-tion state of GANTC motifs has a direct effect on thetranscription of at least four genes the ctrA and dnaAgenes encoding master regulators of the cell cycle(3435) the ftsZ and mipZ genes required for celldivision (36) and most probably the ccrM gene itself(37) The transition from the fully methylated to thehemi-methylated state of a GANTC motif located in cisis sufficient to cause a small but significant change in thetranscriptional activity of the ctrA dnaA and ftsZ pro-moters This supports a model according to which thechange in GANTC methylation state during DNA repli-cation might function to co-ordinate gene expressionchanges with the progression of the replication forksetting the mechanical basis for the regulation ofthe timing of multiple cell cycle events (323435) Werecently showed that an increase in ftsZ transcription issufficient to restore the viability of ccrM cells in richmedium demonstrating that the direct activation of ftsZtranscription by DNA methylation is one of the most im-portant functions of CcrM in C crescentus (36) Theserescued cells still exhibited a residual phenotype Forexample cells were elongated and straighter thanwild-type cells and had shorter stalks These observa-tions suggested that more genes are probably regulatedby CcrM-dependent methylation in C crescentusInterestingly the ccrM gene is not essential anymore forthe viability of C crescentus when cells are cultivated in

slow-growing conditions such as minimal medium thesecells are nevertheless slightly elongated and grow slightlymore slowly than wild-type cells (36) The physiologicalimportance of GANTC methylation is probably notrestricted to C crescentus as the homolog of ccrM wasshown to be essential for viability in fast-growing culturesof each of the species where this was tested (333638ndash40)Here we assess the global influence of GANTC methy-

lation on transcription profiles in C crescentus byanalyzing the transcriptome of a ccrM strain and of astrain in which CcrM is constitutively expressed so thatthe chromosome remains in the fully methylated statethroughout the cell cycle We show that hundreds ofgenes are differentially expressed in both mutant strainscompared with the wild type Genes whose transcriptioncould directly be affected by GANTC methylation arepredicted on the basis of their differential expression inthe mutant strains and through bioinformatic analysis ofGANTC conservation in promoters Our findings reveal astrong link between CcrM-dependent methylation and cellcycle control in C crescentus that is also likely to exist inmany other Alphaproteobacteria

MATERIALS AND METHODS

Phylogenetic analysis

NCBI whole bacterial proteomes were searched forproteins with homology to the CcrM protein fromC crescentus strain CB15 (blastp e-valuelt 1e-10) (41)one strain was selected per genus and the protein with thelowest blastp e-value was used for each individual strainOn the basis of a MUSCLE alignment of the sequences(4243) the subtype b N6-adenine-methyltransferases wereselected using the motif IV [DPPY 6frac14 SPP(YF)] and themotif X [T(QED)KP 6frac14AXFP] to discriminate between

Figure 1 CcrM is a cell cyclendashregulated DNA methyltransferase inC crescentus Upper panel schematic of the C crescentus cell cycleshowing the differentiation of a swarmer cell (SW) into a stalked cell(ST) which then turns into a pre-divisional cell (PD) Lower panelschematic showing the changes in the methylation state of GANTCmotifs on the chromosome as a function of chromosome replicationand cell cycle progression

Nucleic Acids Research 2014 Vol 42 No 6 3721

N6-methyltransferases and N4-methyltransferases (44)A tree was calculated with FastTree (4546) using theWAG matrix for amino-acid substitutions to get ageneral idea of the topology (Supplementary Figure S1B)Our final set comprised all sequences of CcrM-homologswhich had a conserved C-terminal domain ie sequencesfrom Alphaproteobacteria Helicobacter and someChloroflexi with the addition of Haemophilus influenzaeHinfI [from REBASE (8)] on the basis of the FastTreetree two sequences were added as outgroups (fromStreptococcus mitis and Stigmatella aurantiaca) A newalignment was made with MUSCLE and curated withGBlocks 091b (47) with default options and gaps allowedat all positions The final trees were calculated on the basisof the curated alignment using RAxML (48) or MrBayesversion 321 with 200 000 iterations and 2 4 chains(4950) with the WAG matrix for amino-acid substitutionand the Gamma model of substitution rate heterogeneityBranches with support values lt05 were left unresolved(Supplementary Figure S1C)

Distribution of pentanucleotides in the genomes ofAlphaproteobacteria

Bacterial genome sequences were downloaded fromthe NCBI repository GANTC motifs and otherpentanucleotides were counted in the complete genomesequences (fna file) in the protein coding sequences (ffnfile) and in the RNA coding sequences (frn file) Thenumber of GANTC motifs and other pentanucleotidesin intergenic sequences was calculated as followsnumber in the genome (number in protein codingsequences+number in RNA coding sequences) Thefrequency of each nucleotide in each complete genomesequence was calculated as the ratio between the numberof each nucleotide and the total number of nucleotides ofthe whole genome The expected number of a givenpentanucleotide with a central variable N in a genomewas calculated as freq(A)freq(C)freq(G)freq(A)(genome length) where freq(A) is the frequency of As inthe genome for example and lsquogenome lengthrsquo is thetotal number of nucleotides in the considered genomeThe expected number of GANTC motifs and otherpentanucleotides in protein coding RNA coding andnon-coding sequences was calculated as (subsequencelengthgenome sequence length)(total pentanucleotidenumber) where lsquosubsequence lengthrsquo is the length of theprotein coding RNA coding and non-coding sequencesrespectively and the total pentanucleotide number is thetotal number of a given pentanucleotide found in the con-sidered genome As lsquoOther Proteobacteriarsquo we used thegenomes of the following Neisseria gonorrhoeae FA1090Burkholderia mallei ATCC 23344 Nitrosomonas europaeaATCC 19718 Myxococcus xanthus DK1622 Bdellovibriobacteriovorus HD100 Desulfovibrio vulgaris DP4Helicobacter pylori F32 Nautilia profundicola AmHSulfurospirillum deleyianum DSM 6946 E coli str K-12substr DH10B Pseudomonas aeruginosa PAO1 Vibriocholerae O1 biovar El Tor str N16961

Growth conditions and bacterial strains

Three C crescentus strains were used in this study(i) Strain NA1000 (CB15N) is a synchronizable derivativeof the wild-type CB15 strain (5152) (ii) Strain JC362 (53)is a derivative of the NA1000 strain expressing a secondcopy of the ccrM gene under the control of the endogen-ous lacZ promoter (PlacccrM) which is constitutivelyactive (31) and (iii) Strain JC1149 (36) is a derivativeof the NA1000 strain with a deletion of the ccrMgene (ccrM) Cells were cultivated in M2G minimalmedium at 28

C Experiments were all performedusing unsynchronized cell populations because well-synchronized JC1149 populations could not be isolatedprobably due to the slightly abnormal morphology of theccrM cells (36)

Single molecule real time sequencing analysis

Genomic DNA was extracted from cells cultivated toexponential phase using the Qiagen Puregene YeastBactDNA extraction kit with a second ethanol precipitationstep after resuspension the DNA was finally dissolved in10mM TrisndashHCl pH 8 For each DNA sample SingleMolecule Real Time (SMRT) Bell libraries with a 2-kbinsert size were sequenced in 6 SMRT cells and thedata were analyzed with tools provided in the PacificBiosciences SMRT portal using the NC_011916 NA1000genome (NCBI) as a reference Mean depth coverage was182 for strain NA1000 187 for strain JC362 and 37 forstrain JC1149 SMRT confidence scores defined as -10log(p-value) for methylated adenines were typicallyhigh in GANTC motifs for the NA1000 and JC362strains (median is 115 and 145 respectively) and low forthe JC1149 strain (median is 3) For adenines in all GANTCmotifs in the chromosome the inter-pulse duration (IPD)ratio was calculated by dividing the average measured IPDfor the nucleotide by the IPD value predicted in an in silicomodel for non-methylated DNA IPD ratios werecalculated for all adenines in GANTC contexts on bothDNA strands We used the average IPD ratio foradenines at GANTC motifs in the chromosome of strainJC362 to approximate the value for adenines fullymethylated in chromosomes in the whole population(=768) the theoretical average IPD ratio for adenineshemi-methylated in chromosomes in the whole populationwould then be (768+1)2=434 An IPD of 24 corres-ponding to an adenine fully methylated in chromosomesfrom 20 of the population or hemi-methylated in 40of the population was chosen as a threshold to consider thata GANTC motif was under-methylated

Preparation of RNA samples for transcriptome orqRT-PCR analysis

All strains were cultivated in triplicates and independentcultures (NA1000 versus JC1149) or independent biolo-gical samples (NA1000 versus JC362) were used forsubsequent transcriptome and quantitative real-timepolymerase chain reaction (qRT-PCR) analysis Twomilliliters of cell culture aliquots were pelleted frozen inliquid nitrogen and stored at 80C Pellets were

3722 Nucleic Acids Research 2014 Vol 42 No 6

homogenized in 1ml of Trizol reagent and incubated at65C for 10 min Two hundred microliters of chloroformwere added and after 3min at room temperature theextracts were centrifuged for 15min at maximum speedFive hundred fifty microliters of 100 ethanol were addedto 550 ml of aqueous phase from the Trizol treatment andRNA was purified using the Ambion PureLink RNAmini-kit (standard protocol without the lysis step) RNAwas eluted in 40 ml of H2O treated in a 60-ml reaction with10 ml of Invitrogen DNase I Amp Grade for 30min atroom temperature

Transcriptome analysis

Custom microarrays were designed by Nimblegen usingC crescentus NA1000 protein and small RNA coding se-quences When possible three different probes of 60 bpwere designed per gene Microarrays had a 4-plex format(four sub-arrays per chip) each probe was printed inrandomly positioned triplicates in each of the sub-arrays One sub-array was used per biological sampleRNA samples were prepared as described above and 6 mlof EDTA 25mM (Invitrogen) were added for a 10-minincubation at 65C to inactivate the DNase I Sixty micro-liters of lysis buffer from the PureLink RNA kit with 12-mercaptoethanol and 60 ml of 100 ethanol were addedto the reaction and the RNA was purified again using thePureLink standard protocol (without the lysis step) TheRNA was resuspended in 32 ml from which 2 ml were usedfor quality check via Bioanalyzer and quantification withNanodrop cDNA was synthetized from 5 mg of RNAusing the Roche double-stranded cDNA synthesissystem (cat 11011708310001) and the protocol describedin the lsquoRoche cDNA Synthesis System for use withNimbleGen Gene Expression Microarraysrsquo technicalnote Promega random hexamers at the same concentra-tion were used instead of the oligo(dT)15 The double-stranded cDNA was precipitated with isopropanolpurified and resuspended in 40 ml according to theprotocol The whole purified double-stranded cDNA(05ndash1 mg cDNA) was used as input for the standardKlenow-based NimbleGen Cy3 labelling protocol Twomicrograms of labelled cDNA were hybridized 16 h inone sub-array using the standard Nimblegen protocolfor assembly hybridization and washing Arrays werescanned in an Agilent High-Resolution Microarrayscanner at 2 mm resolution Normalized intensities wereextracted from the pictures with the Nimblescan 26software using the standard workflow and defaultoptions The Robust Multi-array Average (RMA)module of the software was used to normalize the intensityvalues for all chips The statistical analysis lsquomutant versuswild typersquo was performed with the R limma package onthe basis of the intensity values of all biological replicatesfor the mutant and wild-type strains Genes with anadjusted Plt 001 were considered significantly up- ordownregulated The logFC value given by the limmafunction was used as the log-ratio shown in Tables

Flow cytometry analysis methods to determine spon-taneous mutation rates and methods for qRT-PCRanalysis are described in Supplementary Information

RESULTS

CcrM homologs are conserved in mostAlphaproteobacteria

The functions of the CcrM family of N6-adeninemethyltransferases have been studied in a limitednumber of Alphaproteobacteria including C crescentusAgrobacterium tumefaciens Rhizobium meliloti andBrucella abortus (3338ndash40) To assess the phylogeneticconservation of CcrM homologs beyond these speciesand more thoroughly than before (54) we searched forproteins with a significant similarity to the C crescentusCcrM protein in all available bacterialproteomes (Supplementary Table S1) We found thatall Alphaproteobacteria except Rickettsiales andMagnetococcales (55) had a CcrM homolog with a highsimilarity score (Figure 2A) As the target recognitiondomain of CcrM is conserved in each of these proteinsthese enzymes most likely also methylate the adenine inGANTC motifs The only exceptions were the bacteriabelonging to the SAR11 clade (eg Pelagibacter ubiqueand related species) that do have a CcrM homologalthough they have sometimes been classified asRickettsiales (5657) and the cicada endosymbiontCandidatus Hodgkinia cicadicola Dsem (Rhizobiale)whose genome does not encode a CcrM homologOutside Alphaproteobacteria the genomes of sporadicspecies and clades across the phylogeny encode a proteinclosely related to CcrM ccrM homologs were onlyfound in close proximity to an endonuclease gene ona chromosome in several Epsilonproteobacteria orGammaproteobacteria (Supplementary Table S2) suggest-ing that CcrM homologs are orphan N6-adeninemethyltransferases in all AlphaproteobacteriaA phylogenetic tree was built using the sequences

of proteins with a high similarity to the C crescentusCcrM protein belonging to the b-class of N6-adeninemethyltransferases and comprising a conservedadditional C-terminal domain specific to the CcrMfamily (Supplementary Figure S1) The most parsimoni-ous scenario compatible with the phylogenetic tree is thatthe gene encoding the ancestor of the CcrM homologentered the genome of an ancestral Alphaproteobacteriumafter the divergence of Magnetoccocales and Rickettsialesand that it was thereafter transmitted vertically to the des-cendent species (Figure 2A) The pervasiveness of verticaltransmission suggests that the CcrM homolog is part ofthe core genome and plays a critical physiological role inthe large and diverse terminal clade of the alphaproteo-bacterial tree

GANTC motifs are under-represented in the genomesof Alphaproteobacteria but over-represented inintergenic regions

Previous studies indicated that GANTC motifs are lessfrequent in the genome of C crescentus than expected ina random sequence of nucleotides and that these motifsare over-represented in intergenic regions these biasesmight be connected to the biological role that DNAmethylation plays in controlling transcription or other


cellular processes in C crescentus (355859) To explorethis possibility more systematically we analyzed thefrequency and distribution of GANTC motifs in all avail-able genomes of Alphaproteobacteria and of a control setof other Proteobacteria We found that GANTC motifswere on average at least 2-fold less frequent than expectedin the genomes of all orders of Alphaproteobacteriaincluding Magnetococcales but excluding Rickettsiales(Figure 2B) In the group of other Proteobacteria this

bias was largely attenuated Notably we also observedthat GANTC motifs showed a distribution bias betweenprotein coding and intergenic sequences in each ordercomprising bacterial species encoding a CcrM homolog(Figure 2C and D) being on average gt15-fold over-represented in intergenic regions and slightly under-represented in coding regions These biases were largelyattenuated in the group of other Proteobacteria lacking aCcrM homolog Overall these findings suggest that

Figure 2 Conservation of CcrM in Alphaproteobacteria and distribution of GANTC motifs in their genomes (A) Schematic showing the conser-vation of CcrM in different orders of Alphaproteobacteria Panels (B) (C) and (D) show the observed-over-expected GANTC ratios using the wholegenome (B) the intergenic sequences (C) and the coding sequences (D) of sequenced bacterial species from different orders of AlphaproteobacterialsquoOther Proteobacteriarsquo correspond to a control of 10 mixed other Proteobacteria The expected number of motifs was calculated for each genometaking the nucleotide composition into account The limits of the box represent the first and third quartiles and the bold line the median of thedistribution In the whole figure orders of bacterial species containing a CcrM homolog are indicated in red


selective pressure limits the overall frequency of GANTCmotifs in protein coding sequences on the genomes ofbacteria encoding a CcrM homolog whereas they aretolerated or favoured in intergenic sequences

To check whether these biases were specific to GANTCmotifs we also analyzed the frequency and distribution ofthe 24 pentanucleotides of the same structure as GANTC(each of the four bases present one time one centralvariable N) (Supplementary Figure S2) Surprisinglyanother one of these pentanucleotides CTNAG pre-sented similar strong frequency biases not only inAlphaproteobacteria encoding CcrM homologs butalso in the control group of other Proteobacteria(Supplementary Figure S3) Thus a bias in the frequencyand distribution of pentanucleotides of that structure canbe found in bacterial genomes independently of theircapacity to be methylated by CcrM homologs indicatingthat the biased distribution of GANTC motifs in thegenomes of CcrM-encoding Alphaproteobacteria mightalso be explained by other factors

The methylation of GANTC motifs in the genome ofC crescentus is strictly dependent on CcrM

The temporal regulation of the methylation ofGANTC motifs seems important in a variety ofAlphaproteobacteria since cells that constitutivelyexpress homologs of CcrM often appear abnormal(3134394060) A system of DNA methylation probesdistributed at a dozen positions along the C crescentuschromosome previously suggested that newly replicatedGANTC motifs stay hemi-methylated until the pre-divisional stage of the C crescentus cell cycle independ-ently of the moment when they got replicated due to thestrict temporal regulation of CcrM concentration duringthe cell cycle (3031) According to this model GANTCmotifs located close to the chromosomal origin shouldstay hemi-methylated for a longer period during the cellcycle than motifs located close to the chromosomalterminus as they are replicated earlier during the cellcycle To confirm this model for each of the GANTCmotifs on the C crescentus chromosome we made useof the recently developed SMRT sequencing technologyof PacBio on genomic DNA extracted from wild-typeccrM (36) or CcrM-over-expressing (31) cells cultivatedto exponential phase This technology measures the timeneeded for a DNA polymerase to incorporate a nucleotideon a template DNA (IPD) which varies depending on themethylation state of the nucleotide found on the templateDNA (61) This method is powerful at detectingN6-methyladenines on bacterial chromosomes (6263)and we used it to estimate the proportion of cells in thebacterial population in which a given GANTC motif ismethylated on either strand We observed a positivecorrelation between the proximity of a GANTC motif tothe chromosomal origin and its probability to be hemi-methylated in a mixed population of the wild-type strain(Figure 3A) We estimated that motifs located close to thechromosomal origin were on average hemi-methylated in80 of the cells while sites located close to the chromo-somal terminus were hemi-methylated in lt10 of the

cells In the ccrM population we found that none ofthe GANTC motifs were fully methylated (Figure 3C)confirming that the methylation of each GANTC motifon the C crescentus chromosome is dependent on CcrMIn the CcrM-over-expressing population we observed thatnearly all GANTC motifs were fully methylated in thewhole population (Figure 3B) showing that the fullymethylated to hemi-methylated switch that takes placefor most of the GANTC motifs on the C crescentuschromosome is dependent on the precise temporal regula-tion of the concentration of CcrM during the cell cycleThese results confirmed at the genomic scale and in aquantitative manner the model previously proposed byZweiger et al for most GANTC motifs (31)Interestingly through our SMRT sequencing analysis

we found 35 exceptions to this rule 24 GANTC motifswere strongly under-methylated and 11 additionalGANTC motifs were asymmetrically methylated (onestrand being more often under-methylated than theother one) in a population of unsynchronized wild-typeC crescentus cells (Supplementary Table S3) A recentstudy using a synchronized population of C crescentuscells demonstrated that most of these motifs remain

Figure 3 CcrM-dependent methylation of GANTC motifs in thegenome of C crescentus Predicted methylation state of the DNA as of the chromosomal population being fully methylated at a particu-lar locus along the chromosome of C crescentus (A) NA1000(B) PlacccrM [JC362] (C) ccrM [JC1149]) We used the SMRTportal to detect methylated motifs in the chromosome of C crescentusand extracted the IPD ratios for adenines in all GANTC motifs (bothstrands) We then calculated the average IPD ratio in 20 kb windowsalong the chromosome and plotted the values according to the chromo-somal co-ordinates (the origin of replication is situated at the junctionbetween 4000 kb and 1 kb) The average IPD ratio for strain JC362 wasequaled to 100 and the theoretical value for hemi-methylated DNA[(value for fully methylated DNA+1)2] as 0 the curve was fittedwith the R loess function


under-methylated throughout the whole cell cycle (64)Here we show that only three of these motifs remainedunder-methylated in cells over-expressing CcrM while theother 32 GANTC motifs were efficiently methylated onboth DNA strandsOverall these results demonstrate that the ccrM

and CcrM-over-expressing strains are accurate tools forexploring the functions of GANTC methylation inC crescentus and the functions of the methylationswitch that occurs at most GANTC motifs over thecourse of the cell cycle of C crescentus

Control of chromosome replication and spontaneousmutation rate in the absence of GANTC methylationin C crescentus

The orphan adenine methyltransferase Dam was shown tobe required for the replication of one of the two chromo-somes in V cholerae (1516) and to be involved in thecontrol of the initiation of chromosome replication inE coli (1465) Several GANTC motifs can be found inthe origin of replication of C crescentus and of otherclosely related species (66) and it was previously shownthat cells over-expressing CcrM accumulate additionalchromosomal copies (31) We thus hypothesized thatCcrM might be required for the regulation of chromosomereplication in C crescentus ensuring that it occurs onlyonce per cell cycle (3031) To test this hypothesis we usedflow cytometry to compare the DNA content of cells inwild-type and ccrM populations treated with rifampicinRifampicin blocks the initiation of DNA replication butnot the elongation of DNA replication so that wild-typeC crescentus cells accumulate either one or two completechromosomes after rifampicin treatment (SupplementaryFigure S4A) We observed that ccrM populationscultivated in minimal medium had a DNA contentprofile similar to wild-type populations (SupplementaryFigure S4B) We concluded that CcrM is neitherrequired for the control of the initiation of chromosomereplication nor for the correct segregation of replicatingchromosomes before cell division unlike Dam in severalenterobacteria (714)In addition to its regulatory roles Dam plays an

important role in DNA MMR in a subset ofGammaproteobacteria (1718) the hemi-methylated stateof GATC motifs behind the replication fork providescritical information to identify the newly replicatedstrand more likely to contain errors that need to berepaired (18) This raised the possibility that the methyla-tion of GANTC motifs by CcrM may similarly be used forDNA repair in C crescentus We therefore compared thespontaneous mutation rates of the wild-type and ccrMstrains by calculating the frequency of rifampicin resistantmutants arising in each population and found that theywere similar (Supplementary Figure S5) This result showsthat methylation of GANTC motifs by CcrM is notneeded for the MMR mechanism in C crescentusCumulatively these observations indicate that CcrM in

Alphaproteobacteria does not share the most conservedfunctions of Dam in Gammaproteobacteria This is con-sistent with the different evolutionary histories of the two

proteins Furthermore we provide evidence below that theimpact of CcrM-dependent methylation on the regulationof gene expression in C crescentus also differs fromthe known impact of Dam-dependent methylation inE coli (19ndash21)

Alterations in gene expression in the absence of GANTCmethylation in C crescentus

To evaluate the impact of CcrM-dependent DNA methy-lation on global gene expression profiles in C crescentuswe used custom-made DNA microarrays to compare thetranscription levels in wild-type and ccrM populationscultivated to exponential phase in minimal medium Wefound that out of the 3932 genes in the C crescentusNA1000 genome for which probes were designed theexpression levels of 388 genes were significantly (correctedStudentrsquos test Plt 001) changed in the ccrM straincompared with the wild-type strain with 152 of the 388genes being affected gt2-fold (Supplementary Table S4)For 17 genes the effects were verified by qRT-PCR andwere essentially consistent with the microarray results(Supplementary Figure S6 and Table 1) We classifiedeach gene strongly misregulated in ccrM cells accordingto the known or predicted function of the protein that itencodes (NCBI single letter COG) (Figure 4A) We foundthat genes encoding proteins involved in DNA replicationrecombination or repair were significantly enriched amongthe genes that were upregulated in the absence of CcrMwhile genes encoding proteins involved in cell motilitywere significantly enriched among the genes that weredownregulated in the absence of CcrM (Figure 4A)

A subset of the transcriptional effects observed in theccrM strain was probably directly due to the absence ofmethylation at promoter regions containing GANTCmotifs (140 misregulated genes contained minimum oneGANTC motif in their promoter region) To assesswhether the transcriptome analysis was likely to revealsuch direct effects of GANTC methylation on transcrip-tion we calculated the probability that significantlymisregulated genes contained a GANTC motif in theirpromoter region Because many cell cyclendashregulatedgenes are controlled by relatively long promoter regionsin C crescentus (67) we looked for the presence ofGANTC motifs up to 200 bp upstream of the annotatedtranslational start codon of each gene We found that theprobability to find a GANTC motif was 18-fold higherthan in a random set of genes (Fisherrsquos exact test Plt 005)(Figure 5A) This value rose to 25-fold when consider-ing only the genes misregulated gt2-fold (Fisherrsquos exacttest Plt 005) There was a clear enrichment in genes con-taining a GANTC motif in their promoter region amongthe genes that were the most significantly up- ordownregulated in the ccrM strain (Figure 5B and C)The conservation of a GANTC motif in the promoterregions of homologous genes in closely related bacteriahas been previously used as an indicator that the methy-lation of the motif may play a role in regulating theactivity of the promoter (36) We thus asked whethergenes strongly misregulated in the ccrM strain had ahigher probability of containing a lsquoconservedrsquo GANTC


Table

1Essentialgenes

containingGANTC

motifs

intheirpromoterregionandsignificantlymisregulatedin

ccrM

cellscomparedwithwild-typeCcrescentuscells

Genename

Knownorpredictedfunction

Log-ratio

MA

Log-ratio

qRT-PCR

CtrA

regulon

DnaA

regulon

GcrA

regulon

GANTC

conservation

Essentialgenes

repressed

inccrM

CCNA_00390

ADP-heptosemdash

LPSheptosyltransferase

085

072

No

No

No

4CCNA_01104

Glycosyltransferase

077

ND

No

No

No

2CCNA_01275

Methylenetetrahydrofolate

dehydrogenase

(NADP+

)methenyltetrahydrofolate

cyclohydrolase

084

ND

No

No

No

1

CCNA_01450

Single-stranded

DNA-specificexonuclease

RecJ

103

120

No

No

Yes

4CCNA_01535

Single-stranded

DNA

bindingprotein

056

ND

No

Yes

No

2CCNA_01590

NAD-dependentDNA

ligase

123

ND

No

No

No

3CCNA_01651

DNA

gyrase

subunit

A114

101

No

No

Yes

5CCNA_01776

Ribonuclease

D068

092

No

No

No

5CCNA_02052

Topoisomerase

IVsubunit

B197

ND

No

No

Yes

3CCNA_02086

Celldivisionprotein

FtsN

109

ND

No

No

Yes

5CCNA_02127

Oxacillin

resistance-associatedprotein

FmtC

193

ND

No

No

Yes

2CCNA_02208

Thymidylate

synthase

103

ND

No

No

No

3CCNA_02246

DivisionplanepositioningATPase

MipZ

052a

254

Yes

Yes

Yes

5CCNA_02256

Mg2+

transporter

MgtE

083

114

No

No

No

5CCNA_02389

Glucosamine-1-phosphate

acetyltransferase

UDP-N

-acetylglucosaminepyrophosphorylase

087

069

No

No

No

5

CCNA_02623

Celldivisionprotein

FtsZ

144

175

Yes

Yes

No

5CCNA_02679

Hypotheticalprotein

107

ND

No

No

No

3CCNA_03607

Ribonucleoside-diphosphate

reductase

subunit

alphaNrdA

047

077

No

Yes

No

5

Essentialgenes

activatedin

ccrM

CCNA_00379

Thioldisulfideinterchangeprotein

DsbA

069

053

No

No

No

5CCNA_00845

Antitoxin

protein

RelB-1

082

ND

No

No

No

1CCNA_00893

Transcriptionalregulatory

protein

053

ND

No

No

No

0CCNA_01946

Tyrosyl-tR

NA

synthetase

089

028

No

No

No

4CCNA_02635

Celldivisionprotein

FtsW

065

ND

Yes

No

No

2CCNA_03130

Cellcycleresponse

regulatorCtrA

046

034

Yes

No

Yes

5CCNA_03471

4-hydroxy-3-m

ethylbut-2-enyldiphosphate

reductase

074

023

No

No

No

4CCNA_03766

16SrR

NA

processingprotein

Rim

M111

ND

No

No

No

3CCNA_03820

Outer-mem

branelipoproteinscarrierprotein

109

ND

No

No

No

3

Genes

wereconsidered

essentialaccordingto

(67)

Thelog-ratiosindicate

theexpressionchangeobserved

usingmicroarrays(M

A)orqRT-PCR

comparingwild-typeand

ccrM

cells

ND

indicatesthatthelog-ratiowasnotmeasuredbyqRT-PCR

forthatgene

aIndicatesthatunspecificlow-signalprobes

wereunfortunately

designed

todetectmipZ

bymicroarrays

TheCtrA

andDnaA

directregulonsweredescribed

in(68)and(69)respectivelyThe

GcrA

regulonwasdescribed

in(70)ThelsquoG

ANTC

conservationrsquocolumnindicatesthenumber

ofspeciesin

whichaGANTC

motifis

foundin

thepromoterregion(200bpupstream

oftheir

translationalstart

codon)ofhomologousgenes

amongfivebacterialspeciesclosely

relatedto

Ccrescentus(C

crescentusCsegnisCK31Phenylobacter

zucineumBrevundim

onassubvibrioides)


motif compared with a random set of genes We foundthat genes containing a conserved GANTC motif weremore frequent among the misregulated genes thanexpected randomly Genes containing a GANTC motifthat was conserved in seven closely related species werefor example five times more frequent among the mostdownregulated genes than expected randomly (Fisherrsquosexact test Plt 005) (Figure 5D) This result indicatesthat promoters that contain conserved GANTC motifsare more likely to be affected by the absence of CcrMThus we predict that genes that are significantlymisregulated in the ccrM strain and that are under thecontrol of a promoter region that contains a conservedGANTC motif have a high probability of being dir-ectly regulated by the methylation state of theirpromoter region (Supplementary Table S4) The ctrAftsZ and mipZ genes which were shown to be directlyregulated by DNA methylation (3536) fit these criteria(Table 1) Additional interesting candidates from ourstudy include for example CCNA_01450 (recJ)CCNA_01651 (gyrA) CCNA_02283 (annotated as anendonuclease) CCNA_02389 (UDP-N-acetylglucosaminepyrophosphorylase) and CCNA_02726 (annotated as anacetyltransferase) (Supplementary Table S4)

To evaluate the major physiological consequences of thetranscriptional changes observed in the absence of CcrM-mediated methylation we first examined the set of genesthat were reported to be essential for the viability ofC crescentus (67) and that were significantly (correctedStudentrsquos test Plt 001) downregulated we found that 31essential genes (Supplementary Table S4) including fourgenes encoding cell division proteins (FtsZ MipZ FtsNand FtsE ftsX encoding FtsX is co-transcribed with ftsEand misregulated at Plt 005) and seven genes encodingproteins involved in DNA replication or repair or in themaintenance of chromosome topology (Ssb RecJ ligaseGyrA Topo IV thymidylate synthase and NrdA) Eachof these 18 essential genes contained a relatively conservedGANTC motif in their promoter region (Table 1) suggest-ing that their expression might be directly activated byCcrM-mediated DNA methylation We also observedthat genes belonging to the GcrA (70) DnaA (69) andCtrA (68) regulons were strongly enriched among themost significantly downregulated genes in the ccrMstrain (Figures 6AndashC) with the GcrA regulon being themost striking case (10-fold enrichment when consideringgenes whose transcription is changed at least 2-fold in theccrM strain) Genes whose expression is temporally

Figure 4 Relative frequency of functional categories among genes found as strongly misregulated during the transcriptome analysis Genes whoseexpression changedgt 2-fold in the ccrM (A) and the CcrM-over-expressing (B) strains were selected Blue and yellow bars represent the set of geneswhose expression is downregulated and upregulated respectively in the corresponding strain Stars dark blue and bright yellow colours indicate asignificant over-representation (Plt 005 Fisherrsquos exact test)


regulated during the cell cycle (71) were also significantlyenriched (Figure 6D) Altogether these observationsindicate a strong connection between DNA methylationby CcrM and cell cycle control considering that GcrADnaA and CtrA are essential master transcriptional regu-lators of the C crescentus cell cycle and that many essen-tial genes might be directly regulated by DNA methylation(Table 1)

Effect of the constitutive over-expression of CcrM onglobal transcription profiles in C crescentus

To evaluate the global impact of CcrM-dependent methy-lation on gene expression in C crescentus we also

analyzed the transcriptional profiles of bacterial popula-tions in which CcrM is expressed at all times in the cellcycle When CcrM is over-expressed the period of post-replicational hemi-methylation of GANTC motifs that ischaracteristic of the wild-type strain is abolished havingpotential consequences on gene expression (Figure 3)We found that the expression levels of 546 genes weresignificantly changed in the strain with constitutive over-expression of CcrM compared with the wild-type strainwith 214 of these genes being affected gt2-fold(Supplementary Table S5) For 15 genes the effects wereverified by qRT-PCR and were essentially consistent withthe microarray results (Supplementary Figure S6)To evaluate whether these effects may result from direct

effects of GANTC methylation on promoter activity wecalculated the probability that significantly misregulatedgenes contained a GANTC motif in their promoter regionand compared this value with the probability obtainedfor a random set of genes We found no significant

Figure 5 Conserved GANTC motifs are enriched upstream of genessignificantly misregulated in the ccrM strain Frequency relative tothe entire genome of genes whose promoter region (here 200 bpupstream of their translational start codon) contains a GANTC motifamong genes significantly misregulated (upregulated or downregulated)(A) significantly downregulated (B) and significantly upregulated (C) inthe ccrM strain compared with the wild-type strain (D) Frequencyrelative to the entire genome of genes whose promoter region(200 bp upstream the translational start codon) contains a GANTCin one or two or in seven bacterial species closely related toC crescentus (C crescentus Caulobacter segnis Caulobacter K31Phenylobacter zucineum Brevundimonas subvibrioides Asticcacaulisexcentricus Maricaulis maris) among genes significantly downregulatedin the ccrM strain Stars indicate that the bias is significant (Plt 005Fisherrsquos exact test)

Figure 6 Involvement of CcrM in the regulation of the C crescentuscell cycle Frequency relative to the entire genome of genes belongingto the direct DnaA regulon (69) (A) the direct CtrA regulon (68)(B) the GcrA regulon (70) (C) or whose expression levels varyduring the cell cycle (71) (D) among genes significantly downregulatedor strongly downregulated (gt2-fold change) in the ccrM strain Starsindicate a significant bias (Plt 005 Fisherrsquos exact test) A comparisonbetween genes significantly misregulated in the ccrM strain andpossible direct targets of GcrA as determined by a ChIP-seq experi-ment (72) is shown in Supplementary Figure S10


(Fisherrsquos test Plt 005) difference even when consideringthe most misregulated genes (Supplementary Figure S7)which suggests that most of the changes in gene expressionobserved in cells over-expressing CcrM result fromindirect rather than direct effects of GANTC methyla-tion on the activity of promotersOnly a few genes involved in the regulation of the cell

cycle were found among the genes that were significantlymisregulated in the CcrM over-expressing strain Insteadgenes encoding proteins involved in inorganic ion trans-port and metabolism were over-represented among themost downregulated genes in this mutant strain whilegenes encoding proteins involved in DNA replicationrecombination and repair were over-represented amongthe most upregulated genes (Figure 4B) Notablyregulons involved in the resistance to different forms ofenvironmental stress such as the Fur (73) SigT (74) andFixKL (75) regulons were over-represented amongmisregulated genes (Figure 7AndashC) Similarly genesupregulated under heavy-metal stress (76) were alsoover-represented (Figure 7D) These observationssuggest that cells with over-expressed CcrM throughoutthe cell cycle exhibit a general and unspecific stressresponse

DISCUSSION

In the present study we analyzed the methylation stateof all GANTC motifs on the chromosome of threeC crescentus strains expressing different levels of theCcrM DNA methyltransferase (Figure 3) and determinedthe global transcriptional changes associated with the dif-ferences in the methylation state We observed thathundreds of genes were misregulated in cells lackingCcrM or in cells in which CcrM is constitutively over-expressed throughout the cell cycle compared with wild-type cells (Supplementary Tables S4 and S5) A significantproportion of the gene expression changes detected in theCcrM-deficient strain appeared to be due to direct methy-lation-mediated gene regulation since conserved GANTCmotifs were more frequently found in the promoterregions of genes misregulated in this strain than inrandom promoter regions (Figure 5 and SupplementaryTable S4) Considering that 80 genes with a GANTCmotif in their promoter region are found among genesstrongly misregulated in the ccrM strain and that thisnumber is 2-fold higher than expected by chance weestimate that 40 of these genes could be directlyregulated by CcrM (some essential candidates are shownin Table 1 and Figure 8) In contrast gene expressionchanges detected in the CcrM over-expressing strainappeared to be mostly due to secondary effects of theconstitutive methylation of the chromosome that yields astress phenotype (Figure 7 and Supplementary Figure S7)Consistent with these findings we found no negative cor-relation between the changes in gene expression observedin ccrM cells and in CcrM-over-expressing cells for mostgenes (Supplementary Figure S8) as would have beenexpected if the absence of methylation and constitutive-methylation had detectable opposite effects on the activity

of many promoters We also observed that genesmisregulated in each strain generally encoded proteinsinvolved in different processes (Figure 4)

In the absence of CcrM-dependent methylationmultiple genes involved in processes that are essential tothe viability of C crescentus were misregulated (Table 1and Supplementary Tables S4) Figure 8 shows 17 essen-tial genes that are good candidates for being directlyactivated by CcrM-dependent methylation as they havea relatively conserved GANTC motif in their promoterregion These genes include many genes encodingproteins required for cell division such as FtsZ FtsNand MipZ and proteins involved in DNA replicationrepair and topology such as the ligase the gyrase andSsb (Figure 8 Table 1 and Supplementary Table S4)We previously showed that the artificial expression ofFtsZ can restore the viability of ccrM cells in fast-growing conditions but that these cells were still longerstraighter and with shorter stalks than wild-type cells (36)

Figure 7 Constitutive over-expression of CcrM yields a stress pheno-type Frequency relative to the entire genome of genes belonging tothe Fur regulon (A) or the SigT regulon (B) among genes significantlymisregulated (upregulated or downregulated) or strongly misregulated(gt2-fold change) in the PlacccrM strain Frequency relative to theentire genome of genes belonging to the FixKL regulon (C) or the setof genes induced under heavy metal stress (D) among genes signifi-cantly misregulated or strongly misregulated (gt2-fold change) in thePlacccrM strain Stars indicate a significant bias (Plt 005 Fisherrsquosexact test)


Our transcriptome analysis suggests that these cells mightbe longer due to insufficient quantities of FtsN or toexcessive quantities of FtsW (Supplementary Table S4the ftsN and ftsW messengers were 21 times less and 16times more abundant in ccrM than in wild-type cellsrespectively) Interestingly ccrM cells still seemed toreplicate their chromosome efficiently and had a normalspontaneous mutation rate (Supplementary Figures S4and S5) despite the downregulation of several genesinvolved in DNA replication or repair and the over-ex-pression of the CtrA inhibitor of replication (Figure 8

and Table 1 and Supplementary Table S4) This resultsuggests that proteins like Ssb or the ligase are not inlimiting quantities for DNA replication and that thepost-transcriptional regulation of CtrA maintains intracel-lular levels of active CtrA low enough for DNA replica-tion to initiate (29) Consistent with our results it waspreviously shown that a C crescentus strain that carriesa unique copy of the ctrA gene under the control of amutant un-methylatable ctrA promoter does not have anabnormal phenotype (35) In addition to these many es-sential genes several genes involved in processes that arerequired for the development of C crescentus were alsomisregulated in the absence of CcrM-dependent methyla-tion For example the staR and creS non-essential genesinvolved in stalk biogenesis (78) and in cell curvature (79)respectively were significantly downregulated in ccrMcells (Supplementary Table S4 the staR and creS messen-gers were 29 and 16 times less abundant in ccrM thanin wild-type cells respectively) and this might provide anexplanation for the additional phenotypes that wereobservedAnother remarkable finding was that genes regulated

by the three global transcriptional regulators of theC crescentus cell cycle GcrA CtrA and DnaA werestrongly over-represented in the set of genes that werethe most misregulated in the absence of CcrM-mediatedmethylation (Figure 6 and Supplementary Figure S9 andS10) These three regulators are essential to the viabilityof C crescentus and they regulate the expression ofhundreds of genes involved in various events of the cellcycle (27) Our observation suggests that C crescentusgenes encoding proteins that are critical for cell cycle pro-gression are often regulated by one or more global regu-lators and by DNA methylation (Figures 6 and 8 andTable 1) showing the strong interconnection betweenCcrM-dependent DNA methylation and the C crescentuscell cycle regulatory network A possible explanation isthat the binding or the activity of one or more of theseglobal regulators is sensitive to the methylation state ofthe DNA at or next to their binding site The consensusbinding sites of CtrA and DnaA do not contain a sequencethat resembles a GANTC motif (6869) As for GcrA itsconsensus binding site is not yet clearly characterized buta recent study demonstrated that GcrA activates the tran-scription of several genes by promoting the recruitmentof the RNA polymerase to promoter regions containingmethylated GANTC motifs that overlapped a GcrAbinding site (72) Interestingly the homologs of GcrAhave the same conservation pattern as CcrM homologs(Figure 2A) The expression of many other membersof the GcrA direct regulon (6872) was nevertheless notsignificantly affected in the CcrM-deficient strain(Supplementary Table S4 and Supplementary Figure S9)suggesting that the activity of GcrA might not always beinfluenced by CcrM-dependent methylation Furtherstudies will be required to test whether the activities ofthe CtrA and DnaA global regulators may also beinfluenced by the methylation state of some promoterregions or if the over-representation of the CtrA andDnaA regulons among genes significantly misregulated

Figure 8 Model for the CcrM-dependent activation of essential genescontaining conserved GANTC motifs in their promoter Genesincluded in this schematic contain a GANTC motif in their promoterregion (200 bp upstream of their translational start codon) which isconserved in a minimum of two out of five bacterial species closelyrelated to C crescentus (Table 1) Genes coloured in orange encodeproteins involved in DNA replication repair or topology Genescoloured in yellow encode proteins involved in cell division Genescoloured in grey encode proteins involved in other functions Solidblack arrows indicate regulatory pathways identified as significantduring the transcriptome analysis using the ccrM strain in thisstudy Dashed black arrows indicate regulatory effects previouslyidentified (3477) but not found as significantly affected using theccrM strain in this study Solid blue red and green arrows indicatethe GcrA- CtrA- and DnaA-dependent regulatory pathways identifiedin (70) (68) and (69) respectively This schematic suggests that manybut not all essential genes activated by CcrM are also co-regulated byDnaA CtrA or GcrA master regulators showing the strong intercon-nection between CcrM-dependent DNA methylation and theC crescentus cell cycle regulatory network


in the ccrM strain is only a consequence of regulation bymore than one regulatorThe chromosomes of cells over-expressing CcrM are

maintained in the fully methylated state throughout thecell cycle (Figure 3) these cells are slightly elongated andtend to accumulate additional copies of the chromosome(31) The microarray analyses revealed that the transcrip-tion levels of many genes are changed in this mutant(Supplementary Table S5) and that cells exhibit a stressresponse (Figure 7) Transcriptional effects directly due tothe elimination of the fully methylated to hemi-methylatedswitch characteristic of the wild-type strain were howevereither rare or so weak that the method was not sensitiveenough to detect them (Supplementary Figure S7)A possible link between the over-replication phenotypeof the strain and the transcriptome can be found in thedownregulation of the gene encoding the PopZ protein(Supplementary Table S5 the popZ messenger was14 times less abundant in CcrM-over-expressing cellsthan in wild-type cells) which is required for the anchor-ing of the two chromosomal origins at opposite cell polesafter the initiation of chromosome replication (8081)Insufficient intracellular levels of PopZ may contributeto the abnormal DNA content in cells over-expressingCcrM as was previously described for PopZ-depletedcells (8081) Other phenotypes may be attributable to aglobal stress response that seems to take place in thesecells (Figure 7)Our SMRT sequencing analysis showed that 35

GANTC motifs along the chromosome were frequentlyunder-methylated on minimum one strand in wild-typecells (Supplementary Table S3) We found that onlythree of these motifs remained under-methylated in cellsover-expressing CcrM To address whether the under-methylation of the other 32 GANTC motifs in wild-typecells may affect gene expression we compared the mRNAlevels of nearby genes in the wild-type and in the CcrM-over-expressing cells We observed a significant differencein expression of two genes these were the CCNA_01150gene of unknown function and the CCNA_03248 geneencoding a TonB-dependent receptor (SupplementaryTables S3 and S5) the CCNA_01150 and theCCNA_03248 messengers were five times more and 13times less abundant in CcrM-over-expressing than inwild-type cells respectively) The difference in expressionmight be linked to the difference in the methylation stateof GANTC motifs in the two strains Similarly under-methylated GATC sites were observed on the chromo-somes of Dam-expressing bacteria and they are sometimesinvolved in epigenetic switches of gene expression(71124ndash2682) These sites often overlap the bindingsites of the Lrp OxyR and Fur global regulators whichwere shown to compete with Dam for the DNA and to besometimes sensitive to its methylation state Interestinglywe found that the predicted Fur regulon was over-represented among the genes that were misregulatedwhen the CcrM enzyme was over-produced inC crescentus (Figure 7) We nevertheless did not findFur-regulated promoters carrying frequently under-methylated GANTC motifs on the C crescentus chromo-some This observation suggests that the C crescentus Fur

protein does not often protect the promoter that itcontrols from CcrM-mediated methylation at least whencells were cultivated in iron-rich medium contrarily towhat was previously observed at the sci1 promoteron the E coli chromosome (82) It is still possible thatCcrM-dependent methylation affects the activity of theC crescentus Fur protein using a mechanism differentfrom the one previously described in E coli (82) An ex-cellent candidate gene that may be regulated by an epigen-etic switch mediated by Fur and DNA methylation inC crescentus is the CCNA_02275 gene as its promotercontains two GANTC sites that overlap a Fur bindingsite and since its messenger was 22 times lessabundant in CcrM-over-expressing than in wild-typecells (Supplementary Table S5) This gene encodes atrans-membrane protein of unknown function and isrepressed by Fur (83) These results suggest that Furmay have a higher affinity for the CCNA_02275promoter when it is in a fully methylated rather than ina hemi-methylated state Additional detailed studies willbe required to understand how this gene might beepigenetically regulated in C crescentus

Overall our study demonstrates that the overlapbetween the roles of Dam-dependent methylation inenterobacteria and the roles of CcrM-dependent methyla-tion in Alphaproteobacteria is restricted In both casesthe methylation of adenines has pleiotropic effects ongene expression but the genes and functions representedin the Dam and CcrM regulons are different InC crescentus but not E coli the methylation ofGANTC motifs may mediate the integration of the cellcycle regulatory circuit with chromosome replication Inaddition we found that CcrM-dependent methylation isnot required to control the initiation of chromosome rep-lication (Supplementary Figure S4) or to correct DNAmismatches during the MMR process (SupplementaryFigure S5) as it is often the case for Dam-dependentmethylation (111214) Dam is generally co-conservedwith MutH which is the protein that recognizes thenewly synthesized non-methylated DNA strand thatneeds to be repaired during methyl-directed MMR (18)Most bacteria lack Dam and MutH like C crescentus andBacillus subtilis (138485) These bacteria probably use amethylation-independent strand recognition mechanismduring MMR which might be spatially coupled with theDNA polymerase complex (85) This study exemplifies theneed to explore the multifaceted use of DNA methylationin a variety of bacterial species

ACCESSION NUMBERS

The microarray data are publicly available at the GEOdatabase (GEO accession numbers GSE52722 andGSE52375 httpswwwncbinlmnihgovgeo)

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online


ACKNOWLEDGEMENTS

We are grateful to Keith Harshman Johann Weberand Hannes Richter from the Lausanne GenomicTechnologies Facility and to Jonas Korlach and TysonClark from the Pacific Biosciences Company (MenloPark CA USA) for helping us during the transcriptomeqRT-PCR and SMRT sequencing analysis We thankmembers of the Collier laboratory for helpful discussions

FUNDING

University of Lausanne Swiss National ScienceFellowships [3100A0_122541 and 31003A_140758 to JC]National Institute of Health Grant [RO1-GM051426 toLS] Funding for open access charge Swiss NationalScience Fellowship [31003A_140758 to JC]

Conflict of interest statement None declared

REFERENCES

1 SchlagmanSL MinerZ FeherZ and HattmanS (1988) TheDNA [adenine-N6]methyltransferase (Dam) of bacteriophage T4Gene 73 517ndash530

2 HoelzerK ShackeltonLA and ParrishCR (2008) Presence androle of cytosine methylation in DNA viruses of animals NucleicAcids Res 36 2825ndash2837

3 BochowS EllimanJ and OwensL (2012) Bacteriophage adeninemethyltransferase a life cycle regulator Modelled using Vibrioharveyi myovirus like J Appl Microbiol 113 1001ndash1013

4 JeltschA (2002) Beyond watson and crick DNA methylationand molecular enzymology of DNA methyltransferasesChembiochem 3 274ndash293

5 OoiSK OrsquoDonnellAH and BestorTH (2009) Mammaliancytosine methylation at a glance J Cell Sci 122 2787ndash2791

6 DavisBM ChaoMC and WaldorMK (2013) Entering the eraof bacterial epigenomics with single molecule real time DNAsequencing Curr Opin Microbiol 16 192ndash198

7 WionD and CasadesusJ (2006) N6-methyl-adenine anepigenetic signal for DNA-protein interactions Nat RevMicrobiol 4 183ndash192

8 RobertsRJ VinczeT PosfaiJ and MacelisD (2010)REBASEndasha database for DNA restriction and modificationenzymes genes and genomes Nucleic Acids Res 38 D234ndashD236

9 JeltschA (2003) Maintenance of species identity and controllingspeciation of bacteria a new function for restrictionmodificationsystems Gene 317 13ndash16

10 MarinusMG and CasadesusJ (2009) Roles of DNA adeninemethylation in host-pathogen interactions mismatch repairtranscriptional regulation and more FEMS Microbiol Rev 33488ndash503

11 LowDA and CasadesusJ (2008) Clocks and switches bacterialgene regulation by DNA adenine methylation Curr OpinMicrobiol 11 106ndash112

12 CasadesusJ and LowD (2006) Epigenetic gene regulation in thebacterial world Microbiol Mol Biol Rev 70 830ndash856

13 BrezellecP HoebekeM HietMS PasekS and FeratJL(2006) DomainSieve a protein domain-based screen that led tothe identification of dam-associated genes with potential link toDNA maintenance Bioinformatics 22 1935ndash1941

14 CollierJ (2009) Epigenetic regulation of the bacterial cell cycleCurr Opin Microbiol 12 722ndash729

15 DemarreG and ChattorajDK (2010) DNA adenine methylationis required to replicate both Vibrio cholerae chromosomes onceper cell cycle PLoS Genet 6 e1000939

16 ValME SkovgaardO Ducos-GalandM BlandMJ andMazelD (2012) Genome engineering in Vibrio cholerae a feasibleapproach to address biological issues PLoS Genet 8 e1002472

17 PukkilaPJ PetersonJ HermanG ModrichP andMeselsonM (1983) Effects of high levels of DNA adeninemethylation on methyl-directed mismatch repair in Escherichiacoli Genetics 104 571ndash582

18 IyerRR PluciennikA BurdettV and ModrichPL (2006)DNA mismatch repair functions and mechanisms Chem Rev106 302ndash323

19 OshimaT WadaC KawagoeY AraT MaedaMMasudaY HiragaS and MoriH (2002) Genome-wide analysisof deoxyadenosine methyltransferase-mediated control of geneexpression in Escherichia coli Mol Microbiol 45 673ndash695

20 Lobner-OlesenA MarinusMG and HansenFG (2003) Roleof SeqA and Dam in Escherichia coli gene expression aglobalmicroarray analysis Proc Natl Acad Sci USA 1004672ndash4677

21 Robbins-MankeJL ZdraveskiZZ MarinusM andEssigmannJM (2005) Analysis of global gene expression anddouble-strand-break formation in DNA adeninemethyltransferase- and mismatch repair-deficient Escherichia coliJ Bacteriol 187 7027ndash7037

22 SeshasayeeAS (2007) An assessment of the role of DNAadenine methyltransferase on gene expression regulation in E coliPLoS One 2 e273

23 BalbontinR RowleyG PucciarelliMG Lopez-GarridoJWormstoneY LucchiniS Garcia-Del PortilloF HintonJCand CasadesusJ (2006) DNA adenine methylation regulatesvirulence gene expression in Salmonella enterica serovarTyphimurium J Bacteriol 188 8160ndash8168

24 van der WoudeMW (2011) Phase variation how to create andcoordinate population diversity Curr Opin Microbiol 14205ndash211

25 van der WoudeM HaleWB and LowDA (1998) Formationof DNA methylation patterns nonmethylated GATC sequences ingut and pap operons J Bacteriol 180 5913ndash5920

26 DaliaAB LazinskiDW and CamilliA (2013) Characterizationof undermethylated sites in Vibrio cholerae J Bacteriol 1952389ndash2399

27 CollierJ and ShapiroL (2007) Spatial complexity and control ofa bacterial cell cycle Curr Opin Biotechnol 18 333ndash340

28 CurtisPD and BrunYV (2010) Getting in the loop regulationof development in Caulobacter crescentus Microbiol Mol BiolRev 74 13ndash41

29 CollierJ (2012) Regulation of chromosomal replication inCaulobacter crescentus Plasmid 67 76ndash87

30 MarczynskiGT (1999) Chromosome methylation andmeasurement of faithful once and only once per cell cyclechromosome replication in Caulobacter crescentus J Bacteriol181 1984ndash1993

31 ZweigerG MarczynskiG and ShapiroL (1994) A CaulobacterDNA methyltransferase that functions only in the predivisionalcell J Mol Biol 235 472ndash485

32 ReisenauerA KahngLS McCollumS and ShapiroL (1999)Bacterial DNA methylation a cell cycle regulator J Bacteriol181 5135ndash5139

33 StephensC ReisenauerA WrightR and ShapiroL (1996)A cell cycle-regulated bacterial DNA methyltransferase is essentialfor viability Proc Natl Acad Sci USA 93 1210ndash1214

34 CollierJ McAdamsHH and ShapiroL (2007) A DNAmethylation ratchet governs progression through a bacterial cellcycle Proc Natl Acad Sci USA 104 17111ndash17116

35 ReisenauerA and ShapiroL (2002) DNA methylation affects thecell cycle transcription of the CtrA global regulator inCaulobacter EMBO J 21 4969ndash4977

36 GonzalezD and CollierJ (2013) DNA methylation by CcrMactivates the transcription of two genes required for the divisionof Caulobacter crescentus Mol Microbiol 88 203ndash218

37 StephensCM ZweigerG and ShapiroL (1995) Coordinate cellcycle control of a Caulobacter DNA methyltransferase and theflagellar genetic hierarchy J Bacteriol 177 1662ndash1669

38 WrightR StephensC and ShapiroL (1997) The CcrM DNAmethyltransferase is widespread in the alpha subdivision ofproteobacteria and its essential functions are conserved inRhizobium meliloti and Caulobacter crescentus J Bacteriol 1795869ndash5877


39 KahngLS and ShapiroL (2001) The CcrM DNAmethyltransferase of Agrobacterium tumefaciens is essential andits activity is cell cycle regulated J Bacteriol 183 3065ndash3075

40 RobertsonGT ReisenauerA WrightR JensenRB JensenAShapiroL and RoopRM II (2000) The Brucella abortus CcrMDNA methyltransferase is essential for viability and itsoverexpression attenuates intracellular replication in murinemacrophages J Bacteriol 182 3482ndash3489

41 AltschulSF GishW MillerW MyersEW and LipmanDJ(1990) Basic local alignment search tool J Mol Biol 215403ndash410

42 EdgarRC (2004) MUSCLE a multiple sequence alignmentmethod with reduced time and space complexity BMCBioinformatics 5 113

43 EdgarRC (2004) MUSCLE multiple sequence alignment withhigh accuracy and high throughput Nucleic Acids Res 321792ndash1797

44 MaloneT BlumenthalRM and ChengX (1995) Structure-guided analysis reveals nine sequence motifs conserved amongDNA amino-methyltransferases and suggests a catalyticmechanism for these enzymes J Mol Biol 253 618ndash632

45 PriceMN DehalPS and ArkinAP (2010) FastTree 2mdashapproximately maximum-likelihood trees for large alignmentsPLoS One 5 e9490

46 PriceMN DehalPS and ArkinAP (2009) FastTreecomputing large minimum evolution trees with profiles instead ofa distance matrix Mol Biol Evol 26 1641ndash1650

47 CastresanaJ (2000) Selection of conserved blocks from multiplealignments for their use in phylogenetic analysis Mol Biol Evol17 540ndash552

48 StamatakisA HooverP and RougemontJ (2008) A rapidbootstrap algorithm for the RAxML web servers Syst Biol 57758ndash771

49 HuelsenbeckJP and RonquistF (2001) MRBAYES bayesianinference of phylogenetic trees Bioinformatics 17 754ndash755

50 RonquistF and HuelsenbeckJP (2003) MrBayes 3 bayesianphylogenetic inference under mixed models Bioinformatics 191572ndash1574

51 EvingerM and AgabianN (1977) Envelope-associated nucleoidfrom Caulobacter crescentus stalked and swarmer cells JBacteriol 132 294ndash301

52 MarksME Castro-RojasCM TeilingC DuL KapatralVWalunasTL and CrossonS (2010) The genetic basis oflaboratory adaptation in Caulobacter crescentus J Bacteriol 1923678ndash3688

53 CollierJ and ShapiroL (2009) Feedback control of DnaA-mediated replication initiation by replisome-associated HdaAprotein in Caulobacter J Bacteriol 191 5706ndash5716

54 BrilliM FondiM FaniR MengoniA FerriLBazzicalupoM and BiondiEG (2010) The diversity andevolution of cell cycle regulation in alpha-proteobacteria acomparative genomic analysis BMC Syst Biol 4 52

55 BazylinskiDA WilliamsTJ LefevreCT BergRJZhangCL BowserSS DeanAJ and BeveridgeTJ (2013)Magnetococcus marinus gen nov sp nov a marinemagnetotactic bacterium that represents a novel lineage(Magnetococcaceae fam nov Magnetococcales ord nov) at thebase of the Alphaproteobacteria Int J Syst Evol Microbiol 63801ndash808

56 ViklundJ EttemaTJ and AnderssonSG (2012) Independentgenome reduction and phylogenetic reclassification of the oceanicSAR11 clade Mol Biol Evol 29 599ndash615

57 GuptaRS and MokA (2007) Phylogenomics and signatureproteins for the alpha proteobacteria and its main groups BMCMicrobiol 7 106

58 NiermanWC FeldblyumTV LaubMT PaulsenITNelsonKE EisenJA HeidelbergJF AlleyMR OhtaNMaddockJR et al (2001) Complete genome sequenceof Caulobacter crescentus Proc Natl Acad Sci USA 984136ndash4141

59 PaulinoLC de MelloMP and OttoboniLM (2002)Differential gene expression in response to copper inAcidithiobacillus ferrooxidans analyzed by RNA arbitrarily primedpolymerase chain reaction Electrophoresis 23 520ndash527

60 WrightR StephensC ZweigerG ShapiroL and AlleyMR(1996) Caulobacter Lon protease has a critical role in cell-cyclecontrol of DNA methylation Genes Dev 10 1532ndash1542

61 FlusbergBA WebsterDR LeeJH TraversKJOlivaresEC ClarkTA KorlachJ and TurnerSW (2010)Direct detection of DNA methylation during single-moleculereal-time sequencing Nat Methods 7 461ndash465

62 ClarkTA MurrayIA MorganRD KislyukAOSpittleKE BoitanoM FomenkovA RobertsRJ andKorlachJ (2012) Characterization of DNA methyltransferasespecificities using single-molecule real-time DNA sequencingNucleic Acids Res 40 e29

63 MurrayIA ClarkTA MorganRD BoitanoM AntonBPLuongK FomenkovA TurnerSW KorlachJ andRobertsRJ (2012) The methylomes of six bacteria Nucleic AcidsRes 40 11450ndash11462

64 KozdonJB MelfiMD LuongK ClarkTA BoitanoMWangS ZhouB GonzalezD CollierJ TurnerSW et al(2013) Global methylation state at base-pair resolution of theCaulobacter genome throughout the cell cycle Proc Natl AcadSci USA 110 E4658ndashE4667

65 NieveraC TorgueJJ GrimwadeJE and LeonardAC (2006)SeqA blocking of DnaA-oriC interactions ensures staged assemblyof the E coli pre-RC Mol Cell 24 581ndash592

66 ShaheenSM OuimetMC and MarczynskiGT (2009)Comparative analysis of Caulobacter chromosome replicationorigins Microbiology 155 1215ndash1225

67 ChristenB AbeliukE CollierJM KalogerakiVSPassarelliB CollerJA FeroMJ McAdamsHH andShapiroL (2011) The essential genome of a bacterium MolSyst Biol 7 528

68 LaubMT ChenSL ShapiroL and McAdamsHH (2002)Genes directly controlled by CtrA a master regulator ofthe Caulobacter cell cycle Proc Natl Acad Sci USA 994632ndash4637

69 HottesAK ShapiroL and McAdamsHH (2005) DnaAcoordinates replication initiation and cell cycle transcription inCaulobacter crescentus Mol Microbiol 58 1340ndash1353

70 HoltzendorffJ HungD BrendeP ReisenauerA ViollierPHMcAdamsHH and ShapiroL (2004) Oscillating globalregulators control the genetic circuit driving a bacterial cell cycleScience 304 983ndash987

71 LaubMT McAdamsHH FeldblyumT FraserCM andShapiroL (2000) Global analysis of the genetic networkcontrolling a bacterial cell cycle Science 290 2144ndash2148

72 FioravantiA FumeauxC MohapatraSS BompardCBrilliM FrandiA CastricV VilleretV ViollierPH andBiondiEG (2013) DNA binding of the cell cycle transcriptionalregulator GcrA depends on N6-adenosine methylation inCaulobacter crescentus and other Alphaproteobacteria PLoSGenet 9 e1003541

73 da Silva NetoJF BrazVS ItalianiVC and MarquesMV(2009) Fur controls iron homeostasis and oxidative stress defensein the oligotrophic alpha-proteobacterium Caulobacter crescentusNucleic Acids Res 37 4812ndash4825

74 Alvarez-MartinezCE LourencoRF BaldiniRL LaubMTand GomesSL (2007) The ECF sigma factor sigma(T) isinvolved in osmotic and oxidative stress responses in Caulobactercrescentus Mol Microbiol 66 1240ndash1255

75 CrossonS McGrathPT StephensC McAdamsHH andShapiroL (2005) Conserved modular design of an oxygensensorysignaling network with species-specific output Proc NatlAcad Sci USA 102 8018ndash8023

76 HuP BrodieEL SuzukiY McAdamsHH andAndersenGL (2005) Whole-genome transcriptional analysis ofheavy metal stresses in Caulobacter crescentus J Bacteriol 1878437ndash8449

77 CollierJ MurraySR and ShapiroL (2006) DnaA couplesDNA replication and the expression of two cell cycle masterregulators EMBO J 25 346ndash356

78 BiondiEG SkerkerJM ArifM PrasolMS PerchukBS andLaubMT (2006) A phosphorelay system controls stalkbiogenesis during cell cycle progression in Caulobacter crescentusMol Microbiol 59 386ndash401


79 AusmeesN KuhnJR and Jacobs-WagnerC (2003) Thebacterial cytoskeleton an intermediate filament-like function incell shape Cell 115 705ndash713

80 BowmanGR ComolliLR ZhuJ EckartM KoenigMDowningKH MoernerWE EarnestT and ShapiroL (2008)A polymeric protein anchors the chromosomal originParBcomplex at a bacterial cell pole Cell 134 945ndash955

81 EbersbachG BriegelA JensenGJ and Jacobs-WagnerC(2008) A self-associating protein critical for chromosomeattachment division and polar organization in caulobacter Cell134 956ndash968

82 BrunetYR BernardCS GavioliM LloubesR andCascalesE (2011) An epigenetic switch involving overlapping fur

and DNA methylation optimizes expression of a type VI secretiongene cluster PLoS Genet 7 e1002205

83 da Silva NetoJF LourencoRF and MarquesMV (2013)Global transcriptional response of Caulobacter crescentus to ironavailability BMC Genomics 14 549

84 Martins-PinheiroM MarquesRC and MenckCF (2007)Genome analysis of DNA repair genes in the alphaproteobacterium Caulobacter crescentus BMC Microbiol 7 17

85 SimmonsLA DaviesBW GrossmanAD and WalkerGC(2008) Beta clamp directs localization of mismatch repair inBacillus subtilis Mol Cell 29 291ndash301


state of the DNA at or near their binding site In somecases the presence of one or more under-methylatedGATC motifs in a promoter is an indication that it isregulated by such a bistable epigenetic switch althoughthere also exists under-methylated GATC motifs that arenot part of epigenetic mechanisms regulating gene expres-sion (2526) Dam is to date the only DNA adeninemethyltransferase whose global effects on gene expressionhave been studied in bacteria

The cell cyclendashregulated methyltransferase CcrMwas first identified in Caulobacter crescentus anAlphaproteobacterium that divides asymmetrically intotwo morphologically different progeny cells thedaughter stalked cell that immediately initiates DNA rep-lication and cell division and the daughter swarmer cellthat first differentiates into a stalked cell before replicationand cell division (Figure 1) (2728) This bacterium initi-ates the replication of its chromosome only once per cellcycle and specifically in the stalked progeny or during theswarmer-to-stalked cell transition (2930) The expressionof CcrM is restricted to the late predivisional stage nearthe end of chromosome replication which implies thatGANTC motifs on the chromosome are supposedlyfully methylated (methylated on both DNA strands) inswarmer cells and then become hemi-methylated(methylated on one strand only) on DNA replication instalked cells and stay so until the end of the replication ofthe chromosome in pre-divisional cells (Figure 1) (30ndash32)Maintaining the chromosome constitutively fullymethylated by over-expressing CcrM leads to morpho-logical and cell cyclendashrelated defects in C crescentus sug-gesting that the temporal regulation of the transition fromthe fully methylated to the hemi-methylated state has bio-logical importance (3133) In C crescentus the methyla-tion state of GANTC motifs has a direct effect on thetranscription of at least four genes the ctrA and dnaAgenes encoding master regulators of the cell cycle(3435) the ftsZ and mipZ genes required for celldivision (36) and most probably the ccrM gene itself(37) The transition from the fully methylated to thehemi-methylated state of a GANTC motif located in cisis sufficient to cause a small but significant change in thetranscriptional activity of the ctrA dnaA and ftsZ pro-moters This supports a model according to which thechange in GANTC methylation state during DNA repli-cation might function to co-ordinate gene expressionchanges with the progression of the replication forksetting the mechanical basis for the regulation ofthe timing of multiple cell cycle events (323435) Werecently showed that an increase in ftsZ transcription issufficient to restore the viability of ccrM cells in richmedium demonstrating that the direct activation of ftsZtranscription by DNA methylation is one of the most im-portant functions of CcrM in C crescentus (36) Theserescued cells still exhibited a residual phenotype Forexample cells were elongated and straighter thanwild-type cells and had shorter stalks These observa-tions suggested that more genes are probably regulatedby CcrM-dependent methylation in C crescentusInterestingly the ccrM gene is not essential anymore forthe viability of C crescentus when cells are cultivated in

slow-growing conditions such as minimal medium thesecells are nevertheless slightly elongated and grow slightlymore slowly than wild-type cells (36) The physiologicalimportance of GANTC methylation is probably notrestricted to C crescentus as the homolog of ccrM wasshown to be essential for viability in fast-growing culturesof each of the species where this was tested (333638ndash40)Here we assess the global influence of GANTC methy-

lation on transcription profiles in C crescentus byanalyzing the transcriptome of a ccrM strain and of astrain in which CcrM is constitutively expressed so thatthe chromosome remains in the fully methylated statethroughout the cell cycle We show that hundreds ofgenes are differentially expressed in both mutant strainscompared with the wild type Genes whose transcriptioncould directly be affected by GANTC methylation arepredicted on the basis of their differential expression inthe mutant strains and through bioinformatic analysis ofGANTC conservation in promoters Our findings reveal astrong link between CcrM-dependent methylation and cellcycle control in C crescentus that is also likely to exist inmany other Alphaproteobacteria

MATERIALS AND METHODS

Phylogenetic analysis

NCBI whole bacterial proteomes were searched forproteins with homology to the CcrM protein fromC crescentus strain CB15 (blastp e-valuelt 1e-10) (41)one strain was selected per genus and the protein with thelowest blastp e-value was used for each individual strainOn the basis of a MUSCLE alignment of the sequences(4243) the subtype b N6-adenine-methyltransferases wereselected using the motif IV [DPPY 6frac14 SPP(YF)] and themotif X [T(QED)KP 6frac14AXFP] to discriminate between

Figure 1 CcrM is a cell cyclendashregulated DNA methyltransferase inC crescentus Upper panel schematic of the C crescentus cell cycleshowing the differentiation of a swarmer cell (SW) into a stalked cell(ST) which then turns into a pre-divisional cell (PD) Lower panelschematic showing the changes in the methylation state of GANTCmotifs on the chromosome as a function of chromosome replicationand cell cycle progression

















RESULTS






























Table

1Essentialgenes

containingGANTC

motifs


ccrM


Genename


Log-ratio

MA

Log-ratio

qRT-PCR

CtrA

regulon

DnaA

regulon

GcrA

regulon

GANTC

conservation

Essentialgenes

repressed

inccrM

CCNA_00390

ADP-heptosemdash


085

072

No

No

No

4CCNA_01104

Glycosyltransferase

077

ND

No

No

No

2CCNA_01275


dehydrogenase

(NADP+


cyclohydrolase

084

ND

No

No

No

1

CCNA_01450

Single-stranded


RecJ

103

120

No

No

Yes

4CCNA_01535

Single-stranded

DNA

bindingprotein

056

ND

No

Yes

No

2CCNA_01590

NAD-dependentDNA

ligase

123

ND

No

No

No

3CCNA_01651

DNA

gyrase

subunit

A114

101

No

No

Yes

5CCNA_01776

Ribonuclease

D068

092

No

No

No

5CCNA_02052

Topoisomerase

IVsubunit

B197

ND

No

No

Yes

3CCNA_02086

Celldivisionprotein

FtsN

109

ND

No

No

Yes

5CCNA_02127

Oxacillin


FmtC

193

ND

No

No

Yes

2CCNA_02208

Thymidylate

synthase

103

ND

No

No

No

3CCNA_02246


MipZ

052a

254

Yes

Yes

Yes

5CCNA_02256

Mg2+

transporter

MgtE

083

114

No

No

No

5CCNA_02389


acetyltransferase

UDP-N


087

069

No

No

No

5

CCNA_02623

Celldivisionprotein

FtsZ

144

175

Yes

Yes

No

5CCNA_02679

Hypotheticalprotein

107

ND

No

No

No

3CCNA_03607


reductase

subunit

alphaNrdA

047

077

No

Yes

No

5

Essentialgenes

activatedin

ccrM

CCNA_00379


DsbA

069

053

No

No

No

5CCNA_00845

Antitoxin

protein

RelB-1

082

ND

No

No

No

1CCNA_00893


protein

053

ND

No

No

No

0CCNA_01946

Tyrosyl-tR

NA

synthetase

089

028

No

No

No

4CCNA_02635

Celldivisionprotein

FtsW

065

ND

Yes

No

No

2CCNA_03130

Cellcycleresponse

regulatorCtrA

046

034

Yes

No

Yes

5CCNA_03471

4-hydroxy-3-m


reductase

074

023

No

No

No

4CCNA_03766

16SrR

NA

processingprotein

Rim

M111

ND

No

No

No

3CCNA_03820

Outer-mem


109

ND

No

No

No

3

Genes

wereconsidered


(67)



usingmicroarrays(M

A)orqRT-PCR


ccrM

cells

ND


forthatgene


wereunfortunately

designed

todetectmipZ

bymicroarrays

TheCtrA

andDnaA



GcrA

regulonwasdescribed

in(70)ThelsquoG

ANTC


ofspeciesin

whichaGANTC

motifis

foundin


oftheir

translationalstart



relatedto

Ccrescentus(C


zucineumBrevundim

onassubvibrioides)
















DISCUSSION
















ACCESSION NUMBERS


SUPPLEMENTARY DATA



ACKNOWLEDGEMENTS


FUNDING



REFERENCES









































































































RESULTS






























Table

1Essentialgenes

containingGANTC

motifs


ccrM


Genename


Log-ratio

MA

Log-ratio

qRT-PCR

CtrA

regulon

DnaA

regulon

GcrA

regulon

GANTC

conservation

Essentialgenes

repressed

inccrM

CCNA_00390

ADP-heptosemdash


085

072

No

No

No

4CCNA_01104

Glycosyltransferase

077

ND

No

No

No

2CCNA_01275


dehydrogenase

(NADP+


cyclohydrolase

084

ND

No

No

No

1

CCNA_01450

Single-stranded


RecJ

103

120

No

No

Yes

4CCNA_01535

Single-stranded

DNA

bindingprotein

056

ND

No

Yes

No

2CCNA_01590

NAD-dependentDNA

ligase

123

ND

No

No

No

3CCNA_01651

DNA

gyrase

subunit

A114

101

No

No

Yes

5CCNA_01776

Ribonuclease

D068

092

No

No

No

5CCNA_02052

Topoisomerase

IVsubunit

B197

ND

No

No

Yes

3CCNA_02086

Celldivisionprotein

FtsN

109

ND

No

No

Yes

5CCNA_02127

Oxacillin


FmtC

193

ND

No

No

Yes

2CCNA_02208

Thymidylate

synthase

103

ND

No

No

No

3CCNA_02246


MipZ

052a

254

Yes

Yes

Yes

5CCNA_02256

Mg2+

transporter

MgtE

083

114

No

No

No

5CCNA_02389


acetyltransferase

UDP-N


087

069

No

No

No

5

CCNA_02623

Celldivisionprotein

FtsZ

144

175

Yes

Yes

No

5CCNA_02679

Hypotheticalprotein

107

ND

No

No

No

3CCNA_03607


reductase

subunit

alphaNrdA

047

077

No

Yes

No

5

Essentialgenes

activatedin

ccrM

CCNA_00379


DsbA

069

053

No

No

No

5CCNA_00845

Antitoxin

protein

RelB-1

082

ND

No

No

No

1CCNA_00893


protein

053

ND

No

No

No

0CCNA_01946

Tyrosyl-tR

NA

synthetase

089

028

No

No

No

4CCNA_02635

Celldivisionprotein

FtsW

065

ND

Yes

No

No

2CCNA_03130

Cellcycleresponse

regulatorCtrA

046

034

Yes

No

Yes

5CCNA_03471

4-hydroxy-3-m


reductase

074

023

No

No

No

4CCNA_03766

16SrR

NA

processingprotein

Rim

M111

ND

No

No

No

3CCNA_03820

Outer-mem


109

ND

No

No

No

3

Genes

wereconsidered


(67)



usingmicroarrays(M

A)orqRT-PCR


ccrM

cells

ND


forthatgene


wereunfortunately

designed

todetectmipZ

bymicroarrays

TheCtrA

andDnaA



GcrA

regulonwasdescribed

in(70)ThelsquoG

ANTC


ofspeciesin

whichaGANTC

motifis

foundin


oftheir

translationalstart



relatedto

Ccrescentus(C


zucineumBrevundim

onassubvibrioides)
















DISCUSSION
















ACCESSION NUMBERS


SUPPLEMENTARY DATA



ACKNOWLEDGEMENTS


FUNDING



REFERENCES

















































































































Table

1Essentialgenes

containingGANTC

motifs


ccrM


Genename


Log-ratio

MA

Log-ratio

qRT-PCR

CtrA

regulon

DnaA

regulon

GcrA

regulon

GANTC

conservation

Essentialgenes

repressed

inccrM

CCNA_00390

ADP-heptosemdash


085

072

No

No

No

4CCNA_01104

Glycosyltransferase

077

ND

No

No

No

2CCNA_01275


dehydrogenase

(NADP+


cyclohydrolase

084

ND

No

No

No

1

CCNA_01450

Single-stranded


RecJ

103

120

No

No

Yes

4CCNA_01535

Single-stranded

DNA

bindingprotein

056

ND

No

Yes

No

2CCNA_01590

NAD-dependentDNA

ligase

123

ND

No

No

No

3CCNA_01651

DNA

gyrase

subunit

A114

101

No

No

Yes

5CCNA_01776

Ribonuclease

D068

092

No

No

No

5CCNA_02052

Topoisomerase

IVsubunit

B197

ND

No

No

Yes

3CCNA_02086

Celldivisionprotein

FtsN

109

ND

No

No

Yes

5CCNA_02127

Oxacillin


FmtC

193

ND

No

No

Yes

2CCNA_02208

Thymidylate

synthase

103

ND

No

No

No

3CCNA_02246


MipZ

052a

254

Yes

Yes

Yes

5CCNA_02256

Mg2+

transporter

MgtE

083

114

No

No

No

5CCNA_02389


acetyltransferase

UDP-N


087

069

No

No

No

5

CCNA_02623

Celldivisionprotein

FtsZ

144

175

Yes

Yes

No

5CCNA_02679

Hypotheticalprotein

107

ND

No

No

No

3CCNA_03607


reductase

subunit

alphaNrdA

047

077

No

Yes

No

5

Essentialgenes

activatedin

ccrM

CCNA_00379


DsbA

069

053

No

No

No

5CCNA_00845

Antitoxin

protein

RelB-1

082

ND

No

No

No

1CCNA_00893


protein

053

ND

No

No

No

0CCNA_01946

Tyrosyl-tR

NA

synthetase

089

028

No

No

No

4CCNA_02635

Celldivisionprotein

FtsW

065

ND

Yes

No

No

2CCNA_03130

Cellcycleresponse

regulatorCtrA

046

034

Yes

No

Yes

5CCNA_03471

4-hydroxy-3-m


reductase

074

023

No

No

No

4CCNA_03766

16SrR

NA

processingprotein

Rim

M111

ND

No

No

No

3CCNA_03820

Outer-mem


109

ND

No

No

No

3

Genes

wereconsidered


(67)



usingmicroarrays(M

A)orqRT-PCR


ccrM

cells

ND


forthatgene


wereunfortunately

designed

todetectmipZ

bymicroarrays

TheCtrA

andDnaA



GcrA

regulonwasdescribed

in(70)ThelsquoG

ANTC


ofspeciesin

whichaGANTC

motifis

foundin


oftheir

translationalstart



relatedto

Ccrescentus(C


zucineumBrevundim

onassubvibrioides)
















DISCUSSION
















ACCESSION NUMBERS


SUPPLEMENTARY DATA



ACKNOWLEDGEMENTS


FUNDING



REFERENCES








































































































DISCUSSION
















ACCESSION NUMBERS


SUPPLEMENTARY DATA



ACKNOWLEDGEMENTS


FUNDING



REFERENCES




































































































ACCESSION NUMBERS


SUPPLEMENTARY DATA



ACKNOWLEDGEMENTS


FUNDING



REFERENCES


























































































ACKNOWLEDGEMENTS


FUNDING



REFERENCES


























































































The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach

Documents

Transcript of The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach