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Comparative analysis of Caulobacter chromosome replication origins

McGill University, Department of Microbiology and Immunology, 3775 University Street, Room 506, Montreal, QC H3A 2B4, Canada

Correspondence
Gregory T. Marczynski
gregory.marczynski{at}mcgill.ca

Caulobacter crescentus (CB15) initiates chromosome replication only in stalked cells and not in swarmers. To better understand this dimorphic control of chromosome replication, we isolated replication origins (oris) from freshwater Caulobacter (FWC) and marine Caulobacter (MCS) species. Previous studies implicated integration host factor (IHF) and CcrM DNA methylation sites in replication control. However, ori IHF and CcrM sites identified in the model FWC CB15 were only conserved among closely related FWCs. DnaA boxes and CtrA binding sites are established CB15 ori components. CtrA is a two-component regulator that blocks chromosome replication selectively in CB15 swarmers. DnaA boxes and CtrA sites were found in five FWC and three MCS oris. Usually, a DnaA box and a CtrA site were paired, suggesting that CtrA binding regulates DnaA activity. We tested this hypothesis by site-directed mutagenesis of an MCS10 ori which contains only one CtrA binding site overlapping a critical DnaA box. This overlapping site is unique in the whole MCS10 genome. Selective DnaA box mutations decreased replication, while selective CtrA binding site mutations increased replication of MCS10 ori plasmids. Therefore, both FWC and MCS oris use CtrA to repress replication. Despite this similarity, phylogenetic analysis unexpectedly shows that CtrA usage evolved separately among these Caulobacter oris. We discuss consensus oris and convergent ori evolution in differentiating bacteria.

The GenBank accession numbers for hemE ori Duf299 sequences of the FWC42, FWC17, FWC18 and MCS18 Caulobacter strains are EU327255, EU327256, EU327257 and EU327258, respectively.

Six supplementary figures, showing FWC ori DNA alignments, MCS ori DNA alignments, MCS10 ori plasmid AR, MCS10 ori plasmid abundance in CB15, MCS10 ori plasmid instability, and antibiotic selection and ori plasmid abundance, and four supplementary tables, listing bacterial strains and plasmids, PCR primers used to amplify replication origins, primers used for site-directed mutagenesis of pMCS10 ori, and DNA sequences analysed, together with supplementary information and references, are available with the online version of this paper.

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