RK2 plasmid dynamics in Caulobacter crescentus cells - two modes of DNA replication initiation

Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work we analyzed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of . Our microscopic observations showed asymmetric distribution of plasmid RK2 foci between the two compartments of predivisional cells, resulting in asymmetric allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per foci is approximately equal in both swarmer and predivisional cells. Analysis of the dynamics of TrfA- complex formation during the Caulobacter cell cycle revealed that TrfA binds primarily during the G1 phase, however plasmid DNA synthesis occurs during the S and G2 phases of the cell cycle. Both and analysis of RK2 replication initiation in cells demonstrated that it is independent of the DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation was also possible.