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 analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical 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 focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2.

It has been demonstrated that plasmids are not randomly distributed but are located symmetrically in mid-cell, or (1/4), (3/4) positions in bacterial cells. In this work we compared the localization of broad-host-range plasmid RK2 mini-replicons, which lack an active partitioning system, in Escherichia coli and Pseudomonas putida cells. In E. coli the location of the plasmid mini-replicon cluster was at the cell poles. In contrast, in Pseudomonas cells, as a result of the interaction of chromosomally encoded ParB protein with RK2 centromere-like sequences, these mini-derivatives were localized in the proximity of mid-cell, or (1/4), (3/4) positions. The expression of the Pseudomonas parAB genes in E. coli resulted in a positional change in the RK2 mini-derivative to the mid-cell or (1/4), (3/4) positions. Moreover, in a P. putida parAB mutant, both RK2 mini-derivatives and the entire RK2 plasmid exhibited disturbances of subcellular localization. These observations raise the possibility that in certain bacteria chromosomally encoded partitioning machinery could affect subcellular plasmid positioning.