Chromosome dynamics in bacteria: triggering replication at opposite location and segregation in opposite direction

Abstract

The accurate onset of chromosome replication and segregation are fundamental for the survival of the cell. In bacteria, regulation of chromosome replication lies primarily at the initiation step. The bacterial replication initiator DnaA recognizes the origin of replication (ori) and opens this double stranded site allowing for the assembly of the DNA replication machinery. Following the onset of replication initiation, the partitioning protein ParA triggers the onset of chromosome segregation by direct interactions with ParB-bound to the centromere. The subcellular organization of ori and centromere are maintained after the completion of each cell cycle. It remains unclear what triggers the onset of these key chromosome regulators DnaA and ParA. One potential scenario is that the microenvironment of where the onset of replication and segregation take place hosts the regulators that trigger the activity of DnaA and ParA. In order to address this, we analyzed whether the activity of DnaA and ParA are restricted to only one site within the cell. In non-dividing cells of the alpha proteobacterium Caulobacter crescentus, ori and centromere are found near the stalked pole. To test DnaA's ability to initiate replication away from the stalked pole, we engineered a strain where movement of ori was induced in the absence of chromosome replication. Our data show that DnaA can initiate replication of the chromosome independently of the subcellular localization of ori. Furthermore, we discovered that the partitioning protein ParA was functional and could segregate the replicated centromere in the opposite direction from the new pole toward the stalked pole. We showed that the organization of the ParA gradient can be completely reconstructed in the opposite orientation by rearranging the location of the centromere. Our data reveal the high flexibility of the machineries that trigger the onset of chromosome replication and segregation in bacteria. Our work also provides insights into the coordination between replication and segregation with the cellular organization of specific chromosomal loci.

ParA and its functions that go beyond chromosome segregation in Caulobacter crescentus

“…Notably, when cells contain sub-optimal levels of DnaA and high levels of ParA, the frequency of chromosome replication initiation increases as evidenced by the higher number of cells displaying two CFP-ParB foci. These data are consistent with our previous observation that parA overexpression results in cells initiating replication at sub-optimal DnaA levels [25].…”

“…Notably, when cells contain sub-optimal levels of DnaA and high levels of ParA, the frequency of chromosome replication initiation increases as evidenced by the higher number of cells displaying two CFP-ParB foci. These data are consistent with our previous observation that parA overexpression results in cells initiating replication at sub-optimal DnaA levels [25]. To investigate a potential mechanism that requires direct protein-protein interactions, we analyzed the localization of ParA and DnaA in cells that over-initiated chromosome replication.…”

“…Caulobacter initiates chromosome replication only once per cell cycle, regardless of the type of growth media (minimal vs. rich). We previously reported an intriguing observation where cells with high levels of the partitioning ParA protein were able to initiate chromosome replication in cells containing insufficient levels of the replication initiator DnaA [25]. This observation suggested a potential link between chromosome replication and chromosome segregation.…”

Stable inheritance of Sinorhizobium meliloti cell growth polarity requires an FtsN-like protein and an amidase

“…In many bacteria with polarly anchored oriC, such as C. crescentus, chromosome segregation relies on movement of the new oriC towards the new cell pole, enabled by interaction of oriCbound ParB with a ParA gradient 52 . In C. crescentus, polar localization of the ParB-ParS complex alone determines the orientation of the ParA gradient, required for oriC migration, independent of the pole descendance 53 . Our observation of successful oriC 2 migration in a major part of the S. meliloti cells with inverted growth polarity suggests that in S. meliloti the ParA gradient may be formed independent of the old-new pole arrangement and localization of the cell wall growth zone.…”