To let go or not to let go: how ParA can impact the release of the chromosomal anchoring in Caulobacter crescentus.

Chromosomal maintenance is vital for the survival of bacteria. In Caulobacter crescentus, chromosome replication initiates at ori and segregation is delayed until the nearby centromere-like region parS is replicated. Our understanding of how this sequence of events is regulated remains limited. The segregation of parS has been shown to involve multiple steps including polar release from anchoring protein PopZ, slow movement and fast ParA-dependent movement to the opposite cell pole. In this study, we demonstrate that ParA's competing attractions from PopZ and from DNA are critical for segregation of parS. Interfering with this balance of attractions-by expressing a variant ParA-R195E unable to bind DNA and thus favoring interactions exclusively between ParA-PopZ-results in cell death. Our data revealed that ParA-R195E's sole interactions with PopZ obstruct PopZ's ability to release the polar anchoring of parS, resulting in cells with multiple parS loci fixed at one cell pole. We show that the inability to separate and segregate multiple parS loci from the pole is specifically dependent on the interaction between ParA and PopZ. Collectively, our results reveal that the initial steps in chromosome segregation are highly regulated.

To let go or not to let go: how ParA can impact the release of the chromosomal anchoring in Caulobacter crescentus.

Chromosomal maintenance is vital for the survival of bacteria. In Caulobacter crescentus, chromosome replication initiates at ori and segregation is delayed until the nearby centromere-like region parS is replicated. Our understanding of how this sequence of events is regulated remains limited. The segregation of parS has been shown to involve multiple steps including polar release from anchoring protein PopZ, slow movement, and fast ParA-dependent movement to opposite cell pole. In this study, we demonstrate that ParA's competing attractions from PopZ and from DNA are critical for segregation of parS. Interfering with this balance of attractions - by expressing a variant ParA-R195E unable to bind DNA and thus favoring interactions exclusively between ParA-PopZ - results in cell death. Our data revealed that ParA-R195E's sole interactions with PopZ obstruct PopZ's ability to release the polar anchoring of parS resulting in cells with multiple parS loci fixed at one cell pole. We show that the inability to separate and segregate multiple parS loci from the pole is specifically dependent on the interaction between ParA and PopZ. Interfering with interactions between PopZ and the partitioning protein ParB, which is the interaction that anchors parS at the cell pole, does not rescue the ability of cells to separate the fixed parS loci when expressing parA-R195E. Thus, ParA and PopZ appear to have a distinct conversation from ParB yet can impact the release of ParB-parS from the anchoring at the cell pole. Collectively, our results reveal that the initial steps in chromosome segregation are highly regulated.

ParA's Impact beyond Chromosome Segregation in Caulobacter crescentus.

Maintaining proper chromosome inheritance after the completion of each cell cycle is paramount for bacterial survival. Mechanistic details remain incomplete for how bacteria manage to retain complete chromosomes after each cell cycle. In this study, we examined the potential roles of the partitioning protein ParA on chromosomal maintenance that go beyond triggering the onset of chromosome segregation in Caulobacter crescentus. Our data revealed that increasing the levels of ParA result in cells with multiple origins of replication in a DnaA-ATP-dependent manner. This ori supernumerary is retained even when expressing variants of ParA that are deficient in promoting chromosome segregation. Our data suggest that in Caulobacter ParA's impact on replication initiation is likely indirect, possibly through the effect of other cell cycle events. Overall, our data provide new insights into the highly interconnected network that drives the forward progression of the bacterial cell cycle. IMPORTANCE The successful generation of a daughter cell containing a complete copy of the chromosome requires the exquisite coordination of major cell cycle events. Any mistake in this coordination can be lethal, making these processes ideal targets for novel antibiotics. In this study, we focused on the coordination between the onset of chromosome replication, and the partitioning protein ParA. We demonstrate that altering the cellular levels of ParA causes cells to accumulate multiple origins of replication in Caulobacter crescentus. Our work provides important insights into the complex regulation involved in the coordination of the bacterial cell cycle.