Sinorhizobium meliloti is alternately capable of colonizing the soil as a free-living bacterium or establishing a chronic intracellular infection with its legume host for the purpose of nitrogen fixation. We previously identified the S. meliloti two-component sensor histidine kinase CbrA as playing an important role in regulating exopolysaccharide production, flagellar motility and symbiosis. Phylogenetic analysis of CbrA has highlighted its evolutionary relatedness to the Caulobacter crescentus sensor histidine kinases PleC and DivJ, which are involved in CtrAdependent cell cycle regulation through the shared response regulator DivK. We therefore became interested in testing whether CbrA plays a role in regulating S. meliloti cell cycle processes. We find the loss of cbrA results in filamentous cell growth accompanied by cells that contain an aberrant genome complement, indicating CbrA plays a role in regulating cell division and possibly DNA segregation. S. meliloti DivK localizes to the old cell pole during distinct phases of the cell cycle in a phosphorylation-dependent manner. Loss of cbrA results in a significantly decreased rate of DivK polar localization when compared with the wild-type, suggesting CbrA helps regulate cell cycle processes by modulating DivK phosphorylation status as a kinase. Consistent with a presumptive decrease in DivK phosphorylation and activity, we also find the steady-state level of CtrA increased in cbrA mutants. Our data therefore demonstrate that CbrA contributes to free-living cell cycle regulation, which in light of its requirement for symbiosis, points to the potential importance of cell cycle regulation for establishing an effective host interaction.

CbrA is a DivJ/PleC-like histidine kinase of DivK that is required for cell cycle progression and symbiosis in the alphaproteobacterium Sinorhizobium meliloti. Loss of cbrA results in increased levels of CtrA as well as its phosphorylation. While many of the known Caulobacter crescentus regulators of CtrA phosphorylation and proteolysis are phylogenetically conserved within S. meliloti, the latter lacks the PopA regulator that is required for CtrA degradation in C. crescentus. In order to investigate whether CtrA proteolysis occurs in S. meliloti, CtrA stability was assessed. During exponential growth, CtrA is unstable and therefore likely to be degraded in a cell cycle-regulated manner. Loss of cbrA significantly increases CtrA stability, but this phenotype is restored to that of the wild type by constitutive ectopic expression of a CpdR1 variant that cannot be phosphorylated (CpdR1 D53A ). Addition of CpdR1 D53A fully suppresses cbrA mutant cell cycle defects, consistent with regulation of CtrA stability playing a key role in mediating proper cell cycle progression in S. meliloti. Importantly, the cbrA mutant symbiosis defect is also suppressed in the presence of CpdR1 D53A . Thus, regulation of CtrA stability by CbrA and CpdR1 is associated with free-living cell cycle outcomes and symbiosis. IMPORTANCEThe cell cycle is a fundamental process required for bacterial growth, reproduction, and developmental differentiation. Our objective is to understand how a two-component signal transduction network directs cell cycle events during free-living growth and host colonization. The Sinorhizobium meliloti nitrogen-fixing symbiosis with plants is associated with novel cell cycle events. This study identifies a link between the regulated stability of an essential response regulator, free-living cell cycle progression, and symbiosis. Sinorhizobium meliloti is a member of the class Alphaproteobacteria that grows free-living in the soil or as a beneficial nitrogen-fixing symbiont in association with legumes in the genera Medicago, Melilotus, and Trigonella. As a free-living organism, S. meliloti undergoes an asymmetric cell division with once-andonly-once DNA replication per cell cycle (1, 2, 3). However, inside its host, this bacterium undergoes differentiation into a bacteroid that includes a novel cell cycle program of repeated DNA replication in the absence of cell division (endoreduplication) (3). The underlying molecular mechanisms that dictate cell cycle progression and differentiation in S. meliloti remain to be explored in detail and therefore represent a novel aspect of symbiont physiology. Known and putative S. meliloti cell cycle regulators are conserved among other members of the Alphaproteobacteria that also specialize in chronic colonization of eukaryotic hosts, in particular rhizobial species in the genera Agrobacterium, Bartonella, and Brucella (2, 4). Thus, a deeper understanding of S. meliloti cell cycle regulation will provide insight into processes that are broadly important to both cell cycle ...

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