Identification of Regulatory Mechanisms that Contribute to Novel Cell Cycle Outcomes During Host-Microbe Symbiosis

Sinorhizobium meliloti has emerged as a key model organism for understanding the link between cell cycle regulation and chronic host colonization because it undertakes novel cell cycle outcomes during symbiosis within the root nodules of legumes. The DivJ-like histidine kinase, CbrA, is a necessary regulator for both symbiosis and cell cycle progression. CbrA indirectly regulates the activity and degradation of CtrA, a master cell cycle transcription factor. We initially isolated a spontaneous ΔcbrA symbiosis suppressor strain from a rare phenotypically wild type root nodule, which contains a mutation that suppresses all symbiotic and free-living cell cycle defects of the ΔcbrA mutant. Whole genome sequencing identified the mutation responsible for ΔcbrA suppression to be located within a PAS domain of divL (divLQ362P), which encodes a modified histidine kinase that likely mediates CbrA regulation of CtrA by serving as a bridge between the CbrA cognate response regulator DivK and the CckA/ChpT phosphorelay. The divLQ362P mutant shows temperature-sensitivity, a slow-growth defect, and a lag in the G2 phase of the cell cycle. However, divLQ362P does not appear to greatly affect endogenous CtrA levels. Using the slow growth phenotype, suppressors of divLQ362P have been isolated in order to better understand the function of the DivL PAS domain in cell cycle regulation. Ten additional ΔcbrA symbiosis suppressor strains were isolated. These ten strains fall into three linkage groups and have been characterized further. One group also suppresses ΔcbrA free-living cell cycle defects via a mutation (divLV787M) in the predicted HATPase_c domain of DivL. The other two groups do not fully suppress ΔcbrA free-living cell cycle defects and appear to be symbiosis-specific. Western blot analysis has revealed these symbiosis-specific suppressors do not restore CtrA levels to that of wild type and retain some cell morphology defects. This work aims to provide evidence of how the free-living cell cycle is connected with the development of symbiosis by using a direct forward genetics approach to probe the symbiosis in the context of the cell cycle