The Sinorhizobium meliloti nitrogen-fixing symbiosis requires CbrA-dependent regulation of a DivL and CckA phosphorelay.

The cell cycle is a fundamental process involved in bacterial reproduction and cellular differentiation. For Sinorhizobium meliloti, cell cycle outcomes depend on its growth environment. This bacterium shows a tight coupling of DNA replication initiation with cell division during free-living growth. In contrast, it undergoes a novel program of endoreduplication and terminal differentiation during symbiosis within its host. While several DivK regulators at the top of its CtrA pathway have been shown to play an important role in this differentiation process, there is a lack of resolution regarding the downstream molecular activities required and whether they could be unique to the symbiosis cell cycle. The DivK kinase CbrA is a negative regulator of CtrA activity and is required for successful symbiosis. In this work, spontaneous symbiosis suppressors of ΔcbrA were identified as alleles of divL and cckA. In addition to rescuing symbiotic development, they restore wild-type cell cycle progression to free-living ΔcbrA cells. Biochemical characterization of the S. meliloti hybrid histidine kinase CckA in vitro demonstrates that it has both kinase and phosphatase activities. Specifically, CckA on its own has autophosphorylation activity, and phosphatase activity is induced by the second messenger c-di-GMP. Importantly, the CckA^A373S suppressor protein of ΔcbrA has a significant loss in kinase activity, and this is predicted to cause decreased CtrA activity in vivo. These findings deepen our understanding of the CbrA regulatory pathway and open new avenues for further molecular characterization of a network pivotal to the free-living cell cycle and symbiotic differentiation of S. meliloti.IMPORTANCESinorhizobium meliloti is a soil bacterium able to form a nitrogen-fixing symbiosis with certain legumes, including the agriculturally important Medicago sativa. It provides ammonia to plants growing in nitrogen-poor soils and is therefore of agricultural and environmental significance as this symbiosis negates the need for industrial fertilizers. Understanding mechanisms governing symbiotic development is essential to either engineer a more effective symbiosis or extend its potential to non-leguminous crops. Here, we identify mutations within cell cycle regulators and find that they control cell cycle outcomes during both symbiosis and free-living growth. As regulators within the CtrA two-component signal transduction pathway, this study deepens our understanding of a regulatory network shaping host colonization, cell cycle differentiation, and symbiosis in an important model organism.