Sinorhizobium meliloti CtrA Stability Is Regulated in a CbrA-Dependent Manner That Is Influenced by CpdR1

Abstract

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 ...

An Adaptor Hierarchy Regulates Proteolysis during a Bacterial Cell Cycle

“…As PopA is responsive to the second messenger cyclic di-GMP, levels of which oscillate during the cell cycle (Abel et al, 2013; Lori et al, 2015), our work suggests that CpdR and RcdA may represent an ancestral adaptor complex that has been co-opted by Caulobacter for coupling second messenger cues to cell cycle progression. In S. meliloti , it is clear that adaptors play a crucial role in the symbiosis transition, where misregulation of CpdR dramatically affects proper nodule formation and plant growth (Kobayashi et al, 2009; Pini et al, 2015; Schallies et al, 2015). In general, bacterial development (such as the morphological transition of Caulobacter or sporulation of Bacillus ) requires changes in proteome composition within a single generation.…”

Regulated Proteolysis in Bacteria:Caulobacter

“…For example, in the plant symbiont Sinorhizobium meliloti , there are two orthologs of CpdR, but only one (CpdR1) exhibits physiological defects when either deleted or overactivated (73). CpdR1 appears to play a role in controlling CtrA stability, which is particularly important in the endoreduplication process during symbiosis (98; 110). Less is know about the role of RcdA in S. meliloti, but depletion of RcdA increases CtrA levels (98) supporting its role in controlling CtrA stability similar to that seen in Caulobacter .…”

Direct regulation of cell cycle regulatory gene expression by NtrX to promoteSinorhizobium meliloticell division

“…Due to the conservation of the CtrA regulatory system in αproteobacteria, some homologous genes encoding components of this system that function in cell cycle regulation, such as ctrA, ccrM, cpdR1, divJ, divK, gcrA, and pleC, have been identified in Sinorhizobium meliloti (the symbiont of Medicago species) (12)(13)(14). Additionally, a few specific genes, such as cbrA, are essential for cell cycle regulation through interplay with the CtrA system (15,16).…”

“…Since S. meliloti and C. crescentus belong to -proteobacteria, based on the research results of C. crescentus, with the aid of DNA sequence homology analysis, many cell cycle regulatory genes such as ctrA, ccrM, cpdR1, divJ, divK, gcrA and pleC, have been identified in S. meliloti (12)(13)(14). In addition, the hybrid histidine kinase CbrA is linked to the CtrA regulatory system, which is an important regulator of cell division in S. meliloti (15,16). However, it is still unclear whether these regulatory proteins conduct environmental nutrition signals (e.g., ammonia nitrogen) and whether they play a regulatory role in the symbiotic process.…”