The Rhizobium etli response regulator CenR is essential for both: Free-life and the rhizobial nitrogen-fixing symbiosis

The canonical two-component systems (TCS) consist of a histidine kinase and a response regulator that work together to control various pathways in bacteria. Rhizobia are rod-shaped, Gram-negative alpha-proteobacteria capable of establishing a nitrogen-fixing symbiosis with compatible legume hosts. These bacteria can live freely in the soil or as intracellular symbionts within root nodules. Here, we characterized an orphan OmpR-type response regulator in Rhizobium etli CE3, which we renamed CenR due to its similarity to CenR proteins known as essential regulators of cell envelope-related functions in alpha-proteobacteria. We identified the cognate histidine kinase encoded by cenK, located in a separate genomic region from cenR. CenR and CenK form a TCS that has not been previously reported in Rhizobium. Our results indicate that the overexpression of cenR as well as the absence of cenK, negatively impacts R. etli growth and cell morphology, while bacteria overexpressing cenR also exhibit uncoordinated cell division. Furthermore, we demonstrated that the CenKR TCS directly or indirectly regulates the expression of essential genes involved in pathways that control cell growth and morphology. Electrophoretic mobility shift assays confirmed that CenR binds directly to the promoter regions of two uncharacterized genes in R. etli. Furthermore, analysis of the R. etli - common bean (Phaseolus vulgaris) symbiosis revealed increased infection threads, reduced leghemoglobin content, and lower nitrogen fixation efficiency in nodules infected by the cenR-overexpressing strain. In conclusion, our findings revealed that the CenKR TCS coordinates important cell cycle events in Rhizobium that are vital for both free-living and symbiotic conditions.