Direct regulation of cell cycle regulatory gene expression by NtrX to promote Sinorhizobium meliloti cell division

Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by a signaling network centered on CtrA. The gene expression of regulatory proteins in the network can be regulated by nutrient signaling systems. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes in the CtrA signaling network. Three groups of S. meliloti ntrX mutants showed similar cell division defects, such as slow growth, abnormal morphology of some cells and delayed DNA synthesis. Quantitative RT-PCR assays indicated that in these mutants, the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZ1 was down-regulated. Western blotting showed that the CtrA and GcrA proteins were apparently increased in the mutants. The 53rd aspartate conserved in NtrX homologs can be phosphorylated in vitro and in vivo. The phosphorylated NtrX protein can bind directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN1-5TTG. Therefore, phosphorylation of NtrX is essential for cell cycle regulation of S. meliloti. We expressed the NtrX protein carrying a phosphorylation site substitution in Agrobacterium tumefaciens and found that the expressed strains had different growth phenotypes, suggesting that NtrX also regulates cell division in this bacterial species. Our findings reveal that NtrX acts as a transcriptional regulator that positively affects bacterial cell division, associated with nitrogen metabolism. IMPORTANCESinorhizobium meliloti infects the host legume alfalfa and induces the formation of nitrogen-fixing nodules. The proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of the interaction between symbiotic partners. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but the molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.