Cyclic mononucleotide- and Clr-dependent gene regulation in Sinorhizobium meliloti

To identify physiological processes affected by cAMP in the plant-symbiotic nitrogen-fixing α-proteobacterium Sinorhizobium meliloti Rm2011, cAMP levels were artificially increased by overexpression of its cognate adenylate/guanylate cyclase gene cyaJ. This resulted in high accumulation of cAMP in the culture supernatant, decreased swimming motility and increased production of succinoglycan, an exopolysaccharide involved in host invasion. Weaker, similar phenotypic changes were induced by overexpression of cyaB and cyaG1. Effects on swimming motility and succinoglycan production were partially dependent on clr encoding a cyclic AMP receptor-like protein. Transcriptome profiling of an cyaJ-overexpressing strain identified 72 upregulated and 82 downregulated genes. A considerable number of upregulated genes are related to polysaccharide biosynthesis and osmotic stress response. These included succinoglycan biosynthesis genes, genes of the putative polysaccharide synthesis nodP2-exoF3 cluster and feuN, the first gene of the operon encoding the FeuNPQ regulatory system. Downregulated genes were mostly related to respiration, central metabolism and swimming motility. Promoter-probe studies in the presence of externally added cAMP revealed 18 novel Clr-cAMP-regulated genes. Moreover, the addition of cGMP into the growth medium also promoted clr-dependent gene regulation. In vitro binding of Clr-cAMP and Clr-cGMP to the promoter regions of SMc02178, SMb20906,SMc04190, SMc00925, SMc01136 and cyaF2 required the DNA motif (A/C/T)GT(T/C)(T/C/A)C (N4) G(G/A)(T/A)ACA. Furthermore, SMc02178, SMb20906,SMc04190and SMc00653 promoters were activated by Clr-cAMP/cGMP in Escherichia coli as heterologous host. These findings suggest direct activation of these 7 genes by Clr-cAMP/cGMP.