The symbiosis regulator CbrA modulates a complex regulatory network affecting the flagellar apparatus and cell envelope proteins

Abstract

Sinorhizobium meliloti participates in a nitrogen-fixing symbiosis with legume plant host species of the genera Medicago, Melilotus, and Trigonella. We recently identified an S. meliloti two-component sensory histidine kinase, CbrA, which is absolutely required to establish a successful symbiosis with Medicago sativa (K. E. Gibson, G. R. Campbell, J. Lloret, and G. C. Walker, J. Bacteriol. 188:4508-4521, 2006). In addition to having a symbiotic defect, the cbrA::Tn5 mutant also has free-living phenotypes that suggest a cell envelope perturbation. Because the bases for these phenotypes are not well understood, we undertook an identification of CbrA-regulated genes. We performed a microarray analysis and compared the transcriptome of the cbrA::Tn5 mutant to that of the wild type. Our global analysis of gene expression identified 162 genes that are differentially expressed in the cbrA::Tn5 mutant, including those encoding proteins involved in motility and chemotaxis, metabolism, and cell envelope function. With regard to those genes with a known role in symbiosis, we observed increased expression of nine genes with overlapping functions in bacterial invasion of its host, which suggests that the mutant could be competent for invasion. Since these CbrA-repressed genes are vital to the invasion process, it appears that down-regulation of CbrA activity is important at this stage of nodule development. In contrast, our previous work showed that CbrA is required for bacteria to establish themselves within the host as nitrogen-fixing symbionts. Therefore, we propose a model in which CbrA functions as a developmental switch during symbiosis.

FIG. 1.

CbrA plays an important role in growth phase regulation of bioS. β-Glucuronidase activity expressed from a bioS′-uidA⁺ fusion was measured from either exponential phase or stationary phase wild-type and cbrA::Tn5 cultures. The uidA⁺ transcription fusion serves as a reporter for promoter activity and also disrupts gene function.

FIG. 2.

CbrA regulates the expression of genes predicted to affect c-di-GMP signaling. β-Glucuronidase activity was measured from stationary phase wild-type and cbrA::Tn5 cultures. The uidA⁺ transcription fusion present in each strain is indicated below the graph. The SMc00888′-uidA⁺ fusion was integrated in order to leave the gene intact while the SMc00887′-uidA⁺ fusion disrupted the native gene.

FIG. 3.

CbrA regulates flagellar motility and chemotaxis. (a) β-Glucuronidase activity was measured from stationary phase wild-type and cbrA::Tn5 cultures. The uidA⁺ transcription fusion present in each strain, indicated below the graph, was integrated into the genome in order to leave gene function intact. (b) Flagellar filaments isolated from wild-type and cbrA::Tn5 stationary phase cultures were analyzed by SDS-PAGE and Coomassie protein detection. (c) The swimming ability of wild-type and cbrA::Tn5 bacteria was assessed after 3 days' incubation on LB/MC medium supplemented with 0.22% agar.

FIG. 4.

Genes encoding secreted outer-membrane and extracellular proteins are highly enriched within the pool of CbrA-regulated genes. The subcellular localization of each protein was predicted using the PSORT algorithm. (a) The percentage of S. meliloti proteins predicted to be localized to each subcellular compartment or secreted extracellularly. (b) The percentage of genes differentially expressed in the cbrA::Tn5 mutant whose protein products are known or predicted to be localized to each subcellular compartment or secreted extracellularly. A few genes discussed in the text have been highlighted.

FIG. 5.

CbrA regulates the expression of genes encoding outer membrane proteins. β-Glucuronidase activity was measured from stationary phase wild-type and cbrA::Tn5 cultures. The uidA⁺ transcription fusion present in each strain is indicated below the graph. The SMc00489 fusion disrupts gene function while the ropB1 fusion leaves the gene intact.