The soil-dwelling ␣-proteobacterium Sinorhizobium meliloti engages in a symbiosis with legumes: S. meliloti elicits the formation of plant root nodules where it converts dinitrogen to ammonia for use by the plant in exchange for plant photosynthate. To study the coordinate differentiation of S. meliloti and its legume partner during nodule development, we designed a custom Affymetrix GeneChip with the complete S. meliloti genome and Ϸ10,000 probe sets for the plant host, Medicago truncatula. Expression profiling of free-living S. meliloti grown with the plant signal molecule luteolin in defined minimal and rich media or of strains altered in the expression of key regulatory proteins (NodD1, NodD3, and RpoN) confirms previous data and identifies previously undescribed regulatory targets. Analyses of root nodules show that this Symbiosis Chip allows the study of gene expression in both partners simultaneously. Our studies detail nearly 5,000 transcriptome changes in symbiosis and document complex transcriptional profiles of S. meliloti in different environments.expression ͉ Medicago ͉ microarray ͉ Sinorhizobium meliloti

ABSTRACTSinorhizobium melilotican live as a soil saprophyte and can engage in a nitrogen-fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma (σ) factors interacting with core RNA polymerase. TheS. melilotigenome encodes 14 of these alternative σ factors, including two putative RpoH (“heat shock”) σ factors. We used custom Affymetrix symbiosis chips to characterize the global transcriptional response ofS. melilotirpoH1,rpoH2, andrpoH1 rpoH2mutants during heat shock and stationary-phase growth. Under these conditions, expression of over 300 genes is dependent onrpoH1andrpoH2. We mapped transcript start sites of 69rpoH-dependent genes using 5′ RACE (5′ rapid amplification of cDNA ends), which allowed us to determine putative RpoH1-dependent, RpoH2-dependent, and dual-promoter (RpoH1- and RpoH2-dependent) consensus sequences that were each used to search the genome for other potential direct targets of RpoH. The inferredS. melilotiRpoH promoter consensus sequences share features ofEscherichia coliRpoH promoters but lack extended −10 motifs.

SummaryA screen for novel symbiotic mutants of the nitrogenfixing legume symbiont Sinorhizobium meliloti uncovered a crucial role for the putative response regulator FeuP in the symbiotic infection process. Transcriptome analysis shows that FeuP controls the transcription of at least 16 genes, including ndvA, which encodes an ATP-dependent exporter of cyclic b glucans. Loss of feuP function gives rise to traits associated with cyclic b glucan biosynthetic defects, including poor growth and motility under hypoosmotic conditions, and the inability to invade plant tissue during the early stages of symbiotic infection. Analysis of cyclic glucans indicates that the feuP mutant is able to synthesize intracellular cyclic b glucans, but is unable to export them. Cyclic b glucan export can be restored to feuP mutant cells by constitutive expression of ndvA; likewise, the symbiotic phenotype of a feuP mutant is rescued by ectopic ndvA expression. We further show that the linked sensor kinase gene, feuQ, is also important for modulating ndvA transcription, and that signalling through the FeuP/FeuQ pathway is responsive to extracellular osmotic conditions, with low osmolarity stimulating ndvA expression.

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.As a model system for studying methods that invasive bacteria employ to transition from a free-living environment to their niche within the host, we explored how the gram-negative bacterium Sinorhizobium meliloti establishes a nitrogen-fixing symbiosis within the roots of leguminous plants such as Medicago sativa.