No abstract

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.

SummarySinorhizobium meliloti is a nitrogen-fixing bacterial symbiont of alfalfa and related legumes. Symbiotic infection by S. meliloti requires an osmosensory twocomponent system composed of the response regulator FeuP and the sensor kinase FeuQ. The FeuPQ pathway positively regulates transcription of multiple genes including ndvA, which encodes the cyclic glucan exporter. Here we show that proper regulation of this signalling pathway is essential for cell viability. Without the small 83 amino acid protein FeuN, S. meliloti cells are unable to grow, and this phenotype is dependent on the FeuPQ pathway. Using Escherichia coli as a heterologous system, we show that expression of feuP and feuQ leads to a dramatic increase in ndvA promoter activity, but that simultaneous expression of feuN abrogates this effect. Random mutagenesis of the feuPQ bicistron revealed a defined region of the FeuQ protein in and around its two predicted transmembrane domains that are required for FeuNdependent signalling modulation. Marker enzyme fusion experiments indicate that most of the FeuN polypeptide is localized to the periplasm. Our data support a model in which FeuN interacts directly with FeuQ to attenuate phosphorylation of FeuP, and that without this activity, hyperactive signalling through FeuPQ results in cessation of growth or death.