Characterization of an alternative ECF-like RNA polymerase sigma factor double mutant of the legume-interacting nitrogen-fixing bacterial symbiont Sinorhizobium meliloti

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The soil-dwelling alpha-proteobacterium Sinorhizobium meliloti can establish a symbiotic interaction with the legume plant Medicago truncatula. During this interaction, root nodules in which the bacteria proliferate and fix atmospheric dinitrogen to make it available for plant metabolism are formed. The invasion of plant cells occurs through root hairs via a specialized structure, the infection thread, built by both plant and bacterial contributions. In the nodule tissue the bacteria are released into the cytoplasm of root cells, still enclosed by a plant-derived membrane. The molecular mechanisms of bacterial release are not well understood to date. To respond to environmental changes and stimuli, organisms need to undergo transcriptional adjustments. In this respect, sigma factors, co-factors for the core RNA polymerase play an important role in recognizing special promoter sets belonging to genes of shared functions. One subgroup of alternative sigma factors comprises the extracytoplasmic function (ECF) sigma factors. In S. meliloti there are ten ECF-type sigma factors of the RpoE kind. Mutant analyses of all single and all possible double mutants revealed that the rpoE6 rpoE8 double mutant is impaired in bacterial release from the infection thread into nodule cells. Also, no nitrogen-fixation takes place in nodules formed by this mutant. The ECF sigma factors from S. meliloti have not been studied in detail before and their corresponding target genes are good candidates to be involved in the release of bacterial cells into plant root cells. The objectives of this research proposal are to perform a detailed phenotypic analysis of the mutant, transcription profiling and the identification of target genes of RpoE6 and RpoE8. The phenotype analyses will include observation of the infection process by various microscopy techniques to reveal all defects of the mutant as well as a general observation of the plant habitus. Differential gene expression studies will provide a large data set of genes directly or indirectly dependent on rpoE6 and/or rpoE8. Subsequent verification of these potential targets and the generation of the corresponding bacterial mutants and their analyses regarding symbiosis performance will provide insight about their role and function at the step of bacterial cell release from the infection thread. This will provide new information about the essential mechanism of how bacteria enter the nodule tissue for final nitrogen-fixation.