The long term goal of our research is to try and understand how the bacterium Rhizobium meliloti establishes a nitrogen-fixing symbiosis with its legume host alfalfa. During symbiosis, a complex developmental program governs the progression of R. meliloti from free-living bacterial cells to intracellular, altered forms called bacteroids. We have previously shown that a broad array of symbiotically essential genes, normally activated at a late stage of development, are also induced by microaerobiosis. Since bacteroids exist within a microaerobic environment inside legume nodules, decreased oxygen availability must constitute a major developmental signal. The specific goal of this research proposal is to try and understand, at the molecular level, how oxygen regulates the expression of various nitrogen fixation (nif or fix) genes. Twenty-three such genes are known, and twenty-one of these are regulated in response to oxygen availability by two recently identified proteins that constitute a new two component regulatory system: FixL (sensor) and FixJ (regulator). We outline plans here to conduct detailed structure/function studies on FixL and FixJ in the belief that these two proteins represent a new and valuable model system for studying oxygen regulation of gene expression. Both FixL and FixJ will be subjected to random and site-specific mutagenesis, deletion analysis, purification, and study in vivo and in vitro. Protein domains or amino acid residues predicted to have specific functional significance based on similarities with other two component systems will be confirmed as such. Of particular interest will be investigations into how the bifunctional regulator FixL senses oxygen and conveys this information to FixJ via phosphorylation/dephosphorylation. The role of membrane attachment and cellular redox state in FixL function will be examined. Features of target promoters that are critical for induction by FixLJ will be identified. Since FixLJ can function in E. coli, studies will initially be carried out in this organism. FixL or FixJ mutants that are generated in E. coli will be examined in R. meliloti for their effects on 02 sensing and bacteroid development using RK2-based broad host range plasmid vectors. Efforts will also be made to identify other genes that are microaerobically induced using a promoter bank cloned into a broad host range plasmid vector carrying a firefly luciferase reporter gene. This promoter bank will also be used to examine global changes in gene expression that occur during the transition of R. meliloti from aerobic to microaerobic growth and from bacterium to bacteroid.
