STUDIES OF THE STRUCTURE, REGULATION AND FUNCTION OF THE FIXABC GENES OF RHIZOBIUM (MELILOTI, NITROGEN FIXATION, PARASPONIA, NIF)

The Gram-negative bacteria of the genera Rhizobium and Bradyrhizobium fix nitrogen in a symbiotic association with nodules on the roots of legumes. The protein products of certain Rhizobium genes are known to be essential for symbiotic nitrogen fixation. This thesis describes the study at the structural, genetic and physiological levels of three such genes, fixA, B and C, from two species, R. meliloti and Parasponia Rhizobium.; I have sequenced the entire fixABC operon of R. meliloti, and determined the operon structure and molecular weights of the gene products. I have also determined that DNA homologous to this operon exists in all other rhizobia examined. In addition, fixABC homology was discovered with Azotobacter vinelandii, a free-living, aerobic, nitrogen fixing species. The fixABC genes do not bear any physical or genetic complementarity, however, with the well-characterized nif genes of the free-living diazotroph Klebsiella pneumoniae.; I also analyzed the expression of the R. meliloti fixA promoter in a non-homologous Eschericia coli background. In an E. coli host, the R. meliloti nifA gene product (positive regulator of nif and fix genes) activated transcription from the R. meliloti nifH (nitrogenase reductase) promoter, but failed to significantly activate the fixA promoter. This effect is believed to be the result of incompatibility between the E. coli RNA polymerase, the E. coli ntrA sigma factor (specific for nitrogen assimilation promoters), and the R. meliloti fixA promoter.; Parasponia Rhizobium derepresses its nitrogen fixation genes ex planta. Its fixB and fixC genes were mutagenized with transposon Tn5. These mutants had a Nif('-) phenotype ex planta and a Fix('-) phenotype in siratro nodules, indicating that the fixB and fixC genes are directly involved in the biochemical process of nitrogen fixation.