Regulation of Azorhizobium caulinodans ORS571 nitrogen fixation (nif/fix) genes.

Biological nitrogen fixation is the microbial process by which atmospheric dinitrogen (N _{<font size="-1">2</font>} ) is reduced to ammonia. In all microbes studied, dinitrogen reduction is catalyzed by a highly conserved enzyme complex, called nitrogenase. The nitrogenase subunits and functions required for nitrogenase assembly and activity are encoded by the nitrogen fixation (nif/fix) genes.Nitrogen-fixing organisms can be roughly divided into two major groups: the free-living nitrogen fixing (diazotrophic) species, such as Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus, and symbiotically N _{<font size="-1">2</font>} fixing organisms, such as Rhizobium species and Bradyrhizobium japonicum.The fate of the ammonia produced by these two distinct groups of nitrogen fixing organisms is quite different. While free-living nitrogen-fixing organisms will assimilate the ammonia produced for their own growth (diazotrophy), the strictly symbiotically nitrogen-fixing organisms (predominantly) excrete the ammonia into the cytoplasm of infected plant cells, to be assimilated by the host. The regulation of nitrogen fixation (nif/fix) gene expression is quite different as well, and has been difficult to compare directly between diazotrophs and strictly symbiotic nitrogen fixing organisms. The discovery of Azorhizobium caulinodans ORS571, a tropical rhizobium capable of both diazotrophy and symbiotic nitrogen fixation in stem- and root nodules induced on its host, the tropical shrub Sesbania rostrata, changed this situation and has allowed a direct comparison of (nif/fix) gene regulation in the diazotrophic versus symbiotic state. This unusual organism was chosen for the research reported in this thesis, which focuses on the expression of the central (nif/fix) regulatory gene nifA , a gene which is essential for diazotrophic growth and symbiotic nitrogen fixation, and which responds to a complex regulatory circuit.In chapter 2 a current review of the regulation of nitrogen fixation genes is presented. The review focuses on the regulation of nifA gene expression and NifA activity in different bacterial species.In chapter 3 the involvement of ntr-mediated control of the nifA promoter via the -24/-12 element is analyzed by site-specific mutagenesis and chimeric nifA - lacZ reporter gene fusions integrated into the A. caulinodans chromosome. The -24/-12 promoter element was shown to be important for nifA gene expression suggesting the involvement of a σ54 (NtrA; RpoN)-type transcription factor in nifA gene regulation.Chapter 4 addresses the involvement of a ntrA(rpoN) -like sigma factor in nifA expression, and reports the cloning and analysis of an A. caulinodans ntrA(rpoN) gene. Although the ntrA(rpoN) gene identified in this study was shown to control the expression of some of the A.caulinodans nif genes (like nifHDK) and nitrate assimilation genes, it did not appear to regulate the expression of nifA , suggesting the presence of an additional ( nifA specific) ntrA ( rpoN )-equivalent gene.To search for trans -acting factors involved in the regulation of the nifA expression, the in vitro interaction of proteins in A. caulinodans crude cell extracts with the nifA regulatory region was studied by gel retardation assays. Chapter 5 presents the binding of (a) sequence-specific protein(s) in extracts of A.caulinodans to the nifA 5' upstream region. However the nature of the protein(s) and the exact location of the binding site in the nifA 5' upstream region remains to be determined.During the preparation of crude cell extracts from A. caulinodans fi xLJ and fixK mutant strains, large amounts of a red/pink, UV fluorescent pigment was observed in the culture medium. Chapter 6 describes the isolation and characterization of this pigment.Chapter 7 describes the isolation and characterization of an leuA -like gene of A.caulinodans which was incidentally identified with an Escherichia coli fnr probe. Comparison of the 5' upstream region of the A. caulinodans leuA gene with the leu operons of other organisms suggest a conserved regulation mechanism for the expression (transcription attenuation), found in many amino acid biosynthetic operons.Finally in chapter 8 the current model for nitrogen fixation gene regulation in A.caulinodans, deduced from the studies presented in this thesis and previous data, is presented