<p>Biological nitrogen fixation is the microbial process by which atmospheric dinitrogen (N <sub><font size="-1">2</font></sub> ) 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 <em>(nif/fix)</em> genes.<p>Nitrogen-fixing organisms can be roughly divided into two major groups: the free-living nitrogen fixing (diazotrophic) species, such as <em>Klebsiella pneumoniae, Azotobacter vinelandii</em> and <em>Rhodobacter capsulatus,</em> and symbiotically N <sub><font size="-1">2</font></sub> fixing organisms, such as <em>Rhizobium</em> species and <em>Bradyrhizobium japonicum.</em><p>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 <em>(nif/fix)</em> 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 <em>Azorhizobium caulinodans</em> ORS571, a tropical rhizobium capable of both diazotrophy and symbiotic nitrogen fixation in stem- and root nodules induced on its host, the tropical shrub <em>Sesbania rostrata,</em> changed this situation and has allowed a direct comparison of <em>(nif/fix)</em> 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 <em>(nif/fix)</em> regulatory gene <em>nifA</em> , a gene which is essential for diazotrophic growth and symbiotic nitrogen fixation, and which responds to a complex regulatory circuit.<p>In chapter 2 a current review of the regulation of nitrogen fixation genes is presented. The review focuses on the regulation of <em>nifA</em> gene expression and NifA activity in different bacterial species.<p>In chapter 3 the involvement of ntr-mediated control of the <em>nifA</em> promoter via the -24/-12 element is analyzed by site-specific mutagenesis and chimeric <em>nifA</em> - <em>lacZ</em> reporter gene fusions integrated into the <em>A. caulinodans</em> chromosome. The -24/-12 promoter element was shown to be important for <em>nifA</em> gene expression suggesting the involvement of a σ54 (NtrA; RpoN)-type transcription factor in <em>nifA</em> gene regulation.<p>Chapter 4 addresses the involvement of a <em>ntrA(rpoN)</em> -like sigma factor in <em>nifA</em> expression, and reports the cloning and analysis of an <em>A. caulinodans ntrA(rpoN)</em> gene. Although the <em>ntrA(rpoN)</em> gene identified in this study was shown to control the expression of some of the <em>A.</em><em>caulinodans nif</em> genes (like <em>nifHDK)</em> and nitrate assimilation genes, it did not appear to regulate the expression of <em>nifA</em> , suggesting the presence of an additional ( <em>nifA</em> specific) <em>ntrA</em> ( <em>rpoN</em> )-equivalent gene.<p>To search for <em>trans</em> -acting factors involved in the regulation of the <em>nifA</em> expression, the <em>in vitro</em> interaction of proteins in <em>A. caulinodans</em> crude cell extracts with the <em>nifA</em> regulatory region was studied by gel retardation assays. Chapter 5 presents the binding of (a) sequence-specific protein(s) in extracts of <em>A.</em><em>caulinodans</em> to the <em>nifA</em> 5' upstream region. However the nature of the protein(s) and the exact location of the binding site in the <em>nifA</em> 5' upstream region remains to be determined.<p>During the preparation of crude cell extracts from <em>A. caulinodans fi</em> xLJ and <em>fixK</em> 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.<p>Chapter 7 describes the isolation and characterization of an <em>leuA</em> -like gene of <em>A.</em><em>caulinodans</em> which was incidentally identified with an <em>Escherichia coli fnr</em> probe. Comparison of the 5' upstream region of the <em>A. caulinodans leuA</em> gene with the <em>leu</em> operons of other organisms suggest a conserved regulation mechanism for the expression (transcription attenuation), found in many amino acid biosynthetic operons.<p>Finally in chapter 8 the current model for nitrogen fixation gene regulation in <em>A.</em><em>caulinodans,</em> deduced from the studies presented in this thesis and previous data, is presented.
|Qualification||Doctor of Philosophy|
|Award date||14 Jan 1994|
|Place of Publication||S.l.|
|Publication status||Published - 1994|
- molecular biology