| The nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae is regulated by the centralized nitrogen regulation (ntr) system of the cell.; The first section of this thesis describes the cloning and transposon Tn5 mutagenesis of the glnA region of K. pneumoniae, and the identification of a gene (glnR), located immediately adjacent to glnA, encoding a trans-acting protein, essential for the positive activation of the nif gene cluster and other nitrogen assimilation genes such as histidine utilization (hut).; The second section of this thesis describes the physical analysis of the K. pneumoniae glnAR region, including the identification of the glnA gene product (glutamine synthetase; 58,000 daltons) and the two gene products of the glnR region, designated glnL(ntrB; 36,000 daltons) and glnG(ntrC; 56,000 daltons), by analogy to the nomenclature used in Escherichia coli. This section also describes the construction of a plasmid carrying a fusion of the glnLG(ntrBC) genes to the strong, inducible (lamda)P(,L) promoter, useful for the amplification of the glnL(ntrB) and glnG(ntrC) gene products.; The third section of this thesis describes the cloning and characterization of the glnF(ntrA) gene of K. pneumoniae. The cloned glnF(ntrA) gene was shown to complement the previously identified GlnA('-)Ntr('-) phenotype of glnF('-)(ntrA('-)) strains of E. coli and K. pneumoniae. This further established the role of the glnF(ntrA) gene product as an essential trans-acting positive activator of ntr regulated genes (such as nif), in addition to the glnG(ntrC) gene product. The glnF(ntrA) gene product was shown to be 84,000 daltons, and its synthesis was shown not to be regulated by fluctuating ammonia concentrations in the cell.; The fourth section of this thesis describes the analysis of a segment of the K. pneumoniae chromosome immediately adjacent to the nif gene cluster. This region has been cloned and contains the promoter-operator and the first two genes of the histidine biosynthetic operon (hisDGO). A precise correlated physical and genetic map of the K. pneumoniae hisDGO has been generated using transposon Tn5 mutagenesis. Finally, observation regarding the insertional specificity of transposon Tn5, the polarity of Tn5 induced insertion mutations and the usefulness of Tn5 mutagenesis in the generation of large collections of insertion mutations in cloned DNA segments are discussed.; The appendices describe the analysis of transposable elements involving the his4C region of Saccharomyces cerevisiae, including physical evidence for a transposable element carrying the his4C gene. |
