CONTROL OF KLEBSIELLA PNEUMONIAE NITROGEN FIXATION (NIF) GENES BY THE NIFA PRODUCT

The products of the nifLA operon regulate the expression of nitrogen fixation (nif) genes in Klebsiella pneumoniae. The first part of this thesis describes regulatory factors that control the activity of the nifLA promoter. Specifically, the products of glnG (ntrC), glnF (ntrA), glnL (ntrB) and possibly glnB are shown or implicated to exert regulatory effects on nifLA expression. This thesis also describes the discovery that the nifA product can substitute for the glnG product in activating a number of genes involved in nitrogen metabolism (eg. glnALG, hut, aut), including its own nifLA promoter. As with the case of the glnG product, activation mediated by the nifA product appears to require the glnF product.; The second section of this thesis describes the structure of the nifLA promoter and a consensus heptameric sequence (TTTTGCA) found among five glnG and nifA regulated promoters examined. For two of these promoters, where the transcriptional start points have been determined, the consensus sequence is located at the -15 region, suggesting that it may be involved in glnG and nifA mediated transcription. In contrast, a promoter that responds to nifA, but not glnG mediated activation, does not contain this heptameric sequence, but instead the sequence CCCTGCA in its -15 region.; The last section of this thesis centers on the development of the P2 helper-dependent phage P4 as a specialized cloning vector for K. pneumoniae. This thesis describes that P4virl, an immunity-insensitive mutant, can replicate autonomously as a multicopy plasmid in E. coli and K. pneumoniae (in the absence of a P2 helper). It also describes experiments that show P4 can integrate site specifically into the K. pneumoniae chromosome, in which the event occurs at a low frequency. In order to convert cloned DNA into single copy states, vectors were constructed derived from P4virl. In addition to antibiotic resistance genes, these derivatives carry a nonsense mutation in its replication gene, and hence cannot replicate in a wild type host. Consequently, this allows direct selection of integrated lysogens. This approach, which involves cloning DNA as recombinant P4 plasmids in host strains that carry nonsense suppressors, and the subsequent integration of a single copy into a wild type host, was demonstrated for certain nif genes which would otherwise exhibit multicopy-associated inhibitory effects on nif expression.