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Title: The molecular biology of ammonia assimilation in the obligate methanotroph Methylococcus capsulatus strain Bath
Author: Cardy, Donald Leonard Nicholas
ISNI:       0000 0001 3519 8430
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1989
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The structural gene (g1nA) encoding the ammonia assimilation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath). Complementation of Escherichia coli and Klebsiella pneumoniae g1nA mutants was demonstrated. In vitro and in vivo expression analysis revealed the cloned g1nA gene to encode a polypeptide with an apparent M_r of 60,000 as determined by PAGE. Expression of the M. capsulatus (Bath) g1nA gene in E. coli was found to be regulated by nitrogen levels in an Ntr+ but not an Ntr− background. This regulation was not observed when the cloned H. capsulatus (Bath) g1nA gene was under the influence of the chloramphenicol acetyl transferase gene of the vector. The nucleotide sequence of the M. capsulatus (Bath) g1nA gene and flanking sequences has also been determined. The gene comprises 1407 bp encoding a polypeptide of M_r 51,717 containing 468 amino acids. The 5' leader region contains three putative promoters. Promoters P1 and P3 resemble the canonical -10 -35 E. coli type promoter. Promoter P2 which is located between P1 and P3, resembles the NtrA dependent promoters of enteric organisms. A potential NtrC-binding site was also determined. The 3' flanking region contained a small putative open reading frame (ORF) encoding a polypeptide of M_r 7022. The identity of this polypeptide remains to be elucidated. Comparisons of g1nA structural genes and GS enzymes at the nucleotide and amino acid levels between M. capsulatus (Bath) and both prokaryotes and eukaryotes have been determined. The presence of ntrA, ntrB, ntrC, g1nB and rpoD homologues in the M. capsulatus (Bath) genome was determined by heterologous hybridization studies. Type I and Type II obligate methanotrophs were also screened for g1nA, ntrC and ntrA homologues. Both Type I and Type II organisms were found to have homologues to each of these gene probes. A portion of a M. capsulatus (Bath) putative ntrC gene has been cloned on a cosmid, pCOS1 and was found to be unlinked to g1nA and lies some 8.5 kb downstream of g1nA. The development of a plasmid transformation and gene transfer system for M. capsulatus (Bath) based on previously published methods has also been assessed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology