NITROGEN METABOLISM IN SALMONELLA TYPHIMURIUM: L-METHIONINE SR-SULFOXIMINE RESISTANT GLUTAMINE SYNTHETASE AND MOLECULAR CLONING OF GDHA, THE STRUCTURAL GENE FOR GLUTAMATE DEHYDROGENASE
Abstract
Genetic and physical studies of glutamine synthetase, glutamate dehydrogenase, and the respective structural genes, glnA and gdhA, were undertaken in order to investigate aspects of ammonia assimilation. Two mutants of S. typhimurium resistant to growth inhibition by the glutamine synthetase transition state analog, L-methionine SR-sulfoximine, were isolated. The altered physiological properties of these strains included a partial growth requirement for glutamine, an inability to grow on limiting nitrogen sources, and elevated transport activities for these compounds. The glnA982 and glnA983 alleles in these strains were located relative to other glnA mutations by transduction and deletion mapping. Enzymic analysis of glutamine synthetase purified from one mutant strain showed it was defective in the reverse (gamma)-glutamyl transferase activity but retained significant biosynthetic activity. Apparent K(,m) values were increased 14-fold for NH(,3) and 4.4-fold for glutamate, suggesting the presence of a specific active site alteration in the mutant enzyme. The gdhA gene of S. typhimurium was cloned into the multi-copy plasmid pBR328. Analysis of the hybrid plasmid, pJB101, and its derivatives located gdhA in a 2.2 kb EcoRI/PvuII region. Glutamate dehydrogenase activities in plasmid-bearing strains were increased over 200-fold relative to the wild-type strain. In vivo labelling experiments showed that the gdhA('+) plasmids encode a 47K M(,R) polypeptide that corresponds to the reported M(,R) of the enzyme subunit. Genetic and in vitro recombination analyses confirm that the direction of gdhA transcription is in a counterclockwise direction on the smallest complementing plasmid. Expression of the galK gene on a gdhA::galK fusion plasmid and the regulation of glutamate dehydrogenase synthesis in plasmid-bearing strains showed that the gdhA regulatory region is present on the cloned gene. The use of these plasmids in studying the control of gdhA expression is discussed.
Degree
Ph.D.
Subject Area
Microbiology
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