GENETIC AND REGULATORY STUDIES OF THE GLT REGION IN SALMONELLA TYPHIMURIUM (GLUTAMATE, SYNTHASE)

MARY JANE MADONNA, Purdue University

Abstract

This thesis describes the isolation and characterization of mutations affecting glutamate synthase (glt). Through the controlled action of glutamate synthase, glutamate dehydrogenase, and glutamine synthetase, nitrogen metabolism is regulated in the cell. Studies presented here will help to understand genetic and regulatory characteristics of glutamate synthase. The location of the genes encoding glutamate synthase was studied. The glt locus was mapped at 69 Units (U) on the S. typhimurium linkage map. These analyses required the use of Tn10 insertions in the glt region. The selections for and the mapping of the Tn10 elements is described. The region designated as glt defined the structural gene locus for glutamate synthase. Deletion mutations were obtained in glt. Point mutations affecting glutamate synthase were mapped against these deletions. The final result, a deletion map of glt, is presented. Strains with polar nonsense mutations and Mucts dl (lac bla) insertions in glt were characterized. Results obtained from SDS-polyacrylamide gel analyses showed that the glutamate synthase subunits were affected in strains with both types of polar mutations. Results indicated that a large proportion of the deletion map defines mutations in the gene encoding the large subunit of glutamate synthase. Also observed in these studies was a polar effect in glt expression. This suggests glt is organized as an operon. Regulation of glt expression was studied in rho ((rho)) mutants. For both S. typhimurium and Escherichia coli, rho mutations caused increased levels of glutamate synthase. The rho mutations also affected glutamate transport; uptake was enhanced in rho strains. Regulation was examined in strains with Mucts dl (lac bla) insertions in glt. Mucts dl (lac bla) phage can create fusions of its lac genes to foreign promoters. Therefore lac expression was monitored. (beta)-Galactosidase activity mimicked the response of glutamate synthase, under different growth conditions. Results of these studies and mapping experiments indicated that lac was under the control of the glt promoter. The isolation and characterization of possible glt promoter mutations is described. Another method was used to characterize regulatory mutations. These mutations had pleiotropic effects on the cell. Glutamate synthase levels were restored to wild-type levels in strains that had formerly reduced activities. The mutations were also suppressors of glnF mutations. In addition, the mutations were near glnF. These results implied that a new regulatory gene involved in the control of nitrogen assimilation has been identified.

Degree

Ph.D.

Subject Area

Microbiology

COinS