REGULATION OF AMMONIA ASSIMILATION IN SALMONELLA TYPHIMURIUM

STUART ALAN ROSENFELD, Purdue University

Abstract

The three primary ammonia assimilatory enzymes in Salmonella typhimurium are glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. Although the biochemical properties of these enzymes are well established, the mechanisms controlling their synthesis are unknown. Since I have been studying the role of glutamate dehydrogenase (GDH) in ammonia assimilation, this thesis emphasizes the isolation and characterization of mutants having altered GDH activities. In order to obtain GDH mutants, I developed a procedure for mutagenizing S. typhimurium using the bacteriophage Mu cts dl(Ap('r),lac). Since S. typhimurium is not normally sensitive to Mu, bacteriophage P1 served as a vector for transferring Mu cts dl(Ap('r),lac) DNA into S. typhimurium cells. This technique yielded several classes of glutamate auxotrophs. Most of the auxotrophs had no GDH activity, suggesting that the Mu had inserted either in the GDH structural gene (gdhA) or in another gene that controlled gdhA expression. A property associated with Mu cts dl(Ap('r),lac) phage is its ability to create single-step fusions of its lac genes to foreign promoters. Several of the insertion mutants had (beta)-galactosidase activity controlled the same as GDH synthesis in wild-type cells. Transduction experiments have shown that these fused Mu insertions are located in the same region of the chromosome as mutations causing thermolabile GDH enzyme activity. These data are supportive of the insertions being within gdhA. Another class of glutamate auxotrophs isolated by Mu mutagenesis had very low but regulated levels of GDH. These auxotrophs reverted to prototrophy at high frequencies but still retained the original Mu insertion, implying that the Mu insertions were not located in gdhA. Detailed mapping data showed that the Mu insertions might be within the gdhA promoter region or in another gene necessary for activating gdhA transcription. Another topic covered in this thesis is the mapping of the gdhA gene. Transductions demonstrated linkage of mutations producing a thermolabile GDH protein to the pncA and nit genes, placing the gdhA locus between 25-30 Units on the S. typhimurium chromosome. Results of additional genetic crosses with various Tn10 insertions established the gene order: pncA-gdhA-nit. Hfr strains were constructed to orient this cluster with outside genes, and conjugation experiments indicated the gene order: pyrD-pncA-gdhA-nit-trp. To characterize this region further, I used the Mu cts dl(Ap('r),lac) insertions within the gdhA gene to identify the direction of gdha transcription and to isolate delection mutations having a variety of endpoints. Transductions of these deletion mutations with gdhA point mutations permitted me to construct a deletion map of this region. A more general aspect of ammonia utilization discussed in this thesis is the effects of hisT mutations. The hisT gene codes for the enzyme which converts two uridine nucleotides to pseudouridine in the anticodon loop of several tRNA species. I examined the consequences of the hisT mutation on the ability of S. typhimurium strains to use various nitrogen sources and found that strains with the hisT('-) mutation grew more rapidly than hisT('+) strains in glucose medium containing either L-arginine or L-proline as the sole nitrogen source. Glutamate synthase activities were lower in hisT mutants than in isogenic hisT('+) controls; however, glutamate dehydrogenase was about three-fold higher in the hisT mutants grown in glucose-arginine medium. The results suggest that the enzymes of nitrogen utilization respond either directly or indirectly to tRNA species affected by the hisT mutation.

Degree

Ph.D.

Subject Area

Microbiology

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