Date of Award
12-1987
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Dale Noel
Second Advisor
Reinhold Mueller
Third Advisor
Sally Hennen
Fourth Advisor
James B. Courtright
Fifth Advisor
Walter W. Fredricks
Abstract
The enzymes and genes of tryptophan biosynthesis have been investigated in order to learn about regulation and gene organization in Zymomonas mobilis. Zymomonas is an unusual genus of Gram-negative bacteria, potentially important for industrial fermentation of ethanol. Zymomonas is also interesting for its unique physiology and its phylogenetic relationships to other bacteria. Knowledge of the regulation and organization of trp genes may be useful for evaluating the significance of the regulation and organization of the other metabolic genes in Z. mobilis. In addition, information on tryptophan biosynthesis in Z. mobilis may lend insight into the evolution of this pathway in bacteria. The first step in the investigation was to isolate tryptophan auxotrophs. Twelve trp mutants were isolated after nitrosoguanidine mutagenesis. Six mutants appeared to be analogous to E. coli trp E mutants which are defective in anthranilate synthase. Five of the mutants did not have tryptophan synthase B activity and were classified as trp B mutants. One mutant lacking tryptophan synthase activity grew on indole and was classified as a trp A mutant. Some of these mutants were used in two experiments to investigate the regulation of tryptophan biosynthesis. First, a trp B mutant accumulated indole only if supplied with growth limiting concentrations of tryptophan. This result suggested that the carbon flow through the pathway was regulated. The second experiment suggested that not all of the trp genes or enzymes are regulated in response to the concentration of tryptophan. Tryptophan synthase and indoleglycerol phosphate synthase did not vary when trp mutants or the wild type were grown on limiting or excess tryptophan. The organization of the Z. mobilis trp genes was examined by genetic cloning, complementation analysis, restricting mapping, and Southern hybridization. Five Z. mobilis trp genes (D,C,F,B, and A) were cloned by complementation of E. coli and P. putida trp mutants. The trp F, B, adnd A genes were clustered, but not closely associated with the trp D and trp C genes. These results suggest that there are at least two trp gene clusters in Z. mobilis.