|Ph.D Thesis||Department of Biology|
|Supervisors:||Prof. Beja Oded|
|Full Thesis text|
Tryptophan pathway genes have been extensively studied in bacteria that can be cultured under laboratory conditions. The present work characterizes trp genes from bacteria living in the stringent environments, the Sargasso Sea and a volcanic vent on Mount Melbourne, Antarctica.
The Sargasso Sea database was analyzed for tryptophan genes. At least 5% of all the amino acid biosynthesis genes are trp genes. Many of the contigs and scaffolds contain whole or split operons that are similar to previously analyzed trp gene organizations. Only two scaffolds discovered within this analysis possess a different operon organization than those previously known. Many marine organisms lack an operon-type organization of these genes or have mini-operons containing 2 trp genes. Analysis of the trpB genes revealed that the dichotomous division between trpB_1 and trpB_2 also occurs in organisms from the Sargasso Sea. One cluster was found to contain sequences distinct from most known trpB sequences.
The trp genes from newly isolated Antarctic bacteria, their enzymes and probable regulatory mechanisms were characterized. It was found that their anthranilate synthase (ASase) achieves its highest activity at 55oC and pH 8.2 and is more active in presence of free ammonia than in presence of L-glutamine. It does not contain a phosphoribosyl transferase subunit (PRTase) and seems to have 2 subunits of trpE and 2 subunits of trpG. PRTase and tryptophan synthase (TSase) have their highest activities under similar conditions. Their Kms and kcats were also determined. PRTase is a large protein that seems to contain several subunits. The size of TSase shows that it most likely contains two ß and two α subunits. The regulation of the tryptophan pathway was found to include feedback inhibition by the end product, L-tryptophan, and coordinate repression-derepression. Tryptophan non -competitively inhibits the ASase activity with a Ki=0.6µM. The trpE, C and B genes were found not to be closely linked.
The present analysis shows that trp genes are widely dispersed among bacterial species. The novel organization of these genes indicates that there is much to be discovered with respect to both the reason for certain gene orders and the regulation of tryptophan biosynthesis in marine bacteria. At the same time it was found that a species living in geographical and spatial isolation has the same genes and uses some of the regulatory mechanisms previously known. The present investigation strengthens the idea that the trp genes all evolved from a common ancestor.