The role of DAHP synthase isozymes in control of carbon flow into the shikimate pathway in bacteria and plants
Abstract
In enteric bacteria there are three isozymes of DAHP synthase, each allowing carbon flow in the shikimate pathway under different physiological conditions. The regulation of two of the three isozymes at the DNA level was examined to explore the conditions that control their expression. The aroF gene, encoding the tyrosine-sensitive DAHP synthase was cloned from Salmonella typhimurium. The regulation of this aroF gene, and of the previously characterized aroF gene of Escherichia coli, was analyzed by construction of fusions between the regulatory regions of these genes and lacZ. Several regions of potential regulatory significance were identified by comparison of the two aroF genes from the enteric bacteria. These regions of similarity include the presumptive Tyr repressor binding sites including the distance between the two binding sites, which is predicted to occupy three turns of $\beta$-form DNA. A palindrome that lies within an untranslated portion of the transcribed aroF DNA was also identified in both aroF genes. The potential regulatory sequences were modified by site directed mutagenesis and the effect on expression determined by measurement of $\beta$-galactosidase activity. An analysis of aroF-lacZ fusions containing different lengths of the coding region of aroF allowed identification of sequences within the aroF coding region that are necessary for efficient expression. Measurement of mRNA levels showed that this regulation is post-transcriptional. Regulation of the aroH gene of E. coli, which encodes the tryptophan-sensitive DAHP synthase, was also analyzed using a lacZ fusion. Control of aroH expression by the Tyr repressor was shown to be separate from the previously reported transcriptional control of aroH expression by the Trp repressor. The regulation by these two repressors is cumulative. Maximal repression requires the corepressors tryptophan and tyrosine, respectively. Solanum tuberosum L. (potato) was shown to have two DAHP synthase activities. The two isozymes were separated by column chromatography and were shown to have different immunological determinants, different organ distributions, and to respond differently to environmental stresses, such as wounding.
Degree
Ph.D.
Advisors
Herrmann, Purdue University.
Subject Area
Biochemistry|Molecular biology|Microbiology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.