The physiology and metabolism of Escherichia coli is perhaps the best understood of any organism (1). Nearly all of the major metabolic pathways have been elucidated, and much of the regulation of these pathways is well understood. There is, however, one significant deficiency in our knowledge: the pathways of L-alanine biosynthesis have not been fully elucidated (2). L-alanine is the second most abundant amino acid found in E. coli protein, is a component of peptidoglycan, is the precursor to another component of peptidoglycan, D-alanine, and is a precursor to biotin.
With the availability of complete genomic sequences, it is now possible to make strong predictions about an organism s physiology and metabolism solely from analysis of its DNA sequence. A key to this analysis is having several model organisms whose metabolism and physiology are well defined. E. coli is one such model organism. Using whole genome comparisons, it has been possible to reconstruct the biosynthetic pathways of all 20 amino acids in E. coli, Haemophilus influenzae, and Bacillus subtilis. However, for L-alanine this required assuming a wider specificity for the aspartate aminotransferase. While this assumption is logical, it is not supported by experimental results (see below). A more recent analysis does use some of the reported enzymes used in L-alanine biosynthesis, but as outlined below, this information is incomplete (4). The fundamental reason that the biosynthesis of L-alanine has not been completely elucidated is that no strict L-alanine auxotroph has yet been isolated. This stems from the fact that there are multiple pathways of L-alanine biosynthesis. Clearly, understanding the synthesis of L-alanine and its regulation is important for a comprehensive understanding of E. coli metabolism. Moreover, understanding L-alanine biosynthesis in E. coli can then be used to predict its biosynthetic pathways in other organisms whose genome has been sequenced.
This project will employ a genetic approach to determine the pathways of L-alanine biosynthesis in E. coli. Mutations that confer an L-alanine requirement will be isolated by either counterselection and screening or by directed mutagenesis of genes of unknown function that could potentially function in L-alanine synthesis (e.g. aminotransferases). Once the genes have been identified, the enzymes encoded by those genes will be purified and their biochemical activities and allosteric regulators identified.
The regulation of the genes will be investigated using fusions of the promoter regions to the lacZc reporter gene. Deletion analysis of the promoter regions will be used to identify the important cis-acting regulatory sites. Additionally, these fusions will be used to determine the involvement of trans-acting factors in regulation. Since L-alanine is synthesized in large part from pyruvate and affects nitrogen metabolism, particular attention will be paid to several global regulatory factors that affect genes involved in carbon and nitrogen metabolism. One gene that has been shown to be involved in L-alanine biosynthesis is avtA, and it has been proposed that the global regulator, Lrp, is involved in its regulation (2). Computer analysis of its promoter region supports this hypothesis, and the influence of Lrp on avtA expression will be thoroughly studied.
Xavier University of Louisiana is a historically black college and university (HBCU). The Biology Department has approximately 1100 undergraduates. It has been successful in training and placing students into medical school, and seeks to build on this success in preparing students to pursue an academic career. This project has been designed to allow undergraduates to be involved in research on a daily basis using modern molecular biology techniques. In particular, most of the strain construction can be performed in short segments of time, allowing students to integrate their research into a demanding academic schedule.