Pseudomonas aeruginosa is an outstanding example of a microbial pathogen which resists antimetabolite therapy. It is likewise uncommonly resistant to analog mimicks of L-tyrosine and L-phenylalanine which are effective false feedback inhibitors in vitro. This resistance is related to the existence of two simultaneously present pathway branchlets to L-phenylalanine in addition to dual branchlets to L-tyrosine. Bacillus subtilis and Euglena gracilis exemplify organisms having completely different post-prephenate pathway sequences, both of which co-exist in P. aeruginosa. Consequently, tightly blocked phenylalanine and tyrosine auxotrophs are not isolated from P. aeruginosa. Growth on fructose as carbon source invokes a limitation of early-pathway flow, presumably by depletion of PEP. This has provided a condition that facilitates the selection of regulatory mutants. A workable strategy for the sequential isolation of structural-gene mutants has also been devised. We propose to obtain mutants deficient in each structural gene of the total aromatic pathway. Deregulated mutants corresponding to enzymes subject to regulation in wild type will be isolated. In many cases sequential mutagenesis will result in strains bearing multiple mutations. The subtle effects of individual mutations will be studied by segregating individual mutations into different strains following genetic recombination. A comparison of physiological and nutritional effects caused by all possible combinations of structural-gene and regulatory mutations will be used to formulate an interpretation of the metabolic significance of the enzymological complexity that characterizes aromatic biosynthesis in P. aeruginosa. An overview of pathway-flow characteristics, pathway arrangement and regulation, and basis of resistance/sensitivity to aromatic antimetabolites will be pursued. We will locate the chromosomal map positions of each structural gene and regulatory gene available for aromatic amino acid biosynthesis as well as for pyocyanine biosynthesis and aromatic amino acid catabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK038309-02
Application #
3237593
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-07-01
Project End
1988-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Schools of Arts and Sciences
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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Xia, T H; Ahmad, S; Zhao, G S et al. (1991) A single cyclohexadienyl dehydratase specifies the prephenate dehydratase and arogenate dehydratase components of one of two independent pathways to L-phenylalanine in Erwinia herbicola. Arch Biochem Biophys 286:461-5
Fischer, R S; Zhao, G; Jensen, R A (1991) Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis. J Gen Microbiol 137:1293-301
Hendry, A T; Bhatnagar, R K; Shanmugam, K T et al. (1990) Exploitation of the broad specificity of the membrane-bound isoenzyme of lactate dehydrogenase for direct selection of null mutants in Neisseria gonorrhoeae. J Gen Microbiol 136:45-50
Bonner, C A; Fischer, R S; Ahmad, S et al. (1990) Remnants of an ancient pathway to L-phenylalanine and L-tyrosine in enteric bacteria: evolutionary implications and biotechnological impact. Appl Environ Microbiol 56:3741-7
Coleman, J B; Casini, A F; Serroni, A et al. (1990) Evidence for the participation of activated oxygen species and the resulting peroxidation of lipids in the killing of cultured hepatocytes by aryl halides. Toxicol Appl Pharmacol 105:393-402

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