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 Arthritis, Diabetes, Digestive and Kidney Diseases (NIADDK)
Type
Research Project (R01)
Project #
5R01AM019447-09
Application #
3151217
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-04-01
Project End
1986-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
9
Fiscal Year
1985
Total Cost
Indirect Cost
Name
State University of NY, Binghamton
Department
Type
Schools of Arts and Sciences
DUNS #
090189965
City
Binghamton
State
NY
Country
United States
Zip Code
13902