The goal of the proposed research is to understand the mechanisms regulating exopolysaccharide synthesis in Pseudomonas cepacia, a bacterium which has emerged as a serious cause of respiratory tract infections in patients with cystic fibrosis. The exopolysaccharide alginic acid has been implicated as a determinant of pathogenicity in Pseudomonas aeruginosa infections, but to our knowledge its production by P. cepacia has not been described. We wish to test the following hypothesis, which is based on our discovery of a direct correlation between deficiency of the enzyme glucose dehydrogenase and ability of P. cepacia to excrete alginate and other exopolysaccharide material. The hypothesis is that operation of the direct oxidative pathway of glucose degradation in this bacterium inhibits exopolysaccharide synthesis and that switchover to an alternative route of glucose dissimilation, the phosphorylative pathway, is important for exopolymer formation. To attain our objectives we will: 1) better define the relationship between exopolymer formation and glucose dehydrogenase deficiency by screening P. cepacia isolates from cystic fibrosis patients for defects in this and/or other enzymes of the direct oxidative pathway, 2) clone the gene encoding glucose dehydrogenase and introduce it into alginate-excreting strains to test its ability to shut off exopolymer formation, 3) gain more information about the route of alginate biosynthesis by isolating and characterizing mutants blocked in this process, and 4) characterize in more detail the exopolymers of several clinical isolates, one of which complexes with the beta-glucan- binding dye calcofluor white and causes it to fluoresce green under ultraviolet light. Corollary experiments will also be carried out to determine the organization of key carbohydrate utilization genes on fragments of the P. cepacia chromosome already cloned into the cosmid vector pLAFR.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI024869-03
Application #
3138119
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1987-07-01
Project End
1990-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Massachusetts Amherst
Department
Type
Schools of Arts and Sciences
DUNS #
153223151
City
Amherst
State
MA
Country
United States
Zip Code
01003