Sulfonamide antibiotics prevent the biosynthesis of reduced folate compounds by inhibiting the production of H2pteroate from 6-hydroxymethylpterin pyrophosphate and p- aminobenzaoate (PABA). A common mode of resistance to sulfonamide antibiotics in E. coli is via mutation of the structural gene encoding H2pteroate synthase, so that the altered enzyme product is a sulfonamide resistant form that discriminates more effectively between PABA and sulfonamides than does the wild type enzyme. Sulfonamide resistance conferred by many R-determinant plasmids isolated from multiple antibiotic resistant clinical E. coli strains is by the same mechanism: expression of a sulfonamide resistant H2pteroate synthase. However, I have shown that sulfonamide resistance in E. coli can be mediated by gene amplification. Although I have not yet determined the identity of the amplified gene, the gene encoding H2pteroate synthase is a prime candidate, since that gene product is the target of sulfonamide inhibition. The sulfonamide resistant strain that I am characterizing has an 8-fold tandemly amplified DNA segment 18 kb in length. The association of an insertion sequence (IS5) with one (or both) endpoints of the amplified DNA suggests a role for site specific (illegitimate) recombination. This event is distinct from other illegitimate amplifications that seem to be independent of IS sequences. This application proposes to determine the mechanism of the gene amplification event, and to characterize H2pteroate synthase structure, function and expression at the genetic level.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI025106-03
Application #
3138458
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1987-07-01
Project End
1991-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
Schools of Arts and Sciences
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Hussein, M J; Green, J M; Nichols, B P (1998) Characterization of mutations that allow p-aminobenzoyl-glutamate utilization by Escherichia coli. J Bacteriol 180:6260-8
Vedantam, G; Guay, G G; Austria, N E et al. (1998) Characterization of mutations contributing to sulfathiazole resistance in Escherichia coli. Antimicrob Agents Chemother 42:88-93
Vedantam, G; Nichols, B P (1998) Characterization of a mutationally altered dihydropteroate synthase contributing to sulfathiazole resistance in Escherichia coli. Microb Drug Resist 4:91-7
Merkel, W K; Nichols, B P (1996) Characterization and sequence of the Escherichia coli panBCD gene cluster. FEMS Microbiol Lett 143:247-52
Green, J M; Merkel, W K; Nichols, B P (1992) Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme. J Bacteriol 174:5317-23
Tran, P V; Nichols, B P (1991) Expression of Escherichia coli pabA. J Bacteriol 173:3680-7
Green, J M; Nichols, B P (1991) p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC. J Biol Chem 266:12971-5
Tran, P V; Bannor, T A; Doktor, S Z et al. (1990) Chromosomal organization and expression of Escherichia coli pabA. J Bacteriol 172:397-410
Hughes, D; Thompson, S; O'Connor, M et al. (1989) Genetic characterization of frameshift suppressors with new decoding properties. J Bacteriol 171:1028-34