Defense Date


Document Type


Degree Name

Master of Science


Microbiology & Immunology

First Advisor

Gordon L. Archer


Trimethoprim resistance(Tp r) is encoded by conjugative plasmids in clinically significant staphylococcal isolates. Two genetically and physically similar plasmids from S. aureus, pG01 and pGOS, have Tp r genes that map in different locations on these plasmids. In order to study the relatedness of the Tp r genes and their products to other known Tp r genes, a 1.2 kb fragment of pG01 and a 4.2 kb fragment of pGOS were cloned in E. coli and used as probes for in situ filter hybridization experiments.

A 500 base pair subclone of the original 1.2 kb fragment containing only the staphylococcal Tp r structural gene, showed no homology with genes from E. coli encoding a dihydrofolate reductase(DHFR) with an altered Tp r binding affinity or the B. subtilis gene for DHFR. Positive hybridization signals were seen with restriction fragments from pG01, pGOS, and plasmid DNA from five other Tp r staphylococci. A 700 bp portion of the original fragment showed homology with several different restriction fragments of EcoRI-digested pGOl and pGOS, suggesting the presence of repeated sequences on both plasmids. These sequences corresponded to areas of the plasmids known to be involoved in deletions which occur during viral transductions.

Lysates of bacteria containing the cloned and native Tp r genes were assayed spectrophotometrically for DHFR activity and compared with activity of E. coli containing genes for DHFR type I and type II. In addition, the Tp Ic 50 (the concentration of Tp required to reduce DHFR activity by 50%) was determined. Tp r staphylococci containing the plasmid-encoded Tp r gene had twenty times higher specific activity than Tp sensitive staphylococci. E. coli containing the cloned staphylococcal gene had DHFR activity equal to that of staphylococcal strains from which the clones were derived and 300 times higher activity than Tp sensitive E. coli. Determination of the Tp Ic 50 showed the staphylococcal protein to be 7000 times more resistant to Tp than the normal cellular DHFR, but four times less resistant to Tp than the DHFR type I and 450 times less resistant than DHFR type II. The staphylococcal Tp r gene product is a protein with DHFR activity that is resistant to Tp inhibition. The gene is expressed in E. coli, but is dissimilar to several previously characterized E. coli Tp r genes.


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