Directed evolution of trimethoprim resistance in Escherichia coli.
Directed evolution is a useful tool in the study of enzymes. It is used in this study to investigate the means by which resistance to the antibiotic trimethoprim develops in dihyrofolate reductase from Escherichia coli. Mutants with clinical levels of resistance were obtained after only three generations. After four generations of directed evolution, several mutants were characterized, along with some point mutants made to investigate amino acid changes of interest. Several mutations were found to grant resistance to trimethoprim, both by reducing the binding affinity of the enzyme for the drug, and by increasing the activity of the enzyme.
Watson
Morgan
M
Research School of Chemistry, Australian National University, Canberra, ACT, Australia. watson@rsc.anu.edu.au
Liu
Jian-Wei
JW
Ollis
David
D
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D017354:Point Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014295:Trimethoprim / Q000378:metabolism / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase