title: ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli.
abstract: OBJECTIVE
  We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in Escherichia coli. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity.
  
  
  METHODS
  ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into E. coli, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured.
  
  
  RESULTS
  Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into E. coli, whereas the mutant remained susceptible to triclosan. Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells.
  
  
  CONCLUSIONS
  ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.
authors.affiliation: Department of Chemistry, University of St. Thomas, St. Paul, Minn., USA.
authors.forename: Taylor L
authors.initials: TL
authors.lastname: Fischer
authors.forename: Robert J
authors.initials: RJ
authors.lastname: White
authors.forename: Katherine F K
authors.initials: KF
authors.lastname: Mares
authors.forename: Devin E
authors.initials: DE
authors.lastname: Molnau
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Donato
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014260:Triclosan / Q000494:pharmacology*
chemical_list: D000890:Anti-Infective Agents; D029968:Escherichia coli Proteins; D014260:Triclosan; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); C493924:fabI protein, E coli; D054889:Fatty Acid Synthase, Type II