Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene.
Mutants with insertion mutations in the Pseudomonas aeruginosa protein F (oprF) gene were created in vivo by Tn1 mutagenesis of the cloned gene in Escherichia coli and in vitro by insertion of the streptomycin resistance-encoding omega fragment into the cloned gene, followed by transfer of the mutated protein F gene back to P. aeruginosa. Homologous recombination into the P. aeruginosa chromosome was driven by a bacteriophage F116L transduction method in the oprF::Tn1 mutants or Tn5-instability in the oprF::omega mutants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting demonstrated that the resultant oprF insertion mutants had lost protein F, whereas restriction digestion and Southern blotting experiments proved that the mutants contained a single chromosomal oprF gene with either Tn1 or omega inserted into it. It has been proposed that protein F has a role in antibiotic uptake in P. aeruginosa. Measurement of antibiotic resistance levels showed small to marginal increases in resistance, compared with that of the parent P. aeruginosa strain, to a variety of beta-lactam antibiotics. Protein F-deficient mutants had altered barrier properties as revealed by a three- to fivefold increase in the uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine.
Woodruff
W A
WA
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Hancock
R E
RE
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000172:deficiency* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007769:Lactams; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010539:Permeability; D018272:Porins; D011550:Pseudomonas aeruginosa / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D007769:Lactams; D018272:Porins