Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa.
Earlier studies proved that Pseudomonas aeruginosa OprD is a specific porin for basic amino acids and imipenem. It was also considered to function as a nonspecific porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose.
Huang
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Department of Microbiology, University of British Columbia, Vancouver, Canada.
Hancock
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D000599:Amino Acids, Diamino / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D005942:Gluconates / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D015378:Imipenem / Q000633:toxicity; D008239:Lysine / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D010957:Plasmids; D018272:Porins; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity
D000599:Amino Acids, Diamino; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D005942:Gluconates; D018272:Porins; C077157:OprD protein, Pseudomonas aeruginosa; D015378:Imipenem; D005947:Glucose; D008239:Lysine; C030691:gluconic acid