Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa

Abstract

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::fQ 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::fQ 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 P-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.

Role of the Novel OprD Family of Porins in Nutrient Uptake in Pseudomonas aeruginosa

“…We first tried to confirm this result for the well-characterized porin OprD. Despite the evidence suggesting a role for OprD in the uptake of basic amino acids (11,20,36), we were unable to detect a growth defect when the oprD mutant was grown in arginine. A growth defect was detected, however, for the opdK mutant grown in limiting concentrations of vanillate.…”

“…When present as a mixture, the amino acids and imipenem compete for the binding site within OprD and raise the MIC to imipenem 16-fold, emphasizing the role of this porin in amino acid uptake (11).…”

“…4), OprD, OpdC, OpdB, and OpdT, are likely candidates for permitting low levels of nonspecific uptake through the outer membrane. Indeed, it has already been shown that OprD can act as a nonspecific channel for the uptake of gluconate (11). Thus, in order to greatly decrease outer membrane permeability, mutants deficient in multiple OprD homologues would have to be generated.…”

“…This facilitated uptake is necessary with nutrient-deficient conditions when uptake through the outer membrane becomes rate limiting for growth and simple diffusion through general porins becomes inefficient. The binding sites of specific porins do not preclude the entry of structurally unrelated compounds, and thus, these channels also display some characteristics common to general porins (11). Specific porins are often induced to higher copy numbers by their substrates.…”

Bacterial outer membrane channel for divalent metal ion acquisition

“…Some channels, called porins, discriminate by size and behave as a molecular sieve with a limit of about 600 Da. Other channels similar to porins bind solutes with low affinity, and thus are more selective but have relativity broad substrate specificities (7,8). Growth phenotypes of bacterial mutants defective in genes encoding low-selectivity channels have not been demonstrated in most cases.…”