Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa

Huang, Hong; Siehnel, Richard; Bellido, F.; Rawling, Eileen G.; Hancock, Robert E. W.

doi:10.1016/0378-1097(92)90347-q

Cited by 20 publications

(27 citation statements)

References 17 publications

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“…By contrast, the product of the gusC gene is an outer membrane protein that can enhance glucuronide uptake but does not transport glucuronide. We have found no indications that GusC acts as a binding protein or interacts directly with GusB, with permeabilized cells, subcellular vesicles, or the purified proteins; there is a significant level of sequence homology of GusC (22.4% amino acid identity with a global alignment) to a porin-like protein of Pseudomonas aeruginosa (27) (accession number AAG03552.1). This may indicate that it acts to facilitate transfer of higher-molecular-weight glucuronides across the outer membrane of E. coli.…”

Section: Resultsmentioning

confidence: 84%

ThegusBCGenes ofEscherichia coliEncode a Glucuronide Transport System

et al. 2005

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Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of ␤-glucuronides with synthetic [14 C]phenyl-1-thio-␤-D-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of ␤-D-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respirationgenerated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

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Section: Resultsmentioning

confidence: 84%

ThegusBCGenes ofEscherichia coliEncode a Glucuronide Transport System

et al. 2005

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“…One turned out to be the oprD structural gene, and the other region (the opdE gene) might encode a protein influencing the expression of OprD (14). In keeping with this hypothesis, the cloned oprD gene in E. coli was expressed poorly from its own promoter (14) and the level of OprD observed in the outer membrane of P. aeruginosa was influenced by both the growth medium and the carbon source (13). All of the OprD-defective mutants of P. aeruginosa studied to date were genetically undefined, and many were from clinical sources.…”

Section: Resultsmentioning

confidence: 99%

“…It is not possible to directly compare these literature studies with the investigation reported here, since these clinical strains had different genetic backgrounds and since different techniques and media were used. Also, it is known that there are at least one regulatory locus (opdE) (14) and one poorly understood multiple-antibiotic resistance locus (nfxC) (10) that can influence oprD expression and imipenem susceptibility. Furthermore, the potential for obtaining double mutants when utilizing clinical strains or when selecting directly with antibiotics has been described previously (41).…”

Section: Discussionmentioning

confidence: 99%

“…E. coli CE1248 (F-recA-56 phoEproABphoR-69 ompB-471 thr leu thi pyrF thy ilvA his lacY argG tonA rspL cod dra utr glpR) (29) was a strain with mutations preventing the production of porins OmpF, OmpC, and PhoE. E. coli CE1248(pBK19R) contained the oprD gene which was cloned as the 2.1-kb BamHI-KpnI fragment into pTZ19R in the same orientation as the lac promoter (14).…”

Section: Methodsmentioning

confidence: 99%

“…To compensate for this low permeability and to permit the uptake of essential nutrients available at low concentrations in the environment, certain substrate-specific transport systems are present in the P. aeruginosa outer membrane. For example, OprP allows the facilitated permeation of phosphate (4), OprB (formerly called protein D1) has a binding site for glucose (13,34), iron-repressible outer membrane proteins (IROMP) permit uptake of Fe3+-siderophore complexes when produced under the conditions of iron deprivation (35), and OprD (formerly called protein D2) (12,14) facilitates the diffusion of basic amino acids and small peptides containing these amino acids (32). These channels may be utilized by compounds which are structural analogs of the natural substrate.…”

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confidence: 99%

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

Huang

Hancock

1993

J Bacteriol

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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.

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Biophysical characterization of OprB, a glucose-inducible porin ofPseudomonas aeruginosa

Wylie

Bernegger-Egli

O'Neil

et al. 1993

J Bioenerg Biomembr

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OprB, a glucose-inducible porin of P. aeruginosa, was characterized by black lipid bilayer analysis and circular dichroism spectroscopy. Black lipid bilayer analysis of OprB revealed a single-channel conductance of 25 pS, the presence of a glucose binding site with a Ks for glucose of 380 +/- 40 mM, and the formation of channels with a strong selection for anions. Analysis of P. aeruginosa OprB circular dichroism spectra revealed a high beta sheet content (40%) which is within the range of that determined for other porins. Values obtained from black lipid bilayer analysis were compared to those previously obtained for OprB of P. putida [Saravolac et al. (1991). J. Bacteriol. 173, 4970-4976] and indicated extensive similarities in the single-channel conductance and glucose-binding properties of these two porins. Immunological and amino terminal sequence analysis revealed a high degree of homology. Of the first 14 amino terminal residues, 12 were identical. A major difference between the two porins was found in their ion selectivity. Whereas P. aeruginosa OprB is anion selective, P. putida OprB and other carbohydrate selective porins are known to be cation selective.

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Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa

Cited by 20 publications

References 17 publications

ThegusBCGenes ofEscherichia coliEncode a Glucuronide Transport System

ThegusBCGenes ofEscherichia coliEncode a Glucuronide Transport System

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

Biophysical characterization of OprB, a glucose-inducible porin ofPseudomonas aeruginosa

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