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

Wylie, John; Bernegger-Egli, Christine; O'Neil, Joe D. J.; Worobec, Elizabeth A.

doi:10.1007/bf01108411

Cited by 27 publications

(34 citation statements)

References 47 publications

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“…It has also been shown that there is active transport of pyruvate across the cytoplasmic membrane in E. coli but not in Pseudomonas (41). Glucose, in contrast, is taken up very efficiently by P. aeruginosa (13,67,73). In E. coli, expression of PDH genes is regulated by a pyruvate-sensitive repressor, PdhR (50,51).…”

Section: Discussionmentioning

confidence: 99%

Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa

1997

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A mutant of Pseudomonas aeruginosa, OT2100, which appeared to be defective in the production of the fluorescent yellow-green siderophore pyoverdine had been isolated previously following transposon mutagenesis (T. R. Merriman and I. L. Lamont, Gene 126:17-23, 1993). DNA from either side of the transposon insertion site was cloned, and the sequence was determined. The mutated gene had strong identity with the dihydrolipoamide acetyltransferase (E2) components of pyruvate dehydrogenase (PDH) from other bacterial species. Enzyme assays revealed that the mutant was defective in the E2 subunit of PDH, preventing assembly of a functional complex. PDH activity in OT2100 cell extracts was restored when extract from an E1 mutant was added. On the basis of this evidence, OT2100 was identified as an aceB or E2 mutant. A second gene, aceA, which is likely to encode the E1 component of PDH, was identified upstream from aceB. Transcriptional analysis revealed that aceA and aceB are expressed as a 5-kb polycistronic transcript from a promoter upstream of aceA. An intergenic region of 146 bp was located between aceA and aceB, and a 2-kb aceB transcript that originated from a promoter in the intergenic region was identified. DNA fragments upstream of aceA and aceB were shown to have promoter activities in P. aeruginosa, although only the aceA promoter was active in Escherichia coli. It is likely that the apparent pyoverdine-deficient phenotype of mutant OT2100 is a consequence of acidification of the growth medium due to accumulation of pyruvic acid in the absence of functional PDH.The pyruvate dehydrogenase (PDH) complex catalyzes the oxidative decarboxylation of pyruvate to form acetyl coenzyme A (acetyl-CoA):The complex contains multiple copies of three enzymatic components: PDH (E1) (EC 1.2.4.1), dihydrolipoamide acetyltransferase (E2) (EC 2.3.1.12), and lipoamide dehydrogenase (E3) (EC 1.8.1.4). In all gram-negative bacteria studied so far, the inner core of the complex consists of 24 E2 molecules arranged with octahedral symmetry (42). The peripheral E1 and E3 subunits are bound to the surface of the E2 core. Genes encoding PDH have been characterized in Escherichia coli. The three components of the complex are encoded by a single operon that includes a regulatory gene (pdhR) and the aceE (E1), aceF (E2), and lpd (E3) structural genes. The genes are expressed as a 7.4-kb polycistronic transcript originating upstream of the pdhR gene at the pyruvate-inducible pdh promoter (51). In addition, a smaller lpd transcript is initiated at the independent lpd promoter upstream of the lpd gene (61). It has been demonstrated that the gene product of pdhR negatively regulates transcription by binding to the pdh promoter in the absence of pyruvate (50, 51).The genes encoding PDH in Pseudomonas aeruginosa have not been characterized, but the enzyme has been purified and some of its properties have been determined (37). The purified complex was found to contain three major and one minor polypeptide components: E1 (M r , 92,500), E2 (major...

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Section: Discussionmentioning

confidence: 99%

Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa

1997

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“…Although the single-channel conductance of OpdH was on average 10 times greater than those of other characterized specific porins of Pseudomonas species, it is considered small compared to those of OmpF and OmpC, the major porins of E. coli (0.23 nS versus 1 to 2 nS) (9,23,25,38,57). Also, of the many compounds that were tested as potential substrates for OpdH, significant growth defects were observed only for two compounds: isocitrate and cis-aconitate.…”

Section: Discussionmentioning

confidence: 99%

Characterization of OpdH, a Pseudomonas aeruginosa Porin Involved in the Uptake of Tricarboxylates

et al. 2007

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The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis-aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions.The outer membrane of the opportunistic pathogen Pseudomonas aeruginosa is intrinsically poorly permeable to many classes of compounds, with a permeability coefficient that is 10-to 500-fold lower than that of Escherichia coli (17,60). Despite the barrier presented by the outer membrane, P. aeruginosa is renowned for its metabolic versatility and is capable of importing and using such diverse compounds as carbohydrates, alcohols, organic acids, aromatic compounds, aliphatic compounds, and amino acids and other nitrogenous compounds as nutrient sources (44).These hydrophilic nutrients enter the cell via outer-membranespanning, water-filled channels called porins. The major P. aeruginosa outer membrane porin is OprF. This protein belongs to a class of porins termed the slow porins, wherein one of two channel sizes is formed (31,32,46). The majority of OprF porins consist of small pores (0.36 nS) that provide rather inefficient uptake routes; however, a small fraction of OprF molecules (approximately 1 out of every 500) form larger channels (2 to 5 nS) that can facilitate the uptake of di-, tri-, and tetrasaccharides. Thus, it is believed that the majority of outer membrane transport occurs through substrate-specific porins (5,11,19).Pseudomonas species possess a large collection of specific porins (48). The most extensively characterized specific porins of P. aeruginosa include the carbohydrate-specific porin OprB (58), the phosphate-specific porin OprP (18), the orthophosphate-specific porin OprO (42), and the basic amino acid-specific porin OprD (50). Generally, these porins tend to form narrow pores that are often occluded by long surface loops (27). The uptake of specific classes of molecules is facilitated through these channels by virtue of substrate-specific binding sites that orient substrates into favorabl...

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“…Previous analyses, including the induction of OprB by growth on glucose (10), the demonstration of facilitated diffusion of glucose into liposomes containing OprB (30), and the verification of a glucose binding site in OprB (35), suggested that OprB would form a glucose-selective porin in the outer membrane of P. aeruginosa. The results presented here verify that in vivo, OprB does facilitate the diffusion of glucose into the cell.…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) used the 11% (wt/vol) gel system of Lugtenberg et al (17). The production of polyclonal antibodies specific for heat-modified OprB and immunodetection protocols have been previously described (35). Bacterial cell envelopes were prepared by the method of Lugtenberg et al (17).…”

Section: Methodsmentioning

confidence: 99%

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The OprB porin plays a central role in carbohydrate uptake in Pseudomonas aeruginosa

Wylie

Worobec

1995

J Bacteriol

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Using interposon mutagenesis, we have generated strains of Pseudomonas aeruginosa which lack or overexpress the substrate-selective OprB porin of this species. A marked decrease or increase in the initial uptake of glucose by these strains verified the role of OprB in facilitating the diffusion of glucose across the outer membrane of P. aeruginosa. However, we also demonstrated that the loss or overexpression of OprB had a similar effect on the uptake of three other sugars able to support the growth of this bacterium (mannitol, glycerol, and fructose). This effect was restricted to carbohydrate transport; arginine uptake was identical in mutant and wild-type strains. These results indicated that OprB cannot be considered strictly a glucoseselective porin; rather, it acts as a central component of carbohydrate transport and is more accurately described as a carbohydrate-selective porin.Diffusion rates across the outer membrane of Pseudomonas aeruginosa are 100 to 500 times lower than those measured for Escherichia coli (37). The low outer membrane permeability of P. aeruginosa has been attributed to the unusual channel-forming properties of the OprF porin, the major, constitutively expressed protein in the outer membrane of this bacterium (9). OprF has some homology with the OmpA protein of enteric bacteria (33) and, like OmpA, shows only limited porin activity (28). In enteric bacteria, the low channel-forming activity of OmpA does not have a major influence on outer membrane permeability, as these bacteria also produce classical general diffusion porins (e.g., OmpF and OmpC). The more efficient permeation of solutes through OmpF and OmpC overshadows the diffusion properties of the OmpA protein. In contrast, the apparent lack of typical general diffusion porins in P. aeruginosa results in the diffusion properties of the outer membrane of this bacterium being determined largely by the relatively inefficient OprF porin.The low outer membrane permeability of P. aeruginosa is compensated for by the presence of several substrate-selective porins (14,21,29). Porins of this type are also present in enteric bacteria, but in these species, substrate-selective porins have generally been associated with the diffusion of relatively large substrates (e.g., LamB, maltodextrins [32]; ScrY, sucrose [25]; TolC, peptides [2]). In the absence of their respective porins, these substrates either do not diffuse or diffuse to only a limited extent, through nonspecific channels. In contrast, the substrate-selective porins of P. aeruginosa facilitate the diffusion of much smaller substrates. In E. coli, diffusion of small substrates, such as glucose or glycerol, is generally believed to occur via nonspecific channels. In P. aeruginosa, if facilitated diffusion across the outer membrane did not occur, the low outer membrane permeability would potentially limit the transport of almost all substrates, regardless of size.Four substrate-selective porins have been identified in P. aeruginosa. The OprD porin facilitates the diffusion of basic ...

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

Cited by 27 publications

References 47 publications

Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa

Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa

Characterization of OpdH, a Pseudomonas aeruginosa Porin Involved in the Uptake of Tricarboxylates

The OprB porin plays a central role in carbohydrate uptake in Pseudomonas aeruginosa

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