OmpA is the principal nonspecific slow porin of Acinetobacter baumannii

Acinetobacter species show high levels of intrinsic resistance to many antibiotics. The major protein species in the outer membrane of Acinetobacter baumannii does not belong to the high-permeability trimeric porin family, which includes Escherichia coli OmpF/OmpC, and instead is a close homolog of E. coli OmpA and Pseudomonas aeruginosa OprF. We characterized the pore-forming function of this OmpA homolog, OmpA(Ab), by a reconstitution assay. OmpA(Ab) produced very low pore-forming activity, about 70-fold lower than that of OmpF and an activity similar to that of E. coli OmpA and P. aeruginosa OprF. The pore size of the OmpA(Ab) channel was similar to that of OprF, i.e., about 2 nm in diameter. The low permeability of OmpA(Ab) is not due to the inactivation of this protein during purification, because the permeability of the whole A. baumannii outer membrane was also very low. Furthermore, the outer membrane permeability to cephalothin and cephaloridine, measured in intact cells, was about 100-fold lower than that of E. coli K-12. The permeability of cephalothin and cephaloridine in A. baumannii was decreased 2- to 3-fold when the ompA(Ab) gene was deleted. These results show that OmpA(Ab) is the major nonspecific channel in A. baumannii. The low permeability of this porin, together with the presence of constitutive β-lactamases and multidrug efflux pumps, such as AdeABC and AdeIJK, appears to be essential for the high levels of intrinsic resistance to a number of antibiotics.     

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Susceptibility and intrinsic tolerance of Pseudomonas aeruginosa to selected plant volatile compounds

AIMS: To examine the causes for variations in sensitivity and intrinsic tolerance of Pseudomonas aeruginosa to plant volatile compounds. METHODS AND RESULTS: Minimum inhibitory concentrations were determined for a selection of volatile phytochemicals against P. aeruginosa using a microdilution assay. Effects on growth were also assessed in 100-ml broth cultures. The two strains of P. aeruginosa included in the study exhibited intrinsic tolerance to all compounds, with the exception of carvacrol and trans-cinnamaldehyde. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased P. aeruginosa sensitivity to all compounds except trans-cinnamaldehyde, implicating an ATP-dependent efflux mechanism in the observed tolerance. Outer membrane integrity following treatment with test compounds was assessed by measuring sensitization to detergents. Only carvacrol caused damage to the outer membrane of P. aeruginosa. CONCLUSIONS: The intrinsic tolerance of P. aeruginosa strains to plant volatile compounds is associated with an active efflux mechanism and the barrier function of the outer membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer an explanation for the intrinsic tolerance to plant volatile compounds exhibited by P. aeruginosa. The study also confirms that the outer membrane-permeabilizing action of carvacrol, previously reported for the gram-negative bacteria Escherichia coli and Salmonella, extends to monoterpene-tolerant strains of P. aeruginosa.     

Pseudomonas aeruginosa outer membrane permeability: isolation of a porin protein F-deficient mutant.

A mutant of Pseudomonas aeruginosa severely deficient in outer membrane protein F levels was isolated by screening heavily mutagenized strains for membrane protein alterations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To provide a basis for phenotypic comparison, three independent spontaneous revertants with normal protein F levels were isolated. Neither the protein F-deficient mutant nor its revertants had gross surface alterations as judged by their sensitivities to 31 phages with diverse receptors and their low degrees of leakage of periplasmic beta-lactamase into the supernatant. Outer membrane permeability was measured in whole cells by examining the rates of hydrolysis of a chromogenic beta-lactam, nitrocefin, by periplasmic RP1-encoded beta-lactamase. It was found that the outer membrane permeabilities of wild-type and protein F revertant strains were similar, but low when compared with those of Escherichia coli and an antibiotic-supersusceptible mutant Z61 of P. aeruginosa. The loss of protein F caused a further significant decrease in outer membrane permeability. The results suggest that protein F is a pore-forming protein in vivo and that only a small proportion, as few as 1 in 400, of the protein F molecules form active functional channels in vivo.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

Identification and characterization of porins in Pseudomonas aeruginosa.

Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chem. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D1, D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa.     

Expression in Caulobacter crescentus of the phosphate-starvation-inducible porin OprP of Pseudomonas aeruginosa

The gene for the phosphate-starvation-inducible outer membrane protein OprP, of Pseudomonas aeruginosa was introduced into Caulobacter crescentus CB2A on a plasmid vector. As is the case in P. aeruginosa and Escherichia coli the oprP gene was inducible under conditions of limiting phosphate in C. crescentus. However, the maximal medium concentration of phosphate which still permitted induction of OprP was lower in C. crescentus (50 microM) than in P. aeruginosa (200 microM). Induction of OprP was coincident with the process of stalk elongation, known to occur in C. crescentus under phosphate starvation conditions. When induced, OprP was localized to the cell envelope and became a major membrane protein, indicating that the Pseudomonas promoter was efficiently recognized in C. crescentus and that the gene product was targeted to the appropriate region of the cell. Our data provide support for the hypothesis that the mechanism for regulation of phosphate-starvation-inducible genes is highly conserved amongst the eubacteria.     

Factors that influence the permeability assay of the outer membrane of Pseudomonas aeruginosa

Brief exposure of Pseudomonas aeruginosa to a temperature of 10 degrees C or lower caused a significant leakage of the periplasmic beta-lactamase into the medium. The extent of leakage increased as the incubation temperature was lowered to 4 degrees C and reached a maximum at 0 degrees C. Cells grown in the presence of beta-lactamase inducers were unsuitable for the permeability assay. It was found that the diffusion rates of beta-lactams through the outer membrane of P. aeruginosa were much lower than those previously reported, as assayed under refined conditions. The diffusion rates of beta-lactams in one of the mutants tested were an order of magnitude lower than those of the other strains, despite the fact that the outer membrane protein profile of the strain appeared to be indistinguishable from those of the others. These results suggest that beta-lactam antibiotics diffuse through the outer membrane of P. aeruginosa, at least partly, through a non-porin pathway.     

The barrier function of the outer membrane of Pseudomonas maltophilia in the diffusion of saccharides and β-lactam antibiotics

This paper reports that the efficiency of solute diffusion through the outer membrane of Pseudomonas maltophilia is roughly 3 to 5% of that of Escherichia coli. This is despite the fact that the outer membrane pore(s) is only a little smaller than that of E. coli. These results suggest that P. maltophilia has a low copy number of porin(s). The outer membrane of antibiotic resistant clinical isolates showed even less efficient permeability towards saccharides and antibiotics than the laboratory strains.     

The role of outer membrane proteins in peptide uptake by Escherichia coli

Abstract The influence of various outer membrane proteins on peptide penetration through the outer membrane in Escherichia coli was assessed by determining peptide transport kinetics in wild type and outer membrane protein-deficient strains. Peptide uptake was measured in whole cells by using a fluorescamine-based assay to monitor continuously the removal of peptides from the medium. Transport data were collected and processed using a microcomputer to give overall K _m and V _max values for peptide transport in each strain. In the mutants, K _m values were changed more markedly then V _max values reflecting an alteration in diffusion through the envelope. This approach shows that porins are involved in facilitating peptide penetration and that the OmpF channel appears to be more important than either OmpC or PhoE proteins. The loss of OmpA protein also decreases outer membrane permeability towards peptides, although whether this protein forms pores itself or exists more to maintain the functional integrity of other proteins is not known.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

Reconstitution of maltose transport in malB mutants of Escherichia coli through calcium-induced disruptions of the outer membrane.

The barrier function of the Escherichia coli outer membrane against low concentrations of maltose in strains missing the lambda receptor was partially overcome by treating the cells for 3 h with 25 mM Ca2+. Kinetic analysis of maltose-transport revealed a Ca2+-induced shift of the apparent Km of the system from about 100 microM in cells pretreated with Tris to about 15 microM in cells pretreated with Tris plus Ca2+. In contrast to maltose transport in untreated cells, that of Ca2+-treated lamB cells was inhibited by molecules with a high molecular weight, such as amylopectin (molecular weight, 20,000), and anti-maltose-binding protein antibodies. In addition, lysozyme was shown to attack Ca2+-treated cells in contrast to untreated cells. The Ca2+-induced permeability increase of the outer membrane allowed reconstitution of maltose transport in a mutant missing the maltose-binding protein with osmotic shock fluid containing the maltose-binding protein. Even though Ca2+-treatment allowed the entry of large molecules, the release of the periplasmic maltose-binding protein or alkaline phosphatase was negligible.     

Outer membrane porin proteins F, P, and D1 of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers.

Native oligomers of three Pseudomonas aeruginosa outer membrane porin proteins and one Escherichia coli porin were demonstrated by using a chemical cross-linking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was cross-linked to dimers in outer membrane and whole-cell cross-linking experiments. Purified preparations of P. aeruginosa proteins F, D1 (glucose induced), and P (phosphate starvation induced) and E. coli protein PhoE (Ic) were also cross-linked to reveal dimers and trimers upon two-dimensional sodium dodecyl sulfate-polyacrylamide electrophoretic analysis. Cross-linking of protein F was abolished by pretreatment of the protein with sodium dodecyl sulfate, indicating that the cross-linked products were due to native associations in the outer membrane.     

Amino acids at positions 3 and 4 determine the membrane specificity of Pseudomonas aeruginosa lipoproteins

Escherichia coli lipoproteins with Asp at position 2 remain in the inner membrane, whereas those having other amino acids are targeted to the outer membrane by the Lol system. However, inner membrane lipoproteins without Asp at position 2 are found in other Gram-negative bacteria. MexA of Pseudomonas aeruginosa, an inner membrane-specific lipoprotein involved in multidrug efflux, has Gly at position 2. To identify the residue or region of MexA that functions as an inner membrane retention signal, we constructed chimeric lipoproteins comprising various regions of MexA and an outer membrane lipoprotein, OprM, and analyzed their membrane localization. Lys and Ser at positions 3 and 4, respectively, were found to be critical for the inner membrane localization of MexA in P. aeruginosa. Substitution of these residues with Leu and Ile, which are present in OprM, was sufficient to target the chimeric lipoprotein to the outer membrane and to abolish the ability of MexA to confer drug resistance. The membrane specificity of a model lipoprotein, lipoMalE, a lipidated variant of the periplasmic maltose-binding protein of E. coli, was also determined by the residues at positions 3 and 4 in P. aeruginosa. In contrast to the widely accepted "+2 rule" for E. coli lipoproteins, these results suggest a new "+3, +4 rule" for lipoprotein sorting in P. aeruginosa, namely, the final destination of lipoproteins is determined by the residues at positions 3 and 4.     

Pore size dependence on growth temperature is a common characteristic of the major outer membrane protein OprF in psychrotrophic and mesophilic Pseudomonas species

Pseudomonas species adapt well to hostile environments, which are often subjected to rapid variations. In these bacteria, the outer membrane plays an important role in the sensing of environmental conditions such as temperature. In previous studies, it has been shown that in the psychrotrophic strain P. fluorescens MF0, the major porin OprF changes its channel size according to the growth conditions and could affect outer membrane permeability. Studies of the channel-forming properties of OprFs from P. putida 01G3 and P. aeruginosa PAO1 in planar lipid bilayers generated similar results. The presence of a cysteine- or proline-rich cluster in the central linker region is not essential for channel size modulations. These findings suggest that OprF could adopt two alternative conformations in the outer membrane and that folding is thermoregulated. In contrast, no difference according to growth temperature was observed for structurally different outer membrane proteins, such as OprE3 from the Pseudomonas OprD family of specific porins. Our results are consistent with the fact that the decrease in channel size observed at low growth temperature is a particular feature of the OprF porin in various psychrotrophic and mesophilic Pseudomonas species isolated from diverse ecological niches. The ability to reduce outer membrane permeability at low growth temperature could provide these bacteria with adaptive advantages.     

Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa

It has previously been shown that myo-inositol hexakisphosphate (myo-InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron-dependent growth inhibition. In this study, the iron transport properties of myo-inositol trisphosphate and tetrakisphosphate regio-isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo-Ins(1,2,3)P3 or myo-InsP6. Iron accumulation from other compounds, notably D/L myo-Ins(1,2,4,5)P4 and another inositol trisphosphate regio-isomer, D-myo-Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate-determining step.     

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.     

Porin of Pseudomonas aeruginosa forms low conductance ion channel in planar lipid bilayers.

Protein E1, a porin of the outer membrane of Pseudomonas aeruginosa, was reconstituted into planar lipid bilayers. Single channel conductance of the protein appeared to be 230 pS (pico siemens) in 1 M KCl-10 mM Hepes, pH7.2. This value is approximately 5 times lower than the conductance of the OmpF channel of Escherichia coli. Conductance increased linearly as the membrane potential was raised from -200 mV to +200 mV, and was nearly proportional to the KCl concentration. These results show that protein E1 is probably a genuine porin in the P. aeruginosa outer membrane supporting the earlier conclusion that protein E1 forms a small channel.     

Transport systems for branched-chain amino acids in Pseudomonas aeruginosa.

The cells of Pseudomonas aeruginosa showed high activity for leucine transport in the absence of Na+, giving a Km value of 0.34 microM. In the presence of Na+, however, two Km values, 0.37 microM (LIV-I system) and 7.6 microM (LIV-II system), were obtained. The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. However, the LIV-II system served for the entry of branched-chain amino acids only. The LIV-II system alone was operative in membrane vesicles, for the transport of branched-chain amino acids in membrane vesicles required Na+ and gave single Km values for the respective amino acids. When cells were osmotically shocked, the activity of the LIV-I system decreased, whereas the LIV-II system remained unaffected. The shock fluid from P. aeruginosa cells showed leucine-binding activity with a dissociation constant of 0.25 microM. The specificity of the activity was very similar to that of the LIV-I system. These results suggest that a leucine-binding protein(s) in the periplasmic space may be required for the transport process via the LIV-I system of P. aeruginosa.