A small diffusion pore in the outer membrane of Pseudomonas aeruginosa

The permeability properties of the outer membrane of Pseudomonas aeruginosa were re-examined, since the reported conclusions are conflicting [Decad, M. G. and Nikaido, H. (1976) J. Bacteriol. 128, 325-336; Caulcott, C. A., Brown, M. R. W. and Gonda, I. (1984) FEMS Microbiol. Lett. 21, 119-123]. On the basis of the experimental evidence to be described below we conclude that the exclusion limit of the outer membrane of P. aeruginosa is smaller than the size of uncharged disaccharides but larger than the size of hexose. This conclusion is based on the following evidence. Penetration of monosaccharides into the expanded periplasm was large and that of disaccharides was small, after the cells were plasmolyzed with 600 mosM NaCl. A significant amount of protein was released after osmotic down-shock of cells treated with the hypertonic monosaccharides but not of cells treated with the hypertonic saccharides larger than disaccharides. Centrifuged pellets of cells treated with hypertonic di, tri and tetrasaccharides weighed about 15-20% less than that of cells treated with the isotonic monosaccharide, suggesting that the osmotic pressure was exerted on the outer membrane causing dehydration and shrinking of the cells. By contrast, cells treated with the hypertonic pentose and hexoses weighed about 0.1% and 6% less, respectively, than cells treated with the isotonic saccharide, suggesting that pentose diffused through the outer membrane freely.     

Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability.

Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.     

In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.     

Transmembrane permeability channels in vesicles reconstituted from single species of porins from Salmonella typhimurium.

Aggregates of the "major" outer membrane proteins, "porins," of Salmonella typhimurium form diffusion channels in reconstituted vesicle membranes. The aggregate consists of three species of porins with apparent molecular weights of 34,000, 35,000, and 36,000 when active aggregates are subjected to sodium dodecyl sulfate-acrylamide gel electrophoresis after heating in the presence of sodium dodecyl sulfate (Nakae, J. Biol. Chem. 251:2176-2178, 1976). Single species of porins were isolated by solubilization of membranes and subsequent gel filtration in the presence of sodium dodecyl sulfate from the mutant strains of Salmonella typhimurium that produced only single species of porin. The single species of porins of either 34,000, 35,000, or 36,000 daltons formed diffusion channels when assayed for sucrose permeability in the vesicle membranes reconstituted from porins, phospholipids, and lipopolysaccharides. The exclusion limits of the pores made of single species of porins were not distinguishable from each other and from the exclusion limits of the pores made of the porin aggregates from the wild-type strain, when the permeability of vesicle membranes to radioactive di-, tri-, and tetrasaccharides and to various sizes of radioactive polyethylene glycol was determined. Porin-deficient mutants produced residual amounts of porin amounting to 1 to 5% that produced by the parent strain. This residual porin made diffusion channels when the isolated porins were incorporated into the vesicle membrane and assayed for permeability of saccharides.     

Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores

The purified outer membrane proteins of Pseudomonas aeruginosa were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and these were tested by the liposome swelling method for the diffusion of saccharides with different Mr. Proteins C (Mr, 70,000), D (Mr, 46,000), and E (Mr, 43,000) were found to confer the monosaccharide-permeable pores in the reconstituted liposome membranes. The membrane vesicles containing proteins F (Mr, 34,000), G (Mr, 25,000), or H (Mr, 19,000) showed no detectable pore-forming activity. The pores formed by proteins C, D, or E appeared to be smaller than that formed by the Escherichia coli porins. The size of the solutes that permeated through the newly identified porins is similar to that through the intact and purified outer membrane of P. aeruginosa (Yoneyama, H., and Nakae, T. (1986) Eur. J. Biochem. 157, 33-38; Yoshihara, E., Gotoh, N., and Nakae, T. (1988) Biochem. Biophys. Res. Commun. 156, 470-476).     

Outer membrane of gram-negative bacteria. XII. Molecular-sieving function of cell wall.

The permeability function the cell wall of gram-negative bacteria such as Salmoenlla was investigated by producing cells with an expanded periplasmic volume, and incubating them with radioactive non-utilizable oligo- and polysaccharides or polyethylene glycols. To quantitative the extent of penetration of these hydrophilic compounds into the periplasm, the radioactivity of the cell pellet was determined after centrifugation. We found that only di- and trisaccharides could fully diffuse into the periplasm, whereas higher-molecular-weight saccharides were nonpenetrable. In addition, low-molecular-weight polyethylene glycols rapidly diffused across the cell wall. Kinetics experiments also showed that both sucrose and raffinose in the periplasm exchanged rapidly with sugars in the medium, even at 0 degrees C. These results suggest that the cell wall acts as a molecular sieve, with an exclusion limit near 550 to 650 daltons for saccharides. We also suggest that the diffusion of these hydrophilic compounds most likely occurs through water-filled pores present in the cell wall of gram-negative bacteria.     

Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier.

A method for separating the outer and inner membranes of Pseudomonas aeruginosa PAO1 in the absence of added ethylenediaminetetraacetic acid was devised. The method yields two outer membrane fractions which show the same protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differ substantially in their relative contents of phospholipids. One of these outer membrane fractions and the inner membrane fraction are less than 4% cross-contaminated, as judged by the content of typical inner and outer membrane markers. The outer membrane contains four major protein bands with apparent molecular weights of 37,000, 35,000, 21,000 and 17,000. Vesicles reconstituted from lipopolysaccharide and phospholipids were impermeable to all saccharides included in the vesicles during vesicle formation. When the vesicles contained outer membrane proteins, they fully retained only those saccharides of greater than 9,000 molecular weight, suggesting that the exclusion limit of the outer membrane of P. aeruginosa for saccharides is substantially larger than the figure (500 to 600 daltons) obtained for certain enteric bacteria. The advantages and potential disadvantages of having an outer membrane with a higher exclusion limit for hydrophilic substances are discussed.     

Electron microscopic visualization of the outer membrane permeability of Bacteroides fragilis

Abstract Electron microscopy of negatively stained Bacteroides fragilis cells treated with hypertonic solutions of di- or trisaccharides showed the shrinkage of whole cells, whereas the pentose- or hexose-treated cells shrank less significantly. Electron microscopy of thin sections of the monosaccharide-treated cells showed plasmolysis. Determination of the diffusion rate by the liposome-swelling method suggested that the apparent exclusion limit of the outer membrane pore is close to the size of uncharged saccharides of approx. M _r 250. These results suggested that the outer membrane of B. fragilis contains small diffusion pores.     

Periplasmic space in Salmonella typhimurium and Escherichia coli.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.     

Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides

Protein D2 of Pseudomonas aeruginosa outer membrane is known to facilitate the specific permeation of imipenem (N-formimdoylthienamycin) across this membrane barrier. We have characterized the binding site in the protein D2 channel by studying the competitive inhibition, by various solutes, of imipenem diffusion into the periplasm. We found that basic amino acids, lysine, arginine, histidine, and ornithine, were effective inhibitors. L- and D-lysine were found to be competitive inhibitors with approximate Ki values of 0.6 and 0.3 mM, respectively. Peptides containing L-lysine at the carboxyl terminus, as well as dipeptides containing L-lysine at the amino terminus, were also able to inhibit the transport. Wild type cells transported tripeptide Thr-Ser-Lys into the periplasm three to four times as rapidly as the mutant cells lacking the D2 protein. These results suggest that protein D2 plays a physiologically significant role in the uptake of basic amino acids and peptides containing these amino acids across the outer membrane of P. aeruginosa.     

Glycosylation is required for outer membrane localization of the lectin LecB in Pseudomonas aeruginosa

The fucose-/mannose-specific lectin LecB from Pseudomonas aeruginosa is transported to the outer membrane; however, the mechanism used is not known so far. Here, we report that LecB is present in the periplasm of P. aeruginosa in two variants of different sizes. Both were functional and could be purified by their affinity to mannose. The difference in size was shown by a specific enzyme assay to be a result of N glycosylation, and inactivation of the glycosylation sites was shown by site-directed mutagenesis. Furthermore, we demonstrate that this glycosylation is required for the transport of LecB.     

The metal dependence of pyoverdine interactions with its outer membrane receptor FpvA

To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 microM AlCl(3) to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.     

Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli

We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli. Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm. Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction. Both were degraded with a half-time of about 40 s. Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes. Processing occurs mainly post-translationally for both the A and the B subunits. However, they show different kinetics of processing and subsequent segregation into the periplasm. Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.     

Isolation and characterization of transposon Tn5-induced mutants of Pseudomonas perfectomarina defective in nitrous oxide respiration.

Outer membranes of Haemophilus influenzae type b were fractionated to yield Triton X-100-insoluble material and lipopolysaccharide and phospholipids. Liposomes reconstituted from lipopolysaccharide and phospholipids were impermeable to sucrose (Mr, 342) and to a high-molecular-weight dextran (average Mr, 6,600). When the Triton X-100-insoluble material was introduced into the reconstituted liposomes, the vesicles became permeable to sucrose, raffinose (Mr, 504), and stachyose (Mr, 666) and fully retained dextrans of Mr greater than 1,500. Inulin (average Mr, 1,400) was tested for its efflux from the reconstituted outer membrane vesicles; 62% of the added inulin was trapped. The molecular weight exclusion limit for the outer membrane of H. influenzae type b was therefore estimated at approximately 1,400. A protein responsible for the transmembrane diffusion of solutes was purified from H. influenzae type b by extraction of whole cells with cetyl trimethyl ammonium bromide. When this extract was passed over DEAE-Sepharose, three protein-containing peaks (I, II, and III) were eluted. Peaks I and II contained mixtures of proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; when tested for their pore-forming properties, these proteins were unable to render liposomes of lipopolysaccharide and phospholipid permeable to sucrose. Peak III contained only one molecular species of protein of molecular weight 40,000; this protein acted as a porin in reconstituted vesicles. The molecular weight exclusion limit for 40,000-molecular-weight protein matched the estimate of approximately 1,400 which was determined for outer membranes. A series of homologous saccharides of increasing degree of polymerization was prepared from agarose by hydrolysis with beta-agarase and fractionation on gel filtration chromatography. These oligosaccharides of Mr, 936, 1,242, 1,548, and 1,854 were assayed for retention by the complete vesicles containing 40-kilodalton protein and lipopolysaccharide and phospholipids. All of these oligosaccharides were lost by efflux through the porin. Since the molecular conformation of the largest oligosaccharide is an elongated semirigid helix, it is suggested that the pore formed by the 40-kilodalton protein does not act as a barrier to the diffusion of this compound.     

Sensitivity of Escherichia coli to various beta-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic beta-lactamases: a quantitative predictive treatment

In Gram-negative bacteria, beta-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic beta-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the beta-lactamases have been studied and their significance in creating beta-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of beta-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC beta-lactamase, or a plasmid-coded TEM-type beta-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter, 'target access index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.     

Structure of arabinogalactan oligosaccharides derived from arabinogalactan-protein of Coffea arabica instant coffee powder

Arabinogalactan-protein, previously isolated from instant coffee powder of Coffea arabica, has been subjected to partial mild acidic and enzymatic hydrolyses. Separation of obtained mixtures by size exclusion and HPLC chromatographies afforded series of oligosaccharides, structure of which were studied by NMR spectroscopy. Mild acidic hydrolysis afforded oligosaccharides without any αAraf substituent while after enzymatic hydrolysis αAraf was found in di-, tri-, and tetrasaccharides. In all cases αAraf was a terminal substituent linked separately to O3, O6, and to both, O3 and O6, of βGal residues. Identification of di-, tri-, and tetrasaccharides containing α-Araf enabled to distinguish in the ¹H NMR spectra αAraf signals linked to O6 and O3 of neighboring βGal unit. Composition of polymeric residues after enzymatic and mild acidic hydrolyses was also analyzed.     

Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.

Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.     

Sensitivity of Escherichia coli to various β-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic β-lactamases: a quantitative predictive treatment

In Gram-negative bacteria, β-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic β-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the β-lactamases have been studied and their significance in creating β-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of β-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC β-lactamase, or a plasmid-coded TEM-type β-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter,‘target access Index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.