Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane.

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane. Determination of the diffusion rates of saccharides and beta-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.     

How does lysozyme penetrate through the bacterial outer membrane?

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme.In the presence of Mg²⁺, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg²⁺ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg²⁺. Mg²⁺ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose.These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.     

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.     

Bypass of receptor-mediated resistance to colicin E3 in Escherichia coli K-12.

Colicin E3 was found to kill, under conditions of osmotic shock, cells lacking a functional outer membrane receptor (bfe). Under such conditions, component A of the colicin, carrying endonucleolytic activity, also killed bfe cells, whereas fragment T2, obtained by tryptic digestion of the colicin and also active endonucleolytically, was inactive. Tolerance to the colicin caused by defects in the outer membrane could be overcome by osmotic shock, whereas tolerance probably caused by an altered plasma membrane could not.     

Porin activity in the osmotic shock fluid of Escherichia coli.

Osmotic shock fluid of Escherichia coli exhibited pore-forming activity. This activity could be followed by an in vitro assay based on the conductivity increase for ions due to the presence of pores in black lipid membranes. The histogram (the distribution of conductivity increments in a single pore experiment) obtained with osmotic shock fluid from E. coli was identical to the histogram obtained by detergent-solubilized porin isolated from the outer membrane. The osmotic shock fluid from porin-negative mutants also exhibited pore activity, although the histogram and ion specificity were different from those of porin. Antibodies raised against detergent-solubilized porin were able to form precipitin lines by the Ouchterlony immunodiffusion technique when shock fluids, but not detergent-solubilized porin, were used. These antibodies prevented the formation of pores when shock fluids contained porin but not when shock fluids obtained from porin-negative mutants were used. Macroscopic membrane conductivity of shock fluids due to porin exhibited a concentration dependence, in contrast to detergent-solubilized porin. These results indicate that the hydrodynamic properties of periplasmic or "soluble" porin are different from those of the detergent-solubilized porin of the outer membrane. Periplasmic porin comprises about 0.7% of total protein in the osmotic shock fluid.     

Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria.

Treatment of isolated yeast mitochondria with high levels (1 mg/ml) of trypsin severely inhibits protein import but does not destroy the integrity of the outer membrane or abolish mitochondrial energy coupling. If the outer membrane of these trypsin-inactivated mitochondria is disrupted by osmotic shock, the resulting mitoplasts are again able to import proteins. Protein import into mitoplasts, like that into intact mitochondria, is energy-dependent; however, whereas import into mitochondria is inhibited by antibody against 45-kd proteins of the outer membrane [Ohba and Schatz, EMBO J., 6, 2109-2115 (1987)], import into mitoplasts not affected by this antibody. Protein import into mitoplasts appears to bypass one or more steps normally occurring at the mitochondrial surface.     

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.     

Localization and assembly into the Escherichia coli envelope of a protein required for entry of colicin A.

Mutations in tolQ, previously designated fii, render cells tolerant to high concentrations of colicin A. In addition, a short deletion in the amino-terminal region of colicin A (amino acid residues 16 to 29) prevents its lethal action, although this protein can still bind the receptor and forms channels in planar lipid bilayers in vitro. These defects in translocation across the outer membrane in the tolQ cells or the colicin A mutant cannot be bypassed by osmotic shock. The TolQ protein, which is constitutively expressed at a low level, was studied in recombinant plasmid constructs allowing the expression of various TolQ fusion proteins under the control of the inducible caa promoter. The TolQ protein was thus "tagged" with an epitope from the colicin A protein for which a monoclonal antibody is available. A fusion protein containing the entire TolQ protein plus the 30 N-terminal residues of colicin A was shown to complement the tolQ mutation. Pulse-chase labeling followed by gradient fractionation indicated that the bulk of the overproduced fusion protein was rapidly incorporated into the inner membrane, with small amounts localized to regions corresponding to the attachment sites between inner and outer membranes and to the outer membrane itself. However, most of the protein was rapidly degraded, leaving only that localized to the attachment sites and the outer membrane remaining at very late times of chase.     

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.     

Biochemical characteristics of the outer membranes of plant mitochondria.

Like the outer membranes of liver mitochondria, those of plant mitochondria are impermeable to cytochrome c when intact and can be ruptured by osmotic shock. Isolated plant outer mitochondrial membranes are also similar to the corresponding liver membranes in terms of phospholipid and sterol content. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis experiments indicate that a single class of proteins (apparent molecular weight 30 000) comprises the bulk of the plant outer membrane protein. There are also considerable amounts of polysaccharide associated with these membranes, which may contribute to their osmotic stability.     

Biochemical characteristics of the outer membranes of plant mitochondria

Like the outer membranes of liver mitochondria, those of plant mitochondria are impermeable to cytochrome c when intact and can be ruptured by osmotic shock. Isolated plant outer mitochondrial membranes are also similar to the corresponding liver membranes in terms of phospholipid and sterol content. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis experiments indicate that a single class of proteins (apparent molecular weight 30 000) comprises the bulk of the plant outer membrane protein. There are also considerable amounts of polysaccharide associated with these membranes, which may contribute to their osmotic stability.     

Effects of Fatty Acid Substitution on the Release of Enzymes by Osmotic Shock

The release of enzymes by osmotic shock from Escherichia coli strain 30E, an unsaturated fatty acid auxotroph, was examined in culture supplemented with either cis- or trans-unsaturated fatty acids. Cultures grown in oleate-supplemented medium release a large fraction of the total cyclic phosphodiesterase, acid hexose phosphatase, and 5'-nucleotidase following osmotic shock. Cultures grown in elaidate-supplemented medium release much less of these same enzymes after shock treatment. Cultures grown with either supplementation show total release of these enzymes upon conversion to spheroplasts, demonstrating that the enzymes are in the periplasmic space in both cases. Cultures grown with either oleate or elaidate as fatty acid source were washed and suspended in medium containing the other isomer. The change from oleate to elaidate resulted in a rapid decrease in ability of the cells to release the three enzymes after osmotic shock so that within a 25% increase in cell mass the culture responded to osmotic shock as would a culture grown overnight in elaidate-supplemented medium. The reverse experiment resulted in a gradual increase in the ability of the cells to respond to osmotic shock. The outer membrane of E. coli is altered by the incorporation of elaidate, as indicated by electron microscopic data.     

A Function of 40 kDa Outer Membrane Protein in Serratia marcescens

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium-binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.     

Energetics underlying the process of long-chain fatty acid transport.

The gram negative bacterium Escherichia coli has evolved a highly specific system for the transport of exogenous long-chain fatty acids (C12-C18) across the cell envelope that requires the outer membrane protein FadL and the inner membrane associated fatty acyl CoA synthetase. The transport of oleate (C18:1) across the cell envelop responds to metabolic energy. In order to define the source of metabolic energy which drives this process, oleate transport was measured in wild-type and ATP synthase-defective (Deltaatp) strains which were (i) subjected to osmotic shock and (ii) starved and energized with glucose or d-lactate in the presence of different metabolic inhibitors. Osmotic shock did not eliminate transport but rather reduced the rate to 33-55% of wild-type levels. These results suggested a periplasmic protein may participate in this process or that osmotic shock disrupts the energized state of the cell which in turn reduces the rate of oleate transport. Transport systems which are osmotically sensitive also require ATP. The process of long-chain fatty acid transport requires ATP generated either by substrate-level or oxidative phosphorylation. Following starvation, the basal rate of transport for wild-type cells was 340.4 pmol/min/mg protein compared to 172.0 pmol/min/mg protein for the Deltaatp cells. When cells are energized with glucose, the rates of transport were increased and comparable (1242.6 and 1293.8 pmol/min/mg protein, respectively). This was in contrast to cells energized with d-lactate in which only the wild-type cells were responsive. The role of ATP is likely due to the ATP requirement of fatty acyl CoA synthetase for catalytic activity. The process of oleate transport is also influenced by the energized state of the inner membrane. In the presence of carbonyl cyanide-m-chlorophenylhydrazone oleate transport is depressed to 30-50% of wild-type levels in wild-type and Deltaatp strains under starvation conditions. These results are mirrored in cells energized with glucose and d-lactate, indicating that an energized membrane is required for optimal levels of oleate transport. These data support the hypothesis that the fatty acid transport system of E. coli responds to both intracellular pools of ATP and an energized membrane for maximal proficiency.     

Physical analysis of light-scattering changes in bovine photoreceptor membrane suspensions.

We have used electron microscopy and model calculations to analyze the physical basis of light-scattering signals from suspensions of photoreceptor membranes. These signals have previously been used to probe interactions between photoactivated rhodopsin (R*) and the peripheral membrane enzyme, GTP-binding protein (G) (Kühn et al., 1981, Proc. Natl. Acad. Sci. USA., 78:6873-6877). Although there is no unique physical interpretation of these signals, we have shown in this study that they were qualitatively unchanged when the rod outer segment fragments (containing stacked disks) were fragmented by sonication or osmotic shock to produce spherical disk membrane vesicles. An exact treatment of the scattering process for spherical vesicles enabled us to evaluate the effects of changing membrane thickness, refractive index, or vesicle diameter. We present a particular redistribution of mass upon R*-G interaction that fits the experimental data.     

Uptake of ferrienterochelin by Escherichia coli: energy dependent stage of uptake.

The uptake of the siderophore-iron complex ferrienterochelin was found to be strongly dependent upon an energized membrane state, as demonstrated by its sensitivity to dinitrophenol, azide, and cyanide. Ferrienterochelin uptake may also be dependent upon phosphate bond energy, as indicated by sensitivity to arsenate and iodoacetic acid. Although the adenosine triphosphatase does not appear to be involved in this energy coupling mechanism, ferrienterochelin uptake was shown to be less dependent upon phosphate bond energy than was glutamine uptake. Sensitivity of ferrienterochelin uptake to osmotic shock was shown to be due to the release of a ferrienterochelin binding compound located in the outer membrane of the cells and probably identical to the colicin B receptor protein.     

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.     

Role of voltage-dependent anion channels in glutathione transport into yeast mitochondria

Glutathione (GSH) is imported into mitochondria from the extra-mitochondrial cytoplasm. Translocation across the inner membrane of mitochondria is thought to occur via the dicarboxylate and 2-oxoglutarate carriers; however, the means by which GSH passes through the outer membrane is unknown. Disruption of the outer membrane of yeast mitochondria using either digitonin or osmotic shock did not alter GSH accumulation as compared with accumulation in intact mitochondria. These results suggested that passage across the outer membrane was not the rate-limiting step in GSH accumulation. Mitochondria isolated from yeast strains with a disruption in the major pore-forming protein of the outer membrane, VDAC1, accumulated GSH to a greater extent than mitochondria isolated from a wild-type strain. Disruption of the gene for VDAC2 did not affect GSH import. Thus, neither VDAC form is essential for GSH translocation into mitochondria, and the participation of another outer membrane channel in GSH import is possible.     

Evidence that F-plasmid proteins TraV, TraK and TraB assemble into an envelope-spanning structure in Escherichia coli

We have examined the role of the F-plasmid TraV outer membrane lipoprotein in the assembly of F-pili. Yeast two-hybrid analysis with a traV bait repeatedly identified traK, which is predicted to encode a periplasmic protein, among positive prey plasmids. A traK bait in turn identified traV and traB, which is predicted to encode an inner membrane protein. A traB bait exclusively identified traK preys. Several additional observations support the hypothesis that TraV, TraK and TraB form a complex in Escherichia coli that spans the cell envelope from the outer membrane (TraV) through the periplasm (TraK) to the inner membrane (TraB). First, two-hybrid analyses indicated that TraV and TraB bind to different TraK segments, as required if TraK bridges a ternary complex. Secondly, all three proteins fractionated with the E. coli outer membrane in tra+ cells. In contrast, TraB fractionated with the inner membrane in traV or traK mutant cells, and TraK appeared in the osmotic shock fluid from the traV mutant. These results are consistent with a TraV-TraK-TraB complex anchored to the outer membrane via the TraV lipoprotein. Further, in traK mutant cells, TraV failed to accumulate to a detectable level, and the TraB level was significantly reduced, suggesting that TraV and TraB must interact with TraK for either protein to accumulate to its normal level. Both TraK and TraV accumulated in traB2[Am] cells; however, the TraB2 amber fragment could be detected by Western blot, and sequence analysis indicated that the fragment retained the TraK-binding domain suggested by yeast two-hybrid analysis. We propose that TraV is the outer membrane anchor for a trans-envelope, Tra protein structure required for the assembly of F-pili and possibly for other events of conjugal DNA transfer.     

Import-defective colicin B derivatives mutated in the TonB box

The pore-forming colicin B is taken up into Escherichia coli by a receptor and TonB-dependent process. The receptor and colicin B both contain a similar amino acid sequence, close to the N-terminal end, termed the TonB box. Point mutations were introduced into the TonB-box region of the colicin B structural gene cba resulting in colicin B derivatives which were partially or totally inactive against E. coli cells. All derivatives still bound to the receptor. An inactive derivative killed cells when translocated across the outer membrane by osmotic shock treatment, and formed pores in planar lipid bilayer membranes identical to the wild-type colicin. Some of the mutations were partially suppressed by mutations in the tonB structural gene. It was concluded that the TonB-box mutations define a region that is involved in the uptake of colicin B across the outer membrane.