Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

An alteration in outer membrane permeability associated with a division lesion in a strain of Salmonella typhimurium.

Salmonella typhimurium strain 4a is a temperature sensitive mutant with defects in both septation and separation. The separation lesion was reversed by phenethylalcohol but this agent failed to allow septation or growth at restrictive temperature. Organisms of strain 4a grown at 42 degrees C were, unlike the parental strain, resistant to lysis by lysozyme plus EDTA and lipopolysaccharide was poorly extracted by EDTA from cultures of strain 4a grown at 42 degrees C. Such cultures may, therefore, be resistant to lysis with lysozyme plus EDTA not because the murein is altered but because the EDTA fails to permeabilize the outer membrane to lysozyme. In confirmation of this, murein isolated from strain 4a after growth at 42 degrees C showed the same sensitivity to lysozyme as murein from the parental strain. In spite of the altered envelope properties of strain 4a after growth at 42 degrees C, no major changes in protein or phospholipid composition have so far been demonstrated.     

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Polysaccharide inducible outer membrane proteins of Bacteroides xylanolyticus X5-1

Little is known about how bacteria degrade structural polysaccharides or the regulatory systems that control this degradation. Bacteroides xylanolyticus X5-1 is a Gram-negative, anaerobic bacterium that can grow on structural polysaccharides such as xylan and pectin. In order to determine the response of this organism to specific substrates,B. xylanolyticus was grown on a variety of mono-, di-, and polysaccharides. Electrophoretic analysis revealed no distinct differences in the polypeptide profile of the inner membrane enrichments of cells grown on different carbohydrates. However, distinct differences in protein composition of outer membrane enrichments were clearly observed. The profiles from cells grown on starch, xylan, and pectin were distinct from each other and their respective monosaccharide. In addition, cells initially grown on xylan did not alter their total membrane protein composition after three generations of growth in medium containing xylan and glucose. Thus,B. xylanolyticus X5-1 altered its outer membrane protein composition in response to specific polysaccharide substrates, but analysis of this specific response revealed no evidence that glucose was preferred over xylan as a substrate.     

Molecular composition of the outer membrane of Escherichia coli and the importance of protein-lipopolysaccharide interactions

Whole cells of Escherichia coli strains 0111, K12 and B as well as the ampicillin-resistant mutant K12 D21 and several lipopolysaccharide (LPS) mutants derived from this strain were analyzed for their molar LPS content per mg dry weight. An increase of the LPS concentration in some LPS mutants was substantiated by analyzing isolated cell walls and relating the molar LPS content to the murein subunit as measure of cell surface area. The increase of LPS was paralleled by increasing amounts of phospholipid while the overall protein content in the outer membrane decreased.According to the pattern of major outer membrane proteins in the various strains and the respective LPS structures, protein-LPS interactions are discussed as important requirements for outer membrane assembly and stability.Abbreviations LPS lipopolysaccharide - SDS sodium dodecyl-sulfate Dedicated to Dr. Otto Lüderitz on the occasion of his 60th birthday     

Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.     

Effect of bacteriophage P1 lysogeny on lipopolysaccharide composition and the lambda receptor of Escherichia coli.

The outer membrane of Escherichia coli was altered as a consequence of lysogeny by bacteriophages P1 and P1 cmts. The predominant change was a reduction in the size of lipopolysaccharide to a heptose-deficient form. P1 cmts lysogens were still sensitive to several bacteriophages but were resistant to lambda vir. Neither whole cells nor solubilized outer membranes from P1 cmts lysogens were able to inactivate lambda vir, and 32P-labeled lambda vir was unable to adsorb to P1 cmts lysogens. P1 cmts lysogens were also affected in maltose transport. The level of periplasmic maltose-binding protein was reduced somewhat, but there was no significant reduction in the level of the outer membrane lambda receptor (LamB). These membrane abnormalities were all corrected in strains cured of P1 cmts. It is suggested that P1 cmts affects lipopolysaccharide biosynthesis by a phage conversion mechanism and consequently the function of the lambda receptor.     

Antigenic variation and increase in pathogenicity in Pseudomonas aeruginosa as a result of growth on glucose or N-hexadecane as sole carbon source.

When Pseudomonas aeruginosa is grown on glucose as opposed to n-hexadecane as the sole carbon source, the antigenicity, virulence, and protein composition of the outer membrane are altered. The hydrocarbon-grown cells demonstrate a 3-log increase in virulence over the glucose-grown cells (in mice). There also appears to be an additional protein present in the outer membrane of the n-hexadecane-grown cells. This protein may contribute to the observed antigenic differences between the two cell types.     

Changes in the composition of anionic membrane phospholipids influence the protein secretion and biogenesis of cell envelope in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD 102 and HDL11 with controlled synthesis of anionic phospholipids, phosphatidylglycerol, and cardiolipin. Inactivation of the pgsA gene encoding the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. A conforming increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of lipopolysaccharide and lipopolyprotein of the outer membrane that determine the membrane barrier properties. The results obtained testify that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between the protein secretion and biogenesis of cell envelope components.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.     

Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins.     

Role of outer membrane components in the nonspecific binding ofEscherichia coli to cellulose

The involvement of lipopolysaccharide and outer membrane proteins in the binding ofEscherichia coli to cellulose was investigated. Cellulose binding was assayed in defined strains with or without O-antigenic polysaccharide and in mutants with defects in lipopolysaccharide core synthesis. Binding was also tested in strains lacking major outer membrane proteins. Optimal cellulose binding was exhibited by rough strains and was reduced to various extents in the presence of different O-antigens. Core defects also reduced but did not abolish binding to cellulose. Reduced binding was also found in mutants lacking OmpC protein, but OmpC/OmpA double mutants orompB mutants lacking OmpC and OmpF were not affected. Mutants with reduced cellulose binding were also isolated directly through selection of nonbinding populations after chromatography on cellulose columns. Each of the independent isolates derived fromE. coli K12 with reduced cellulose binding had multiple mutations, with additional phenotypic changes such as phage resistance, increased sensitivity to bile salts, or altered patterns of outer membrane proteins. These results suggest that no single receptor that could be altered by mutation was responsible for the binding ofE. coli to cellulose. Rather, the nonspecific binding of cellulose was more likely to be due to interaction with, or the combined activity of, several integral outer membrane components that could be masked by O-antigen.     

Morphological changes of synchronized Campylobacter jejuni populations during growth in single phase liquid culture

Campylobacter jejuni strains demonstrate a variety of growth phase-linked distinct morphological forms when grown in liquid culture. The typical spiral form of the organism, evident during logarithmic phase, undergoes elongation during stationary phase before becoming coccoid via the formation of membrane blebs and budded forms in decline phase. Cellular elongation and coccoid formation occurred despite the inhibition of protein synthesis and without a detectable change in the protein components of the inner and outer cell membranes.     

The dilution rate affects the outer membrane protein and lipopolysaccharide composition of Haemophilus influenzae type b grown under iron limitation.

When Haemophilus influenzae type b was grown under iron limitation in continuous culture, the dilution rate affected the outer membrane protein and lipopolysaccharide composition. Investigations of the effect of the reduced availability of iron or other environmental parameters on these surface components should be controlled for growth rate.     

Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease.

Saccharomyces cerevisiae strains are often host to several types of cytoplasmic double-stranded RNA (dsRNA) genomes, some of which are encapsidated by the L-A dsRNA product, an 86,000-dalton coat protein. Here we present the finding that nuclear recessive mutations in the NUC1 gene, which encodes the major nonspecific nuclease of yeast mitochondria, resulted in at least a 10-fold increase in amounts of the L-A dsRNA and its encoded coat protein. The effect of nuc1 mutations on L-A abundance was completely suppressed in strains that also hosted the killer-toxin-encoding M dsRNA. Both NUC1 and nuc1 strains containing the L-A genome exhibited an increase in coat protein abundance and a concomitant increase in L-A dsRNA when the cells were grown on a nonfermentable carbon source rather than on glucose, an effect independent of the increase in coat protein due to nuc1 mutations or to the absence of M. The increase in L-A expression in nuc1 strains was similar to that observed in strains with mutations in the nuclear gene encoding the most abundant outer mitochondrial membrane protein, porin. nuc1 mutations did not affect the level of porin in the mitochondrial outer membrane. Since the effect of mutations in nuc1 was to alter the copy number of the L-A coat protein genome rather than to change the level of the M toxin genome (as do mak and ski mutations), these mutations define a new class of nuclear genes affecting yeast dsRNA abundance.     

Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane.

Neisseria meningitidis group B serotype 2 strain M986 contains two predominant outer membrane proteins, with apparent molecular weights of 41,000 (protein b) and 28,000 (protein e). Heating of outer membrane vesicles at 56 degrees C for 20 min caused much of b** to disaggregate and denature into b (41,000 daltons). In contrast, protein e could be rapidly solubilized by SDS at room temperature into its monomeric state (e*), but it was not converted to its final higher apparent molecular weight of 28,000 (e) unless heated at 100 degrees C for 2 min. We propose that protein b exists in the membrane as trimers or tetramers in a transmembrane configuration and that protein e exists as subunits on the exterior surface of the outer membrane and has a highly ordered tertiary structure.     

Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.     

Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis

A panel of monoclonal antibodies (MAb) was generated against Chlamydia trachomatis serovar B, an etiologic agent of blinding trachoma. The specificities of MAb were determined by dot blot assay by using viable elementary bodies of 13 C. trachomatis serovars and two C. psittaci strains. The dot blot assay was used to identify those antigens that were unique and immunoaccessible on the chlamydial surface. MAb were identified that recognized bi-specific (serovars B and Ba) or subspecies-specific (various B complex serovars) surface-exposed antigenic determinants that were either resistant or sensitive to heat denaturation (56 degrees C, 30 min). All of the MAb recognized the major outer membrane protein as determined by either immunoblotting or radioimmunoprecipitation. MAb specific for immunoaccessible major outer membrane protein epitopes protected mice from toxic death after i.v. injection of B serovar elementary bodies and neutralized the infectivity of the organism for monkey eyes. In contrast, MAb reactive against non-immunoaccessible subspecies- or species-specific major outer membrane protein epitopes or against an immunoaccessible genus-specific epitope located on chlamydial lipopolysaccharide did not protect mice from toxic death or neutralize infectivity of the parasite for monkey eyes. These data suggest that those major outer membrane protein antigenic determinants that are serovar or serogroup specific and are accessible to antibody on the chlamydial cell surface may be useful as a recombinant subunit vaccine for trachoma.