New major outer membrane proteins found in an Escherichia coli tolF mutant resistant to bacteriophage TuIb.

Cell envelopes prepared from an Escherichia coli tolF strain selected as resistant to phage TuIb contained a new major outer membrane protein related to outer membrane proteins Ia and Ib. The strain that produces this protein is a tolF par double mutant but contains an additional mutation leading to the production of the new major outer membrane protein. Antibiotic sensitivity lost as a result of the tolF mutation is regained in strains that contain the new major outer membrane protein. This indicates that this protein functions to restore the selective permeability of the outer membrane to low-molecular-weight hydrophilic molecules.     

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.     

Solubilization, isolation, and immunochemical characterization of the major outer membrane protein from Rhodopseudomonas sphaeroides

Solubilization of the major outer membrane protein of Rhodopseudomonas sphaeroides, and subsequent isolation, has been achieved by both non-detergent- and detergent-based methods. The protein was differentially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-soluble protein was purified to homogeneity by a combination of DEAE-Sephadex chromatography and preparative electrophoretic techniques. Similar to the peptidoglycan-associated proteins of other Gram-negative bacteria, the protein was also purified by differential temperature extraction of the outer membrane in the presence of sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. Immunochemical analysis of the proteins isolated by the two techniques established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody directed against the major outer membrane protein reacted with the three high molecular weight aggregates present in the outer membrane which we have previously shown to be composed of the major outer membrane protein and three nonidentical small molecular weight proteins.     

The identification of outer membrane proteins and flagella of Campylobacter jejuni

The outer membrane proteins of five clinical isolates of Campylobacter jejuni were identified by 125I-surface labelling and SDS-PAGE of outer membrane preparations. All isolates expressed a major outer membrane protein of variable molecular weight (43 000-46 000: 43K-46K). Several constant surface proteins were also identified including a 27K protein which was surface-exposed and acid-extractable but was not present in the outer membrane preparations. Isolated flagella comprised a major 62K protein and a minor 87K protein. Both proteins were absent in an aflagellate variant. The 62K protein was immunoblotted and immunoprecipitated by rabbit anti-flagella antisera.     

Characterization of outer membrane proteins in Chlamydia trachomatis LGV serovar L2

We used a photoactivatable, lipophilic reagent, 3'-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies of Chlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.     

Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp

Significant differences in cysteine-containing proteins and detergent-related solubility properties were observed between outer membrane protein complexes of reproductive (reticulate body) and infective (elementary body) forms of Chlamydia psittaci (6BC). Elementary bodies harvested at 48 h postinfection possessed a 40-kilodalton major outer membrane protein and three extraordinarily cysteine-rich outer membrane proteins of 62, 59, and 12 kilodaltons, all of which were not solubilized by sodium dodecyl sulfate in the absence of thiol reagents. Intracellularly dividing reticulate bodies harvested at 21 h postinfection were severely deficient in the cysteine-rich proteins but possessed almost as much major outer membrane protein as did the elementary bodies. Most of the major outer membrane protein of reticulate bodies was solubilized by sodium dodecyl sulfate and was present in envelopes as monomers, although a proportion formed disulfide-cross-linked oligomers. By 21 to 24 h postinfection, reticulate bodies commenced synthesis of the cysteine-rich proteins which were found in outer membranes as disulfide-cross-linked complexes. The outer membranes of reticulate bodies of Chlamydia trachomatis (LGV434) also were found to be deficient in cysteine-rich proteins and to be more susceptible to dissociation in sodium dodecyl sulfate than were outer membranes of elementary bodies.     

Localization of proteolytic activity in the outer membrane of Escherichia coli.

An enzyme in the cytoplasmic membrane, nitrate reductase, can be solubilized by heating membranes to 60 degrees C for 10 min at alkaline pH. A protease in the cell envelope has been shown to be responsible for this solubilization. The localization of this protease in the outer membrane was demonstrated by separating the outer membrane from the cytoplasmic membrane, adding back various forms of outer membrane protein to the cytoplasmic membrane, and following the increase in nitrate reductase solubilization with increasing amounts of outer membrane proteins. This solubilization is accompanied by the cleavage of one of the subunits of nitrate reductase and is inhibited by the protease inhibitor p-aminobenzamidine. Analysis of membrane proteins synthesized by cells grown in the presence of various amounts of p-aminobenzamidine revealed that p-aminobenzamidine affects the synthesis of the major outer membrane proteins but has little effect on the synthesis of cytoplasmic membrane proteins. When outer membrane is reacted with the protease inhibitor [3H]diisopropylfluorophosphate, a single protein in the outer membrane is labeled. Since the interaction with diisopropylfluorophosphate is inhibited by p-aminobenzamidine, it is suggested that this single outer membrane protein is responsible for the in vitro solubilization of nitrate reductase and the in vivo processing of the major outer membrane proteins.     

A dominant sulfhydryl-containing protein in the outer membrane of Neisseria gonorrhoeae.

By using a method that labels sulfhydryl-containing proteins in situ, we have detected a major outer membrane protein of Neisseria gonorrhoeae at 41 kDa. A protein of this molecular mass has not previously been shown to be a major outer membrane protein in gonococci. In addition, a minor protein rich in cysteinyl residues was detected at 31.5 kDa.     

Composition of major outer membrane proteins of Vibrio vulnificus isolates: effect of different growth media and iron deficiency

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl-nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron-deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl-nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.     

A major outer membrane protein of Serratia marcescens which was easily solubilized and had a capacity to bind to calcium

Easily solubilized major outer membrane protein was found in Serratia marcescens. The protein was originally obtained as a membrane-associated, insoluble protein in the outer membrane when the cells were slightly disrupted. However, the amount of this protein in the outer membrane gradually decreased with the time of sonication. The decrease was not due to decomposition of the protein but to solubilization into a soluble fraction after a long period of disruption. The molecular weight of this protein was 47 kDa and it bound calcium. Another 40 kDa calcium binding protein was also found in the outer membrane of S. marcescens.     

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.     

Heat modifiability and detergent solubility of outer membrane proteins of Rhodopseudomonas sphaeroides

The outer membrane fraction from Rhodopseudomonas sphaeroides was isolated by isopycnic density centrifugation. The purity of this fraction was assayed by several methods. When the outer membrane fraction obtained after French press lysis of cells was compared with the outer membrane fragments released during spheroplast formation, the polypeptide profiles were identical. Detergent solubilization of membrane fractions showed that Triton X-100 nonselectively solubilizes both the cytoplasmic membrane and the outer membrane, whereas Deriphat 160 selectively solubilizes the cytoplasmic membrane. Several outer membrane polypeptides, including the major outer membrane protein, exhibited changes in electrophoretic mobility that depended upon the temperature of solubilization in sodium dodecyl sulfate. Solubilization at room temperature in the presence of ions reproduced the effect of thermal denaturation on the major outer membrane polypeptide.     

Freeze etch morphology of outer membrane mutants of Escherichia coli K12

Freeze etching showed that the loss of each of the major outer membrane proteins b, c or d in mutants of Escherichia coli K12 does not influence the morphology of fracture faces of the outer membrane.Mutants that possess a heptose-deficient lipopolysaccharide and which in addition are deficient in one or more major outer membrane proteins exhibit a reduction in the number of intramembranous particles of the outer membrane.Moreover it was shown that lipid phase transitions induce a lateral lipid protein separation in the outer membrane, similar to that found in the cytoplasmic membrane.     

The surface properties of Neisseria gonorrhoeae: topographical distribution of the outer membrane protein antigens.

Gonococci were labelled with 125I using the lactoperoxidase system. The amount of label incorporated was similar with all strains including those which appeared capsulated. Electrophoresis on sodium dodecyl sulphate-polyacrylamide gels revealed that the major proteins labelled were those found in outer membrane preparations. Comparison of variants of one strain showed that the major outer membrane protein (protein I) was always present and heavily labelled. The second major protein (protein II) was present in variable amounts but labelling was proportional to the amount present. A third protein (III) was only present in outer membranes from a freshly isolated variant but was present in whole cells of each strain. Protein III was not labelled in whole cells but was labelled in outer membrane preparations suggesting that many membranes have their inner surface exposed. The labelling of a strain adapted to growth in guinea-pig chambers failed to reveal any new major surface proteins. The results demonstrate the variation in surface topography possible with variants of one strain of gonococcus but show that one major protein antigen is always expressed on the surface.     

Isolation and Characterization of Three Porin-Like Proteins from Vibrio vulnificus: Effect of Different Growth Media on Their Production

The effect of the culture media on the composition of the outer membrane protein of Vibrio vulnificus strain 393 from human blood was examined. Only one major outer membrane protein, with an apparent molecular weight of 37,000 (37K protein) and 34,000 (34K protein), was formed in the cells grown in 3% NaCl-BHI broth and chemically defined medium, respectively. The production of one major outer membrane protein was also observed in other isolates from humans and asari clam when they were grown in 3% NaCl-BHI broth. On the other hand, three major outer membrane proteins, with apparent molecular weights of 48,000 (48K protein), 37,000 (37K protein), and 34,000 (34K protein), were produced in the cells grown in 3% NaCl-nutrient broth. Three proteins, 48K, 37K, and 34K from strain 393, were purified and the amino acid compositions were determined. Although there was a little difference in the composition of amino acid among three proteins, the amino acid compositions of the three porin-like proteins showed characteristic properties of the porins of Escherichia coli and Salmonella typhimurium. Immunoblot analysis of the outer membrane proteins from four vibrios, E. coli, and S. typhimurium using monospecific antisera against these three porin-like proteins showed that only the antiserum against 37K protein cross-reacted with the outer membrane proteins from all the strains tested.     

衣原体主要外膜蛋白的研究进展

衣原体感染与多种慢性疾病密切相关,其主要外膜蛋白(MOMP)是一种多功能蛋白,分别与外膜结构的稳定性、生长代谢调节、抗原性和毒力密切相关。随着沙眼衣原体和肺炎衣原体基因组测序的完成,人们得以揭示其重要的生物合成、代谢途径,确定调控机制及其与致病的相关性。利用分子生物学技术在分子水平分析衣原体主要外膜蛋白的结构、抗原表位,对于免疫防御、免疫病理和免疫诊断均有重要意义。本文综述了衣原体主要外膜蛋白的分子结构、基因特性、抗原表位与应用前景。     

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.     

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis.

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.     

Strain-specific variation in the protein and lipopolysaccharide composition of the group B meningococcal outer membrane.

Variation in the protein and lipopolysaccharide composition of the meningococcal outer membrane may be due to either serotype differences or to changes in cultural conditions. There are 12 antigenically distinct serotypes of group B meningococci, and these are associated with distinct major outer membrane protein patterns on sodium dodecyl sulfate-polyacrylamide gels. In most strains the predominant outer membrane protein carries the serotype-specific determinant. Certain strains, when grown under similar conditions in different media showed an altered membrane composition. The type 2 strain, M986, grown in modified Frantz medium-A, had a reduced amount of the major 41,000-dalton protein while a 28,000-dalton protein predominated. The altered protein composition may be related to changes in cell metabolism as reflected by the pH of the medium after growth. Growth of the organism in Frantz medium-B caused a negligible drop in pH and the 41,000-dalton protein remained predominant. There was also variation associated with changes in the growth rate. Increasing the aeration caused a concomitant increase in growth rate and cell yield. We observed two quantitative changes in outer membrane proteins in four of seven strains examined: (i) where only a single major protein changed (three strains), and (ii) where an increase in one protein component was associated with a decrease in another protein (one strain). When the strains were grown in tryptic soy broth (Difco Laboratories, Detroit, Mich.) with either high or low aeration, the total protein in the outer membrane remained constant. In contrast, with high aeration there was a significant increase in lipopolysaccharide. These studies suggest that the cell surface proteins may be altered by the organism to meet a variety of environmental conditions.     

Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp. strain 109D

The ability of Bdellovibrio sp. to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired. Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp. could acquire the OmpC protein in the absence of the OmpF protein. However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane. The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp. exhibited a kinetic and temporal preference for the OmpF protein. Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency.