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.     

Deletions or duplications in the BtuB protein affect its level in the outer membrane of Escherichia coli.

The Escherichia coli btuB product is an outer membrane protein that mediates the TonB-coupled active transport of cobalamins and the uptake of the E colicins and bacteriophage BF23. The roles of various segments of the BtuB protein in its function or cellular localization were investigated by analysis of several genetic constructs. Hybrid proteins in which various lengths from the amino terminus of BtuB were linked to alkaline phosphatase (btuB::phoA genes) were all secreted across the cytoplasmic membrane. The BtuB-PhoA proteins that carried up to 327 amino acids of BtuB appeared to reside in the periplasmic space, whereas hybrid proteins containing at least 399 amino acids of BtuB were associated with the outer membrane. Eleven in-frame internal deletion mutations that spanned more than half of the mature sequence were prepared by combining appropriate restriction fragments from btuB variants with 6-bp linker insertions. None of the deleted proteins was able to complement any BtuB functions, and only three of them were detectable in the outer membrane, suggesting that most of the deletions affected sequences needed for stable association with the outer membrane. Duplications covering the same portions of BtuB were prepared in the same manner. All of these partial duplication variants complemented all BtuB functions, although some gave substantially reduced levels of activity. These proteins were found in the outer membrane, although some were subject to proteolytic cleavage within or near the duplicated segment. These results indicate that the insertion of BtuB into the outer membrane requires the presence of several regions of teh BtuB protein and that the presence of extra or redundant segments of the protein can be tolerated during its insertion and function.     

Expression of hydroxamate and phenolate siderophores by Shigella flexneri.

Shigella flexneri strains were assayed for the ability to synthesize and utilize phenolate and hydroxamate siderophores. The hydroxamate aerobactin was synthesized by all isolates tested, whereas phenolates were only rarely produced. Expression of aerobactin was accompanied by production of a single iron-regulated outer membrane protein (Mr = 74,000). This protein was not produced by a mutant defective in aerobactin utilization and may serve as the aerobactin receptor. Phenolate (enterobactin)-producing strains synthesized three additional outer membrane proteins (Mr = 74,000, 81,000, and 83,000) in response to iron starvation. These proteins are the same apparent size as those produced by Escherichia coli K-12 strains. Ent sequences are apparently present in strains which do not synthesize this compound. Although normally silent, ent genes can be activated in Ent- strains to produce Ent+ variants. These laboratory variants are phenotypically indistinguishable from clinical Ent+ isolates.     

Cultivation of Escherichia coli to high cell densities in a dialysis reactor

High cell density cultivation of Escherichia coli on a glycerol-based mineral medium was studied. The cultivation was done in a dialysis reactor composed of two chambers. The inner chamber is formed and separated from an outer chamber by a membrane. Fresh medium was continuously exchanged with medium in the outer chamber so that both glycerol and other components of the medium were supplied to the inner chamber through the membrane. Inhibitory substances diffused from the inner to the outer chamber and were subsequently removed with effluent from the outer chamber. Initially, mathematical models were used to describe the process. The optimal cultivation parameters, such as the initial glycerol concentrations in the two chambers, the desired transport rate across the membrane, glycerol concentration in the feed/dialysing medium, and the time to start the medium exchange, were determined from preliminary experiments and calculations. The actual cultivation results agreed very well with the model predictions. A very high cell concentration of 174 g dry weight/1 was obtained. This cell concentration is within the range of the maximum theoretical concentration of E. coli in culture broth (160–200 g/l).Dedicated to the 60th birthday of Prof. Dr. D. Vortmeyer Correspondence to: H. Märkl     

The role of the protein with molecular weight of 22 kD in the phagocytosis of vaccine strain of Yersinia pestis EV

The comparative study of different stages of the phagocytosis of vaccine strain Y. pestis EV and its achromogenic variants (AV), has been carried out with the use of peritoneal macrophages as an in vitro experimental model. As revealed in this study, AV whose outer membrane contains no protein with a molecular weight of 22 kD exhibit lower capacity for adherence and for being ingested by phagocytes than the initial strain. The absence of this protein does not inhibit the multiplication of AV inside phagocytes, leading to incomplete phagocytosis, which is characteristic of the initial strain. The suggestion is made that the 22 kD protein may be one of the adhesion factors, necessary for ensuring the initial stages of phagocytosis.     

The effect of protein II and pili on the interaction of Neisseria gonorrhoeae with human polymorphonuclear leucocytes

Colonial variants of Neisseria gonorrhoeae strain P9 expressing different pili and/or outer membrane protein II (P.II) were investigated with respect to their interaction with human polymorphonuclear leucocytes (PMN). Two assay systems were used. A phagocytic killing assay measured the intracellular survival of gonococci, and PMN chemiluminescence (CL) was used to determine the initial surface interactions. All variants expressing P.II were killed effectively by PMN and also greatly stimulated PMN CL. The P.II- variants, on the other hand, were resistant to phagocytic killing and stimulated a much lower CL response. The presence of different P.II species was associated with different CL profiles and therefore different modes of interaction with the PMN membrane. A P.II-specific monoclonal IgG was opsonic and greatly increased PMN CL in contrast to F(ab')2 prepared from the same antibody, which inhibited it, thus confirming the role of P.II in the PMN interaction. Phagocytic killing assays revealed that with the loss of P.II, gonococcal variants acquired resistance to killing. Comparison of piliated and non-piliated pairs of variants with the same P.II profile showed that PMN-gonococcal interactions are dominated by the nature of the P.II species present whereas pili have little effect.     

Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH.     

放线共生放线杆菌粗糙型与光滑型菌落的主要外膜蛋白

目的：观察放线共生放线杆菌粗糙型与光滑型菌株菌体蛋白表达上的差异。方法：聚丙稀酶胺凝胶电泳观察两型细菌全细胞蛋白及超高速离心提取的细菌主要外膜蛋白差异。结果：全细胞蛋白电泳两型细菌蛋白带无明显差异;提取的主要外膜蛋白电泳粗糙型存在18、29、45kDa蛋白带,实验室参考菌株不存在,临床光滑型菌株存在少量,光滑型实验室参考菌株存在的蛋白带在所有实验菌株中均存在。结论：18、29、45kDa蛋白带可能与放线共生放线杆菌粗糙型菌株相关。     

Variation in the expression of pili and outer membrane protein by Neisseria meningitidis during the course of meningococcal infection

The occurrence of antigenic shift during meningococcal infection has been investigated by comparison of paired isolates obtained from the blood, cerebrospinal fluid or nasopharynx of patients. Isolates from any individual produced identical DNA 'fingerprints' and showed stability in expression of both class 2 outer membrane protein and an antigen common to pathogenic Neisseria, confirming their origin as a single strain. One of the four strains examined produced variants which differed in the molecular mass of their class 5 outer membrane proteins. Three of the strains produced pili containing the epitope recognized by monoclonal antibody SM1 and two of these gave rise to variants which expressed pili of differing subunit molecular masses. The two variants of the remaining strain produced pilins lacking the common epitope detected by antibody SM1 but radioimmune precipitation with polyclonal anti-pilus antiserum revealed that variation in the molecular mass of the pilin expressed also occurred with this second class of pili. Antigenic variation in expression of both class 5 outer membrane proteins and pili therefore appears to be a common occurrence during meningococcal infection.     

Altered outer membrane protein in different colonial types of Neisseria gonorrhoeae.

Dark-colored colony types of Neisseria gonorrhoeae (T3 and dark variants of T1 and T2) had markedly increased amounts of an approximately 28,000-dalton outer membrane protein, as compared with light-colored colony types (T4 and light variants of T1 and T2). The presence of this protein appeared to be unrelated to piliation. The apparent molecular weight of this protein on sodium dodecyl sulfate-polyacrylamide gels varied, depending on methods used to solubilize envelope proteins. In view of the location of this protein on the outer membrane, this protein could be important to the pathogenicity or antigenicity of the organism as well as to colonial characteristics in vitro.     

Antigenic variation of outer membrane protein II in colonial variants of Neisseria gonorrhoeae P9

Antibodies were detected by an enzyme-linked immunosorbent assay (ELISA) in sera from rabbits immunized wtih outer membranes from colonial opacity variants in Neisseria gonorrhoeae P9. ELISA-inhibition experiments with purified antigens revealed approximately equal proportions of antibodies directed against each of the three major surface antigens, lipopolysaccharide, the major outer membrane protein (protein I) and protein II, the variable protein associated with colonial opacity. Inhibition experiments with intact gonococci showed considerable antigenic diversity which could be correlated with differences between the protein II species present. Despite their considerable structural homology, different protein II species from colonial variants of the same strain showed little cross-reactivity with specific anti-protein II sera, thus demonstrating the considerable variation in that part of the antigen which is exposed on the surface of the gonococcus and is closely involved in pathogenic mechanisms.     

In vivo membrane assembly of split variants of the E.coli outer membrane protein OmpA.

The two-domain, 325 residue outer membrane protein OmpA of Escherichia coli is a well-established model for the study of membrane assembly. The N-terminal domain, consisting of approximately 170 amino acid residues, is embedded in the membrane, presumably in the form of a beta-barrel consisting of eight antiparallel transmembrane beta-strands. A set of 16 gene variants carrying deletions in the membrane-embedded domain of OmpA was constructed. When pairs of these mutant genes were co-expressed in E.coli, it was found that a functional OmpA protein could be assembled efficiently from two complementary protein fragments. Assembly was found when the polypeptide chain was split at the second or third periplasmic turn. All four protein termini were located in the periplasmic space. Interestingly, duplication of transmembrane strands five and six led to a variant with an unusual topology: the N-terminus of one fragment and the C-terminus of the other fragment were exposed at the cell surface. This is the first demonstration of correct membrane assembly of split beta-structured membrane proteins. These findings are important for a better understanding of their folding/assembly pathway and may have implications for the development of artificial outer membrane proteins and for the cell surface display of heterologous peptides or proteins.     

Insertion of atToc34 into the chloroplastic outer membrane is assisted by at least two proteinaceous components in the import system.

Toc34 is a member of the outer membrane translocon complex that mediates the initial stage of protein import into chloroplasts. Toc34, like most outer membrane proteins, is synthesized in the cytosol at its mature size without a cleavable transit peptide. The majority of outer membrane proteins do not require thermolysin-sensitive components on the chloroplastic surface or ATP for their insertion into the outer membrane. However, different results have been obtained concerning the factors required for Toc34 insertion into the outer membrane. Using an Arabidopsis homologue of pea Toc34, atToc34, we show that the insertion of atToc34 was greatly reduced by thermolysin pretreatment of chloroplasts as assayed either by protease digestion or by alkaline extraction. The insertion was also dependent on the presence of ATP or GTP. A mutant of atToc34 with the GTP-binding domain deleted still required ATP for optimal insertion, indicating that ATP was used by other protein components in the import system. The ATP-supported insertion was observed even in thermolysin-pretreated chloroplasts, suggesting that the protein component responsible for ATP-stimulated insertion is a different protein from the thermolysin-sensitive component that assists atToc34 insertion.     

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Ferrichrome-promoted iron uptake in Escherichia coli K12 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coli resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.     

Body structure of marine sponges

Summary Specimens of Haliclona elegans (Bowerbank, 1866) are covered by a thin, double layered dermal membrane extending over large subdermal spaces. The pores in the dermal membrane are formed by single porocytes with one or sometimes several pores in the center of the cell. The subjacent tissue shows a faintly developed mesenchyme and numerous big choanocyte chambers projecting into lacunar spaces of the incurrent canal system. The outer surface of the chambers is directly covered by the pinacocyte epithelium of the incurrent canal wall, which also separates them completely from the mesenchyme. Water influx into the chambers is guaranteed by prosopylar openings in the pinacocyte cover at the outer chamber surface. The chambers are connected to the excurrent canal system in the eurypylous way by wide apopyles, each of which is surrounded by a small ring of flagellated cone cells. About 15% of the choanocyte chambers in H. elegans contain central cells, which are thought to derive from migrating pinacocytes of the canal systems.     

The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.     

Connection of outer membrane protein composition in Yersinia pestis cells with intrinsic plasmids]

A set of isogenic derivatives of Yersinia pestis EV strain was obtained including the variants harbouring the different compositions of Yersinia own plasmids. The protein profiles of outer membranes of the set of strains were defined. The polyacrylamide gel electrophoresis has shown the small 6.1 Md plasmid to code an outer membrane protein with mol mass 29 kDa, different from pesticin I, while the heavy 60.0 Md plasmid encodes the 15-16 kDa polypeptide different from monomers of F1 and T-antigens of plague microbe.     

Demonstration of a Ferric Vibrioferrin-Binding Protein in the Outer Membrane of Vibrio parahaemolyticus

Under iron-restricted conditions, Vibrio parahaemolyticus produces a siderophore, vibrioferrin, accompanying expression of two major outer membrane proteins of 78 and 83 kDa. Autoradiographic analysis of nondenaturing polyacrylamide gel electrophoregrams of outer membrane preparations previously incubated with [⁵⁵Fe]ferric vibrioferrin revealed a single radiolabeled band, in which the 78-kDa protein was detected predominantly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum against the purified 78-kDa protein partially inhibited Fe-VF binding to isolated OMPs. The 78-kDa protein was cleaved by the treatment of whole cells with proteinase K, indicating that a portion of this protein is exposed on the surface of the outer membrane. The treated cells lost most of their iron uptake activity mediated by vibrioferrin. These results suggest that the ferric vibrioferrin-binding protein of 78 kDa may function as the receptor for ferric vibrioferrin involved in the initial step of vibrioferrin-mediated iron uptake. Immunoblot analysis using the antiserum against the 78-kDa protein demonstrated that the molecular mass and antigenic properties of the protein were highly conserved among V. parahaemolyticus strains examined. The antiserum also recognized an iron-repressible outer membrane protein of 78 kDa from iron-restricted V. alginolyticus strains, some of which appeared to produce vibrioferrin.     

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.