Dynamic state of concanavalin A receptor interactions on fibroblast surfaces.

Cultured normal and transformed fibroblasts were treated "in situ" by the concanavalin A-peroxidase labelling technique. It is known that peroxidase recognizes only a fraction of the bound lectin depending on the cell type. Kinetics studies revealed that 80 to 95 percent of the peroxidase and only 10 percent of the lectin are released from the cell surface when the labelled cells were reincubated at 37 degrees C. It is shown that it is mostly the concanavalin traced by peroxidase that is released and also that the lectin and the enzyme are shed as a complex or concomitantly. Consequently, the shedding pattern of the enzyme is used to demonstrate heterogeneity in the lectin binding sites; there are two main components labelled by concanavalin and peroxidase, one which has a short period (from 6 to 16 min) and another one with a much longer one (1.3 to 3 h). It is shown that when cells are incubated at 37 degrees C after a lectin treatment, secondary binding forces occur between the lectin and cell surface components which render the lectin unavailable for inhibiting sugars. Under the same conditions, some peroxidase can still be bound and a slight agglutination can still occur.

Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus.

We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus. The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg. D. africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme indicates the presence of three [4Fe-4S]2+/1- centers/subunits. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV. The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor. The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process. D. africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme. POR was shown to be located in the cytoplasm by immunogold labelling.

Construction, expression and release of hybrid colicins.

Colicins A and E1 are two pore-forming colicins sharing homology in their C-terminal domains but not in their N-terminal or central domains. Using site-directed mutagenesis, restriction sites were inserted at the proper locations to allow recombination of these domains. Six different constructs were obtained. All these proteins were expressed in Escherichia coli and properly recognized by monoclonal antibodies directed against epitopes located in different domains of colicin A. Out of the six hybrids, only two were released to the extracellular medium. Immunocytolocalization indicated that some of the hybrids aggregated within the cytoplasm. With some hybrids, the defect in release was related to a defect in synthesis of the lysis protein that normally promotes release.

Immunological analysis of porin polymorphism in Escherichia coli B and K-12.

Two sets of monoclonal antibodies (MoF type I and MoF type II) directed against the OmpF protein were used to analyze the immunological reactivity of the major outer membrane porins of E. coli B and K-12. All these antibodies present a specificity to the native OmpF protein. In addition, among the type II antibodies, MoF 18, 19 and 20 could recognize an epitope present on both monomeric and trimeric forms of the porin as demonstrated by immunoblotting analyses. The use of two different screening methods led to the isolation of two different sets of MoF, one specific for a native conformation accessible only on E. coli B strain and the second directed against epitopes present on OmpF of the two strains, B and K-12. These various responses are discussed in relation to the lipopolysaccharide binding to OmpF and with respect to the screening test used.

Immunological evidence for differences in the exposed regions of OmpF porins from Escherichia coli B and K-12.

Nine monoclonal antibodies (MoF 0-8) directed against the native form (trimeric) of outer membrane protein OmpF of Escherichia coli B were obtained and characterized. All these antibodies bind to OmpF porin in intact E. coli B cells but not OmpF from E. coli K-12 cells which only differ at positions 66, 117 and 262 in the sequence. These antibodies exhibit a specificity to the native form, failing to recognize the denatured form in a liquid immunorecognition assay. Four tested antibodies are able to protect against colicin A, a bacteriotoxin using OmpF as receptor. One monoclonal antibody (MoF 0) is specific to the external topology of native porin in the outer membrane and three antibodies could recognize epitopes present in each conformation of subunits of trimer form. It is concluded that the region around the 66th and more probably around the 262nd amino acids are involved in cell-surface exposed epitopes. Moreover, these results support the assumption that the conformation of protruding regions of OmpF from E. coli B and K-12 are different.

Gastric lipase and pepsinogen during the ontogenesis of rabbit gastric glands.

In rabbit stomach, gastric lipase activity level was found to increase from birth to 30 days old (weaning), and then decreased. In contrast, pepsin activity only appeared between 30 to 45 days old, and increased till to the adult level. It was observed that maturation of gastric glands in cardial mucosa was a downward elongation process from the mitotic cell pool. These mitotic cells were always found in the neck of the gastric glands, corresponding to the bottom of the gland at 6 days old and to the mid-zone of the gland in adult. Location of rabbit gastric lipase (RGL) cells in cardial glands varied with age and was found along the pit of the gastric glands at 6 days old. The extent of this cellular location decreased with age, whereas a second RGL cell zone appeared below the mitotic cell area at 18 and 30 days old. At 45 days old, the pepsinogen cells appeared in the bottom of the gland, and consequently the RGL cells were located in the mid-zone of the gastric glands, between mitotic cells (neck of the gland) and pepsinogen cells (lower part of the gland). Ultrastructural study of cardial gastric glands revealed different morphologies of the secretion granules in the cells along the gastric glands. In 6-day-old rabbits, secretory granules were found uniformly electron dense in the bottom of the glands and were RGL-labeled by the immunogold technique. In the medium part of the glands, granules appeared biphasic, with a clear and a dense part, and RGL labeling was confined to the electron-dense part.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of rabbit gastric lipase and pepsinogen.

Lipase and pepsin activities were determined in rabbit gastric biopsy specimens. Lipase activity was found to be restricted to a small part of the fundic mucosa, near the cardia, whereas pepsin activity spread over about two thirds of the total fundic area, overlapping that of lipase. The cells producing these two enzymes were labeled by immunofluorescence using polyclonal antibodies against rabbit gastric lipase (RGL) or antibodies against rabbit pepsinogen. The immunocytochemical localization showed unequivocally that RGL and pepsinogen, which were both present in the cardial area, were in fact located in different gastric cells. The cells producing pepsinogen were in the lower base of the gastric fundic glands, whereas the cells producing RGL were in the upper base of the same glands. The cells producing pepsinogen and RGL showed no significant morphological differences. In the part of the fundic area, where only pepsin activity was detected, cells producing pepsinogen covered both the lower and the upper base of the gastric glands. No chief cells were observed in the antral mucosa. RGL and pepsinogen could represent useful gastric enzyme markers for cellular differentiation studies.

Improving the stability of a foreign protein in the periplasmic space of Escherichia coli.

An efficient expression/export vector comprising the entire phoS (phosphate binding protein) gene fused to a synthetic gene encoding the human growth hormone releasing factor (mhGRF) has recently been constructed [1]. The hybrid protein (PhoS-mhGRF) was exported to the periplasmic space. However, in this location proteolytic degradation occurred at the C-terminal region. Phenylmethylsulfonyl fluoride (PMSF) increased the stability of the hybrid protein indicating that a serine protease may be involved in the proteolytic cleavage. The correct export and subsequent degradation of the recombinant protein in the periplasmic space were demonstrated in situ using double immunogold labeling on ultrathin sections. Using a phoS-based expression/export vector in the presence of PMSF, 2-4 mg of hybrid protein per liter of culture could be obtained.

Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli.

Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coli B and K-12, have been used to study the assembly and the topology of the protein. This paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies. A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows us to propose a rough model including 2 antigenic sites. The second part is focused on the assembly of the OmpF protein in the outer membrane. Various forms, precursor, unassembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process. The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope. The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly.

A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli.

Inducible hybrid genes encoding two large domains, a periplasmic domain consisting of the PhoS sequence and an outer membrane domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesized hybrid polypeptides are correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivity toward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogold labelling on ultrathin cryosections. Insertion of a hydrophobic sequence between PhoS and OmpF improves the trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimeric conformation which is recognized by specific antibodies. Moreover, the deletion of the C-terminal region impairs the functional insertion of the OmpF domain; this indicates that the last membrane-spanning region of OmpF is necessary for the correct folding and orientation of the protein in the outer membrane.

Colicin A lysis protein promotes extracellular release of active human growth hormone accumulated in Escherichia coli cytoplasm.

The colicin A lysis protein (Cal) was used to direct the extracellular release of recombinant proteins produced in Escherichia coli. The cal gene, under the control of its inducible promoter, was introduced into an expression vector encoding the human growth hormone devoid of its signal sequence (Met-hGH). Cal and Met-hGH were simultaneously expressed at two different levels of Met-hGH induction. The results indicate that Cal causes the excretion of non-aggregated Met-hGH from the cytoplasm to the culture medium and that the Met-hGH is correctly folded since the released Met-hGH is antigenically indistinguishable from the authentic mature hGH and is biologically active in binding to specific receptor sites.

Cytoplasmic and periplasmic expression of a synthetic gene for ferredoxin in Escherichia coli.

A synthetic gene coding for a modified ferredoxin II of Desulfovibrio desulfuricans Norway strain was assembled from 10 oligonucleotides. This gene was cloned into various expression vectors allowing either cytoplasmic expression or export to the periplasmic space. In the latter case, two different constructs were made, each of which contained the OmpA signal peptide: one of these constructs contained 3 additional N-terminal amino acids as compared to the wild-type ferredoxin (56 amino acid residues). The expression of proteins encoded by the 3 constructs was assayed in E coli and the proteins were localized by cell fractionation and immunogold labelling. A low percentage of the periplasmic ferredoxin (approximately 5%) was secreted to the medium in the absence of cell lysis. The recombinant ferredoxin was purified and found to be correctly processed by the leader peptidase. However, due to the high cysteine content intramolecular and intermolecular disulfide bonds were formed and prevented binding of [4Fe-4S] clusters. Reconstitution experiments using these recombinant proteins are in progress.

Colicin N interaction with sensitive Escherichia coli cells: in situ and kinetic approaches.

The kinetics of colicin N binding to the Escherichia coli surface, comprising the recognition of and association with cell-surface-exposed sites of OmpF porin, is a rapid event taking place during the first seconds of incubation. Immunogold labelling demonstrates the membrane localization of the colicin N bound to sensitive cells. Analyses of colicin-induced efflux indicate a short lag before the onset of cytoplasmic K+ release. This delay reflects the time necessary for translocation from the external side and pore-forming insertion into the cytoplasmic membrane.

Involvement of a single periplasmic hydrogenase for both hydrogen uptake and production in some Desulfovibrio species.

Various sulphate-reducing bacteria differing in the number of genes encoding hydrogenase were shown to ferment lactate in coculture with Methanospirillum hungatei, in the absence of sulphate. The efficiency of interspecies H2 transfer carried out by these species of sulphate-reducing bacteria does not appear to correlate with the distribution of genes coding for hydrogenase. Desulfovibrio vulgaris Groningen, which possesses only the gene for [NiFe] hydrogenase, oxidizes hydrogen in the presence of sulphate and produces some hydrogen during fermentation of pyruvate without electron acceptor. The hydrogenase of D. vulgaris was purified and characterized. It exhibits a molecular mass of 87 kDa and is composed of two different subunits (60 and 28 kDa). D. vulgaris hydrogenase contains 10.6 iron atoms, 0.9 nickel atom and 12 acid-labile sulphur atoms/molecule, and the absorption spectrum of the enzyme is characteristic of an iron-sulphur protein. Maximal H2 uptake and H2 evolution activities were 332 and 230 units/mg protein, respectively. D. vulgaris cells contain exclusively the [NiFe] hydrogenase, whatever the growth conditions, as shown by biochemical and immunological studies. Immunocytolocalization in ultrathin frozen sections of cells grown on lactate and sulphate, on H2 and sulphate and on pyruvate showed that the [NiFe] hydrogenase was located in the periplasmic space. Labelling was enhanced in cells grown on H2 and sulphate and on pyruvate. The results enable us to conclude that D. vulgaris Groningen contains a single hydrogenase of the [NiFe] type, located in the periplasmic space like that described for D. gigas. This enzyme appears to be involved in both H2 uptake and H2 production, depending on the growth conditions.

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?

The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.

Purification and reconstitution into liposomes of an integral membrane protein conferring immunity to colicin A.

The immunity protein to colicin A protects producing cells from the action of this pore-forming toxin. It is located into the cytoplasmic membrane. This protein has been 'tagged' with an epitope from the colicin A protein for which a monoclonal antibody is available. The fusion protein (named VL1) has been purified after extraction from the membrane in two steps using a chromatofocusing and an immunoadsorbant chromatography. The purified protein has then been reconstituted into lipid vesicles.

Structural properties of the tubular appendage spinae from marine bacterium Roseobacter sp. strain YSCB.

Spinae are tubular surface appendages broadly found in Gram-negative bacteria. Little is known about their architecture, function or origin. Here, we report structural characterization of the spinae from marine bacteria Roseobacter sp. YSCB. Electron cryo-tomography revealed that a single filament winds into a hollow flared base with progressive change to a cylinder. Proteinase K unwound the spinae into proteolysis-resistant filaments. Thermal treatment ripped the spinae into ribbons that were melted with prolonged heating. Circular dichroism spectroscopy revealed a dominant beta-structure of the spinae. Differential scanning calorimetry analyses showed three endothermic transformations at 50-85°C, 98°C and 123°C, respectively. The heating almost completely disintegrated the spinae, abolished the 98°C transition and destroyed the beta-structure. Infrared spectroscopy identified the amide I spectrum maximum at a position similar to that of amyloid fibrils. Therefore, the spinae distinguish from other bacterial appendages, e.g. flagella and stalks, in both the structure and mechanism of assembly.

Exclusive localization of colicin A in cell cytoplasm of producing bacteria.

The production of colicin A in Citrobacter freundii and in Escherichia coli was studied. After induction with low concentrations of mitomycin C, these organisms differed with regards to cell growth, cell viability, and kinetics of colicin A biosynthesis. Despite these differences, immunoferritin labelling on ultra-thin sections of induced frozen cells demonstrated that colicin A was located exclusively within the cell cytoplasm in both types of bacteria. By using protein markers, it was shown that at no time after induction was colicin A accumulated in the periplasmic space or in inner or outer membranes. These results were confirmed by a biochemical approach. For at least 3 h after induction, colicin A remained associated with producing cells and no colicin A activity was found in the periplasmic space. These results are discussed with reference to the synthesis and export of other bacteriocins.