Expression, immunochemical characterization and localization of the Mycobacterium tuberculosis protein p27.

Rv2108 is a gene of the PPE family of Mycobacterium tuberculosis specific for this bacterial complex and that may encode a putative protein p27. This gene was amplified, inserted into bacterial vectors, sequenced, and expressed as a recombinant protein. Specific antibodies to this protein were generated and used for immunochemical characterization and cellular localization. Mass spectrometric analysis of the expressed protein revealed a molecule that corresponded to the p27 putative protein. The expressed protein was immunologically active, and reacted with antibodies from tuberculosis patient sera. Specific immunoblot analysis confirmed the presence of the p27 antigen in Mycobacterium bovis BCG strain and in human clinical isolates of M. tuberculosis, but not in other mycobacteria tested. Western blot and immunoelectron microscopic analysis of BCG strain indicated that the p27 protein is localized in the membrane of the cell. The specific expression of the p27 protein in the M. tuberculosis complex could provide a novel specific complimentary diagnostic test for the presence of and infection with M. tuberculosis.

Cloning and sequencing of the central region of the flagellin gene from the Gram-positive bacterium Clostridium tyrobutyricum ATCC 25755.

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum fiagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain. A PCR-restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation.

Evidence for an heterogeneous glycosylation of the Clostridium tyrobutyricum ATCC 25755 flagellin.

Glycosylation analysis of the flagellin from the Gram-positive species Clostridium tyrobutyricum has been supplemented. Amino acid analysis of the glycopeptides obtained after pronase digestion of flagellin indicated that O-glycosylation which was previously demonstrated after nonreductive beta-elimination, probably occurred via the hydroxyl group of serine. Otherwise, beta-elimination partly deglycosylated flagellin. After this treatment carbohydrates were still linked to protein as shown by a digoxigenin-hydrazide labelling. Therefore, in addition to linkages via serine, alkaline resistant linkages exist on the flagellin and some glycans may be linked to the protein core via the amide nitrogen of asparagine or via the hydroxyl group of tyrosine. Furthermore, according to an immunological analysis, glycans attached to flagellin via alkaline sensitive linkages may be different from those attached via alkaline resistant linkages.

Partial analysis of the flagellar antigenic determinant recognized by a monoclonal antibody to Clostridium tyrobutyricum.

In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D-glucose and N-acetyl-glucosamine in a molar ration of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D-glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The over-all stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain alpha (1-->4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.

Biochemical and immunological analyses of the flagellin of Clostridium tyrobutyricum ATCC 25755.

The monoclonal antibody 21E7-B12 (IgG3) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and beta-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.

Monoclonal antibody detection of Clostridium microcolonies directly on membrane used for milk filtration.

The normal procedure for bacterial colony detection requires a nitrocellulose transfer step after membrane filtration and culture to prevent the development of a high background during the immunodetection. In this paper, we describe a modification of the basic protocol that omits the transfer step and reduces the risk of background. Previous observations indicated that interactions between milk components (principally cream) and membrane are responsible for the high non-specific staining observed. Experiments were performed to remove lipid components or to block the membrane binding sites before milk filtration. Samples of milks of different origin (collected at different times of the year) and different membranes were tested. The results obtained showed that removing lipids did not significantly improve the test but, on the contrary, led to an antigen diffusion. Incubation of the membrane in 0.1% (w/v) of Tween 20 in phosphate-buffered saline before milk filtration prevented non-specific binding, and allowed performance of the detection without any noticeable background.

Production of monoclonal antibodies against the outer cell wall of Clostridium tyrobutyricum.

Several hybridoma cell lines producing murine monoclonal antibodies (mAbs) directed to the Clostridium tyrobutyricum outer cell wall have been established and characterized. Whole bacteria, crude extract of cell wall, and polysaccharide fraction of crude extract have been used as immunogens. The immunizations were performed either in vivo or in vitro after priming in vivo. Amongst the clones obtained, six hybridoma cell lines were selected. Four mAbs recognized only the immunizing strain (ATCC 25755), while two mAbs recognized all the C. tyrobutyricum tested strains. Three mAbs were IgM, one IgG3, and two IgG1 isotypes. The antigens (proteins or polysaccharides) recognized by these mAbs have been characterized by Western Blot. These mAbs could be used for an early detection of C. tyrobutyricum in milk.

Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody.

A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.

Histone-induced changes in the cellular growth of synchronized Chlamydomonas reinhardii are due to histone H1.

The cellular growth of Chlamydomonas reinhardii is modified by the addition of a total exogenous histone fraction. These modifications may be related to chloroplast DNA replication; they are different according to the different classes of histones. The H1 subfraction seems to be responsible for the effect of the total histone fraction.

[Inhibitory effect of exogenous histones on cellular growth and DNA synthesis in Chlamydomonas reinhardtii]. / Action inhibitrice des histones exogénes sur la croissance cellulaire et sur la synthése d' ADN chez Chlamydomonas reinhardtii.

The addition of exogenous histones to synchronous culture of Chlamydomonas reinhardtii, at the beginning of the cell cycle, leads to the death of the cells. The same amount of histones added in the middle of the cycle, only blocks the cell division. The mechanism of this inhibition effect of the histones could involve a block at the level of the chloroplast DNA replication.

Isolation and partial characterization of Chlamydomonas reinhardtii chromatin.

The use of a cell wall less mutant of Chlamydomonas reinhardtii allowed the isolation of the nuclei of this organism. The study of the extracted desoxyribonucleoprotein shows that a set of established properties of the chromatin of higher eucaryotes do not apply to this material. This is particularly the case for the failure to sediment in a good yield. This could be due to a low content in basic proteins of the chromatin of Chlamydomonas.