Detection of diphtheria toxin in culture supernates of Corynebacterium diphtheriae and C. ulcerans by immunoassay with monoclonal antibody.

Monoclonal antibody (MAb) to diphtheria toxin was produced in mouse hybridomas, and shown by ELISA to be of sub-class IgG1. Hybridomas were inoculated into mice to produce ascitic fluid from which MAb was purified by caprylic acid. The MAb was shown by immunoblotting to be directed against the A fragment of the toxin and also against the intact toxin molecule. After conjugation with fluorescein isothiocyanate, it was used in an immunoassay to detect toxin in culture supernates of Corynebacterium diphtheriae and C. ulcerans. The assay correlated well with the Elek test and with virulence in guinea-pigs; but it gave occasional false positive results, probably by binding of MAb to defective toxin.

Nonspecific stimulation of host defense by Corynebacterium kutscheri. II. Isolation of the active moiety.

The isolation and determination of biological activities of the active component of Corynebacterium kutscheri were attempted in the present investigation. The antitumor effect was confined to the subcellular particle fraction of this bacterium and was associated with a molecule of glycoprotein nature (40,000-38,000 Daltons) isolated from this fraction by affinity chromatography with concanavalin A-Sepharose 4B. This substance exerted mitogenic activity on C3H/HeJ splenocytes and T cells, stimulatory activity on macrophages, and further exhibited antitumor effect on P388 leukemia in CDF1 mice. The Winn assay disclosed that the antitumor effect induced by this substance was dependent on L3T4+ T cells. Furthermore, both the mitogenic and antitumor activity of this moiety were resistant to heating at 100 degrees C for 30 min or RNase digestion, but sensitive to trypsin digestion, or low or high pH. These results indicate that the antitumor effect of C. kutscheri is attributable to the heat-stable glycoprotein moiety which can directly stimulate T cells and macrophages.

Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii.

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.

Analysis of mycolic acids from a group of corynebacteria by capillary gas chromatography and mass spectrometry.

The cell wall of leprosy-derived corynebacteria (a group of 'diphtheroids' isolated from human leprosy lesions and patients' blood) was previously shown to contain, in addition to peptidoglycan and arabinogalactan, mycolic acids. These alpha-branched beta-hydroxy fatty acids were attributed to the corynomycolic group, according to their RF in monodimensional thin-layer chromatography. In the present work, mycolic acids from leprosy-derived and reference corynebacteria have been fractionated by monodimensional and bidimensional thin-layer chromatography and by gas chromatography. Pyrolyzed mycolic acids have been analyzed on conventional packed columns, whereas intact methyl esters of mycolic acids with free and silylated beta-hydroxyl group have been analyzed on capillary columns, and their structure has been established by mass spectrometry. In all leprosy-derived corynebacteria, some 20 components containing 24-36 carbon atoms and 0-4 double bonds were obtained. The three major groups had 32, 34 and 36 carbons, and the frequency of unsaturated versus saturated chains increased proportionally to the molecular weight. For comparison, the main components of a reference corynebacterium. Corynebacterium diphtheriae PW8, had 30 and 32 carbons, and their hydrocarbon chains were essentially saturated. This work confirms the relative chemical homogeneity of different leprosy-derived corynebacteria and describes some peculiar traits in the chemical structure of this group of organisms. In addition, it shows the complexity of the mycolic acid fraction of corynebacterial cell wall and suggests that the mycolic acid pattern is a sort of fingerprint of each bacterial strain grown under standard conditions. Finally, the fractionation of intact corynomycolic acid methyl esters with free or silylated beta-hydroxyl group by capillary gas chromatography proved to be the best analytical procedure at present available for resolving this complex mixture of corynomycolate isomers. Structural determination of silylated samples by mass spectrometry is preferred because they have more diagnostic fragments.

The search for antiviral agents among corynebacteria.

Extracts of strains of Corynebacterium diphtheriae var. mitis avirulent, C. hofmannii, C. xerosis, C. parvum and an unidentified Corynebacterium species were tested for antiviral activity against laboratory strains of echovirus type 11, adenovirus type 2 and vaccinia, and a recent isolate of herpes simplex virus. Unlike a previous study, no appreciable antiviral activity was detected in the extracts tested

Profile analysis of total mycolic acids from skin corynebacteria and from named Corynebacterium strains by gas-liquid chromatography and gas-liquid chromatography/mass spectrometry.

Gas-liquid chromatography (g.l.c.) was investigated as a potential tool in the classification and identification of Cornyebacterium strains isolated from human skin, on the basis of the g.l.c. profile of the trimethylsilyl derivatives of their mycolic acid methyl esters. The g.l.c. patterns of five skin corynebacteria were compared with those of reference strains Corynebacterium diphtheriae PW8 and Corynebacterium xerosis NCTC 9755 and NCTC 7929. Further compositional information was obtained by gas-liquid chromatography/mass spectrometry (g.l.c./m.s.) of mycolates from C. diphtheriae PW8 and two of the skin isolates. In addition to identifying and examining the individual mycolate species and comparing the differences in mycolate profile between skin corynebacteria and between reference strains, a limited assessment was made of the possibility of distinguishing between organisms at both strain and species levels on the basis of mycolic acid composition as revealed by g.l.c. "fingerprinting".

Zeatin ribonucleosides in the transfer ribonucleic acid of Rhizobium leguminosarum, Agrobacterium tumefaciens, Corynebacterium fascians, and Erwinia amylovora.

Until recently, the presence in transfer ribonucleic acid (tRNA) of the hydroxylated cytokinin ribosylzeatin [N6-(4-hydroxy-3-methylbut-2-enyl)adenosine]was thought to be unique to higher plants. This extension of work from several laboratories indicates the presence of 2-methylthioribosylzeatin in the tRNA of the plant-associated bacteria Rhizobium leguminosarum, Agrobacterium tumefaciens, and Corynebacterium fascians, but not in that of Erwinia amylovora. This cytokinin has the cis configuration, as is normally found in the tRNA's of plants. The tRNA thionucleotide patterns in these bacteria are different from those of Escherichia coli, Bacillus subtilis, and Salmonella typhimurium, which contain the unhydroxylated analogs of ribosylzeatin or 2-methylthioribosylzeatin.

Chemical structure of the peptidoglycan, its modifiability and relation to the biological activity.

The peptidoglycan is a heteropolymer composed of polysaccharide chains which are cross-linked through short peptides. The polysaccharide moiety (glycan) is made up of beta-1,4 glycosidically linked N-acetylglucosamine and N-acylmuramic acid residues. The carboxyl group of muramic acid is usually substituted by a peptide which consists of alternating L- and D-amino acids. These peptide subunits are cross-linked between the diamino acid in position 3 and the C-terminal D-alanine in position 4 of an adjacent peptide subunit either in a direct way or via an interpeptide bridge (Group A). In some coryneform bacteria the cross-linkage extends from the alpha-carboxyl group of D-glutamic acid in position 2 to D-alanine of a neighbouring peptide subunit (Group B). In the latter case a diamino acid is always found in the interpeptide bridge. A chemical modification of the peptidoglycan may occur in some bacteria due to growth in a quite unbalanced medium. The influence of glycine-rich and glycine-deficient growth medium on the chemical structure of the peptidoglycan of S. aureus will be discussed. Inhibiting concentrations of penicillin, glycine or D-amino acids can also modify the peptidoglycan. Further modification can occur through different extraction procedures which are used to obtain a clean peptidoglycan free of non-peptidoglycan cell wall material. Little is known about the molecular basis of the biological activity. The chemical composition is at least important for the antigenic determinants. The lysozyme susceptibility and the size of the preparation may be other crucial points for the biological activity of the peptidoglycan.

Amino acid sequence of the threonine-containing mureins of coryneform bacteria.

In a study of the mureins of coryneform bacteria (Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium, Erysipelothrix), 21 threonine-containing strains were found. In several of the strains the amino acid and amino sugar composition of the murein was muramic acid (Mur), glucosamine (GlcNH(2)), d-Glu, l-Lys, l-Thr, and Ala in a molar ratio of 1:1:1:1:1:4 or 5, and in several other strains it was Mur, GlcNH(2), d-Glu, l-Lys, l-Thr, Ala, and l-Ser in a molar ratio of 1:1:1:1:1:3:1. The amino acid sequence of the mureins was determined by analyzing the oligopeptides derived from partial acid hydrolysates. It was shown that there were five different murein types. The peptide subunits attached to the muramic acid are the same, namely l-Ala-d-GluNH(2)-l-Lys-d-Ala. In one strain, the alpha-carboxyl group of d-Glu is substituted by d-alanine amide. The interpeptide bridges of the different types consist of the peptides l-Ala-l-Thr-l-Ala, l-Ala-l-Thr, l-Ala-l-Ala-l-Thr, l-Ala-l-Ala-l-Ala-l-Thr, or l-Ala-l-Thr-l-Ser which are bound through their C-termini (l-Ala, l-Thr, l-Ser) to the epsilon-amino group of l-Lys of one peptide subunit and by their N-termini (l-Ala) to the C-terminal d-Ala of an adjacent peptide subunit. Determination of the N- and C-terminal groups in the mureins showed that about 15 to 30% of the interpeptide bridges are not cross-linked.

Electron microscopic examination of Corynebacterium ovis.

Corynebacterium ovis (C. pseudotuberculosis) was examined by electron microscopy after being subjected to various methods of fixation. The organism exhibited a fine structure similar to other corynebacterial species in the appearance of its cell wall, plasma membrane, nuclear apparatus, cytoplasmic matrix, wealth and complexity of intracytoplasmic membrane systems, and polyphosphate granules. An outstanding structural feature was the existence of an electron-dense, floccular layer external to the cell wall which both ligroin and acetone-methanol extractions demonstrated to be the previously postulated surface lipid of this organism. The only variations in structure evident between virulent and attenuated strains was a quantitative difference in the thickness and appearance of the surface lipid. The observation of this layer provided a basis for explaining the surface properties of C. ovis, with particular respect to its clumping capacity in suspension, the waxiness of its growth on solid media, and its ability to grow as a pellicle on suitable liquid media. The variation in the visible amount of surface lipid between the virulent and avirulent strains adequately explained the divergence of these three surface properties between the strains.