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The presence of fats and oils in sewage has been related to the formation of stable foams in activated sludge treatment systems. Foam forming microbes can utilise and, in some cases, store lipid substrates. Since surface lipids would confer the hydrophobicity necessary for flotation on the sludge biomass, the extractable lipids in foaming and non-foaming biomass samples were examined. Both pure mono-cultures and sludge samples were used. The results showed that, whilst there were some differences in the lipid profiles of the mono-cultures, the different sludge types did not show any significant pattern or variation which could be used as a lipid-based explanation for foam formation.

Crystals of diphtheria toxin have been obtained in vapor diffusion experiments from concentrated solutions of potassium tartrate as the precipitating agent. The crystals are trigonal with unit cell dimensions a = b = 97.90 A and c = 100.30 A. The space group is P31 12 or its enantiomorph. The unit cell contains 6 molecules of molecular weight 62,000 with 1 molecule/asymmetric unit. The crystals diffract beyond 2.8 A resolution and are able to withstand more than 100 h of x-ray exposure. The complete structure analysis is in progress.

Several methods for epidemiological typing of Corynebacterium diphtheriae were compared with the well accepted phage typing analysis. For this purpose, isolates from outbreaks of diphtheria in specific areas of the FRG and Sweden were analyzed by phage typing, their bacterial polypeptide profiles were examined and their phage-DNA restriction enzyme patterns were compared. All techniques were able to identify whether certain outbreaks were epidemiologically linked or not. Phage typing and phage-DNA restriction enzyme fragment analysis were limited in their application to lysogenic strains, whereas bacterial polypeptide analysis was universally applicable. Analysis of bacterial polypeptides was superior to all methods, especially in terms of speed and simplicity.

The gasochromatic method was applied to the study of the cellular fatty acids composition in diphtheria and nonpathogenic corynebacteria (diphtheroids and psendo diptheria bacillus). Marked differences in the content of unsaturated fatty acids were revealed in them. Thus, palmito leic acid served the preponderant unsaturated fatty acid in Corynebacteria diphtheriae, and unsaturated fatty acids with 18 carbon atoms (octadeconoic and linoleic)--in nonpathogenic corynebacteria. The mentioned changes permit use this sign as differential. When grown on Loeffler's medium all the corynebacteria under study had a similar fatty acid composition characterized by the prevalence of unsaturated fatty acids with 18 carbon atoms. On the basis of studying the fatty acid spectrum of the nutrient media used it is supposed that one of the factors determining the revealed dependence of the corynebacterial fatty acid composition on the culture medium was the fatty acid composition of the latter.

The isolation of a 'palmitone lipid' from Corynebacterium diphtheriae is described. The use of a temporary hydrophobic protecting group allows the obtaining of the lipid in free and pure form. Structural studies by chemical degradation and mass spectrometry allow one to propose structure Ic for this compound, namely 6-(2-tetradecyl 3-keto octadecanoyl)-alpha-D-trehalose. This structure was confirmed by chemical synthesis.

The adjuvant activity of cell wall skeletons (mycolic acid-arabino-galactan-mucopeptide, CWS) prepared from the cells of mycobacteria, nocardia and corynebacteria was examined in vivo in mice and guinea pigs. The cell wall skeletons of Mycobacterium bovis BCG (BCG-CWS), Nocardia asteroides 131 and Corynebacterium diphtheriae PWC suspended in Freund's incomplete adjuvant (FIA) as water-in-oil emulsions showed potent adjuvant activity on the formation of circulating antibody and cell-mediated immunity to bovine serum albumin (BSA), sheep erythrocytes (SRBC) and sulfanylazo-bovine serum albumin (SA-BSA) in mice and guinea pigs. After acetylation or acid treatment, BCG-CWS retained its adjuvant activity, but the activity of BCG-CWS was destroyed completely by alkaline treatment. The cell wall constituents, arabinose-mycolate and arabino-galactan, prepared from BCG-CWS showed no adjuvant activity. It was also shown that BCG-CWS suspended in phosphate buffered saline or associated with oil droplets augmented cell-mediated cytotoxicity in which thymus-derived lymphocytes (T-cells) are effector cells.

Polyacrylamide gel electrophoresis of cell proteins was investigated as a possible typing method for Corynebacterium diphtheriae. A method was developed using stock strains which were representatives of the five gravis serotypes described by Robinson & Peeney (1936). This technique was then applied to recent isolates sent to our laboratory for identification.

The composition and structure of a preparation of 'cord factor' (di-beta-hydroxy acyl trehaloses) from Corynebacterium diphtheriae have been determined by a combination mass spectral ionization methods. The methods were tested by means of synthetic 6,6'-dicorynomycolate of alpha-D-trehalose prior to their use on natural products. The determination of the molecular weight of the components and the estimation of their relative abundance in the natural mixture were made by field desorption mass spectrometry and cationization by means of cesium iodide. At least 22 molecular species differing from the chain length and the unsaturation degree were detected (carbon numbers C76 to C66). The position of acylation and the nature of the acyl chains were obtained from the electron impact mass spectra of the trimethylsilyl derivatives. The trehalose molecular was found to be esterified by a complex mixture of corynomycolic acids (3-hydroxy 2-alkyl fatty acids) which were present as saturated, mono-unsaturated and di-unsaturated homologues (carbon numbers C32 to C24). The 6 and 6' sites of acylation were the only detectable ones.

The hybridization of DNA of 19 C. diphtheriae v. mitis strains with DNA of 3H-labeled C. diphtheriae v. mitis strain C7(beta) tox+ resulted in revealing four homology levels: I (102-98%), II (78-68%), III (61-50%) and IV (42-25%). Homology levels I and II differed from one another on the level of species. In the hybridization of DNA of the same strains with DNA of 3H-labeled C. diphtheriae v. gravis (group II) strain 165(1) tox+ three homology levels were revealed; levels I and II differed from one another on the level of the genus. In the first and second series of the experiments the distribution of DNA of the above strains on these levels not always coincided. The genomes of C. diphtheriae v. mitis strains 7111 tox+ and 132 tox+ were shown to differ from those of both reference strains on the level of the genus. In strains C7(beta) tox+ and PW-8 tox+ Massachusetts the genetic similarity of their genomes on the level of species was disclosed. Among C. diphtheriae v. mitis and intermedius strains at least 5 homology levels were revealed; this fact suggests the presence of at least 5 bacterial species among them. The relationship of strains on the level of species within groups I and II of C. diphtheriae v. gravis was confirmed: DNA of the related strains belonging to the corresponding group were always to be found within the same homology level. Also confirmed was the fact that the strains of groups I and II, as well as strain C7(beta) tox+, differed on the level of species.(ABSTRACT TRUNCATED AT 250 WORDS)

This article presents a review of current experience with the restriction enzyme technique in the epidemiology of bacterial infections. It is concluded that this technique has great potential, and that its practical value has been proved under a number of different epidemiologic conditions.

Electron microscopy and microprobe X-ray analysis were used to study metachromatic inclusions of Spirillum itersonii , Corynebacterium diphtheriae, and Micrococcus luteus. In situ metachromatic inclusions were electron dense and contained phosphorus and divalent cations. Metachromatic inclusions isolated by anion-exchange column chromatography and by isoosmolar Metrizamide density gradient centrifugation were similar in composition to in situ inclusions.

The chemical characteristics of 6 batches of the preparation, obtained from the cell wall of C. diphtheriae grown in liquid and solid culture media, with respect to their content of nitrogen, hexoses, pentoses, total amino sugars, lipids and to the possible admixture of nucleic acids are presented. From the results of the chemical analysis of these batches their standardization according to the ratio of total amino sugars and pentoses to total nitrogen in C. diphtheriae cell-wall preparation is proposed.

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".

The exotoxins produced by ten strains of C. ulcerans (two human, six bovine and two equine) have been studied. On the criteria of toxin-antitoxin neutralisation and immunoprecipitation tests using highly specific diphtheria and C. ovis antitoxins with crude toxic filtrates, (NH4)2SO4 concentrates, and partially purified chromatographic preparations of these, together with the presence or absence of inhibition of the action of staphylococcal beta-haemolysin, and the reaction produced when injected intradermally into rabbits, two toxins could be identified, namely diphtheria toxin and C. ovis toxin. There was no evidence for the production of a third toxin specific for C. ulcerans. Five strains produced both diphtheria and C. ovis toxins. In four diphtheria toxin predominated, but in the fifth C. ovis toxin predominated. Two strains produced only diphtheria toxin and two only C. ovis toxin, though there was good but not complete evidence that a third strain (Revell) also fell into this latter group. Considerable variation occurred in the concentration of each toxin and, where both were present, in the proportion of each.

The mass spectra of the trimethylsilyl ethers of the four positional isomers of methyl-O-palmitoyl-alpha-D-glucopyranoside have been studied, and the structures of the principal ions assigned by the use of exact mass measurements and deuterium labelling on the aliphatic chain, the trimethylsilyl group and the glucosidic methyl group. The origin of some fragments has been elucidated by analysis of reactions of metastable ions. The mass spectra of the different isomers exhibit major differences, which depend upon the presence or absence of the aliphatic chain. These results indicate that this is a useful method for determining the position of an acyl group in a methyl glucoside. The applicability of this mass spectrometric method in structural determination of unknown acylated sugars is discussed. The structure of a corynomycoloyl-alpha-D-trehalose, isolated from Corynebacterium diphtheriae, has been determined by mass spectrometry as an application of the method. The molecular weight of this compound has been determined by field desorption mass spectrometry (cationization method), and the study of the electron impact spectrum of the trimethylsilyl derivatives clearly demonstrates that the corynomycolic acid is linked by an ester group to position 6 of the trehalose molecule.

Whole-organism methoanolysates of bacterionemae contained mycolic acids in addition to other long-chain fatty acids. These mycolic acids were similar in general structure and overall size to those found in strains of Corynebacterium diphtheriae and Corynebacterium xerosis. The long-chain fatty acids of bacterionemae, mainly straight-chain saturated and unsaturated acids, were similar to those of certain coryneform bacteria including C. diphtheriae. On the basis of these lipid data, and results of earlier studies, we recommend that the genus Bacterionema be transferred from the family Actinomycetaceae to the Coryneform Group of Bacteria.

Seven new nontoxinogenic (tox) mutants of corynebacteriophage beta were isolated. Strains of Cornyebacterium diphtheriae C 7 lysogenic for these tox mutants of beta were tested for their ability to produce extracellular diphtherial toxin or proteins (CRMs) that cross-react immunologically with toxin. By using a sensitive reversed passive hemagglutination assay for toxin antigen, three of the tox mutants were phenotypically CRM+ and four were CRM-. The molecular weights of the CRMs produced by mutants beta tox-1, beta tox-2, and beta tox-3 were determined to be approximately 20,000, 26,000, and 34,000, respectively, by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The 26,000 and 34,000-dalton CRMs had nicotinamide adenine dinucleotide: elongation factor 2 adenosine diphosphate ribose transferase activity, but the 20,000-dalton CRM did not. These three CRMs correspond to amino-terminal fragments of diphtherial toxin and appear to be formed by chain termination during protein synthesis directed by phages with nonsense mutations in the structural gene for diphtherial toxin. No complementation was observed between independently isolated tox mutants of phage beta. The positions of four tox markers on the vegetative genetic map of phage beta were determined, and the orientation of transcription of the structural gene for diphtherial toxin with respect to other markers on the genetic map of phage beta was established.

A method for isolation of immunochemically active proteins from Corynebacterium diphtheria membranes was elaborated. The proteins were solubilized with the nonionic detergent NP-40 and gel-filtered through an Ultrogel AcA-34 column under denaturating conditions. The purified proteins (Mr = 64 kD) were antigenically active in a solid phase radioimmunoassay with human antidiphtheria antibodies.

Electron-dense inclusion bodies were found in most Plesiomonas shigelloides cells, regardless of the incubation time. At the 4-hr incubation period, the size of inclusion bodies was distributed in the range of 50 to 150 nm in diameter, and at the logarithmic phase of growth it increased up to a size visible by light microscope. By an electron microprobe X-ray analysis, phosphorus, potassium, and magnesium were detected in the inclusion bodies which confirms the assumption of Pastian and Bromel (Appl. Environ. Microbiol. 47: 216 (1984] that the inclusion bodies have a very similar elemental composition to the polyphosphate granules of C. diphtheriae.

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

Diphtheria toxin splits into two fragments when heated at 100 C for 10 min in a phosphate buffer. The separated fragments have molecular weights of 24,000 and 39,000, respectively. These molecular weights are similar to those of the A and B fragments found in diphtheria toxin preparations after thiol reduction. Since the separation of toxin into fragments is not complete, it is likely that only nicked toxin molecules having a cleaved peptide bond are split by heating. When toxin is suspended in phosphate buffer at pH 6.4, the B-like fragment precipitates, but at pH 7.8 it does not. Heated toxin is unable to intoxicate sensitive cells or cause a necrodermal response in animals. Fragment A produced by heating is active in inhibiting cell-free protein synthesis. It is able to intoxicate both HeLa and L cells when the uptake of the fragment is facilitated by addition of polyornithine to the cultures. Fragment B produced by heating is involved with binding to the cell surface. It is able to delay the action of toxin on KB cell cultures preincubated with fragment B.