Clostridia

Am J Vet Res, 1977 Jul, 38(7), 1069 - 73
Isolation and identification of anaerobes in the veterinary diagnostic laboratory; Berkhoff GA et al.; In a 1-year survey (1975 to 1976) of anaerobic bacteria recovered from diseased animals, anaerobes were as follows: Clostridium spp, 50%; gram-positive nonspore-forming anaerobic bacilli, 19%; gram-negative anaerobic bacilli, 19%; Actinomyces spp, 10%; and anaerobic cocci, 1% . Anaerobes were in approximately 61% of the specimens that were culturally positive for any bacteria . Approximately 25% of the specimens did not yield any bacteria (sterile specimens) . The method for isolating and identifying anaerobes was based upon the use of reducible solid mediums and was specially designed for veterinary diagnostic laboratories . Emphasis was placed on the selection, collection, and transportation of specimens.

Hoppe Seylers Z Physiol Chem, 1977 Jul, 358(7), 789 - 95
Immobilized Clostridium perfringens neuraminidase . Substrate cleavage and enzyme release during incubation; Parker TL et al.; Pure Clostridium perfringens neuraminidase was immobilized on Sepharose 4 B, azido-Sepharose 4 B and controlled pore glass (CPG)- glycophase using different coupling procedures . The immobilized enzyme showed increased stability under various conditions relative to the soluble enzyme . The low release of active enzyme from the supports under incubation conditions was quantitated using a highly sensitive radioactive assay . The activity of the immobilized enzyme was dependent on the nature of the support and the substrate . Activity decreased with increasing substrate molecular weight, but the enzyme showed improved cleavage with GD1a micelles and human erythrocytes, substrates having ordered surface properties . Uses of immobilized neuraminidase in biochemistry and cell biology are considered and evaluated relative to the measured release of enzyme from the supports reported and to the molecular size and organization of possible substrates.

Can J Microbiol, 1977 Jul, 23(7), 908 - 15
Enterotoxin formation by Clostridium perfringens type A studied by the use of fluorescent antibody; Niilo L; Fluorescein isothiocyanate-conjugated antibody to purified enterotoxin of Clostridium perfringens was used to study the intracellular formation of enterotoxin by this organism . Enterotoxin was detected at 4 h of growth at the end of the cell containing forespore . With the development of the spore, enterotoxin accumulation continued and involved the entire length of the cell until its lysis with the release of enterotoxin and mature spore . The spores did not contain demonstrable enterotoxin . Only a certain number of the sporulated cells of the enterotoxigenic strains studied produced this toxin . The amount of enterotoxin produced varied with sporulation percentage, and between strains and individual cells.

Can J Microbiol, 1977 Jul, 23(7), 884 - 92
ICMSF methods studies . VIII . Comparative study for the enumeration of Clostridium perfringens in foods; Hauschild AH et al.; Four methods were compared in an international comparative study for the enumeration of Clostridium perfringens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserin) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods . The confirmed C . perfringens counts were slightly lower for D than for A-C . The percentages of presumptive colonies confirmed as C . perfringens were essentially the same in each method . The relative numbers of nonspecific colonies were the lowest in C, followed by B, D, and A . The methods were also compared for simplicity and for aspects associated with the recognition and selection of presumptive colonies.

Appl Environ Microbiol, 1977 Jul, 34(1), 30 - 3
Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media; Odlaug TE et al.; Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days . The spore count was determined at different intervals over the 180-day storage period . There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C . The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.

Eur J Nucl Med, 1977 Jun 30, 2(2), 117 - 20
Enhancement of incorporation of 131iododeoxyuridine into tumors after application of Clostridium oncolyticum s . butyricum (M 55) . Animal experiments on positive scintigraphic localization of tumors; Volm M et al.; The application of spores of the bacterium Clostridium oncolyticum s . butyricum (M 55) results in an increased incorporation of 131Iododeoxyuridine in tumors (amelanotic melanoma Fortner III of the golden hamster and Brown-Pearce carcinoma of the rabbit) . This leads to an improved scintigraphic demonstration of the tumors.

J Cell Sci, 1977 Jun, 25, 335 - 54
Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos . III . Characterization of galactose- and glucosamine-labelled materials; Johnson KE; Normal Rana pipiens gastrulae show more incorporation of isotopically-labelled galactose and glucosamine into TCA-insoluble materials than blastulae . Interspecific hybrid embryos which undergo developmental arrest at the onset of gastrulation often synthesize reduced amounts of galactose- and glucosamine-labelled materials . These materials are high-molecular weight, but not collagen . After pronase digestion, labelled materials elute in the void volume of Sephadex G-50 . Labelled materials migrate slowly on cellulose acetate, bind to several kinds of anion exchangers and elute at low ionic strength, are precipitated by cetyl pyridinium chloride (CPC) when mixed with carrier compounds, and are not degraded by Clostridium neuraminidase or Streptomyces, leech, and bovine testicular hyaluronidases.

Appl Environ Microbiol, 1977 Jun, 33(6), 1287 - 8
Production and counting of spores of Clostridium chauvoei; Bagadi HO; The concentration and viability of spores produced by four different strains of Clostridium chauvoei (C . feseri) grown in a modified medium for 18 days are described . The medium yielded enough viable spores for experimental work.

Appl Environ Microbiol, 1977 Jun, 33(6), 1270 - 4
Hydrogen utilization by clostridia in sewage sludge; Ohwaki K et al.; A sporeformer morphologically different but physiologically similar to Clostridium aceticum Wieringa was isolated from sewage sludge . It used large amounts of H2 and CO2, converting them chiefly to acetic acid . Growth occurs anaerobically on yeast extract alone, but after the nutrients in yeast extract are used, growth continues at a reduced rate, supported by the conversion of the gases to acetate.

J Bacteriol, 1977 Jun, 130(3), 1084 - 90
A four-iron, four-sulfide ferredoxin with high thermostability from Clostridium thermoaceticum; Yang SS et al.; A ferredoxin containing only one {Fe4S4} cluster was purified from Clostridium thermoaceticum . It has a molecular weight of about 7,300, a partial specific volume of 0.67, and an isoelectric point of 3.25 . Its absorption spectrum has two maxima at 390 nm (epsilon = 16.8 X 10(3)M-1cm-1) and at 280 nm (epsilon = 24.2 X 10(3)M-1cm-1) . The absorption at 390 nm is almost half that of other clostridial ferredoxins, which have two {Fe4S4} clusters, and is similar to other ferredoxins with only one {Fe4S4} cluster . The ferredoxin had high thermal stability and retained over 50% of its activity after treatment at 80 degrees C . It functions in the transfer of electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP), which indicates the presence of pyruvate:ferredoxin oxidoreductase and reduced ferredoxin-NADP reductase in C, thermoaceticum . NADPH is used in the synthesis of acetate from CO2 in this organism.

Onderstepoort J Vet Res, 1977 Jun, 44(2), 53 - 4
The taxonomic position of Clostridium botulinum type c; Jansen BC et al.; Experimental evidence is produced to justify abandoning the practice of subdividing Costridium botulinum Type C into type Calpha and Cbeta.

J Gen Microbiol, 1977 Jun, 100(2), 395 - 401
Clostridium sporogenes isolates and their relationship to C . botulinum based on deoxyribonucleic acid reassociation; Nakamura S et al.; Sixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type A190 . Sporulation was observed in most of 21 umbonate and rhizoidal colony-forming strains (colony-type I strains), but not in most of the 41 strains with convex and circular or crenate colonies with a mat to semi-glossy surface (colony-type II strains) . More than half of the latter strains showed much higher heat resistance than the rhizoidal colony-forming strains . The DNA isolated from colony-type II strains was 81% or more homologous to C . botulinum A190 DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A190 DNA . Colony-type I strains differed from C . botulinum by 30 to 40% DNA homology and the DNA duplexes formed between these strains and strain A190 showed deltaT m(e) values of 7-0 degrees C when compared with the T m(e) of homologous DNA duplexes of strain A190.

Can J Microbiol, 1977 Jun, 23(6), 829 - 32
Enumeration of Clostridium botulinum spores in meats by a pour-plate procedure; Hauschild AH et al.; Colonies of Clostridium botulinum could be easily distinguished from meat particles by supplementing Wynne agar with 0.4% egg yolk . The pour-plate method was suitable for enumeration of C . botulinum, provided the medium was covered with a layer of agar containing 0.01% dithiothreitol . Viable counts of heat-treated spores were consistently higher in Wynne agar supplemented with egg yolk (Wynne-EY agar) than in Wynne agar alone.

Biokhimiia, 1977 Jun, 42(6), 1077 - 82
{The actions of low-molecular fragments of substrate and their analogs on the activity of isoenzymes of phospholipase C from Clostridium perfringens}; Litvinko NM et al.; The interaction of the lecithin molecule fragments and their analogues with phospholipase C Cl . perfringens was studied by gel-diffusion in agarose-lecithin gels . It was found intense inhibition of phospholipase C activity in the presence of cathionic compounds; this phenomenon shows the existence of anionic centre in the active site of enzyme . The esteric centre is probably hydrophobic nature and is not capable to bind the negatively charged groups . However, phosphoserine, phosphothreonine, gamma-aminobutyric, aspartic and glutamic acids can interact with an additional cathionic centre, whose location in phospholipase C differs from that in pancreatic phospholipase A2.

Am J Vet Res, 1977 Jun, 38(6), 857 - 61
Reproduction and treatment of necrotic enteritis in broilers; Truscott RB et al.; A change in ration from one containing 50% fish meal to a standard chick starter containing 10(8) Clostridium perfringens/g of feed resulted in the production of necrotic enteritis in 2-week-old broiler chicks . Lincomycin added to the inoculated ration at a concentration of 20 g/907.2 kg significantly reduced mortality in the chicks . Inoculation of broth cultures of C perfringens directly into the duodenum, using a surgically inserted tetrafluoroethylene resin tube, indicated a relationship existed between the number of C perfringens inoculated and the gross lesions . The disease could be consistently produced by this technique.

Appl Environ Microbiol, 1977 Jun, 33(6), 1295 - 7
Identification of intermediates formed during the degradation of hexachlorocyclohexanes by Clostridium sphenoides; Heritage AD et al.; Washed cell suspensions of Clostridium sphenoides degraded the alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane via delta-3,4,5,6-tetrachloro-1-cyclohexene and the gamma-isomer via gamma-3,4,5,6-tetrachloro-1-cyclohexene . Both intermediates were further metabolized to unknown substances . The tetrachlorocyclohexene intermediates were identified by gas chromatography and mass spectrometry.

Infect Immun, 1977 Jun, 16(3), 910 - 4
Correlation between oral toxicity and in vitro stability of Clostridium botulinum type A and B toxins of different molecular sizes; Sugii S et al.; The in vitro sensitivity to acid and pepsin differed markedly among Clostridium botulinum type A and B toxins of different molecular sizes . The larger the molecular size of the toxin, the higher the resistance to these agents . Tye B derivative toxin was rapidly inactivated, but the progenitor toxins resisted in vitro exposure to rat intestinal juice . The molecular dissociation of the progenitor toxins did not occur in rat intestinal juice of pH 7.0, but did occur in a buffer solution of the same pH . The oral toxicity may depend mostly on the stability of toxin molecules in the stomach and, to a less extent, in the intestine . The present results seem to justify the conclusion that C . botulinum type A and B progenitor toxins with molecular sizes larger than 16S are more potent oral toxins than 12S progenitor toxins.

Biochim Biophys Acta, 1977 May 27, 492(1), 215 - 24
The proteolytic action of bacterial proteases from Clostridium histolyticum on bovine fibrinogen; Tranqui L et al.; Bacterial proteases from Clostridium histolyticum bring about the polymerization of fibrinogen into structured fibres, the period being 9 nm when the ionic strength (I) of the solution is between 0.1 and 0.2 . At I=0.3 no polymerization occurs, but the successive actions of bacterial proteases and thrombin induce polymerization, the fibres obtained showing a periodic structure: the major period is 23 nm with two minor striations . The striation pattern is different from that obtained with fibrin which shows a major period of 23 nm with three minor striations . The degree of degradation of fibrinogen monitored by disc gel electrophoresis shows that the Aalpha polypeptide chain (mol . wt.=68 000) is degraded into Aalpha1 (mol . wt.=32 000) and Aalpha2 (mol . wt.=29 000), whilst the mobility of Bbeta increases and gamma remains unchanged; the molecular weight is estimated between 272 000 and 269 000 . These results emphasize that the charge distribution differences which are compatible with the loss of different portions of the molecule, lead to variations in fibre structures.

Lancet, 1977 May 14, 1(8020), 1046 - 8
Hospital outbreak of clostridium perfringens food-poisoning; Thomas M et al.; An outbreak of Clostridium perfringens (C.welchii) food-poisoning affected a third of the patients in a large hospital, and one frail patient died . C.perfringens type A, serotype 1, was isolated from 46 (61 per cent) of 76 patients examined and from food, and a new serotype (61) was isolated from 16 . The attack-rate among patients who ate a minced-ham dish was 78 per cent . The cooking and storage of this mince was faulty: cuts of meat were much too large, they were kept at room temperature too long before refrigeration, and after cooking they were put into mincers used also for raw meat . C.perfringens type A, serotype 1, was isolated from meat scraps in a mincer . Final reheating was inadequate to destroy even vegetative bacteria, and multiplication may have occurred during slow distribution to the wards . Outbreaks of C.perfringens fool-poisoning are common in hospitals, and some underlying problems are discussed . A plea is made for the Food Hygiene Regulations to apply to National Health Service premises and for simple but effective reforms in institutional catering management.

JAMA, 1977 May 2, 237(18), 1946 - 51
Infant botulism . Epidemiological, clinical, and laboratory aspects; Arnon SS et al.; Clostridium botulinum organisms and toxin were identified in the feces of six infants, aged 5 to 20 weeks, who had illnesses clinically consistent with botulism . Five of the infants lived in California and became ill within a six-month period in 1976; one infant became ill in New Jersey in 1975 . Three cases were type A botulism, and three were type B . No source of ingested botulinal toxin could be found in any case . However, one infant with type B botulism had ingested a food containing C botulinum type B organisms, and no toxin was found in it . The clinical findings in these cases include constipation, weak sucking and crying ability, pooled oral secretions, cranial nerve deficits, generalized weakness, and, on occasion, sudden apnea . A characteristic electromyographic pattern termed "brief, small, abundant, motor-unit action potentials" (BSAP) was observed . The sources of C botulinum toxin for these six infants is thought to have been in vivo (gastrointestinal) production following ingestion of C botulinum organisms . Studies are underway to determine the full clinical spectrum, incidence, and potential public health importance of this infectious disease newly recognized in infants.

Res Vet Sci, 1977 May, 22(3), 343 - 6
Observations on the possible invasiveness of Clostridium botulinum for waterfowl; Graham JM et al.; Twenty-four ducklings given multiple doses of Clostridium botulinum type C spores and toxin per os and 27 affected waterfowl from four natural outbreaks of the disease were examined bacteriologically . No evidence of invasion of the blood or liver was found in any bird and it is suggested that invasiveness and toxigenesis in internal organs are probably of little, if any, importance in the causation of botulism in waterfowl.

J Assoc Off Anal Chem, 1977 May, 60(3), 563 - 9
An improved cooked meat medium for the detection of Clostridium botulinum; Quagliaro DA; A new medium composed of cooked meat and fluid thioglycolate broth was tested with 20 proteolytic and 11 saccharolytic strains of Clostridium botulinum . All of the proteolytic strains produced a black ring at the surface of the broth, presumably due to hydrogen sulfide production, while the saccharolytic strains produced white opaque rings . Since the black ring is formed rapidly and is easily seen, the new medium may be useful for the rapid identification of proteolysis in unknown isolates . Growth and toxin production were better in the new medium than in the usual cooked meat medium or in cooked meat medium plus 0.5% glucose . These results suggest that the new medium will be useful in facilitating identification of C . botulinum.

J Assoc Off Anal Chem, 1977 May, 60(3), 541 - 5
Collaborative study of a method for the detection of Clostridium botulinum and its toxins in foods; Kautter DA et al.; The mouse toxicity and protection technique for the detection and identification of Clostridium botulinum and its toxins in foods was collaboratively studied by 11 laboratories . Each laboratory received 4 samples of cream of mushroom soup; 2 contained spores and toxin of C . botulinum type A, 1 contained spores and toxin of C . botulinum type E, and 1 contained spores of C . sporogenes . The media used were cooked meat medium (beef heart or chopped liver broth) and trypticase peptone glucose yeast extract broth with trypsin . The results indicate that this method has a high degree of repeatability and reproducibility . All 11 laboratories correctly identified the toxins and the nontoxic sample in the food and detected and identified the viable spores in the samples by means of the subsequent cultures . This method has been adopted as official first action.

Ann Sclavo, 1977 May-Jun, 19(3), 494 - 501
{Two outbreaks of "Clostridium perfringens" food poisoning: epidemiological remarks (author's transl)}; Caroli G et al.; Two outbreaks of Cl . perfringens food poisoning which occurred in Florence during 1976 have been described . The first one involved three hundred primary school children; processed re-heated turkey meat was thought to have been the vehicle of infection in the school meal . The clinical symptoms consisted of mild diarrhoea in all cases and the duration of the illness was about 12 hours . The possible part played by food storage temperature, post-cooking periods and food trolleys in the spread of infection is discussed . The other outbreak interested three people who ate a dish with gravy in a restaurant; one of these suffered severe haemorrhagic enteritis and died after two weeks . Necroscopy was performed and the results of post-mortem examination as well as histological and bacteriological findings certified that the cause of death was severe enteritis (Necrotizing enteritis) in elderly debilitated patient (with diabetes, chronic bronchitis, arteriosclerosis and previously gastroresected).

Zh Mikrobiol Epidemiol Immunobiol, 1977 May, (5), 71 - 4
{Characteristics of the immunologic response of animals to Clostridium perfringens anatoxin}; Petrov RV et al.; Experiments were conducted on guinea pigs, rabbits and mice (mongrel and inbred); immunogenic properties of Cl . perfringens toxoids of different purity were studied . Toxin neutralization and passive hemagglutination tests were used to determine the antitoxic immunity level . It appeared that in the immunization of guinea pigs and rabbits the degree of immunogenicity of the preparations increased with the elevation of their specific activity . Under the same conditions both the mongrel and the inbred mice displayed the maximum immune response in the immunization with the least purified preparations, and the minimum after the injection of a highly purified antigen.

J Infect Dis, 1977 May, 135(5), 850 - 4
Anaerobic bacteria in biliary disease in elderly patients; Shimada K et al.; Gallbladder bile from 52 elderly subjects who had undergone biliary tract surgery was examined for the presence of bacteria . Twelve patients had sterile bile, 18 specimens of bile yielded anaerobes as well as aerobes, and 22 yielded aerobic bacteria only . Escherichia coli was the most commonly isolated organism (30 strains) . Bacteroides fragilis was the most frequently encountered anaerobic bacterium and was found in 15 patients . The Klebsiella-Enterobacter group was the second most commonly isolated group and B . fragilis was third . Clostridium perfringens was recovered in 10 specimens of bile . Anaerobic bacteria were recovered more frequently in patients with ductal obstruction . The relatively frequent isolation of anaerobes, especially of B . fragilis, in this study may be related to the anaerobic techniques used, to the age of the patients, and to the high incidence of pigment stones among the subjects.

Can J Microbiol, 1977 May, 23(5), 601 - 6
Clostridium perfringens--specific lysin; Nakamura S et al.; A mitomycin C induced lysate of Clostridium perfringens strain KZ219 was lytic to 50 strains of C . perfringens of types A-E, and three strains of C . plagarum . The lysin was active against only 2 out of 87 strains of 51 other clostridial species . The optimum pH of the lytic agent was 5.5 . The activity was largely inactivated by proteolytic enzymes, and nearly completely inactivated by heating at 60 degrees C for 5 min.

J Mol Evol, 1977 Apr 29, 9(2), 111 - 9
Phylogenetic studies of two rubredoxins from sulfate reducing bacteria; Vogel H et al.; The sequences of two rubredoxins isolated from the sulfate reducing bacteria: Desulfovibrio vulgaris and Desulfovibrio gigas have been elucidated . They have similar sequences but many more differences occur than would be expected from two bacteria of the same genus . Of the 52 sites, only 37 are occupied by identical residues . The primary structures are compared with those of the anaerobic bacteria rubredoxins of Clostridium pasteurianum, Micrococcus aerogenes, Pseudomonas oleovorans and Peptostreptococcus elsdenii: only 12 identities are found, mostly in the two clusters that contain two iron-bound cysteines each . A phylogenetic tree based on the primary structures is presented and possible relations with plant and bacterial ferredoxins are discussed . A secondary and tertiary structure stereochemically compatible with the sequence data, is proposed.

J Biol Chem, 1977 Apr 25, 252(8), 2657 - 61
Evidence for separate enzymes of pyruvate decarboxylation and pyruvate synthesis in soluble extracts of Clostridium pasteurianum; Bush RS et al.; Additional evidence to that already presented (Sauer, F . D., Bush, R . S., and Stevenson, I . L . (1976) Biochim . Biophys . Acta 445, 518-520) suggests that pyruvate-ferredoxin oxidoreductase isolated from Clostridium pasteurianum consists of two separate enzymes: (a) pyruvate lyase, which catalyzes the CoA and electron acceptor-dependent decarboxylation of pyruvate, and (b) pyruvate synthase, which catalyzes the reduced ferredoxin-dependent carboxylation of acetyl-CoA to pyruvate . The enzymes separated on Sephadex G-200 and with acrylamide gel electrophoresis but complete separation of one enzyme free of the other was not achieved . Extensive purification procedures were not used because both enzymes are unstable . The results confirm published reports that pyruvate lyase contains thiamin and a chromophore which participates in electron transfer . Pyruvate synthase, however, did not appear to be a thiamin enzyme and there was no evidence to indicate participation of an enzyme chromophore in the pyruvate synthase reaction.

J Biol Chem, 1977 Apr 10, 252(7), 2245 - 53
Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring 1H nuclear magnetic resonances in clostridial-type ferredoxins; Packer EL et al.; We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques . We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C . acidi-urici ferredoxin . The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to {4Fe-4S} clusters in synthetic inorganic analogues . In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position . In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position . This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the {4Fe-4S} cluster . The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins . This suggests that the molecular environments of the corresponding cysteinyl residues are identical . Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ . This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the {4Fe-4S} clusters or differences in charge density at the cysteinyl beta protons or both . The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O . The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C . acidi-urici ferredoxin . This was done by comparing the 1H NMR spectra of C . acidi-urici {(3',5'-2H2)Tyr}ferredoxin and C . acidi-urici {PHE2}ferredoxin with that of C . acidi-urici native ferredoxin.

Biochemistry, 1977 Apr 5, 16(7), 1467 - 73
Benzo{a}yrenedione/benzo{a}pyrenediol oxidation-reduction couples and the generation of reactive reduced molecular oxygen; Lorentzen RJ et al.; The ability of the isomeric quinone metabolites of benzo{a}pyrene, benzo{a}pyrene-6,12-dione, benzo{a}pyrene-1,6-dione, and benzo{a}pyrene-3,6-dione to undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo{a}pyrenediols and intermediate semiquinone radicals has been characterized . Under anaerobic conditions, all three benzo{a}pyrenediones are easily reduced to benzo{a}pyrenediols, even by mild biological agents such as NAD(P)H, cysteamine, and glutathione . The benzo{a}pyrenediols, in turn, are very rapidly autoxidized to the benzo{a}pyrenediones when exposed to air . Substantial amounts of hydrogen peroxide are produced during these autoxidations, and other reactive reduced oxygen species, such as the superoxide and hydroxyl radicals, are probably formed transiently as well . The benzo{a}pyrenediol-benzo{a}pyrenedione interconversions proceed by one-electron steps; the corresponsing semiquinone radicals can be monitored by electron spin resonance spectroscopy as inter mediates during these reactions carried out at high pH . Benzo{a}pyrenediones induce DNA strand scission when incubated with bacteriophage T7 DNA . This damage is modified by conditions which indicate that reduced oxygen species propagate the free-radical reactions responsible for the strand scission . Benzo{a}pyrenediones are electron-acceptor substrates for NADH dehydrogenase from Clostridium kluyveri . Catalytic amounds of these benzo{a}pyrene metabolites, together with this respiratory enzyme function as cyclic oxidation-reduction couples which link NADH and molecular oxygen in the continuous production of hydrogen peroxide . These data, together with preliminary results with cells in culture, indicate that benzo{a}pyrenediones are potentially harmful metabolites of benzo{a}pyrene, acting by processes which lead to their regeneration rather than depletion; nucleic acid and call damage is probably produced by the reactive reduced oxygen species resulting from such regenerative oxidation-reduction cycles.

Ann Microbiol (Paris), 1977 Apr, 128A(3), 267 - 76
{Taxonomic characters of "Clostridium tyrobutyricum" (author's transl)}; Roux C et al.; The taxonomic characters of 77 strains of Clostridium tyrobutyricum have been studied and compared to those of known strains . This work shows the insufficiency of the Bergey's Manual (8th edition) nine characters distinguishing Clostridium groupe I to identify C . tyrobutyricum . To be more representative of the species, addition of one or two characters to this key of determination and some modifications to the complementary description of C . tyrobutyricum in this manual are proposed.

Appl Environ Microbiol, 1977 Apr, 33(4), 955 - 62
Citrate, a specific substrate for the isolation of Clostridium sphenoides; Walther R et al.; With a medium containing citrate as the carbon and energy source, 10 clostridial strains were isolated from various mud samples . Characterization of these strains revealed that they all belonged to the same species, Clostridium sphenoides . Strains of this organism obtained from culture collections were also able to grow citrate, whereas 15 other clostridial species tested were not . Citrate was fermented by C . sphenoides to acetate, ethanol, carbon dioxide, and hydrogen . Experiments with stereospecifically 14C-labeled citrate indicated that citrate lyase was involved in citrate degradation.

Can J Comp Med, 1977 Apr, 41(2), 188 - 94
Inactivation of Clostridium haemolyticum toxic fluids and their antigenicity; Lozano EA; One hundred fifty-one isolates of Clostridium haemolyticum were examined for consistent toxin production following repeated serial transfers in laboratory media . Most of these isolates produced only small amounts of toxic materials and serial transfers appeared to reduce toxigenic characteristics . Eleven of the isolates consistenly produced measurable amounts of toxic materials . One of these isolates was used for production of toxic fluids that were concentrated by lyophilization and reconstitution to a smaller volume or by precipitation with ammonium sulphate followed by dialysis against water and glycerol . Known amounts of these substances were inactivated with formalin, heat, beta-propiolactone, ultra-violet irradiation and glutathione . The resulting toxoids were inoculated into guinea pigs and most were judged to be nonimmunogenic because the animals were unable to resist dermal challenge . Toxic materials with added glycine were inactivated with formaldehyde as readily as those without the amino acid but the resulting toxoids were immunogenic while those prepared without the amino acid were not.

Appl Environ Microbiol, 1977 Apr, 33(4), 963 - 6
Improved procedure for crystallization of Clostridium botulinum type A toxic complexes; Sugiyama H et al.; A modified protocol for the preparation of botulinum type A toxic crystals is decribed . The essential change was the substitution of chromatography on diethylaminoethyl-Sephadex for the step in which toxin was precipitated with ethanol . Colored materials and nucleic acids were removed by the chromatography so that crystal lots of greater uniformity could be produced . Significantly more of toxin in the starting culture was recovered as crystals . The lots of crystals obtained initially by either the original or the modified method contained an antigen that was eliminated when the lots were recrystallized.

Infect Immun, 1977 Apr, 16(1), 107 - 9
Oral toxicities of Clostridium botulinum toxins in response to molecular size; Ohishi I et al.; Clostridium botulinum type A, B, and F toxins of different molecular sizes were fed to mice to compare the oral toxicities . The progenitor toxin, a complex of a toxic and nontoxic component, of any type was higher in oral toxicity to mice than the dissociated toxic component or the derivative toxin . The former may no doubt play a more important role in the pathogenesis of food-borne botulism . The higher oral toxicity possessed by the progenitor toxin, including the exceptionally high one found with type B-L toxin, can be explained solely by the protection afforded by the nontoxic component attached to the toxic component . The possibility of the highest oral toxicity of type B-L toxin to humans is discussed.

Am J Dis Child, 1977 Apr, 131(4), 445 - 6
Otogenous tetanus: a sequelae of chronic ear infections; Fischer GW et al.; Eight patients had bacteriologically confirmed otogenous tetanus and all survived, suggesting that this may be a less severe form of the disease . Clostridium organisms most probably secondarily infect the purulent ear discharge after contamination by dirty cloth or fingers . Since most cases of tetanus are seen first by pediatricians or family physicians, they should be familiar with this potential source . Adequate tetanus immunization predisposed to chronic otorrhea.

Avian Dis, 1977 Apr-Jun, 21(2), 256 - 63
The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens; Al-Sheikhly F et al.; The intraduodenal administration of large numbers of Clostridium perfringens cells harvested from broth cultures and resuspended in PBS or fresh sterile thioglycollate broth produced a very mild form of necrotic enteritis . Administering an appropriate number of cells in culture supernatant, however, produced typical field-type disease . Alpha toxin was shown to be the significant toxin recoverable from broth-culture supernatant fluids . Requirements to produce the disease are minor intestinal damage and sufficient numbers of toxigenic C . perfringens in the intestine.

Avian Dis, 1977 Apr-Jun, 21(2), 241 - 55
The pathology of necrotic enteritis of chickens following infusion of crude toxins of Clostridium perfringens into the duodenum; Al-Sheikhly F et al.; Necrotic enteritis was produced in 4-week-old chickens with bacteria-free crude toxins of Clostridium perfringens . Typical gross lesions could be seen as early as 3 hr after intraduodenal infusion of toxin was begun . Early microscopic lesions were observed at 20 minutes, progressing with time to involve most of the mucosa . Death was related to the amount of toxin administered . Because systemic absorption of toxin is likely, acutely affected birds may die from toxemia.

Avian Dis, 1977 Apr-Jun, 21(2), 230 - 40
The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum; Al-Sheikhly F et al.; Necrotic enteritis was consistently reproduced when enough active broth culture of Clostridium perfringens type A was infused intraduodenally . Typical lesions of necrotic enteritis were seen as early as 5 hr after infusion was begun . The histologic lesions observed at 1 hr were characterized by edema in the lamina propria and desquamation of epithelial cells . Large numbers of clostridia were seen among these sloughed cells . Coagulation necrosis of the tips of villi became evident at 3 hr and was marked at 5 hr . Many clumps of clostridia were obvious among the necrotic tissue . At 8 and 12 hr the necrotic lesions extended to involve most of the villus structures . Morphologically abnormal erythrocytes were evident in the visceral organs at 12 hr.

Mikrobiologiia, 1977 Mar-Apr, 46(2), 346 - 51
{Variable forms of Clostridium felsineum}; Avrova NP; Variants of five types, differing in morphology of their colonies, resulted from spontaneous variability of Clostridium felsineum, the main agent involved in flax retting, when it was grown in a liquid medium and then on a solid medium . Morphological, cultural, and biochemical properties of the variants are described . The variants of the second and third types were less stable than those of the first type in the course of passages on growth media . The rate of spore formation was higher in the variants of the first type whereas the activity of pectolytic enzymes was greater in the variants of the second type.

Hoppe Seylers Z Physiol Chem, 1977 Mar, 358(3), 339 - 52
{On cobyrinic acid biosynthesis . Novel methylated hydroporphyrins and their role in cobyrinis acid formation (author's transl)}; Deeg R et al.; Clostridium tetanomorphum and Propionibacterium shermanii were examined for intermediates in the synthetic pathway uroporphyrinogen III leads to cobyrinic acid . The isolation of two novel methylated hydroporphyrins, whose methyl groups are derived from S-adenosyl-L-methionine, is described . Spectroscopic (field desorption-mass, visible absorption) und electrophoretic studies as well as incorporation of labelled substrates indicate that they are analogues of a dihyrouroporphyrin and a tetrahydrouroporphyrin with adjacent reduced rings . Field desorption spectra of the {C2H3}- and {CH3}-tetrahydrouroporphyrin analogues show that the compound contains two methyl groups; it is concluded that the chlorine-like compound has one methyl group . Dehydrogenation experiments indicate that the methyl groups are located at one beta-carbon of the reduced rings . Incorporation experiments suggest that the tetrahydrouroporphyrin-like compound is an intermediate in cobyrinic acid biosynthesis . Studies on the utilization of a heptacarboxyporphyrinogen from C . tetanomorphum for cobyrinic acid formation are also described.

J Infect Dis, 1977 Mar, 135 Suppl, S7 - 12
In vitro susceptibility of anaerobes: comparison of clindamycin and other antimicrobial agents; Sutter VL; The in vitro activity of clindamycin against anaerobic bacteria was compared with that of other antimicrobial agents . Clindamycin remained effective against most anaerobic bacteria found in infections, although resistance to the drug was found in certain groups, such as Peptococcus and Clostridium species.

Can J Microbiol, 1977 Mar, 23(3), 346 - 53
Studies on some characteristics of hydrogen production by cell-free extracts of rumen anaerobic bacteria; Joyner AE et al.; Hydrogen production was studied in the following rumen anaerobes: Bacteroides clostridiiformis, Butyrivibrio fibrisolvens, Enbacterium limosum, Fusobacterium necrophorum, Megasphaera elsdenii, Ruminococcus albus, and Ruminococcus flavefaciens . Clostridium pasteurianum and Escherichia coli were included for comparative purposes . Hydrogen production from dithionite, dithionite-reduced methyl viologen, pyruvate, and formate was determined . All species tested produced hydrogen from dithionite-reduce methyl viologen, but only C . pasteurianum, B . clostridiiformis, E . limosum, and M . elsdenii produced hydrogen from dithionite . All species except E . coli produced hydrogen from pyruvate, but activity was low or absent in extracts of E . limosum, F . necrophorum, R . albus, and R . flavefaciens unless methyl viologen was added . Hydrogen was produced from formate only by E . coli, B . clostridiiformis, E . limosum, F . necrophorum, and R . flavefaciens . Extracts were subjected to ultracentrifugation in an effort to determine the solubility of hydrogenase . The hydrogenase of all species except E . coli appeared to be soluble, although variable amounts of hydrogenase activity were detected in the pellet . Treatment of extracts of the rumen microbial species with DEAE-cellulose resulted in loss ofhydrogen production from pyruvate . Activity was restored by the addition of methyl viologen . It is concluded that hydrogen production in these rumen microorganisms is similar to that in the saccharolytic clostridia.

Infect Immun, 1977 Mar, 15(3), 999 - 1001
Effect of Clostridium perfrongens enterotoxin in mitochondrial respiration; McDonel JL et al.; Clostridium perfringens enterotoxin caused a 26 to 41% reduction in the rate of oxygen consumption by rat liver mitochondria when various tricarboxylic acid cycle intermediates were used as substrate . However, P/O ratios were unaltered.

Infect Immun, 1977 Mar, 15(3), 765 - 71
Capsular polysaccharide of Clostridium perfringens Hobbs 9; Cherniak R et al.; Several closely related capsular polysaccharides were isolated from a strain of Clostridium perfringens Hobbs 9 type A by extraction of encapsulated cells with cold 0.85% NaCl . The soluble polymers were precipitated with alcohol and purified by (NH4)2SO4 fractionation, enzymatic digestion with papain and ribonuclease, and chromatography on diethylaminoethyl-Sephadex A25 . The polysaccharides were composed mainly of glucose, galactose, and galactosamine . The major fraction contained these constituents (representing 77% of the dry weight) in a molar ratio of 1:1.6:1.1 . All of the fractions contained phosphate and peptide material that was not removed during purification . The polysaccharides were closely related but not identical as indicated by double-diffusion-in-gel experiments . Immunoelectrophoresis in agarose demonstrated that the polysaccharides had identical mobilities and that no resolution into additional fractions occurred . The immunological activity of all the purified polysaccharides was destroyed by periodate oxidation but was unaffected by protease.

Mikrobiologiia, 1977 Mar-Apr, 46(2), 311 - 7
{Ecologic features of cultures of the species Clostridium gürfelii}; Vozniakovskaia IuM et al.; The Clostridium cultures decomposing pectin were isolated from flax straw during flax retting . The activity was manifested only at high pH values, the optimum pH being 6.7--8.2 . Such ecological adaptation of the cultures is determined by the properties of the pectolytic enzyme complex which they synthesize . The complex does not contained polygalacturonase (the optimum pH 5.0) but has a high activity of pectate transeliminase (the optimum pH 8.0--8.4) . The cultures differ from Clostridium felsineum which possesses a high activity of polygalacturonase and therefore can decompose pectin in acid medium . By their morphological, cultural and physiological properties, the bacteria are classed as Clostridium gufelii (Prevot, 1966).

Vet Rec, 1977 Feb 5, 100(6), 106 - 9
Avian botulism and the high prevalence of Clostridium botulinum in the Norflok Broads; Borland ED et al.; An account is given of a severe outbreak of type C botulism in waterfowl that occurred on the Norfolk Broads during the exceptionally warm summer of 1975 . Forty-five mud samples were collected from 22 well distributed aquatic sites representing a considerable proportion of the total number of Broads . All samples except one (ie, 97-8 per cent) were shown to contain Clostridium botulinum and 58 per cent contained more than one type of the organism . Types B, C and E were demonstrated in 62-2 per cent, 51-1 per cent and 60 per cent of samples respectively . Recent surveys, made by identical methods, of aquatic environments in the London area and the Camargue (France) showed prevalences of Cl botulinum of 72-5 per cent and 4-5 per cent respectively . It seems likely that the Norfolk Broads will continue to present a risk to waterfowl from botulism in future hot summers.

Appl Environ Microbiol, 1977 Feb, 33(2), 289 - 97
Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum; Weimer PJ et al.; The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C . thermocellum/Methanobacterium thermoautotrophicum cocultures was studied . All cultures were grown under anaerobic conditions in batch culture at 60 degrees C . When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture . Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture . Monocultures produced primarily ethanol, acetic acid, H2 and CO2 . Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture . In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion . Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture . Fermentation of cellobiose was more rapid than that of cellulose . In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.

Biull Eksp Biol Med, 1977 Feb, 83(2), 198 - 200
{Immune response of mice of different inbred lines to Clostridium oedematiens anatoxin}; Petrov RV et al.; Mice belonging to a number of inbred strains were immunized intradermally with Cl . oedematiens alpha-toxoid . The immunization was repeated 30 days later . On the 20th and the 30th days after the first injection and on the 10th day after the second one the antibody level against the toxoid was determined in the blood of mice by the passive hemagglutination test . The maximum response to the primary immunization was observed in the mice of the C3H strain, and the minimum one--in mice of the DBA/2 strain; the difference was more than 30-fold . The rest of the strains used in the test (A,CBA, BALB/c, AKR, CC57BR) displayed an intermediate level of the immune response . The differences reduced after the repeated immunization . The immune response to this antigen in mice is supposed to be genetically controlled.

Appl Environ Microbiol, 1977 Feb, 33(2), 400 - 5
Isolation and molecular size of Clostridium botulinum type C toxin; Syuto B et al.; A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin . The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein . The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain . The molecular weights of the subunits were approximately 98,000 and 53,000 . When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).

J Bacteriol, 1977 Feb, 129(2), 1148 - 50
Biochemical properties of Clostridium bifermentans spores; Hausenbauer JM et al.; As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue . As in spores of Bacillus species, germination of C . bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid . In contrast to spores of Bacillus species, dormant spores of C . bifermentans contained little free amino acid.

J Hyg (Lond), 1977 Feb, 78(1), 39 - 41
A comparison of the distribution of Clostridium botulinum in soil and in lake mud; Smith GR et al.; In 1975, 25 soil samples were collected from the London area . Of these, 20 were obtained 200-300 yards from 20 lakes that had been shown in 1974 to contain mud contaminated with one or more of types of B, C, D, and E of clostridium botulinum . By means of a technique comparable with that use for the examination of mud, the 20 soil samples were found negative . The remaining 5 soil samples, obtained from sites that were not in close proximity to lakes, were also negative except for one that contained type B.

J Hyg (Lond), 1977 Feb, 78(1), 33 - 7
The low prevalence of Clostridium botulinum in the lakes, marshes and waterways of the Camargue; Smith GR et al.; Mud samples collected in June 1975 from the lakes, marshes and waterways of the Camargue were examined for Clostridium botulinum . The Grand Rhone and Petit Rhone were shown to contain types B and E, but of 44 samples taken from well distributed sites on the Ile de la Carmargue, only two (4-5%) were positive and these contained type E alone . The survey indicated a much lower prevalence of Cl . botulinum than any encountered in recent surveys of inland aquatic environments elsewhere.

Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 547 - 51
Identification of the iron-sulfur center in trimethylamine dehydrogenase; Hill CL et al.; Trimethylamine dehydrogenase {trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7} from a facultative methylotroph bacterium has a molecular weight of 147,000 and contains two types of prosthetic groups, one a covalently bound organic chromophore of uncertain structure and the other containing iron and labile sulfur (S*) . The structure of the Fe-S* center has been investigated by reactions of the enzyme with sodium mersalyl, o-xylyl-alpha,alpha'-dithiol, and p-methoxybenzenethiol in a 4:1 vol/vol hexamethylphosphoramide/water reaction medium, which destabilizes tertiary structure . Mersalyl treatment results in reduction of visible absorbance consistent with the presence of a 4-Fe center of the ferredoxin type . Reaction with thiols effects partial bleaching of the organic chromophore, as established by separate studies of a detached chromophore peptide, and results in removal (extrusion) of the core unit of the Fe-s* center in the form of the complexes {Fe4S*4(S2-o-xylyl)2}n2n- and {Fe4S*4(SC6H4OMe)4}2-, which were identified by absorption spectra . These results, in conjunction with control extrusion reactions of oxidized ferredoxins from spinach and Clostridium pasteurianum, establish that trimethylamine dehydrogenase contains one Fe4S*4 core unit most probably present as a ferredoxin-type, cysteinate-ligated cluster {Fe4S*4(S-Cys)4}.

Appl Environ Microbiol, 1977 Feb, 33(2), 254 - 6
Amorphous ferrous sulfide as a reducing agent for culture of anaerobes; Brock TD et al.; Amorphous ferrous sulfide, prepared by reacting ferrous ammonium sulfate and sodium sulfide, is an excellent reducing agent for the culture of anaerobes . It reduces resazurin and reacts much more rapidly with O2 than does either soluble sulfide (HS)- or cysteine . One of the end products of the oxidation of ferrous sulfide with O2 is red and serves as an indicator for the oxygen contamination of a culture medium . Amorphous ferrous sulfide served as a suitable reducing agent for the growth of species of Methanobacterium or Clostridium . Its use is recommended for enrichment or culture of anaerobes (e.g . autotrophs, fermentative organisms) from sediments and other habitats were organic reducing agents are undesirable and where soluble sulfide might be toxic.

Acta Pathol Microbiol Scand {B}, 1977 Feb, 85B(1), 89 - 94
Chemical modification and characterization of enterotoxin from clostridium perfringens type A; Granum PE et al.; Enterotoxin from Clostridium perfringens type A has been purified . The enterotoxin was shown to be heat-labile, but re-activation of heat treated enterotoxin did occur down to an activity of 15 per cent of the native enterotoxin . The molecular weight was shown to be 34,000 by ultracentrifugation, and the molecular weight did not change significantly after treatment with 0.1 M beta-mercaptoethanol and 6 M guanidine hydrochloride . It was concluded that the enterotoxin consists of one single polypeptide chain . The enterotoxin did not lose any activity after treatment with iodoacetic acid and iodoacetamide, but a complete loss of activity was observed after succinylation.

J Bacteriol, 1977 Feb, 129(2), 843 - 9
Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A; Labbe RG et al.; Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens . When used at a concentration that inhibited {14C}uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells . At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation . When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence . Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat . The half-life of enterotoxin RNA was estimated to be at least 58 min . When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained . The two peaks corresponded to enterotoxin and another spore coat protein(s) . Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA.

Can J Microbiol, 1977 Feb, 23(2), 152 - 60
{Study of the NADH and NADPH-ferredoxin oxidoreductase activities in Clostridium acetobutylicum}; Petitdemange H et al.; The NADH and NADPH-ferredoxin oxidoreductase have been studied in Clostridium acetobutylicum . Acetyl-CoA is an obligatory activator of NADH-ferredoxin reductase activity and NADH a competitive inhibitor of ferredoxin-NAD+ reductase activity . These regulations are the same when C . acetoburylicum moves from 'butylic-type metabolism' to 'butyric-type metabolism'; this demonstrates that NADH-ferredoxin oxidoreductase cna, through its reversible action, meet the very different cell needs imposed by these two types of culture . The physiological function of the clostridial NADPH-ferredoxin oxidoreductase was anabolic as it has been with other clostridia.

Biochim Biophys Acta, 1977 Jan 25, 490(1), 120 - 31
Comparative immunochemistry of bacterial, algal and plant ferredoxins; Tel-Or E et al.; 1 . Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus, the 2 {4Fe-4S} ferredoxin from Clostridium pasteurianum, and the 2Fe-2S ferredoxins from the blue-green algia Spirulina maxima, the green alga Scenedesmus obliquus, and the higher plant Beta vulgaris . The antibodies were tested for immunoprecipitation activity with seven bacterial, twelve blue-green algal, six eukaryotic algal and six higher plant ferredoxins . 2 . Antibodies to the bacterial ferredoxins reacted to a significant extent only with their homologous proteins . On the other hand, antibodies to the plant and algal ferredoxins showed cross-reaction with other ferredoxins . There was a correlation between the degrees of immunoprecipitation and the similarity in amino acid sequences . These results suggest that the method can be used as a marker in taxonomic studies . 3 . The interaction of the antibodies with the five native ferredoxins was compared with the reactions with their apoproteins . In each case the degree of interaction was different . This behaviour was interpreted as due to an influence of tertiary structure on the antibody-antigen interaction.

Biochemistry, 1977 Jan 11, 16(1), 30 - 3
Influence of cationic triphenylmethane dyes upon DNA polymerization and product hydrolysis by Escherichia coli polymerase I; Wolfe AD; The cationic triphenylmethane dyes crystal violet, methyl green, and malachite green inhibited DNA synthesis catalyzed by Escherichia coli B polymerase I (polymerase I) . Lower concentrations of the dyes inhibited DNA replication as a direct function of the proportion of AT to GC in the DNA of Clostridium perfringens, Escherichia coli, and Micrococcus lysodeikticus . When the intercalant proflavin was employed, the GC-rich micrococcal DNA was most severely inhibited . In addition, both the triphenylmethanes and proflavin inhibited product hydrolysis catalyzed by polymerase I.

J Biol Chem, 1977 Jan 10, 252(1), 20 - 31
Purification and properties of 3-keto-5-aminohexanoate cleavage enzyme from a lysine-fermenting Clostridium; Yorifuji T et al.; The lysine-fermenting Clostridium SB4 is shown to contain a new type of beta-keto acid-degrading enzyme that converts 3-keto-5-aminohexanoate and acetyl-CoA reversibly to L-3-aminobutyryl-CoA and acetoacetate . Following the development of a sensitive radiochemical assay the enzyme was purified 175-fold to about 90% homogeneity in 44% yield . The specific activity of the purified enzyme is 44 IU/mg of protein at 30 degrees . The equilibrium constant for the forward reaction was found to be 0.68 at 30 degrees and pH 7.0, corresponding to a deltaG0' of 0.23 kcal/mol . The enzyme is highly substrate-specific . Of several substrate analogs tested in the forward and back reactions only beta-alanyl-CoA and D-3-aminobutyryl-CoA are utilized about 130% and 1.7% as fast as L-3-aminobutyryl-CoA, respectively . The product formed from beta-alanyl-CoA and acetoacetate is a neutral beta-keto acid, presumably 3-keto-5-aminopentanoic acid; its borohydride reduction product was partially characterized as a hydroxy-amino acid by various chromatographic and ion exchange methods . The activity of the purified enzyme is increased about 5-fold by addition of 0.1 mM Co2+ and to a lesser extent by Mn2+ . Activity is inhibited by orthophosphate, thiol reagents, and EDTA; however, exposure of the enzyme to the latter compound prior to addition of Co2+ increases activity, presumably by removing competing divalent cations . Tracer experiments have shown that carbon atoms 1 and 2 of acetoacetate are derived from carbon atoms 1 and 2 of 3-keto-5-aminohexanoate whereas carbon atoms 3 and 4 are derived from acetyl-CoA . The amino acid moiety of 3-aminobutyryl-CoA is derived from carbon atoms 3 to 6 of 3-keto-5-aminohexanoate . Since no evidence for covalent enzyme-substrate intermediates could be obtained by the study of four possible group exchange reactions, a concerted reaction between 3-keto-5-aminohexanoate and acetyl-CoA is considered . The enzyme has a molecular weight of about 97,000 and probably contains four identical subunits . The relatively high specific activity of the enzyme in extracts of Clostridium SB4 indicates it functions in the main pathway of lysine degradation . This relatively stable enzyme provides a convenient and specific method for the quantitative estimation of nanomolar amounts of L- and D-3-aminobutyryl-CoA and beta-alanyl-CoA.

Biochem J, 1977 Jan 1, 161(1), 37 - 47
Preparation and characterization of armadillo submandibular glycoproteins; Wu AM et al.; The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species . Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch . Biochem . Biophys . 124, 41-50) . The composition of glycoprotein A is the simplest one among the known mucus glycoproteins . Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B . These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine . Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B . For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J . Biol . Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids . The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins . Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B . The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex . For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core . The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase . Glycoprotein B has chemical properties similar to those of glycoprotein A . However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75 . Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses . The apparent mol.wts . of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.

Zentralbl Bakteriol {Orig A}, 1977, 237(2-3), 358 - 71
{Susceptibility to Thiamphenicol and Chloramphenicol of Anaerobic Bacteria (author's transl)}; Werner H et al.; The in vitro susceptibility to thiamphenicol and chloramphenicol of 272 anaerobes, most of which were recent clinical isolated, was determined by broth dilution tests, With chloramphenicol, 133 anaerobic gram-negative non-sporing rods (48 Bacteroides fragilis, 13 B . thetaiotaomicron, 14 B . oralis, 16 Sphaerophorus varius etc.) had MIC values of 0.03 through 16 microng/ml . Very similar results (MIC, 0.06-16 microng/ml) were obtained with thiamphenicol . In concentrations of 4 microng/ml or less chloramphenicol inhibited 90.2% and thiamphenicol 78.95% of the strains . Strains with only moderate sensitivity to both antibiotics (MIC, 8 or 16 microng/ml) belonged to B . fragilis or other Bacteroides species . Members of the Fusobacterium and Sphaerophorus group were susceptible to less than or equal to 2 microng chloramphenicol/ml and less than or equal to 4 microng thiamphenicol/ml respectively . With both antibiotics, 102 strains of gram-positive non-sporing anaerobes (P . acnes, Peptostreptococcus spp., Peptococcus spp.) were susceptible to less than or equal to 8 microng/ml . Of 37 Clostridium isolates, 35 (belonging to C . perfringens, C . septicum, C . cadaveris etc.) were inhibited by concentrations of 8 microng/ml or less of chloramphenicol and thiamphenicol . Only one strain each of C . perfringens and Clostridium sp . had an MIC of 16 microng thiamphenicol/ml . Accordingly, resistance to thiamphenicol or chloramphenicol was not observed . A standardized monodisk agardiffusion test was performed on 40 Bacteroidaceae, 18 Peptococcaceae and 20 C, (P.) acnes strains . Only a poor correlation was observed between MIC and zone size for thiamphenicol and chloramphenicol . Therefore, inhibition zone diameter measurement cannot be regarded as a reliable method to detect chloramphenicol or thiamphenicol resistance in anaerobes . Thiamphenicol, which is virtually as active against anaerobes as chloramphenicol but lacks serious toxicity, may well play an important role in the therapy of various anaerobic infections.

Ann Ist Super Sanita, 1977, 13(4), 805 - 32
{Studies of the microbial content of raw materials used in pharmaceutical preparations}; Bonomi E et al.; The microbiological controls (determination of the total and mycetic bacterial count; isolation of enterococci, Clostridium, Pseudomonas aeruginosa, Staph . aureus, E . coli, Salmonella and other Enterobacteriaceae) executed on 1517 samples of 100 varous raw materials, have ascertained the presence of a microbial contamination varying to matters, with presence also of pathogenic or hygienically undesirable microrganisms . The more contaminated substances have resulted to be those organic either of vegetable or animal origin . Sensible differences have been noted between the various lots and according to the various suppliers . The conservation trials under various conditions of temperature (15-22-37 degrees C) and humidity (30 degrees - 60 degrees - 90 degrees of relative humidity) with opened or closed recipients, have demonstrated the possibility of remarkable increases of the number of the microrganisms in the raw materials with increase of temperature and humitidy, especially in the non hermatically closed recipients.

Arzneimittelforschung, 1977, 27(12), 2263 - 5
Susceptibility to erythromycin of anaerobes of the genera Bacteroides, Fusobacterium, Sphaerophorus, Veillonella, Clostridium, Corynebacterium, Peptococcus, Peptostreptococcus; Werner H et al.; The minimal inhibitory concentrations (MIC) of erythromycin were determined by broth dilution tests for 313 anaerobic strains, most of which were clinical isolates . All the gram-positive anaerobes tested (84 Peptococcaceae, including 21 Peptostreptococcus anaerobius and 15 Peptococcus variabilis; 65 Corynebacterium acnes and 29 Clostridium strains, including 13 C . perfringens) were sensitive (MIC values 0.012 through 3.12 microgram erythromycin/ml); so were 111 cultures of gram-negative anaerobes (52 Bacteroides fragilis, 12 B . thetaiotaomicron, 7 B . vulgatus, 13 B . oralis, 4 B . melaninogenicus, 10 Sphaerophorus necrophorus, 2 Veillonella sp., 11 members of other species) . Erythromycin at concentrations of 6.25 through 200.0 microgram/ml was active against 24 strains (1 B . fragilis, 4 Fusobacterium fusiforme, 9 Sph . freundi, 10 Sph . varius) . The present results are compared to the limited number of reports existing with regard to the susceptibility of anaerobes to erythromycin.

Zentralbl Bakteriol Parasitenkd Infektionskr Hyg, 1977, 132(7), 659 - 65
Microbial society and its activity in the soil; Prasad M; In this study, with the help of the buried slide method, a qualitative observation of the microsociological association in the soil was assessed . Fungal mycelium cohabiting with bacterial forms, rods as well as cocci, individual colonies of Azotobacter-type cells, and certain algae of Hantzschia species were recorded in a central European typical garden soil . The use of CHOLODNY-STRUGGER combination method was helpful in ascertaining the decaying nature of a soil nematode and also fungal mycelium which, during the process of decomposition, served there as a substrate for bacterial multiplication . Colony of Clostridium-type rod-shaped bacteria existed between the soil particles, acting as living bridges . When examined under fluorescent microscope, the bacterial forms emitted shining fluorescence of green colour which in effect presented them as lighted objects, prominent against the dull red background of soil objects and other decaying substances.

South Med J, 1977 Jan, 70(1), 5 - 7
Type A botulism from commercially canned beef stew; Blake PA et al.; Two of three persons who ate lunch together became ill with symptoms characteristic of botulism . One died before botulism was suspected and before specimens could be collected for laboratory testing, but a serum specimen from the other patient, who survived, yielded botulinal toxin, type A . The third person remained asymptomatic, but Clostridium botulinum type A was cultured from his stool . The three persons had shared two canned foods: home-canned green beans and commercially canned beef stew . The green beans were initially assumed to be the cause of the outbreak . However, the empty stew can was recovered from the garbage, and washings from the can yielded C botulinum, type A, and its toxin.

Z Allg Mikrobiol, 1977, 17(7), 501 - 6
Dissimilatory nitrate reduction in Clostridium tertium; Hasan M et al.; Fermentation balance studies were carried out on Clostridium tertium grown with and without nitrate in the medium . Nitrate reduction increased the efficiency of energy produced from glucose by permitting the utilization of additional sites of substrate level phosphorylation . The effect was even more dramatic in C . tertium than in C . perfringens, with increased cell yields of about 30% being observed in the former compared with 20% in the latter . Unlike C . perfringens, C . tertium responded to the presence of nitrate in the medium with an increased growth rate . A slight increase in the Y ATP of these cultures was also observed, and quantitatively, this appeared to be consistent with the prediction of Stouthammer and Bettenhaussen that Y ATP will vary with the growth rate . Thus, C . tertium, like C . perfringens, was able to use nitrate as an electron acceptor in conjunction with its energy metabolism, suggesting that this may be widespread among the nitrate-reducing anaerobes.

Exp Pathol (Jena), 1977, 13(6), 296 - 301
Effect of microwave radiation on cells treated with membrane-injuring substances; Szmigielski S et al.; WISH cells grown in vitro were pretreated with subcytotoxic concentrations of digitonin, cortisol and purified bacterial toxins -- staphylococcal beta-haemolysin or Clostridium perfringens alpha-toxin and irradiated with 3 GHz electromagnetic wave (microwaves) at the field power densities 5 or 40 mW/cm2 . At 40 mW/cm2 increase in temperature of the culture medium of about 2-3 degrees C was noted, while at 5 mW/cm2 no detectable increase in temperature was found . Control and pretreated WISH cells after irradiation in the microwave field were used for evaluation of their viability, incorporation of tritiated thymidine, glycine and uridine and level of intracellular cyclic AMP . Irradiation with microwaves resulted in lowering of thymidine and glycine incorporation along with changes in the intracellular amount of cAMP (decrease in cells exposed to 5 mW/cm2 and increase in those exposed to 40 mW/cm2) . Under both conditions viability of the cultures was normal . Pretreatment of cells with digitonin or purified bacterial toxins followed by irradiation with microwaves resulted in enhancement of the cytotoxic effect with lowering of cell viability, especially after exposition to power density of 40 mW/cm2 . Cortisol led to decrease in 3H-glycine and 3H-uridine incorporation into WISH cells, but did not influence the reaction of the cells to microwave radiation . In view of the results presented it may be concluded that substances injuring cell membranes sensitize cell cultures to electromagnetic radiation of the microwave range and may enhance the specific (non-thermal) effect of microwaves.

J Biochem (Tokyo), 1977 Jan, 81(1), 115 - 26
Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens; Yamakawa Y et al.; 1 . Phospholipase C{EC 3.1.4.3} was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100 . During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms . 2 . The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C . perfringens) antitoxin, indicating the homogeneity of the preparation . 3 . The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported . 4 . The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150 . From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C . perfringens phospholipase C can be accounted for . 5 . For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions . In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin . The effects of various divalent cations on the enzymatic hydrolysis were also investigated . 6 . The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.

Can J Comp Med, 1977 Jan, 41(1), 112 - 6
{Necrotic enteritis in broiler chickens . III . Study of the factors favoring the multiplication of Clostridium perfringens and the experimental transmission of the disease}; Bernier G et al.; Necrotic enteritis was reproduced experimentally in two week old broiler chickens by intravenous injection and also by oral administration of a pure culture of Clostridium perfringens . In the first experiment, gross and microscopic intestinal lesions, typical of necrotic enteritis, were observed in all diseased birds and mortality was obtained only in the group of birds that were injected with 0.4 ml or more of the pure culture of the microorganism . In the second experiment, the highest mortality was noted in the group of birds that received orally, in addition to the Clostridium culture, a solution of sodium bicarbonate, to obtain an alkaline intestinal content and opium to decrease the intestinal peristaltism . The gross and microscopie intestinal lesions of the diseased and killed birds were more severe than those observed in the other groups and were similar to those encountered in field outbreaks of necrotic enteritis.

Am J Public Health, 1977 Jan, 67(1), 44 - 9
A progressive approach to the problem of foodborne infections; Zaki MH et al.; Qualitative and quantitative bacteriological examinations of 100 samples of perishable foods from 39 retail stores were performed to determine the presence of bacterial contaminants and to explore the feasibility of establishing and utilizing microbiological standards in enforcement . Forty-six per cent of the samples had standard plate counts in excess of 100,000 per gram, 17 per cent showed coliform organisms in excess of 100 per gram, 20 per cent revealed the presence of Staphylococcus aureus and 2 per cent Clostridium perfringens . None of the shell fish samples grew Vibrio parahaemolyticus . The bacteriological findings are discussed in relation to pertinent variables and the use of microbiological standards for potentially hazardous foods is explored . All 450 retail food establishments in a selected area of Western Suffolk County (New York) were subjected to comprehensive study, using a scoring system developed by the Food and Drug Administration . Initial inspections revealed 32 per cent as having one or more major violations . Follow-up inspections were performed to insure compliance and most violations were corrected within four weeks . Six months later all establishments were reinspected . The scoring system was found to have limited value . Half the establishments with major violations on initial inspection had major violations six months later as compared to less than a quarter of those with no initial major violation.

Chemotherapy, 1977, 23(4), 227 - 35
Concentrations of tinidazole in body fluids and tissues in gynaecological patients; Ripa T et al.; The concentrations of tinidazole in various tissues and body fluids were studied in gynaecological patients after a single 2g oral dose . Tinidazole was determined by the agar-diffusion technique using a strain of Clostridium bifermentans . Reliable estimates of concentrations down to 0.5 microng/ml could be obtained . Dichloromethane extraction of tinidazole added to various tissues in known amounts gave a recovery of 100 +/- 10% . Peak serum values of 32-52 microng/ml were reached 3-6h after the administration . The concentrations in peritoneal fluid, obtained at operation 8.5-15h after the intake, varied between 16 and 40 microng/ml . Specimens from the Fallopian tubes yielded 15-26 microng tinidazole/g tissue; similar levels were obtained specimens from myometrium, endometrium, portio, vaginal secretions, omental fat, and cutis . It is concluded that, with the given dose, tinidazole concentrations are achieved in fluids and tissues of the female genital tract that are far in excess of those that should be therapeutical in infections caused by microorganisms know to respond to nitroimidazole treatment.

Acta Microbiol Pol, 1977, 26(3), 285 - 94
Purification of Clostridium toxoids; Buchowicz I et al.; A two-step fractionation procedure was applied for purification and concentration of the individual Clostridium toxoids . The toxoids were precipitated with hydrochloric acid in the presence of sodium sextametaphosphate, then antigenic fractions were separated from inactive contaminants by Sephadex G-75 filtration . Specific activity of the preparations thus obtained, as determined by Mancini radial immunodiffusion, was 150--565 binding units per mg of protein nitrogen for Clostridium perfringens toxoid, 204--352 binding units for Clostridium oedematiens toxoid and 26.6 -- 51.2 binding units for Clostridium septicum toxoid.

Eur J Biochem, 1977 Jan, 72(2), 247 - 50
Synthesis of stereospecifically deuterated phenylalamines and determination of their configuration; Bartl K et al.; 1 . Starting from trans-cinnamic acid a chiral (-)3-phenyl-{2,3-2H}propionic acid has been synthesized using Clostridium kiuyveri cells as catalyst . 2 . The chiral dideuterated acid has been converted by chemical methods to a mixture of (2R) and (2S)-phenyl{2,3-2H}-alanine . 3 . By means of 1H nuclear magnetic resonance spectroscopy and the action of D and L-amino acid oxidase the configuration of the phenylalamine has been shown to be (2R, 3S) and (2S, 3S), respectively . The labelled phenylalanine is thus sterically and isotopically homogenous at position 3 but heterogenous at position 2.

Biochem J, 1976 Dec 15, 160(3), 475 - 9
Validation of a simple radiochemical assay measuring hydrolysis of choline-labelled microsomal phosphatidylcholine by phospholipase C . pH-dependence; Cater BR et al.; Selective hydrolysis of phosphatidylcholine species, which are selectively radioactively labelled in vivo, does not appear to interfere with a radiochemical assay for hydrolysis of microsomal phosphatidylcholine by C-type phospholipases from Bacillus cereus or Clostridium perfringens . Both phospholipases substantially hydrolysed phosphatidylcholine over the pH range 4.0-10.0.

J Biol Chem, 1976 Dec 10, 251(23), 7398 - 404
Gonadotropin receptors in plasma membranes of bovine corpus luteum . I . Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors; Azhar S et al.; The ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D . Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect . Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus . Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition . The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively . Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity . The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex . The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes . Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor . Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38) . Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively.

Proc Natl Acad Sci U S A, 1976 Dec, 73(12), 4369 - 73
Interactions of heterologous nitrogenase components that generate catalytically inactive complexes; Emerich DW et al.; A unique method is described for inhibiting nitrogenase . When Clostridium pasteurianum nitrogenase is assayed in the presence of the Mo-Fe protein of Azotobacter vinelandii, all the characteristic activities of nitrogenase are inhibited . C . pasteurianum nitrogenase is unaffected by the Fe protein of A . vinelandii . The Fe protein, but not the Mo-Fe protein of C . pasteurianum, inhibits A . vinelandii nitrogenase . Both inhibitions described result from the formation of an inactive complex of A . vinelandii Mo-Fe protein and C . pasteurianum Fe protein . Complex formation requires active components, as oxygen-denatured proteins are ineffective . The results for titration of components of the complex against each other and kinetic data each indicate that the inactive complex consists of two molecules of C . pasteurianum Fe protein per molecule of A . vinelandii Mo-Fe protein . The results of kinetic experiments suggest that the Fe protein from each organism competes for the same site(s) on the A . vinelandii Mo-Fe protein . The Fe protein of C . pasteurianum will form an active or an inactive complex with the Mo-Fe proteins from six different organisms . Inhibition by nitrogenase components that form inactive complexes provides numeroius ways to study the mechanism of nitrogenase action.

Strahlentherapie, 1976 Dec, 152(6), 537 - 41
{Tumor hyperthermia using high frequency for increase of oncolysis by clostridium butyricum (M 55)}; Dietzel F et al.; In solid experimental tumors hypoxic cells preferably are inactivated by means of a short-termed radio-frequency treatment with eddy-current fields . Hence follows an increase of the necro-biotic areas . The presence of anoxic-necrobiotic areas is indispensable for the termination of certain anaerobic spores such as Clostridium butyricum s . oncolyticum (M 55) . Local tumor hyperthermy is tested as a technique in altogether 861 mice bearing neck tumors, in order to enhance the germination of oncolytic Clostridia in tumors differing by their mode of formation and rate of growth . In all the three test systems used (Ehrlich solid carcinoma, Harding-Pasey-melanoma, fibrosarcoma induced by methylcholanthrene), the oncolysis being brought about by Clostridia can be intensified significantly by means of a short-termed warming of the tumor up to temperatures of 42 to 44 degress C using radio-frequency.

Onderstepoort J Vet Res, 1976 Dec, 43(4), 165 - 73
The antibody response of cattle to Clostridium botulinum types C and D toxoids; Jansen BC et al.; The resistance of cattle with varying serum-antitoxin titres was determined by per os challenge . The results proved that a solid immunity can be produced against C . botulinum toxins C1 and D . The immune response of cattle to various quantities of C . botulinum C1 and D toxoids, aluminium-phosphate-adsorbed and in water-in-oil emulsion was investigated . The response to antigen in water-in-oil emulsion was far superior to the other when they were used for primary and secondary stimuli . When cattle had been given a solid basic immunity with 2 injections of antigen in water-in-oil emulsion, essentially the same booster effect was obtained with antigen in water-in-oil emulsion and in aqueous solution . Only some of the animals injected intramuscularly with antigens in water-in-oil emulsion developed local lesions . These lesions were not large and their histological picture indicated a noticeable decline in severity within 20 weeks . A case is thus made out for the use of C . botulinum C1 and D toxoids in water-in-oil emulsion for the primary and secondary stimuli and an aqueous solution of these antigens for any booster stimulus as an improved method of protecting cattle against botulism.

Jpn J Med Sci Biol, 1976 Dec, 29(6), 335 - 49
Production and properties of theta-toxin of Clostridium perfringens with special reference to lethal activity; Soda S et al.; theta-Toxin of Clostridium perfringens produced in a synthetic medium showed high toxicity . The mouse was killed with 5-10 hemolytic units of the toxin . Neutralization experiments showed that lethal and hemolytic activities were due to the same toxic entity . The amount of theta-toxin expressed in lethal activity reached more than 30% that of alpha-toxin in the synthetic medium SM67 . Although the activities of alpha-and theta-toxins were not additive in terms of LD50, increase in the ratio of theta-toxin to alpha-toxin resulted in reduction of the survival time of the mouse injected with a lethal dosis of the mixture of the two toxins when compared with alpha-toxin alone . Unlike those produced in other media, the hemolytic and lethal activities of theta-toxin produced in the synthetic medium was not activated by reduction with thioglycollate, even after partial purification . In other respects, the toxin was not different significantly from those reported in the past . The toxic form was detected also in complex medium . It was suggested that theta-toxin may be produced as a nascent entity.

Appl Environ Microbiol, 1976 Dec, 32(6), 803 - 7
"Phoenix phenomenon" in the growth of Clostridium perfringens; Shoemaker SP et al.; The "Phoenix phenomenon" was observed with Clostridium perfringens Hobbs' serological type 9 (HT9) in a cooked-meat medium at 81.7 degrees C by a decrease in plate count (phase I), followed by an increase in count to the intiial level (phase II) and a continued increase above the initial count (phase III) . The effects of sporulation, age of inoculum, assay medium, anaerobiosis, diluent, and growth inhibitor were studied . This phenomenon was reproduced in experiments with sporulation-negative mutants derived from HT9 inocula of various cell ages, and different assay media (sulfite-iron agar, tryptose-soytone-yeast extract agar, prereduced peptone-yeast extract agar, prereduced veal agar, and veal agar) . When strict anaerobic conditions were employed, it was necessary to increase the heating temperature to 52.3 degrees C to observe the phenomenon . The phenomenon was eliminated at 52.3 degrees C when a combination of strict anaerobic conditions, prereduced media, and prereduced veal diluent was employed . The addition of nalidixic acid at the minimum point of the growth curve (end of phase I) had no effect on the appearance of phase II; however, phase III was completely inhibited . This indicated that phases I and II were an injury-recovery process.

Acta Pathol Microbiol Scand {B}, 1976 Dec, 84B(6), 414 - 20
An enterotoxin produced by Clostridium perfringens type D . Purification by affinity chromatography; Uemura T et al.; Clostridium perfringens type D 9867 produced enterotoxin immunologically identical to that produced by types A and C which are responsible for C . perfringens food poisoning . Enterotoxin from type C 5386 and type D 9867 was produced in Duncan og Strong sporulation medium (DS medium) . The supernatant fluid from DS-cultures was used for purification of the enterotoxin by affinity chromatography on a monospecific anti-enterotoxin-coupled CNBr activated-Sepharose 4B and activated CH-Sepharose 4B column . The enterotoxin purified by this one-step procedure proved to be of a purity comparable to that obtained by conventional methods, and possessed lethal activity in mice.

Zentralbl Bakteriol {Orig B}, 1976 Dec, 163(5-6), 433 - 40
The ultrastructure of oncolytic spores found on pig skin; Althoff J et al.; A new type of clostridium spore, derived from melanotic hamster tumors treated with pig skin extracts, was examined in a lyophylized form by electron microscopy (EM) . Scanning EM indicated that two out of five preparations contained only spores . The spores exhibited oval forms (1.5 X 1.0 MU)with elongated, sometimes infolded, poles indicating an overlying membrane . These structures corresponded to the exosporium as seen by transmission EM . The size of the spores, including the cortex (350-550 A), inner membranes, and cytoplasm averaged 5100 X 5900 A . Cytochemical findings suggest that acid mucopolysaccharides and polypeptides containing N-acetyl-neuraminic acid are localized within the exterior and interior layers of the exosporium and at the surface of the outer coat, which could have a tumor inhibiting effect.

Biochim Biophys Acta, 1976 Nov 19, 450(2), 142 - 53
3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens; Macdonald IA et al.; 25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts . All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase . In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate . The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups . Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products . These enzyme systems appear to be constitutive rather than inducible . In contrast to C . perfringens . Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity . pH studies of the C . perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively . Kinetic studies gave Km estimates of approx . 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes . 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates) . There was no activity against 3beta-OH-containing steroids . The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.

Biochim Biophys Acta, 1976 Nov 8, 452(1), 42 - 58
Nitrogenase IX . Effect of the MgATP generator on the catalytic and EPR properties of the enzyme in vitro; Davis LC et al.; Nitrogenase(nitrogen:(acceptor) oxidoreduction, EC 1.7.99.2) of Clostridium pasteuranium is very sensitive to the ratio of MgADP/MgATP in dithionite oxidation assays . Variation of concentration of creatine kinase, an ATP-regenerating enzyme, can be used to control the ratio of ADP/ATP and thereby the dithionite oxidation activity of nitrogenase . The in vitro properties of nitrogenase support the suggestion of Haaker (Haaker, H., deKok, A . and Veeger, C . (1974) Biochim . Biophys . Acta 357, 344-357) that in vivo the nucleotide ratio and not the electron supply normally regulates nitrogenase activity . In EPR experiments it has been shown that the "steady state" varies as a function of the concentration of creatine kinase . The spectral differences are interpreted as being a function of the ratio of MgADP/MgATP obtained in the pseudo steady-state condition, which occurs as a result of variation in relative rates of ATP-utilizing and ATP-generating reactions, that is, the relative nitrogenase and creatine kinase activities . Implications of these finding for interpretation of previously reported kinetic and EPR studies are discussed.

Biochim Biophys Acta, 1976 Nov 8, 452(1), 262 - 70
Coenzyme B-12-dependent reactions . Part IV . Observations on the purification of ethanolamine ammonia-lyase; Joblin KN et al.; Purification of ethanolamine ammonia-lyase (EC 4.3.1.7) from a Clostridium sp . grown at the University of Sussex, U.K . and the National Institutes of Health, U.S.A., has been compared and an improved isotopic assay for the enzyme has been developed . Successful purification of this enzyme from Sussex-grown cells requires modification of the published procedure (Kaplan and Stadtman (1968) J . Biol, Chem . 243, 1787-1793) principally a 70% decrease in volume during precipitation with 0.4 M NaCl . This modification also increases the yield from N.I.H.-grown cells . Purified enzyme, resolved of inactive cobalamins, has the same high specific activity from both sources and behaves in the same way on disc gel electrophoresis . Sussex enzyme, before resolution, has less than 20% of the specific activity of unresolved N.I.H . enzyme and contains over 50% more inactive cobalamin . The bound cobalamin from both preparations has been identified as a "base-on" Co11 psi-cobalamin.

Gastroenterology, 1976 Nov, 71(5), 793 - 6
The importance of clostridia in experimental intestinal strangulation; Yale CE et al.; Hemorrhagic and ischemic strangulation of closed intestinal segments was sutdied in germ-free rats individually monocontaminated with one of eight separate species of clostridia . Ischemic strangulation was found to be as deadly as hemorrhagic strangulation in the presence of clostridia . Some clostridia were very toxic whereas others were relatively innocuous . The clostridia, ranked in the order of decreasing lethality, were: Clostridium perfringens type A, C . septicum, C . histolyticum, C . haemolyticum, C . bifermantans, C . sporogenes, C . tertium, and C . novyi type A . All species were readily established in the gastrointestinal tract of the nonoperated germ-free rat . The clostridia appear to be one of the most potentially lethal groups of organisms commonly found in the intestinal tract of human beings and animals.

J Clin Pathol, 1976 Nov, 29(11), 1011 - 3
Characteristics of a strain of Clostridium carnis causing septicaemia in a young infant; Wort AJ et al.; Clostridium carnis is a species which is only rarely isolated from man or animals and is occasionally found in the soil . This paper is an account of a single isolate found in blood cultures obtained from an 8-week-old boy who was suffering from gastroenteritis.

Injury, 1976 Nov, 8(2), 117 - 9
Chronic Clostridium septicum infection of a tibial fracture: a case report; Lucas HK et al.; An open transverse fracture of the mid-shaft of the tibia of a professional footballer became infected by Clostridium septicum and, after early compression plating, required surgical intervention on three further occasions and extensive antibiotic treatment before healing occurred . Clostridial infection is a recognized complication of open fractures contaminated with soil, and the necrotizing toxins produced by the C . septicum were probably responsible for the persistence of this infection . Infection occurred in less than 1 per cent of our series of 215 operations of compression plating of fresh fractures of the tibial shaft . Infection by clostridium species is a serious complication of open fractures . This patient did not show the spreading inflammation and necrosis, or the marked systemic upset, characteristic of acute clostridial infection, but persistent local infection necessitated prolonged surgical and antibiotic treatment.

Appl Environ Microbiol, 1976 Nov, 32(5), 685 - 8
Inactivation of Clostridium botulinum toxin by ruminal microbes from cattle and sheep; Allison MJ et al.; Toxin from Clostridium botulinum type C was rapidly inactivated during incubation in vitro with ruminal contents from either a cow or a sheep . Fractions of ruminal contents from which cells had been removed by high-speed centrifugation did not inactivate toxin . Inactivation was associated with fractions containing bacteria, whereas fractions containing protozoa and relatively few bacteria were much less active . This activity may help explain the relatively greater tolerance by ruminants to oral doses of botulinum toxin than to toxin administered by other routes . The results are also pertinent to assays for botulinum toxin from gastrointestinal samples obtained postmortem.

Ann Clin Lab Sci, 1976 Nov-Dec, 6(6), 537 - 9
A rapid glutamic decarboxylase test for identification of bacteria; Freier PA et al.; A simple rapid glutamic decarboxylase test is described . This test was found useful in the identification of Escherichia coli, Shigella sp., Providencia alcalifaciens, Clostridium perfringens and Bacteroides fragilis.

Ann Microbiol (Paris), 1976 Nov-Dec, 127B(4), 509 - 24
{Purification and some properties of "Clostridium perfringens" delta toxin (author's transl)}; Tixier G et al.; Delta toxin, a hemolytic exocellular protein excreted by C . perfringens type C has been purified to homogeneity, assessed by polyacrylamide disc gel electrophoresis . Purification steps involved successively calcium phosphate gel formation in culture supernatant fluid, salting-out of unadsorbed material by ammonium sulfate to 50 % saturation, isoelectric focusing and gel filtration on Sephadex G75 . Purified toxin appears as a basic protein occuring in two forms with isoelectric points of 8.8 and 9.4 as disclosed by isoelectric focusing . Molecular weight estimated by SDS-polyacrylamide disc gel electrophoresis was found to be close to 42,000 for the two forms . The lytic activity of delta toxin is inhibited by Ca++ and EDTA . The toxin is activated by short-term treatment with low concentration of trypsin.

J Med Microbiol, 1976 Nov, 9(4), 475 - 85
A serotyping system for Clostridium welchii (C . perfringens) type A, and studies on the type-specific antigens; Hughes JA et al.; A serotyping scheme for Clostridium welchii (C . perfringens) type A employing 57 antisera has been used to investigate the epidemiology of 153 food-poisoning outbreaks and 32 cases of gas gangrene and other clinical infections . Respectively 65% and 59% of the isolates were typable, and in 55% of the food-poisoning outbreaks the causative serotypes were established . Isolation and reporting methods that would render the typing scheme of even greater epidemiological value are described . The type-specific antigen was shown to reside in the capsule and to be lost from strains that had become rough . Development of roughness and its prevention are described . A great range of antisera and an internationally acceptable serotyping scheme is expected after integration of this set with those developed independently in America and Japan.

Appl Environ Microbiol, 1976 Nov, 32(5), 711 - 5
Growth and toxin production by Clostridium botulinum in moldy tomato juice; Huhtanen CN et al.; Tomato juice inoculated with Cladosporium sp . or Penicillium sp . developed pH gradients with the upper portions near the mold mats having pH values near neutrality and the lower portions remaining more acid . Clostridium botulinum spores in these moldy tomato juices germinated, grew out, and produced toxin.

Lancet, 1976 Oct 30, 2(7992), 934 - 6
Infant botulism . Identification of Clostridium botulinum and its toxins in faeces; Midura TF et al.; Clostridium botulinum and its toxin were identified in the faeces of four infants, aged 6 to 13 weeks, who had symptoms consistent with botulism . Two cases had type-A toxin and two cases had type-B toxin present in their faeces . No toxin was detectable in sera C . botulinum and toxin could be recovered from faeces more than 8 weeks after admission to hospital . All four cases occurred within a 6-month period . The source of the toxin in these infants may have been in-vivo production from ingested organisms.

J Membr Biol, 1976 Oct 20, 29(1-2), 179 - 84
On the question of an electrokinetic requirement for phospholipase C action; Dawson RM et al.; The phospholipase C of clostridium welchii (alpha toxin) has an absolute requirement for trace quantities of Ca2+ . It attacks pure phosphatidylcholine particles (smectic mesophases) having a close-packed bilayer structure only when their surface zeta potential is made positive by the addition of certain divalent ions (e.g., Ca2+) to the aqueous phase or by the presence of low concentrations of long chain cations to the lipid . Alternatively, if the rotational freedom of individual phospholipid molecules is increased by the insertion of short n-alkanols (e.g., hexanol) into the bilayer or when a monolayer of the substrate at an air/water interface is expended, enzymic hydrolysis can occur without any requirement for a net postive charge on the surface.

Biochemistry, 1976 Oct 19, 15(21), 4559 - 65
Histone redistribution and conformational effect on chromatin induced by formaldehyde; Polacow I et al.; Histone redistributions between endogenous DNA in calf thymus chromatin and exogenous DNA from Clostridium perfringens (69% A + T) or from Micrococcus luteus (30% A + T) induced by 0.6 M NaCl or by 2% formaldehyde were studied by thermal denaturation . The observed redistribution occurred on histone Hl when the exogenous DNA was (A + T)-richer than the DNA in chromatin, and when the mixture was exposed to 0.6 M NaCl or formaldehyde . When a (G + C)-richer DNA was added as the acceptor for histones, no substantial transfer of histones from chromatin DNA to exogenous DNA was found . Thus the activation energy of histone dissociation from chromatin DNA seems to be substantially lowered by 0.6 M NaCl or formaldehyde such that histones (mostly histone Hl) can be dissociated and bind the (A + T)-richer DNA and form a more stable complex . It is suggested that the formaldehyde effect on histones may be due to the loss of positive charges on lysine and arginin residues (probably more on lysine than on arginine) in histones after their rapid reaction with formaldehyde . Formaldehyde treatment of chromatin also distorts the DNA conformation, as revealed by circular dichroism (CD) studies . This structural effect occurs mainly on those base pairs bound by histones other than Hl, or within the chromatin subunit . Histone redistribution is treated as a thermodynamic phenomenon of histone binding to DNA . The validity of using formaldehyde to study chromatin structure is discussed.

Biochemistry, 1976 Oct 19, 15(21), 4736 - 41
Collagenase enzymes from Clostridium: characterization of individual enzymes; Lwebuga-Mukasa JS et al.; Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized . The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen . The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00 . Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose . The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity . At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000 . The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain . All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive . Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell.

Lancet, 1976 Oct 2, 2(7988), 715 - 6
Necrotising enterocolitis of the newborn--is it gas-gangrene of the bowel?
Pedersen PV, Hansen FH, Halveg AB, Christiansen ED.
Necrotising enterocolitis (N.E.C.) of the newborn is thought to be caused by ischaemia of the bowel . This would favour the conversion of clostridial spores, which can occur very early in the intestinal tract of newborns, to toxin-producing, invading bacilli . The histology of resected gut specimens from 6 of 7 N.E.C . patients who had undergone operation was similar to that in cases of gas-gangrene of the bowel and that in experimentally provoked pneumatosis cystoides intestinalis . In one case Clostridium perfringens type A was cultured in great number by anaerobic technique . The clostridia in these cases may have played an important role in the development of N.E.C.

Angiology, 1976 Oct, 27(10), 602 - 9
Gas-forming clostridial mycotic aneurysm of the abdominal aorta . A case report; Babenco GO et al.; Gas-forming mycotic aneurysms are extremely rare . A case is reported in which rupture of a gas-forming mycotic aneurysm of the distal abdominal aorta due to Clostridium paraputrificum occurred in an elderly male with a myeloproliferative disorder and a necrotic carcinoma of the colon.

Can J Microbiol, 1976 Oct, 22(10), 1579 - 83
Stability of mRNA from the Clostridium sporogenes phage F1; Taylor DE et al.; Polyacrylamide gel electrophoresis was used to study the decay of individual species of mRNA in F1, a bacteriophage specific for the obligate anaerobie Clostridium sporogenes . Immediate early mRNA species had a half-life of 3.5 min, while delayed early and late mRNA had a half-life of between 6 and 8 min.

Am J Clin Pathol, 1976 Oct, 66(4), 737 - 42
Laboratory diagnosis of botulism complicated by pyridostigmine treatment of the patient . A method for selectively removing interfering substances from clinical specimens; Horwitz MA et al.; In August 1974, a case of botulism occurred; home-canned potatoes and peas containing Clostridium botulinum, type A, were strongly incriminated as the vehicle of transmission . C . botulinum, type A, was isolated from a stool specimen of the patient, but the mouse neutralization test for botulinal toxin could not be completed because the stool extract contained a highly toxic, heat-stable substance that rapidly killed mice . Historical and laboratory evidence indicated that the substance was pyridostigmine bromide, a low-molecular-weight drug with which the patient had been treated after her disease was misdiagnosed as myasthenia gravis . A generally applicable method employing dialysis by which toxic SUBSTANCED of low molecular weigth could be selectively removed from specimens without diminishing the potency of botulinal toxin contained in them was developed . Dialysis rendered a pyridostigmine solution, a stool extract from the patient with botulism, and a stool extract from a person taking pyridostigmine virtually nontoxic to mice . Dialysis did not significantly alter the toxicity to mice of crude botulinal toxin; it selectively eliminated all or almost all pyridostigmine toxicity from a pyridostigmine-botulinal toxin mixture without altering the toxicity of the botulinal toxin.

Ann Microbiol (Paris), 1976 Oct, 127B(3), 283 - 94
{Identification of two plasmids isolated from a bacteriocinogenic strain of Clostridium perfringens}; Ionesco H et al.; Two plasmids were found and studied in the bacteriocinogenic strain N5 of Clostridium perfringens: one is a bacteriocinogenic factor (MW=5.7 X 10(6)) and the other a cryptic plasmid (MW 32.4 X 10(6)) . Simultaneous loss of the ability to produce bacteriocin and of the bacteriocin resistance in a "cured" variant corresponds to the loss of the bacteriocinogenic plasmid DNA . The syntheses of haemolysin 0 and phospholipase C do not seem to be coded for by the cryptic plasmid.

Jpn J Med Sci Biol, 1976 Oct, 29(5), 255 - 63
In vitro aggregation of platelets induced by alpha-toxin (phospholipase C) of Clostridium perfringens; Sugahara T et al.; Highly purified alpha-toxin (phospholipase C) of Clostridium perfringens prepared by affinity chromatography on agarose-linked egg-yolk lipoprotein induced the in vitro aggregation of platelets of an irreversible type . The aggregation started after a time lag, the length of which depended on the concentration of the toxin; the reciprocal of the time lag was found to be directly proportional to the toxin concentration . Using this assay method, we demonstrated that the platelet-aggregating activity of alpha-toxin reached minimum at around 70 C but heating at higher temperatures inactivated it to a lesser extent; the same anomaly in heat inactivation was observed with phospholipase C activity possessed by the toxin . By subjecting purified alpha-toxin to isoelectric focusing, four molecular forms were isolated, all of which were associated with both the platelet-aggregating and phospholipase C activities . From all these results we concluded that the entity responsible for the platelet-aggregating activity is identical with alpha-toxin (phospholipase C).

Nucleic Acids Res, 1976 Oct, 3(10), 2443 - 50
Conformations of A,T-rich DNAs; Selsing E et al.; DNAs from the genomes of Clostridium perfringens and Cytophaga johnsonii display orthodox A-DNA and B-DNA structures despite their high (A+L) nucleotide content . Unique structures, such as those found for synthetic DNAs having specific special sequences, do therefore not necessarily occur for DNAs having more random base sequence even if these have unusual base compositions . Clostridium perfringens DNA exhibits unusual structural properties only prior to purification by gel filtration.

Avian Dis, 1976 Oct-Dec, 20(4), 774 - 6
Clostridium perfringens infection in turkey poults; Eleazer TH et al.; Necropsy of dead and sick 5-day-old poults from a flock of 12,500 Broad-Breasted White turkeys having high early mortality (685 the first week) revealed rather large inflamed retained yolk sacs with semiliquid contents, ascites, and swollen greenish-colored livers . Cultures of livers and yolk sacs were negative by standard aerobic procedures for salmonella and any other bacterial pathogens . At the beginning of the 4th week, more poults were necropsied and found to have swollen livers with necrotic foci grossly visible, and a necrotic inflammation of the midintestinal tract extending anteriorly from attachment of the yolk stalk . Clostridium perfringens was isolated from livers and yolk remnants by anaerobic culture.

Clin Nephrol, 1976 Oct, 6(4), 451 - 2
Acute intravascular hemolysis with minimal renal impairment in Clostridium perfringens infection; Slotki IN et al.; We report here a case of post-abortal clostridium prefringens infection in which there was severe intravascular hemolysis with black urine, but only minor abnormalities of the clotting mechanism and mild renal failure . The patient recovered following supportive therapy only.

Can J Microbiol, 1976 Oct, 22(10), 1497 - 501
Lecithinase-negative variants of Clostridium perfringens; the identity of C . plagarum with C . perfringens; Nakamura S et al.; Identity of Clostridium plagarum (Prevot) com . nov . (1938) with C . perfringens was demonstrated in the tests for the cultural and biochemical properties, DNA-DNA homology, susceptibility to C . perfringens-specific lysin, complementary synthesis of C . perfringens theta-toxin between C . plagarum and theta-toxin-negative mutants of C . perfringens, as well as the tests for gas-chromatographic patterns.

J Gen Microbiol, 1976 Oct, 96(2), 401 - 7
The effect of hydrogen peroxide on spores of Clostridium bifermentans; Bayliss CE et al.; The effect of hydrogen peroxide on the germination, colony formation and structure of spores of Clostridium bifermentans was examined . Treatment with 0.35 M-hydrogen peroxide increased the germination rate at 25 degrees C but increasing the temperature or concentration of hydrogen peroxide decreased both the germination rate and colony formation . The presence of Cu2+ increased the lethal effect of hydrogen peroxide on colony formation as much as 3000-fold . Pre-incubation of spores with Cu2+ before treatment with hydrogen peroxide produced a similar increase, but this could be eliminated by washing the spores with dilute spores--apparently from the coat--and treatment with dithiothreitol, which also removes spore-coat protein, increased the lethal effect of hydrogen peroxide 500-fold, suggesting that spore-coat protein has a protective effect against hydrogen peroxide.

J Pharm Sci, 1976 Oct, 65(10), 1471 - 6
Electrochemistry of drug action I: electrooreduction of ferredoxins; Chien YW; Ferredoxin serves as an electron carrier in the oxidation-reduction system in anaerobic microorganisms, transferring electrons from a low potential donor to electron-accepting biochemicals . The anaerobicidal activity of some drugs may be due to their interference with the electron transport function of ferredoxin . Two types of ferredoxins (isolated from Clostridium pasteurianum and spinach) were studied, and their electrochemical reduction and biochemical properties were analyzed using a sensitive ac polarographic technique . The reduction potential of both ferredoxins was linearly related to pH . The mechanisms of electron transport in ferredoxin molecules were found to be related to their sulfur-iron bonds . The dissociation of the sulfur-iron bonds resulted in the formation of a free sulfhydryl group and the interruption of the electroactivity of ferredoxin . This sulfur-iron dissociation process was found to be pH dependent . The electroreduction of ferredoxins was an energy-requiring, pH-dependent process.

Biochim Biophys Acta, 1976 Sep 28, 446(1), 301 - 9
Renaturation of formyltetrahydrofolate synthetase from urea and guanidinium chloride solutions; Garrison CK et al.; Formyltetrahydrofolate synthetase from Clostridium cylindrosporum and Clostridium acidi-urici was denatured in 6 M urea and 4 M guanidinium chloride . Viscometric, fluorimetric and ultracentrifugal measurements were used to determine that the protein is completely unfolded under these conditions . The polypeptide chains refold upon dilution of the denaturant-protein solutions to give final concentrations of 0.5 M urea or 0.1 M guanidinium chloride . In the presence of NH4+, but not in its absence, the refolded proteins associate to produce the catalytically active tetramer . Refolding and reassociation were followed by measuring changes in protein fluorescence and by determination of sedimentation constants . Under most conditions 80% of the enzymic activity is recovered.

Biochim Biophys Acta, 1976 Sep 14, 445(2), 518 - 20
The separation of pyruvate-ferredoxin oxidoreductase from Clostridium pasteurianum into two enzymes catalyzing different reactions; Sauer FD et al.; The ferredoxin requiring cleavage of pyruvate to acetyl-CoA and CO2 is catalyzed by pyruvate ferredoxin oxidoreductase (pyruvate:ferredoxin oxidoreductase (CoA-acetylating):, EC 1.2.7.1) . The same enzyme is thought to catalyze the reversal of this reaction, i.e . the synthesis of pyruvate from acetyl-CoA and CO2 in the presence of reduced ferredoxin . Evidence is presented that the forward and reverse reactions are catalyzed not by one, but by two proteins that are clearly separable by Sephadex G-200 gel filtration.

Arch Microbiol, 1976 Sep 1, 109(3), 315 - 7
The use of stable sulfur isotope labelling to elucidate sulfur metabolism by Clostridium pasteurianum; McCready RG et al.; An unique isotope labelling experiment was conducted whereby mixtures of sulfate and sulfite of different isotopic compositions were metabolized by Clostridium pasteurianum . The results showed during reduction of 1 mM SO3= plus 1 mM SO4=, essentially all evolved H2S arose from the sulfite whereas in the case of cellular sulfur, 85% was derived from sulfite and the remainder from sulfate.

Infect Immun, 1976 Sep, 14(3), 793 - 803
Relationship of bacteriophages to alpha toxin production in Clostridium novyi types A and B; Eklund MW et al.; The relationship of specific bacteriophages to the production of the lethal alpha toxin in Clostridium novyi types A and B was investigated . When type A strain 5771 reverted to the phage-sensitive state, it ceased to produce alpha toxin but continued to produce the gamma and epsilon antigens . This "nontoxigenic" culture, therefore, more closely resembled C . botulinum types C and D than the other C . novyi types . Phage-sensitive type B strains also ceased to produce the alpha toxin but continued to produce the beta toxin, and therefore very colesly resembled C . novyi type D (C . haemolyticum) . Alpha toxin was again produced when the phage-sensitive cultures were reinfected with the respective tox+ phages . Alpha toxin production could also be induced in the "nontoxigenic" phage-sensitive derivatives from type B strain 8024 by tox+ phages isolated from other strains of type B . tox- phages were also isolated, but they did not affect alpha toxin production . The tox+ phages also caused a marked change in the colonial morphology of type B strains . In this report we present evidence that alpha toxin production by C . novyi type A strain 5771 and type B strain 8024 depends upon the continued presence and participation of specific bacteriophages designated as NA1tox+ and NB1tox+, respectively.

Infect Immun, 1976 Sep, 14(3), 680 - 6
Molecular forms of neurotoxins in proteolytic Clostridium botulinum type B cultures; Dasgupta BR et al.; A modified purification method was used to isolate the neurotoxin of proteolytic Clostridium botulinum type B strain Lamanna . The preparation was found to be a mixture of two protein forms . They were of molecular weight 152,000 and could not be separated by ion-exchange chromatography or electrophoresis in polyacrylamide gel . One was a single polypeptide chain, and the other was a dichain molecule (nicked toxin) held together by an interchain disulfide bond(s) . Trypsinization increased the toxicity of the toxin preparation and converted the single-chain molecules into dichain forms that were indistinguishable from the endogenously generated nicked toxin . A protease of the type B culture, with substrate specificity similar to that of trypsin, did not change detectably the molecular form of unnicked type E toxin, although toxicity was increased . Higher toxicity was obtained when unnicked type E was trypsinized; the resulting preparation contained only nicked toxin molecules.

Infect Immun, 1976 Sep, 14(3), 597 - 602
Phage conversion to hemagglutinin production in Clostridium botulinum types C and D; Oguma K et al.; Five toxigenic strains of Clostridium botulinum types C and D were incubated at 37 degrees C for 7 days in 15 ml of the following media: LYG medium, cooked-meat medium, egg meat medium, and N-Z-amine medium . The supernatants of these cultures were tested for hemagglutinin production with 1% erythrocytes obtained from mice, guinea pigs, chickens, sheep, monkeys, and humans . Four toxigenic strains produced hemagglutinin . The highest hemagglutinin titer was obtained with a combination of human erythrocytes and cultures incubated in LYG medium . When the same experiment was carried out with many nontoxigenic strains, hemagglutination was observed in only one strain, C-N71 . Strains producing hemagglutinin also produced phages . The phages obtained from toxin- and hemagglutinin-producing strains converted nontoxigenic indicator strains to produce both toxin and hemagglutinin . The phage obtained from a toxin-positive hemagglutinin-negative strain could only induce cultures to produce toxin, and the phage from a toxin-negative hemagglutinin-positive strain could only induce production of hemagglutinin . These studies suggest that the production of hemagglutinin by C . botulinum types C and D is governed by bacteriophages and that hemagglutinin production can be transmitted separately or concomitantly with toxin production.

Appl Environ Microbiol, 1976 Sep, 32(3), 409 - 16
Beneficial effect of catalase treatment on growth of Clostridium perfringens; Harmon SM et al.; Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase . Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected . Plate counts of C . perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively . Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability . Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls . Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase . These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C . perfringens on these media.

Can J Microbiol, 1976 Sep, 22(9), 1410 - 4
Anomalous aggregation of Clostridium perfringens enterotoxin under dissociating conditions; Enders GL Jr et al.; Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides . Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide . No conditions capable of inhibiting this phenomenon were found . Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons . Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein . The evidence presented indicates possible detergent induced structural alterations of the protein.

Ann Clin Lab Sci, 1976 Sep-Oct, 6(5), 381 - 99
Laboratory diagnosis of foodborne diseases; Kazal HL; Many bacterial species are responsible for sporadic cases and outbreaks of foodborne intoxication and infection . The foodborne diseases are classified on the basis of the pathogenetic mechanisms involved into four categories: performed toxin, enterotoxin formed in the colonized small intestine, mucosal invasion (enterocolitis) and mucosal invasion with bacteremia . Invasive and toxigenic strains of enteropathogenic Escherichia coli are discussed . In vivo test systems for the identification of enterotoxigenic organisms and tissue culture assays for the heat-labile enterotoxin of E . coli are described . Current laboratory methods for the diagnosis of foodborne diseases of major public health interest are reviewed - botulism, staphylococcal intoxication, Clostridium perfringens enteritis, salmonellosis, enteropathogenic E . coli infection, Vibrio parahaemolyticus infection and Bacillus cereus enteritis . The role of the laboratory in the epidemiologic surveillance and investigation of foodborne diseases is emphasized.

Agents Actions, 1976 Sep, 6(5), 627 - 35
Haemorrhagic and inflammatory properties of collagenase from C . histolyticum; Vargaftig BB et al.; Collagenase from Clostridium histolyticum induced haemorrhages when applied to the surface of dog lung; it exerted a similar effect on mouse lung when injected intrathoracically . Injected into rat paws, bacterial collagenase induced haemorrhage and oedema . Effects of collagenase were prevented by several procedures that inhibit collagenolytic activity (heating at various temperatures and incubation with metal-complexing agents such as EDTA, penicillamine and dithiothreitol) . Protein protease inhibitors, dexamethasone and standard acidic anti-inflammatory drugs had only a slight or no effect on collagenase-induced haemorrhages; dexamethasone and acidic anti-inflammatory drugs blocked collagenase-induced oedema . Inhibition of endogenous kinin-releasing mechanisms by administration of hexadimethrine, a recognized inhibitor of the activation of clotting Factor XII, and depletion of kininogen by administration of carrageenin blocked collagenase-induced oedema . Collagenase did not increase permeability of rat skin vessels, nor did it release potential inflammatory mediators, such as bradykinin or prostaglandins, from plasma or platelets . Bacterial collagenase-induced haemorrhage presumably resulted from enzymatic destruction of membranous structures; at least a portion of the inflammatory response may be due to activation of a kinin-like system.

S Afr Med J, 1976 Aug 28, 50(3F), 1435 - 8
Anaerobic infections in hospital practice; Appelbaum PC et al.; The role of anaerobes in the pathogenesis of infections was investigated . Anaerobes were isolated from 0,25% of blood cultures and from 15,8% of other specimens tested; in 15,1% of cases where anaerobes were isolated, no aerobes were found . The strains most commonly encountered were Bacteroides fragilis, B . melaninogenicus, Peptostreptococcus anaerobius and Clostridium perfringens . Sensitivity tests in vitro showed all organisms to be sensitive to clindamycin, metronidazole and chloramphenicol, while a large proportion were resistant to penicillin G and smaller numbers were resistant to tetracycline.

Vet Rec, 1976 Aug 7, 99(6), 98 - 9
Suspected botulism in foxhounds; Darke PG et al.; An outbreak of acute paralysis in a pack of foxhounds, which followed the ingestion of raw and partially cooked meat, was almost certainly due to botulism . Botulinal toxin was detected in the serum of one of three hounds which recovered, and Clostridium botulinum type C was present in a sample of meat remaining from a batch fed to the hounds.

Proc Natl Acad Sci U S A, 1976 Aug, 73(8), 2659 - 63
Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety; Cone JE et al.; A small, heat-stable selenoprotein, one of the components of the glycine reductase complex, was labeled with 75Se by growth of Clostridium sticklandii in the presence of Na2 75SeO3 . The selenium-containing moiety, which is essential for the biological activity of the protein, was shown to be a selenocysteine residue . It was isolated as its Se-carboxymethyl, Se-carboxyethyl, and Se-aminoethyl derivatives from digests of the pure 75Se-labeled protein that had been reduced and treated with the various alkylating agents prior to hydrolysis . In each instance the 75Se-labeled moiety obtained from an alkylated protein sample and the corresponding alkyl derivative of authentic selenocysteine were indistinguishable . Several studies of the native selenoprotein detected a chromophore (UVmax 238nm) that appeared upon reduction of the protein with KBH4 and rapidly disappeared upon exposure to oxygen . This oxygen-labile chromophore is thought to be the ionized -SeH group of the selenocysteine residue.

J Bacteriol, 1976 Aug, 127(2), 770 - 9
Regulation of molybdate transport by Clostridium pasteurianum; Elliott BB et al.; The regulation of the molybdate (MoO42-) transport activity of Clostridium pasteurianum has been studied by observing the effects of NH3, carbamyl phosphate, MoO42-, and chloramphenicol on the ability of cells to take up MoO42- . Compared with cells fixing N2, cells grown in the presence of 1 mM NH3 are greater than 95% repressed for MoO42- transport . Uptake activity begins to increase just before NH exhaustion (under Ar or N2) and continues to increase throughout the lag period as cells shift from NH3-growing to N2-fixing conditions . When cells are shifted from N2-fixing to NH3-growing conditions the transport activity per fixed number of cells decreases by increase of bells in absence of transport synthesis . Carbamyl phosphate (greater than or equal to 15 mM) but not NH3 inhibits 58% of the in vitro uptake activity . When 1 mM carbamyl phosphate is added just before the exhaustion of NH3, the transport activity, measured 2 h later, is 100% repressed . Cells grown in the presence of high MoO42- (1mM) are 80% repressed for MoO42- transport . Synthesis of the MoO42- transport system is also completely stopped when chloramphenicol (300 mug/ml) is added just before the exhaustion oNH 3 from the medium . These findings demonstrate that the ability of cells to transport MoO42- is dependent upon new protein synthesis and can be repressed by high levels of substrate . The regulation of MoO42- uptake by NH3 or carbamyl phosphate closely parallels the regulation of nitrogenase activity . Activity of neither nitrogenase component (Fe protein or MoFe protein) was detected even 3 h after the exhaustion of the NH3 if either MoO42- was absent or if WO42- was present in place of MoO42- . The duration of the diauxic lag increases with decreasing concentration of MoO42- in the medium . If no MoO42- is present the lag continues indefinitely . If MoO42- is added late in the lag period, growth under N2-fixing conditions resumes but only after a normal induction period.

J Gen Microbiol, 1976 Aug, 96(2), 297 - 312
The apparent ATP requirement for nitrogen fixation in growing Klebsiella pneumoniae; Hill S; The apparent ATP requirement for N2 fixation in Klebsiella pneumoniae was high (the ATP/N2 molar ratio was 29 when estimated in anaerobic glucose-limited chemostat cultures) compared with that determined previously in Azotobacter chroococcum and in Clostridium pasteurianum . The high value was probably not due to unfavourable temperature, phosphate concentration or pH . The apparent ATP requirement for N2 fixation was probably no lower in 02-limited chemostat cultures than in anaerobic glucose-limited chemostat cultures . When hydrogen was introduced into the atmosphere over the anaerobic glucose-limited chemostate culture, there was an increase in the apparent ATP requirement for N2 fixation and in the activity of nitrogenase in intact organisms . A comparison of these increases suggests that some ATP is wasted by the ATP-dependent H2-evolving activity of nitrogenase.

J Biochem (Tokyo), 1976 Aug, 80(2), 195 - 201
Characterization of one neutral and two acidic glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU); Sugahara K et al.; One neutral and two acidic glycoasparagines were isolated from the urine of patients with aspartylglycosylaminuria (AGU) . The neutral one was identified as beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn . The acidic ones were composed of 1 mole of sialic acid and 2 moles each of galactose and N-acetylglucosamine, attached to asparagine, and were isomeric with respect to the position of sialic acid attachment since they produced the same glycoasparagine on incubation with the neuraminidase {EC 3.2.1.18} from Clostridium perfringens . The structure of the resulting sialic acid-free glycoasparagine was determined to be beta-Gal-beta-GlcNAc-beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn based mainly on the results of sequential enzymatic degradations.

Am J Dis Child, 1976 Aug, 130(8), 877 - 9
Meningitis due to combined infections . Association of Haemophilus influenzae type B and Clostridium perfringens; Gehrz RC et al.; Haemophilus influenzae type B and Clostridium perfringens were recovered simultaneously from the cerebrospinal fluid of a patient with purulent meningitis . No antecedent history of head trauma was present to explain the coexistence of the anaerobe with Haemophilus organisms . A review of the literature on mixed meningitis indicates that no previous cases of anaerobes have been reported in uncomplicated meningitis due to multiple organisms . In addition, the recovery of clostridia is extremely unusual in the absence of an identifiable portal of entry . We have identified two additional cases of clostridia infection in the central nervous system and recommend that anaerobic organisms be considered in selected cases of meningitis.

Biochem J, 1976 Aug 1, 157(2), 439 - 47
Nitrogenases from Klebsiella pneumoniae and Clostridium pasteurianum . Kinetic investigations of cross-reactions as a probe of the enzyme mechanism; Smith BE et al.; In combination with the Mo-Fe protein of nitrogenase from Klebsiella pneumoniae, the Fe protein of nitrogenase from Clostridium pasteurianum forms an active enzyme with novel properties different from those of either of the homologous nitrogenases . The steady-state rates of reduction of acetylene and H+ are 12% of those of the homologous system from C.pasteurianim . Acetylene reductase activity exhibited an approx . 10min lag at 30 degrees C before the rate of reduction became linear, consistent with a once-only activation step being necessary for acetylene reduction to occur . No such lag was observed for H2 evolution . The activity with N2 as a reducible substrate was very low, implying that acetylene reductase activity is not necessarily an accurate indication of nitrogen-fixing ability . This is of particular relevance to studies on mutant and agronomically important organisms . Stopped-flow spectrophotometric studies showed unimolecular electron transfer from the Fe protein to the Mo-Fe protein to occur at the same rate (k2 = 2.5 X 10(2)s-1) and with the same dependence on ATP concentration (apparent KD = 400 muM) as with the homologous Klebsiella nitrogenase . However, an ATP/2e ratio of 50 was obtained for H2 evolution, indicating that ATP hydrolysis had been uncoupled from electron transfer to substrate . These data indicate that ATP has at least two roles in the mechanism of nitrogenase action . The combination of the Mo-Fe protein of nitrogenase of C.pasteurianim and the Fe protein of K.pneumoniae were inactive in all the above reactions, except for a weak adenosine triphosphatase activity, 0.5% of that of the homologous K.pneumoniae system.

Jpn J Microbiol, 1976 Aug, 20(4), 287 - 92
Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia; Takumi K et al.; The cell wall peptodoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined . The petidoglycan of C . botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81 . All other types of C . botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type . The peptidoglycans of Clostridium bifermentans and Clostridium histoloyticum contained DAP but they differed from those of C . botulinum in the molar ratio of alanine to glutamic acid . The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90 . On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine . In spite of the difference in amino acid composition of peptidoglycans among the clostridia, the quantitative precipitin test demonstrated that antiserum against C . botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other clostridia as well as various types of C . botulinum.

J Bacteriol, 1976 Aug, 127(2), 863 - 73
Purification of properties of dihydroorotase, a zinc-containing metalloenzyme in Clostridium oroticum; Taylor WH et al.; Dihydroorotase +4,5-L-dihydro-orotate amidohydrolase {EC 3.5.2.3}), which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate, has been purified from orotate-grown Clostridium oroticum . The enzyme is homogeneous when subjected to polyacrylamide gel electrophoresis and is stable at pH 7.6 in 0.3 M NaCl containing 10 muM ZnSO4 . The enzyme has a molecular weight of approximately 110,000 . Sodium dodecyl sulfate gel electrophoresis, using three different buffer systems, indicated the enzyme is composed of two subunits, each having a molecular weight of 55,000 . Dihydroorotase is shown by atomic absorption spectroscopy to be a zinc-containing metalloenzyme with 4 g-atoms of zinc per 110,000 g of protein . The pH optima for the conversion of N-carbamyl-L-aspartate to L-dihydroorotate and for L-dihydroorotate to N-carbamyl-L-aspartate are pH 6.0 and 8.2, respectively . The Km values for N-carbamyl-L-aspartate and for L-dihydroorotate are 0.13 and 0.07 mM, respectively . Inhibitor studies indicate that zinc may be involved in the catalytic activity of the enzyme.

J Biol Chem, 1976 Jul 10, 251(13), 4159 - 61
Formyltetrahydrofolate synthetase-catalyzed formation of ATP from carbamyl phosphate and ADP . Evidence for a formyl phosphate intermediate in the enzyme's catalytic mechanism; Buttlaire DH et al.; Formyltetrahydrofolate synthetase from Clostridium cylindrosporum catalyzes phosphoryl transfer from carbamyl phosphate in ADP to form ATP . The phosphoryl transfer reaction has an obligatory requirement for tetrahydrofolate presumably as a cofactor for a proper conformation of the active site . Carbamyl phosphate is an analog of formyl phosphate-a potential intermediate in the normal enzymic reaction . The ability of the enzyme to promote synthesis of ATP from carbamyl phosphate and ADP supports a stepwise chemical reaction mechanism for the enzyme in which formyl phosphate participates as a tightly bound intermediate.

J Gen Microbiol, 1976 Jul, 95(1), 67 - 77
Butyricin 7423: a bacteriocin produced by Clostridium butyricum NCIB7423; Clarke DJ et al.; Butyricin 7423 is a trypsin-sensitive bacteriocin produced by Clostridium butyricum NCIB7423 and active against some other species of Clostridium . It has a bactericidal but non-lytic action on growing cultures of Clostridium pasteurianum . The primary action of butyricin 7423 appears to be at the cell membrane . Thus treatment of C . pasteurianum with an excess of butyricin altered the permeability of its cell membrane, allowing the release of several metabolites and ions . Efflux of rubidium ions from organisms preloaded with 86Rb+ was particularly rapid and extensive . The action of butyricin 7423 on Clostridium species seems to differ from that of other clostridocins, such as the boticins and perfringocin 28, and more closely resembles the mode of action on susceptible (aerobic) organisms of bacteriocins such as staphylococcin 1580 or colicins A, E1, I and K.

Appl Environ Microbiol, 1976 Jul, 32(1), 172 - 8
Influence of postirradiation incubation temperature on recovery of radiation-injured Clostridium botulinum 62A spores; Chowdhury MS et al.; The number of colonies formed by unirradiated Clostridium botulinum 62A spores was independent of temperature, in the range from 20 to 45 degrees C (in 5 degrees C increments); no colonies developed at 50 degrees C . Spores irradiated at 1.2 or 1.4 Mrads produced more macrocolonies at 40 degrees C than at higher or lower temperatures . Apparently, radiation-injured spores were capable of repair of 40 degrees C than at the other temperatures studied . More than 99% of the radiation (1.2 Mrads) survivors were injured and were unable to form macrocolonies in the presence of 5% NaCl . The germinated radiation-injured spores were also sensitive to dilution, resulting in the loss of viability of 77 to 79% of the radiation survivors . At 30 and 40 degrees C, the irradiated spores did not differ significantly in the extent of germination (greater than 99% at both 30 and 40 degrees C), emergence (64% at 30 degrees C and 67% at 40 degrees C), and the maximum number of emerged cells that started to elongate (69% at 30 degrees C and 79% at 40 degrees C) . However, elongation was remarkably more extensive at 40 degrees C than at 30 degrees C . Many elongated cells lysed within 48 h at 30 degrees C, indicating an impaired repair mechanism . If the radiation-injured spores were incubated at 40 degrees C in the recovery (repair) medium for 8 to 10 h, they germinated, emerged, and elongated extensively and were capable of repair . If, after 8 to 10 h at 40 degrees C, these cultures were shifted to 30 degrees C, the recovery at 30 increased by more than eightfold, resulting in similar colony counts at 30 and 40 degrees C . Thus, repair appeared to be associated with outgrowth . Repair did not occur in the presence of chloramphenicol at 40 degrees C, whereas penicillin had no effect, suggesting that the repair involved protein synthesis but did not require multiplication.

Avian Dis, 1976 Jul-Sep, 20(3), 525 - 33
Fluorescent antibody test in diagnosis of ulcerative enteritis; Berkhoff GA et al.; A direct fluorescent antibody (FA) test to demonstrate the presence of Clostridium colinum in cryostat sections of liver and intestine from chickens with ulcerative enteritis ("quail disease") was investigated . Both field cases and experimentally infected birds were studied by FA, bacterial isolation of the clostridium, and histopathology . The FA test proved highly specific for Cl . colinum . It was concluded that the described direct FA procedure offers simplicity and speed over bacterial isolation procedures for etiologic diagnosis of ulcerative enteritis.

Am J Med Technol, 1976 Jul, 42(7), 233 - 7
A modified plate holding system for anaerobic cultures; Flournoy DJ et al.; A modified plate holding system for anaerobes has been developed which: 1) effectively reduces the volume of oxygen-free gas required, 2) promotes earlier bacterial growth, and 3) monitors the anaerobic atmosphere of the holding vessel and the rate of gas flow . Cultures of Bacteroides fragilis, Clostridium perfringens, C . septicum, Fusobacterium nucleatum, Peptococcus sp . and Propionibacterium sp . were evaluated for optimal growth . All cultures grew as well as or better when preincubated in the holding system for one to three hours then incubated in a GasPak system for the remainder of the 24 hours, as compared to incubation only in the GasPak or holding system for the entire 24-hour period.

Antibiotiki, 1976 Jul, 21(7), 613 - 7
{Effect of the physiological activity of Clostridium cells on their sensitivity to antibiotics}; Poliak MS et al.; Actively multiplicating cells of C1 . perfringens proved to be more sensitive to 7-chlor-7-desoxylincomycin and rifampicin than the cells in the phase of the population dying . The bactericidal effect of the antibiotics on Clostridia vegetating at a temperature range within 37--4degrees was studied . Determination of the content of higher fatty acids in the cultivation medium with the method of gas chromatography showed that the metabolic processes in the bacterial cells went on at a temperature of 4degrees . Sensitivity of Clostridia to antibiotics at 20 and 4degrees lowered . However, all antibiotics inhibited the cell viability under such conditions . The inhibitors of the intracellular protein synthesis, i . e . rifampicin, 7-chlor-7-desoxylincomycin and morphocycline proved to be most active . The effect of beta-lactame antibiotics, i . e . cephaloridine and benzylpenicillin was reliable though lower.

Arch Intern Med, 1976 Jul, 136(7), 788 - 91
Gas gangrene: review of 34 cases; Caplan ES et al.; High morbidity and mortality continue to result from gas gangrene, despite the use of aggressive modes of therapy . Between 1967 and 1973, 34 patients with gas gangrene were seen at the University of Maryland Hospital; 11 (32.3%) died . Clostridium perfringens was recovered from the wounds in 79% of the cases and from the blood in 15% . Eighty-five percent of the wounds contained one or more organisms in addition to C perfringens, with as many as seven organisms recovered from some wounds . Twenty-nine patients received hyperbaric oxygen treatments, as well as the more conventional antibiotic drugs; it was not possible to assess the value of this added therapy . Gangrene of the abdominal wall resulted in a higher (50%) mortality than gangrene of an extremity (24%) . Presence of normal or depressed white blood cell counts, decreased platelet counts, and abnormal renal or liver functions all denoted a poor prognosis.

Appl Environ Microbiol, 1976 Jul, 32(1), 145 - 58
Observations on bacteriophages of Clostridium botulinum type C isolates from different sources and the role of certain phages in toxigenicity; Hariharan H et al.; Twenty strains of Clostridium botulinum type C, including 12 isolates from avian sources with varying toxigenic properties, were examined by electron microscope for the presence of bacteriophages . All toxigenic strains were infected with one or two types of phages . Three types of phages designated large, small, and intermediate were observed . Most of the strains carried the large and small phage, with the large phage being present in much greater numbers . Since there is evidence that highly toxigenic strains of C . botulinum type C are responsible for large outbreaks of botulism in wild birds, the phenomenon of toxigenic variation among the type C strains was investigated . Experiments were carried out employing a broth medium on a phagefree nontoxigenic strain for elucidating the role of bacteriophages in toxigenicity . All phage suspensions contained large phages, with the exception of one that caused conversion . The exception was a preparation containing an intermediate type of phage . Phages from different strains produced cultures of varying toxigenic characteristics . By employing a tube-lytic test and an agar-overlay-phage assay technique, it was determined that whenever the phage-bacterium relationship resulted in an initial high degree of lysis, the potency of toxin in the culture was weak . It appeared that in highly toxigenic strains, the phage-bacterium relationship is characterized by a stable lysogenic type of association . It was also found that in a highly toxigenic converted culture the percentage of toxigenic cells was 100, whereas in hypotoxigenic culture the percentage was only 20.

Appl Environ Microbiol, 1976 Jul, 32(1), 121 - 4
Effect of acid and salt concentration in fresh-pack pickles on the growth of Clostridium botulinum spores; Ito KA et al.; The addition of various amounts of acetic acid to pureed cucumbers inoculated with Clostridium botulinum spores has shown that outgrowth is inhibited at pH 4.8 but not at pH 5.0 . Inoculation experiments with whole cucumbers showed that as little as 0.9% acetic acid in the brine was sufficient to prevent outgrowth from spore inocula as high as 10(6)/cucumber . It was further shown that the rapid rate of acetic acid penetration into fresh-pack pickles prevents the growth of any C . botulinum spores that may be present.

Can J Microbiol, 1976 Jul, 22(7), 953 - 9
Stable L-forms of Clostridium perfringens and their growth on glass surfaces; Mahony DE et al.; L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin . After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent . At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules . Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form . Sucrose could also be withdrawn from the medium . The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined . L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.

Science, 1976 Jun 25, 192(4246), 1343 - 4
Ribonucleotide reductase in blue-green algae: dependence on adenosylcobalamin; Gleason FK et al.; Ten species of freshwater blue-green algae exhibit an adenosylcobalamin-dependent ribonucleotide reductase, thuse explaining the requirement for cobalt by these organisms . The evidence suggests a phylogenetic affinity between the cyanophytes and bacteria, such as Clostridium and Rhizobium, and the euglenoid flagellates, which also use the cofactor-dependent reductase . In contrast, the ribonucleotide reductase reaction in the few green algae surveyed shows no dependence on cobalamins.

Biochemistry, 1976 Jun 15, 15(12), 2633 - 41
Oxidation-reduction properties of several low potential iron-sulfur proteins and of methylviologen; Stombaugh NA et al.; Apparent oxidation-reduction potentials at pH 7.0 and 25 degrees C were determined using the H2-hydrogenase system with ferredoxins from the following sources: Clostridium pasteurianum, -403 mV; C tartarovorum, -424 mV; C . acidi-urici, -434 mV; Peptococcus aerogenes, -427 mV; Chromatium D, -482 mV (pH 8.0); B . polymyxa, Fd I, -377 mV, and Fd II, -422 mV; and spinach, -428 mV . The pH dependence of these values was variable, ranging from -2 to -24 mV/pH unit increase for different ferredoxins . Over the range of buffer concentrations between 0.05 and 0.2 M, the potentials did not vary significantly . The number of electrons transferred during reduction (as determined by integrations of EPR spectra and by dithionite titration) is 2 for the first five proteins, while potentiometric data for all the cases fit a Nernst equation for which n = 1 . The E degrees' value for the redox indicator methylviologen at pH 7.4 was found to be -460 mV, according to both the H2-hydrogenase system and cyclic voltammetry, significantly different from the value previously reported at higher pH's . Additionally, the presence of C . pasteuranum ferredoxin appears to shift the E degrees value of methylviologen to even more negative values . An analysis of sources of error inherent with potential determinations with H2 and hydrogenase is presented . The electronic and EPR spectra of P . aerogenes ferredoxin, for which the x-ray structure has been published, are given here . It appears that the determination of potentials of ferredoxin and other low-potential porteins with the H2-hydrogenase system affords certain experimental advantages over alternative methods currently employed with these and similar substances.

Biochim Biophys Acta, 1976 Jun 8, 430(3), 434 - 44
Properties and function of clostridial membrane ATPase; Riebeling V et al.; ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum . About 70% of the total activity was found in the particulate fraction . The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+ . Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect . The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate . Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ({S}0.5 v = 1.3 mM) and maximal activity (120 U/g) . The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+ . Solubilization, however, led to instability of the enzyme . The clostridial solubilized and membrane-bound ATPase showed different properties similar to the "allotopic" properties of mitochondrial and other bacterial ATPases . The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD) . DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect . The membrane-bound ATPase could not be stimulated by trypsin pretreatment . Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C . pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation . A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.

J Clin Invest, 1976 Jun, 57(6), 1419 - 25
The effect of phospholipase C on human blood platelets; Otnaess AB et al.; The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied . Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis . Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C . A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment . Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine . Sphingomyelin was not a substrate for the enzyme under the conditions used . The platelets contained no detectable endogenous phospholipase C activity . The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin . Total platelet factor 3 releasable by freezing and thawing was reduced . Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed . When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred . Sphingomyelinase alone gave no aggregation . The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens . The distribution of phospholipids in the platelet membrane is discussed.

Hoppe Seylers Z Physiol Chem, 1976 Jun, 357(6), 839 - 53
Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens; Nees S et al.; Clostridium perfringens cells were cultivated on a large scale using an automatic system . 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution . The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel . Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein . 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis . Contaminating enzyme activities were not detected . 4) The isoelectric point of pH 4.7 was found for the enzyme . At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined . The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2 . The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction . Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM . 5) An average molecular weight of 99200 was determined for the native enzyme using different methods . After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits . The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts . 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction . The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule . It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis . A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.

Appl Environ Microbiol, 1976 Jun, 31(6), 826 - 30
Bacteriological quality and shelf life of ground beef; Emswiler BS et al.; The bacteriological quality of unfrozen raw ground beef was evaluated after 0, 3, 6, 9, 12, 15, and 18 days of storage at 29 +/- 1 F (-1.7 +/- 0.6 C) . At the time of fabrication, all of the ground beef samples contained 10(6) or fewer total aerobic and psychrotrophic bacteria/g; 81% contained 100 or fewer coliforms/g; 94% contained 100 or fewer Escherichia coli/g; and all of the samples contained 100 or fewer coagulase-positive Staphylococcus aureus and Clostridium perfringens/g . Total aerobic and psychrotrophic bacteria increased by 1 log between 3 and 18 days of storage . Coliform and E . coli counts decreased during storage, whereas coagulase-positive S . aureus and C . perfringens counts did not change significantly . These data indicate that meat processors, wholesalers, and retailers could improve the bacteriological quality and prolong the shelf life of ground beef packaged in oxygen-impermeable film if the temperature of product never exceeded 29 +/- 1 F (-1.7 +/- 0.6 C).

Biochim Biophys Acta, 1976 May 20, 434(1), 244 - 57
Purification and properties of paramagnetic protein from Clostridium pasteurianum W5; Cardenas J et al.; The purification to homogeneity of the non-heme iron protein, sometimes referred to as either "red protein" or "paramagnetic protein", from Clostridium pasteurianum W5 extracts is described and its physicochemical properties studied . This paramagnetic protein (g= 1.94) has a molecular weight of about 25000 and contains two iron and two acid-labile sulfur atoms per mol of protein . Its midpoint potential at pH 7.5, as determined by electron paramagnetic resonance titration, is -300 mV . Optical circular dichroism and electron paramagnetic resonance spectra of the paramagnetic protein are similar to those of two iron-two acid-labile sulfur ferredoxins . The biochemical reduction of the purified protein was also studied.

Biochim Biophys Acta, 1976 May 20, 434(1), 4 - 17
Non-heme iron proteins . The amino acid sequence of rubredoxin from Desulfovibrio vulgaris; Bruschi M; A non-heme iron protein, rubredoxin has been isolated from the sulfate-reducing bacterium, Desulfovibrio vulgaris, strain Hildenborough . The complete amino acid sequence has been established . The 52 amino acid residues of the protein were aligned with the aid of tryptic and chymotryptic peptides and of a fragment produced by cleavage of the Asn-Gly bond (22-23) by hydroxylamine . The sequence of the first 30 residues of the molecule was determined using an automatic sequenator, after removal of the N-terminal methionine by CNBr . In comparing this sequence with those of Micrococcus aerogenes, Clostridium pasteurianum and Peptostreptococcus elsdenii rubredoxins, a high degree of mutation was observed between these homologous proteins . It has been shown that 20 amino acid residues occurred in identical positions . The locations of the four cysteine residues were found to be invariable . A crystallographic study of the Desulfovibrio vulgaris rubredoxin is in progress.

Eur J Biochem, 1976 May 17, 65(1), 107 - 11
Cytidine diphosphate diglyceride of bovine brain . Positional distribution of fatty acids and analysis of major molecular species; Thompson W et al.; A method is described for the isolation of CDP-diglyceride from bovine brain . Yields of the product ranged from 9.2-15.5 mumol per kilogram of tissue, which corresponds to about 1% of the level of phosphatidic acid . Mild alkaline hydrolysis of the product gave three water-soluble phosphate esters which had the same electrophoretic mobilities as CMP, CDP-glycerol and glycerol 3-phosphate . The liponucleotide was quantitatively hydrolysed by CDP-diglyceride hydrolase from Escherichia coli to phosphatidic acid and CMP . No dCMP was recovered in enzymatic or alkaline hydrolysates and it is concluded there can be little or no dCDP-diglyceride in bovine brain . Brain CDP-diglyceride was similar to phosphatidylinositol in that in both lipids stearate was the major saturated fatty acid and arachidonate the most abundant unsaturated fatty acid . This differed significantly from the fatty acid patterns of other metabolically related phospholipids, phosphatidic acid and cardiolipin . Brain CDP-diglyceride was hydrolysed with phospholipase C from Clostridium welchii with the liberation of the diglyceride moiety in high yield . Treatment of the diglyceride with pancreatic lipase showed CDP-diglyceride with the asymmetric distribution of fatty acids characteristic of most mammalian phospholipids, saturated fatty acids being found mostly at position 1 and polyunsaturated fatty acids at position 2 . The derived diglyceride acetates were separated into different molecular species by argentation thin-layer chromatography . These analyses showed that 1-stearoyl, 2-arachidonoyl was the major species of brain CDP-diglyceride.

Tijdschr Diergeneeskd, 1976 May 15, 101(10), 548 - 58
}Acute severe mastitis in cows of the Dutch-Friesian Breed (author's transl)}; Osinga A et al.; One hundred cases of severe acute mastitis are reported . Bacteriological examination of the milk was negative in 23% of the cases . E . coli was most frequently found to be the causative organism (23%), followed by St . aureus (19%), C . pyogenes (10%), Str . dysgalactiae (9%), Str . agalactiae (5%) and a number of other pathogenic agents such as atypical streptococci, Str . uberis, haemolytic streptococci, Aerobacter aerogenes, Kl . pneumoniae, a clostridium and P . multocida . In several cases, a tentative diagnosis may be based on the clinical symptoms, which is then found to be correct on bacteriological examination of the milk . The prognosis will vary with the causative organism . Treatment was life-saving in more than 90% of the cases, the milk-producing capacity of the affected quarter being completely preserved in 36% and partly preserved in 27% of the cases.

Biochim Biophys Acta, 1976 May 13, 429(3), 1036 - 40
Lactate racemase . Hydroxylamine-dependent 18O exchange of the alpha-hydroxyl of lactic acid; Pepple JS et al.; The lactic acid racemase (EC 5.1.2.1) derived from Clostridium butylicum catalyzes the racemization of the alpha-18O label . The proposed alpha-carbonyl intermediate for the enzyme-catalyzed reaction has been previously shown to be trapped as an enzyme-bound oxime in the presence of hydroxylamine . This report demonstrates that the formation of the inactive enzyme-bound oxime, followed by reactivation in the presence of an excess of competing free carbonyl (pyruvic acid) results in a complete loss of the alpha-18O label from an original alpha-18O-labeled lactic acid.

Eur J Biochem, 1976 May 1, 64(2), 565 - 72
Coordinated action of pectinesterase and polygalacturonate lyase complex of Clostridium multifermentans; Sheiman MI et al.; The polygalacturonate lyase and pectinesterase activities of Clostridium multifermentans, both produced extracellularly when the organism grows on pectin or polygalacturonate, have been suggested to be associated in a single complex . Both enzymic sites act on their respective substrates by single-chain action patterns, as shown by equivalent release of terminal tritium label and total product throughout the reaction . From these results, the Km and V of the lyase, and the amount of lyase activity present, we calculate the steady-state concentration of lyase substrate expected during action of the two sites on pectin if the sites are independent . No such steady-state concentration of lyase substrate was observed . Therefore, we conclude that the two types of active site act in a coordinated manner; the polysaccharide chain passes from the esterase site to the lyase site without intermediate dissociation and rebinding . This 'molecular disassembly line' constituted by the two sites may represent a system of general significance in synthesis and degradation of biological polymers.

Can J Microbiol, 1976 May, 22(5), 673 - 6
Urease-negative strains of Clostridium sordellii; Nakamura S et al.; Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C . sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose . The test for cell wall sugar components of urease-negative and -positive strains of C . sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.

J Assoc Off Anal Chem, 1976 May, 59(3), 602 - 5
Phosphorus pentoxide as a drying agent for bacterial culture extracts analyzed by gas-liquid chromatography; Finne G et al.; The procedure for gas chromatographic analysis of metabolic products of microbial fermentation includes solvent extraction of the aqueous growth media, drying of the extract, and direct chromatographic analysis of the solvent . In this study, 2 drying agents, magnesium sulfate and phosphorus pentoxide, were compared . Both were effective in removing water; however, phosphorus pentoxide removed water more completely and at a faster rate than magnesium sulfate . When a thermal conductivity detector is used, it is important to completely remove water from the solvent to prevent interference with volatile acids and alcohols . When water is present, short-chain alcohols (C2-C5) are eluted together with the water, causing peak overlap and shoulder separations . Phosphorus pentoxide quickly and effectively removed water so that a baseline was established following the solvent front on the chromatogram . The use of phosphorus pentoxide is particularly advantageous for identification or fermentation studies on Clostridium and Propionibacterium when rapid identification is desired or when large numbers of cultures are to be tested.

Atherosclerosis, 1976 May-Jun, 23(3), 429 - 36
Hemodynamically-induced increase in soluble collagen in the anastomosed veins of experimental arteriovenous fistulae; Smith RA et al.; External jugular veins that had been subjected to the hemodynamic stresses produced by experimental arteriovenous anastomosis developed 2% increased total protein contents and 17% increased collagen contents . When the stressed veins were homogenized and extracted with saline solutions, statistically significant increases in the saline-soluble proteins and in the saline-soluble collagen (87% and 267%, respectively) were observed . Increased amounts of low molecular weight peptides were found in the extracts . A fraction of these peptides could be degraded by Clostridium collagenase . The saline extract also contained proteins which resembled by their amino-acid composition the acidic structural proteins of the connective tissues . Additonally, in 3 dogs so tested, changes were found in the hydroxylation and glycosylation of lysine from gelatin extracts as well as in the lysine and desmosine contents of the insoluble elastin fractions . This is the first demonstration of a hemodynamically induced increase in the saline solubility of connective tissue proteins in the absence of dietary manipulations.

J Bacteriol, 1976 May, 126(2), 869 - 82
Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits; Sleytr UB et al.; Cell walls of Clostridium thermohydrosulfuricum and C . thermosaccharolyticum have a two-layered structure, consisting of a thin, lysozyme-sensitive murein layer and a surface (S) layer composed of hexagonally or tetragonally arranged subunits . The subunits can be removed from the murein layer by treatment of cell wall preparations, are composed of a fragile, pH-sensitive monolayer of macromolecular subunits . In both organisms the first stage of the cell division process involves only the plasma membrane and the murein layer . During the subsequent cell separation, a surplus of S-layer subunits appears at the site of division, and consequently the newly formed cell poles remain completely covered by the s layer throughout the separation process . In autolyzed cells an additional layer of subunits assembles on extended areas of the inside of the mucopeptide layer . These observations indicate that the biological function of the S layer depends on its ability to maintain a complete covering of the cell surface at all stages of cell growth and division.

J Med Microbiol, 1976 May, 9(2), 129 - 36
Clostridium difficile: isolation and characteristics; Hafiz S et al.; Clostridium difficile can be grown readily in Reinforced Clostridial Medium (RCM) containing 0-1-0-4% of o-, m- or p-cresol, or phenol . We recommend 0-2% of phenol or p-cresol in RCM for the isolation of this organism . The characteristic "cornfield" growth in RCM in 25-ml Universal containers is described . Glucose, fructose, galactose, mannose, raffinose, aesculin and mannitol are fermented with production of acid and gas; maltose, sucrose, glycogen, soluble starch and sorbitol are fermented with production of acid only . Lactose and rice starch are not fermented by any strain, and DL-methionine is not attacked . Nitrate is reduced to nitrite . Hydrogen sulphide and indole are not produced . Gelatin is attacked by all strains, but in some cases prolonged incubation is required . Hyaluronidase is produced, but not deoxyribonuclease . A lethal toxin appears to be produced . Strains possess shared and strain-specific antigens.

Prikl Biokhim Mikrobiol, 1976 May-Jun, 12(3), 449 - 53
{Soluble proteins of the vegetative cells and spores of Clostridium botulinum type B and their toxicity}; Nikolaeva SA; By disc microelectrophoresis in polyacrylamide gel proteins of vegetative cells and spores of Clostridium botulinum type B were investigated at different growth phases and toxicity of protein zones was measured . Protein components of vegetative cells varied in their electrophoretic mobility, depending on the growth phase, and differed significantly from spore proteins . Spores contained 1% toxin of vegetative cells . Electrophoregrams of intrasporal proteins showed a toxic zone with an approximate molecular weight of 10(5)-10(6).

Appl Environ Microbiol, 1976 May, 31(5), 723 - 30
Incidence of Salmonella spp., Clostridium botulinum, and Vibrio parahaemolyticus in an estuary; Sayler GS et al.; A study of the incidence of Salmonella spp., Vibrio parahaemolyticus-like organisms, and clostridium botulinum in samples collected at five stations located in the Upper Chesapeake Bay, a major estuary on the Atlantic Coast of the United States, was conducted in December 1973 through December 1974 . C . botulinum types B and E were detected in 12.3% of the total sediment samples examined . V . parahaemolyticus was recovered from 10.4% of a total of 86 water, sediment, and suspended sediment samples . Of 131 samples examined for the presence of Salmonella spp., approximately 3% were found to be positive for serologically confirmed Salmonella isolates . Shellfish examined during the investigation were also found to be free of enteric pathogens . The low frequency of occurrence of V . parahaemolyticus was attributed to the low salinities encountered at the sites included in the study . A low incidence of Salmonella spp . in the Upper Chesapeake Bay samples was found, whereas the distribution of C . botulinum appeared to be both random and autochthonous . A strong relationship between presence of potential pathogens and other generally accepted microbiological indicators of pollution was not observed.

Can J Microbiol, 1976 May, 22(5), 603 - 10
Temperate phages of Clostridium perfringens type C1; Grant RB et al.; Four phages isolated from carrier strains of Clostridium perfringens type C belong to two classes . The three phages of class I, c1, c3, and c4, and homoimmune and serologically closely related . The phage of class II, c5, is heteroimmune to the class I phages and not related to them serologically . Transduction experiments with several of the phages were negative . Mutants of the indicator strain with surface alterations occurred spontaneously in stock cultures . Electron micrographs show the phages of each class to be distinct yet similar, having polyhedral heads of about the same diameter 55 nm, and long, flexible tails without sheaths or collars . Phages c4 and c5 were characterized for their lysogenic properties . Phage c4 was inducible with mitomycin C . Both c4 and c5 were temperate viruses by the test of stability of their respective lysogens to phage-specific antisera.

J Assoc Off Anal Chem, 1976 May, 59(3), 606 - 12
Collaborative study of an improved method for the enumeration and confirmation of Clostridium perfringens in foods; Harmon SM; A collaborative study was conducted in 10 laboratories to evaluate the performance of a new method for the enumeration of vegetative cells of Clostridium perfringens in foods . Results obtained by the new method were compared with results from the official first action method, 46.049-46.053 . Per cent recoveries of 4 C . perfringens strains from inoculated roast beef samples were higher and more consistent in tryptose-sulfite-cycloserine (TSC) agar with or without added egg yolk than in sulfite-polymyxin-sulfadiazine (SPS) agar, specified in the official first action method . The confirmatory technique utilized in the new method was also found to be more reliable than the technique described in the official first action method . Based on the collaborative results, the new method with TSC agar for enumeration and a modified motility-nitrate medium together with a lactose-gelatin medium for confirmation of C . perfringens has been adopted as official first action to replace 46.049-46.053.

J Bacteriol, 1976 May, 126(2), 845 - 51
Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: gel filtration of mutant and reverant activities; Rood JI et al.; Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity . The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities . These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins . Models that explain the genetic basis for these results are discussed.

J Bacteriol, 1976 May, 126(2), 831 - 44
Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins; Rood JI et al.; Biochemical characterization of hemagglutinin and sialidase activities from Clostridium perfringens strain CN3870 revealed that this strain produced three sialidase enzymes that were separable to gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis . The molecular weights of sialidase I, II, and III activities were 310,000 +/- 10,000, 105,000 +/- 4,000 and 64,000 +/- 2,000, respectively, the first figure being an approximate value only.

J Biol Chem, 1976 Apr 25, 251(8), 2435 - 9
Purification and properties of proline reductase from Clostridium sticklandii; Seto B et al.; Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents . The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000 . A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition . Amino acid analysis showed a preponderance of acidic amino acids . No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis . A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein . No molybdenum, iron, or selenium was found in the pure protein . Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro . Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.

J Biol Chem, 1976 Apr 25, 251(8), 2499 - 510
Specificity of bacterial ribosomes and messenger ribonucleic acids in protein synthesis reactions in vitro; Stallcup MR et al.; Ribosomes from two Gram-negative bacteria translated f2 RNA, T4 early mRNA, mRNA from three Gran-negative bacteria, and mRNA from six Gram-positive bacteria; ribosomes from three Gram-positive bacteria translated mRNA from the Gram-positive strains, but did not translate the other mRNAs . Ribosomes from the Gram-negative bacterium Escherichia coli translated synthetic poly(U,G) but ribosomes from the Gram-positive bacterium Clostridium pasteurianum translated poly(U,G) very poorly, mRNA from Gram-negative bacteria was translated only in the presence of a high salt ribosomal wash containing initiation factors . mRNA from Gram-positive bacteria and synthetic poly(U,G) were translated much more efficiently when wash components were present, but were also translated to a small, but significant, extent in the absence of wash components . The translation specificity of each type of ribosome was independent of the source of ribosomal wash components . When the radioactively labeled products of in vitro protein synthesis were analyzed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography, it was found that each different bacterial and phage RNA preparation directed the synthesis of a unique set of polypeptide products of discrete sizes . Three different types of ribosomes were used to translate each of several Gram-positive bacterial messenger preparations; the overall patterns of products obtained with a given mRNA are similar, but some differences in the products formed or the relative amounts of the various products synthesized can be detected.

Biochim Biophys Acta, 1976 Apr 23, 428(2), 312 - 20
Chemical reactivity of labile sulfur of iron-sulfur proteins . The reaction of triphenyl phosphine; Manabe T et al.; The reaction of triphenyl phosphine to iron-sulfur proteins from adrenal cortex mitochondria, spinach chloroplasts, and Clostridium pasteurianum was investigated . As ethanol concentrations in the reaction mixture increased, the rate of the reaction decreased . In the simultaneous presence of 1 M KC1 and 5 M urea, the reaction rate reached at maximum . Under these conditions the initial rates of the decolorization reaction by the phosphine were found to be 8.7, 0.88, and 1.8 nmol of ferredoxin per min at 25 degrees C for adrenal, spinach, and clostridial ferredoxins, respectively . The kinetic curves for the reaction of the phosphine sulfide formation, the loss of labile sulfur, and the deterioriation of visible absorption showed a similar pattern with a comparable rate . During this reaction, the complete reduction of ferric ions present in ferredoxin was observed with a fast rate under either aerobic or anaerobic conditions . These results suggest that the iron atoms in ferredoxin are first reduced by the intramolecular reductants in the presence of triphenyl phosphine with the concomitant formation of S2-2, which then reacts with triphenyl phosphine resulting in the formation of triphenyl phosphine sulfide.

Sem Hop, 1976 Apr 23, 52(16), 999 - 1005
{Clostridium perfringens infection . Problems posed excluding resuscitation}; Lagarde P et al.; The authors report 18 cases of Clostridium perfringens infection and discuss the main clinical and etiological aspects apart from gynecological forms and those usually requiring intensive care . Pathological foci, digestive infection and the problem of bacteremia are discussed together with appropriate treatment.

Eur J Biochem, 1976 Apr 15, 64(1), 255 - 62
The enzyme complex citramalate lyase from Clostridium tetanomorphum; Buckel W et al.; 1 . The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts . However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride . Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C . tetanomorphum or Klebsiella aerogenes . 2 . Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate . Using ido{2-(14)C}acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme . The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism . 3 . Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K . aerogenes . The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation . Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts . These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme . 4 . Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase . The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier . 5 . The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase . Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria . A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.

J Biol Chem, 1976 Apr 10, 251(7), 1859 - 63
Leucine 2,3-aminomutase, an enzyme of leucine catabolism; Poston JM; The initial step in the fermentation of leucine to acetate, isobutyrate, and ammonia by Clostridium sporogenes is the B12 coenzyme-dependent conversion of alpha-leucine to beta-leucine (3-amino-4-methylpentanoate) . The amino group migration reaction, catalyzed by leucine 2,3-aminomutase, is reversible and is inhibited by intrinsic factor . The enzyme activity has been found in several clostridia, in rat, sheep, rhesus, and African green monkey livers, and in human leukocytes.

Arch Microbiol, 1976 Apr 1, 107(3), 283 - 8
The end products of the metabolism of aromatic amino acids by Clostridia; Elsden SR et al.; The end products of the metabolism of phenylalanine, tyrosine and tryptophan by growing cultures of clostridia have been identified . The species used were Clostridium aminovalericum; C . bifermentans; C . botulinum proteolytic type A; C . botulinum proteolytic type B; C . cochlearium; C . difficile; C . ghoni; C . histolyticum; C . lentoputrescens; C . limosum; C . lituseburense; C . malenomenatum; C . mangenoti; C . propionicum; C . putrefaciens; C . sordellii; C . sporogenes; C . sporosphaeroides; C . sticklandii; C . subterminale; C . tetani; C . tetanomorphum . The mixture of aromatic compounds formed, which depended upon the species, included phenyl acetic acid, phenyl propionic acid, phenyl lactic acid, phenol, p-cresol, p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, indole, indole acetic acid and indole propionic acid.

Surg Neurol, 1976 Apr, 5(4), 253 - 4
Pellet-gun brain wound complicated by Clostridium Perfringens meningitis; DeWeese WO et al.; A ten-year-old male was hospitalized for a pellet-gun wound to the brain . He developed Clostridial meningitis within eighteen hours in spite of radical debridement and prophylactic antibiotics . However, successful recovery was obtained with high levels of penicillin and chloromycetin antibiotic therapy.

J Gen Microbiol, 1976 Apr, 93(2), 388 - 96
Changes in spores of Clostridium bifermentans caused by treatment with hydrogen peroxide and cations; Waites WM et al.; Spores of Clostridium bifermentans were treated with hydrogen peroxide until their peripheries had lost refractility . The centres of such spores only retained refractility at acid pH . Adding monovalent cations or increasing the pH caused the treated spores to lose their remaining refractility and decreased the turbidity of spore suspensions . Divalent cations prevented or reversed this loss of central refractility and decreased the fall in turbidity . Calcium ions also prevented but did not reverse the loss of central refractility which occurred on drying or applying pressure . Electron micrographs of spores treated with hydrogen peroxide showed that the cortex was depleted or absent and that the loss of central refractility was accompanied by protoplast swelling . It is suggested that divalent cations make spores resistant to drying and pressure by cross-linking negatively charged groups within the protoplast, and that together with hydrogen ions they neutralize the negatively charged groups, thus preventing the swelling of the protoplast, loss of refractility and fall in extinction which occur when divalent cations are replaced by monovalent cations.

J Bacteriol, 1976 Apr, 126(1), 377 - 83
Chemical characterization of the regularly arranged surface layers of Clostridium thermosaccharolyticum and Clostridium thermohydrosulfuricum; Sleytr UB et al.; Clostridum thermosaccharolyticum and Clostridium thermohydrosulfuricum possess as outermost cell wall layer a tetragonal or hexagonal ordered array of macromolecules . The subunits of the surface layer can be detached from isolated cell walls with urea (8M) or guanidine-HCl (4 to 5 M) . Triton X-100, dithiothreitol, ethylenediaminetetracetate, and KCl (3 M) had no visible effect on the regular arrays . Sodium dodecyl sulfate-polyacrylamide electrophroesis showed that, in both organisms, the surface layer is composed of glycoprotein of molecular weight 140,000 . The glycoprotein from both microorganisms has a predominantly acidic amino acid composition and an acidic isoelectric point after isoelectric focusing on polyacrylamide gels . The glycocomponent is composed of glucose, galactose, mannose, and rhamnose.

Gann, 1976 Apr, 67(2), 275 - 7
Effect of Clostridium toxoids, especially of Clostridium perfringens toxoid, on mouse transplanted tumors; Sasaki T et al.; The antitumor activities of toxoids of Clostridium perfringens, C . nouyi, C . septicum, and C . tetani were tested against sarcoma-180 and Ehrlich carcinoma . Among them, C . perfringens toxoid showed a high antitumor activity against the growth of the implanted sarcoma-180 ascites form . The results of the inhibiting effect of C . perfringens toxoid on Nakahara-Tokuzen-Fukuoka (NTF) reticulum cell sarcoma and methylcholanthrene-induced fibrosarcoma were also described.

Hoppe Seylers Z Physiol Chem, 1976 Apr, 357(4), 535 - 41
On the mechanism and some properties of vinylacetyl-CoA delta-isomerase of Clostridium kluyveri; Schleicher E et al.; Vinylacetyl-CoA delta-isomerase from Clostridium kluyveri grown on ethanol/acetate was purified 32-fold . The enzyme is rather labile . All experiments were conducted with the substrate analog thioester of vinylacetic acid and N-acetylchysteamine (vinylacetyl-SEtNAc (1 f)) . 3-Butinoyl-SEtNAc is a strong inhibitor of the isomerase . The hydrogen transfer from the alpha-position of vinylacetyl-SEtNAc to the gamma-position of 2-butenoyl-SEtNAc (1f leads to 2 f) occurs partially intramolecularly (40-50%) as shown by experiments in 3HOH/H2O, 2H2O and 3HOH/2H2O as well as by experiments with {2,3-3H}vinylacetyl-SEtNAc . Only 0.07 atoms of tritium are incorporated into the gamma-position of 2f when the isomerisation takes place in 3HOH/H2O . The extent of intramolecularity is in agreement with results of experiments conducted in 2H2O with whole cells {4} . The reaction 1f leads to 2f shows no or only negligiebl reversibility and at least no considerable isotope effect.

Appl Environ Microbiol, 1976 Apr, 31(4), 492 - 8
Chemical manipulation of the heat resistance of Clostridium botulinum spores; Alderton G et al.; The chemical forms of Clostridium botulinum 62A and 213B were prepared, and their heat resistances were determined in several heating media, including some low-acid foods . The heat resistance of C . botulinum spores can be manipulated up and down by changing chemical forms between the resistant calcium form and the sensitive hydrogen form . The resistant chemical form of type B spores has about three times the classical PO4 resistance at 235 F (112.8 C) . As measured in peas and asparagus, both types of C . botulinum spores came directly from the culture at only a small fraction of the potential heat resistance shown by the same spores when chemically converted to the resistant form . The resistant spore form of both types (62A and 213B), when present in a low-acid food, can be sensitized to heating at the normal pH of the food.

J Biol Chem, 1976 Mar 25, 251(6), 1675 - 82
Derivatives of Clostridium acidi-urici ferredoxin containing altered amino acid sequences . Semisynthetic synthesis, biological activity, and stability; Lode ET et al.; The semisynthetic syntheses and some properties of derivatives of Clostridium acidi-urici ferredoxin that contain amino acid deletions or replacements in the peptide chain are described . All 16 stable derivatives prepared, with the exception of {Trp2}ferredoxin, were fully active as electron carriers in the two enzymatic assay systems tested: the phosphoroclastic system and the ferrodoxin-dependent reduction of cytochrome c . E1Trp1}Ferredoxin had 70% of the activity of native ferredoxin in both assay systems . The stability in aerobic solution of {Ala1}ferredoxin, which had had its natural alanyl NH2-terminal residue removed and then replaced chemically, is the same as that of the native ferrodoxin (half-life of approximately 54 days) . The relative stabilities of derivatives with a replacement or deletion of the NH2-terminal residue are as follows: {Ala1}- greater than or equal to {Phe1}-, {Lys1}-, { Pro1}-, {Leu1}- greater than {Met1}- greater than {Gly1}- greater than {Glu1}- greater than des-Ala1-ferrodoxin . The data indicate that a large bulky residue, but not a negatively charged residue, is tolerated in position 1 of the peptide chain and the greatly decreased stability (half-life = 1 day) of des-Ala1-ferredoxin confirms the importance of the NH2-terminal residue for the stability of the protein . The relative stabilities of derivatives containing Ala1, but including a replacement for the normal Tyr2, are as follows: Native greater than {Trp2}- greater than or equal to {Phe2}- greater than {His2}- greater than {Leu2}- greater than {Pro2}ferredoxin . {Gly2}- and des-Ala1-Tyr2-apoferredoxin did not form stable derivatives upon reconstitution with iron and sulfide, nor did {3-NO2-Tyr2, 30}- and {Leu2,3-NO2-Tyr30}apoferredoxins . Other relatively stable and fully active derivatives prepared included: {3-NH2-Tyr30}-, {3-F-Phe2}-, and {2-F-Phe2}ferredoxin . The behavior of these various derivatives demonstrates the importance of the peptide chain for the stability of C . acidi-urici ferredoxin and shows that the activity of ferredoxin can be altered by a single amino acid substitution in the peptide chain.

J Biol Chem, 1976 Mar 25, 251(6), 1683 - 7
Apparent oxidation-reduction potential of Clostridium acidi-urici ferredoxin . Effect of pH, ionic strength, and amino acid replacements; Lode ET et al.; The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase . The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V . In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase . A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes . This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate . Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts . This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables . The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V . Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C . acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined . Under similar conditions, the Emp of most of the 13 derivatives examined, including those of {Leu2}- and{3-NH2-Tyr30}ferredoxin, is approximately the same as that of native ferredoxin . However, the Emp of {His2}ferredoxin is approximately 15 mv more positive, whereas that of {Trp2}ferredoxin is 22 mv more negative than that of native C . acidi-urici ferredoxin . Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives . It is suggested that the changes observed in the Emp of C . acidi-urici ferredoxin are caused by protein conformational changes.

Eur J Biochem, 1976 Mar 16, 63(1), 271 - 87
An extracellular aminopeptidase from Clostridium histolyticum; Kessler E et al.; An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity . Absence of endopeptidase activity in the purified preparation was demonstrated . Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme . Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated . From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer . Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides . The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme . The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin . Low rates of hydrolysis was observed for charged residues, and amides of amino acids . Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X . The highest Kcat values are observed with proline and alanine . In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues . The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+ . The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively . The optimal temperature is 40 degrees C . Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.

Biochem J, 1976 Mar 15, 154(3), 725 - 9
Partial purification of a dicyclohexylcarbodi-imide-sensitive membrane adenosine triphosphatase complex from the obligately anaerobic bacterium Clostridium Pasteurianum; Clarke DJ et al.; The membrane adenosine triphosphatase complex of vegetatively growing Clostridium pasteurianum, solublized with Triton X-100, has been recovered as a significantly purified particulate preparation that is still sensitive to inhibition by dicyclohexylcarbodiimide and butyricin 7423.

J Clin Microbiol, 1976 Mar, 3(3), 318 - 23
Anaerobic infections in children: a prospective survey; Thirumoorthi MC et al.; Over an 18-month period, cultures from 95 infants and children yielded 146 anaerobic organisms in 110 clinical specimens . Bacteroides was the most frequently isolated anaerobe, followed by Propionibacterium and Clostridium species . Intra-abdominal sources, soft tissues, and blood were the three major sources (82%) of isolation of anaerobes . Whereas most patients (58%) were over 5 years of age and only 11% were newborns, anaerobic infections constituted a rather uniform proportion of all infections, regardless of sources, in all age groups . Anaerobes accounted for only 2.9% of all positive cultures encountered from the various sources . Rates of recovery of anaerobes from intra-abdominal sources were significantly the highest, and from soft-tissue infections they were significantly the lowest . The anaerobic bacteremias observed were of no clinical significance when Propionibacterium species were isolated; however, recovery of other anaerobes from the blood, and primarily Bacteroides species, was usually associated with clinical disease . Except in blood cultures, anaerobes almost invariably coexisted with facultative bacteria.

Am Fam Physician, 1976 Mar, 13(3), 76 - 80
Tetanus; Hughes JH et al.; While the incidence of clinical tetanus had decreased greatly, efforts to eradicate the disease will depend mainly upon continued stress on prophylaxis . Recent cases demonstrate the need for both continued vigil in prophylaxis and sensitivity to the early signs of the clinical disease . The fundamentals of treatment are: neutralize formed toxin, eradicate Clostridium tetani, debride tissues adequately, control spasm and start immunization . Complications are legion . Mortality is highest in the very young and in the elderly.

Surgery, 1976 Mar, 79(3), 313 - 24
Anaerobic infections in surgery: clinical review; Anderson CB et al.; Anaerobic bacteria are being recognized with increasing frequency as important micro-organisms in surgical infections . Clostridium, Bacteriodes, Fusobacterium, and Peptostreptococcus are the clinically prominent pathological anaerobes . All are commensals and, consequently, most anaerobic infections are endogenous in origin . In the colon, anaerobes are 1,000 times more prevalent than aerobes . This has important implications regarding the management of gastrointestinal tract operations and the treatment of infections originating from the bowel . Typical anaerobic infections include gas gangrene, brain abscess, oral infections, putrid lung abscesses, intra-abdominal abscesses, and wound infections following gynecologic and bowel surgery, perirectal abscesses, postabortal infections, and septic thrombophlebitis . Infections with anaerobic organisms must be suspected when there is feculent odor and/or gas production following gynecologic or bowel surgery, when there are organisms on gram staining but no growth on aerobic cultures, or when septicemia is associated with repeatedly negative blood cultures . Debridement and drainage constitute the main stay of treatment . All anaerobes are sensitive to chloramphenicol and clindamycin and all but Bacteroides fragils are sensitive to penicillin . Identification of anaerobes requires proper specimen sampling, immediate culturing on prereduced media, and careful gram staining of clinical material . The frequency of anaerobic organisms in surgical infections generally is not recognized by many surgeons; their importance needs to be stressed in the future.

Can J Microbiol, 1976 Mar, 22(3), 435 - 7
Studies on mode of action of a bacteriocin from Clostridium septicum; Schallehn G et al.; A bacteriocin was found in the supernatant fluid of Clostridium septicum strain Ovinus . Sensitivity to the bacteriocin was confined to other strains of C . septicum and to strains of C . chauvoei; the other Gram-positive and Gram-negative bacteria tested for sensitivity were unaffected by the bacteriocin . The bacteriocin killed sensitive cells rapidly but cell lysis did not appear to be involved . The bacteriocin inhibited protein and RNA synthesis immediately after its addition to sensitive cells; DNA synthesis was inhibited 10 min later.

Zentralbl Bakteriol {Orig B}, 1976 Mar, 161(5-6), 527 - 33
{On N2-fixing clostridia and bacilli from soils (author's transl)}; Hammann R et al.; In total 30 nitrogen-fixing, saccharolytic Clostridia and 4 nitrogen-fixing bacilli, all freshly isolated from gleyed soils, were screened for sensitivity to 20 antibiotics . The isolates were compared in their sensitivity with 5 type cultures representing the species Clostridium butyricum, C . saccharobutyricum (2 strains) C . multifermentans and C . sporogenes . Generally speaking, both clostridia and bacilli are sensitive to the same antibiotics (Table 2) . In addition, the nitrogen-fixing bacilli belonging to Bacillus polymyxa and B . macerans showed sensitivity to neomycin and kanamycin . Except for the species C . tyrobutyricum, none of the various saccharolytic Clostridium species could be distinguished by differences in sensitivity to antibiotics . Differential methods are given in Table 3.

Obstet Gynecol, 1976 Mar, 47(3), 337 - 41
Clostridium perfringens infection complicating chemotherapy for choriocarcinoma; Lacey CG et al.; A case of Clostridium perfringens sepsis and gas gangrene complicating chemotherapy for gestational choriocarcinoma is reported . The infection was eradicated using antibiotics, surgery, and hyperbaric oxygen therapy . The pathophysiology, diagnosis, and treatment of this unusual but often lethal complication are reviewed.

Infect Immun, 1976 Mar, 13(3), 987 - 9
Comparison of progenitor toxins of nonproteolytic with those of proteolytic Clostridium botulinum Type B; Miyazaki S et al.; A nonproteolytic strain of Clostridium botulinum type B produces two toxins of different molecular weight (16S and 12S) that are indistinguishable from the corresponding toxins of a proteolytic strain in molecular weight and construction but differ in potential toxicity, activation ratio, and hemagglutinability . Successful hybridization between the toxic and nontoxic components (both7S) of 12S toxins of biologically heterologous type B strains confirmed the physico-chemical similarity between the toxic as well as the nontoxic components.

Appl Environ Microbiol, 1976 Mar, 31(3), 342 - 8
Inhibitory effects of H2 on growth of Clostridium cellobioparum; Chung KT; Hydrogen inhibits the growth of hydrogen-producing Clostridium cellobioparum, but not of Escherichia coli or Bacteroides ruminicola . The inhibition is reversible . When hydrogen was removed either by palladium black or by gassing out the tube, glucose utilization increased as did optical density and hydrogen production of C . cellobioparum . Removal of the H2 by methanogenic bacteria favors the growth of C . cellobioparum . Grown with Methanobacterium ruminantium in various concentrations of glucose, the Clostridium reaches a higher optical density and produces more H2 and a higher viable cell count . The cell yield is also higher than in pure culture . In mixed culture, C . cellobioparum produces more acetic acid and less lactic acid, ethanol, and butyric acid than in pure culture . The significance of this metabolic shift and hydrogen utilization in methanogenesis is discussed.

J Clin Pathol, 1976 Mar, 29(3), 185 - 6
Significance of the isolation of Clostridium welchii from routine blood cultures; Ahmad FJ et al.; Clostridium welchii has been demonstrated in approximately 20% of contact plates taken from the antecubital fossa of 185 inpatients and outpatients and healthy staff . The highest incidence was in a group of 40 very ill patients . The isolation of the organism from blood cultures is not always of clinical significance . Skin preparation as at present practised is often inadequate to remove the spores when contamination is relatively heavy, for example, in bedridden patients.

Infect Immun, 1976 Mar, 13(3), 855 - 60
Antigenicity of converting phages obtained from Clostridium botulinum types C and D; Oguma K et al.; Phage conversion of toxigenicity in Clostridium botulinum types C and D was accomplished by using nontoxigenic strains and phages purified from plaques . Although the morphology of the converting phages seemed to be the same, they were divided into three groups on the basis of their conversion spectrum . The first group consists of phages obtained from toxogenic strains C-Stockholm and C-468 . The second group consists of phages from strains D-1873 and C-203 . The third group consists of phages from strains D-South African and D-4947 . These converting phages were also classified into the same three groups by a neutralization test with specific antiphage sera . Cross-neutralization, however, was observed between phages belonging to group 1 and group 2,by both the neutralization test of converting ability and by a plaque experiment in which the surviving rates of phages were calculated after treatment with each antiphage serum . The antigenic differences among these converting phages should probably comprise one of the reasons for the existence of the specific infection spectrum in C . botulinum types C and D.

Hum Pathol, 1976 Mar, 7(2), 177 - 86
Isolation and identification of anaerobic bacteria; Rosenblatt JE; Anaerobic bacteria make up a predominant part of the normal human flora . Adequate specimen collection must avoid contamination with this flora . Suitable methods include thoracentesis, transtracheal aspiration, needle and syringe aspiration of closed abscesses, and endocervical aspiration of intrauterine pus . Swabs are generally unsuitable . Sputum, voided urine, vaginal secretions, and specimens contaminated with feces are not cultured anaerobically . Specimens should be transported in an oxygen-free container . There are several efficient methods for culturing anaerobes, including the simple and inexpensive GasPak jar . The average clinical laboratory may not be able to afford the time, personnel, and equipment necessary for complete identification of all anaerobes isolated . However, the identification of certain ones, including Bacteroides fragilis and Clostridium perfringens, is relatively simple . Primary emphasis should be placed on the rapid recognition of the presence of obligate anaerobes in a culture and the immediate reporting of all available information to the clinician . Subsequent reports can provide as complete an identification as is possible for each laboratory.

Appl Environ Microbiol, 1976 Mar, 31(3), 455 - 7
Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A; Labbe R et al.; Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium . In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters.

Zentralbl Bakteriol {Orig A}, 1976 Mar, 234(2), 243 - 6
{About the influence of beta-toxin of Clostridium perfringens (type C) on the motorics of intestine segments (in vitro) . I . Communication}; Parnas J; The Beta-Toxin of Clostridium perfringens (Type C) was introduced intra lumen to jejunum and ileum segments of rabbits, then examined by pharmacologic method (in vitro) . The Beta-Toxin showed a paralysing activity on the motorics of the intestine which fact may be in relation to the paralysing activity of this toxin in dysenteria in piglets.

Res Vet Sci, 1976 Mar, 20(2), 225 - 6
The effect of enterotoxin of Clostridium welchii (perfringens) type A on fowls; Niilo L; Lethal doses of enterotoxin of Clostridium welchii (perfringens) type A injected intravenously into young fowls caused immediate lassitude, with partial recovery, followed by death seven to 35 h after inoculation . Lesions found were ascites, hydropericardium, oedema of the muscles, hepatic congestion, urate deposits on the peritoneum and the pericardium, and intestinal hyperaemia . Sublethal doses produced no clinical signs or lesion . The LD50 of this enterotoxin was 74-84 mug/kg of body weight.

J Immunol, 1976 Mar, 116(3), 661 - 8
Lysis of tumor cells by antibody and complement . VI . Enhanced killing of enzyme-pretreated tumor cells; Boyle MD et al.; The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement . Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement . The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors . The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme . Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens . The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination . Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.

Zentralbl Bakteriol {Orig A}, 1976 Mar, 234(2), 247 - 59
{Extraction and concentration of Clostridium botulinum toxins from specimens (author's transl)}; Sonnenschein B et al.; In order to detect minimal amounts of Clostridium botulinum toxins in animal tissue or food specimens it is necessary to use an extraction method which results in concentration of the botulinal toxins . In the present examinations, artificially contaminated canned beans were used to develop a suitable procedure for extraction and concentration of botulinal toxins A-E . The procedure consisted of 4 steps: 1 . Canned beans were diluted 1:2 with 0.1 m phosphate buffer pH 6.0 . 2 . The diluted material was homogenised with an "Ultra-Turrax" homogeniser for 20 sec . 3 . The monogenised material was centrifuged at 4000 rpm for 30 min . 4 . 15 ml of supernatant was concentrated using a "Millipore ultrafiltration chamber" (with a membrane capable of excluding all material with a molecular weight above 25,000) . A pressure of 1.5 atmospheres was applied until the terminal volume was 0.5 ml . Following extraction and concentration, the samples were assayed for botulinal toxin in mice . Using this assay the concentration of the five toxins were shown to be as follows: Type A toxin: 19.0-fold toxin concentration Type B toxin: 14.8-fold toxin concentration Type C toxin: 20.6-fold toxin concentration Type D toxin: 28.2-fold toxin concentration Type E toxin: 112.2-fold toxin concentration

Biochim Biophys Acta, 1976 Feb 24, 421(2), 334 - 7
Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group; Petitdemange H et al.; NADH and NADPH-ferredoxin oxidoreductases have been studied in Clostridium acetobutylicum, Cl . tyrobutyricum and Cl . pasteurianum . The study of the distribution and regulation of these enzymatic activities in well-defined culture conditions, reveals that the essential function of NADPH-ferredoxin oxidoreductase is to produce NADPH, while NADH-ferredoxin oxidoreductase can, depending on cellular conditions, produce or oxidize NADH . When these Clostridia use glycolysis, regulation of the NADH-ferredoxin oxidoreductase by acetyl-CoA (obligatory activator of NADH-ferroxin reductase activity) and by NADH (competitive inhibitor of ferredoxin-NAD+ reductase activity) allow the enzymes to function correlatively with glyceraldehyde-3-phosphate dehydrogenase and thus control the levels of NAD+ and NADH in the cell . In Cl . tyrobutyricum and Cl . pasteurianum, the ferredoxin-NADP+ reductase activities are regulated by NAD+ and NADH in accordance with the intracellular concentrations of these coenzymes . In Cl . tyrobutyricum growing on pyruvate/acetate, NADH and NADPH-ferredoxin reductase activities cannot be detected; only the ferredoxin-NAD+ and ferredoxin-NADP+ reductase activities are found . In this Clostridium, regulation of the ferredoxin-NADP+ reductase activity is the same whether it is grown on glucose or pyruvate . Contrary to this, the ferredoxin-NAD+ reductase activity undergoes a drastic change, since NADH no longer controls the enzymatic activity . In this case regulation is no longer necessary, since glyceraldehyde-3-phosphate dehydrogenase does not function.

Med Biol, 1976 Feb, 54(1), 39 - 49
Cytotoxic effects on the plasma membrane of human diploid fibroblasts--a comparative study of leakage tests; Thelestam M et al.; Confluent monolayers of human diploid lung fibroblasts were treated with cytolytic agents . The induced membrane damage was investigated by different test systems . Changes of membrane permeability were compared with morphological alterations . Four tests employed leakage of cytoplasmic markers of different sizes as criteria of membrane damage . Radioactive markers of the following decreasing size order were used: {3H}RNA (MW greater than 200,000) greater than 51Cr greater than {3H}nucleotides greater than {1-14C}alpha-amino-isobutyric acid (AIB, MW 103) . Uptake of trypan blue was employed as a fifth criterior of changed membrane permeability . A comparison between the tests indicated the following order of sensitivity for detection of membrane damage: leakage of AIB-label greater than leakage of nucleotide label greater than leakage of 51Cr-label=uptake of trypan blue= morphological changes greater than leakage of RNA-label . Two distinct types of leakage patterns were evident: 1 . Upon incubation with Triton X-100 all four release curves coincided . This was considered as representing large functional "holes" . 2 . With the polyene amphotericin B the smallest marker (AIB) was released to a strikingly greater extent than other markers . This was regarded as representing very small functional "holes" . Melittin from bee venom and theta-toxin from Clostridium perfringens induced leakage patterns of two intermediate types . The results indicate that a combination of several different size markers may be useful for characterizing induced membrane lesions.

Can J Microbiol, 1976 Feb, 22(2), 269 - 75
Morphological modifications of cells of Clostridium pasteurianum caused by growth on sulfite; McCready RG et al.; The morphology of Clostridium pasteurianum cells grown on 10(-2) M SO32- showed significant alteration in cell shape and the absence of the electron translucent reserve polysaccharide (amylopectin) when compared to sulfate-grown cells . At the lower sulfite concentrations (10(-3) and 10(-4)M SO32-) the cells showed the cytoplasmic changes noted above but the cell shapes were not modified.

J Clin Microbiol, 1976 Feb, 3(2), 180 - 5
Electron capture gas chromatography study of the acid and alcohol products of Clostridium septicum and Clostridium chauvoei; Brooks JB et al.; The metabolic products produced by several strains of Clostridium septicum obtained from patients and animals, along with strains of Clostridium chauvoei, were studied in chopped meat glucose medium by electron capture gas-liquid chromatography (EC-GLC) . The strains of C . septicum and C . chauvoei were shown to comprise five different metabolic groups . Both the EC-GLC study and the O and H antigenic study performed previously showed that strains of C . septicum comprise a heterogeneous group . One type of metabolic profile was found only in strains of C . chauvoei . The O antigen types and EC-GLC metabolic types of C . septicum correlated fairly well in isolates from cancer patients but not in stock culture and animal isolates.

Hoppe Seylers Z Physiol Chem, 1976 Feb, 357(2), 147 - 52
{On uroporphyrinogen formation: Studies with 1-aminomethyl-3, 8, 13, 18-tetra(2-carboxyethyl)-2, 7, 12, 17-tetracarboxymethylbilinogen (author's transl)}; Dauner HO et al.; The preparation of the aminomethyl-bilinogen which results from formal "head to tail" condensation of porphobilinogen is described . The chemical cyclocondensation of this compound at pH 7.4 yields uroporphyrinogen I . Enzymatic studies with enzyme preparations from Propionibacterium shermanii, which synthesize uroporphyrinogens from porphobilinogen, show that the rate of cyclisation is increased by these enzymes and indicate that the bilinogen also might be used for uroporphyrinogen III formation . This is also suggested by studies on the formation of cobyrinic acid from {4-14C}5-aminolevulinate via uroporphyrinogen III in the presence of the aminomethylbilinogen by cell-free extracts from Clostridium tetanomorphum.

Arch Microbiol, 1976 Feb, 107(1), 25 - 31
The electron transport to nitrogenase in Mycobacterium flavum; Bothe H et al.; 1 . Two ferredoxin-type iron-sulfur proteins have been isolated from Mycobacterium flavum 301 grown under nitrogen-fixing, iron-sufficient conditions . No flavodoxin was observed . 2 . These ferredoxins are apparently soluble: they were present in the supernatant fraction after disrupting by decompression . Only small amounts were present in particulate fractions . 3 . The two ferredoxins were separated by chromatography on DEAE-cellulose, Sephadex or electrophoresis . 4 . Both ferredoxins mediated the transfer of electrons from illuminated spinach chloroplasts to a nitrogenase preparation to reduce acetylene . Ferredoxin II was specifically about five times more active than ferredoxin I . Ferredoxin II was also active in the photosynthetic NADP+-reduction whereas ferredoxin I was not . 5 . Both ferredoxins were reversibly reduced by either sodium dithionite, illuminated spinach chloroplasts or hydrogen plus hydrogenase from Clostridium pasteurianum . 6 . Attempts to determine the primary electron donor for nitrogen fixation in Mycobacterium flavum were unsuccessful . Acetylene reduction in Mycobacterium extracts was obtained only with sodium dithionite or illuminated spinach chloroplasts as electron donors . The reduction of the electron carrier (e.g . ferredoxin) rather than the transfer of electrons from the reduced carrier to nitrogenase was rate-limiting.

J Infect Dis, 1976 Feb, 133(2), 199 - 201
Current status of passive immunity to diphtheria and tetanus in the newborn; Nathenson G et al.; The extent of transplacentally derived immunity to the toxins of Corynebacterium diphtheriae and Clostridium tetani was assessed by a hemagglutination technique using the cord sera of a group of infants recently born in Bronx, New York . Protective levels of antitoxin to C . diphtheriae were found in 64% of the infants . Tetanus antitoxin was present in sufficient quantity for protection in 38% of the cord sera . While this degree of immunity reepresents an improvement over that achieved 20 years ago and probably reflects the effects of immunization procedures, further attention to the maintenance of immunizations in women of childbearing age would improve these statistics.

Appl Environ Microbiol, 1976 Feb, 31(2), 234 - 42
Toxoid of Clostridium botulinum type F: purification and immunogenicity studies; Hatheway CL; Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography . Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods . The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days . Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin . The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered . The toxoid prepared from the most highly purified toxin (method {iii}) conferred the highest immunity in guinea pigs at a given dose level . A relation between serum antitoxin level and resistance to challenge was observed . At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin . A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.

Appl Environ Microbiol, 1976 Feb, 31(2), 208 - 12
Effect of sodium nitrite inhibition on intracellular thiol groups and on the activity of certain glycolytic enzymes in Clostridium perfringens; O'Leary V et al.; Activities of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) (GAP-DH) and aldolase (EC 4.1.2.13) in cells of Clostridium perfringens that had been inhibited with sodium nitrite were investigated . A complete loss in GAP-DH activity and a 67% decrease in aldolase activity were observed when growth of C . perfringens was inhibited . There was also a 91% decrease in the concentration of free sulfhydryl groups of soluble cellular components . Dithiothreitol restored some activity to inactive GAP-DH from sodium nitrite-inhibited cells, indicating that a loss of reduced sulfhydryl groups was involved in the inactivation of the enzyme . The evidence presented suggests that sodium nitrite inhibition of C . perfringens may involve an interaction of sodium nitrite as nitrous acid with sulfhydryl-containing constituents of the bacterial cell.

Jpn J Exp Med, 1976 Feb, 46(1), 45 - 50
Clostridium perfringens exotoxins . IV . Inhibition of the theta-toxin induced hemolysis by steroids and related compounds; Hase J et al.; 1 . The inhibitory powers of thirty two samples of steroids and their related compounds on the theta-toxin induced hemolysis were assayed . Apparent I50 value of cholesterol, the most potent inhibitor amoung them, was 0.022 muM . 2 . Inhibitory powers of sterol acetates were not more than one tenth of those of the corresponding sterol, and steroids without 3-hydroxyl group hardly inhibited the hemolysis . These results suggest that 3-hydroxyl group of sterols plays an important role in the inhibition . 3 . The inhibitory power of sterols were strongly affected by steric situation of 3-hydroxyl group for the face of their tetracyclic rings . From these results it is suggested that theta-toxin binds to beta-side of the tetracyclic rings but not to alpha-side . 4 . A methylsterol and pentacyclic triterpens with 3-hydroxyl group also inhibited the hemolysis, but steroids and their related compounds either with a hydrophylic or without a hydrophobic group at their ring D had little, if any, inhibitory power.

Infect Immun, 1976 Feb, 13(2), 337 - 44
Evidence for a one-hit theory in the immune bactericidal reaction and demonstration of a multi-hit response for hemolysis by streptolysin O and Clostridium perfringens theta-toxin; Inoue K et al.; An analytical method was developed for estimating the number of hits necessary to lyse or kill cells in which various concentrations of the cells are treated with a constant amount of the lytic or killing agent in a constant reaction volume . The reaction may be due to a single-component agent or occur by a sequential chain of reactions due to a multi-component agent, even including side, abortive, or counter-reactions . It was clearly shown by this method that immune bactericidal reactions followed a one-hit theory . It was shown by this method that streptolysin O required four or five hits for hemolysis and Clostridium perfringens theta-toxin required two hits . These results were confirmed by both logarithmic dose-response and survival analyses . It was also shown that streptolysin O and theta-toxin can act complementarily on accumulation of the hits for hemolysis.

J Bacteriol, 1976 Feb, 125(2), 444 - 52
Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens; Labbe RG et al.; The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined . Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium . The rate of RNA synthesis decreased as sporulation progressed . Deoxyadenosine increased uptake of {14C}uracil and {14C}thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml . Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation . The rate of protein breakdown during vegetative growth was 1%/h . During sporulation this rate increased to 4.7%/h . When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h . If added at 3 h the rate decreased to 2.1%/h . The role of proteases in this process is discussed.

J Bacteriol, 1976 Feb, 125(2), 429 - 34
Spore membrane(s) as the site of damage within heated Clostridium perfringens spores; Flowers RS et al.; Clostridium perfringens spores were injured by ultrahigh-temperature treatment at 105 C for 5 min . Injury was manifested as an increased sensitivity to polymyxin and neomycin . Since many of the survivors could not germinate normally the ultrahigh-temperature-treated spores were sensitized to and germinated by lysozyme . Polymyxin reportedly acts upon the cell membrane . Neomycin may inhibit protein synthesis and has surface-active properties . Injured spores were increasingly sensitive to known surface-active agents, sodium lauryl sulfate, sodium deoxycholate, and Roccal, a quaternary ammonium compound . Injured spores sensitive to polymyxin and neomycin also were osmotically fragile and died during outgrowth in a liquid medium unless the medium was supplemented with 20% sucrose, 10% dextran, or 10% polyvinylpyrrolidone . The results suggested that a spore structure destined to become cell membrane or cell wall was the site of injury . Repair of injury during outgrowth in the presence of protein, deoxyribonucleic acid, ribonucleic acid and cell wall synthesis inhibitors was consistent with this hypothesis.

Proc Soc Exp Biol Med, 1976 Feb, 151(2), 260 - 3
The size pH, and redox potential of the cecum in mice associated with various microbial floras; Celesk RA et al.; Cecal size and in situ redox potential and pH of cecal contents were determined in conventionally reared mice and mice reared under a variety of gnotobiotic conditions: germfree, monoassociated with a cecal Clostridium sp., hexaflora-associated and thermoduric polyflora-associated . The mean Eh was approximately +200 mV in germfree and -200 mV in conventional mice . The Eh was close to zero in the monoassociated mice, thus occupying a position intermediate between the germfree and conventional mice . The potentials observed in the hexaflora and the thermoduric flora groups were indistinguishable from those of conventional animals . The degree of normalization was more advanced with respect to the redox potential than to the cecal size in the various gnotobiotic groups . In the thermoduric polyflora-associated group, normalization was observed in both cecal size and redox potential . This demonstrates that normalization can be accomplished with a relatively simplified microflora, at least with regard to the parameters studied.

J Biol Chem, 1976 Jan 25, 251(2), 493 - 7
Properties of ecto-(inoganic) pyrophosphatase of nervous system cells in culture . Activation upon partial release of sialic acid from the cell surface; Stefanovic V et al.; Clonal line NN hamster astroblasts and clonal line N18 neuroblasts were treated with phospholipase C-free, protease-free, and hemolysin-free Clostridium perfringens sialidase, at a low level (5 X 10(-3) units/ml) so as to maintain cell intactness and to avoid spurious protein effects . A rapid, regular release of sialic acid was achieved . An approximately 9-fold increase in ecto-pyrophosphatase activity could be brought about by action of C . perfringens sialidase for 10 min . Since the sialidase preparations were employed at a level which gave a very low concentration of extraneous protein, and the preparations were free of demonstrable phospholipase C and protease activities, these effects appear to relate specifically to removal of cell surface sialic acid . Neutral p-nitrophenyl phosphatase was activated under the same conditions, but activity remained low compared with pyrophosphatase . Progress curves for activation of the two enzymes were dissimilar . Ecto-pyrophosphatase of NN and N18 cells had an absolute requirement for Mg2+ both before and after removal of cell surface sialic acid . In the presence of near optimum Mg2+ (5 mM), other divalent cations were inhibitory at a low level (10(-1)mM) . The effect of Mg2+ concentration, as well as inorganic pyrophosphate concentration, upon ecto-pyrophosphatase activity was shown to obey Michaelis-Menten kinetics for the control activity and for the sialidase-enhanced activity of both cell types . Km for Mg2+ and for pyrophosphate remained constant upon ecto-pyrophosphatase enhancement by sialic acid removal; increase in enzymatic activity was accounted for entirely by an increase in Vmax.

Vet Rec, 1976 Jan 17, 98(3), 49 - 50
Initial studies on "six months disease" in sheep; Pyakural S et al.; Enterotoxaemia in sheep due to Clostridium welchii type D was indicated by field and laboratory investigations in Nepal . Morphological, cultural, biochemical, biological and toxin-producing characteristics observed were used to type the isolates . In anaerobic meat medium, all isolates produced pinkish discoloration of meat . All the strains fermented lactose, maltose, dextrose and sucrose whereas, salicine was fermented only by 17 strains . All but five strains were MR negative . Out of 200 isolated, 166 produced both alpha and epsilon toxins and the remaining 34 non-toxogenic strains are likely to be variants which have lost their toxogenicity . Epidemiologically the local name "Six months disease" and enterotoxaemia are considered to be identical diseases.

Lancet, 1976 Jan 17, 1(7951), 125 - 6
Pathogenesis of enteritis necroticans in Papula New Guinea; Lawrence G et al.; It is suggested that enteritis necroticans occurs in the people of the Highlands of Papua New Guinea because they have low levels of digestive proteases in the intestinal lumen . These low protease levels are the result of a low-protein diet and the presence of heat-stable trypsin inhibitors in sweet potatato, the dietary staple . Low proteolytic activity allows the B toxin of Clostridium welchii type C, which is extremely susceptible to proteolysis, to initiate the disease.

J Wildl Dis, 1976 Jan, 12(1), 116 - 26
A relationship between avian carcasses and living invertebrates in the epizootiology of avian botulism; Duncan RM et al.; A survey of the sources of Clostridium botulinum type C toxin possibly utilized as food by aquatic birds in an epizootic area of avian botulism in northern Utah showed that living aquatic and terrestrial invertebrates normally found in close association with dead, decomposing birds commonly carried the toxin . Of 461 samples associated with 21 species of avian carcasses, 198 were toxin-positive . Invertebrate species not normally scavengers of vertebrate tissues were less commonly and less highly toxic, particularly when captured 30 cm or more from a carcass; six of 237 samples of such aquatic invertebrates low-level toxin . Of the species tested, blow fly larvae (Calliphoridae) were the most consistently and highly toxic, although others, particularly adult and larval stages of several species of beetles (Coleoptera), contained toxin at levels probably significant in the epizootiology of the disease . An estimated 0.05 to 0.25 g of the most toxic fly larvae or 15 g of the most toxic beetles tested carried a mediam lethal dose for an adult mallard duck . Examination of stomach contents of aquatic birds dead of botulism showed that some had consumed invertebrates.

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