Repression of toxin production by tryptophan in Clostridium botulinum type E

Of the seven amino acids required by Clostridium botulinum type E, tryptophan is the most essential and may provide the cell with nitrogen. The addition of excess tryptophan (10–20 mM) or other nitrogenous nutrients to minimal growth medium markedly decreased toxin formation but did not affect growth in C. botulinum type E. On the other hand, the addition of an enzymatic digest of casein (NZ Case) stimulated toxin formation and overcame repression by tryptophan. Immunoblots of proteins in culture fluids using antibodies to type E toxin indicated that tryptophan-repressed cultures produced less neurotoxin protein. Inhibitors of neurotoxin did not accumulate in cultures grown in minimal medium supplemented with high tryptophan. The results suggest that tryptophan availability in foods or in the intestine may be important for toxin formation by C. botulinum type E.     

Clostridium halophilium sp. nov. and C. litorale sp. nov., an obligate halophilic and a marine species degrading betaine in the Stickland reaction

Two new mesophilic, sporeforming, gram-positive, strictly anaerobic, rod-shaped bacteria were isolated which utilized betaine in the Stickland reaction. Strain M1 was obtained from pasteurized hypersaline sediments. Cells were motile rods and formed spherical terminal spores. Betaine was used with hydrogen and several amino acids as electron donors. In addition, several carbohydrates served as substrates. Growth required 1.5% NaCl with an optimum at 6.0% NaCl. The guanine plus cytosine content of the DNA was 26.9%. This strain is described as a new species, Clostridium halophilum.Strain W6 was isolated from marine sediments. Cells were motile rods and formed ovoid, subterminal spores. Betaine was used with hydrogen and several amino acids as electron donors. Carbohydrates were not fermented. Growth optimum was at 1.0% NaCl. The guanine plus cytosine content of the DNA was 26.1%. This strain is described as a new species, Clostridium litorale.Non standard abbreviations DMG N,N-dimethylglycine - TMA trimethylamine - PY peptone-yeast extract - PYG peptone-yeast extract-glucose     

Slostridium homopropionicum sp. nov., a new strict anaerobe growing with 2-, 3-, or 4-hydroxybutyrate

From anoxic sewage sludge a new strictly anaerobic, spore-forming bacterium was isolated with 2-hydroxybutyrate as sole substrate. 2-, 3-, and 4-hydroxybutyrate, 4-chlorobutyrate, crotonate, vinylacetate, and pyruvate were fermented to acetate and butyrate. Fructose was converted to acetate, butyrate, butanol, and H₂. Lactate and acrylate were fermented to acetate and propionate. Cells pregrown with lactate fermented 2-hydroxybutyrate to butyrate, propionate and acetate. No inorganic electron acceptors were reduced. The DNA base ratio was 32.0±1.0 mol % and was similar to that of Clostridium propionicum, which was determined to be 35.3±0.5 mol %. Strain LuHBu1 is described as type strain of a new species, Clostridium homopropionicum sp. nov. Another isolate obtained from marine sediment degraded 2-and 3-hydroxybutyrate to acetate and butyrate and was in some respects similar to the known species Ilyobacter polytropus.     

Mutation of Clostridium thermocellum in the presence of certain carbon sources

Abstract When a cellobiose-grown inoculum of Clostridium thermocellum was transferred to either glucose or fructose as the sole carbon sourcem growth occurred only after a long lag of 180–200 h. We established that sugar uptake and phosphorylation were not limiting growth nor was the lag period the time take for a physiological adaptation process or for the growth of a mutant carried over in the cellibiose-grown incoculum. It became apparent that a mutation was occuring during the lag period in response to the selection pressure exerted by the presence of glucose or fructose as the sole carbon source. Once growth occurred on glucose and fructose, the cells could be transferred to cellobiose and back to glucose or fructose without exhibiting the long lag period. The change was stable over several transfers in the respective sugars.     

New approach for selective separation of dilute products from simulated clostridial fermentation broths using cyclodextrins

The ability of cyclodextrins (CD) to form crystalline insoluble complexes with organics was explored in this study in view of a selective separation of dilute products obtainable from three clostridial fermentation systems. To this purpose, a product or a product mixture at a concentration of 0.150 mM each were treated with alpha-CD or beta-CD (0.150 mM) in aqueous solutions as well as in a nutrient broth as a simulated fermentation medium. In the acetone-butanol-ethanol system, and in the butanol-iso-propanol system, alpha-CD was found to precipitate selectively 48% and 46% butanol after 1 h agitation at 30 degrees C. However, beta-CD was found to be superior for the butyric acid-acetic acid system because it selectively precipitated 100% butyric acid under the same conditions. Cooling the three product system with alpha-CD to 4 degrees C for 24 h increased significantly the crop of precipitates but decreased the selectivity for either butanol or butyric acid. Cyclodextrins were thus shown to offer potentially a new exciting possibility for downstream processing of low-concentration fermentation products.     

Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum

The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specfic activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 M coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60°C. Kinetic constants were measured in the forward reaction. The apparent K _m for pyruvate and coenzyme A were 322 M and 3.7 M, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.Non-standard abbreviations NAD(H) nicotinamide adenine dinucleotide (reduced) - NADP(H) nicotinamide adenine dinucleotide phosphate (reduced) - DTE dithioerythritol - PMS phenazine methosulfate - NBT nitro blue tetrazolium chloride - DMSO dimethyl sulfoxide - DCPIP dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide - TTC triphenyltetrazolium chloride - FAD flavin adenine dinucleotide - FMN flavin mononucleotide     

Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum

The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred at high frequency (10^-2–10^-3 per recipient, selecting for tetracycline resistance) to the Gram-positive anaerobe Clostridium acetobutylicum. Selection in the presence of two antibiotics (tetracycline and erythromycin) with the plasmids pAM 180 and pINY1275 yielded only low numbers of transconjugants (10^-8 per recipient). Matings were done by combining liquid and filter mating procedures under anaerobic conditions. No plasmid DNA could be detected in the transconjugants selected on a minimal medium in the presence of tetracycline. DNA-DNA hybridization experiments with restricted chromosomal DNA using biotinylated pAM120::Tn916 as probe revealed the presence of homologous sequences in the transconjugants but not in Clostridium acetobutylicum wild type. The transconjugants were used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec. diacetylactis. In both cases tetracycline-resistant strains were found. Transfer frequencies in these experiments were less than 10^-7 per recipient.     

16S rRNA gene sequence‐based analysis of clostridia related to conversion of germfree mice to the normal state

Aims: To determine phylogenetic groups of clostridia inhabiting the mouse intestine that are essential for normalization of germfree (GF) mice. Methods and Results: Using both the culture method and cloning, clostridia inhabiting the mouse intestine were isolated, and phylogenetic analysis based on 16S rRNA gene sequences was carried out. As a result, the isolates were found to have novel sequences, and no isolate was determined to be identical to previously known identified clostridia. Although the taxonomy of mouse intestinal clostridia was complex, many of them belonged to Clostridium clusters XIVa and IV in conventional (CV) and limited flora mice and ex‐germfree mice administered chloroform‐treated CV mouse faeces. The clostridia that belonged to cluster XIVa were most often present and showed the highest diversity. Conclusions: Clostridia belonging clusters XIVa and IV are dominant in the mouse intestine as in other gut ecosystems. The novel groups in these clusters are essential for normalization of GF mice. Significance and Impact of the Study: The results of this study can be applied in the strict control of mouse intestinal microbiota and will provide important information for normalization of GF mice and also for research on microbiology of the mouse intestine.     

Inhibition of the glucosyltransferase activity of clostridial Rho/Ras-glucosylating toxins by castanospermine

Castanospermine was identified as an inhibitor of the Rho/Ras-glucosylating Clostridium sordellii lethal toxin and Clostridium difficile toxin B. Microinjection of castanospermine into embryonic bovine lung cells prevented the cytotoxic effects of toxins. The crystal structure of the glucosyltransferase domain of C. sordellii lethal toxin in complex with castanospermine, UDP and a calcium ion was solved at a resolution of 2.3A. The inhibitor binds in a conformation that brings its four hydroxyl groups and its N-atom almost exactly in the positions of the four hydroxyls and of the ring oxygen of the glucosyl moiety of UDP-glucose, respectively.     

Clostridium paraputrificum M-21发酵制氢培养条件研究

利用类腐败梭状芽孢杆菌M-21(Clostridium paraputn M-21),在37℃、150r／min条件下发酵制氢,以葡萄糖为碳源,蛋白胨为氮源,lmol的葡萄糖可以产生1．05mol的氢气,最终所产气体中有70％H2和30％c02(体积百分数)。最优初始pH范围为7．0—7．5,少量乙酸的存在对氢气的生成有促进作用,若大量存在,会严重抑制茵的生长。在C／N质量比为1．0时,所产氢气体积最多,1g葡萄糖可产生75mL Hz(常温常压)。在高温83℃下对种茵预处理30s促进孢子的萌发,会缩短发酵产氢的时间。以淀粉为碳源,所产氢气体积略微高于葡萄糖,1g淀粉可产生78mL H2(常温常压);以蔗糖为碳源,所产氢气体积略微低于葡萄糖,1g蔗糖可产生72mL H2(常温常压)。该茵不能降解利用羧甲基纤维素,木质素磺酸钠,及纸浆等。