Disarming Clostridium difficile

In this issue, Puriet al. (2010) present inhibitors that prevent the autocatalytic activation of the clostridial toxin TcdB in vivo. Their approach is likely to provide guidance for the development of novel drugs targeting virulence factors and thereby rendering bacterial pathogens innocuous.     

Fast purification of Clostridium sordellii cytotoxin.

We have developed a rapid method for the purification of proteins, combining titration curve analysis with a two-step column chromatographic procedure. We have used this approach to purify the cytotoxin (L toxin) from Clostridium sordellii. We have also determined the amino acid composition of this cytotoxin. This toxin has a pI value of 4.20 and an Mr of 260,000, reduction of which results in a band of Mr 43,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Since both the proteins of Mr 260,000 and 43,000 are recognized by the polyclonal anti-C. sordellii L toxin, which neutralizes the L toxin cytotoxicity, we propose a hexameric structure for the protein of Mr 260,000, each subunit being Mr 43,000.     

Total Synthesis of Five Lipoteichoic acids of Clostridium difficile

The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interest in preventive strategies, such as vaccines based on (synthetic) cell wall antigens. Recently, the structure of the lipoteichoic acid (LTA) of this species has been elucidated. Additionally, this LTA was found to induce the formation of protective antibodies against C. difficile in rabbits and mice. The LTA from C. difficile is isolated as a microheterogenous mixture, differing in size and composition, impeding any structure–activity relationship studies. To ensure reliable biological results, pure and well‐defined synthetic samples are required. In this work the total synthesis of LTAs from C. difficile with defined chain length is described and the initial biological results are presented.     

Fermentation of xylose into acetic acid by Clostridium thermoaceticum

For optimum fermentation, fermenting xylose into acetic acid by Clostridium thermoaceticum (ATCC 49707) requires adaptation of the strain to xylose medium. Exposed to a mixture of glucose and xylose, it preferentially consumesxylose over glucose. The initial concentration of xylose in the medium affects the final concentration and the yield of acetic acid. Batch fermentation of 20 g/L of xylose with 5g/L of yeast extract as the nitrogen source results in a maximum acetate concentration of 15.2 g/L and yield of 0.76 g of acid/g of xylose. Corn steep liquor (CLS) is a good substitute for yeast extract and results in similar fermentation profiles. The organism consumes fructose, xylose, and glucose from a mixture of sugars in batch fermentation. Arabinose, mannose, and galactose are consumed only slightly. This organism loses viability on fed-batch operation, even with supplementation of all the required nutrients. In fed-batch fermentation with CSL supplementation, d-xylulose (an intermediate in the xylose metabolic pathway) accumulates in large quantities.     

Decomposition of phosphatidylethanolamine with phospholipase C from Clostridium perfringens

Conclusions The optimum conditions for the enzymatic decomposition of phosphatidylethanolamine with the phospholipase C fromClostridium perfringens have been described. When a sonically irradiated aqueous suspension of lipid containing zinc ions is used, 70–80% decomposition is achieved.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 2, pp. 422–424, February, 1967.     

Influence of redox potential on product distribution in Clostridium thermosuccinogenes

Clostridium thermosuccinogenes are the only known anaerobic thermophilic bacteria that ferment inulin to succinate and acetate as major products and formate, lactate, and ethanol as minor products. In this study, organic acid production in 2-L fermentations having an initially low (−300 to −330 mV) or high (−220 to −250 mV) redox potential was compared for two strains of C. thermosuccinogenes (DSM 5808 and DSM 5809). Although DSM 5809 consistently provided higher succinate yield, high variability in results was attributed to the absence of redox control during the fermentations, and, therefore, fermentations at three controlled redox potentials (−240, −275, and −310 mV) were conducted. At an intermediate redox potential (−275 mV), the succinate yield was the greatest (0.36 g of succinate/g of hexose unit), whereas ethanol yield was the least (0.02 g/g). Redox potential did not significantly affect acetate or lactate formation. At controlled redox potential of −275 mV, the growth of DSM 5809 on three substrates was also compared: inulin, fructose, and glucose. DSM 5809 had similar growth rates when inulin (0.20/h) or glucose (0.21/h) was the carbon source but grew more slowly when fructose (0.16/h) was the carbon source. Also, the specific rate of utilization of fructose by DSM 5809 was higher (0.89 g of fructose/[g of biomass·h]) compared to glucose (0.53 g/[g·h]) or inulin (0.55 g/[g·h]). Succinate was the major product formed by DSM 5809 fermenting inulin (0.50 g/[g·h]) or glucose (0.36 g/[g·h]), and ethanol was the principal product when DSM 5809 fermented fructose (0.54 g/[g·h]).     

Electromicrobial transformations using the pyruvate synthase system of Clostridium sporogenes

A bioelectrochemical method by which the enzymology of reductive carboxylations (RCOOH + CO₃ + 6 [H] → RCH₂COOH + 2 H₂O) could be investigated is described. This method was used for a detailed study of the enyzmology of the overall reaction (viz. acetyl phosphate to pyruvate) catalysed by pyruvate synthase in Clostridium sporogenes. The same method could be utilised to harness such reductive carboxylations for commercial biotransformations of xenobiotics. By adjusting the reaction conditions it was possible to alter the proportions of the products synthesised, and to synthesise compounds more reduced and/or with a greater number of carbon atoms than pyruvate.     

Purification of isoenzyme I of phospholipase C from Clostridium perfringens

V. I. Lenin Tashkent State University. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 888–890, November–December, 1988.     

Clostridium difficile toxin B: characterization and sequence of three peptides

The cytotoxin, also named toxin B, was isolated from a toxigenic strain of Clostridium difficile, purified to homogeneity and partially characterized. The purification procedure included ultrafiltration followed by anion-exchange chromatography. We noticed that a non-specific nucleic material eluted with the protein during the purification. The presence of these nucleic acids appeared to be important for the toxic activity of the protein. Some characteristics of the cytotoxin were examined, especially the amino acid composition and the sequence of three tryptic fragments.     

应用气相色谱法鉴定艰难梭菌

应用国产气相色谱仪,选用不锈钢色谱柱,在色谱柱柱温１４０℃、检测室温度１９０℃下采用１５％改性聚乙二醇固定液（ＦＦＡＰ）,以氮气作载体,将从临床肿瘤病人粪便中分离的５株艰难梭菌（Ｃｌｏｓｔｒｉｄｉｕｍｄｉｆｆｉｃｉｌｅ）菌株与１株参考株的脂肪酸代谢产物气相色谱图进行了对比分析,发现分离株与参考株的色谱图非常相似：均出现乙酸、异丁酸、丁酸、异戊酸、异己酸、丙酸、戊酸峰。其中乙酸、丁酸、异己酸峰较高;其次为异丁酸及异戊酸峰;而丙酸及戊酸峰则较低。     

l-Selective dipeptide synthesis using novel thermophilic enzyme from Clostridium sp.

A novel, inexpensive, thermophilic protease-type enzyme isolated from Clostridium thermohydrosulfuricum was used for dipeptide synthesis. The enzyme showed broad substrate selectivity and enantioselectivity towards l-amino acids in peptide bond formation.     

A possible oligosaccharide-conjugate vaccine candidate for Clostridium difficile is antigenic and immunogenic

Nosocomial infections with the Gram-positive pathogen Clostridium difficile pose a major risk for hospitalized patients and result in significant costs to health care systems. Here, we present the chemical synthesis of a PS-II hapten of a cell wall polysaccharide of hypervirulent ribotype 027 of C. difficile. Mice were immunized with a conjugate consisting of the synthetic hexasaccharide and the diphtheria toxoid variant CRM(197). The immunogenicity of the glycan repeating unit was demonstrated by the presence of specific IgG antibodies in the serum of immunized mice. Murine monoclonal antibodies interact with the synthetic hexasaccharide, as determined by microarray analysis. Finally, we found that specific IgA antibodies in the stool of hospital patients infected with C. difficile recognize the synthetic PS-II hexasaccharide hapten.     

Mode of action of Clostridium perfringens initiation protein (spore-lytic enzyme)

The extracellular initiation protein (IP; spore germination enzyme) produced by Clostridium perfringens was further purified and characterized. IP hydrolysed spore cortical fragments with the release of free amino groups. End group analysis of hydrolysed fragments indicated the presence of N-terminal alanine but no reducing sugars. Molecular weight analysis of IP- and lysozyme-treated fluorescamine-labelled cortical fragments indicated that IP acts only on peptidoglycan chains containing cross-linked peptide subunits. IP failed to hydrolyse a number of nitrophenyl-conjugated glucopyranosides and galactopyranosides. The results indicate that IP is an N-acetylmuramyl-L-alanine amidase.     

Electron Paramagnetic Resonance Spectroscopy of the Iron-Molybdenum Cofactor of Clostridium pasteurianum Nitrogenase

We report a computer simulation study of the electron paramagnetic resonance (EPR) spectral line shape of the iron-molybdenum cofactor of nitrogenase. The unusually broad and asymmetric line shape of the EPR spectrum can be interpreted in terms of a distribution of zero-field splitting parameters called D-strain. The best fit simulations were computed using D = 2.5 cm(-1) and E = 0.317 cm(-1) and distributions in D and E approximated by Gaussians of half-widths 0.446 cm(-1) and 0.108 cm(-1), respectively. The value of D estimated in the present work is smaller than previous estimates by others but consistent with the temperature dependence of the EPR spectrum. The large D-strain is most likely caused by an ensemble of nearly isoenergetic conformational states and should not be considered as being indicative of chemical inhomogeneity.     

Correlation between hydrogen bond lengths and reduction potentials in Clostridium pasteurianum rubredoxin

15N NMR hyperfine-shift data were collected for wild-type and site-specific mutant (V44I, V44A, and V44G) Clostridium pasteurianum rubredoxins in the oxidized state. Whereas most of the (15)N NMR signals did not exhibit large systematic changes upon mutation of residue 44, the signal from the backbone nitrogen of residue 44 itself (arrows) shifted by approximately 400 ppm. These shifts were used to determine the lengths of the hydrogen bond between the backbone amide of residue 44 and the side-chain sulfur of cysteine-44, which is covalently ligated to the iron of the metal center. The results, which demonstrated that this hydrogen bond is shorter in mutants with higher reduction potential, point to the importance of hydrogen bonds in modulating the reduction potential of iron-sulfur proteins.     

Immobilisation of Clostridium spp. for production of solvents and organic acids

This review summarises the high potential of immobilised cells systems for the fermentative production of compounds, mainly produced by representatives of the Clostridium genus. Microorganisms of Clostridium species are recognised as good producers of a wide range of chemicals in almost every sector of industry. The combination of this microorganism with its immobilisation opens up new possibilities and renders the fermentation process more sophisticated than in a free-cell system. This review provides a comprehensive summary of techniques used in immobilisation of Clostridium species with regard to specific products and types of fermentation. In addition, comparisons of particular types of immobilisation techniques used in fermentation processes are summarised by specific products.