Clostridia in soil of the Antarctica.

From the soil in the area around the Syowa Station, the East Ongul Island, the Antarctica, a total of 193 strains of clostridia were isolated and identified. It was surprising that the soil samples taken from the places which were considered to be scarcely contaminated by human beings and animals contained many clostridia. One hundred and fifty-five strains were assigned to 11 species, including C. perfringens, C. bifermentans, C. sordellii, C. sporogenes, C. plagarum, C. paraperfringens, C. septicum, C. tertium, C. cadaveris, C. butyricum and C. felsineum, but 38 strains remained unidentified. C. perfringens, C. bifermentans and C. sordellii were isolated very frequently and C. sporogenes less frequently. All the strains of C. sordellii were nonpathogenic and had almost the same characteristics as those of C. bifermentans except for the attitude in the urease test. The peculiar distribution and characteristics of the clostridia in the Antarctic soil were discussed in comparison with those found in the soil in Japan.     

Pseudomembranous colitis: isolation of two species of cytotoxic clostridia and successful treatment with vancomycin.

Lincomycin-resistant Clostridium sporogenes obtained from the stools of a patient with lincomycin-associated pseudomembranous colitis produced a heat-stable cytotoxin in low titre when grown in chopped meat medium. Vancomycin eradicated this strain and all other clostridia, and controlled the symptoms. When diarrhea recurred 7 days after treatment with vancomycin was stopped, clostridia including C. sporogenes and C. difficile were again isolated. The C. difficile produced a heat-labile cytotoxin in high titre that was unaffected by growth in various media and induced colitis in hamsters. Treatment with vancomycin, to which all the clostridia were sensitive, eradicated both toxic species and controlled the diarrhea. Antibiotic-induced pseudomembranous colitis may be associated with more than one species of toxin-producing clostridia. Vancomycin therapy should be continued for 10 days or more in patients with severe disease to eradicate the responsible organism.     

产溶剂梭菌分子遗传操作技术研究进展

产溶剂梭菌是一类重要的工业微生物.通过遗传改造以优化产溶剂梭菌的发酵性能一直是溶剂制造技术研究的重要课题,但长期受限于该类菌并不完善的遗传操作工具,未见明显突破.近年来,随着TargeTron基因中断、大片段基因整合等新技术和新方法的出现,其分子遗传改造已取得较大进展.文中对产溶剂梭菌的分子遗传操作工具研究进展进行了总结,并指出了现有技术在效率及全面性方面的不足.基于此,今后应进一步优化现有的梭菌基因失活技术,如建立基于同源重组的基因删除和替换;同时也应发展新的分子操作技术,如基因组多位点共编辑、多拷贝定点和随机整合等.     

Diversity of mesophilic clostridia in Costa Rican soils

Costa Rica is located in the Tropic, one of the most biologically diverse regions of the world; its soil is an important epicenter of biodiversity and Clostridium spp. are among the most frequent bacteria. The diversity of clostridia in Costa Rican soils and its possible association with geographic zone, pH or type of soil was studied in 117 soil samples: 18 from the Atlantic Zone, 30 from the Central Plateau, 30 from the Dry Pacific, 13 from the North Zone, and 26 from the South Pacific. The pH and the mesophilic clostridia species were determined for each sample. For bacterial isolation, a selective methodology for spores and pre-reduced anaerobically sterilized media were used. A total of 1945 strains of clostridia were isolated, 98% were identified and corresponded to 54 species; the most frequent species were C. subterminale (56%), C. oceanicum (51%), C. bifermentans and C. glycolicum (50%, each), C. sporogenes (49%), and C. sordellii (42%). An average of 7.1 species per sample was obtained; the Atlantic Zone showed the greatest diversity: 8.6 species per sample and a total of 45 species. Except for C. chauvoei, all described toxigenic clostridia species were isolated; C. sordellii (42%) and C. perfringens (38%) were the most frequent. No statistical relation could be established between geographic zone or type of soil and any species, showing that clostridia had a high adaptation capability to grow in different soil conditions; only some clostridia were isolated from very acidic samples while others from soils with a wide range of pH. In general, a uniform distribution of most species and a high variety of clostridia in Costa Rican soils were observed, in agreement with the high biodiversity described for other living beings in this country.     

A rapid test for the presumptive identification of Clostridium perfringens

A novel rapid method for the identification of colonies of Clostridium perfringens (key iD Lab M Ltd. Bury, UK) was evaluated. The method consists of a test strip containing substrates for pre-formed enzymes selected for optimum differentiation of C. perfringens from other clostridia. One hundred and forty-six strains of clostridia were tested using the key iD strip. The strip successfully confirmed the identity of all 73 strains of C. perfringens tested, and differentiated these from 73 strains of 20 other clostridial species. C. absonum and C. baratii, spedes which are very similar to C. perfringens, could also be differentiated by this method. The key iD strip is recommended for laboratories as a rapid alternative to more conventional tests for presumptive identification of C. perfringens.     

Molecular biological detection and characterization of Clostridium populations in municipal landfill sites

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.     

食气梭菌的研究进展

食气梭菌是一类主要的化能自养微生物,可利用二氧化碳(CO_2)和一氧化碳(CO)合成多种化学品和燃料,具有良好的工业应用前景。天然的食气梭菌吸收、固定和转化一碳气体速率较慢,能量代谢效率低且高值产物种类少。近年来,随着组学、分子遗传学工具以及生化分析技术的快速发展,食气梭菌的生理代谢特点及其相关分子机制、代谢工程设计、改造和发酵工艺等方面都得到广泛而深入的研究。本文针对近年来食气梭菌的研究进展进行了梳理和总结,以期能为这类重要工业微生物的基础和应用研究,以及一碳气体的生物转化利用提供参考。     

Iron reduction and gley formation by nitrogen-fixing clostridia

Summary Studies on iron reduction and the mechanism of gley formation by nitrogen-fixing clostridia are reported. Up to 10⁶ cells/g soil of anaerobic, nitrogen-fixing clostridia, capable of reducing iron (III)-oxide, were counted in samples taken from various top soils. In a gleyed subsoil as many as 10⁵ bacteria per g soil, capable of reducing and fixing nitrogen, were enumerated using the most probable number technique. In general, the ratio of the auxotrophic iron reducing clostridia (glucose+yeast extract fermenters) to the prototrophic iron reducing flora (glucose fermenters) was found much larger in the top soil samples than in those derived from various gleyed subsoils.An enrichment method for the isolation of nitrogen-fixing, iron reducing clostridia of the butyric-butyl type is described. The iron reducing capacity of this type of clostridia as well as of Clostridium pasteurianum was determined quantitatively. Generally, the presence of soil or soil extract enhanced the amount of dissolved ferrous iron, both with butyric acid fermenters and with Cl. pasteurianum.When enriched iron reducing clostridia were incubated anaerobically under N₂-atmosphere in a sterile, red-colored, lateritic type of soil with glucose, intense gleying occurred within a few days. Microscopic observations indicated the presence of sporeforming bacteria of the Clostridium butyricum type or related species.The biological and chemical mechanism of gley formation is discussed.This research was started at the Institut für Landwirtschaftliche Mikrobiologie, Justus Liebig-Universität, Giessen, Germany.     

Metabolic regulation in solventogenic clostridia: regulators,mechanisms and engineering

Solventogenic clostridia, a group of important industrial microorganisms, have exceptional substrate and product diversity, capable of producing a series of two-carbon and even long-chain chemicals and fuels by using various substrates, including sugars, cellulose and hemicellulose, and C1 gases. For the sake of in-depth understanding and engineering these anaerobic microorganisms for broader applications, studies on metabolic regulation of solventogenic clostridia had been extensively carried out during the past ten years, based on the rapid development of various genetic tools. To date, a number of regulators that are essential for cell physiological and metabolic processes have been identified in clostridia, and the relevant mechanisms have also been dissected, providing a wealth of valuable information for metabolic engineering. Here, we reviewed the latest research progresses on the metabolic regulation for chemical production and substrate utilization in solventogenic clostridia, by focusing on three typical Clostridium species, the saccharolytic C. acetobutylicum and C. beijerinckii, as well as the gas-fermenting C. ljungdahlii. On this basis, future directions in the study and remodeling of clostridial regulation systems, were proposed for effective modification of these industrially important anaerobes.     

Molecular Biological Detection and Characterization of Clostridium Populations in Municipal Landfill Sites

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.     

Acetogenic and sulfate-reducing bacteria inhabiting the rhizoplane and deep cortex cells of the sea grass Halodule wrightii.

Recent declines in sea grass distribution underscore the importance of understanding microbial community structure-function relationships in sea grass rhizospheres that might affect the viability of these plants. Phospholipid fatty acid analyses showed that sulfate-reducing bacteria and clostridia were enriched in sediments colonized by the sea grasses Halodule wrightii and Thalassia testudinum compared to an adjacent unvegetated sediment. Most-probable-number analyses found that in contrast to butyrate-producing clostridia, acetogens and acetate-utilizing sulfate reducers were enriched by an order of magnitude in rhizosphere sediments. Although sea grass roots are oxygenated in the daytime, colorimetric root incubation studies demonstrated that acetogenic O-demethylation and sulfidogenic iron precipitation activities were tightly associated with washed, sediment-free H. wrightii roots. This suggests that the associated anaerobes are able to tolerate exposure to oxygen. To localize and quantify the anaerobic microbial colonization, root thin sections were hybridized with newly developed (33)P-labeled probes that targeted (i) low-G+C-content gram-positive bacteria, (ii) cluster I species of clostridia, (iii) species of Acetobacterium, and (iv) species of Desulfovibrio. Microautoradiography revealed intercellular colonization of the roots by Acetobacterium and Desulfovibrio species. Acetogenic bacteria occurred mostly in the rhizoplane and outermost cortex cell layers, and high numbers of sulfate reducers were detected on all epidermal cells and inward, colonizing some 60% of the deepest cortex cells. Approximately 30% of epidermal cells were colonized by bacteria that hybridized with an archaeal probe, strongly suggesting the presence of methanogens. Obligate anaerobes within the roots might contribute to the vitality of sea grasses and other aquatic plants and to the biogeochemistry of the surrounding sediment.     

Superoxide dismutase in anaerobes: survey.

Superoxide dismutase (SOD) was present in 23 of 28 strains of the genus Bacteroides tested. Several clostridia, anaerobic cocci, and anaerobic, grampositive, nonsporing rods contained measureable SOD, but the frequency of SOD occurrence was much lower than in the bacteroides. These data indicate that there is a large variation in SOD levels between genera and among species within a genus of anaerobic bacteria. There was also no correlation between source of isolate, SOD levels, and presumed pathogenicity of the isolate.     

Differentiation of the main species of Clostridium by gas chromatography

For the first time in the USSR the properties of microorganisms of the genus Clostridium have been studied with the use of the gas-chromatographic techniques. The analysis of the quantitative and qualitative composition of extracellular alcohols and carboxylic acids in 99 museum and newly isolated strains of 18 Clostridium species has made it possible to classify these microorganisms with 7 sharply differing groups. The above techniques permit the classification of clostridia with one of the groups within 2 hours if the microbial cultures have been grown in glucose-containing peptone yeast medium.     

Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia.

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.     

Clostridial iron-sulphur proteins

Iron-sulfur proteins are ubiquitous catalysts of a wide range of biological reactions, and are particularly abundant in clostridia which lack the ability to synthesize hemes. The development of research on these metalloproteins has therefore been strongly associated with biochemical investigations of clostridial metabolism. Major breakthroughs in the field, from the first isolation of an iron-sulfur protein in 1962, to the recent determination of an Fe-hydrogenase structure, have been made with clostridia. These data, as well as others obtained through studies on clostridia, are transferable to many other bioenergetic machineries, due to the strong phylogenetic conservation of some important components. For instance, clear homologies exist between constituents of the anaerobic electron transfer chains in clostridia and aerobic respiratory chains. The contribution of iron-sulfur proteins to the biotechnological and medical significance of clostridia is also discussed. Structural and functional genomics are expected to bring forth a wealth of novel data on clostridia and iron-sulfur proteins.     

Clostridia from Grana Cheese

Forty strains of proteolytic and saccharolytic clostridia isolated from Grana cheese were re-identified by DNA-DNA homology. Thirty culture collection strains were also examined for comparison. Organisms of the species Clostridium tyrobutyricum which present variability in phenotypic characteristics were found to constitute a homogeneous group, genetically unrelated to all the reference or competitor strains used. The proteolytic clostridia from cheese show a high level of homology with the reference strains C.sporogenes ATCC 319 and C. sporogenes ATCC 3584 and negligible homology with C. bifermentans ATCC 19299. Three strains with phenotypic characters very similar to those of the species C. butyricum present a level of DNA homology with C. butyricum ATCC 19398 ranging from 61–71%. In the light of the results obtained some taxonomic conclusions have been drawn.     

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats

AIMS: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. METHODS AND RESULTS: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. CONCLUSIONS: The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.     

Reduction of uranium(VI) to uranium(IV) by clostridia

Several different species of clostridia reduced U(VI) to U(IV) to various degrees. The optimal pH for U(VI) reduction is 5 to 6 in most cases; a Clostridium sp. showed the highest rate at pH 4. Nitrate did not affect U(VI) reduction, indicating that this process in clostridia is nitrate independent.     

16S rDNA analysis reveals phylogenetic diversity among the polysaccharolytic clostridia

Abstract Small subunit rDNA sequences were determined for 13 mesophilic, polysaccharolytic, mainly cellulolytic species of the genus Clostridium and one cellulolytic Eubacterium specues. Sequences were compared to those of 36 representatives of mesophilic and thermophilic clostridia, including those of nine thermophilic polysaccharolytic species published previously. The majority of strains group with 23S rRNA clusters I and III, while the others group with the thermophilic polysaccharolytic clostridia, i.e. C. stercorarium, C. thermolacticum and C. thermocellum . Lack of close genetic relationships between the various polysaccharolytic species is unexpected and may indicate that these biotechnologically important organisms differ with respect to the enzymology of polysaccharolytic degradation as well.