Changes in the cell wall of Clostridium species following passage in animals

Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice. We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae. Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates. A thick granular wall prior to animal inoculation was shown only in C. perfringens, C. butyricum, and C. difficile. This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K. pneumoniae or B. fragilis.     

Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction

A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types A to F, a type E toxin-producing C. butyricum strain, and a type F toxin-producing C. baratii strain, but no PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR-based detection method can be used for the rapid diagnosis of botulism.     

Utilization of (E)-2-butenoate (Crotonate) by Clostridium kluyveri and some other Clostridium species

Clostridium La 1 obtained from a Clostridium kluyveri culture was compared with a typical C. kluyvery strain (DSM 555). The former grows on crotonate and is unable to use ethanol-acetate as carbon sources. The latter grows on crotonate only after long adaptation periods. Resting cells of both strains show also pronounced differences in the fermentation of crotonate. This holds even for C. kluyveri grown on crotonate. Besides several other differences the most striking is that there is no hybridization between the DNA of both strains.Crotonate seems not to be a very special carbon source since C. butyricum and C. pasteurianum grow on crotonate medium supplemented by peptone and yeast extract.Non Standard Abbreviations EA-medium ethanol and acetate as carbon source - C-medium crotonate as carbon source - DSM Deutsche Sammlung von Mikroorganismen     

The fermentation of glycerol byClostridium butyricum LMG 1212t2 and 1213t1 andC. pasteurianum LMG 3285

Summary In batch culture on reiinforced clostridial medium strain-dependent product profiles from glycerol revealed unusual fermentation products such as propionate and n-propanol with Clostridium butyricum LMG 1213t₁, and 1,3-propanediol with C. butyricum LMG 1212t₂ and C. pasteurianium LMG 3285. Only the latter two strains were able to grow on glycerol in a minimal medium. Nicotinamide adenine dinucleotide (NAD⁺)-dependent dehydrogenase activities were detected with 1,3-propanediol and n-butanol as substrate (the latter only after a lag period) in cell-free extracts of C. butyricum LMG 1212t₂ and with 1,3-propanediol, n-butanol and ethanol in cell-free extracts of C. pasteurianum LMG 3285. The data indicated the existance of a specific 1,3-propanediol dehydrogenase in both organisms. In a chemostat, C. butyricum LMG 1212t₂ converted 65% of the glycerol supplied as sole carbon and energy source to 1,3-propanediol without H₂ production. Increasing concentration of acetate in the inflow medium resulted in less 1,3-propanediol and more butyrate and H₂ production. C. pasteurianum LMG 3285 converted somewhat more than half of the glycerol supplied as sole energy and carbon source to n-butanol with significant concomitant H₂ production. This fermentation pattern was hardly affected by acetate as co-substrate. Offprint requests to: P. De Vos     

Proliferation of diversified clostridial species during biological soil disinfestation incorporated with plant biomass under various conditions

Biological soil disinfestation (BSD) involves the anaerobic decomposition of plant biomass by microbial communities leading to control of plant pathogens. We analyzed bacterial communities in soil of a model experiment of BSD, as affected by biomass incorporation under various conditions, to find out the major anaerobic bacterial groups which emerged after BSD treatments. The soil was treated with Brassica juncea plants, wheat bran, or Avena strigosa plants, irrigated at 20 or 30 % moisture content and incubated at 25–30 °C for 17 days. The population of Fusarium oxysporum f. sp. spinaciae incorporated at the start of the experiment declined markedly for some BSD conditions and rather high concentrations of acetate and butyrate were detected from these BSD-treated soils. The polymerase chain reaction-denaturing gradient gel electrophoresis analysis based on the V3 region of 16S rRNA gene sequences from the soil DNA revealed that bacterial profiles greatly changed according to the treatment conditions. Based on the clone library analysis, phylogenetically diverse clostridial species appeared exceedingly dominant in the bacterial community of BSD soil incorporated with Brassica plants or wheat bran, in which the pathogen was suppressed completely. Species in the class Clostridia such as Clostridium saccharobutylicum, Clostridium acetobutylicum, Clostridium xylanovorans, Oxobacter pfennigii, Clostridium pasteurianum, Clostridium sufflavum, Clostridium cylindrosporum, etc. were commonly recognized as closely related species of the dominant clone groups from these soil samples.     

24株鸡源丁酸梭菌的分离鉴定及耐药基因与毒力基因携带情况

[目的]旨在对鸡源丁酸梭菌进行分离鉴定与安全性评估.[方法]利用厌氧培养方法对源自汶上芦花鸡与SPF鸡粪便样品进行丁酸梭菌的分离与纯化,挑选可疑菌落进行微生物质谱鉴定,进一步通过16S rRNA基因测序进行鉴定,16S rRNA测序结果与NCBI核苷酸数据库中丁酸梭菌的16S rRNA序列进行同源性分析;同时,进行所有...     

Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis

Summary Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.     

Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers

Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen‐producing strains. Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster‐specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. Conclusions: The newly designed [FeFe] hydrogenase cluster‐specific primers, targeting the cluster‐conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. Significance and Impact of the Study: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus.     

Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.     

Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii

Several solvent-producing clostridia, including Clostridium acetobutylicum and C. beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of ¹⁵N₂ into cellular material. The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron–molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C. acetobutylicum or C. beijerinckii or both. The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVω and nifVα genes. The corresponding nif genes of these three clostridial species are highly related to each other. However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C. pasteurianum. However, the nifN-B and nifVω genes of C. pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C. acetobutylicum. C. acetobutylicum and C. beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species. Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 281–286. Received 02 September 2000/ Accepted in revised form 22 November 2000     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Fermentation of raw glycerol to 1,3-propanediol by new strains ofClostridium butyricum

Industrial glycerol obtained through the transesterification process using rapeseed oil did not support growth of several strains ofClostridium butyricum obtained from bacterial culture collections. Ten new strains ofC. butyricum were obtained from mud samples from a river, a stagnant pond, and a dry canal. These new isolates fermented the commercial glycerol and produced 1,3-propanediol as a major fermentation product with concomitant production of acetic and butyric acids. Four of the ten isolates were able to grow on industrial glycerol obtained from rapeseed oil. One strain,C. butyricum E5, was very resistant to high levels of glycerol and 1,3-propanediol. Using fed-batch fermentation, 109 g L^–1 of industrial glycerol were converted into 58 g of 1,3-propanediol, 2.2 g of acetate and 6.1 g of butyrate per liter.     

Genetic manipulation of butyrate formation pathways in Clostridium butyricum

Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H₂, it can produce acids (lactic, acetic and butyric acids) and CO₂, as well as a small amount of ethanol. It has been proposed that elimination of competing pathways, such as the butyrate formation pathway, should increase H₂ yields in Clostridium species. However, the application of this strategy has been hindered by the unavailability of genetic tools for these organisms. In this study, we successfully transferred a plasmid (pMTL007) to C. butyricum by inter-specific conjugation with Escherichia coli and disrupted hbd, the gene encoding β-hydroxybutyryl-CoA dehydrogenase in C. butyricum. Fermentation data showed that inactivation of hbd in C. butyricum eliminated the butyrate formation pathway, resulting in a significant increase in ethanol production and an obvious decrease in H₂ yield compared with the wild type strain. However, under low partial pressure of H₂, the hbd-deficient strain showed increased H₂ production with the simultaneous decrease of ethanol production, indicating that H₂ production by C. butyricum may compete for NADH with the ethanol formation pathway. Together with the discovery of a potential bifurcating hydrogenase, this study extends our understanding of the mechanism of H₂ production by C. butyricum.     

Heterologous expression of clostridial hydrogenase in the Cyanobacterium synechococcus PCC7942

The Clostridium pasteurianum hydrogenase I has been expressed in the cyanobacterium Synechococcus PCC7942. The Shine-Dalgarno sequence of the structural gene encoding hydrogenase I from C. pasteurianum was changed to that of the cat (chloramphenicol acetyltransferase) gene. The hydrogenase gene was cloned downstream of a strong promoter, isolated from Synechococcus PCC7942, with the cat gene as a reporter gene. Expression of clostridial hydrogenase was confirmed by Western and Northern blot analyses in Synechococcus and Escherichia coli, whereas in vivo/in vitro measurements and activity staining of soluble proteins separated on non-denaturing polyacrylamide gels revealed functional expression of hydrogenase only in cyanobacterial cells. The changed Shine-Dalgarno sequence appeared to be essential for the functional expression of clostridial hydrogenase in Synechococcus, but had no influence on the expression and activity of clostridial hydrogenase expressed in E. coli.     

Distribution of the Rubredoxin Gene Among the Clostridium butyricum Species

With PCR methods, the rubredoxin gene was systematically identified among 11 strains of Clostridium butyricum; this ubiquity means major functions in the metabolism of the Clostridia. The 11 PCR products allowed deduction of a sequence of 26 amino acids corresponding to positions 11–36 of the rubredoxin. They all contained the tyrosines at positions 11 and 13 and the phenylalanine at position 30 characteristic of the rubredoxin, but differed at positions 14–17, 20, 25, 29, and 31, allowing determination of three types of rubredoxins among these 11 strains of C. butyricum. Received: 13 October 1998 / Accepted: 23 November 1998     

Hydrogenase Activity in Deeply Buried Sediments of the Arctic and North Atlantic Oceans

The goal of this study was to measure hydrogenase activity in oceanic sediments of the Lomonosov Ridge (Arctic Ocean) and of the Porcupine Seabight (North Atlantic), and to understand how its distribution varied depending on geochemical and lithological characteristics of the sediment. Hydrogenase activity was found at all sites, and absolute values and downhole distributions varied widely within and between sites. At the Lomonosov Ridge, hydrogenase was below detection in the top 190 meters of sediment, but high levels were measured in the organic carbon-rich layers below this depth. Activity at Challenger Mound, Porcupine Seabight (Site 1317) was one to two orders of magnitude higher than the upslope or downslope sites (1318 and 1316, respectively), and was higher in the mound than below the mound base. Consistent with the interpretation of hydrogenase as an indicator of microbial activity, cells were present in all hydrogenase-positive samples or in nearby horizons. Cell-specific activity values were as much as 1000 fold lower than those of cultured Clostridium pasteurianum, with the values from the highest activity sediments approaching those of C. pasteurianum more closely. This suggests that little adaptation in hydrogenase activity might be necessary to support H₂ metabolism in deeply buried sediments that contain relatively high levels of substrates.     

Characterization of Neurotoxigenic Clostridium butyricum Strain by DNA Hybridization Test and by in vivo and in vitro Germination Tests of Spores

The germination of spores of a neurotoxigenic Clostridium butyricum strain (BL 6340), which was isolated from infant botulism in Italy, and that of a non-toxigenic C. butyricum type strain (NCIB 7423) were studied. The spores of BL 6340 strain were killed at 80 C for 10 min, and required the mixture of L-alanine, L-lactate, glucose and bicarbonate for their optimal germination. These characteristics are the same as those of Clostridium botulinum type E strain, but different from those of NCIB 7423 strain. In a hybridization test, however, the labeled DNAs extracted from NCIB 7423 strain highly (98%) hybridized to the DNAs of the BL 6340 strain, but little (45%) to the DNAs of C. botulinum type E strain. The biochemical properties of the BL 6340 and NCIB 7423 strains were identical, but different from those of C. botulinum type E. These data confirmed that the BL 6340 strain belongs to C. butyricum species, but that only its characteristics of toxin production, its minimum requirements for germination, and the behavior of its spores to heat treatment are the same as those of C. botulinum type E. When conventionally raised suckling mice were injected with 5 × 10⁷ spores of BL 6340 strain intra- or orogastrically, botulism was not observed. However, 8- to 13-day-old mice had type E botulinum toxin in the large intestine 3 days after introduction of its spores.     

Isolation of a new butanol-producing Clostridium strain: High level of hemicellulosic activity and structure of solventogenesis genes of a new Clostridium saccharobutylicum isolate

New isolates of solventogenic bacteria exhibited high hemicellulolytic activity. They produced butanol and acetone with high selectivity for butanol (about 80% of butanol from the total solvent yield). Their 16S rDNA sequence was 99% identical to that of Clostridium saccharobutylicum. The genes responsible for the last steps of solventogenesis and encoding crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, 3-hydroxybutyryl-CoA dehydrogenase, alcohol dehydrogenase, CoA-transferase (subunits A and B), acetoacetate decarboxylase, and aldehyde dehydrogenase were identified in the new C. saccharobutylicum strain Ox29 and cloned into Escherichia coli. The genes for crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, and 3-hydroxybutyryl-CoA dehydrogenase composed the bcs-operon. A monocistronic operon containing the alcohol dehydrogenase gene was located downstream of the bcs-operon. Genes for aldehyde dehydrogenase, CoA-transferase (subunits A and B), and acetoacetate decarboxylase composed the sol-operon. The gene sequences and the gene order within the sol- and bcs-operons of C. saccharobutylicum Ox29 were most similar to those of Clostridium beijerinckii. The activity of some of the bcs-operon genes, expressed in heterologous E. coli, was determined.