| 1. |
Dhalluin A,
Lemée L,
Pestel-Caron M,
Mory F,
Leluan G,
Lemeland JF,
Pons JL,
( 2003 ) Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene. PMID : 12747415 : DOI : 10.1078/072320203322337362 Abstract >>
Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.
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2. |
Nakayama J,
Akkermans AD,
De Vos WM,
( 2003 ) High-throughput PCR screening of genes for three-component regulatory system putatively involved in quorum sensing from low-G + C gram-positive bacteria. PMID : 12723594 : DOI : 10.1271/bbb.67.480 Abstract >>
Quorum sensing of gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.
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3. |
Raynaud C,
Sarçabal P,
Meynial-Salles I,
Croux C,
Soucaille P,
( 2003 ) Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum. PMID : 12704244 : DOI : 10.1073/pnas.0734105100 PMC : PMC154289 Abstract >>
The genes encoding the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum VPI1718 were characterized from a molecular and a biochemical point of view. This operon is composed of three genes, dhaB1, dhaB2, and dhaT. When grown in a vitamin B12-free mineral medium with glycerol as carbon source, Escherichia coli expressing dhaB1, dhaB2, and dhaT produces 1,3-PD and high glycerol dehydratase and 1,3-PD dehydrogenase activities. dhaB1 and dhaB2 encode, respectively, a new type of glycerol dehydratase and its activator protein. The deduced proteins DhaB1 and DhaB2, with calculated molecular masses of 88,074 and 34,149 Da, respectively, showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases and pyruvate formate lyases activating enzymes and their homologues. The 1,158-bp dhaT gene codes for a 1,3-PD dehydrogenase with a calculated molecular mass of 41,558 Da, revealing a high level of identity with other DhaT proteins from natural 1,3-PD producers. The expression of the 1,3-PD operon in C. butyricum is regulated at the transcriptional level, and this regulation seems to involve a two-component signal transduction system DhaASDhaA, which may have a similar function to DhaR, a transcriptional regulator found in other natural 1,3-PD producers. The discovery of a glycerol dehydratase, coenzyme B12 independent, should significantly influence the development of an economical vitamin B12-free biological process for the production of 1,3-PD from renewable resources.
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4. |
Wang X,
Maegawa T,
Karasawa T,
Kozaki S,
Tsukamoto K,
Gyobu Y,
Yamakawa K,
Oguma K,
Sakaguchi Y,
Nakamura S,
( 2000 ) Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains. PMID : 11055954 : DOI : 10.1128/aem.66.11.4992-4997.2000 PMC : PMC92410 Abstract >>
Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.
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5. |
Wiedmann M,
Arcuri EF,
( 2000 ) Phylogeny and functional conservation of sigma(E) in endospore-forming bacteria. PMID : 10878124 : DOI : 10.1099/00221287-146-7-1593 Abstract >>
Conservation of the sporulation processes between Bacillus spp. and Clostridium spp. was investigated through evolutionary and complementation analyses of sigma(E). Alignment of partial predicted sigma(E) amino acid sequences from three Bacillus spp., Paenibacillus polymyxa and five Clostridium spp. revealed that amino acid residues previously reported to be involved in promoter utilization (M124, E119 and N120) and strand opening (C117) are conserved among all these species. Phylogenetic analyses of various sigma factor sequences from endospore-forming bacteria revealed that homologues of sigma(E), sigma(K) and sigma(G) clustered together regardless of genus, suggesting a common origin of sporulation sigma factors. The functional equivalence between Clostridium acetobutylicum sigma(E) and Bacillus subtilis sigma(E) was investigated by complementing a non-polar B. subtilis sigma(E) null mutant with the spoIIG operon from either B. subtilis (spoIIG(Bs)) or C. acetobutylicum (spoIIG(Ca)). Single-copy integration of spoIIG(Bs) into the amyE locus of the sigma(E) null mutant completely restored the wild-type sporulation phenotype, while spoIIG(Ca) only partially restored sporulation. Maximal expression of spoIIG(Ca)-lacZ occurred approximately 12 h later than maximal expression of spoIIG(Bs)-lacZ. Differences in temporal expression patterns for spoIIG(Ca) and spoIIG(Bs) in the B. subtilis background may at least partially explain the observed sporulation complementation phenotypes. This study suggests a common phylogenetic ancestor for sigma(E) in Bacillus spp. and Clostridium spp., although regulation of sigma(E) expression may differ in these two genera.
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6. |
Amine J,
Raval G,
Petitdemange H,
( 1999 ) Distribution of the rubredoxin gene among the Clostridium butyricum species. PMID : 10355113 : Abstract >>
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.
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7. |
Poulet S,
Hauser D,
Quanz M,
Niemann H,
Popoff MR,
( 1992 ) Sequences of the botulinal neurotoxin E derived from Clostridium botulinum type E (strain Beluga) and Clostridium butyricum (strains ATCC 43181 and ATCC 43755). PMID : 1543481 : DOI : 10.1016/0006-291x(92)91615-w Abstract >>
Recently, it has been shown that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755), isolated from cases of infant botulism, produce a botulinal neurotoxin type E (BoNT/E). Here we have determined the nucleotide sequences of the BoNT/E genes of these two C. butyricum strains and from C. botulinum E strain Beluga. We show that the sequences of the BoNT/E genes from the two C. butyricum strains are identical and differ in only 64 positions resulting in 39 amino acid changes (97% identity at the amino acid level) from that derived from C. botulinum. Our data suggest a transfer of the BoNT/E gene from C. botulinum to the originally nontoxigenic C. butyricum strains.
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8. |
Hill JE,
Penny SL,
Crowell KG,
Goh SH,
Hemmingsen SM,
( 2004 ) cpnDB: a chaperonin sequence database. PMID : 15289485 : DOI : 10.1101/gr.2649204 PMC : PMC509277 Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
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9. |
O'Brien JR,
Raynaud C,
Croux C,
Girbal L,
Soucaille P,
Lanzilotta WN,
( 2004 ) Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization. PMID : 15096031 : DOI : 10.1021/bi035930k Abstract >>
The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.
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10. |
Franciosa G,
Pourshaban M,
De Luca A,
Buccino A,
Dallapiccola B,
Aureli P,
( 2004 ) Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography. PMID : 15240298 : DOI : 10.1128/AEM.70.7.4170-4176.2004 PMC : PMC444775 Abstract >>
Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.
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11. |
Huggins AS,
Bannam TL,
Rood JI,
( 1992 ) Comparative sequence analysis of the catB gene from Clostridium butyricum. PMID : 1489203 : DOI : 10.1128/aac.36.11.2548 PMC : PMC284373 Abstract >>
Sequence analysis of the Clostridium butyricum chloramphenicol acetyltransferase (CAT) gene, catB, showed that it encoded a CAT monomer of 219 amino acids with a molecular weight of 26,114. Comparison of the deduced amino acid sequence of the CATB monomer to those of sixteen other CATs showed that it was most closely related to the CATQ monomer from Clostridium perfringens.
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12. |
Calusinska M,
Joris B,
Wilmotte A,
( 2011 ) Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers. PMID : 21838748 : DOI : 10.1111/j.1472-765X.2011.03135.x Abstract >>
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. 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. 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. 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.
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13. |
Benson MA,
Fu Z,
Kim JJ,
Baldwin MR,
( 2011 ) Unique ganglioside recognition strategies for clostridial neurotoxins. PMID : 21849494 : DOI : 10.1074/jbc.M111.272054 PMC : PMC3190786 Abstract >>
Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E �K H �K SXWY �K G, with additional stabilizing interactions provided by two arginine residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.
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14. |
Raynaud C,
Lee J,
Sarçabal P,
Croux C,
Meynial-Salles I,
Soucaille P,
( 2011 ) Molecular characterization of the glycerol-oxidative pathway of Clostridium butyricum VPI 1718. PMID : 21478343 : DOI : 10.1128/JB.00112-11 PMC : PMC3133205 Abstract >>
The glycerol oxidative pathway of Clostridium butyricum VPI 1718 plays an important role in glycerol dissimilation. We isolated, sequenced, and characterized the region coding for the glycerol oxidation pathway. Five open reading frames (ORFs) were identified: dhaR, encoding a putative transcriptional regulator; dhaD (1,142 bp), encoding a glycerol dehydrogenase; and dhaK (995 bp), dhaL (629 bp), and dhaM (386 bp), encoding a phosphoenolpyruvate (PEP)-dependent dihydroxyacetone (DHA) kinase enzyme complex. Northern blot analysis demonstrated that the last four genes are transcribed as a 3.2-kb polycistronic operon only in glycerol-metabolizing cultures, indicating that the expression of this operon is regulated at the transcriptional level. The transcriptional start site of the operon was determined by primer extension, and the promoter region was deduced. The glycerol dehydrogenase activity of DhaD and the PEP-dependent DHA kinase activity of DhaKLM were demonstrated by heterologous expression in different Escherichia coli mutants. Based on our complementation experiments, we proposed that the HPr phosphoryl carrier protein and His9 residue of the DhaM subunit are involved in the phosphoryl transfer to dihydroxyacetone-phosphate. DhaR, a potential regulator of this operon, was found to contain conserved transmitter and receiver domains that are characteristic of two-component systems present in the AraC family. To the best of our knowledge, this is the first molecular characterization of a glycerol oxidation pathway in a Gram-positive bacterium.
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15. |
Cai G,
Jin B,
Saint C,
Monis P,
( 2011 ) Genetic manipulation of butyrate formation pathways in Clostridium butyricum. PMID : 21787814 : DOI : 10.1016/j.jbiotec.2011.07.004 Abstract >>
Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H?, it can produce acids (lactic, acetic and butyric acids) and CO?, 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 H? 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 �]-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 H? yield compared with the wild type strain. However, under low partial pressure of H?, the hbd-deficient strain showed increased H? production with the simultaneous decrease of ethanol production, indicating that H? 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 H? production by C. butyricum.
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16. |
Ferraris L,
Butel MJ,
Aires J,
( 2010 ) Antimicrobial susceptibility and resistance determinants of Clostridium butyricum isolates from preterm infants. PMID : 20822886 : DOI : 10.1016/j.ijantimicag.2010.07.005 Abstract >>
This study reports the antibiotic susceptibility and genetic resistance determinants of 39 Clostridium butyricum strains isolated from the faeces of preterm infants as well as one reference strain. Results showed that all the strains were susceptible to cefoxitin, imipenem, vancomycin, tigecycline, metronidazole, chloramphenicol and linezolid. Resistance was observed to clindamycin (100%), penicillin G, amoxicillin and piperacillin (15%), tetracycline (7.5%) and erythromycin (5%). Investigation of the genetic basis of the observed resistance phenotypes showed that resistance to penicillin was due to �]-lactamase activity and that resistance to tetracycline involved tet(O) or tet(O/32/O) homologue genes. Clindamycin and erythromycin resistance may involve another genetic determinant, different from those commonly described for clostridia.
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17. |
Nakanishi S,
Tanaka M,
( 2010 ) Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli. PMID : 19840859 : DOI : 10.1016/j.anaerobe.2009.10.002 Abstract >>
A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588)--a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.
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18. |
Agarwal R,
Swaminathan S,
( 2008 ) SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E. PMID : 18658150 : DOI : 10.1074/jbc.M803756200 PMC : PMC2533769 Abstract >>
Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.
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19. |
Wang MY,
Olson BH,
Chang JS,
( 2007 ) Improving PCR and qPCR detection of hydrogenase A (hydA) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin. PMID : 17909787 : DOI : 10.1007/s00253-007-1196-1 Abstract >>
Detection of hydA genes of Clostridia spp. using degenerative and species specific primers for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to polymerase chain reaction (PCR) and quantitative PCR (qPCR) reactions. BSA concentrations ranging from 100 to 400 ng/microl were examined using pure cultures and a variety of environmental samples as test targets. A BSA concentration of 100 ng/microl, which is lower than previously reported in the literature, was found to be most effective in improving the detection limit. The brightness of amplicons with 100 ng/mul BSA increased in ethidium bromide-treated gels, the minimum detection limit with BSA was at least one log greater, and cycle threshold (C(T)) values were lower than without BSA in qPCR indicating improved detection of target deoxyribonucleic acid for most samples tested. Although amplicon visualization was improved at BSA concentrations greater than or equal to 100 ng/microl, gene copy numbers detected by qPCR were less, C(T) values were increased, and T(m) values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that BSA at 100 ng/microl reduced the variability of peak areas and T(m) values.
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20. |
Bouvet P,
Ferraris L,
Dauphin B,
Popoff MR,
Butel MJ,
Aires J,
( 2014 ) 16S rRNA gene sequencing, multilocus sequence analysis, and mass spectrometry identification of the proposed new species "Clostridium neonatale". PMID : 25232167 : DOI : 10.1128/JCM.00477-14 PMC : PMC4313276 Abstract >>
In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, "Clostridium neonatale." To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies.
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21. |
Dover N,
Barash JR,
Burke JN,
Hill KK,
Detter JC,
Arnon SS,
( 2014 ) Arrangement of the Clostridium baratii F7 toxin gene cluster with identification of a �m factor that recognizes the botulinum toxin gene cluster promoters. PMID : 24853378 : DOI : 10.1371/journal.pone.0097983 PMC : PMC4031146 Abstract >>
Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative �m factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted �m factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the �m70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.
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22. |
( 1997 ) Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-alpha-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli. PMID : 9353929 : DOI : 10.1099/00221287-143-10-3287 Abstract >>
An Escherichia coli clone was detected in a Clostridium butyricum NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1.8 kb NspHI-Scal fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57,184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequences identity was apparent with the 4-alpha-glucano-transferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and E. coli (16.2%). The clostridial gene (designated maIQ) was followed by a second ORF which, through its homology to the equivalent enzymes of E. coli and S. pneumoniae, was deduced to encode maltodextrin phosphorylase (MaIP). The translation stop codon of MaIQ overlapped the translation start codon of the putative maIP gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-alpha-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant C. butyricum enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the E. coli MaIQ protein, but differs significantly from the glucanotransferase of Thermotoga maritima, which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase provides C. butyricum with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves.
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23. |
( 1997 ) A gene (plsD) from Clostridium butyricum that functionally substitutes for the sn-glycerol-3-phosphate acyltransferase gene (plsB) of Escherichia coli. PMID : 9393688 : DOI : 10.1128/jb.179.23.7257-7263.1997 PMC : PMC179674 Abstract >>
The sn-glycerol-3-phosphate acyltransferase (plsB) of Escherichia coli is a key regulatory enzyme that catalyzes the first committed step in phospholipid biosynthesis. We report the initial characterization of a novel gene (termed plsD) from Clostridium butyricum, cloned based on its ability to complement the sn-glycerol-3-phosphate auxotrophic phenotype of a plsB mutant strain of E. coli. Unlike the 83-kDa PlsB acyltransferase from E. coli, the predicted plsD open reading frame encoded a protein of 26.5 kDa. Two regions of strong homology to other lipid acyltransferases, including PlsB and PlsC analogs from mammals, plants, yeast, and bacteria, were identified. PlsD was most closely related to the 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC) gene family but did not complement the growth of plsC(Ts) mutants. An in vivo metabolic labeling experiment using a plsB plsX plsC(Ts) strain of E. coli confirmed that the plsD expression restored the ability of the cells to synthesize 1-acyl-glycerol-3-phosphate. However, glycerol-3-phosphate acyltransferase activity was not detected in vitro in assays using either acyl-acyl carrier protein or acyl coenzyme A as the substrate.
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24. |
( 1993 ) 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. PMID : 8355622 : DOI : 10.1111/j.1348-0421.1993.tb03227.x Abstract >>
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.
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25. |
Brown DP,
Ganova-Raeva L,
Green BD,
Wilkinson SR,
Young M,
Youngman P,
( 1994 ) Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain. PMID : 7885226 : DOI : 10.1111/j.1365-2958.1994.tb02176.x Abstract >>
Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis. It is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-containing surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spoIVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria.
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26. |
Benson AM,
Mower HF,
Yasunobu KT,
( 1966 ) The amino acid sequence of Clostridium butyricum ferredoxin. PMID : 5227671 : DOI : 10.1073/pnas.55.6.1532 PMC : PMC224355 Abstract >>
N/A
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27. |
Hegge JW,
Swarts DC,
Chandradoss SD,
Cui TJ,
Kneppers J,
Jinek M,
Joo C,
van der Oost J,
( 2019 ) DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute. PMID : 31069393 : DOI : 10.1093/nar/gkz306 PMC : PMC6582352 Abstract >>
Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (?65�XC) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37�XC). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.
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28. |
( 2013 ) Genetic diversity of the flagellin genes of Clostridium botulinum groups I and II. PMID : 23603687 : DOI : 10.1128/AEM.00686-13 PMC : PMC3697585 Abstract >>
Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37�XC) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30�XC). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum, subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum.
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29. |
( 1998 ) The glycyl radical enzyme TdcE can replace pyruvate formate-lyase in glucose fermentation. PMID : 9657990 : PMC : PMC107315 Abstract >>
Mutants of Escherichia coli unable to synthesize a functional pyruvate formate-lyase (PFL) are severely impaired in their capacity to grow by glucose fermentation. In a functional complementation assay designed to isolate the pfl gene from Clostridium butyricum, we fortuitously identified a gene that did not encode a PFL but nonetheless was able to complement the phenotypic defects caused by an E. coli pfl mutation. The clostridial gene encoded a basic 14. 5-kDa protein (TcbC) which, based on amino acid similarity and analysis of immediately adjacent DNA sequences, was part of a transposase exhibiting extensive similarity to the product of the site-specific transposon Tn554 from Staphylococcus aureus. Our studies revealed that the clostridial TcbC protein activated the transcription of the E. coli tdcABCDEFG operon, which encodes an anaerobic L-threonine-degradative pathway. Normally, anaerobic synthesis of the pathway is optimal when E. coli grows in the absence of catabolite-repressing sugars and in the presence of L-threonine. Although anaerobic control of pathway synthesis was maintained, TcbC alleviated glucose repression. One of the products encoded by the tdc operon, TdcE, has recently been shown to be a 2-keto acid formate-lyase (C. Hesslinger, S. A. Fairhurst, and G. Sawers, Mol. Microbiol. 27:477-492, 1998) that can accept pyruvate as an enzyme substrate. Here we show that TdcE is directly responsible for the restoration of fermentative growth to pfl mutants.
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30. |
( 1998 ) Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types. PMID : 9465394 : DOI : 10.1111/j.1574-6968.1998.tb12823.x Abstract >>
The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I-III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X1 (435 bp) and ORF-X2 (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.
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