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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.

Systematic and applied microbiology 26 (1)
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.
KeywordMeSH Terms
Genes, Bacterial
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
2. Tsuge  H, Nagahama  M, Nishimura  H, Hisatsune  J, Sakaguchi  Y, Itogawa  Y, Katunuma  N, Sakurai  J,     ( 2003 )

Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.

Journal of molecular biology 325 (3)
PMID : 12498797  :   DOI  :   10.1016/s0022-2836(02)01247-0    
Abstract >>
Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT. The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380. Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity. At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase. Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction.
KeywordMeSH Terms
Mutagenesis, Site-Directed
Protein Conformation
3. Calcutt  MJ, Hsieh  HY, Chapman  LF, Smith  DS,     ( 2002 )

Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens.

FEMS microbiology letters 214 (1)
PMID : 12204375  :   DOI  :   10.1111/j.1574-6968.2002.tb11327.x    
Abstract >>
The Clostridium perfringens gene encoding the previously characterized alpha-N-acetylgalactosaminidase (alphaNAG) was identified by protein microsequencing and database searching. The alphaNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted alpha-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic alphaNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected alphaNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial alphaNAG-encoding gene and represents an important step in the development of recombinant alphaNAG as a tool in the enzymatic conversion of blood group antigens.
KeywordMeSH Terms
Cloning, Molecular
Hexosaminidases
4. Miyamoto  K, Chakrabarti  G, Morino  Y, McClane  BA,     ( 2002 )

Organization of the plasmid cpe Locus in Clostridium perfringens type A isolates.

Infection and immunity 70 (8)
PMID : 12117935  :   DOI  :   10.1128/iai.70.8.4261-4272.2002     PMC  :   PMC128129    
Abstract >>
Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C. perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene. In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates. Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid. The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus. Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested. Similar dcm sequences were also detected in several cpe-negative C. perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene. Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C. perfringens plasmid. The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C. perfringens type A strains.
KeywordMeSH Terms
5. Justin  N, Walker  N, Bullifent  HL, Songer  G, Bueschel  DM, Jost  H, Naylor  C, Miller  J, Moss  DS, Titball  RW, Basak  AK,     ( 2002 )

The first strain of Clostridium perfringens isolated from an avian source has an alpha-toxin with divergent structural and kinetic properties.

Biochemistry 41 (20)
PMID : 12009886  :   DOI  :   10.1021/bi012015v    
Abstract >>
Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene. It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens. Previously characterized alpha-toxins from different strains of C. perfringens are almost identical in sequence and biochemical properties. We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C. perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains. The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations. The SWCP structure is in an open-form conformation, with three zinc ions in the active site. This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site. The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures. We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin. This will provide essential information when developing an effective vaccine that will protect against C. perfringens infection in a wide range of domestic livestock.
KeywordMeSH Terms
Calcium-Binding Proteins
6. Briolat  V, Reysset  G,     ( 2002 )

Identification of the Clostridium perfringens genes involved in the adaptive response to oxidative stress.

Journal of bacteriology 184 (9)
PMID : 11948145  :   DOI  :   10.1128/jb.184.9.2333-2343.2002     PMC  :   PMC134984    
Abstract >>
Clostridium perfringens is a ubiquitous gram-positive pathogen that is present in the air, soil, animals, and humans. Although C. perfringens is strictly anaerobic, vegetative and stationary cells can survive in a growth-arrested stage in the presence of oxygen and/or low concentrations of superoxide and hydroxyl radicals. Indeed, it possesses an adaptive response to oxidative stress, which can be activated in both aerobic and anaerobic conditions. To identify the genes involved in this oxidative stress response, C. perfringens strain 13 mutants were generated by Tn916 insertional mutagenesis and screened for resistance or sensitivity to various oxidative stresses. Three of the 12 sensitive mutants examined harbored an independently inserted single copy of the transposon in the same operon as two genes orthologous to the ydaD and ycdF genes of Bacillus subtilis, which encode a putative NADPH dehydrogenase. Complementation experiments and knockout experiments demonstrated that these genes are both required for efficient resistance to oxidative stress in C. perfringens and are probably responsible for the production of NADPH, which is required for maintenance of the intracellular redox balance in growth-arrested cells. Other Tn916 disrupted genes were also shown to play important roles in the oxidative stress response. This is the first time that some of these genes (e.g., a gene encoding an ATP-dependent RNA helicase, the beta-glucuronidase gene, and the gene encoding the atypical iron sulfur prismane protein) have been shown to be involved in the oxidative response.
KeywordMeSH Terms
Genes, Bacterial
7. Ashida  H, Maskos  K, Li  SC, Li  YT,     ( 2002 )

Characterization of a novel endo-beta-galactosidase specific for releasing the disaccharide GlcNAc alpha 1-->4Gal from glycoconjugates.

Biochemistry 41 (7)
PMID : 11841232  :   DOI  :   10.1021/bi011940e    
Abstract >>
In contrast to the beta-linked GlcNAc, the alpha-linked GlcNAc has not been commonly found in glycoconjugates. We have recently revealed the presence of an unusual endo-beta-galactosidase (Endo-beta-Gal(GnGa)) in Clostridium perfringens capable of releasing GlcNAcalpha1-->4Gal from glycans expressed in the gastric mucous cell-type mucin [Ashida, H., Anderson, K., Nakayama, J., Maskos, K., Chou, C.-W., Cole, R. B., Li, S.-C., and Li, Y.-T. (2001) J. Biol. Chem. 276, 28226-28232]. To characterize Endo-beta-Gal(GnGa), we have cloned its gene, gngC, from the genomic DNA library prepared from C. perfringens ATCC10543. The gene encodes 420 amino acid residues including a 17-residue signal peptide at the N-terminus. Using pUC18, we were able to prepare 25 mg of the fully active and pure recombinant Endo-beta-Gal(GnGa) from 1 L of Escherichia coli DH5alpha culture, which was 170 times higher than that produced by the original clostridial strain. Endo-beta-Gal(GnGa) shares a low but significant sequence similarity with two other endo-beta-galactosidases (16-21% amino acid identity). It also shows some similarity with bacterial 1,3-1,4-beta-glucan 4-glucanohydrolases of the glycoside hydrolase family 16. Endo-beta-Gal(GnGa) was found to contain the EXDX(X)E sequence (Glu-168 to Glu-173), that has been identified as the catalytic motif of families 16 and 7 retaining glycoside hydrolases. We have used site-directed mutagenesis to show that Glu-168 and Glu-173 were essential for the Endo-beta-Gal(GnGa) activity. By NMR spectroscopy, Endo-beta-Gal(GnGa) was found to act as a retaining enzyme.
KeywordMeSH Terms
Glycoside Hydrolases
8. Sasaki  Y, Yamamoto  K, Tamura  Y, Takahashi  T,     ( 2001 )

Tetracycline-resistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema.

Veterinary microbiology 83 (1)
PMID : 11524166  :   DOI  :   10.1016/s0378-1135(01)00402-3    
Abstract >>
The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined. The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema. Five (22%) of 23 C. septicum strains, five (71%) of seven C. perfringens strains and all strains of C. sordellii showed resistance to oxytetracycline. These oxytetracycline-resistant strains carried tetracycline-resistance genes [tetA(P), tetA408(P), tetB(P) and tetM]. The sequences of the tetracycline-resistance genes of some C. septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species. This is the first report of resistance of C. septicum to tetracycline.
KeywordMeSH Terms
9. Shimamoto  S, Moriyama  R, Sugimoto  K, Miyata  S, Makino  S,     ( 2001 )

Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity.

Journal of bacteriology 183 (12)
PMID : 11371539  :   DOI  :   10.1128/JB.183.12.3742-3751.2001     PMC  :   PMC95252    
Abstract >>
A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.
KeywordMeSH Terms
Heat-Shock Proteins
10. Roberts  AP, Johanesen  PA, Lyras  D, Mullany  P, Rood  JI,     ( 2001 )

Comparison of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the CW459tet(M) element from Clostridium perfringens shows that they have similar conjugation regions but different insertion and excision modules.

Microbiology (Reading, England) 147 (Pt 5)
PMID : 11320127  :   DOI  :   10.1099/00221287-147-5-1243    
Abstract >>
Comparative analysis of the conjugative transposons Tn5397 from Clostridium difficile and Tn916 from Enterococcus faecalis, and the CW459tet(M) element from Clostridium perfringens, has revealed that these tetracycline-resistance elements are closely related. All three elements contain the tet(M) resistance gene and have sequence similarity throughout their central region. However, they have very different integration/excision modules. Instead of the int and xis genes that are found in Tn916, Tn5397 has a large resolvase gene, tndX. The C. perfringens element encodes the putative Int459 protein, which is a member of the integrase family of site-specific recombinases but is not closely related to Int from Tn916. Based on these studies it is concluded that the clostridial elements have a modular genetic organization and were derived independently from distinct mobile genetic elements.
KeywordMeSH Terms
Viral Proteins
11. Yokoyama  I, Kurosawa  N, Morozumi  K, Kobayashi  T, Muramatsu  H, Ogawa  H,     ( 2000 )

Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney.

The Journal of biological chemistry 275 (25)
PMID : 10858461  :   DOI  :   10.1074/jbc.M001888200    
Abstract >>
Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.
KeywordMeSH Terms
Glycoside Hydrolases
12. Wiedmann  M, Arcuri  EF,     ( 2000 )

Phylogeny and functional conservation of sigma(E) in endospore-forming bacteria.

Microbiology (Reading, England) 146 (Pt 7) (N/A)
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.
KeywordMeSH Terms
13. Jepson  M, Titball  R,     ( 2000 )

Structure and function of clostridial phospholipases C.

Microbes and infection 2 (10)
PMID : 11008117  :  
Abstract >>
A range of clostridial species produce phospholipases C. The zinc metallo phospholipases C have related sequences but different properties. All of these enzymes may be arranged, like alpha-toxin as two-domain proteins. Differences in enzymatic, haemolytic and toxic properties may be explained by differences in amino acids at key positions.
KeywordMeSH Terms
Type C Phospholipases
14. Yaguchi  H, Swe  T, Cole  ST, Ohtani  K, Banu  S,     ( 2000 )

Identification of novel VirR/VirS-regulated genes in Clostridium perfringens.

Molecular microbiology 35 (4)
PMID : 10692162  :   DOI  :   10.1046/j.1365-2958.2000.01760.x    
Abstract >>
Novel genes that are regulated in Clostridium perfringens by the two-component regulatory system, VirR/VirS, were identified using a differential display method. A plasmid library was constructed from C. perfringens chromosomal DNA, and the plasmids were hybridized with cDNA probes prepared from total RNA of wild-type strain 13 and its virR mutant derivative TS133. Three clones were identified that carry newly identified VirR/VirS-regulated genes, two of which were positively regulated and one of which was negatively regulated. Genes located on the identified clones were deduced by nucleotide sequencing, and the target genes of the VirR/VirS system were identified with a set of Northern hybridizations. A 4.9 kb mRNA transcribing the metB (cystathionine gamma-synthase), cysK (cysteine synthase) and ygaG (hypothetical protein) genes was negatively regulated, whereas 1.6 and 6.0 kb transcripts encoding ptp (protein tyrosine phosphatase) and cpd (2',3'-cyclic nucleotide 2'-phosphodiesterase) respectively, were shown to be positively regulated by the VirR/VirS system. The other gene, hyp7, whose transcript was positively regulated by the VirR/VirS system, was shown to activate the transcription of the colA (kappa-toxin) and plc (alpha-toxin) genes, but not the pfoA (theta-toxin) gene in C. perfringens. These results suggested that the global regulatory system VirR/VirS could regulate various genes, other than toxin genes, both positively and negatively and that the hyp7 gene might encode a novel regulatory factor for toxin production in C. perfringens.
KeywordMeSH Terms
15. Sun  Y, Katayama  S, Taniguchi  Y, Fujinaga  K,     ( 1999 )

Analysis of genes involved in nitrate reduction in Clostridium perfringens.

Microbiology (Reading, England) 145 (Pt 12) (N/A)
PMID : 10627036  :   DOI  :   10.1099/00221287-145-12-3377    
Abstract >>
We have conducted the genetic analysis of fermentative nitrate reduction in Clostridium perfringens, a strict anaerobic bacterium. Nitrate reductase (NarA) was purified from the cytoplasmic fraction of the organism. Using a degenerate primer designed from its N-terminal amino acid sequence, a 9.5 kb fragment containing seven ORFs was cloned. The molecular mass and N-terminal amino acid sequence predicted from the nucleotide sequence of ORF 4 coincided with those determined for the purified NarA, indicating that ORF 4 corresponds to a narA gene. ORFs 5 and 6 encode a 15.4 kDa ferredoxin-like protein containing four iron-sulfur clusters and a 45 kDa protein homologous to NADH oxidase, respectively. Analyses involving primer extension and Northern blotting revealed that these three ORFs are transcribed as a polycistronic message. The ORF 5- and ORF 6-encoded proteins were shown by immunoblotting to be synthesized by cells grown in the presence of nitrate. Thus, these two proteins are likely to function as electron-transfer components in nitrate reduction in C perfringens. The 9.5 kb fragment and a downstream region of 6.1 kb do not contain any genes involved in nitrate uptake or nitrite reduction. Instead, all 5 ORFs downstream of ORF 6 are homologous to genes reported for molybdopterin biosynthesis, unlike the genomic organization already determined for the respiratory and assimilatory nitrate-reduction systems. The evolutionary relationships between these two nitrate-reduction systems and the fermentative one based on the results of comparative genetic analysis are discussed.
KeywordMeSH Terms
Operon
16. Matsushita  O, Katayama  S, Miyata  S, Taniguchi  Y, Kaji  M,     ( 1999 )

The hydA gene encoding the H(2)-evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene.

FEMS microbiology letters 181 (2)
PMID : 10585557  :   DOI  :   10.1111/j.1574-6968.1999.tb08863.x    
Abstract >>
A putative hydrogenase (hydA) gene of Clostridium perfringens encodes a protein with strong identity to Clostridium pasteurianum hydrogenase I. Disruption of the hydA gene abolished H(2) productivity, confirming its function. A putative butyrate kinase gene (buk) is adjacent to the hydA gene. When cultures were grown in medium with glucose, 1.8-kb hydA and 2.1-kb buk transcripts and a 3. 9-kb transcript hybridized with both hydA and buk-probe were detectable in all the exponential growth phases. In medium without glucose, these transcripts were decreased rapidly after the mid-exponential phase. These results suggest that the transcription of these two genes is probably regulated by a similar mechanism in response to glucose availability.
KeywordMeSH Terms
Genes, Bacterial
17. Teuber  M, Geissmann  TA,     ( 1999 )

Transcriptional analysis of the rubrerythrin and superoxide dismutase genes of Clostridium perfringens.

Journal of bacteriology 181 (22)
PMID : 10559182  :   PMC  :   PMC94191    
Abstract >>
We cloned and sequenced a 2.7-kb fragment of chromosomal DNA from Clostridium perfringens containing the superoxide dismutase-encoding gene, sod. Previously, rubrerythrin from C. perfringens had been isolated and its gene (rbr) had been cloned (Y. Lehmann, L. Meile, and M. Teuber, J. Bacteriol. 178:7152-7158, 1996). Northern blot experiments revealed a length of approximately 800 bases for each transcript of rbr and sod of C. perfringens. Thus, rbr and sod each represent a monocistronic operon. Their transcription start points were located by primer extension analyses. sod transcription was shown to depend on the growth phase, and it reached a maximum during the transition from log phase to stationary phase. Neither sod nor rbr transcription was influenced by oxidative stress.
KeywordMeSH Terms
Transcription, Genetic
18. Stirewalt  VL, Melville  SB,     ( 1999 )

Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens.

Journal of bacteriology 181 (15)
PMID : 10419949  :   PMC  :   PMC103582    
Abstract >>
Clostridium perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from C. perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned. The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78. The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C. perfringens is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose. With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which beta-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.
KeywordMeSH Terms
Bacterial Proteins
Gene Expression Regulation, Bacterial
Operon
Transcription, Genetic
19. Hunter  SE, Clarke  IN, Kelly  DC, Titball  RW,     ( 1992 )

Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli.

Infection and immunity 60 (1)
PMID : 1729175  :   PMC  :   PMC257509    
Abstract >>
The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin.
KeywordMeSH Terms
20. Li  J, Miyamoto  K, McClane  BA,     ( 2007 )

Comparison of virulence plasmids among Clostridium perfringens type E isolates.

Infection and immunity 75 (4)
PMID : 17261608  :   DOI  :   10.1128/IAI.01981-06     PMC  :   PMC1865703    
Abstract >>
Clostridium perfringens type E isolates produce iota-toxin, which is encoded by iap and ibp genes. Using Southern blot analyses, the current study identified iap/ibp plasmids of approximately 97 or approximately 135 kb among eight type E isolates. For most of these isolates, their iap/ibp plasmid also encoded urease and lambda-toxin. However, the beta2-toxin gene, if present, was on a different plasmid from the iap/ibp plasmid. For all isolates, the iap/ibp plasmid carried a tcp locus, strongly suggesting that these plasmids are conjugative. Overlapping PCR analyses demonstrated some similarity between the iap/ibp plasmids and enterotoxin-encoding plasmids of type A isolates. Additional PCR analyses demonstrated that the iap/ibp locus is located near dcm sequences, an apparent plasmid hot spot for toxin gene insertion, and that two IS1151-related sequences are present in the iap/ibp locus. To begin testing whether those IS1151-like sequences can mobilize iap/ibp genes, a PCR assay was performed that amplifies a product only from circular DNA forms that could represent transposition intermediates. This PCR assay detected circular forms containing iap/ibp genes and silent enterotoxin gene sequences, with or without an IS1151-like sequence. Collectively, these results suggest that a mobile genetic element carrying iap/ibp has inserted onto a tcp-carrying enterotoxin plasmid in a type A isolate, creating a progenitor iap/ibp plasmid. That plasmid then spread via conjugation to other isolates, converting them to type E. Further iap/ibp plasmid diversity occurred when either the iap/ibp genes later remobilized and inserted onto other conjugative plasmids or some iap/ibp plasmids acquired additional DNA sequences.
KeywordMeSH Terms
21. Lebrun  M, Filée  P, Mousset  B, Desmecht  D, Galleni  M, Mainil  JG, Linden  A,     ( 2007 )

The expression of Clostridium perfringens consensus beta2 toxin is associated with bovine enterotoxaemia syndrome.

Veterinary microbiology 120 (1��2��)
PMID : 17126502  :   DOI  :   10.1016/j.vetmic.2006.10.020    
Abstract >>
Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2, is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves (n=14, 87 isolates) and from a group of enterotoxaemic calves (n=8, 41 isolates). PCR results revealed the exclusive presence of the typical "consensus"cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
22. Fernandez-Miyakawa  ME, Fisher  DJ, Poon  R, Sayeed  S, Adams  V, Rood  JI, McClane  BA, Uzal  FA,     ( 2007 )

Both epsilon-toxin and beta-toxin are important for the lethal properties of Clostridium perfringens type B isolates in the mouse intravenous injection model.

Infection and immunity 75 (3)
PMID : 17210666  :   DOI  :   10.1128/IAI.01672-06     PMC  :   PMC1828578    
Abstract >>
Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.
KeywordMeSH Terms
23. Bannam  TL, Teng  WL, Bulach  D, Lyras  D, Rood  JI,     ( 2006 )

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens.

Journal of bacteriology 188 (1��13��)
PMID : 16788202  :   DOI  :   10.1128/JB.00298-06     PMC  :   PMC1483020    
Abstract >>
Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.
KeywordMeSH Terms
Conjugation, Genetic
DNA Transposable Elements
Replicon
24. Johansson  A, Aspan  A, Bagge  E, Båverud  V, Engström  BE, Johansson  KE,     ( 2006 )

Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge.

BMC microbiology 6 (N/A)
PMID : 16737528  :   DOI  :   10.1186/1471-2180-6-47     PMC  :   PMC1513381    
Abstract >>
Clostridium perfringens, a serious pathogen, causes enteric diseases in domestic animals and food poisoning in humans. The epidemiological relationship between C. perfringens isolates from the same source has previously been investigated chiefly by pulsed-field gel electrophoresis (PFGE). In this study the genetic diversity of C. perfringens isolated from various animals, from food poisoning outbreaks and from sludge was investigated. We used PFGE to examine the genetic diversity of 95 C. perfringens type A isolates from eight different sources. The isolates were also examined for the presence of the beta2 toxin gene (cpb2) and the enterotoxin gene (cpe). The cpb2 gene from the 28 cpb2-positive isolates was also partially sequenced (519 bp, corresponding to positions 188 to 706 in the consensus cpb2 sequence). The results of PFGE revealed a wide genetic diversity among the C. perfringens type A isolates. The genetic relatedness of the isolates ranged from 58 to 100% and 56 distinct PFGE types were identified. Almost all clusters with similar patterns comprised isolates with a known epidemiological correlation. Most of the isolates from pig, horse and sheep carried the cpb2 gene. All isolates originating from food poisoning outbreaks carried the cpe gene and three of these also carried cpb2. Two evolutionary different populations were identified by sequence analysis of the partially sequenced cpb2 genes from our study and cpb2 sequences previously deposited in GenBank. As revealed by PFGE, there was a wide genetic diversity among C. perfringens isolates from different sources. Epidemiologically related isolates showed a high genetic similarity, as expected, while isolates with no obvious epidemiological relationship expressed a lesser degree of genetic similarity. The wide diversity revealed by PFGE was not reflected in the 16S rRNA sequences, which had a considerable degree of sequence similarity. Sequence comparison of the partially sequenced cpb2 gene revealed two genetically different populations. This is to our knowledge the first study in which the genetic diversity of C. perfringens isolates both from different animals species, from food poisoning outbreaks and from sludge has been investigated.
KeywordMeSH Terms
Genetic Variation
25. Fisher  DJ, Fernandez-Miyakawa  ME, Sayeed  S, Poon  R, Adams  V, Rood  JI, Uzal  FA, McClane  BA,     ( 2006 )

Dissecting the contributions of Clostridium perfringens type C toxins to lethality in the mouse intravenous injection model.

Infection and immunity 74 (9)
PMID : 16926413  :   DOI  :   10.1128/IAI.00534-06     PMC  :   PMC1594841    
Abstract >>
The gram-positive anaerobe Clostridium perfringens produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals. C. perfringens type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (plc), perfringolysin O (pfoA), beta toxin (cpb), and sometimes beta2 toxin (cpb2) and/or enterotoxin (cpe). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia.
KeywordMeSH Terms
26. Titball  RW, Naylor  CE, Basak  AK,     ( 1999 )

The Clostridium perfringens alpha-toxin.

Anaerobe 5 (2)
PMID : 16887662  :   DOI  :   10.1006/anae.1999.0191    
Abstract >>
The gene encoding the alpha-(cpa) is present in all strains of Clostridium perfringens, and the purified alpha-toxin has been shown to be a zinc-containing phospholipase C enzyme, which is preferentially active towards phosphatidylcholine and sphingomyelin. The alpha-toxin is haemolytic as a result if its ability to hydrolyse cell membrane phospholipids and this activity distinguishes it from many other related zinc-metallophospholipases C. Recent studies have shown that the alpha-toxin is the major virulence determinant in cases of gas gangrene, and the toxin might play a role in several other diseases of animals and man as diverse as necrotic enteritis in chickens and Crohn's disease in man. In gas gangrene the toxin appears to have three major roles in the pathogenesis of disease. First, it is able to cause mistrafficking of neutrophils, such that they do not enter infected tissues. Second, the toxin is able to cause vasoconstriction and platelet aggregation which might reduce the blood supply to infected tissues. Finally, the toxin is able to detrimentally modulate host cell metabolism by activating the arachidonic acid cascade and protein kinase C. The molecular structure of the alpha-toxin reveals a two domain protein. The amino-terminal domain contains the phospholipase C active site which contains zinc ions. The carboxyterminal domain is a paralogue of lipid binding domains found in eukaryotes and appears to bind phospholipids in a calcium-dependent manner. Immunisation with the non-toxic carboxyterminal domain induces protection against the alpha-toxin and gas gangrene and this polypeptide might be exploited as a vaccine. Other workers have exploited the entire toxin as the basis of an anti-tumour system.
KeywordMeSH Terms
27. Jost  BH, Trinh  HT, Songer  JG,     ( 2006 )

Clonal relationships among Clostridium perfringens of porcine origin as determined by multilocus sequence typing.

Veterinary microbiology 116 (1��3��)
PMID : 16650661  :   DOI  :   10.1016/j.vetmic.2006.03.025    
Abstract >>
Clostridium perfringens is ubiquitous in the environment and the intestinal tracts of most mammals, but this organism also causes gas gangrene and enteritis in human and animal hosts. While expression of specific toxins correlates with specific disease in certain hosts, the other factors involved in commensalism and host pathogenesis have not been clearly identified. A multilocus sequence typing (MLST) scheme was developed for C. perfringens with the aim of grouping isolates with respect to disease presentation and/or host preference. Sequence data were obtained from one virulence and seven housekeeping genes for 132 C. perfringens isolates that comprised all five toxin types and were isolated from 10 host species. Eighty sequence types (STs) were identified, with the majority (75%) containing only one isolate. eBURST analysis identified three clonal complexes, which contained 59.1% of the isolates. Clonal complex (CC) 1 contained 31, predominantly type A isolates from diverse host species. Clonal complex 2 contained 75% of the bovine type E isolates examined in this study. Clonal complex 3 consisted predominantly of porcine type A and type C isolates. Interestingly, these porcine isolates (n=32) all carried consensus cpb2 and cna genes, encoding beta2 toxin and CpCna, a collagen binding protein, respectively. This compares to carriage of both these genes by only 3.6% of porcine isolates not present in clonal complex 3 (n=28). The data obtained indicates that MLST may be used to identify host species relationships with respect to these C. perfringens isolates.
KeywordMeSH Terms
28. Iddar  A, Valverde  F, Assobhei  O, Serrano  A, Soukri  A,     ( 2005 )

Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria.

International microbiology : the official journal of the Spanish Society for Microbiology 8 (4)
PMID : 16562377  :  
Abstract >>
The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.
KeywordMeSH Terms
29. Rooney  AP, Swezey  JL, Friedman  R, Hecht  DW, Maddox  CW,     ( 2006 )

Analysis of core housekeeping and virulence genes reveals cryptic lineages of Clostridium perfringens that are associated with distinct disease presentations.

Genetics 172 (4)
PMID : 16489222  :   DOI  :   10.1534/genetics.105.054601     PMC  :   PMC1456398    
Abstract >>
Clostridium perfringens is an important human and animal pathogen that causes a number of diseases that vary in their etiology and severity. Differences between strains regarding toxin gene composition and toxin production partly explain why some strains cause radically different diseases than others. However, they do not provide a complete explanation. The purpose of this study was to determine if there is a phylogenetic component that explains the variance in C. perfringens strain virulence by assessing patterns of genetic polymorphism in genes (colA gyrA, plc, pfoS, and rplL) that form part of the core genome in 248 type A strains. We found that purifying selection plays a central role in shaping the patterns of nucleotide substitution and polymorphism in both housekeeping and virulence genes. In contrast, recombination was found to be a significant factor only for the virulence genes plc and colA and the housekeeping gene gyrA. Finally, we found that the strains grouped into five distinct evolutionary lineages that show evidence of host adaptation and the early stages of speciation. The discovery of these previously unknown lineages and their association with distinct disease presentations carries important implications for human and veterinary clostridial disease epidemiology and provides important insights into the pathways through which virulence has evolved in C. perfringens.
KeywordMeSH Terms
30. Miyamoto  K, Fisher  DJ, Li  J, Sayeed  S, Akimoto  S, McClane  BA,     ( 2006 )

Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates.

Journal of bacteriology 188 (4)
PMID : 16452442  :   DOI  :   10.1128/JB.188.4.1585-1598.2006     PMC  :   PMC1367241    
Abstract >>
Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.
KeywordMeSH Terms
31. Thompson  DR, Parreira  VR, Kulkarni  RR, Prescott  JF,     ( 2006 )

Live attenuated vaccine-based control of necrotic enteritis of broiler chickens.

Veterinary microbiology 113 (1��2��)
PMID : 16289639  :   DOI  :   10.1016/j.vetmic.2005.10.015    
Abstract >>
A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains. In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize ("infection-immunization") chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.
KeywordMeSH Terms
Bacterial Vaccines
Chickens
32. Fisher  DJ, Miyamoto  K, Harrison  B, Akimoto  S, Sarker  MR, McClane  BA,     ( 2005 )

Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene.

Molecular microbiology 56 (3)
PMID : 15819629  :   DOI  :   10.1111/j.1365-2958.2005.04573.x    
Abstract >>
Clostridium perfringens type A isolates carrying an enterotoxin (cpe) gene are an important cause of human gastrointestinal diseases, including food poisoning, antibiotic-associated diarrhoea (AAD) and sporadic diarrhoea (SD). Using polymerase chain reaction (PCR), the current study determined that the cpb2 gene encoding the recently discovered beta2 toxin is present in <15% of food poisoning isolates, which typically carry a chromosomal cpe gene. However, >75% of AAD/SD isolates, which usually carry a plasmid cpe gene, tested cpb2(+) by PCR. Western blot analysis demonstrated that >97% of those cpb2(+)/cpe(+) AAD/SD isolates can produce CPB2. Additional PCR analyses, sequencing studies and pulsed field gel electrophoresis experiments determined that AAD/SD isolates carry cpb2 and cpe on the same plasmid when IS1151 sequences are present downstream of cpe, but cpb2 and cpe are located on different plasmids in AAD/SD isolates where IS1470-like sequences are present downstream of cpe. Those analyses also demonstrated that two different CPB2 variants (named CPB2h1 or CPB2h2) can be produced by AAD/SD isolates, dependent on whether IS1470-like or IS1151 sequences are present downstream of their cpe gene. CPB2h1 is approximately 10-fold more cytotoxic for CaCo-2 cells than is CPB2h2. Collectively, these results suggest that CPB2 could be an accessory toxin in C. perfringens enterotoxin (CPE)-associated AAD/SD.
KeywordMeSH Terms
33. Greco  G, Madio  A, Martella  V, Campolo  M, Corrente  M, Buonavoglia  D, Buonavoglia  C,     ( 2005 )

Enterotoxemia associated with beta2 toxin-producing Clostridium perfringens type A in two Asiatic black bears (Selenarctos thibetanus).

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 17 (2)
PMID : 15825503  :   DOI  :   10.1177/104063870501700216    
Abstract >>
Beta2 (beta2) toxin-producing Clostridium perfringens type A strains were found to be associated with necrotic and hemorrhagic intestinal lesions in 2 Asiatic black bears (Selenarctos thibetanus) that died suddenly. Ten isolates were obtained from the liver, lungs, heart, and small and large intestine of the animals and were examined by multiplex polymerase chain reaction for the genes encoding the 4 lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types as well as for the genes encoding enterotoxin and the novel beta2-toxin for subclassification. In addition, the cpb2 sequence of the 10 isolates was different from the published sequence of cpb2 of pig type C isolate CWC245, whereas it was highly similar to the cpb2 sequence of the C. perfringens type A strain 13. This finding suggests the existence of 2 cpb2 subtypes. This is the first report of enterotoxemia associated with the presence of C. perfringens producing beta2-toxin in the tissues and intestinal content of Asiatic black bears.
KeywordMeSH Terms
Ursidae
34. Tempel  W, Liu  ZJ, Horanyi  PS, Deng  L, Lee  D, Newton  MG, Rose  JP, Ashida  H, Li  SC, Li  YT, Wang  BC,     ( 2005 )

Three-dimensional structure of GlcNAcalpha1-4Gal releasing endo-beta-galactosidase from Clostridium perfringens.

Proteins 59 (1)
PMID : 15688452  :   DOI  :   10.1002/prot.20363    
Abstract >>
N/A
KeywordMeSH Terms
35. Vilei  EM, Schlatter  Y, Perreten  V, Straub  R, Popoff  MR, Gibert  M, Gröne  A, Frey  J,     ( 2005 )

Antibiotic-induced expression of a cryptic cpb2 gene in equine beta2-toxigenic Clostridium perfringens.

Molecular microbiology 57 (6)
PMID : 16135225  :   DOI  :   10.1111/j.1365-2958.2005.04789.x    
Abstract >>
The cpb2 gene of beta2-toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non-porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non-porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full beta2-toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant beta2-toxin showed the absence of expression of the beta2-toxin in equine and the other non-porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the beta2-toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by beta2-toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic-induced ribosomal frameshifting.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
36. Anderson  KM, Ashida  H, Maskos  K, Dell  A, Li  SC, Li  YT,     ( 2005 )

A clostridial endo-beta-galactosidase that cleaves both blood group A and B glycotopes: the first member of a new glycoside hydrolase family, GH98.

The Journal of biological chemistry 280 (9)
PMID : 15618227  :   DOI  :   10.1074/jbc.M414099200    
Abstract >>
We have isolated an endo-beta-galactosidase designated E-ABase from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide (A-Tri; GalNAcalpha1-->3(Fucalpha1-->2)Gal) and B trisaccharide (B-Tri; Galalpha1-->3(Fucalpha1-->2)Gal) from glycoconjugates containing blood group A and B glycotopes, respectively. We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism. This gene was found to be identical to the CPE0329 gene of C. perfringens strain 13, whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 996-1001). Since the amino acid sequence of E-ABase does not bear detectable similarity to any of the 97 existing families of glycoside hydrolases, we have proposed to assign this unusual enzyme to a new family, GH98. We also expressed eabC in Escherichia coli BL21(DE3) and obtained 27 mg of fully active recombinant E-ABase from 1 liter of culture. Recombinant E-ABase not only destroyed the blood group A and B antigenicity of human type A and B erythrocytes, but also released A-Tri and B-Tri from blood group A(+)- and B(+)- containing glycoconjugates. The structures of A-Tri and B-Tri liberated from A(+) porcine gastric mucin and B(+) human ovarian cyst glycoprotein were established by NMR spectroscopy. The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A- and B-containing glycoconju-gates as well as for identifying other glycosidases belonging to the new GH98 family.
KeywordMeSH Terms
37. Jost  BH, Billington  SJ, Trinh  HT, Bueschel  DM, Songer  JG,     ( 2005 )

Atypical cpb2 genes, encoding beta2-toxin in Clostridium perfringens isolates of nonporcine origin.

Infection and immunity 73 (1)
PMID : 15618211  :   DOI  :   10.1128/IAI.73.1.652-656.2005     PMC  :   PMC538998    
Abstract >>
Beta2-toxin, encoded by cpb2, is implicated in the pathogenesis of Clostridium perfringens enteritis. However, cpb2 genes from nonporcine C. perfringens isolates were not always expressed, at least in vitro. Nucleotide sequencing identified atypical cpb2 genes with 70.2 to 70.7% DNA identity to previously identified (consensus) cpb2. Atypical beta2-toxin displayed 62.3% identity and 80.4% similarity to consensus beta2-toxin. No porcine type C isolates (n = 16) and only 3.3% of porcine type A isolates (n = 60) carried atypical cpb2 genes. However, 88.5% of nonporcine isolates carried atypical cpb2 (n = 78), but beta2-toxin was not expressed. Almost half of the nonporcine consensus cpb2 genes (44.4%) carried a frameshift mutation (n = 9), resulting in an absence of beta2-toxin expression. These findings strengthen the role of beta2-toxin in the pathogenesis of enteritis in neonatal pigs. However, the identification of apparently nonexpressed, atypical cpb2 genes raises the question of whether this protein plays the same role in enteritis in other animal species.
KeywordMeSH Terms
38. Varga  J, Stirewalt  VL, Melville  SB,     ( 2004 )

The CcpA protein is necessary for efficient sporulation and enterotoxin gene (cpe) regulation in Clostridium perfringens.

Journal of bacteriology 186 (16)
PMID : 15292123  :   DOI  :   10.1128/JB.186.16.5221-5229.2004     PMC  :   PMC490932    
Abstract >>
Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens, which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme beta-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).
KeywordMeSH Terms
39. Sloan  J, Warner  TA, Scott  PT, Bannam  TL, Berryman  DI, Rood  JI,     ( 1992 )

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid.

Plasmid 27 (3)
PMID : 1513878  :  
Abstract >>
A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.
KeywordMeSH Terms
Genetic Vectors
Plasmids
40. Cole  AR, Gibert  M, Popoff  M, Moss  DS, Titball  RW, Basak  AK,     ( 2004 )

Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin.

Nature structural & molecular biology 11 (8)
PMID : 15258571  :   DOI  :   10.1038/nsmb804    
Abstract >>
Epsilon-toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.
KeywordMeSH Terms
41. Johansson  A, Greko  C, Engström  BE, Karlsson  M,     ( 2004 )

Antimicrobial susceptibility of Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes.

Veterinary microbiology 99 (3��4��)
PMID : 15066727  :   DOI  :   10.1016/j.vetmic.2004.01.009    
Abstract >>
This study was undertaken to determine the in vitro susceptibility of Clostridium perfringens, isolated from poultry to antimicrobials used in poultry production. The minimal inhibitory concentration (MIC) of eight antimicrobials, including the ionophoric coccidiostat narasin, was determined for 102 C. perfringens isolates, 58 from Sweden, 24 from Norway and 20 from Denmark. Susceptibility to each antimicrobial compound was determined by broth microdilution. The isolates were obtained from broilers (89), laying hens (9) and turkeys (4), affected by necrotic enteritis (NE) or by C. perfringens associated hepatitis (CPH), and from healthy broilers. All strains, regardless of origin, proved inherently susceptible to ampicillin, narasin, avilamycin, erythromycin and vancomycin. A low frequency of resistance to virginiamycin and bacitracin was also found. Resistance to tetracycline was found in strains isolated in all three countries; Sweden (76%), Denmark (10%) and Norway (29%). In 80% of the tetracycline-resistant isolates, the two resistance genes tetA(P) and tetB(P) were amplified by PCR whereas in 20% only the tetA(P) gene was detected. No tetM gene amplicon was obtained from any of the tetracycline-resistant isolates. The uniform susceptibility to narasin revealed in this study shows that the substance can still be used to control clostridiosis. In this study, C. perfringens also showed a low degree of resistance to most other antimicrobials tested. Despite the small amounts of tetracycline used in poultry, a considerable degree of resistance to tetracycline was found in C. perfringens isolates from Swedish broilers.
KeywordMeSH Terms
Chickens
Turkeys
42. Huang  IH, Waters  M, Grau  RR, Sarker  MR,     ( 2004 )

Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A.

FEMS microbiology letters 233 (2)
PMID : 15063491  :   DOI  :   10.1016/j.femsle.2004.02.014    
Abstract >>
This study identified a functional spo0A ORF in enterotoxigenic Clostridium perfringens type A. To evaluate the function of spo0A, an isogenic spo0A knock-out mutant was constructed. The spo0A mutant was unable to form endospores and produce enterotoxin, however, these defects could be restored by complementing the mutant with a recombinant plasmid carrying the wild-type spo0A gene. These results provide evidence that spo0A expression is essential for sporulation and enterotoxin production in C. perfringens.
KeywordMeSH Terms
43. Holck  AL, Blom  H,     ( 1992 )

The nucleotide sequence of a putative membrane transport gene from Clostridium perfringens.

DNA sequence : the journal of DNA sequencing and mapping 3 (3)
PMID : 1472712  :  
Abstract >>
A gene from Clostridium perfringens encoding a highly hydrophobic polypeptide of M(r) = 30,698 was cloned and sequenced. The polypeptide showed similarities with the inner membrane proteins of binding protein-dependent transport systems of the gram negative enterobacteria. The sequence homology to the UgpE and MalG proteins of Escherichia coli was 27.6%, or 60.4 and 52.2%, respectively, taking into account the conservative amino acid changes. The polypeptide contained the EAA---G---------(I/V)-LP motif of the gram negative transport proteins. This motif may thus be very old, as clostridia and E. coli separated some 1.5 x 10(9) years ago. The similarities suggest a common evolutionary origin of these genes.
KeywordMeSH Terms
Genes, Bacterial
44. Li  J, Chen  J, Vidal  JE, McClane  BA,     ( 2011 )

The Agr-like quorum-sensing system regulates sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A non-food-borne human gastrointestinal disease strain F5603.

Infection and immunity 79 (6)
PMID : 21464088  :   DOI  :   10.1128/IAI.00169-11     PMC  :   PMC3125842    
Abstract >>
Clostridium perfringens type A strains producing enterotoxin (CPE) cause one of the most common bacterial food-borne illnesses, as well as many cases of non-food-borne human gastrointestinal disease. Recent studies have shown that an Agr-like quorum-sensing system controls production of chromosomally encoded alpha-toxin and perfringolysin O by C. perfringens, as well as sporulation by Clostridium botulinum and Clostridium sporogenes. The current study explored whether the Agr-like quorum-sensing system also regulates sporulation and production of two plasmid-encoded toxins (CPE and beta2 toxin) that may contribute to the pathogenesis of non-food-borne human gastrointestinal disease strain F5603. An isogenic agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, providing the first evidence that, in C. perfringens, this system can control production of plasmid-encoded toxins as well as chromosomally encoded toxins. This mutant also showed reduced production of alpha-toxin and perfringolysin O during vegetative growth. Importantly, when cultured in sporulation medium, the mutant failed to efficiently form spores and was blocked for CPE production. Complementation partially or fully reversed all phenotypic changes in the mutant, confirming that they were specifically due to inactivation of the agr locus. Western blots suggest that this loss of sporulation and sporulation-specific CPE production for the agrB null mutant involves, at least in part, Agr-mediated regulation of production of Spo0A and alternative sigma factors, which are essential for C. perfringens sporulation.
KeywordMeSH Terms
45. Fujita  M, Tsuchida  A, Hirata  A, Kobayashi  N, Goto  K, Osumi  K, Hirose  Y, Nakayama  J, Yamanoi  T, Ashida  H, Mizuno  M,     ( 2011 )

Glycoside hydrolase family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAc alpha1,4Gal beta1R at the non-reducing terminus of O-glycans in gastric mucin.

The Journal of biological chemistry 286 (8)
PMID : 21177247  :   DOI  :   10.1074/jbc.M110.206722     PMC  :   PMC3057825    
Abstract >>
In mammals, �\-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. �\-N-acetylglucosaminidases (�\GNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal �\GNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of �\GNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human �\GNase, by chemically synthesizing a series of disaccharide substrates containing �\-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAc�\1,4Gal�]1pMP and GlcNAc�\1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAc�\1,2Gal�]1pMP, GlcNAc�\1,3Gal�]1pMP, GlcNAc�\1,6Gal�]1pMP, and GlcNAc�\1,4GlcA�]1pMP substrates, this enzyme may represent a specific glycosidase required for degrading �\-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAc�\1,4Gal�]1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered �\-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.
KeywordMeSH Terms
46. Garofolo  G, Galante  D, Serrecchia  L, Buonavoglia  D, Fasanella  A,     ( 2011 )

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum.

Journal of microbiological methods 84 (2)
PMID : 21182874  :   DOI  :   10.1016/j.mimet.2010.12.017    
Abstract >>
In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.
KeywordMeSH Terms
47. Hibberd  MC, Neumann  AP, Rehberger  TG, Siragusa  GR,     ( 2011 )

Multilocus sequence typing subtypes of poultry Clostridium perfringens isolates demonstrate disease niche partitioning.

Journal of clinical microbiology 49 (4)
PMID : 21270221  :   DOI  :   10.1128/JCM.01884-10     PMC  :   PMC3122852    
Abstract >>
Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.
KeywordMeSH Terms
Bacterial Typing Techniques
Multilocus Sequence Typing
48. Lepp  D, Roxas  B, Parreira  VR, Marri  PR, Rosey  EL, Gong  J, Songer  JG, Vedantam  G, Prescott  JF,     ( 2010 )

Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis.

PloS one 5 (5)
PMID : 20532244  :   DOI  :   10.1371/journal.pone.0010795     PMC  :   PMC2879425    
Abstract >>
Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes approximately 85 and approximately 70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne.
KeywordMeSH Terms
49. Li  J, Miyamoto  K, Sayeed  S, McClane  BA,     ( 2010 )

Organization of the cpe locus in CPE-positive clostridium perfringens type C and D isolates.

PloS one 5 (6)
PMID : 20532170  :   DOI  :   10.1371/journal.pone.0010932     PMC  :   PMC2880595    
Abstract >>
Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity.
KeywordMeSH Terms
Genes, Bacterial
50. Bannam  TL, Rood  JI,     ( 1991 )

Relationship between the Clostridium perfringens catQ gene product and chloramphenicol acetyltransferases from other bacteria.

Antimicrobial agents and chemotherapy 35 (3)
PMID : 2039197  :   DOI  :   10.1128/aac.35.3.471     PMC  :   PMC245034    
Abstract >>
The nucleotide sequence of the Clostridium perfringens chloramphenicol acetyltransferase (CAT)-encoding resistance determinant, catQ, was determined. An open reading frame encoding a protein of 219 amino acids with a molecular weight of 26,014 was identified. Although catQ was expressed constitutively, sequences similar in structure to those found upstream of inducible cat genes were observed. The catQ gene was distinct from the C. perfringens catP determinant. The deduced CATQ monomer had considerable amino acid sequence conservation compared with CATP (53% similarity) and other known CAT proteins (39 to 53%). Phylogenetic analysis revealed that the CATQ monomer was as closely related to CAT proteins from Staphylococcus aureus and Campylobacter coli as it was to CAT monomers from the clostridia.
KeywordMeSH Terms
51. Abildgaard  L, Sondergaard  TE, Engberg  RM, Schramm  A, Højberg  O,     ( 2010 )

In vitro production of necrotic enteritis toxin B, NetB, by netB-positive and netB-negative Clostridium perfringens originating from healthy and diseased broiler chickens.

Veterinary microbiology 144 (1��2��)
PMID : 20092968  :   DOI  :   10.1016/j.vetmic.2009.12.036    
Abstract >>
The Clostridium perfringens necrotic enteritis toxin B, NetB, was recently proposed as a new key virulence factor for the development of necrotic enteritis (NE) in broilers. The aim of the present study was to investigate the presence of the netB gene and the in vitro production of the NetB toxin in a well characterized collection of 48 C. perfringens Type A isolates, obtained from Danish broiler flocks. The investigation revealed netB gene prevalences of approx. 50% and 60% among isolates from diseased (NE) and healthy flocks, respectively. Only minor nucleotide variations were observed between the isolates in the coding sequence (CDS) of the netB gene, and the promoter region was observed to be completely conserved. However, in vitro NetB production was only observed in 4 out of 14 netB-positive C. perfringens isolates recovered from healthy birds, whereas 12 out of 13 netB-positive isolates from NE birds were shown to produce the NetB toxin. It is therefore proposed that genotype, i.e. presence of the netB gene, in itself is inadequate for predicting virulence of C. perfringens, and future investigations should focus on the bacterial phenotypes; the regulatory mechanisms involved in the expression of NetB, and potentially also other toxins, and its implications for the virulence of individual C. perfringens strains.
KeywordMeSH Terms
Polymorphism, Single Nucleotide
52. Vidal  JE, Chen  J, Li  J, McClane  BA,     ( 2009 )

Use of an EZ-Tn5-based random mutagenesis system to identify a novel toxin regulatory locus in Clostridium perfringens strain 13.

PloS one 4 (7)
PMID : 19597556  :   DOI  :   10.1371/journal.pone.0006232     PMC  :   PMC2706048    
Abstract >>
Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens. An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 DeltaagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 DeltaagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins. The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.
KeywordMeSH Terms
DNA Transposable Elements
Mutagenesis
53. Li  J, Paredes-Sabja  D, Sarker  MR, McClane  BA,     ( 2009 )

Further characterization of Clostridium perfringens small acid soluble protein-4 (Ssp4) properties and expression.

PloS one 4 (7)
PMID : 19609432  :   DOI  :   10.1371/journal.pone.0006249     PMC  :   PMC2706996    
Abstract >>
Clostridium perfringens type A food poisoning (FP) is usually caused by C. perfringens type A strains that carry a chromosomal enterotoxin gene (cpe) and produce spores with exceptional resistance against heat and nitrites. Previous studies showed that the extreme resistance of spores made by most FP strains is mediated, in large part, by a variant of small acid soluble protein 4 (Ssp4) that has Asp at residue 36; in contrast, the sensitive spores made by other C. perfringens type A isolates contain an Ssp4 variant with Gly at residue 36. The current study has further characterized Ssp4 properties and expression. Spores made by cpe-positive type C and D strains were found to contain the Ssp4 variant with Gly at residue 36 and were shown to be heat- and nitrite-sensitive; this finding may help to explain why cpe-positive type C and D isolates rarely cause food poisoning. Saturation mutagenesis indicated that both amino acid size and charge at Ssp4 residue 36 are important for DNA binding and for spore resistance. C. perfringens Ssp2 was shown to bind preferentially to GC-rich DNA on gel-shift assays, while Ssp4 preferred binding to AT-rich DNA sequences. Maximal spore heat and nitrite resistance required production of all four C. perfringens Ssps, indicating that these Ssps act cooperatively to protect the spore's DNA, perhaps by binding to different chromosomal sequences. The Ssp4 variant with Asp at residue 36 was also shown to facilitate exceptional spore survival at freezer and refrigerator temperatures. Finally, Ssp4 expression was shown to be dependent upon Spo0A, a master regulator. Collectively, these results provide additional support for the importance of Ssps, particularly the Ssp4 variant with Asp at residue 36, for the extreme spore resistance phenotype that likely contributes to C. perfringens type A food poisoning transmission.
KeywordMeSH Terms
54. Abildgaard  L, Schramm  A, Rudi  K, Højberg  O,     ( 2009 )

Dynamics of plc gene transcription and alpha-toxin production during growth of Clostridium perfringens strains with contrasting alpha-toxin production.

Veterinary microbiology 139 (1��2��)
PMID : 19559545  :   DOI  :   10.1016/j.vetmic.2009.05.014    
Abstract >>
The aim of the present study was to investigate transcription dynamics of the alpha-toxin-encoding plc gene relative to two housekeeping genes (gyrA and rplL) in batch cultures of three Clostridium perfringens strains with low, intermediate, and high levels of alpha-toxin production, respectively. The plc transcript level was always low in the low alpha-toxin producing strain. For the two other strains, plc transcription showed an inducible pattern and reached a maximum level in the late exponential growth phase. The transcription levels were however inversely correlated to alpha-toxin production for the two strains. We propose that this discrepancy is due to differences in plc translation rates between the strains and that strain-specific translational rates therefore must be determined before alpha-toxin production can be extrapolated from transcript levels in C. perfringens.
KeywordMeSH Terms
55. Keyburn  AL, Yan  XX, Bannam  TL, Van Immerseel  F, Rood  JI, Moore  RJ,     ( N/A )

Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin.

Veterinary research 41 (2)
PMID : 19931005  :   DOI  :   10.1051/vetres/2009069     PMC  :   PMC2797654    
Abstract >>
A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis.
KeywordMeSH Terms
56. Deguchi  A, Miyamoto  K, Kuwahara  T, Miki  Y, Kaneko  I, Li  J, McClane  BA, Akimoto  S,     ( 2009 )

Genetic characterization of type A enterotoxigenic Clostridium perfringens strains.

PloS one 4 (5)
PMID : 19479065  :   DOI  :   10.1371/journal.pone.0005598     PMC  :   PMC2682570    
Abstract >>
Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains.
KeywordMeSH Terms
57. Lyras  D, Adams  V, Ballard  SA, Teng  WL, Howarth  PM, Crellin  PK, Bannam  TL, Songer  JG, Rood  JI,     ( 2009 )

tISCpe8, an IS1595-family lincomycin resistance element located on a conjugative plasmid in Clostridium perfringens.

Journal of bacteriology 191 (20)
PMID : 19684139  :   DOI  :   10.1128/JB.00668-09     PMC  :   PMC2753038    
Abstract >>
Clostridium perfringens is a normal gastrointestinal organism that is a reservoir for antibiotic resistance genes and can potentially act as a source from which mobile elements and their associated resistance determinants can be transferred to other bacterial pathogens. Lincomycin resistance in C. perfringens is common and is usually encoded by erm genes that confer macrolide-lincosamide-streptogramin B resistance. In this study we identified strains that are lincomycin resistant but erythromycin sensitive and showed that the lincomycin resistance determinant was plasmid borne and could be transferred to other C. perfringens isolates by conjugation. The plasmid, pJIR2774, is the first conjugative C. perfringens R-plasmid to be identified that does not confer tetracycline resistance. Further analysis showed that resistance was encoded by the lnuP gene, which encoded a putative lincosamide nucleotidyltransferase and was located on tISCpe8, a functional transposable genetic element that was a member of the IS1595 family of transposon-like insertion sequences. This element had significant similarity to the mobilizable lincomycin resistance element tISSag10 from Streptococcus agalactiae. Like tISSag10, tISCpe8 carries a functional origin of transfer within the resistance gene, allowing the element to be mobilized by the conjugative transposon Tn916. The similarity of these elements and the finding that they both contain an oriT-like region support the hypothesis that conjugation may result in the movement of DNA modules that are not obviously mobile since they are not linked to conjugation or mobilization functions. This process likely plays a significant role in bacterial adaptation and evolution.
KeywordMeSH Terms
58. Abildgaard  L, Engberg  RM, Pedersen  K, Schramm  A, Hojberg  O,     ( 2009 )

Sequence variation in the alpha-toxin encoding plc gene of Clostridium perfringens strains isolated from diseased and healthy chickens.

Veterinary microbiology 136 (3��4��)
PMID : 19070974  :   DOI  :   10.1016/j.vetmic.2008.11.001    
Abstract >>
The aim of the present study was to analyse the genetic diversity of the alpha-toxin encoding plc gene and the variation in alpha-toxin production of Clostridium perfringens type A strains isolated from presumably healthy chickens and chickens suffering from either necrotic enteritis (NE) or cholangio-hepatitis. The alpha-toxin encoding plc genes from 60 different pulsed-field gel electrophoresis (PFGE) types (strains) of C. perfringens were sequenced and translated in silico to amino acid sequences and the alpha-toxin production was investigated in batch cultures of 45 of the strains using an enzyme-linked immunosorbent assay (ELISA) approach. Overall, the truncated amino acid sequences showed close similarity (>98% at the amino acid level) to previously reported sequences from chicken-derived C. perfringens isolates. Variations were however observed in 23 out of 379 aa positions leading to the definition of 26 different alpha-toxin sequence types among the 60 strains. Moreover, a type II intron of 834 non-coding nucleotides was identified in the plc gene of three of the investigated strains. The in vitro alpha-toxin production investigated in 45 of the strains, including the three harbouring the intron, revealed no correlation between PFGE type, alpha-toxin sequence type, health status of the host chickens and level of alpha-toxin production. It is therefore concluded that neither plc gene type nor alpha-toxin production level seems to correlate to origin (healthy or diseased chicken) of the C. perfringens strains.
KeywordMeSH Terms
Chickens
59. Ashida  H, Maki  R, Ozawa  H, Tani  Y, Kiyohara  M, Fujita  M, Imamura  A, Ishida  H, Kiso  M, Yamamoto  K,     ( 2008 )

Characterization of two different endo-alpha-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens.

Glycobiology 18 (9)
PMID : 18559962  :   DOI  :   10.1093/glycob/cwn053    
Abstract >>
Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene engBF encoding endo-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415-37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl-alpha-glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.
KeywordMeSH Terms
60. Chalmers  G, Bruce  HL, Hunter  DB, Parreira  VR, Kulkarni  RR, Jiang  YF, Prescott  JF, Boerlin  P,     ( 2008 )

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations.

Journal of clinical microbiology 46 (12)
PMID : 18945840  :   DOI  :   10.1128/JCM.01548-08     PMC  :   PMC2593256    
Abstract >>
Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.
KeywordMeSH Terms
Disease Outbreaks
61. Miki  Y, Miyamoto  K, Kaneko-Hirano  I, Fujiuchi  K, Akimoto  S,     ( 2008 )

Prevalence and characterization of enterotoxin gene-carrying Clostridium perfringens isolates from retail meat products in Japan.

Applied and environmental microbiology 74 (17)
PMID : 18606797  :   DOI  :   10.1128/AEM.00783-08     PMC  :   PMC2546627    
Abstract >>
Clostridium perfringens is an important anaerobic pathogen causing food-borne gastrointestinal (GI) diseases in humans and animals. It is thought that C. perfringens food poisoning isolates typically carry the enterotoxin gene (cpe) on their chromosome, while isolates from other GI diseases, such as antibiotic-associated diarrhea, carry cpe on a transferable plasmid. However, food-borne GI disease outbreaks associated with C. perfringens isolates carrying plasmid-borne cpe (plasmid cpe isolates) were recently reported in Japan and Europe. To investigate whether retail food can be a reservoir for food poisoning generally, we evaluated Japanese retail meat products for the presence of two genotypes of enterotoxigenic C. perfringens. Our results demonstrated that approximately 70% of the Japanese retail raw meat samples tested were contaminated with low numbers of C. perfringens bacteria and 4% were contaminated with cpe-positive C. perfringens. Most of the cpe-positive C. perfringens isolates obtained from Japanese retail meat carried cpe on a plasmid. The plasmid cpe isolates exhibited lower spore heat resistance than did chromosomal cpe isolates. Collectively, these plasmid cpe isolates might be causative agents of food poisoning when foods are contaminated with these isolates from equipment and/or the environment after cooking, or they may survive in food that has not been cooked at a high enough temperature.
KeywordMeSH Terms
Food Microbiology
62. Tsuge  H, Nagahama  M, Oda  M, Iwamoto  S, Utsunomiya  H, Marquez  VE, Katunuma  N, Nishizawa  M, Sakurai  J,     ( 2008 )

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin.

Proceedings of the National Academy of Sciences of the United States of America 105 (21)
PMID : 18490658  :   DOI  :   10.1073/pnas.0801215105     PMC  :   PMC2387182    
Abstract >>
The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.
KeywordMeSH Terms
63. Miyamoto  K, Li  J, Sayeed  S, Akimoto  S, McClane  BA,     ( 2008 )

Sequencing and diversity analyses reveal extensive similarities between some epsilon-toxin-encoding plasmids and the pCPF5603 Clostridium perfringens enterotoxin plasmid.

Journal of bacteriology 190 (21)
PMID : 18776010  :   DOI  :   10.1128/JB.00939-08     PMC  :   PMC2580689    
Abstract >>
Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.
KeywordMeSH Terms
Genetic Variation
64. Li  J, McClane  BA,     ( 2008 )

A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

PLoS pathogens 4 (5)
PMID : 18451983  :   DOI  :   10.1371/journal.ppat.1000056     PMC  :   PMC2323104    
Abstract >>
Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.
KeywordMeSH Terms
65. Keyburn  AL, Boyce  JD, Vaz  P, Bannam  TL, Ford  ME, Parker  D, Di Rubbo  A, Rood  JI, Moore  RJ,     ( 2008 )

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens.

PLoS pathogens 4 (2)
PMID : 18266469  :   DOI  :   10.1371/journal.ppat.0040026     PMC  :   PMC2233674    
Abstract >>
For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.
KeywordMeSH Terms
66. Newstead  SL, Potter  JA, Wilson  JC, Xu  G, Chien  CH, Watts  AG, Withers  SG, Taylor  GL,     ( 2008 )

The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates.

The Journal of biological chemistry 283 (14)
PMID : 18218621  :   DOI  :   10.1074/jbc.M710247200     PMC  :   PMC2431023    
Abstract >>
Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97A resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97A resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2A resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested.
KeywordMeSH Terms
67. Sandrini  MP, Clausen  AR, On  SL, Aarestrup  FM, Munch-Petersen  B, Piskur  J,     ( 2007 )

Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner.

The Journal of antimicrobial chemotherapy 60 (3)
PMID : 17615154  :   DOI  :   10.1093/jac/dkm240    
Abstract >>
To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Several FDA-approved nucleoside analogue drugs were screened for their potential bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes. These genes were tested for their ability to increase the susceptibility of a dNK-deficient E. coli strain to various analogues. We overexpressed, purified and characterized the substrate specificity and kinetic properties of the recombinant enzymes from S. enterica and B. cereus. The tested Gram-negative bacteria were susceptible to 3'-azido-3'-deoxythymidine (AZT) in the concentration range 0.032-31.6 microM except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concentrations. Purified recombinant S. enterica thymidine kinase phosphorylated AZT efficiently with a Km of 73.3 microM and k(cat)/Km of 6.6 x 10(4) s(-1) M(-1) and is the activator of this drug in vivo. 2',2'-Difluoro-2'-deoxycytidine (gemcitabine) was a potent antibiotic against Gram-positive bacteria in the concentration range between 0.001 and 1.0 microM. The B. cereus deoxyadenosine kinase had a Km for gemcitabine of 33.5 microM and k(cat)/Km of 5.1 x 10(3) s(-1) M(-1) and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes. Bacterial dNKs efficiently activate nucleoside analogues in a species-specific manner. Therefore, nucleoside analogues have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria.
KeywordMeSH Terms
68. van Asten  AJ, Allaart  JG, Meeles  AD, Gloudemans  PW, Houwers  DJ, Gröne  A,     ( 2008 )

A new PCR followed by MboI digestion for the detection of all variants of the Clostridium perfringens cpb2 gene.

Veterinary microbiology 127 (3��4��)
PMID : 17980519  :   DOI  :   10.1016/j.vetmic.2007.08.035    
Abstract >>
Clostridium perfringens which is a causative agent of several diseases in animals and humans is capable of producing a variety of toxins. Isolates are typed into five types on the basis of the presence of one or more of the four major toxins genes, i.e. cpa, cpb, etx, and iap. A decade ago another toxin termed beta2 (beta2) and its gene (cpb2) were identified. Two alleles of cpb2 are known and a possible link between differences in gene expression and allelic variation has been reported. A correlation between the level of expression and the origin of the isolates has also been suggested. The demonstration and typing of the cpb2 gene in the genome of isolates can be seen as a vital part of research on the role of the beta2 toxin in the pathogenesis of disease. This study describes a PCR with a single primer set which in contrast to published primer sets recognizes both alleles. Subsequent restriction enzyme analysis of the PCR product enables typing of the alleles. Applying this protocol on a total of 102 isolates, a sub-variant was found which occurred only in C. perfringens isolates from pigs and appeared to be the predominant variant found in C. perfringens isolates from this species.
KeywordMeSH Terms
Genetic Variation
69. Chalmers  G, Martin  SW, Hunter  DB, Prescott  JF, Weber  LJ, Boerlin  P,     ( 2008 )

Genetic diversity of Clostridium perfringens isolated from healthy broiler chickens at a commercial farm.

Veterinary microbiology 127 (1��2��)
PMID : 17888591  :   DOI  :   10.1016/j.vetmic.2007.08.008    
Abstract >>
Clostridium perfringens is an important commensal and bacterial pathogen of many animal species. It has particular significance in poultry, where it may cause necrotic enteritis. Our objective was to characterize the population diversity of C. perfringens colonizing healthy birds, and to observe how diversity changed over time. Isolates were obtained from broiler chicken cecal samples in two barns on a single farm, on days 7, 14, 22, 27, 30 and 34 of a single 42-day rearing cycle. Bacitracin was used as a feed additive in one of the barns and withdrawn from the second barn for the duration of the experiment. Each isolate was typed using pulsed-field gel electrophoresis (PFGE) using SmaI restriction endonuclease. A total of 205 cecal isolates from 49 birds were typed, as well as 93 isolates from the barn environment (bedding, drinking water and feces). Eight major PFGE types and 17 subtypes were found in the 298 total isolates. The results show that an optimal sampling strategy would involve a large number of birds, with only a few isolates sampled per bird. The diversity of C. perfringens in this study appears to be low within a single bird, and increases as the bird matures. There was no significant difference in genetic diversity between the two barns. In addition, isolates from fresh fecal samples appear to represent the cecal C. perfringens population accurately, although this was not proven statistically. Antimicrobial susceptibility testing was performed on selected isolates (n=41) representing a cross-section of PFGE types. Based on minimum inhibitory concentration distributions, 95% of the isolates tested were deemed resistant to bacitracin, with a 16 microg/mL breakpoint. Three new cpb2 (beta2 toxin gene) variants were found in the study.
KeywordMeSH Terms
Genetic Variation
70. Yu  Q, Lepp  D, Mehdizadeh Gohari  I, Wu  T, Zhou  H, Yin  X, Yu  H, Prescott  JF, Nie  SP, Xie  MY, Gong  J,     ( 2017 )

The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry.

Infection and immunity 85 (6)
PMID : 28373356  :   DOI  :   10.1128/IAI.00975-16     PMC  :   PMC5442616    
Abstract >>
Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.
KeywordMeSH Terms
Agr-like quorum sensing
Clostridium perfringens
LuxS
NetB
VirS/VirR
necrotic enteritis
poultry
quorum sensing
Quorum Sensing
71. Van Damme-Jongsten  M, Wernars  K, Notermans  S,     ( 1989 )

Cloning and sequencing of the Clostridium perfringens enterotoxin gene.

Antonie van Leeuwenhoek 56 (2)
PMID : 2802575  :   DOI  :   10.1007/bf00399981    
Abstract >>
Several gene banks of Clostridium perfringens in E. coli were constructed. Using a mixture of synthetic 29-mer DNA probes clones were selected containing inserts from the C. perfringens gene coding for the enterotoxin. This has allowed sequencing of the complete gene and its flanking regions. The decuded amino acid sequence (320 a.a.) was found to differ at several sites from the sequence published previously by others. Two 40-mer DNA-probes were used to detect the toxin gene in C. perfringens strains isolated from the faeces of different non-symptomatic animals. Only 6% of the strains were found to possess the gene.
KeywordMeSH Terms
72. Nakano  V, Ignacio  A, Llanco  L, Bueris  V, Sircili  MP, Avila-Campos  MJ,     ( 2017 )

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes.

Anaerobe 44 (N/A)
PMID : 28238845  :   DOI  :   10.1016/j.anaerobe.2017.02.017    
Abstract >>
Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.
KeywordMeSH Terms
Clostridium perfringens
Multilocus sequence typing [MLST]
Phylogenetic
Sequence types [ST]
Genotype
Multilocus Sequence Typing
73. Toniti  W, Yoshida  T, Tsurumura  T, Irikura  D, Monma  C, Kamata  Y, Tsuge  H,     ( 2017 )

Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.

PloS one 12 (2)
PMID : 28199340  :   DOI  :   10.1371/journal.pone.0171278     PMC  :   PMC5310789    
Abstract >>
Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia-actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both �\- and �]/�^-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both �\- and �]/�^-actin, but its sensitivity towards �]/�^-actin is 36% compared with �\-actin. Our results contrast to that only C2-I ADP-ribosylates �]/�^-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both �\- and �]/�^-actin.
KeywordMeSH Terms
74. Shinoda  T, Shinya  N, Ito  K, Ohsawa  N, Terada  T, Hirata  K, Kawano  Y, Yamamoto  M, Kimura-Someya  T, Yokoyama  S, Shirouzu  M,     ( 2016 )

Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin.

Scientific reports 6 (N/A)
PMID : 27647526  :   DOI  :   10.1038/srep33632     PMC  :   PMC5028891    
Abstract >>
The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-? resolution crystal structure of the cell-free synthesized human claudin-4?C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4?C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs.
KeywordMeSH Terms
75. Kawahara  K, Yonogi  S, Munetomo  R, Oki  H, Yoshida  T, Kumeda  Y, Matsuda  S, Kodama  T, Ohkubo  T, Iida  T, Nakamura  S,     ( 2016 )

Crystal structure of the ADP-ribosylating component of BEC, the binary enterotoxin of Clostridium perfringens.

Biochemical and biophysical research communications 480 (2)
PMID : 27751850  :   DOI  :   10.1016/j.bbrc.2016.10.042    
Abstract >>
Binary enterotoxin of Clostridium perfringens (BEC), consisting of the components BECa and BECb, was recently identified as a novel enterotoxin produced by C. perfringens that causes acute gastroenteritis in humans. Although the detailed mechanism of cell intoxication by BEC remains to be defined, BECa shows both NAD+-glycohydrolase and actin ADP-ribosyltransferase activities in the presence of NAD+. In this study, we determined the first crystal structure of BECa in its apo-state and in complex with NADH. The structure of BECa shows striking resemblance with other binary actin ADP-ribosylating toxins (ADPRTs), especially in terms of its overall protein fold and mechanisms of substrate recognition. We present a detailed picture of interactions between BECa and NADH, including bound water molecules located near the C1'-N glycosidic bond of NADH and the catalytically important ADP-ribosylating turn-turn (ARTT) loop. We observed that the conformational rearrangement of the ARTT loop, possibly triggered by a conformational change involving a conserved tyrosine residue coupled with substrate binding, plays a crucial role in catalysis by properly positioning a catalytic glutamate residue in the E-X-E motif of the ARTT loop in contact with the nucleophile. Our results for BECa provide insight into the common catalytic mechanism of the family of binary actin ADPRTs.
KeywordMeSH Terms
Binary enterotoxin
C. perfringens
Protein structure
X-ray crystallography
76. Al-Riyami  B, ?stok  FI, Stott  K, Chirgadze  DY, Christie  G,     ( 2016 )

The crystal structure of Clostridium perfringens SleM, a muramidase involved in cortical hydrolysis during spore germination.

Proteins 84 (11)
PMID : 27488615  :   DOI  :   10.1002/prot.25112    
Abstract >>
Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the C. perfringens S40 SleM protein at 1.8 ?. SleM comprises an N-terminal catalytic domain that adopts an irregular �\/�]-barrel fold that is common to GH25 family lysozymes, plus a C-terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Proteins 2016; 84:1681-1689. ? 2016 Wiley Periodicals, Inc.
KeywordMeSH Terms
GH25 family
cortex lytic enzyme
peptidoglycan lysin
spore
77. Morra  S, Mongili  B, Maurelli  S, Gilardi  G, Valetti  F,     ( 2016 )

Isolation and characterization of a new [FeFe]-hydrogenase from Clostridium perfringens.

Biotechnology and applied biochemistry 63 (3)
PMID : 25851509  :   DOI  :   10.1002/bab.1382    
Abstract >>
This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron-sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645 �� 16 s(-1) . The optimal conditions for catalysis are in the pH range 6.5-8.0 and at the temperature of 50 �XC. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO-inhibited enzyme has a spectrum coherent with the Hox -CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox ; upon reduction with H2 , vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox -CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster.
KeywordMeSH Terms
Clostridium perfringens
H-cluster
[FeFe]-hydrogenases
bio-hydrogen
iron-sulfur centers
recombinant expression
78. Mehdizadeh Gohari  I, Parreira  VR, Nowell  VJ, Nicholson  VM, Oliphant  K, Prescott  JF,     ( 2015 )

A novel pore-forming toxin in type A Clostridium perfringens is associated with both fatal canine hemorrhagic gastroenteritis and fatal foal necrotizing enterocolitis.

PloS one 10 (4)
PMID : 25853427  :   DOI  :   10.1371/journal.pone.0122684     PMC  :   PMC4390311    
Abstract >>
A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatants of an isolate made from a dog and from a foal that died from these diseases were both found to be highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro. There was a highly significant association between the presence of netF with type A strains isolated from cases of canine acute hemorrhagic gastroenteritis and foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal population; some canine and equine netF-positive isolates were genetically indistinguishable. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity. The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.
KeywordMeSH Terms
79. Mehdizadeh Gohari  I, Kropinski  AM, Weese  SJ, Parreira  VR, Whitehead  AE, Boerlin  P, Prescott  JF,     ( 2016 )

Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis.

PloS one 11 (2)
PMID : 26859667  :   DOI  :   10.1371/journal.pone.0148344     PMC  :   PMC4747519    
Abstract >>
The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.
KeywordMeSH Terms
80. Theoret  JR, Uzal  FA, McClane  BA,     ( 2015 )

Identification and characterization of Clostridium perfringens beta toxin variants with differing trypsin sensitivity and in vitro cytotoxicity activity.

Infection and immunity 83 (4)
PMID : 25643999  :   DOI  :   10.1128/IAI.02864-14     PMC  :   PMC4363432    
Abstract >>
By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.
KeywordMeSH Terms
81. Li  J, Freedman  JC, McClane  BA,     ( 2015 )

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718.

Journal of bacteriology 197 (20)
PMID : 26260460  :   DOI  :   10.1128/JB.00349-15     PMC  :   PMC4573732    
Abstract >>
Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. Clostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using single ccpA or codY null mutants and a ccpA codY double null mutant showed that codY and ccpA regulate ETX production independently of one another but that ccpA also affects codY transcription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulate etx transcription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producing C. perfringens strains, via CcpA and CodY, to upregulate ETX production and cause disease.
KeywordMeSH Terms
82. Saitoh  Y, Suzuki  H, Tani  K, Nishikawa  K, Irie  K, Ogura  Y, Tamura  A, Tsukita  S, Fujiyoshi  Y,     ( 2015 )

Tight junctions. Structural insight into tight junction disassembly by Clostridium perfringens enterotoxin.

Science (New York, N.Y.) 347 (6223)
PMID : 25678664  :   DOI  :   10.1126/science.1261833    
Abstract >>
The C-terminal region of Clostridium perfringens enterotoxin (C-CPE) can bind to specific claudins, resulting in the disintegration of tight junctions (TJs) and an increase in the paracellular permeability across epithelial cell sheets. Here we present the structure of mammalian claudin-19 in complex with C-CPE at 3.7 ? resolution. The structure shows that C-CPE forms extensive hydrophobic and hydrophilic interactions with the two extracellular segments of claudin-19. The claudin-19/C-CPE complex shows no density of a short extracellular helix that is critical for claudins to assemble into TJ strands. The helix displacement may thus underlie C-CPE-mediated disassembly of TJs.
KeywordMeSH Terms
83. Wade  B, Keyburn  AL, Seemann  T, Rood  JI, Moore  RJ,     ( 2015 )

Binding of Clostridium perfringens to collagen correlates with the ability to cause necrotic enteritis in chickens.

Veterinary microbiology 180 (3��4��)
PMID : 26455806  :   DOI  :   10.1016/j.vetmic.2015.09.019    
Abstract >>
This study investigated the ability of Clostridium perfringens isolates derived from chickens to bind to collagen types I-V and gelatin. In total 21 strains from three distinct backgrounds were studied: (i) virulent strains isolated from birds suffering from necrotic enteritis, (ii) avirulent strains isolated from birds suffering from necrotic enteritis and (iii) strains isolated from healthy birds. All strains isolated from diseased birds had been assessed for virulence in a disease induction model. The virulent isolates all displayed collagen binding ability. However, most strains in the other two classes showed negligible binding to collagen. The prevalence of a previously described C. perfringens putative collagen adhesin-encoding gene was investigated by PCR screening. It was found that five of the strains carried the putative collagen adhesin-encoding gene and that all of these strains were virulent isolates. Based on these studies it is postulated that collagen adhesion may play a role in the pathogenesis of necrotic enteritis.
KeywordMeSH Terms
Adhesin
Adhesion
Clostridium perfringens
Collagen
Necrotic enteritis
Poultry
Bacterial Adhesion
84. Timbermont  L, De Smet  L, Van Nieuwerburgh  F, Parreira  VR, Van Driessche  G, Haesebrouck  F, Ducatelle  R, Prescott  J, Deforce  D, Devreese  B, Van Immerseel  F,     ( 2014 )

Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis.

Veterinary research 45 (N/A)
PMID : 24708344  :   DOI  :   10.1186/1297-9716-45-40     PMC  :   PMC3992141    
Abstract >>
Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.
KeywordMeSH Terms
Chickens
85. Garnier  T, Cole  ST,     ( 1988 )

Studies of UV-inducible promoters from Clostridium perfringens in vivo and in vitro.

Molecular microbiology 2 (5)
PMID : 2460717  :   DOI  :   10.1111/j.1365-2958.1988.tb00069.x    
Abstract >>
Expression of a 4 kb segment of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens is inducible by UV-irradiation. DNA sequence analysis revealed that this region contains three genes: uviA, uviB and bcn encoding the bacteriocin BCN5. Biochemical studies with mRNAs showed that expression was controlled at the transcriptional level and that the genes were organized in two independent transcriptional units, uviAB and bcn, both directed by tandem promoters inducible by UV light. The bcn gene is transcribed from three promoters (P1, P2, P3) while transcription of uviAB is directed by two promoters (P4, P5). With the exception of P4, which bears some resemblance to the consensus eubacterial promoter sequence, none of these promoters was recognized in vitro by the major forms of RNA polymerase from C. perfringens, Bacillus subtilis or Escherichia coli. Promoters P1, P3 and P5, which show striking homology with each other, contain unusual sequences in the '-35' and '-10' regions known to be recognized by RNA polymerase and this might indicate positive control.
KeywordMeSH Terms
Gene Expression Regulation
Promoter Regions, Genetic
86. Yonogi  S, Matsuda  S, Kawai  T, Yoda  T, Harada  T, Kumeda  Y, Gotoh  K, Hiyoshi  H, Nakamura  S, Kodama  T, Iida  T,     ( 2014 )

BEC, a novel enterotoxin of Clostridium perfringens found in human clinical isolates from acute gastroenteritis outbreaks.

Infection and immunity 82 (6)
PMID : 24664508  :   DOI  :   10.1128/IAI.01759-14     PMC  :   PMC4019177    
Abstract >>
Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.
KeywordMeSH Terms
87. Steffen  C, Matzura  H,     ( 1989 )

Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens.

Gene 75 (2)
PMID : 2541053  :   DOI  :   10.1016/0378-1119(89)90282-5    
Abstract >>
The nucleotide sequence of a CmR determinant, located on the Clostridium perfringens plasmid pIP401, was determined and its gene product was identified as chloramphenicol acetyltransferase (CAT). The cat structural gene is preceded by transcription-initiation signals characteristic for Escherichia coli sigma 70 or Bacillus subtilis sigma 43 promoters. By promoter probing in the heterologous hosts the direction of transcription of the clostridial cat gene was analysed and the cat mRNA start point was determined in vitro using the RNA polymerases of E. coli and B. subtilis. Comparison of the amino acid sequences of C. perfringens CAT and other CAT proteins of Gram-positive and Gram-negative origin shows a remarkable degree of homology between the various enzymes.
KeywordMeSH Terms
Genes, Bacterial
Transcription, Genetic
88. Shearer  AG, Altman  T, Rhee  CD,     ( 2014 )

Finding sequences for over 270 orphan enzymes.

PloS one 9 (5)
PMID : 24826896  :   DOI  :   10.1371/journal.pone.0097250     PMC  :   PMC4020792    
Abstract >>
Despite advances in sequencing technology, there are still significant numbers of well-characterized enzymatic activities for which there are no known associated sequences. These 'orphan enzymes' represent glaring holes in our biological understanding, and it is a top priority to reunite them with their coding sequences. Here we report a methodology for resolving orphan enzymes through a combination of database search and literature review. Using this method we were able to reconnect over 270 orphan enzymes with their corresponding sequence. This success points toward how we can systematically eliminate the remaining orphan enzymes and prevent the introduction of future orphan enzymes.
KeywordMeSH Terms
89. Huyet  J, Naylor  CE, Savva  CG, Gibert  M, Popoff  MR, Basak  AK,     ( 2013 )

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation.

PloS one 8 (6)
PMID : 23805259  :   DOI  :   10.1371/journal.pone.0066673     PMC  :   PMC3689675    
Abstract >>
Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus �]-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 ? crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal �\-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.
KeywordMeSH Terms
90. Siqueira  FF, Almeida  MO, Barroca  TM, Horta  CC, Carmo  AO, Silva  RO, Pires  PS, Lobato  FC, Kalapothakis  E,     ( 2012 )

Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

Veterinary microbiology 159 (3��4��)
PMID : 22560738  :   DOI  :   10.1016/j.vetmic.2012.04.012    
Abstract >>
Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.
KeywordMeSH Terms
Environmental Microbiology
Polymorphism, Genetic
91. Porter  CJ, Bantwal  R, Bannam  TL, Rosado  CJ, Pearce  MC, Adams  V, Lyras  D, Whisstock  JC, Rood  JI,     ( 2012 )

The conjugation protein TcpC from Clostridium perfringens is structurally related to the type IV secretion system protein VirB8 from Gram-negative bacteria.

Molecular microbiology 83 (2)
PMID : 22150951  :   DOI  :   10.1111/j.1365-2958.2011.07930.x    
Abstract >>
Bacterial conjugation is important for the acquisition of virulence and antibiotic resistance genes. We investigated the mechanism of conjugation in Gram-positive pathogens using a model plasmid pCW3 from Clostridium perfringens. pCW3 encodes tetracycline resistance and contains the tcp locus, which is essential for conjugation. We showed that the unique TcpC protein (359 amino acids, 41 kDa) was required for efficient conjugative transfer, localized to the cell membrane independently of other conjugation proteins, and that membrane localization was important for its function, oligomerization and interaction with the conjugation proteins TcpA, TcpH and TcpG. The crystal structure of the C-terminal component of TcpC (TcpC(99-359)) was determined to 1.8-? resolution. TcpC(99-359) contained two NTF2-like domains separated by a short linker. Unexpectedly, comparative structural analysis showed that each of these domains was structurally homologous to the periplasmic region of VirB8, a component of the type IV secretion system from Agrobacterium tumefaciens. Bacterial two-hybrid studies revealed that the C-terminal domain was critical for interactions with other conjugation proteins. The N-terminal region of TcpC was required for efficient conjugation, oligomerization and protein-protein interactions. We conclude that by forming oligomeric complexes, TcpC contributes to the stability and integrity of the conjugation apparatus, facilitating efficient pCW3 transfer.
KeywordMeSH Terms
92. Vidal  JE, Ma  M, Saputo  J, Garcia  J, Uzal  FA, McClane  BA,     ( 2012 )

Evidence that the Agr-like quorum sensing system regulates the toxin production, cytotoxicity and pathogenicity of Clostridium perfringens type C isolate CN3685.

Molecular microbiology 83 (1)
PMID : 22150719  :   DOI  :   10.1111/j.1365-2958.2011.07925.x     PMC  :   PMC3285497    
Abstract >>
Clostridium perfringens possesses at least two functional quorum sensing (QS) systems, i.e. an Agr-like system and a LuxS-dependent AI-2 system. Both of those QS systems can reportedly control in vitro toxin production by C. perfringens but their importance for virulence has not been evaluated. Therefore, the current study assessed whether these QS systems might regulate the pathogenicity of CN3685, a C. perfringens type C strain. Since type C isolates cause both haemorrhagic necrotic enteritis and fatal enterotoxemias (where toxins produced in the intestines are absorbed into the circulation to target other internal organs), the ability of isogenic agrB or luxS mutants to cause necrotizing enteritis in rabbit small intestinal loops or enterotoxemic lethality in mice was evaluated. Results obtained strongly suggest that the Agr-like QS system, but not the LuxS-dependent AI-2 QS system, is required for CN3685 to cause haemorrhagic necrotizing enteritis, apparently because the Agr-like system regulates the production of beta toxin, which is essential for causing this pathology. The Agr-like system, but not the LuxS-mediated AI-2 system, was also important for CN3685 to cause fatal enterotoxemia. These results provide the first direct evidence supporting a role for any QS system in clostridial infections.
KeywordMeSH Terms
Quorum Sensing
93. Miyamoto  K, Yumine  N, Mimura  K, Nagahama  M, Li  J, McClane  BA, Akimoto  S,     ( 2011 )

Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene.

PloS one 6 (5)
PMID : 21655254  :   DOI  :   10.1371/journal.pone.0020376     PMC  :   PMC3105049    
Abstract >>
Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ?65 kb. Complete sequence analysis of the ?65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ?65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.
KeywordMeSH Terms
94. Bannam  TL, Yan  XX, Harrison  PF, Seemann  T, Keyburn  AL, Stubenrauch  C, Weeramantri  LH, Cheung  JK, McClane  BA, Boyce  JD, Moore  RJ, Rood  JI,     ( 2011 )

Necrotic enteritis-derived Clostridium perfringens strain with three closely related independently conjugative toxin and antibiotic resistance plasmids.

mBio 2 (5)
PMID : 21954306  :   DOI  :   10.1128/mBio.00190-11     PMC  :   PMC3181468    
Abstract >>
The pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netB was insertionally inactivated by the chloramphenicol and thiamphenicol resistance gene catP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and the netB gene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 from C. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained the netB gene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2 gene, which encodes a different pore-forming toxin, beta2 toxin. The anaerobic bacterium Clostridium perfringens can cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries the netB gene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids from C. perfringens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 toxin, and the other encoded tetracycline resistance. To our knowledge, this is the first report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.
KeywordMeSH Terms
Drug Resistance, Bacterial
Plasmids
95. Chen  J, Rood  JI, McClane  BA,     ( 2011 )

Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system.

mBio 2 (6)
PMID : 22167225  :   DOI  :   10.1128/mBio.00275-11     PMC  :   PMC3236063    
Abstract >>
Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. IMPORTANCE Mechanisms by which Clostridium perfringens type B and D strains regulate production of epsilon-toxin (ETX), a CDC class B select toxin, are poorly understood. Production of several other toxins expressed by C. perfringens is wholly or partially regulated by both the Agr-like quorum-sensing (QS) system and the VirS/VirR two-component regulatory system, so the present study tested whether ETX expression by type D strain CN3718 also requires these regulatory systems. The agr operon was shown to be essential for signaling CN3718 to produce ETX in broth culture or to upregulate ETX production upon close contact with enterocyte-like Caco-2 cells, which may have pathogenic relevance since ETX is produced intestinally. However, ETX production remained at wild-type levels after inactivation of the VirS/VirR system in CN3718. These findings provide the first information regarding regulation of ETX production and suggest Agr-like QS toxin production regulation in C. perfringens does not always require the VirS/VirR system.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
96.     ( 1997 )

Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene.

Journal of bacteriology 179 (10)
PMID : 9150212  :   DOI  :   10.1128/jb.179.10.3181-3187.1997     PMC  :   PMC179095    
Abstract >>
The exudate of fully germinated spores of Clostridium perfringens S40 in 0.15 M KCI-50 mM potassium phosphate (pH 7.0) was found to contain another spore-lytic enzyme in addition to the germination-specific amidase previously characterized (S. Miyata, R. Moriyama, N. Miyahara, and S. Makino, Microbiology 141:2643-2650, 1995). The lytic enzyme was purified to homogeneity by anion-exchange chromatography and shown to be a muramidase which requires divalent cations (Ca2+, Mg2+, or Mn2+) for its activity. The enzyme was inactivated by sulfhydryl reagents, and sodium thioglycolate reversed the inactivation by Hg2+. The muramidase hydrolyzed isolated spore cortical fragments from a variety of wild-type organisms but had minimal activity on decoated spores and isolated cell walls. However, the enzyme was not capable of digesting isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam in its cortical peptidoglycan. This indicates that the enzyme recognizes the delta-lactam residue peculiar to spore peptidoglycan, suggesting an involvement of the enzyme in spore germination. Immunochemical studies indicated that the muramidase in its mature form is localized on the exterior of the cortex layer in the dormant spore. A gene encoding the muramidase, sleM, was cloned into Escherichia coli, and the nucleotide sequence was determined. The gene encoded a protein of 321 amino acids with a deduced molecular weight of 36,358. The deduced amino acid sequence of the sleM gene indicated that the enzyme is produced in a mature form. It was suggested that the muramidase belongs to a separate group within the lysozyme family typified by the fungus Chalaropsis lysozyme. A possible mechanism for cortex degradation in C. perfringens S40 spores is discussed.
KeywordMeSH Terms
Genes, Bacterial
97.     ( 1997 )

Clostridium perfringens urease genes are plasmid borne.

Infection and immunity 65 (6)
PMID : 9169769  :   PMC  :   PMC175321    
Abstract >>
Although many bacteria are ureolytic, and in some cases urease acts as a virulence factor, the urease phenotype has not been analyzed in the anaerobic pathogen Clostridium perfringens. In this study, approximately 2% of C. perfringens strains, representing the principal biotypes, were found to harbor the urease structural genes, ureABC, and these were localized on large plasmids that often encode, in addition, the lethal epsilon or iota toxins or the enterotoxin. This represents the first report of a plasmid-encoded urease in a gram-positive bacterium. The C. perfringens enzyme was highly similar to the ureases of other bacteria and cross-reacted with antibodies raised against the urease purified from Helicobacter pylori. Urease production was inhibited by urea and induced under growth conditions where the availability of nitrogen sources was limiting. To date, this form of regulation has been observed only for chromosomal ureABC genes.
KeywordMeSH Terms
Plasmids
98.     ( 1997 )

The Clostridium perfringens enterotoxin gene is on a transposable element in type A human food poisoning strains.

Microbiology (Reading, England) 143 (Pt 7) (N/A)
PMID : 9245800  :   DOI  :   10.1099/00221287-143-7-2109    
Abstract >>
The Clostridium perfringens enterotoxin gene (cpe) is rarely found in naturally isolated strains. In human food poisoning strains, cpe is found on the chromosome, and is located episomally in animal isolates. Observations that the gene was somewhat unstable and could be gained or lost suggested that the gene was on a mobile element. An IS200-like element, IS1469, is almost always upstream of cpe. A new insertion element was identified, IS1470, a member of the IS30 family, which is found both up-an downstream of cpe in the type A strain NCTC 8239. PCR results confirmed that this configuration was conserved in type A human food poisoning strains. The enterotoxin gene was on a 6.3 kb transposon which, in addition to the two flanking copies of IS1470, included IS1469 and two 1 kb stretches, one on each side of cpe, with no open reading frames. Results indicated that 14 bp was copied from the genome during insertion. Details of the configuration of DNA in this transposon are presented, and the possible connection of this transposon with the movement of the enterotoxin gene is discussed.
KeywordMeSH Terms
Food Microbiology
Genes, Bacterial
99.     ( 1997 )

Collagenase gene (colA) is located in the 3'-flanking region of the perfringolysin O (pfoA) locus in Clostridium perfringens.

FEMS microbiology letters 146 (1)
PMID : 9053381  :   DOI  :   10.1111/j.1574-6968.1997.tb10186.x    
Abstract >>
The 3'-flanking region of the beta-galactosidase gene (pbg), which is located downstream of the perfringolysin O gene (pfoA), and the 5'-flanking region of the collagenase gene (colA) of Clostridium perfringens strains NCTC8237 and 13, respectively, were analyzed. Southern analysis revealed that the colA gene is located 6.5 kb downstream of the pbg gene in the chromosome of C. perfringens. Sequence analysis showed that between the pbg and colA genes were the arcABDC and ahrC genes, whose putative products were quite similar to enzymes of the arginine deiminase pathway of Pseudomonas aeruginosa and the arginine repressor/activator of Bacillus subtilis, respectively. It is concluded that the genomic structure of the pfoA-colA region consists of pfoR-pfoA-ORF54-pbg-arcABDC-ahrC-colA.
KeywordMeSH Terms
Genes, Bacterial
100.     ( 1993 )

Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli.

Infection and immunity 61 (8)
PMID : 8335373  :   PMC  :   PMC281020    
Abstract >>
A complete copy of the gene (cpe) encoding Clostridium perfringens enterotoxin (CPE), an important virulence factor involved in C. perfringens food poisoning and other gastrointestinal illnesses, has been cloned, sequenced, and expressed in Escherichia coli. The cpe gene was shown to encode a 319-amino-acid polypeptide with a deduced molecular weight of 35,317. There was no consensus sequence for a typical signal peptide present in the 5' region of cpe. Cell lysates from recombinant cpe-positive E. coli were shown by quantitative immunoblot analysis to contain moderate amounts of CPE, and this recombinant CPE was equal to native CPE in cytotoxicity for mammalian Vero cells. CPE expression in recombinant E. coli appeared to be largely driven from a clostridial promoter. Immunoblot analysis also demonstrated very low levels of CPE in vegetative cell lysates of enterotoxin-positive C. perfringens. However, when the same C. perfringens strain was induced to sporulate, much stronger CPE expression was detected in these sporulating cells than in either vegetative C. perfringens cells or recombinant E. coli. Collectively, these results strongly suggest that sporulation is not essential for cpe expression, but sporulation does facilitate high-level cpe expression.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
101.     ( 1993 )

IS1151, an IS-like element of Clostridium perfringens.

Nucleic acids research 21 (2)
PMID : 8382797  :   DOI  :   10.1093/nar/21.2.352     PMC  :   PMC309114    
Abstract >>
N/A
KeywordMeSH Terms
DNA Transposable Elements
102.     ( 1993 )

Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus.

Infection and immunity 61 (9)
PMID : 8359918  :   PMC  :   PMC281100    
Abstract >>
Oligonucleotide probes designed on the basis of the N-terminal sequence of Clostridium perfringens beta-toxin were used to isolate the encoding gene (cpb). The nucleotide sequence of cpb was determined, and on the basis of DNA hybridization experiments it was shown that the gene is found only in type B and C strains of C. perfringens. The deduced amino acid sequence of the beta-toxin revealed homology with the alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. The beta-toxin purified from C. perfringens appeared to exist in monomeric and multimeric forms. Recombinant beta-toxin, produced in Escherichia coli, appeared to be mainly in the multimeric form.
KeywordMeSH Terms
Clostridium perfringens
Staphylococcus aureus
103.     ( 1993 )

Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli.

Infection and immunity 61 (12)
PMID : 8225592  :   PMC  :   PMC281295    
Abstract >>
The iota toxin which is produced by Clostridium perfringens type E, is a binary toxin consisting of two independent polypeptides: Ia, which is an ADP-ribosyltransferase, and Ib, which is involved in the binding and internalization of the toxin into the cell. Two degenerate oligonucleotide probes deduced from partial amino acid sequence of each component of C. spiroforme toxin, which is closely related to the iota toxin, were used to clone three overlapping DNA fragments containing the iota-toxin genes from C. perfringens type E plasmid DNA. Two genes, in the same orientation, coding for Ia (387 amino acids) and Ib (875 amino acids) and separated by 243 noncoding nucleotides were identified. A predicted signal peptide was found for each component, and the secreted Ib displays two domains, the propeptide (172 amino acids) and the mature protein (664 amino acids). The Ia gene has been expressed in Escherichia coli and C. perfringens, under the control of its own promoter. The recombinant polypeptide obtained was recognized by Ia antibodies and ADP-ribosylated actin. The expression of the Ib gene was obtained in E. coli harboring a recombinant plasmid encompassing the putative promoter upstream of the Ia gene and the Ia and Ib genes. Two residues which have been found to be involved in the NAD+ binding site of diphtheria and pseudomonas toxins are conserved in the predicted Ia sequence (Glu-14 and Trp-19). The predicted amino acid Ib sequence shows 33.9% identity with and 54.4% similarity to the protective antigen of the anthrax toxin complex. In particular, the central region of Ib, which contains a predicted transmembrane segment (Leu-292 to Ser-308), presents 45% identity with the corresponding protective antigen sequence which is involved in the translocation of the toxin across the cell membrane.
KeywordMeSH Terms
ADP Ribose Transferases
Antigens, Bacterial
Genes, Bacterial
104.     ( 1994 )

The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline-resistance determinants.

Molecular microbiology 11 (2)
PMID : 8170402  :   DOI  :   10.1111/j.1365-2958.1994.tb00320.x    
Abstract >>
The complete nucleotide sequence and mechanism of action of the tetracycline-resistance determinant, Tet P, from Clostridium perfringens has been determined. Analysis of the 4.4 kb of sequence data revealed the presence of two open reading frames, designated as tetA(P) and tetB(P). The tetA(P) gene appears to encode a 420 amino acid protein (molecular weight 46,079) with twelve transmembrane domains. This gene was shown to be responsible for the active efflux of tetracycline from resistant cells. Although there was some amino acid sequence similarity between the putative TetA(P) protein and other tetracycline efflux proteins, analysis suggested that TetA(P) represented a different type of efflux protein. The tetB(P) gene would encode a putative 652 amino acid protein (molecular weight 72,639) with significant sequence similarity to Tet(M)-like cytoplasmic proteins that specify a ribosomal-protection tetracycline-resistance mechanism. In both C. perfringens and Escherichia coli, tetB(P) encoded low-level resistance to tetracycline and minocycline whereas tetA(P) only conferred tetracycline resistance. The tetA(P) and tetB(P) genes appeared to be linked in an operon, which represented a novel genetic arrangement for tetracycline-resistance determinants. It is proposed that tetB(P) evolved from the conjugative transfer into C. perfringens of a tet(M)-like gene from another bacterium.
KeywordMeSH Terms
Genes, Bacterial
105.     ( 1994 )

Cloning and sequence analysis of ermQ, the predominant macrolide-lincosamide-streptogramin B resistance gene in Clostridium perfringens.

Antimicrobial agents and chemotherapy 38 (5)
PMID : 8067735  :   DOI  :   10.1128/aac.38.5.1041     PMC  :   PMC188147    
Abstract >>
The erythromycin resistance determinant from Clostridium perfringens JIR100 has been cloned, sequenced, and shown to be expressed in Escherichia coli. An open reading frame with sequence similarity to erm genes from other bacteria was identified and designated the ermQ gene. On the basis of comparative sequence analysis, it was concluded that the ermQ gene represented a new Erm hybridization class, designated ErmQ. Genes belonging to the ErmQ class were found to be widespread in C. perfringens, since 30 of 38 macrolide-lincosamide-streptogramin B-resistant C. perfringens strains, from diverse sources, hybridized to an ermQ-specific gene probe. The ermQ gene therefore represents the most common erythromycin resistance determinant in C. perfringens.
KeywordMeSH Terms
Macrolides
106.     ( 1994 )

A complex array of Hpr consensus DNA recognition sequences proximal to the enterotoxin gene in Clostridium perfringens type A.

Microbiology (Reading, England) 140 (Pt 1) (N/A)
PMID : 8162194  :   DOI  :   10.1099/13500872-140-1-97    
Abstract >>
Enterotoxin production in Clostridium perfringens is both strain dependent and sporulation associated. Underlying these phenotypic observations must lie a genetic and molecular explanation and the principal keys will be held within the DNA sequence both upstream and downstream of the structural gene cpe. In accordance with the above we have sequenced 4.1 kbp of DNA upstream of cpe in the type strain NCTC 8239. A region of DNA extending up to 1.5 kb 5' to cpe is conserved in all enterotoxin-positive strains. This region contains a putative ORF with substantial homology to an ORF in the Salmonella typhimurium IS200 insertion element and, in addition, contains multiple perfect consensus DNA-binding sequences for the Bacillus subtilis transition state regulator Hpr. The detailed structural elements revealed by the sequence analysis are presented and used to develop a new perspective on the molecular basis of enterotoxin production in this important food-poisoning bacterium.
KeywordMeSH Terms
107.     ( 1994 )

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens.

Molecular microbiology 12 (5)
PMID : 8052128  :   DOI  :   10.1111/j.1365-2958.1994.tb01063.x    
Abstract >>
The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb HindIII fragment in the C. perfringens chromosome. A 4.3kb PstI fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb HindIII fragment, or the 4.3 kb PstI fragment, resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The PstI fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30,140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51,274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved in virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.
KeywordMeSH Terms
Genes, Bacterial
Mutagenesis, Insertional
108.     ( 1994 )

The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens.

Journal of bacteriology 176 (6)
PMID : 8132455  :   DOI  :   10.1128/jb.176.6.1616-1623.1994     PMC  :   PMC205246    
Abstract >>
The perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was cloned into an Escherichia coli-C. perfringens shuttle vector, and the pfoA gene was expressed in mutants of C. perfringens 13 which lacked the production of perfringolysin O. One group (SI117) could express the pfoA gene, and the other (SI112) could not. A mutation in the regulatory system for pfoA gene expression was suspected in SI112. A chromosomal DNA library constructed from strain 13 was transformed into strain SI112 to identify the regulatory gene(s) for the pfoA gene. Five strains of 10,000 transformants restored perfringolysin O production. All contained a 2.5-kb DNA fragment. This fragment activated the transcription of the pfoA gene and also restored the production of collagenase (kappa-toxin) and hemagglutinin in strain SI112. Deletion analysis showed that a 1.25-kb region was sufficient for the trans activity, and sequence analysis disclosed that open reading frame 2 (ORF2) was located in this region. A homology search for the deduced amino acid sequence revealed that ORF2 was homologous to a response regulator in a two-component signal transduction system. ORF2 was designated virR, and it is suggested that the virR gene plays an important role in the pathogenicity of C. perfringens.
KeywordMeSH Terms
109.     ( 1996 )

Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease.

Infection and immunity 64 (1)
PMID : 8557345  :   PMC  :   PMC173750    
Abstract >>
The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography. The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease. The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells. Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da. The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family. The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component. It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice. These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C. perfringens.
KeywordMeSH Terms
Genes, Bacterial
110.     ( 1995 )

Sequence analysis of flanking regions of the pfoA gene of Clostridium perfringens: beta-galactosidase gene (pbg) is located in the 3'-flanking region.

Microbiology and immunology 39 (9)
PMID : 8577281  :   DOI  :   10.1111/j.1348-0421.1995.tb03256.x    
Abstract >>
The 3'-flanking region of the perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking. A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane-bound transporter proteins of other bacteria and the beta-galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively. ORF80 was named the pbg gene. Clones which showed beta-galactosidase activities were selected from a lambda FIXII genomic library of C. perfringens by blue plaque screening using X-Gal as a substrate. Four clones whose plaques showed blue appearances were obtained. Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct beta-galactosidase genes in C. perfringens. The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a beta-galactosidase of C. perfringens.
KeywordMeSH Terms
Genes, Bacterial
111.     ( 1994 )

Molecular genetic analysis of the nagH gene encoding a hyaluronidase of Clostridium perfringens.

Molecular & general genetics : MGG 243 (2)
PMID : 8177218  :   DOI  :   10.1007/bf00280319    
Abstract >>
A recombinant lambda phage was identified in a Clostridium perfringens genomic library by means of its ability to hydrolyse the fluorescent substrate 4-methyl-umbelliferyl-beta-D-glucosaminide, isolated and shown to encode an endo-beta-N-acetylglucosaminidase. This enzyme, NagH, is also known as hyaluronidase, or Mu toxin, a putative virulence factor which is likely to act on connective tissue during gas gangrene. Nucleotide sequence analysis allowed the primary structure to be deduced and showed hyaluronidase to be a large exported protein of 114,392 Daltons and an enzyme of this size, endowed with the corresponding activities, was partially purified from C. perfringens. Hyaluronidase seems to be organised into two domains, an N-terminal region comprising 700 amino acids bearing the active site and a 300-residue C-terminal segment, containing three copies of an extended motif. Two other reading frames, linked to nagH, also appear to encode proteins with sugar-binding motifs.
KeywordMeSH Terms
Genes, Bacterial
112. Cornillot  E, Saint-Joanis  B, Daube  G, Katayama  S, Granum  PE, Canard  B, Cole  ST,     ( 1995 )

The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne.

Molecular microbiology 15 (4)
PMID : 7783636  :   DOI  :   10.1111/j.1365-2958.1995.tb02373.x    
Abstract >>
The location of the cpe gene, encoding the enterotoxin responsible for food poisoning in humans, has been studied in a series of enterotoxigenic Clostridium perfringens strains by means of pulsed field gel electrophoresis of genomic DNA. The cpe gene was found at the same chromosomal locus in strains associated with food poisoning in humans and was shown to be linked to a repetitive sequence, the HindIII repeat, and an open reading frame, ORF3, that may be part of an insertion sequence. In contrast, when the strains originated from domesticated livestock cpe was located on a large episome where it was often close to a copy of the transposable element IS1151. In these cases, the HindIII repeat was not linked to the cpe gene although this was generally preceded by ORF3.
KeywordMeSH Terms
113. Traving  C, Schauer  R, Roggentin  P,     ( 1994 )

Gene structure of the 'large' sialidase isoenzyme from Clostridium perfringens A99 and its relationship with other clostridial nanH proteins.

Glycoconjugate journal 11 (2)
PMID : 7804004  :  
Abstract >>
Clostridium perfringens possesses two sialidase isoenzymes of different molecular weight. Almost 90% of the gene encoding the 'large' form was found on a 3.1 kb chromosomal fragment (Sau3AI) of strain A99 by hybridization with probes developed from the N-terminal protein sequence and from commonly conserved sialidase motifs ('Asp-boxes'), whereas the remaining 3'-terminal part was detected on a 2.1 kb fragment (Hind III) of chromosomal DNA. After combination of both fragments, the resulting E. coli clones expressed sialidase activity, the properties of the recombinant sialidase corresponding with those of the wild type enzyme. The entire chromosomal fragment of 3665 bp encompasses the complete sialidase gene of 2082 bp corresponding to 694 amino acids, from which a molecular weight of 72,956 for the mature protein can be deduced. The first 41 amino acids are mostly hydrophobic and probably represent a signal peptide. The sialidase structural gene follows a non-coding region with an inverted repeat and a ribosome-binding site. Upstream from the regulatory region, another open reading frame (ORF) was detected. The 3'-terminus of the sialidase structural gene is directly followed by a further ORF of unknown function, which possibly encodes a putative permease or the acylneuraminate pyruvate-lyase involved in sialic acid catabolism. The primary structure of the 'large' isoenzyme is very similar to the sialidase of Clostridium septicum (55% identical amino acids), whereas the homology with the 'small' form of the same species is comparatively low (26%).
KeywordMeSH Terms
Genes, Bacterial
114.     ( 1994 )

Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis.

Infection and immunity 62 (12)
PMID : 7960138  :   PMC  :   PMC303301    
Abstract >>
Clostridium perfringens is a source of food poisoning in humans and animals because of production of a potent enterotoxin (CPE). To study the regulation of the cpe gene in C. perfringens, we cloned and sequenced the cpe promoter regions and N-terminal domains from three strains. The cpe promoter region from one strain contained a 45-bp insertion compared with previously published sequences. This insertion was also found in two (of five) other Cpe+ strains. cpe gene expression in C. perfringens was measured by using translational fusions of each promoter type to the Escherichia coli gusA gene, which codes for beta-glucuronidase. For either promoter type, cpe-gusA expression was undetectable throughout exponential growth but increased dramatically at the beginning of the stationary phase. To measure cpe expression in Bacillus subtilis, cpe-gusA fusions were integrated into the B. subtilis chromosome. Both types of promoter exhibited moderate expression during exponential growth; cpe expression increased threefold at the beginning of the stationary phase. Transcriptional start sites were determined by primer extension and in vitro transcription assays. For C. perfringens, both types of promoter gave the same 5' end, 197 bp upstream of the translation start (50 bp downstream of the 45-bp insertion). In B. subtilis, however, the 5' end was internal to the 45-bp insertion, suggesting the use of a different promoter than that utilized by C. perfringens.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
115. Steinthorsdottir  V, Fridriksdottir  V, Gunnarsson  E, Andrésson  OS, Steinporsdóttir  N/A, Frithriksdóttir  V,     ( 1995 )

Expression and purification of Clostridium perfringens beta-toxin glutathione S-transferase fusion protein.

FEMS microbiology letters 130 (2��3��)
PMID : 7649450  :   DOI  :   10.1111/j.1574-6968.1995.tb07731.x    
Abstract >>
The beta-toxin gene from Clostridium perfringens type C was cloned and expressed as a glutathione S-transferase fusion protein in Escherichia coli. The DNA sequence was determined and compared to the type B sequence. Two nucleotide differences were found in the protein coding sequence, resulting in one amino acid difference between the two proteins. The purified beta-toxin fusion protein is not toxic in mice, but rabbit antiserum raised against it neutralises the toxic effect of C. perfringens type C culture filtrate in mice.
KeywordMeSH Terms
116. Berryman  DI, Rood  JI,     ( 1995 )

The closely related ermB-ermAM genes from Clostridium perfringens, Enterococcus faecalis (pAM beta 1), and Streptococcus agalactiae (pIP501) are flanked by variants of a directly repeated sequence.

Antimicrobial agents and chemotherapy 39 (8)
PMID : 7486927  :   DOI  :   10.1128/aac.39.8.1830     PMC  :   PMC162834    
Abstract >>
The Clostridium perfringens macrolide-lincosamide-streptogramin B resistance gene, ermBP, was sequenced and shown to be identical to the ermB-ermAM gene from the promiscuous Enterococcus faecalis plasmid pAM beta 1 and to have at least 98% nucleotide sequence identity to other ermB-ermAM genes. Flanking the ermBP structural gene were almost identical directly repeated 1,341-bp sequences (DR1 and DR2). These repeats potentially encoded a 298 (or 284)-amino-acid protein that had sequence similarity to chromosomal and plasmid partitioning proteins. The pAM beta 1 and Streptococcus agalactiae (pIP501) erm determinants appeared to have DR2 but had similar internal 973- or 956-bp deletions in DR1, respectively. Some of the other ermB-ermAM class determinants had small portions of DR1, but none had complete copies. It is postulated that the C. perfringens ermBP determinant was derived from an enterococcal or streptococcal determinant that had complete copies of both DR1 and DR2.
KeywordMeSH Terms
Genes, Bacterial
Repetitive Sequences, Nucleic Acid
117. Miyata  S, Moriyama  R, Miyahara  N, Makino  S,     ( 1995 )

A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterization.

Microbiology (Reading, England) 141 (Pt 10) (N/A)
PMID : 7582025  :   DOI  :   10.1099/13500872-141-10-2643    
Abstract >>
Antiserum was raised against a 31 kDa spore-cortex-lytic enzyme, which is released during germination of Clostridium perfringens S40 spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE indicated that the 31 kDa enzyme is spore-specific and that the enzyme in the dormant spore exists as a 36 kDa protein which has no cortex-lytic activity. A gene encoding the 31 kDa enzyme, sleC, was cloned into Escherichia coli using a synthetic oligonucleotide as a hybridization probe and the nucleotide sequence of the entire gene was determined. The N-terminal amino acid sequence of the 36 kDa protein was found in this reading frame, confirming that the 36 kDa protein is a pro-form of the 31 kDa enzyme. The deduced amino acid sequence indicated that the 31 kDa enzyme is produced as a precursor, comprising three portions; an N-terminal prepro-sequence (114 amino acid residues), a pro-sequence (35 amino acid residues) and a mature enzyme (289 amino acid residues). It is suggested that the 36 kDa pro-enzyme is non-covalently attached to the exterior of the cortex layer, and that the proform is processed to release the active enzyme during germination.
KeywordMeSH Terms
Genes, Bacterial
118. Richardson  M, Granum  PE,     ( 1983 )

Sequence of the amino-terminal part of enterotoxin from Clostridium perfringens type A: identification of points of trypsin activation.

Infection and immunity 40 (3)
PMID : 6303961  :   PMC  :   PMC348143    
Abstract >>
The sequence of the first 66 amino acids of the amino-terminal part of the enterotoxin from Clostridium perfringens type A is presented. The trypsin activation of the enterotoxin involves hydrolysis of Lys15-Glu16 and Lys25-Thr26 bonds. The N-terminal sequence of the trypsin-activated enterotoxin has limited homology with the sequence of the N-terminal region of the cholera toxin B subunit.
KeywordMeSH Terms
Enterotoxins
119. Poyart  C, Berche  P, Trieu-Cuot  P,     ( 1995 )

Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers.

FEMS microbiology letters 131 (1)
PMID : 7557308  :   DOI  :   10.1016/0378-1097(95)00232-t    
Abstract >>
An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.
KeywordMeSH Terms
120. Bannam  TL, Crellin  PK, Rood  JI,     ( 1995 )

Molecular genetics of the chloramphenicol-resistance transposon Tn4451 from Clostridium perfringens: the TnpX site-specific recombinase excises a circular transposon molecule.

Molecular microbiology 16 (3)
PMID : 7565113  :   DOI  :   10.1111/j.1365-2958.1995.tb02417.x    
Abstract >>
The chloramphenicol-resistance transposon Tn4451 undergoes precise conjugative deletion from its parent plasmid plP401 in Clostridium perfringens and precise spontaneous excision from multicopy plasmids in Escherichia coli. The complete nucleotide sequence of the 6338 bp transposon was determined and it was found to encode six genes. Genetic analysis demonstrated that the largest Tn4451-encoded gene, tnpX, was required for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnpX gene was completely stable in both organisms. Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans-acting site-specific recombinase. Allelic exchange was used to introduce the tnpX delta 1 allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens. It was also shown by hybridization and polymerase chain reaction (PCR) studies that TnpX-mediated transposon excision resulted in the formation of a circular form of the transposon. The TnpX recombinase was unique because it potentially contained the motifs of two independent site-specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/invertase domain of TnpX was likely to be involved in the excision process by catalysing the formation of a 2 bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at the junction of the circular form. The other Tn4451-encoded genes include tnpZ, which appears to encode a second potential site-specific recombinase. This protein has similarity to plasmid-encoded Mob/Pre proteins, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.
KeywordMeSH Terms
Integrases
121. Richardson  M, Granum  PE,     ( 1985 )

The amino acid sequence of the enterotoxin from Clostridium perfringens type A.

FEBS letters 182 (2)
PMID : 3920076  :   DOI  :   10.1016/0014-5793(85)80358-6    
Abstract >>
The amino acid sequence of the enterotoxin from Clostridium perfringens type A was determined by analysis of peptides derived from the protein by digestion with trypsin chymotrypsin, thermolysin, pepsin, a lysine-specific protease. S. aureus V8 protease and a proline-specific protease, and fragments generated by cleavage with cyanogen bromide or by dilute acetic acid in 7 M guanidine HCl. The sequence which is complete except for the definite order of 3 small peptides between residues 88 and 103 consists of 309 amino acids and contains a correction to our preliminary announcement [(1984) FEMS Symp. 24, 329-330].
KeywordMeSH Terms
Enterotoxins
Serine Endopeptidases
122. Traore  DAK, Wisniewski  JA, Flanigan  SF, Conroy  PJ, Panjikar  S, Mok  YF, Lao  C, Griffin  MDW, Adams  V, Rood  JI, Whisstock  JC,     ( 2018 )

Crystal structure of TcpK in complex with oriT DNA of the antibiotic resistance plasmid pCW3.

Nature communications 9 (1)
PMID : 30213934  :   DOI  :   10.1038/s41467-018-06096-2     PMC  :   PMC6137059    
Abstract >>
Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK-TcpK box complex reveals a binding mode centered on and around the �]-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding.
KeywordMeSH Terms
Crystallography, X-Ray
123. Nguyen  TTT, Fujimura  Y, Mimura  I, Fujii  Y, Nguyen  NL, Arakawa  K, Morita  H,     ( 2018 )

Cultivable butyrate-producing bacteria of elderly Japanese diagnosed with Alzheimer's disease.

Journal of microbiology (Seoul, Korea) 56 (10)
PMID : 30136260  :   DOI  :   10.1007/s12275-018-8297-7    
Abstract >>
The group of butyrate-producing bacteria within the human gut microbiome may be associated with positive effects on memory improvement, according to previous studies on dementia-associated diseases. Here, fecal samples of four elderly Japanese diagnosed with Alzheimer's disease (AD) were used to isolate butyrate-producing bacteria. 226 isolates were randomly picked, their 16S rRNA genes were sequenced, and assigned into sixty OTUs (operational taxonomic units) based on BLASTn results. Four isolates with less than 97% homology to known sequences were considered as unique OTUs of potentially butyrate-producing bacteria. In addition, 12 potential butyrate-producing isolates were selected from the remaining 56 OTUs based on scan-searching against the PubMed and the ScienceDirect databases. Those belonged to the phylum Bacteroidetes and to the clostridial clusters I, IV, XI, XV, XIVa within the phylum Firmicutes. 15 out of the 16 isolates were indeed able to produce butyrate in culture as determined by high-performance liquid chromatography with UV detection. Furthermore, encoding genes for butyrate formation in these bacteria were identified by sequencing of degenerately primed PCR products and included the genes for butyrate kinase (buk), butyryl-CoA: acetate CoAtransferase (but), CoA-transferase-related, and propionate CoA-transferase. The results showed that eight isolates possessed buk, while five isolates possessed but. The CoA-transfer-related gene was identified as butyryl-CoA:4-hydroxybutyrate CoA transferase (4-hbt) in four strains. No strains contained the propionate CoA-transferase gene. The biochemical and butyrate-producing pathways analyses of butyrate producers presented in this study may help to characterize the butyrate-producing bacterial community in the gut of AD patients.
KeywordMeSH Terms
16S rRNA gene sequencing
Alzheimer’s disease
butyrate-producing bacteria
gut microbiota
short-chain fatty acids
16S rRNA gene sequencing
Alzheimer’s disease
butyrate-producing bacteria
gut microbiota
short-chain fatty acids
16S rRNA gene sequencing
Alzheimer’s disease
butyrate-producing bacteria
gut microbiota
short-chain fatty acids
16S rRNA gene sequencing
Alzheimer’s disease
butyrate-producing bacteria
gut microbiota
short-chain fatty acids
16S rRNA gene sequencing
Alzheimer’s disease
butyrate-producing bacteria
gut microbiota
short-chain fatty acids
Gastrointestinal Microbiome
124. Lacey  JA, Allnutt  TR, Vezina  B, Van  TTH, Stent  T, Han  X, Rood  JI, Wade  B, Keyburn  AL, Seemann  T, Chen  H, Haring  V, Johanesen  PA, Lyras  D, Moore  RJ,     ( 2018 )

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens.

BMC genomics 19 (1)
PMID : 29788909  :   DOI  :   10.1186/s12864-018-4771-1     PMC  :   PMC5964661    
Abstract >>
Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome. Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis. The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.
KeywordMeSH Terms
Adhesion
Capsule
Clostridium perfringens
Genome
Necrotic enteritis
Pangenome
Prophage
Evolution, Molecular
Genetic Variation
125.     ( 2013 )

Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens.

mBio 4 (1)
PMID : 23386432  :   DOI  :   10.1128/mBio.00019-13     PMC  :   PMC3565830    
Abstract >>
Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a �]-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 ?. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a �]-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 ?, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.
KeywordMeSH Terms
126. Theoret  JR, Li  J, Navarro  MA, Garcia  JP, Uzal  FA, McClane  BA,     ( 2018 )

Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin.

Infection and immunity 86 (1)
PMID : 29038129  :   DOI  :   10.1128/IAI.00730-17     PMC  :   PMC5736825    
Abstract >>
Many Clostridium perfringens strains produce NanI as their major sialidase. Previous studies showed that NanI could potentiate C. perfringens epsilon toxin cytotoxicity by enhancing the binding of this toxin to host cells. The present study first determined that NanI exerts similar cytotoxicity-enhancing effects on C. perfringens enterotoxin and beta toxin, which are also important toxins for C. perfringens diseases (enteritis and enterotoxemia) originating in the gastrointestinal (GI) tract. Building upon previous work demonstrating that purified trypsin can activate NanI activity, this study next determined that purified chymotrypsin or mouse intestinal fluids can also activate NanI activity. Amino acid sequencing then showed that this effect involves the N-terminal processing of the NanI protein. Recombinant NanI (rNanI) species corresponding to major chymotrypsin- or small intestinal fluid-generated NanI fragments possessed more sialidase activity than did full-length rNanI, further supporting the proteolytic activation of NanI activity. rNanI species corresponding to proteolysis products also promoted the cytotoxic activity and binding of enterotoxin and beta toxin more strongly than did full-length rNanI. Since enterotoxin and beta toxin are produced in the intestines during human and animal disease, these findings suggest that intestinal proteases may enhance NanI activity, which in turn could further potentiate the activity of intestinally active toxins during disease. Coupling these new results with previous findings demonstrating that NanI is important for the adherence of C. perfringens to enterocyte-like cells, NanI sialidase is now emerging as a potential auxiliary virulence factor for C. perfringens enteritis and enterotoxemia.
KeywordMeSH Terms
Clostridium perfringens
beta toxin
enterotoxin
sialidase
127. Roggentin  P, Rothe  B, Lottspeich  F, Schauer  R,     ( 1988 )

Cloning and sequencing of a Clostridium perfringens sialidase gene.

FEBS letters 238 (1)
PMID : 2901987  :   DOI  :   10.1016/0014-5793(88)80219-9    
Abstract >>
Escherichia coli was transformed with pUC vectors containing Sau3A restriction fragments (RF) of Clostridium perfringens DNA. Two clones expressed sialidase activity when assayed with the fluorogenic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid. A synthetic oligonucleotide representing the N-terminus of the expressed enzyme hybridized with the clostridial insert and with a corresponding 2.1 kb Sau3A RF of the C. perfringens genome. The insert reduced to 1.4 kb, which still encoded active sialidase, has been sequenced. The structural gene encodes 382 amino acids representing an Mr of 42770. A hydrophobic leader sequence is absent. Upstream from the initiation codon ATG, a GA-rich region is found and considered as the Shine-Dalgarno sequence. Homology with the N-terminus of the Vibrio cholerae sialidase gene and with viral sialidase sequences was not found.
KeywordMeSH Terms
Cloning, Molecular
Genes
Genes, Bacterial
128. Garnier  T, Saurin  W, Cole  ST,     ( 1987 )

Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for prokaryotic site-specific recombinases.

Molecular microbiology 1 (3)
PMID : 2896291  :   DOI  :   10.1111/j.1365-2958.1987.tb01944.x    
Abstract >>
Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as pIP404 which, in addition to a set of UV-inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site-specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram-negative and Gram-positive bacteria as well as enzymes involved in site-specific DNA inversion. A likely role for the RES protein would be to stabilize pIP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site-specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Streptococcus faecalis.
KeywordMeSH Terms
Biological Evolution
Genes
Genes, Bacterial
Plasmids
129. Garnier  T, Cole  ST,     ( 1986 )

Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene.

Journal of bacteriology 168 (3)
PMID : 2877971  :   DOI  :   10.1128/jb.168.3.1189-1196.1986     PMC  :   PMC213621    
Abstract >>
The bacteriocinogenic plasmid pIP404 from Clostridium perfringens was isolated and cloned in Escherichia coli, and its physical map was deduced. Expression of the bcn gene, encoding bacteriocin BCN5, is inducible by UV irradiation of C. perfringens and thus resembles the SOS-regulated bacteriocin genes of enteric bacteria. The location of bcn on pIP404 was established by a dot-blot procedure, using specific hybridization probes to analyze mRNA samples from induced and uninduced cultures. From the nucleotide sequence of its gene, the molecular weight of BCN5 was deduced to be 96,591, and a protein of this size was secreted by bacteriocin-producing cultures of C. perfringens. The primary structure of the protein suggests that it may function as an ionophore, since a hydrophobic domain, resembling those of the ionophoric colicins, is present at the COOH terminus. No bacteriocin activity could be detected in E. coli harboring plasmids bearing the bcn gene, even when the transcriptional and translational signals were replaced by those of lacZ. A possible explanation may be found in the unusual codon usage of the adenine-thymine-rich bcn gene, as this shows a preference for codons with a high adenine-plus-thymine content, especially in the wobble position. Many of the frequently used codons correspond to those recognized by minor tRNA species in E. coli. Consequently, bcn expression might be limited by tRNA availability in this bacterium.
KeywordMeSH Terms
Genes, Bacterial
Plasmids
130.     ( 2012 )

Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly.

Anaerobe 18 (5)
PMID : 22982043  :   DOI  :   10.1016/j.anaerobe.2012.09.001     PMC  :   PMC3478100    
Abstract >>
Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation.
KeywordMeSH Terms
Protein Processing, Post-Translational
131.     ( 2012 )

Sequence of two plasmids from Clostridium perfringens chicken necrotic enteritis isolates and comparison with C. perfringens conjugative plasmids.

PloS one 7 (11)
PMID : 23189158  :   DOI  :   10.1371/journal.pone.0049753     PMC  :   PMC3506638    
Abstract >>
Twenty-six isolates of Clostridium perfringens of different MLST types from chickens with necrotic enteritis (NE) (15 netB-positive) or from healthy chickens (6 netB-positive, 5 netB-negative) were found to contain 1-4 large plasmids, with most netB-positive isolates containing 3 large and variably sized plasmids which were more numerous and larger than plasmids in netB-negative isolates. NetB and cpb2 were found on different plasmids consistent with previous studies. The pathogenicity locus NELoc1, which includes netB, was largely conserved in these plasmids whereas NeLoc3, present in the cpb2 containing plasmids, was less well conserved. A netB-positive and a cpb2-positive plasmid were likely to be conjugative, and the plasmids were completely sequenced. Both plasmids possessed the intact tcp conjugative region characteristic of C. perfringens conjugative plasmids. Comparative genomic analysis of nine CpCPs, including the two plasmids described here, showed extensive gene rearrangements including pathogenicity locus and accessory gene insertions around rather than within the backbone region. The pattern that emerges from this analysis is that the major toxin-containing regions of the variety of virulence-associated CpCPs are organized as complex pathogenicity loci. How these different but related CpCPs can co-exist in the same host has been an unanswered question. Analysis of the replication-partition region of these plasmids suggests that this region controls plasmid incompatibility, and that CpCPs can be grouped into at least four incompatibility groups.
KeywordMeSH Terms
132.     ( 2012 )

Genotypic and phenotypic characterization of Clostridium perfringens isolates from Darmbrand cases in post-World War II Germany.

Infection and immunity 80 (12)
PMID : 23027533  :   DOI  :   10.1128/IAI.00818-12     PMC  :   PMC3497428    
Abstract >>
Clostridium perfringens type C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates are cpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores with D(100) values (the time that a culture must be kept at 100�XC to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomal cpe gene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both the cpe and cpb genes can be mobilized in Darmbrand isolates. These results suggest that C. perfringens type A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomal cpe gene.
KeywordMeSH Terms
World War II
133.     ( 2013 )

Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex.

Proceedings of the National Academy of Sciences of the United States of America 110 (11)
PMID : 23382240  :   DOI  :   10.1073/pnas.1217227110     PMC  :   PMC3600452    
Abstract >>
Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD(+) analog �]TAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD(+)-bound form (NAD(+)-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD(+)-Ia-actin complex. Here we report high-resolution structures of NAD(+)-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD(+) under different conditions. The structures of NAD(+)-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the �]TAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases.
KeywordMeSH Terms
Protein Processing, Post-Translational
134.     ( 2013 )

Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens.

The Journal of biological chemistry 288 (5)
PMID : 23239883  :   DOI  :   10.1074/jbc.M112.430223     PMC  :   PMC3561570    
Abstract >>
NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 ?. The heptameric assembly shares structural homology to the staphylococcal �\-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.
KeywordMeSH Terms
135.     ( 2013 )

Sudden death syndrome in adult cows associated with Clostridium perfringens type E.

Anaerobe 20 (N/A)
PMID : 23354004  :   DOI  :   10.1016/j.anaerobe.2013.01.001    
Abstract >>
Clostridium perfringens type E is considered a rare toxinotype and an infrequent cause of enterotoxemia of lambs, calves, and rabbits. Until now, only cases of young animal of C. perfringens type E bovine enterotoxemia, characterized by hemorrhagic enteritis and sudden death, have been reported. The present report details the genotypic characterization of C. perfringens type E isolates obtained from intestinal samples of adult cattle during an outbreak of enterotoxemia in Argentina. The sequences of several housekeeping genes of these isolates were analyzed and compared with those obtained from calves in North America showing a clonal unique lineage.
KeywordMeSH Terms
136.     ( 1998 )

Clostridium perfringens type E animal enteritis isolates with highly conserved, silent enterotoxin gene sequences.

Infection and immunity 66 (9)
PMID : 9712814  :   PMC  :   PMC108552    
Abstract >>
Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express alpha and iota toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the iota toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries iota toxin genes, to several different type A C. perfringens isolates.
KeywordMeSH Terms
Conserved Sequence
137.     ( 1998 )

The Clostridium perfringens enterotoxin from equine isolates; its characterization, sequence and role in foal diarrhoea.

Epidemiology and infection 120 (2)
PMID : 9593490  :   DOI  :   10.1017/s0950268897008534     PMC  :   PMC2809390    
Abstract >>
During a survey of foal diarrhoea between 1991 and 1994, Clostridium perfringens was significantly associated with disease with 56% of cases infected [1]. The contribution of enterotoxigenic C. perfringens to this association, was assessed by use of the reverse passive latex agglutination test for enterotoxin (RPLA; Oxoid Unipath) and vero cell toxicity neutralized by antitoxin on stored faecal samples and sporulated faecal isolates of C. perfringens. Polymerase chain reaction (PCR1) based on the DNA sequence for the whole enterotoxin gene [2] yielded a fragment from an equine isolate of the anticipated size which, cloned into plasmid M13 phage, had a sequence essentially identical to the published sequence. Consequently, all faecal isolates were also tested by PCR1 and for a part of the enterotoxin gene (PCR2). Significant association with diarrhoea (controls not in contact with cases) was found with positive RPLA tests on faeces (OR = 13, P = 0.002) and isolates (OR = 4.57, P = < 0.0001), vero cell toxicity of isolates (OR = 1.78, P = 0.026), and PCR1 (OR = nd, P = 0.029) but not PCR2 or vero cell toxicity of faeces. Significant association with diarrhoea was also found for isolates negative by RPLA (OR = 3.91; CI 2.05-7.57; P < 0.0001) or PCR1 (OR = 4.81; CI 2.84-8.20; P < 0.0001). Many of the isolates from RPLA positive faeces and verotoxic isolates were PCR negative and no evidence could be found for the presence of the enterotoxin gene in a random selection of RPLA positive/PCR negative isolates by gene probe on chromosomal DNA and PCR reaction product or vero cell toxicity neutralized by specific antiserum. Failure of the vero cell toxicity on faeces to be associated with diarrhoea or for cytotoxicity of cultures and RPLA on cultures to agree with the PCRs was believed to be related to the presence of other cytotoxins, the inherent cytotoxicity of equine faeces and to the poor specificity of the commercial antiserum used in the test. Enterotoxigenic C. perfringens could not account for the overall association of C. perfringens with foal diarrhoea because (a) cultures positive by PCR, RPLA or cytotoxicity were not significantly more common amongst isolates from cases than controls; and (b) the proportion of isolates from cases positive by PCR (PCR1 or PCR2) was too small at 9.7%.
KeywordMeSH Terms
Animals, Newborn
Clostridium perfringens
138.     ( 1997 )

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99.

Glycoconjugate journal 14 (7)
PMID : 9511987  :  
Abstract >>
The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.
KeywordMeSH Terms

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