The Structural Gene for Tetanus Neurotoxin Is on a Plasmid

Finn, CW; Silver, RP; Habig, William H.; Hardegree, M. C.; Zon, Gerald; Garon, Claude F.

doi:10.1126/science.6326263

Cited by 102 publications

(42 citation statements)

References 7 publications

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“…This observation has led to the suggestion that the presence of the toxin gene cluster may be associated with a transposable genetic element (3). In other clostridial species, toxins are known to be encoded by mobile genetic elements such as bacteriophages and plasmids (6,9,10,31).Following publication of the genome of ⌽CD119 (15), the genome of a second C. difficile temperate phage (⌽C2, a member of the Myoviridae) was published (13). More recently, eight temperate phages were characterized from six different C. difficile isolates, including the hypervirulent strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) (11).…”

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Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile

Govind

Vediyappan

Rolfe

et al. 2009

J Virol

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Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by ⌽CD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in ⌽CD119 lysogens. During this study we found that repR, a putative repressor gene of ⌽CD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in ⌽CD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen.Clostridium difficile, a gram-positive, anaerobic, spore-forming bacterium, has been identified as one of the major causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile produces toxins A and B that damage intestinal mucosa and cause fluid accumulation in the colon (1). The toxin genes tcdA and tcdB, along with accessory genes tcdR, tcdC, and tcdE, are part of a 19.6-kb pathogenicity locus (PaLoc). Toxin genes tcdA and tcdB are positively regulated by TcdR (previously TxeR) (27), and tcdC is involved in the negative regulation of toxin genes (16,29). In pathogenic C. difficile strains, the PaLoc is present at identical locations in the chromosome, whereas it is completely absent in nontoxinogenic strains. This observation has led to the suggestion that the presence of the toxin gene cluster may be associated with a transposable genetic element (3). In other clostridial species, toxins are known to be encoded by mobile genetic elements such as bacteriophages and plasmids (6,9,10,31).Following publication of the genome of ⌽CD119 (15), the genome of a second C. difficile temperate phage (⌽C2, a member of the Myoviridae) was published (13). More recently, eight temperate phages were characterized from six different C. difficile isolates, including the hypervirulent strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) (11). In addition, the multidrug-resistant C. difficile strain CD630 was found to harbor two highly related prophages (13, 39) as part of its mosaic genome, where nearly 11% is made of mobile genetic elements. Thus, it appears that C. difficile strains often harbor temperate phage(s) as part of their genetic makeup. No direct evidence of lysogenic conversion of a nontoxinogenic C. difficile strain to toxin production was shown. However...

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Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile

Govind

Vediyappan

Rolfe

et al. 2009

J Virol

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“…So far, genetic information on C. tetani is mainly restricted to the nucleotide sequences of the tetanus toxin TeTx and of its direct transcriptional activator TetR, both of which are encoded on a plasmid, designated pCL1 in the strain Massachusetts (7)(8)(9)(10). The identification and analysis of all genes in the genome of C. tetani will contribute to our understanding of the lifestyle switch from a harmless soil bacterium to a potentially devastating neurosystem-damaging organism after entering and infecting a mammalian host.…”

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The genome sequence of Clostridium tetani , the causative agent of tetanus disease

Brüggemann

Bäumer

Fricke

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

326

270

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Tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. The manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of Ϸ1 ng͞kg. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the World Health Organization, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The causative agent of tetanus disease is Clostridium tetani, an anaerobic spore-forming bacterium, whose natural habitat is soil, dust, and intestinal tracts of various animals. Here we report the complete genome sequence of toxigenic C. tetani E88, a variant of strain Massachusetts. The genome consists of a 2,799,250-bp chromosome encoding 2,372 ORFs. The tetanus toxin and a collagenase are encoded on a 74,082-bp plasmid, containing 61 ORFs. Additional virulencerelated factors could be identified, such as an array of surfacelayer and adhesion proteins (35 ORFs), some of them unique to C. tetani. Comparative genomics with the genomes of Clostridium perfringens, the causative agent of gas gangrene, and Clostridium acetobutylicum, a nonpathogenic solvent producer, revealed a remarkable capacity of C. tetani: The organism can rely on an extensive sodium ion bioenergetics. Additional candidate genes involved in the establishment and maintenance of a pathogenic lifestyle of C. tetani are presented.

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“…This observation has led to the suggestion that the presence of the PaLoc may be associated with a transposable element (5). In other clostridial species, toxins are known to be encoded by mobile elements such as bacteriophages and plasmids (10,11). However, while there is no direct evidence of lysogenic conversion in C. difficile strains, Tan et al have demonstrated homology between tcdE, a gene located within the PaLoc of C. difficile, and phage holin genes (33).…”

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Genomic Organization and Molecular Characterization of Clostridium difficile Bacteriophage ΦCD119

2006

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In this study, we have isolated a temperate phage (⌽CD119) from a pathogenic Clostridium difficile strain and sequenced and annotated its genome. This virus has an icosahedral capsid and a contractile tail covered by a sheath and contains a double-stranded DNA genome. It belongs to the Myoviridae family of the tailed phages and the order Caudovirales. The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. The DNA sequence of this phage consists of 53,325 bp, which carries 79 putative open reading frames (ORFs). A function could be assigned to 23 putative gene products, based upon bioinformatic analyses. The ⌽CD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The ⌽CD119 attachment site attP lies in a noncoding region close to the putative integrase (int) gene. We have identified the phage integration site on the C. difficile chromosome (attB) located in a noncoding region just upstream of gene gltP, which encodes a carrier protein for glutamate and aspartate. This genetic analysis represents the first complete DNA sequence and annotation of a C. difficile phage.Clostridium difficile, a gram-positive, spore-forming, anaerobic bacillus, is the leading cause of nosocomial diarrhea associated with antibiotic therapy (2). C. difficile causes a variety of diarrheal syndromes, including diarrhea, nonspecific colitis, and pseudomembranous colitis, all of which vary widely in severity (2). Pathogenic C. difficile can produce two major toxins, toxin A, an enterotoxin, and toxin B, a cytotoxin, that are causative agents of diarrhea and colitis (4). Variation in the severity of symptoms of C. difficile-associated disease has been attributed in part to the level of toxin production by the infecting strain(s) (4). The toxin genes, tcdA and tcdB, are part of a 19.6-kb pathogenicity locus (PaLoc), which is present at identical locations in the chromosomes of pathogenic C. difficile strains but is missing from the nontoxinogenic strains. This observation has led to the suggestion that the presence of the PaLoc may be associated with a transposable element (5). In other clostridial species, toxins are known to be encoded by mobile elements such as bacteriophages and plasmids (10, 11). However, while there is no direct evidence of lysogenic conversion in C. difficile strains, Tan et al. have demonstrated homology between tcdE, a gene located within the PaLoc of C. difficile, and phage holin genes (33). In another study, Goh et al. analyzed the effect of bacteriophage infection on toxin production and found an increased toxin B production in some lysogens (12). The evolutionary aspects of the PaLoc and its relationship with C. difficile phages are not known. Detailed characterization of C. difficile phages is necessary to understand their genetics and their potential relationship with the PaLoc of C. difficile. In this study, one of our g...

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The Structural Gene for Tetanus Neurotoxin Is on a Plasmid

Cited by 102 publications

References 7 publications

Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile

Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile

The genome sequence of Clostridium tetani , the causative agent of tetanus disease

Genomic Organization and Molecular Characterization of Clostridium difficile Bacteriophage ΦCD119

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