Analysis of the Neurotoxin Complex Genes in Clostridium botulinum A1-A4 and B1 Strains: BoNT/A3, /Ba4 and /B1 Clusters Are Located within Plasmids

Smith, Theresa J.; Hill, Karen K.; Foley, Brian; Detter, John C.; Munk, A. Christine; Bruce, David; Doggett, Norman A.; Smith, Leonard A.; Marks, James D.; Xie, Gary; Brettin, Thomas

doi:10.1371/journal.pone.0001271

Cited by 152 publications

(154 citation statements)

References 59 publications

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“…BOTULINUM GROUP I GENOTYPING BY 15-LOCUS VNTR 4253 (2,10,14,23,25,26, and 35 RU) were observed for cbms04; four new alleles (7,8,13, and 15 RU) were observed for cbms05; three new alleles (3, 3.5, and 5 RU) were observed for cbms07; one new allele (2 RU) was observed for cbms11; and four new alleles (3, 10, 11, and 12 RU) were observed for cbms14.…”

Section: Vol 49 2011mentioning

confidence: 98%

“…The clustering of populations using these genotyping methods is only partially congruent with BoNT gene lineage, suggesting that BoNT genes have evolved separately from the bacterial chromosome through horizontal genetic transfer events (2,4,8,10,11). This hypothesis is supported by the discovery, in some C. botulinum strains, of transposable insertion sequences (IS) close to the BoNT genes and of BoNT gene sequences in plasmids (8,24,25,29).…”

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confidence: 99%

“…In support of this hypothesis, IS elements were discovered in the flanking regions of the BoNT gene in some genomes and/or the BoNT gene was found in plasmids in some C. botulinum strains. IS elements and plasmids are involved in the mobilization of DNA sequences among different strains (8,11,24,25,29). IS elements allow the transfer of genes between chromosomes and plasmids, and plasmids can mediate the exchange of genetic material between individuals (27).…”

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Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

Fillo¹,

Giordani²,

Anniballi

et al. 2011

J Clin Microbiol

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Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

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Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

Fillo¹,

Giordani²,

Anniballi

et al. 2011

J Clin Microbiol

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“…Pulsed-field gel electrophoresis (PFGE) and Southern analysis of strain H04402 065, performed according to the method of Franciosa et al (6), showed that the neurotoxin gene cluster was chromosomally located, as with type A1 neurotoxin genes (10,12), while some type A2 and all types A3 and A4 are plasmid borne (6,12). DNA sequencing of this region revealed a truncated, nonfunctional type B3 gene (7) in the 3Ј flank.…”

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confidence: 99%

Further Characterization of Proteolytic Clostridium botulinum Type A5 Reveals that Neurotoxin Formation Is Unaffected by Loss of the cntR ( botR ) Promoter Sigma Factor Binding Site

Carter

Mason

Grant³

et al. 2010

J Clin Microbiol

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“…This toxin is encoded by the bont gene as part of a cluster that includes nontoxic accessory genes (10). Two main gene clusters are known: the hemagglutinincontaining (haϩ) cluster in toxin serotypes A1, B, C, D, and G and the orfX cluster in toxin serotypes A, E, and F (7,10). In addition, some Clostridium botulinum type A strains (e.g., NCTC2916) contain a silent type B gene in their chromosome, designated A(B).…”

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Novel Clostridium botulinum Toxin Gene Arrangement with Subtype A5 and Partial Subtype B3 Botulinum Neurotoxin Genes

2009

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ᰔBotulinum neurotoxin, the most poisonous substance known and a potential biothreat agent (1), causes human and animal botulism worldwide (4,9). This toxin is encoded by the bont gene as part of a cluster that includes nontoxic accessory genes (10). Two main gene clusters are known: the hemagglutinincontaining (haϩ) cluster in toxin serotypes A1, B, C, D, and G and the orfX cluster in toxin serotypes A, E, and F (7, 10). In addition, some Clostridium botulinum type A strains (e.g., NCTC2916) contain a silent type B gene in their chromosome, designated A(B). The silent B toxin genes described to date contain the full length of the active type B gene cluster and one or more mutations that result in an unexpressed type B neurotoxin (3,8).Here we report the first infant botulism case resulting from a rare bont/A5 subtype, which we unexpectedly found to be part of a novel neurotoxin type A/B gene arrangement. Our bont/A5 gene sequence differs by 2 bp from the bont/A5 sequence previously identified in four wound botulism cases (2). The California infant strain, designated IBCA94-0216, was characterized by sequencing the toxin complex genes and their flanking regions (GenBank accession number FJ959094). Sequence analysis confirmed that the bont/A5 is located within a hemagglutinin complex (haϩ) that contains the genes ha70, ha17, ha33, botR, ntnh, and bont/A5 (Fig. 1). This type of toxin cluster is commonly associated with strains bont/A1 and bont/B but is not found in strains bont/A2, bont/A3, and bont/A4 (7, 10). The IBCA94-0216 neurotoxin gene cluster has an unexpected 76-bp deletion between the genes ha33 and botR. This is the first example of such a deletion.Downstream from bont/A5 are two copies of transposases belonging to the is3 family, a structure identical to the downstream flanking sequence of the type A1 Hall neurotoxin gene cluster ( Fig. 1) (10). Interestingly, 2,041 bp downstream from the bont/A5 gene and immediately following the second is3 is a partial bont/B gene that is only 65% of a full-length bont/B (6). The partial bont/B gene lacks the nucleotide sequence coding for the light chain of the toxin and begins at nucleotide 1362 of the 3,876-nucleotide full-length toxin gene. Comparisons of this region (1,362 to 3,876 nucleotides) revealed it is most similar to the rare bont/B3 subtype (5). The entire IBCA94-0216 bont/A/B cluster is flanked by a 5Ј flagellin gene and a 3Ј transposase, as is found in the silent type B gene cluster of strain NCTC2916 (7).Nucleic acid pairwise identities (using the Kimura two-parameter method in MEGA4 software) of the toxin cluster genes show that the IBCA94-0216 A5 neurotoxin and ntnh genes are most similar to the type A1 Hall neurotoxin and ntnh genes, respectively, while the hemagglutinin genes are most similar to the hemagglutinin genes of type B strains. This mosaic gene arrangement and the presence of the partial type B neurotoxin gene lead us to speculate that some form of genetic rearrangement occurred between type A and type B neurotoxin gene clusters in the c...

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Analysis of the Neurotoxin Complex Genes in Clostridium botulinum A1-A4 and B1 Strains: BoNT/A3, /Ba4 and /B1 Clusters Are Located within Plasmids

Cited by 152 publications

References 59 publications

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

Further Characterization of Proteolytic Clostridium botulinum Type A5 Reveals that Neurotoxin Formation Is Unaffected by Loss of the cntR ( botR ) Promoter Sigma Factor Binding Site

Novel Clostridium botulinum Toxin Gene Arrangement with Subtype A5 and Partial Subtype B3 Botulinum Neurotoxin Genes

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