Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.     

The complete amino acid sequence of the Clostridium botulinum type-E neurotoxin, derived by nucleotide-sequence analysis of the encoding gene.

The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR). Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene. Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively. Comparisons with partial amino acid sequences of the light chain of BoNT/E from C. botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case. Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity. The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

Sequences of the botulinal neurotoxin E derived from Clostridium botulinum type E (strain Beluga) and Clostridium butyricum (strains ATCC 43181 and ATCC 43755).

Recently, it has been shown that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755), isolated from cases of infant botulism, produce a botulinal neurotoxin type E (BoNT/E). Here we have determined the nucleotide sequences of the BoNT/E genes of these two C. butyricum strains and from C. botulinum E strain Beluga. We show that the sequences of the BoNT/E genes from the two C. butyricum strains are identical and differ in only 64 positions resulting in 39 amino acid changes (97% identity at the amino acid level) from that derived from C. botulinum. Our data suggest a transfer of the BoNT/E gene from C. botulinum to the originally nontoxigenic C. butyricum strains.     

Purification, modeling, and analysis of botulinum neurotoxin subtype A5 (BoNT/A5) from Clostridium botulinum strain A661222

A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences--specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.     

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.     

Characterization of the functional activity of botulinum neurotoxin subtype B6

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A–G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C‐terminal heavy chain (H_C) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously‐characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. H_C/B1 and H_C/B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its H_c.domain.

     

Molecular Characterization of the Clusters of Genes Encoding the Botulinum Neurotoxin Complex in Clostridium botulinum (Clostridium argentinense) Type G and Nonproteolytic Clostridium botulinum Type B

The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes. Received: 28 January 1997 / Accepted: 24 March 1997     

Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and /B1 clusters are located within plasmids

Background

Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A–G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression.

Methodology/Principal Findings

Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid.

Conclusions/Significance

Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum.     

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

Nucleotide sequence of the gene coding for non-proteolyticClostridium botulinum type B neurotoxin: Comparison with other clostridial neurotoxins

The neurotoxin gene of non-proteolyticClostridium botulinum type B (strain Eklund 17B) was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated with primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 3 end of the gene was obtained by using primers designed to the determined sequence of non-proteolytic BoNT/B and a published downstream region of BoNT/B gene from a proteolytic strain. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated the toxin gene encodes a protein of 1291 amino acid residues. Comparative alignment of the derived BoNT/B sequence with those of other published botulinal neurotoxins revealed highest sequence relatedness with BoNT/B of proteolyticC. botulinum. The sequence identity between non-proteolytic and proteolytic BoNT/B was 97.7% for the light chain (corresponding to 10 amino acid changes) and 90.2% for the heavy chain (corresponding to 81 amino acid changes), with most differences occurring at the C-terminal end. A genealogical tree constructed from all known botulinal neurotoxin sequences revealed marked topological differences with a phylogenetic tree ofC. botulinum types based upon small-subunit (16S) ribosomal RNA sequences.     

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

The complete genome sequence of Clostridium botulinum F str. 230613, insertion sites, and recombination of BoNT gene clusters

The genomic DNA of Clostridium botulinum F str. 230613 includes a chromosome (3?993?083?bp, 3502 coding sequences (CDs)) and a plasmid (17?531?bp, 25 CDs). The arrangement of the botulinum neurotoxin serotype F (BoNT/F) gene cluster, a 15-kb (or longer) fragment including the bont gene and other relevant genes, and its different insertion sites in C. botulinum A2 and C. botulinum F were formulated. Mobile elements and virulence factors were analysed. We also found a cell adhesion and pectin lyase domain-containing protein, which may function in attaching to the host and as a pectin lyase. The nine BoNT gene clusters of group I C. botulinum strains were located at three sites in the chromosome of C. botulinum F str. 230613. This study showed the inserting inclination of BoNT/A1 tend to have gene clusters inserted at site 3, BoNT/F at site 2, and BoNT/A2 at site 1. Additionally, we found the recombination event between the BoNT gene clusters of sites 2 and 3, a mechanism that contributed to the diversity of the BoNT gene cluster arrangement.     

Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA⁺ OrfX⁻) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA⁻ OrfX⁺) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA⁺ OrfX⁻ cluster (69A and 32A) and one strain with the HA⁻ OrfX⁺ cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.