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     

Characterization of the Genes Encoding the Botulinum Neurotoxin Complex in a Strain of Clostridium botulinum Producing Type B and F Neurotoxins

The organization of the clusters of genes encoding proteins of the botulinum neurotoxin (BoNT) progenitor complex was elucidated in a strain of Clostridium botulinum producing type B and F neurotoxins. With PCR and sequencing strategies, the type B BoNT-gene cluster was found to be composed of genes encoding BoNT/B, nontoxic nonhemagglutinin component (NTNH), P-21, and the hemagglutinins HA-33, HA-17, and HA-70, whereas the type F BoNT-gene cluster has genes encoding BoNT/F, NTNH, P-47, and P-21. Comparative sequence analysis showed that BoNT/F in type BF strain 3281 shares highest homology with BoNT/F of non-proteolytic (group II) C. botulinum whereas NTNH and P-21 in the type F cluster of strain 3281 are more similar to the corresponding proteins in proteolytic (group I) type F C. botulinum. These findings indicate diverse evolutionary origins for genes encoding BoNT/F and its associated non-toxic proteins, although the genes are contiguous. By contrast, sequence comparisons indicate that genes encoding BoNT/B and associated non-toxic proteins in strain 3281 possess a similar evolutionary origin. It was demonstrated that the genes present in the BoNT/B gene cluster of this type BF strain show exceptionally high homology with the equivalent genes in the silent BoNT/B gene cluster of C. botulinum type A(B), possibly indicating their common ancestry. Received: 30 March 1998 / Accepted: 21 May 1998     

Presumptive identification of common adenovirus serotypes by the development of differential cytopathic effects in the human lung carcinoma (A549) cell culture

Abstract The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published. The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141049 Da. The sequence identity between the C. barati ATCC43756 and non-proteolytic C. botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain. This is much lower than reported identities for the type E neurotoxins from C. botulinum and C. butyricum (96% identity between light chains and 98.8% between the heavy chains). Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C. barati . An ORF upstream of the toxin coding region was revealed. This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C. botulinum type C neurotoxin.     

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.     

Conserved structure of genes encoding components of botulinum neurotoxin complex M and the sequence of the gene coding for the nontoxic component in nonproteolyticClostridium botulinum type F

For investigation of the genes of proteins associated in vivo with botulinum neurotoxin (BoNT), polymerase chain reaction (PCR) experiments were carried out with oligonucleotide primers designed to regions of the nontoxic-nonhemagglutinin (NTNH) gene ofClostridium botulinum type C. The primers were used to amplify a DNA fragment from genomic DNA ofC. botulinum types A, B, E, F, G and toxigenic strains ofClostridium barati andClostridium butyricum. The amplified product from all of these strains hybridized with an internal oligonucleotide probe, whereas all nontoxigenic clostridia tested gave no PCR product and showed no reaction with the probe. TheNTNH gene was shown to be located upstream of the gene encoding BoNT, thereby revealing a conserved structure for genes encoding the proteins of the M complex of the progenitor botulinum toxin in these organisms. The sequence of theNTNH gene of nonproteolyticC. botulinum type F was determined by PCR amplification and sequencing of overlapping cloned fragments. NTNH/F showed 71% and 61% identity with NTNH ofC. botulinum type E and type C respectively.     

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.     

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.     

Gene Organization and Sequence Determination of the Two Botulinum Neurotoxin Gene Clusters in Clostridium botulinum Type A(B) Strain NCTC 2916

The gene organization and nucleotide sequence of the type A and B BoNT-gene clusters in Clostridium botulinum strain NCTC 2916 were studied. The aim was to clarify the organization of genes within C. botulinum type A strains possessing an unexpressed BoNT/B gene. The BoNT/A-gene cluster includes genes encoding BoNT, NTNH and a part of P-47 (the gene for this protein was reported in strains of C. botulinum types E and F). Clustered with the silent BoNT/B gene were genes encoding NTNH, P-21 and HA-33. Sequencing analysis of the NTNHs revealed the presence of 471 amino acids identical in the type B and A gene clusters. This gene organization contrasts markedly with the purported organization in strain NCTC 2916 described by Henderson et al. (FEMS Microbiol. Lett. 140, 151–158). In type A(B) strain NCTC 2916, the neurotoxin gene is of type BoNT/A1 within a gene cluster that has identical organization to that found in BoNT/A2 type strains; these observations may be significant in establishing the origin of the BoNT-gene cluster. Received: 28 July 1997 / Accepted: 15 October 1997     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

Neurotoxin Gene Clusters in Clostridium botulinum Type Ab Strains

There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont/B nucleotide sequences, suggesting that they may have arisen from separate recombination events.Clostridium botulinum is a gram-positive anaerobic bacterium that produces an extremely potent toxin, the botulinum neurotoxin (BoNT). There are seven serologically distinct types of BoNT (serotypes A through G). Although most strains of C. botulinum express a single toxin serotype, some isolates have been shown to produce more than one, namely, Ab, Af, Ba, and Bf (11). In addition, many strains designated type A by mouse bioassay harbor nucleotide sequences for both type A and B toxins (6). These strains have been designated A(B) to indicate the presence of the bont/B gene without type B-specific toxicity.Based on phylogenetic analysis of the neurotoxin gene sequences, four subtypes have been identified within serotype A and five subtypes within serotype B (12). The neurotoxin gene nucleotide sequences of these subtypes differ by up to 8%, and the predicted amino acid sequences differ by up to 16%. In addition, the genes encoding components of the toxin complexes are arranged in clusters that differ in composition and organization (14) (Fig. ​(Fig.1).1). The toxin gene cluster of subtype A1 (termed ha cluster) includes the gene encoding the nontoxic nonhemagglutinin (ntnh), a regulatory gene (botR), and an operon encoding three hemagglutinins (ha70, ha33, and ha17). The toxin gene clusters containing bont/A2 or bont/A3 (termed orfX cluster) include the ntnh and p21 (analogous to botR) genes and several genes of unknown function (orfX1, orfX2, orfX3, and p47). Type Ba and A(B) strains contain two sets of neurotoxin cluster genes in which ha70, ha33, and ha17 are associated with the bont/B gene, and orfX1, orfX2, orfX3, and p47 are associated with the bont/A gene. In addition, some A1 strains contain a neurotoxin gene cluster that is similar to those in A2 and A3, but the bont/A nucleotide sequence is 99.9% identical to that in other A1 strains. These strains have been designated HA⁻ Orfx⁺ A1 (14). The neurotoxin gene cluster in type B strains includes the ntnh, botR, ha70, ha33, and ha17 genes. Notably, no differences in the neurotoxin gene cluster arrangements among the subtypes within serotype B have been reported.Open in a separate windowFIG. 1.Toxin gene cluster arrangements for BoNT type A-producing strains, including Ab, A(B), and Ba strains.Although several studies have described the organization and the nucleotide sequences of the neurotoxin gene cluster components among type A and B strains [including type Ba and A(B) strains], there is limited information regarding the diversity of the neurotoxin cluster genes among C. botulinum type Ab strains. The nucleotide sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene of C. botulinum type Ab strain CDC588 have been previously reported; strain CDC1436 harbors a bont/A2 gene, and both strains CDC1436 and CDC588 harbor a bont/bvB gene (12, 15). Four additional type Ab strains from Italy have been analyzed by a restriction fragment length polymorphism method to determine the bont/A and bont/B subtypes (7, 9). To the best of our knowledge, the complete nucleotide sequences of the neurotoxin gene clusters in C. botulinum type Ab strains have not been reported. Thus, the objective of this study was to analyze the neurotoxin gene cluster composition in three C. botulinum type Ab strains ({"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, CDC588, and CDC1436) available in the CDC strain collection. We report differences in bont/A gene sequence among type Ab strains, including the identification of a novel bont/A nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, and describe differences in the organization of the neurotoxin gene clusters among these strains.     

Sequence Diversity of Genes Encoding Botulinum Neurotoxin Type F

Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was composed exclusively of bont/F genes from C. baratii strains. The region of the bont/F5 gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although the bont/F5 nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.Botulism is a potentially fatal disease caused solely by the action of serologically distinct neurotoxins (BoNT/A, -B, -C, -D, -E, -F, or -G) which prevent acetylcholine release at neuromuscular junctions, resulting in paralysis. Food-borne botulism may result from the ingestion of a preformed toxin that is produced in inadequately preserved food. Under certain conditions, botulinum neurotoxin-producing Clostridium sp. may colonize and produce toxin in wounds (wound botulism) or in the intestine (infant botulism or adult colonization). Globally, human botulism cases are associated with botulinum neurotoxin serotypes A, B, E, and rarely F. The Centers for Disease Control and Prevention (CDC) maintains active surveillance for botulism cases in the United States. Of 1,269 U.S. cases of botulism reported to the CDC between 1981 and 2002, approximately 1% were due to type F toxin (13). An additional 10 cases of type F botulism were reported to the CDC from 2003 to 2007 (http://www.cdc.gov/nationalsurveillance/botulism_surveillance.html).Type F botulism was first described in 1960 following an outbreak occurring in Denmark involving liver paste (30). The organism isolated in this outbreak metabolically resembled proteolytic Clostridium botulinum strains of types A and B. In a subsequent outbreak, type F toxin was found to be produced by a nonproteolytic C. botulinum strain isolated from venison jerky (29). Bivalent toxin-producing strains have been described, including Bf strains isolated from infants in the United States and England (1, 16, 17, 35) and an Af strain isolated from individuals in Argentina with food-borne botulism (11). Bivalent strains may produce higher titers of one toxin type, which are denoted with a capital letter. The only reported organism isolated from infants with botulism due to type F toxin alone (i.e., not associated with additional serotypes as in bivalent strains) is Clostridium baratii (2, 14, 24). In addition, C. baratii type F has been isolated from adults with botulism (28) as well as suspect foods associated with botulism cases (15; CDC, unpublished data).Botulinum neurotoxin genes (bont) are typically found within toxin gene clusters that include other genes encoding components of the toxin complex (ha70, ha17, ha33, ntnh), regulatory proteins (botR), or proteins with unknown functions (p47, orfX1, orfX2, orfX3). Two general toxin gene cluster arrangements have been described, including the orfX cluster (orfX3-orfX2-orfX1-botR-p47-ntnh-bont) and the ha cluster (ha70-ha17-ha33-botR-ntnh-bont) (21, 22). The bont/F genes of type F and type Bf strains examined by Hill et al. (21) were found in an orfX cluster.The amino acid sequence identities of the BoNT serotypes A to G range from approximately 35 to 70% (36). In addition, within nearly all toxin serotypes, various levels of amino acid sequence variation have been observed, resulting in the identification of toxin subtypes (20, 36, 37).Although a limited number of genes encoding type F botulinum neurotoxin have been sequenced, a comparison of sequences available in public databases indicates that significant diversity exists within this serotype. The nucleotide sequence identity of the type F neurotoxin gene from the proteolytic strain Langeland differs from that of the gene in the nonproteolytic strain 202F by 7%. The type F gene from C. baratii strain ATCC 43756 differs from those of Langeland and 202F by 18% and 20%, respectively. Although the bivalent (Bf) strain CDC3281 is phenotypically proteolytic, the toxin gene shows greater similarity to those from nonproteolytic strains (34). In addition to metabolic differences observed between proteolytic and nonproteolytic C. botulinum strains as well as C. baratii, these organisms are phylogenetically distinct based on differences among their 16S rRNA sequences (5, 20).In order to define the degree of genetic diversity among strains encoding botulinum neurotoxin type F, we sequenced the bont/F gene and partially characterized the toxin gene cluster by using a panel of 33 strains with diverse origins. These strains were selected from those available in the CDC culture collection as well as several isolated in Argentina. The only reported Af strains have been isolated in Argentina. Among 68 outbreaks of serotype-confirmed food-borne botulism in Argentina between 1922 and 2007, type F was isolated in two outbreaks, and type Af was isolated in one outbreak. In addition, Lúquez et al. (26) reported isolation of type F and Af strains from Argentine soils.Here, we report that analysis of the bont/F genes from the strains examined in this study revealed a high degree of nucleotide sequence heterogeneity and the identification of seven type F subtypes (F1 to F7). In addition, the nucleotide sequence of one subtype (F5) has not been previously reported and contains evidence of recombination compared to the other subtypes.     

Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813

The molecular composition of the purified progenitor toxin produced by a Clostridium botulinum type C strain 6813 (C-6813) was analyzed. The strain produced two types of progenitor toxins (M and L). Purified L toxin is formed by conjugation of the M toxin (composed of a neurotoxin and a non-toxic nonhemagglutinin) with additional hemagglutinin (HA) components. The dual cleavage sites at loop region of the dichain structure neurotoxin were identified between Arg444-Ser445 and Lys449-Thr450 by the analyses of C-terminal of the light chain and N-terminal of the heavy chain. Analysis of partial amino acid sequences of fragments generated by limited proteolysis of the neurotoxin has shown to that the neurotoxin protein produced by C-6813 was a hybrid molecule composed of type C and D neurotoxins as previously reported. HA components consist of a mixture of several subcomponents with molecular weights of 70-, 55-, 33-, 26~21- and 17-kDa. The N-terminal amino acid sequences of 70-, 55-, and 26~21-kDa proteins indicated that the 70-kDa protein was intact HA-70 gene product, and other 55- and 26~21-kDa proteins were derived from the 70-kDa protein by modification with proteolysis after translation of HA-70 gene. Furthermore, several amino acid differences were exhibited in the amino acid sequence as compared with the deduced sequence from the nucleotide sequence of the HA-70 gene which was common among type C (strains C-St and C-468) and D progenitor toxins (strains D-CB16 and D-1873).     

Molecular characterization of the protease from Clostridium botulinum serotype C responsible for nicking in botulinum neurotoxin complex

A protease was purified from the culture medium of Clostridium botulinum serotype C strain Stockholm (C-St). The purified protease belonged to the cysteine protease family based on assays for enzyme inhibitors, activators and kinetic parameters. The protease formed a binary complex consisting of 41- and 17-kDa proteins held together non-covalently. The DNA sequence encoding the protease gene was shown to be a single open reading frame of 1593 nucleotides, predicting 530 amino acid residues including a signal peptide. The N-terminal region of the native enzyme underwent further proteolytic modification after processing by a signal peptidase. The protease introduced intermolecular cleavage into an intact single chain botulinum neurotoxin (BoNT) at a specific site. Homology modeling and docking simulation of C-St BoNT and C-St protease demonstrated that the specific nicking-site of the BoNT appears to fit into the deep pocket in the active site of the protease.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

Analysis of the Botulinum Neurotoxin Type F Gene Clusters in Proteolytic and Nonproteolytic Clostridium botulinum and Clostridium barati

Comparison of genes encoding type F botulinum neurotoxin progenitor complex in strains of proteolytic Clostridium botulinum strain Langeland, nonproteolytic Clostridium botulinum strain 202F, and Clostridium barati strain ATCC 43256 reveals an identical organization of genes encoding a protein of molecular mass of approx. 47 kDa (P-47), nontoxic-nonhemagglutinin (NTNH) and botulinum toxin (BoNT). Although homology between the protein components of the complexes encoded by these different species all producing botulinum neurotoxin type F is considerable (approx. 69–88% identity), exceptionally high homology is observed between the C-termini of the P-47s (approx. 96% identity) and the NTNHs (approx. 94% identity) encoded by Clostridium botulinum type F strain Langeland and Clostridium botulinum type A strain Kyoto. Such a region of extremely high sequence identity is strongly indicative of recombination in these strains synthesizing botulinum neurotoxins of different antigenic types. Received: 13 April 1998 / Accepted: 9 May 1998     

Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples

An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.