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.     

Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

A collection of 36 Clostridium botulinum type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to botE and orfX1 in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.     

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     

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     

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected.     

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     

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     

A型肉毒神经毒素基因的PCR检测

目的:建立快速筛查A型肉毒毒素的PCR方法。方法:根据GenBank中报道的肉毒毒素基因序列,综合应用多种生物软件分析设计特异的检测引物,从提取的基因组DNA、热裂解产物和菌液等不同形式的模板中扩增大小为457bp的A型肉毒毒素特异基因片段,以肉毒梭菌其他血清型及破伤风梭菌为对照。结果:检测方法无交叉反应,灵敏度可达10pgDNA,3×103个菌。结论:建立的检测方法特异性强、灵敏度高,可以用于A型肉毒毒素基因的快速筛查。     

Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Two-Component Systems Are Involved in the Regulation of Botulinum Neurotoxin Synthesis in Clostridium botulinum Type A Strain Hall

Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.     

The Differentiation of Various Strains of Clostridium botulinum and Transformation Experiments with Type E

The colonial forms and other characteristics of toxigenic and less toxigenic (hypotoxigenic) variants of Clostridium botulinum types A, B, C, D and E are described. In addition to differences in the appearance of the colonies the hypotoxigenic variants were able to produce larger numbers of spores. Transformation from one form to the other was found when they were grown in Robertson's meat broth to which sterile culture filtrates (boiled or unboiled) from the other form was added. A transformation from hypotoxigenic variants to toxigenic forms was also found when hypotoxigenic forms were grown in Robertson's medium to which the sterile culture filtrate (boiled or unboiled) of a strain of Cl. welchii had been added. The possibility of contamination of the cultures is emphasized.
A final appraisal of the situation must be reserved until experiments with single cell isolates have been performed.     

Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)^?1cm^?1. These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.     

Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.     

Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.     

Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

Boticin B is a heat-stable bacteriocin produced by Clostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene, btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing the HindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.