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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 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 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 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.
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 (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 CDC41370, and describe differences in the organization of the neurotoxin gene clusters among these strains.
C. botulinum strains were grown anaerobically at 35°C in Trypticase-peptone-glucose-yeast extract (TPGY) medium. Strains used in this study are identified in Table Table11.
TPGY cultures of C. botulinum strains were incubated overnight at 35°C. Cultures were centrifuged for 10 min at 4,000 rpm in a swinging bucket centrifuge to pellet the bacteria. The pellet was resuspended in 300 μl of Tris-EDTA buffer containing 30 mg of lysozyme/ml (Sigma, St. Louis, MO) and incubated for 30 min at 37°C. The MasterPure DNA purification kit (Epicenter, Madison, WI) was used for subsequent DNA extraction steps as follows. Three hundred microliters of 2× tissue and cell lysis buffer containing 200 μg of RNase A (Qiagen, Valencia, CA) was added to the bacterial suspension, followed by 350 μl of MPC protein precipitation buffer. The suspension was centrifuged for 10 min at 4,000 rpm. Genomic DNA was precipitated by adding 1 ml of isopropanol and resuspended in Tris-EDTA buffer. DNA was stored at 4°C until analysis.
The neurotoxin cluster genes from strain CDC41370 were sequenced in their entirety by PCR amplifying overlapping fragments using high-fidelity Platinum Taq (Invitrogen, Carlsbad, CA). PCR amplifications were performed in a staggered manner, and each gene within the toxin gene cluster was sequenced in at least three overlapping segments. PCR conditions were as follows: 94°C for 5 min, followed by 40 cycles at 94°C for 30 s, 50°C for 30 s, and an extension step at 68°C for 1 to 3 min, depending on the size of the products. The reaction included a final extension at 72°C for 5 min. PCR amplicons were purified using the UltraClean PCR clean-up kit (Mo Bio, Carlsbad, CA) and then sequenced using an Applied Biosystems 3730 DNA analyzer. The amplification and sequencing primers are listed in Table S1 in the supplemental material.
Sequence assembly and phylogenetic analysis of the neurotoxin cluster genes were performed using the software BioNumerics 5.10 (Applied Maths, Austin, TX), and multiple sequence alignments were generated by using MULTALIN (http://bioinfo.genotoul.fr/multalin/). Comparative analysis of the bont/A gene sequences was performed using the software SimPlot (http://sray.med.som.jhmi.edu/SCRoftware/simplot/). The GenBank accession numbers of previously published sequences are shown in Table Table22.
PCR assays were designed to target internal fragments of toxin gene cluster genes ha17, ha34, ha70, orfX1, orfX2, orfX3, and p21, using primers identified in Table S1 in the supplemental material. PCR conditions consisted of an initial denaturing step at 94°C for 5 min followed by 40 cycles at 94°C for 30 s, 50°C for 30 s, and an extension step at 72°C for 30 s (for PCR targeting ha17, ha70, orfX1, or orfX2), 45 s (for PCR targeting ha34 or orfX3), 90 s (for PCR targeting the p21-orfX1 spacing), or 3 min (for PCR targeting the orfX2-orfX3 spacing). All reactions included a final extension at 72°C for 5 min.
Sequences of the bont and neurotoxin cluster genes were determined and deposited in the GenBank database under the accession numbers listed in Table Table22.
The full-length coding sequences of bont/A and bont/B genes from C. botulinum strain CDC41370 and the sequence of the bont/A gene from C. botulinum strain CDC588 were amplified in overlapping segments by PCR and then sequenced. These nucleotide sequences were compared with those of the bont/A and bont/B genes of type A(B) strains sequenced in this study (strains CDC5178, CDC3517, CDC28184, CDC48706, CDC4893, CDC1727, and CDC5277), and with other bont/A and bont/B gene sequences previously published.
The bont/A nucleotide sequence from strain CDC41370 differed from subtypes A1 through A4 by 2 to 7%, and the amino acid sequences differed by 4% to 14% (Table (Table3).3). Compared with previously published nucleotide sequences, the bont/A gene from strain CDC41370 was most similar to subtype A1 (97.8% nucleotide identity), and it clustered separately from other subtypes (Fig. (Fig.2).2). Type Ab strain CDC588 and all five A(B) strains sequenced during this study shared identical bont/A1 nucleotide sequences with each other and with previously reported sequences of A(B) strains, all of which differed from other subtype A1 genes by only 2 nucleotides (Fig. (Fig.22).
While the bont/B nucleotide sequence from strain CDC41370 showed 98.2% identity to that of other bivalent strains, the gene showed greater identity (99.7%) to the sequence from subtype B1 strains (Table (Table44 and Fig. Fig.3).3). When the bont/B nucleotide sequence from the six A(B) strains sequenced in this study were compared with that of subtype B1, all of the sequences harbored the substitution of a T at nucleotide 384, resulting in a stop codon at amino acid position 128. An additional 82 nucleotide changes were observed in these silent bont/B genes. The majority of these nucleotide changes (n = 62) were also found in the previously reported bivalent bont/B gene of type Ab and Ba strains (data not shown) (12, 15).
Comparative analysis of the bont/A gene from strain CDC41370 with other bont/A subtypes suggested that the gene is derived from recombination events between bont/A1, bont/A2, and other serotype A gene sequences (Fig. (Fig.4).4). The bont/A sequence from strain CDC41370 was highly related to the sequence of subtype A1 (99.7% identity) from positions 1 through 2040. The region between bases 2041 and 2491 was 100% identical to bont/A2, and the nucleotides at positions 2492 to 2842 were 99.1% identical to subtype bont/A1. The region between bases 2843 and 3410 showed a high identity with bont/A2 (98.8% identity). The last 480 nucleotides were similar to bont/A1 (95.6%), although 19 unique nucleotide polymorphisms were found in this region.
The variation observed in the nucleotide sequence of the bont/A gene of strain CDC41370 compared to that of the other bont/A subtypes resulted in 18 unique amino acid changes in the predicted protein, of which 9 changes were nonconservative. The 18 unique amino acid changes in strain CDC41370 were located primarily in the binding domain and consisted of the following residues: A414 (catalytic domain); S555, D567, and H581 (translocation domain); N885, E955, A1006, S1139, S1141, T1142, L1143, L1144, G1153, N1187, K1254, Y1259, I1273, and K1294 (binding domain).
The full-length coding sequences of the neurotoxin gene clusters from C. botulinum strain CDC41370 were amplified in overlapping segments by PCR and then sequenced. This strain contained two sets of neurotoxin cluster genes, similar to what has been described in A(B) and Ba strains (14, 15), in which the ha cluster is associated with the bont/B gene and the orfX cluster is associated with the bont/A gene (Fig. (Fig.11).
Comparisons of the bont/A gene clusters (13,903 to 14,973 bp, including bont/A, ntnh, p47, p21, orfX1, orfX2, and orfX3 genes) showed that the nucleotide sequence of the bont/A gene cluster of strain CDC41370 was most similar to that of the toxin gene cluster of subtype A2 (Fig. (Fig.5).5). However, the results were variable when each gene was compared to previously reported sequences in subtypes A1 to A4, including A(B) strains (Table (Table3).3). The orfX3 nucleotide sequence of strain CDC41370 was 99.7% identical to the sequence of subtype A4, whereas the orfX2 and orfX1 nucleotide sequences were 99.2% and 97% identical to the sequences of A(B) strains. The p47 nucleotide sequence was 99.4% identical to the sequence of subtype A3, and the ntnh gene sequence showed 98.6% identity with the sequence of subtype A2. The p21 gene sequence was the most variable among the sequences within the orfX cluster reported here and differed from other previously reported subtypes by 7.5% to 12.3% at the nucleotide level.
The nucleotide and amino acid sequences of the neurotoxin cluster genes associated with the bont/B gene in strain CDC41370 were highly similar (91.5% to 100% nucleotide identity) to those of the neurotoxin cluster genes associated with the bont/B gene in A(B), Ba, and B1 strains (Table (Table44).
Although strain CDC41370 contained the same two sets of neurotoxin cluster genes as did A(B) and Ba strains, it harbored neither the 0.6-kb insertion sequence element between the orfX2 and orfX3 genes, as found in A(B) strains (5), nor the 1.2-kb insertion sequence element between the p21 and orfX1 genes found in subtypes A2, A3, and A4 (14).
The organization of the neurotoxin cluster genes in the other two Ab strains (CDC1436 and CDC588) and seven A(B) strains (CDC28184, CDC1727, CDC3517, CDC4893, CDC5178, CDC5277, and CDC48706) was analyzed using PCR assays designed to target internal fragments of each gene and specific intergenic regions. Although all strains contained bont/A, bont/B, ntnh, ha70, ha17, ha33, botR, p21, p47, orfX1, orfX2, and orfX3 genes, the intergenic spacing between the p21 and orfX1 genes and between the orfX2 and orfX3 genes varied among these strains. Strain CDC588 and all seven A(B) strains contained the 0.6-kb insertion between orfX2 and orfX3, but strain CDC1436 had neither the 1.2-kb insertion between p21 and orfX1, reported for subtypes A2, A3, and A4 (14), nor the 0.6-kb insertion between orfX2 and orfX3, as found in A(B) strains (5).
In this study, we sequenced bont/A, bont/B, and the associated neurotoxin cluster genes of C. botulinum type Ab strain CDC41370 and the bont/A gene of C. botulinum type Ab strain CDC588. These nucleotide sequences were compared with bont/A and bont/B sequences from other strains, including types Ab, A(B), and Ba. In addition, we analyzed the organization of the neurotoxin gene clusters in C. botulinum type Ab strains CDC588 and CDC1436.
The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. In contrast, the bont/A gene from strain CDC1436 is identical to that of BoNT/A2 strains (12). The bont/A nucleotide sequence from strain CDC41370 was most similar to bont/A1, although it differed from this subtype by 2.2%, and the predicted amino acid sequence differed by 4.3%. Among strains of subtype A1, little nucleotide sequence variation has been shown; i.e., bont/A nucleotide sequences in A(B) strains harbor only two nucleotide differences from those of other A1 strains (12), and bont/A nucleotide sequences in HA− Orfx+ A1 strains differ from those of other A1 strains by 4 to 5 nucleotides (18). In comparison, the difference of 87 nucleotides observed in the bont/A nucleotide sequence of strain CDC41370 is remarkable; however, none of the changes in the predicted amino acid sequence observed in strain CDC41370 localized to regions encoding the known conserved functional motifs (1). These variations could cause modifications in the structure of the toxin, with consequent implications such as differences in the binding affinity for neuronal cell receptors or in BoNT antitoxin binding, thereby having an effect on the potency of a given antitoxin. Traditionally, botulinum toxin types (serotypes A through G) have been defined by specific neutralization with antitoxins derived from hyperimmunized animals, and some subtypes have been shown to have differential binding of monoclonal antibodies (10, 16, 17, 20). For example, subtypes A1 and A2 present differences in monoclonal antibody binding affinity, despite the fact that these subtypes share 90% of amino acid sequence identity.
Toxin subtypes were historically identified through monoclonal antibody analyses, but this approach is labor-intensive. Currently, subtypes are defined based on the identification of separate clusters by using phylogenetic analysis of the bont gene sequence (12). For example, bont/E1 and bont/E2 are classified as different subtypes, although they differ by only 1% at the amino acid level. Carter et al. (2) recently described a bont/A gene nucleotide sequence in strains from heroin-associated wound botulism cases in the United Kingdom that differs from that from subtype A1 by 1.4%, and they proposed that this unique sequence be classified as subtype A5. According to these criteria, the bont/A gene sequence of strain CDC41370 could represent a different BoNT/A subtype since the bont gene nucleotide sequence differs by 2.2% from the reported A1 sequence. However, the impact of the variation observed in the bont/A sequence of this strain on functional properties of the toxin is unknown. Moreover, the probability of describing additional unique sequences will likely increase as more diverse strains are examined, questioning the value of clustering neurotoxin gene sequences into subtypes.
Comparative analysis of bont/A from strain CDC41370 with bont/A1 to bont/A4 suggests that this gene is derived from recombinant events involving bont/A1, bont/A2, and probably other bont/A genes. Similarly, subtype A2 could represent a recombination event between bont/A1 and bont/A3 (12). In addition, recombination in the ntnh gene of certain type A and B strains has been reported (3). These findings indicate that recombination events have likely contributed to the diversity among the BoNT serotypes. Further study of the mechanisms producing such diversity in the neurotoxin gene cluster may shed light on the role of such genes, if any, on toxin production and potency.
Previously reported bont/B nucleotide sequences from bivalent strains harboring two bont genes, i.e., A(B), Ba, Bf, and Ab, are highly similar and represent a separate bont/B subtype (bivalent B) (12). Interestingly, the bont/B nucleotide sequence from strain CDC41370 showed higher identity to the sequence from subtype B1 than to other bivalent strains. The bont/B in strain CDC41370 differed from the bivalent B subtype by up to 2.3%, suggesting a different origin of this gene in strain CDC41370 than that in other bivalent strains. In addition, two C. botulinum type Ab strains from Italy exhibited B1 and B3 subtypes using a PCR-restriction fragment length polymorphism method (9). These findings support the hypothesis of mobilization of the bont/B gene among bivalent strains.
Among A(B) strains, the nucleotide sequence analysis of bont/A and bont/B genes showed a relatively low variability. The A(B) strains sequenced during this study harbored identical bont/A nucleotide sequences to that of previously published sequences in other A(B) strains (12). All of the bont/B nucleotide sequences from A(B) strains examined in this study and others (13) harbored a substitution of a T at nucleotide 384, resulting in a premature stop codon. Although accumulation of nucleotide changes after nonsense mutations may be expected, the nucleotide changes in the bont/B sequences were conserved among the A(B) strains examined. Moreover, most of the nucleotide changes observed in these silent bont/B genes were also identified in the previously reported bont/B gene of type Ab and Ba strains. These data suggest that A(B) strains have highly conserved bont/A and bont/B gene sequences. In contrast, strains producing two toxin types (e.g., types Ab and Ba) showed comparatively more variability because at least four different bont/A sequences and three different bont/B sequences have been described in these bivalent strains.
In addition to bont/A and bont/B genes, we sequenced the neurotoxin cluster genes of the C. botulinum strain CDC41370 and compared these sequences to known sequences from types A and B. To our knowledge, this is the first report of the complete nucleotide sequences of both neurotoxin gene clusters in a type Ab strain. In addition, we analyzed the content of the neurotoxin gene cluster in two other type Ab strains (strains CDC588 and CDC1436) and seven A(B) strains (CDC28184, CDC1727, CDC5178, CDC4893, and CDC5277) by PCR assays. As expected (4, 15, 21), all 10 strains contained bont/A, bont/B, ntnh, ha70, ha17, ha33, botR, p21, p47, orfX1, orfX2, and orfX3. However, a difference in intergenic spacing of p21-orfX1 and orfX2-orfX3 was observed among these strains. Strain CDC588 and all six A(B) strains contained a 0.6-kb insertion between orfX2 and orfX3. This has been also described in other A(B) and HA− OrfX+ A1 strains (5, 14, 18). These intergenic spacing sequences contain partial insertion sequence (IS) elements, suggesting a role in gene mobility (4). Various partial IS elements have been described within the orfX cluster in A2, A3, Ba, and A(B) strains (5, 19). However, two type Ab strains analyzed in this study (strains CDC1436 and CDC41370) do not have the IS elements residing within these intergenic sequences. The lack of these potential IS elements has been also reported for the subtype A2 strain Mascarpone (8). The significance of these partial IS elements in the evolution of the neurotoxin gene cluster remains unclear.
Analysis of the nucleotide sequence of the orfX cluster genes associated with bont/A in strain CDC41370 showed variable results compared to the orfX cluster genes of various subtypes. The nucleotide sequence of the orfX3 gene of strain CDC41370 was more similar to that of subtype A4, whereas the orfX2 and orfX1 nucleotide sequences had the highest similarity with those of A(B) strains. The sequence of the p21 gene was the most divergent among the sequences within the orfX cluster. The p47 gene of strain CDC41370 was more similar to the sequence of subtype A3, and the ntnh gene sequence showed high identity with that of subtype A2. Genetic variability in the orfX cluster sequences has been also demonstrated among all the bont/A subtypes (14). Differences in toxin gene cluster arrangement among various subtypes have been described only for type A subtypes. In contrast, the neurotoxin cluster genes associated with the bont/B gene seemed to be less diverse than the orfX cluster. The nucleotide and amino acid sequences of the neurotoxin cluster genes associated with the bont/B gene in strain CDC41370 were highly similar to those of the ha cluster in A(B), Ba, and B1 strains.
BoNT-producing Clostridium strains are genetically and phenotypically diverse (3). C. botulinum strains can be grouped into four phylogenetically distinct lineages, and rare strains of Clostridium butyricum and Clostridium baratii are able to produce BoNT/E and BoNT/F, respectively. Based on these observations, a revision of the nomenclature and classification within this species has been proposed (3). More recently, various molecular analyses have demonstrated a high degree of variability among the BoNT-producing strains, the bont genes, and the neurotoxin-associated protein-encoding genes (12, 14). The description of novel arrangements of the neurotoxin cluster genes and variations in the bont gene sequence, as described in this study and elsewhere, suggests that the nomenclature for BoNT subtypes requires revision. The value of clustering neurotoxin gene sequences into subtypes may diminish as additional variants of the bont sequences are identified and differences among clusters become less evident. In addition, the description of novel toxin gene cluster arrangements increases the complexity of subtype nomenclature, e.g., HA− Orfx+ A1 strains. Thus, the use of only neurotoxin gene sequences as the basis for subtype nomenclature may not be accurate. Additional molecular characterization of BoNT-producing strains and analysis of the functional impact of differences in toxin gene sequences may help to improve the classification of such diverse strains and their associated toxins.
We thank the Division of Food-borne, Bacterial, and Mycotic Diseases Sequencing Facility (CDC) for DNA sequencing and the Biotechnology Core Facility (CDC) for oligonucleotide synthesis.
This publication was supported by funds made available from the Centers for Disease Control and Prevention, Coordinating Office for Terrorism Preparedness and Emergency Response.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
Published ahead of print on 14 August 2009.
†Supplemental material for this article may be found at http://aem.asm.org/.