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Journal of Bacteriology, December 2007, p. 8643-8650, Vol. 189, No. 23
0021-9193/07/$08.00+0     doi:10.1128/JB.00784-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Sequencing the Botulinum Neurotoxin Gene and Related Genes in Clostridium botulinum Type E Strains Reveals orfx3 and a Novel Type E Neurotoxin Subtype

Ying Chen,^1,2 Hannu Korkeala,¹ Johannes Aarnikunnas,³ and Miia Lindström^1*

Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland,¹ College of Chemistry and Biology, Yantai University, Yantai, People's Republic of China,² Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Finland³

Received 20 May 2007/ Accepted 6 September 2007

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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.

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Clostridium botulinum strains produce one or two of the lethal botulinum neurotoxins (BoNTs) designated A to G according to antigenic properties. These toxins are produced as a progenitor complex consisting of BoNT, hemagglutinin (HA), nontoxic non-HA (NTNH), and probably other uncharacterized components, such as P47 and OrfX (14, 15). The genes encoding these proteins are linked as a cluster and vary in composition and structure among different serotypes and strains. In general, the ntnh and bont genes are located conjointly and arranged in the downstream region of the gene cluster, whereas the upstream regions vary between different types and subtypes of C. botulinum (15).

C. botulinum type E predominates in aquatic environments. It differs from the terrestrial toxinotypes of C. botulinum in the arrangement of the neurotoxin gene cluster. Kubota et al. showed that in C. botulinum strain Iwanai, the orfx2, orfx1, and p47 genes are arranged sequentially in the upstream region of the type E neurotoxin gene cluster (19). The partial orfx2 (676 bp) was found in the distal upstream region in the same orientation as orfx1, whereas p47 was located immediately upstream of ntnh and had the opposite orientation to orfx2 and orfx1. The type E neurotoxin-producing Clostridium butyricum strain BL6340 was further shown to be identical to C. botulinum Iwanai in toxin gene arrangement (19). To date, no more sequences of type E neurotoxin gene clusters are available, and the sequences of the complete orfx2 gene and the further upstream region of orfx2 in type E strains are unknown. Type A2 and F strains have orfx2, orfx1, and p47 genes similar to those of type E but with a regulator gene, botR, located between the orfx1 and p47 genes (15). Recently, a new gene, orfx3, in addition to the complete orfx2 gene, was identified in types A1 and A2 (4).

The nucleotide sequence of bont/E has been published for 36 strains (11, 22, 28, 30). BoNTs are composed of a light chain (50 kDa) and a heavy chain (100 kDa), linked by a disulfide bond. BoNTs produced by different types of C. botulinum share homologies in sequence and structure (20, 23). The light chain is a metalloprotease, while the C- and N-terminal halves of the heavy chain are associated with neurospecific binding and translocation into the nerve cell cytosol, respectively (14). Any variation in amino acid sequence in these functional domains is sufficient to influence the structure, function, and antigenic properties of the BoNT complex (18, 20, 25). Based on amino acid sequences of the neurotoxin, three type E C. botulinum and 13 neurotoxigenic C. butyricum strains were classified into three subtypes (25), and 23 C. botulinum and 13 C. butyricum strains were later classified into five subtypes based on the nucleotide sequences of the bont/E gene (11).

In this study, three C. botulinum type E strains isolated in Finland, Germany, and the United States were chosen for sequencing the neurotoxin gene cluster. Furthermore, bont/E genes were sequenced for 11 strains of mainly Finnish origin. The neurotoxin gene cluster was composed of the bont/E, ntnh, p47, orfx1, orfx2, and orfx3 genes. The complete orfx2 and partial orfx3 genes are reported for the first time in C. botulinum type E. Based on nucleotide sequences, bont/E genes in the currently sequenced and previously published C. botulinum strains and other BoNT/E-producing clostridia were classified into six subtypes. A previously unidentified subtype consisted of three Finnish C. botulinum type E strains.

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Bacterial strains, media, and growth conditions. A total of 14 C. botulinum type E strains were analyzed in this study. Strains K3, K8, K14, K15, K35, K36, K44, K51, K117, and K119 were Finnish fish isolates, while strain S16 was isolated from sea sediment in Finland. Strains K81 and K101 were German fish isolates. Strain 31-2570 of an unknown source was isolated in the United States. Strains K35, K81, and 31-2570 were sequenced for the entire type E neurotoxin gene cluster. The other 11 strains were sequenced for the bont/E gene. All strains, apart from 31-2570, have been isolated in our laboratory and were selected for this study based on the wide geographical and genetic diversity revealed by pulsed-field gel electrophoresis (10), randomly amplified polymorphic DNA (13), and amplified fragment length polymorphism (16) analyses. Egg yolk agar plates were used to subculture these strains and confirm their purity. A single typical colony was picked and inoculated into 10 ml of anaerobic tryptone-peptone-glucose-yeast extract (Difco, Detroit, MI) broth. All cultures were incubated overnight at 30°C under anaerobic conditions.

PCR amplification. Genomic DNA was extracted from C. botulinum cultures as described by Keto-Timonen et al. (16). All primers for amplification and sequencing are listed in Table 1.

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TABLE 1. Primers used for sequencing C. botulinum type E strains

When the genes corresponding to previously published ones were being sequenced, PCR was applied to amplify suitable DNA fragments using the primers targeted to the published genes (9, 19, 30). To consecutively sequence the whole gene cluster, all adjacent fragments were amplified in an overlapping fashion.

For sequencing the region upstream of the known genes, inverse PCR was used to amplify the unknown fragment with primers orfx1E-r1 and orfx2E-f2 in the K35 strain (24). The genomic DNA (27 µg) was cut with restriction enzyme HindIII (New England Biolabs, Inc., Ipswich, MA) at 37°C overnight and then ligated with T4 DNA ligase (New England Biolabs, Inc.) to form circular DNA at a DNA concentration of 3 ng/µl. The circular DNA was used as a template at a concentration of 2.5 ng/µl (26). After the upstream region of the orfx2 gene in K35 was sequenced, the primer orfx2E-r4 was designed and used in combination with orfx2E-f2 to amplify the upstream region of the neurotoxin gene cluster in strains K81 and 31-2570.

In sequencing the previously unidentified upstream region of the gene cluster, primers targeted to the consensus sequence of orfx3 from NCTC 2916 and Kyoto-F (4) were successfully used. This method was considered justified since high similarities were observed in the nucleotide sequences and arrangements of the orfx2, orfx1, and p47 genes between the type E strains sequenced here and the previously published type A strains NCTC 2916 and Kyoto-F (4).

The PCRs were conducted using standard protocols (24). Amplification conditions consisted of 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and polymerization at 72°C for 80 s (for 4 min if the fragment was 2 kb or more), followed by a final extension at 72°C for 3 min (for 10 min if the fragment was 2 kb or more). Titanium Taq DNA polymerase (Clontech Laboratories, Inc., Mountain View, CA) was used in all PCRs. Amplification was performed with a PTC-200 thermal cycler (MJ Research, Watertown, MA).

DNA sequencing. Template DNA was purified from PCR products using a QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany) or a Montage PCR96 cleanup kit (Millipore Corp., Billerica, MA). Sequencing PCR was conducted using an ABI BigDye Terminator 3.1 cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions. After precipitation with ethanol or purification with an Agencourt CleanSEQ kit (Agencourt Bioscience Corp., Beverly, MA), the sequencing samples were loaded onto an ABI 310 genetic analyzer or an ABI 3730 genetic analyzer, respectively. The former method was used to sequence the entire neurotoxin gene clusters, and the latter method was used to sequence the 11 neurotoxin genes for more efficient throughput.

Sequence analysis. The sequences of the entire neurotoxin gene clusters and bont/E were assembled and aligned by Sequencher software (Gene Codes, Ann Arbor, MI). Bidirectional sequence information from two independent PCRs was used, and the sequences were considered final when threefold coverage for each gene was obtained. Sequences were edited by BioEdit software (v. 7.0.5) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), and open reading frames were predicted manually and via ORF Finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Alignment and comparison of two or more sequences were performed using Align (http://www.ebi.ac.uk/emboss/align/index.html) or ClustalW software (3).

Type E neurotoxin genes from 33 C. botulinum type E strains, including the 14 genes sequenced in this study and 19 previously published ones, were compared using ClustalW software (3). The GenBank accession numbers of the previously published sequences are shown in Table 2.

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TABLE 2. Sequences for comparison of BoNT/E gene clusters among C. botulinum and C. butyricum strains

Nucleotide sequence accession numbers. The sequence data reported in this paper have been submitted to the EMBL Nucleotide Sequence Database under accession numbers AM695752 through AM695765 and are listed in Table 2.

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Structure and organization of BoNT/E gene cluster in three type E strains. The neurotoxin gene clusters of C. botulinum K35, K81, and 31-2570 consisted of six genes and were each 12,908 bp long (Table 3). For the first time, the entire nucleotide sequences of orfx2 (2,247 bp) and a partial orfx3 gene (1,355 bp), located immediately upstream of orfx2 and having the same orientation as orfx2, were determined for the type E BoNT gene cluster (Fig. 1). The structures of the neurotoxin gene clusters in the three C. botulinum strains were identical to each other and, apart from the novel elements, to published ones.

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TABLE 3. Open reading frames in the neurotoxin gene cluster of C. botulinum type E strains K35, K81, and 31-2570

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FIG. 1. Schematic representation of the neurotoxin gene cluster in Clostridium botulinum type E, A1, and A2 strains. The gene orientation is shown by arrows. Only orfx3 in the type E gene cluster is a partial gene. Type A1 and A2 neurotoxin gene clusters are derived from C. botulinum A1 strain NCTC 2916 (GenBank accession numbers AY497357, Y14238, and X52066) and Kyoto-F (accession numbers AY497358, AB004778, X96493, X87974, and X73423).

K81 and 31-2570 had identical sequences for p47, orfx1, orfx2, and orfx3. These two strains were also highly similar in their bont/E and ntnh sequences. K35 differed from the other two strains in bont/E and orfx2, with 98% and 99.1% identities, respectively; however, K35 was highly identical to K81 and 31-2570 in the p47, orfx1, and orfx3 gene sequences (Table 4). Marked differences were observed in the GC content of the six genes in the bont/E cluster, with orfx1 showing the lowest and the partial orfx3 gene the highest proportions of GC content (Table 3).

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TABLE 4. Identity of BoNT/E gene clusters and member genes among C. botulinum K35, K81, and 31-2570

Diversity of type E neurotoxin genes. The bont/E genes from a total of 33 strains of BoNT/E-producing clostridia were analyzed (Table 2). These strains included the 14 C. botulinum type E strains of mainly Finnish origin described in this study and 19 C. botulinum and C. butyricum strains with bont/E sequences available in GenBank. The 33 strains could be classified into six subtypes based on the nucleotide sequences of bont/E (Fig. 2). Five Finnish and four other C. botulinum strains of diverse origin formed subtype E1. All of these strains were identical in their bont/E gene sequences, with the exception of C. botulinum Beluga (GenBank accession number X62089), which showed a 1% difference. Subtype E2 included one C. botulinum strain E544 (EF028404) alone, while subtype E3 included six C. botulinum strains from Finland, Germany, and the United States. Except for strain 31-2570, the strains in subtype E3 had 100% identical bont/E genes. All C. butyricum strains formed subtypes E4 and E5, with E4 including ATCC 43755 (X62088), BL5262 (AB088207), and BL6340 (AB039264). Subtype E5 was a uniform group containing 11 C. butyricum strains. A previously unidentified subtype designated E6 included three C. botulinum strains of Finnish origin. Of these, K36 and S16 had 100% sequence identity for bont/E.

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FIG. 2. The bont/E genes from 14 Clostridium botulinum type E strains sequenced here and the 19 previously published ones were compared using ClustalW software. Six distinct neurotoxin subtypes were described and designated E1 to E6. C. botulinum strains fell into subtypes E1, E2, E3, and E6, while subtypes E4 and E5 consisted of C. butyricum strains alone. Three Finnish isolates formed a previously unidentified neurotoxin subtype, E6.

The homology analysis of the nucleotide and amino acid sequences of BoNT/E within and between the six subtypes is shown in Table 5. Strains within a subtype had only 1% differences in their nucleotide and amino acid sequences, while 1 to 3% and 1 to 5% differences in nucleotide and amino acid sequences, respectively, were observed between the four subtypes of C. botulinum. Between the C. botulinum and C. butyricum subtypes, 2 to 3% and 3 to 6% differences in the nucleotide and amino acid sequences, respectively, were observed. Moreover, the two C. butyricum subtypes had corresponding differences of 3% and 5% in their nucleotide and amino acid sequences.

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TABLE 5. Homology of nucleotide and amino acid sequences for BoNT/E genes from representative strains of each subtype

Homology of ntnh and p47. Among C. botulinum type E strains K35, K81, 31-2570, and Mashike (GenBank accession number D12697), the ntnh genes were in general 99% homologous in nucleotide and corresponding amino acid sequences; however, strains K35 and Mashike shared 98% amino acid sequence homology for ntnh. Furthermore, the differences between the four C. botulinum strains and C. butyricum BL6340 were 1 to 2% in nucleotide sequences and 2% in amino acid sequences.

All the BoNT/E-producing clostridia shared a high homology in the nucleotide and amino acid sequences for p47. C. botulinum K35 shared 99% homology in the nucleotide and amino acid sequences for p47 with K81, 31-2570, Iwanai (accession number D88418), and Alaska (U70780, previously named p48), the four strains being identical in p47 sequence. With C. butyricum BL6340 (D88419), all C. botulinum type E strains had 98 to 99% and 98% homologies in nucleotide and amino acid sequences, respectively (Table 6).

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TABLE 6. Homology of nucleotide and amino acid sequences for the p47 gene and the orfx1 gene among types E and A neurotoxin-producing clostridia

The p47 genes in the BoNT/E-producing clostridia were markedly different from those in C. botulinum type A strains NCTC 2916 (AY497357), Kyoto-F (X96493), and Mascarpone (DQ310546). The differences in nucleotide and amino acid sequences were 15 to 21% and 19 to 29%, respectively (Table 6).

Homology of orfx1, orfx2, and orfx3. No difference was found in orfx1 among C. botulinum type E strains K35, K81, 31-2570, and Iwanai (D88418). However, differences of 21 to 22% and 28% in nucleotide and amino acid sequences, respectively, for orfx1 were observed among type E, type A1, and type A2 strains (Table 6).

For orfx2, there were only differences of 1% and 2% in the nucleotide and amino acid sequences, respectively, among the C. botulinum type E strains (Table 7), while again considerable differences were observed among strain types E, A1, and A2. Moreover, for orfx3, high homologies (99 to 100%) of nucleotide and amino acid sequences were present among C. botulinum type E strains, while homologies of only 81% and 75 to 76% were observed among strain types E, A1, and A2 (Table 7).

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TABLE 7. Homology of nucleotide and amino acid sequences for the orfx2 gene and the orfx3 gene among types E and A neurotoxin-producing clostridia

Analysis of amino acid sequence of BoNT/E. A total of 149 variable sites were found in the alignment of the BoNT/E amino acid sequences of seven strains representing the different subtypes (Table 8). The variable sites were distributed mainly in the light chain (37.6%) and the C-terminal half of the heavy chain (43.0%), while less variation was observed in the N-terminal half of the heavy chain (19.4%). No variation was seen in the conserved motifs of the functional domains. Compared with the consensus sequence, the sequences of the C. butyricum strains forming subtype E5 had maximum variability, with all their variable residues being within the heavy chain. By contrast, the C. botulinum K8 sequence had no variation at all. Sequences of strains within the new subtype E6 had the second highest variability distributed along the functional domains of the light chain and the N- and C-terminal halves of the heavy chain. The number and distribution of variable amino acid residues for subtype E6 differed markedly from those of the other subtypes.

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TABLE 8. Numbers of variable amino acid residues in BoNT/E among the representative strains of different subtypes

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To our knowledge, we are the first to report partial orfx3 and complete orfx2 genes in the BoNT type E gene cluster of three C. botulinum strains. Apart from orfx3, the other components of the neurotoxin gene cluster were nearly identical to the published sequences of C. botulinum Iwanai and C. butyricum BL6340 in composition, size, and organization (19).

In addition to BoNT/E-producing clostridia, orfx1, orfx2, orfx3, and p47 have been found in some type A1 and A2 strains (4, 6). These genes show tremendous similarities among type E, A1, and A2 toxin gene clusters in nucleotide sequence, size, and gap distance (Fig. 1). An interesting feature of our type E strains and the A2 strain Mascarpone (6) is the lack of potential insertion elements detected in the intergenic regions of the neurotoxin gene cluster of type A1 strain NCTC 2916 and type A2 strain Kyoto-F (4). The striking similarities, together with evidence of transposable elements, suggest that gene transfer and recombination have occurred among type A and E neurotoxin-producing clostridia, supporting the theory of gene transfer playing a role in the evolutionary history of the BoNT gene cluster (2). It is tempting to speculate that the lack of insertion elements in our type E strains and strain Mascarpone (6) indicates that these orfx1-, orfx2-, and orfx3-carrying strains represent a conserved ancestor in the evolutionary history of the neurotoxin gene cluster. However, the variable GC content among the neurotoxin genes, particularly orfx1, orfx2, and orfx3, undermines this theory and may suggest the emergence of these genes from a more heterogeneous origin.

We did not detect definite promoters for orfx1, orfx2, and orfx3 in the three type E strains. Dineen et al. found that in type A2 strain Kyoto-F, orfx1, orfx2, and orfx3 were transcribed as a polycistronic transcript from a conserved promoter 1,179 bases upstream of the orfx1 start codon and speculated that the corresponding gene products had a coordinated role in the production and expression of the neurotoxin complex (4). Whether the orfx1, orfx2, and orfx3 genes play a role in the production of the biologically active BoNT/E complex or whether these genes have lost their meaning through evolution, both possibilities warrant further investigation.

Six subtypes of the type E neurotoxin gene were found among 33 BoNT/E-producing clostridia, based on nucleotide sequences. Five of the subtypes (E1 through E5) are in agreement with a previous subdivision by Hill et al. based on nucleotide sequences (11). While most Finnish C. botulinum strains fell into two subtypes, E1 and E3, the three Finnish strains K35, K36, and S16 formed a new subtype, designated E6, based on their nucleotide sequences. The E6 strains clearly differed from the other subtypes (by 3 to 6%) in their amino acid sequences of BoNT/E. Although the overall variation in BoNT/E sequences seems to be lower than that of, for instance, BoNT/A, this level of amino acid variability has been shown to affect the antigenic and biological properties of the neurotoxin (17, 27). Since C. botulinum type E strains are frequently found in the Baltic Sea region and in Finnish freshwaters and have been shown to possess a strikingly high biodiversity (10, 13, 16), even more BoNT/E subtypes likely exist among the Finnish C. botulinum population. Hence, further investigations of C. botulinum strains are warranted.

In the alignment of the BoNT/E amino acid sequences determined in this study or published previously, we found that most of the 149 variable sites were distributed mainly along the catalytic and binding domains. Strains representing different subtypes showed distinct distributions for the variable amino acid residues. For example, C. botulinum strain 31-2570 in subtype E3 showed variation only within its catalytic domain. In BoNT/E, this domain residing in the light chain specifically recognizes and cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), and thus blocks neurotransmission in the synaptic vesicle (12, 29). However, the highly conserved zinc-binding motif of HELIHSLH, responsible for protease activity in the catalytic domain (1, 8, 20), was unchanged in the compared BoNT/E sequences.

As opposed to subtype E3, the subtype E5 strain C. butyricum LCL155 had all of its variable amino acids within the heavy chain, most of them in the C-terminal binding domain. The high variability in this domain results in a variety of surface structures that can act as neurospecific epitopes, conferring a unique antigenicity and binding to receptors in nerve cells (14, 18, 20). Despite the large number of variable amino acid residues in the binding domain, the conserved motif YLTHMRD, responsible for receptor binding in BoNT/E (18, 20), was unaltered in the BoNT/E sequences analyzed.

A relatively low level of variation among the BoNT/E amino acid sequences was observed in the translocation domain at the N terminus of the heavy chain. With the exception of one amino acid shift from Glu to Val (position 637) in C. butyricum LCL155 (accession number AB037704), no variation was observed in the hydrophobic domain (positions 624 to 645). The translocation domain is assumed to sense environmental variations and is essential for the channel formation in the endocytosis vesicle membrane for further translocation into the nerve cell cytosol (20). Any changes in the hydrophobic domain will probably affect the channel structure and impair the potential spanning function (18, 30).

As discussed above, the strains of the new subtype E6 were 3 to 6% different from the other subtypes in their BoNT/E amino acid sequences. Although the conserved motifs were not altered, variation was evenly distributed in all three functional domains. Tsukamoto et al. reported the neurotoxin of C. butyricum LCL155 to possess different binding activity, toxicity, and antigenicity than those of C. botulinum 35396 and C. butyricum BL5262 (27), with similar levels of amino acid alterations in the heavy chain alone. Considering that subtype E6 had similar levels of alterations along the entire neurotoxin, it may possess specific antigenic structures and biological properties.

In conclusion, we sequenced the BoNT/E gene cluster in three C. botulinum type E strains and the bont/E genes in 11 strains of mainly Finnish origin. The partial orfx3 and complete orfx2 genes were identified in BoNT/E-producing clostridia for the first time. The structure and organization of the neurotoxin gene cluster in type E strains were identical among strains K35, K81, and 31-2570. Based on the alignment of bont/E gene sequences, a total of six neurotoxin subtypes were found among BoNT/E-producing clostridia. A new subtype, E6, consisting of Finnish strains, was observed.

 
The work was supported by the Academy of Finland (206319).

We thank Hanna Korpunen for technical assistance.

 
* Corresponding author. Mailing address: Department of Food and Environmental Hygiene, P.O. Box 66, FIN-00014, University of Helsinki, Finland. Phone: 358-9-19157107. Fax: 358-9-19157101. E-mail: miia.lindstrom{at}helsinki.fi

Published ahead of print on 28 September 2007.

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Journal of Bacteriology, December 2007, p. 8643-8650, Vol. 189, No. 23
0021-9193/07/$08.00+0     doi:10.1128/JB.00784-07
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