Mol Genet Genomics (2014) 289:1267–1274 DOI 10.1007/s00438-014-0887-4

ORIGINAL PAPER

Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan Koji Hosomi · Yoshihiko Sakaguchi · Tomoko Kohda · Kazuyoshi Gotoh · Daisuke Motooka · Shota Nakamura · Kaoru Umeda · Tetsuya Iida · Shunji Kozaki · Masafumi Mukamoto 

Received: 19 May 2014 / Accepted: 5 July 2014 / Published online: 23 August 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C. botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C. botulinum. Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded Communicated by S. Hohmann. Electronic supplementary material  The online version of this article (doi:10.1007/s00438-014-0887-4) contains supplementary material, which is available to authorized users. K. Hosomi · T. Kohda · S. Kozaki · M. Mukamoto (*)  Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1‑58 Rinku Orai Kita, Izumisano, Osaka 598‑8531, Japan e-mail: [email protected]‑u.ac.jp Y. Sakaguchi  Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan K. Gotoh · D. Motooka · S. Nakamura · T. Iida  Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan K. Umeda  Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences, Osaka, Japan

DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C. botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C. botulinum. Keywords  Infant botulism · Plasmid · Nucleotide sequence · Subtype B2 neurotoxin

Introduction Clostridium botulinum is a Gram-positive, spore-forming, anaerobic, rod-shaped bacterium that produces botulinum neurotoxins (BoNTs) that cause serious paralytic illness known as ‘botulism’ (Arnon et al. 2001). Botulinum neurotoxin genes are known to exist in mobile genetic elements such as plasmids and bacteriophages or in chromosomes (Alouf et al. 1999; Eklund et al. 1971; Peck 2009). Mobile genetic elements containing the bont gene are important for the evolution and pathogenicity of C. botulinum. We attempted to determine complete nucleotide sequence of a plasmid containing the bont gene in C. botulinum. Botulism is characterized by a potentially fatal paralytic condition in humans and various animal species. Botulism has been separated into three forms: food-borne botulism, infant botulism, and wound botulism. Infant botulism mainly affects children up to 6 months old, and its

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symptoms are characterized by constipation, generalized weakness, and various neurological disorders (Fox et al. 2005). BoNT is synthesized as a single chain polypeptide and cleaved by endogenous or exogenous proteases to form a disulfide-linked double-chain molecule composed of a 50-kDa light chain and a 100-kDa heavy chain (Montecucco et al. 1995). BoNT is produced as a progenitor neurotoxin complex of two or more protein components (Raffestin et al. 2004). All toxins contain a nontoxic-nonhemagglutinin protein (NTNH), while some toxins have hemagglutinin (HA) proteins that may facilitate the absorption of BoNT from the intestines into the bloodstream (Matsumura et al. 2008; Fujinaga et al. 2009). BoNTs have been divided into seven serotypes (types A through G) based on their antigenic specificities. In addition, a novel serotype (type H) was recently reported (Barash et al. 2013; Dover et al. 2013). More consideration, however, may be needed to establish the position as serotype H since there is no reports on characteristics of purified neurotoxin. The organisms producing BoNTs have been separated into four genetic and physiological groups: group I (proteolytic C. botulinum) strains produce BoNT/A, /B, /F, or/H; group II (nonproteolytic C. botulinum) strains produce BoNT/B, /E, or/F; group III strains produce BoNT/C or/D; and group IV strains produce BoNT/G. In addition, some strains of Clostridium butyricum and Clostridium baratii also produce BoNT/E and/F, respectively (Hatheway 1990; Sugiyama 1980). Each BoNT can be classified into subtypes according to the diversity of their amino acid sequences. For example, BoNT/A and/B have been divided into at least five subtypes (subtypes A1, A2, A3, A4, and A5) and seven subtypes [subtypes B1, B2, B3, B4 (nonproteolytic), B5 (bivalent), B6, and B7], respectively (Hill et al. 2013). The complete whole genome sequence of group I C. botulinum type A was first reported by Sebaihia (2007). To date, the complete whole genome sequences of several types of C. botulinum strains have been reported, and at least five complete nucleotide sequences of the plasmids containing bont genes have been revealed (Peck 2009). Furthermore, some scaffolds or contigs have been added to the DNA database (http://www.ncbi.nlm.nih.gov/genome/726). The complete whole genome sequences of the group I C. botulinum type B strains Okra and 657 were reported in 2007 (Smith et al. 2007). Genomic analysis revealed that each bont/B gene was located on large plasmids that were ~149 kb (pCLD containing the bont/B1 gene in strain Okra) and 270 kb (pCLJ containing the bont/B5 gene in strain 657). These genomic analyses revealed two distinct arrangements of the neurotoxin complex genes in different types of C. botulinum. The ha cluster

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(ha70-ha17-ha33-botR-ntnh-bont complex genes) was detected in subtype A1 strains and type B strains, while the orfX cluster (orfX3-orfX2-orfX1-[botR]-p47-ntnh-bont complex genes) was found in subtypes A2, A3, and A4, type F, subtypes E1 and E3, and BoNT/E-producing C. butyricum strains (Hill et al. 2009). Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. As described in previous studies, strain 111 exhibited different antigenic and biological features from those of two type B strains, Okra (presumably isolated from food-borne botulism in USA) (Lamanna et al. 1947) and 657 (isolated from infant botulism in USA) (Edmond et al. 1977). Phylogenetic analysis showed that the bont/B genes of strains 111, Okra, and 657 could be classified into the separate groups of subtypes B2, B1, and B5 (bivalent), respectively (Umeda et al. 2009). BoNT/B2 produced by strain 111 exhibited different antigenic properties to those of authentic BoNT/ B1 produced by strain Okra (Ihara et al. 2003; Kohda et al. 2007; Kozaki et al. 1998). Genotyping using pulsed field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) revealed that strain 111 was distinct from strain Okra (Umeda et al. 2013). Group I C. botulinum type B strains were investigated for the stability of toxigenicity and the bont/B gene on serial passages (Umeda et al. 2012). The toxigenicity of strains with the bont/B gene located on these plasmids was lost or decreased during serial passages. Isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In strain Okra, the toxicity of the culture was reduced with an increase in the number of passages, whereas the plasmid containing the bont/B1 gene was stably retained after 30 passages. The stability of these plasmids containing the bont/B gene was different between strains 111 and Okra. The plasmid in strain Okra was highly stable, while that in strain 111 was not. The complete nucleotide sequences of two plasmids containing the bont/B1 and/B5 genes in strains Okra and 657, respectively, have already been determined. In the present study, we determined the complete nucleotide sequence of the plasmid containing the bont/B2 gene in C. botulinum strain 111 in order to provide an insight into the differences in the toxigenic stability and evolution of the bont/B gene in C. botulinum.

Materials and methods Bacterial strains Clostridium botulinum type B strain 111 was isolated from the first case of type B infant botulism in Japan, which

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occurred in Ishikawa Prefecture in 1995 (Kakinuma et al. 1996; Yamakawa et al. 1997). Strain 111 was provided by Prof. S. Nakamura (Kanazawa University School of Medicine, Ishikawa, Japan). Purification of the plasmid The bacterium was grown in TPGY medium (5 % trypticase peptone, 0.5 % Bacto peptone, 2 % yeast extract, 0.4 % glucose, and 0.1 % cysteine-HCl, pH 7.4) at 30 °C. Plasmid DNA was separated from chromosomal DNA in PFGE and extracted from the agarose gel using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). PFGE plugs were prepared as described by Hielm et al. (1998). The plugs were electrophoresed in a CHEF-MAPPER apparatus (Bio-Rad Laboratories, Hercules, USA) through a 1 % SeaKem Gold agarose gel (Lonza, Rockland, USA) in 0.5× Tris–borate-EDTA buffer at 14 °C and 6 V/cm. The switching times were ramped from 0.5 to 40 s. Southern blot analysis was performed as described by Umeda et al. (2009). The DNA probe was prepared using a pair of primers (3Fw, 5′-AAGGCTTCGGGGGTATAAT-3′; 2Rv, 5′-CCCTCAGAATCTTCAACGA-3′). Sequence analysis The nucleotide sequence of the plasmid in strain 111 was determined using the shotgun sequencing method and Illumina MiSeq (Illumina, San Diego, USA). Plasmid DNA was sonicated and treated with BAL31 exonuclease (TaKaRa, Otsu, Japan) and the Mighty Cloning Reagent Set (TaKaRa). DNA fragments of 1–2 kb were purified by agarose gel electrophoresis. Purified fragments were ligated with pTS1 (NIPPON GENE, Tokyo, Japan) digested with Hinc II using the DNA Ligation Kit (TaKaRa). The recombinant DNAs were transformed from Escherichia coli strain JM109 to prepare random shotgun libraries and available transformants were selected on LB agar supplemented with 100 µg/ml ampicillin and 5-bromo-4-chloro3-indolyl-D-galactoside (X-gal). The inserted DNA fragments were amplified by PCR using Ex-taq (TaKaRa) and a pair of primers (pGEM-1f, 5′-TCTTCGCTATTACGCCAGCTGG-3′; pGEM-1r, 5′-ATAACAATTTCACACAGGAAACAGC-3′) with 30 cycles at 94 °C for 30 s, 50 °C for 30 s, 68 °C for 2 min. After the treatment with Exo/ SAP-IT (Affymetrix, Cleveland, USA), each PCR product was used directly as a template for DNA sequencing. The sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, USA), and sequences were determined by the 3730xl DNA Analyzer or 3130 Genetic Analyzer (Applied Biosystems). As a sequencing forward primer, pGME-2f, 5′-GCATGGTACCACGCGTACGTAA-3′ was

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used regularly. The sequences obtained were assembled by SEQUENCHER DNA sequencing software (version 4.10, Gene codes). The sequences of gap regions were determined by the primer walking method using custom primers or by sequencing selected clones using a reverse primer (pGEM-2r, 5′-ACTAAAGGGAGAGAGCTCCTGC-3′) (Nakayama et al. 2000). To ensure confidence and high redundancy in the sequence of the plasmid, the purified plasmid DNA was analyzed using Illumina MiSeq. Annotation and computer analysis Potential protein-encoding regions [open reading frames (ORFs)] were predicted using Microbial Genome Annotation Pipeline (MiGAP, National Institute of Genetics, Mishima, Japan) and each ORF was reviewed manually for the presence of a ribosomal binding sequence. Functional annotation was based on homology searches against the GenBank nonredundant protein sequence database using the program BLASTP implemented at NCBI. A feature map was described using in silico Molecular Cloning (Genomics edition version 3.2, In Silico biology, Yokohama, Japan). Genome matcher (http://www.ige.tohoku. ac.jp/joho/gmProject/gmdownloadJP.html, Ohtsubo et al. 2008) was used to compare plasmid synteny. Construction of phylogenetic trees Amino acid sequences were aligned using the ClustalW program (version 1.83). A phylogenetic tree with 1,000 bootstrap replications was constructed by the neighborjoining method, and genetic distances were calculated by the Kimura two-parameter method (Efron et al. 1996). The resulting tree was drawn with NJplot software. Nucleotide sequence accession number The nucleotide sequence determined in this work was submitted to the DDBJ database and may be found under accession number AB855771.

Results Genetic features of pCB111 The plasmid containing the bont/B2 gene in strain 111 was detected by PFGE and Southern blot analysis (Supplementary Fig. 1). The plasmid (named pCB111) was circular 265,575 bp double-stranded DNA (Table 1; Supplementary Fig. 2). The average G+C content was 25.5 %, which was very similar to that of the large plasmids in the C. botulinum subtype B1 strain Okra (25.4 %),

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Fig. 1  Plasmid organization of pCB111. The horizontal arrows represent ORFs identified for pCB111 and the ORF numbers are indicated above the arrows. ORFs were categorized in nine groups according to their predicted functions

subtype B5 strain 657 (25.6 %), subtype A3 strain Loch Maree (25.6 %), and subtype B4 strain Eklund17B (25.0 %) (Smith et al. 2007). pCB111 contained 332 predicted ORFs, but no genes for tRNA. A total of 26 % (85 gene products) of these ORFs were functionally assigned based on sequence homology to known proteins. According to their predicted functions, ORFs were categorized into nine groups: proteins involved in the BoNT/B2 complex, proteins involved in DNA metabolism (DNA replication, recombination, and repair), proteins involved in conjugative plasmid transfer, RNA polymerase sigma factors, transposases, proteins involved in sporulation, CRISPR-associated proteins, proteins with other functions, and hypothetical proteins. ORFs were designated as CB111_001 to CB111_332 (i.e., successive numbers) (Fig. 1; Supplementary Table 1).

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pCB111 had genes predicted to encode DNA polymerase III subunits (CB111_026, 029, 085) and DNA helicase II (CB111_121, 211) for replication, as well as DNA primase (CB111_001) and single-strand DNA binding protein (CB111_036, 086) for DNA metabolism. Therefore, pCB111 appeared to be capable of independent replication. The gene products of at least 11 ORFs were predicted to be involved in conjugative plasmid transfer (CB111_183, 185, 187, 188, 193, 196, 197, 201, 202, 213, 214). For example, the ATPases (CB111_185,197, 201, 202, 214) involved in coupling or substrate translocation processes and pilus assembly proteins (CB111_187, 188, 193, 213) were predicted. Therefore, pCB111 appeared to be capable of conjugative plasmid transfer. Nine of the ORFs were predicted to encode enzymes such as transposases or recombinases. Five of them

Mol Genet Genomics (2014) 289:1267–1274 Fig. 2  Phylogenetic tree based on mobile element enzyme amino acid sequences. CB111_320 was aligned to highly homologous proteins (transposase, recombinase, or integrase) identified in Genbank by BLAST. CB111_320 is indicated in red, the top ten matches are indicated in blue. The numbers on each branch indicate bootstrap values (>950) for the cluster supported by that branch

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(CB111_060, 089, 113, 114, 143) belonged to the IS200/ IS605 family found in Clostridia (Mahillon et al. 1998). The others (CB111_320, 321, 322, 323) contained conserved domains on site-specific tyrosine recombinase XerC/D, phage integrase, or transposase Tn554. Three (CB111_320, 321, 322) appeared be functional, while one (CB111_323) was truncated. The gene homologues of these ORFs have not yet been identified in Clostridia. Gene homologues were detected in Alkaliphilus metalliredigens strain QYMF and their arrangements were commonly conserved (Fig. 2; Supplementary Fig. 3). A. metalliredigens is a Gram-positive, metal-reducing, endospore-forming, anaerobic, rod-shaped bacterium that was isolated from alkaline pH leachate pond in California, USA (Ye et al. 2004). Comparison among plasmids containing bont genes pCB111 containing the bont/B2 gene was compared with pCLJ containing the bont/B5 and/A4 genes (~270 kb), pCLD containing the bont/B1 gene (~149 kb), and pCLK containing the bont/A3 gene (~267 kb) (Fig. 3a). These four plasmids shared significant sequence homology. pCB111 in particular was similar to pCLJ (71 % identity of the total length) and pCLD (69 % identity of the total length). The homology of regions containing many transposases was lower than that of other regions. The upstream region including the bont/A4 complex genes in pCLJ was not detected in pCB111. The numbers of ORFs in pCB111, pCLJ, and pCLD were 332, 323 and 191, respectively. Of these ORFs, the gene products that were functionally predicted or identified were 85, 116, and 43 ORFs, respectively. One hundred ORFs commonly existed among these three plasmids. Twenty ORFs were present in both pCLJ and pCLD (high

CB111_320 Alkaliphilus metalliredigens (YP_001319431.1) Lachnospiraceae bacterium (WP_009264729.1) Bacillus marmarensis (WP_022629640.1) 1000 Bacillus oceanisediminis (WP_019379926.1) Bacillus alcalophilus (WP_003322313.1) Bacillus sp. (WP_023613797.1) Geobacillus thermodenitrificans (YP_001126169.1) 1000 Geobacillus sp. (YP_003989912.1) C. botulinum B6 str. Osaka05 (GAE02618.1) 996 C. botulinum Ba4 str. 657 (YP_002862889.1) C. botulinum A3 str. Loch Maree (YP_001787247.1) Clostridium difficile (WP_009902775.1) Clostridium butyricum (CAA82325.1) Cyanothece sp. (WP_008277887.1) Staphylococcus aureus (WP_001015611.1) XerC Bacillus cereus (WP_016118542.1) XerC Bacillus subtilis (NP_390232.2) XerD Vibrio cholerae (NP_232049.1) XerD

stability), but were absent in pCB111 (low stability). One gene product of the 20 ORFs was similar to the Deathon-curing family (Doc) protein that comprised a plasmid addiction system. Although the doc gene was located on both pCLD and pCLJ, another agent that comprised the plasmid addiction system was not detected in these two plasmids. Therefore, the mechanism underlying plasmid stability remains unclear. Regions flanking the bont/B2 gene The bont/B2 complex genes (CB111_288, 289, 290, 291, 292, 293) contained a ha cluster (arranged as ha70-ha17ha33-botR-ntnh-bont) that was detected in subtype A1, B1, B4, and B5 strains. Partial insertion sequence elements (IS elements) (belonging to the IS256 family) and a truncated flagellin gene were located upstream and downstream of the bont/B2 complex genes, respectively. Upstream and downstream (~9,400 and 3,500 bp, respectively) of the bont/B2 complex genes shared homology with those of the bont/B1 and bont/B5 complex genes (Fig. 3b).

Discussion Botulinum neurotoxin genes are known to be located on chromosomes, plasmids, or bacteriophages. The bont/A, /B, /E and/F genes were previously shown to be located on chromosomes or plasmids in group I and II C. botulinum strains (Peck 2009). BoNT/C and/D were found to be encoded by bacteriophages in group III C. botulinum (Eklund et al. 1971; Sakaguchi et al. 2005). Type C and D strains cured of their phages produced neither BoNT/C nor BoNT/D, respectively. In group IV C. botulinum, the bont/G gene was found to be located on a plasmid (Alouf

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(a) pCLK (LochMaree, A3) 266,785 bp

bont/A pCLJ (657, Ba4) 270,022 bp

bont/B pCB111 (111, B2) 265,575 bp

bont/B pCLD (Okra, B1) 148,780 bp

(b)

Flagellin’

bont/B2 IS256’

Flagellin’

bont/B5 IS256’

pCB111

pCLJ homologous region in pCLK (A3), pCLJ (B5), pCLD (B1)

homologous region in pCLK (A3), pCLJ (B5), pCLD (B1)

Fig. 3  Plasmid synteny among pCB111, pCLJ, pCLD, and pCLK. a The panel illustrates regions of similarity (indicated in pink) shared among these plasmids. The gray arrows symbolize ORFs and transposases are indicated in green. b The panel expands upstream and

downstream regions including neurotoxin type B (bont/B). Neurotoxin complex genes are indicated in red arrows and the two homologous regions are conserved in all four plasmids (in yellow) (Smith et al. 2007)

et al. 1999). Sequence analysis of pCLD, pCLJ, and pCLK in group I C. botulinum strains (Smith et al. 2007) revealed that these plasmids shared significant sequence homology and appeared to be capable of independent replication, because they encoded replication proteins such as DNA polymerase III subunits and DNA helicase II. Sequence comparisons showed that the bont/B1 and/B5 complex genes in pCLD and pCLJ, respectively, and non-homologous region (including intact flagellin gene) in pCLK were flanked upstream and downstream by homologous regions of ~3,500 and 9,400 bp, respectively. In the present study, pCB111 shared significant sequence homology with pCLD, pCLJ, and pCLK, and exhibited a similar genetic organization (Fig. 3a). Furthermore, the gene sequences of DNA polymerase III and DNA helicase II, involved in replication, were highly conserved among pCB111, pCLD, pCLJ, and pCLK. Therefore, these four plasmids appeared to be capable of independent replication by the same replication mechanism. In addition, the homologous regions flanking

the bont/B1 and/B5 genes and partial flagellin genes were conserved upstream and downstream of the bont/B2 complex genes in pCB111 (Fig. 3b). This result suggested that these plasmids had a common ancestor, although strain 111 exhibited different genotype from strains Okra and 657 in molecular epidemiological analyses. As described previously (Hill et al. 2009), bont genes were not randomly located, but were found in some specific regions of the chromosome or plasmid in C. botulinum. IS elements and transposon-associated proteins that could facilitate the horizontal transfer of bont genes were present in C. botulinum. Some recombination, insertion, and horizontal gene transfer events appeared to have occurred. The site-specific recombinases XerC and XerD have been used to mediate the integration and excision of phage genomes into and out of their host chromosomes (Huber et al. 2002), invert DNA segments, and maintain the monomeric state of circular DNA molecules (Das et al. 2013). In this study, the three gene products of CB111_320, 321 and 322 were

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predicted to be site-specific recombinase XerC/D, phage integrase, or transposase Tn554-like proteins in pCB111. The gene homologues of these ORFs were not detected on the plasmid in other C. botulinum, but were found on the chromosome in A. metalliredigens (Fig. 2; Supplementary Fig. 3). This is the first report to show that three complete site-specific recombinase-like protein-encoding genes were clustered on a plasmid of C. botulinum (Fig. 1). These results have provided a novel insight into the mechanisms responsible for the horizontal transfer of bont genes in C. botulinum. The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms such as a site-specific recombination system, active partition process, and plasmid addiction system (Zielenkiewicz et al. 2001). The site-specific recombination system ensures that plasmid oligomers resolve into monomers (Cornet et al. 1994). The active partition process precisely distributes plasmid copies to each daughter cell at division. The plasmid addiction system selectively kills or reduces the growth of plasmidfree descendant cells. It involves a stable toxin and unstable antidote. The activity of the toxin is prevented by the antidote in plasmid-bearing cells. The toxin is activated by the degradation of the antidote and then kills plasmid-free cells. In a previous study (Umeda et al. 2012), the stability of the plasmids pCLD and pCB111 was shown to vary in strains Okra and 111, respectively. The stability of the plasmid (pCLJ) containing the bont/B5 gene in strain 657 was investigated in the present study based on the study by Umeda et al. (2012) (data not shown). pCLJ was stably retained in strain 657 after 30 passages, which was similar to pCLD. Genes encoding the site-specific recombinases XerC and XerD-like proteins were conserved on chromosomes in strains Okra and 657. PCR amplification confirmed that each gene predicted to encode the enzymes XerC and XerD, which are involved in the site-specific recombination system, was conserved on chromosomes in strain 111 (data not shown). Genes predicted to encode active partition process-associated proteins such as Soj and Spo0J in B. subtilis (Yamaichi et al. 2000) were not detected in pCLD or pCLJ. A gene predicted to encode a Death-on-curing family protein that functions as the toxin in the plasmid addiction system was commonly present in pCLJ and pCLD, but not in pCB111. However, genes encoding the antidote in the plasmid addiction system were absent in pCLD and pCLJ. Known active partition process and plasmid addiction systems were not preserved in pCLD and pCLJ, and the site-specific recombination system could not contribute to maintain plasmids stably in strains 111, Okra, or 657. Further studies based on the nucleotide sequence of pCB111 will reveal the mechanism responsible for the stable maintenance of the plasmid in C. botulinum.

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This study provides the nucleotide sequence and organization of the large plasmid (pCB111) containing the bont/B2 gene in group I C. botulinum type B strain 111. Since pCB111 shared significant sequence homology with pCLD, pCLJ, and pCLK (containing the bont/B1, /B5, and A3 genes, respectively), we think that bont gene transfer has been mediated between C. botulinum by plasmids derived from a common ancestor. This sequence information will be useful in future studies that investigate the unknown mechanisms of plasmid replication, horizontal transfer of the bont gene, and stable maintenance of the plasmid in C. botulinum.

References Alouf JE, Freer JH (1999) The comprehensive sourcebook of bacterial protein toxins. In: Popoff MR, Marvaud JC (eds) Structural and genomic features of clostridial neurotoxins, 2nd edn. Academic Press, San Diego, pp 174–201 Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Hauer J, Layton M, Lillibridge S, Osterholm MT, O’Toole T, Parker G, Perl TM, Russell PK, Swerdlow DL, Tonat K (2001) Botulinum toxin as a biological weapon: medical and public health management. J Am Med Assoc 285(8):1059–1070 Barash JR, Arnon SS (2013) A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins. J Infect Dis 209(2):183–191. doi:10.1093/infdis/jit449 Cornet F, Mortier I, Patte J, Louarn JM (1994) Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif. J Bacteriol 176(11):3188–3195 Das B, Martinez E, Midonet C, Barre FX (2013) Integrative mobile elements exploiting Xer recombination. Trends Microbiol 21(1):23–30. doi:10.1016/j.tim.2012.10.003 Dover N, Barash JR, Hill KK, Xie G, Arnon SS (2013) Molecular characterization of a novel botulinum neurotoxin type h gene. J Infect Dis 209(2):192–202. doi:10.1093/infdis/jit450 Edmond BJ, Guerra FA, Blake J, Hempler S (1977) Case of infant botulism in Texas. Tex Med 73(10):85–88 Efron B, Halloran E, Holmes S (1996) Bootstrap confidence levels for phylogenetic trees. Proc Natl Acad Sci USA 93(23):13429–13434 Eklund MW, Poysky FT, Reed SM, Smith CA (1971) Bacteriophage and the toxigenicity of Clostridium botulinum type C. Science 172(3982):480–482 Fox CK, Keet CA, Strober JB (2005) Recent advances in infant botulism. Pediatr Neurol 32(3):149–154 Fujinaga Y, Matsumura T, Jin Y, Takegahara Y, Sugawara Y (2009) A novel function of botulinum toxin-associated proteins: HA proteins disrupt intestinal epithelial barrier to increase toxin absorption. Toxicon 54(5):583–586. doi:10.1016/j.toxicon.2008.11.014 Hatheway CL (1990) Toxigenic clostridia. Clin Microbiol Rev 3(1):66–98 Hielm S, Bjorkroth J, Hyytia E, Korkeala H (1998) Genomic analysis of Clostridium botulinum group II by pulsed-field gel electrophoresis. Appl Environ Microbiol 64(2):703–708 Hill KK, Smith TJ (2013) Genetic diversity within clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes. Curr Top Microbiol Immunol 364:1–20. doi:10.1007/978-3-642-33570-9_1

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1274 Hill KK, Xie G, Foley BT, Smith TJ, Munk AC, Bruce D, Smith LA, Brettin TS, Detter JC (2009) Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains. BMC Biol 7:66. doi:10.1186/1741-7007-7-66 Huber KE, Waldor MK (2002) Filamentous phage integration requires the host recombinases XerC and XerD. Nature 417(6889):656–659 Ihara H, Kohda T, Morimoto F, Tsukamoto K, Karasawa T, Nakamura S, Mukamoto M, Kozaki S (2003) Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity. Biochim Biophys Acta 1625(1):19–26 Kakinuma H, Maruyama H, Takahashi H, Yamakawa K, Nakamura S (1996) The first case of type B infant botulism in Japan. Acta Paediatr Jpn 38(5):541–543 Kohda T, Ihara H, Seto Y, Tsutsuki H, Mukamoto M, Kozaki S (2007) Differential contribution of the residues in C-terminal half of the heavy chain of botulinum neurotoxin type B to its binding to the ganglioside GT1b and the synaptotagmin 2/GT1b complex. Microb Pathog 42(2–3):72–79 Kozaki S, Kamata Y, Nishiki T, Kakinuma H, Maruyama H, Takahashi H, Karasawa T, Yamakawa K, Nakamura S (1998) Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan. Infect Immun 66(10):4811–4816 Lamanna C, Glassman HN (1947) The isolation of type B Botulinum Toxin. J Bacteriol 54(5):575–584 Mahillon J, Chandler M (1998) Insertion sequences. Microbiol Mol Biol Rev 62(3):725–774 Matsumura T, Jin Y, Kabumoto Y, Takegahara Y, Oguma K, Lencer WI, Fujinaga Y (2008) The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption. Cell Microbiol 10(2):355–364 Montecucco C, Schiavo G (1995) Structure and function of tetanus and botulinum neurotoxins. Q Rev Biophys 28(4):423–472 Nakayama K, Takashima K, Ishihara H, Shinomiya T, Kageyama M, Kanaya S, Ohnishi M, Murata T, Mori H, Hayashi T (2000) The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Mol Microbiol 38(2):213–231 Ohtsubo Y, Ikeda-Ohtsubo W, Nagata Y, Tsuda M (2008) GenomeMatcher: a graphical user interface for DNA sequence comparison. BMC Bioinform 9:376. doi:10.1186/1471-2105-9-376 Peck MW (2009) Biology and genomic analysis of Clostridium botulinum. Adv Microb Physi 55(183–265):320. doi:10.1016/ S0065-2911(09)05503-9 Raffestin S, Marvaud JC, Cerrato R, Dupuy B, Popoff MR (2004) Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani. Anaerobe 10(2):93–100 Sakaguchi Y, Hayashi T, Kurokawa K, Nakayama K, Oshima K, Fujinaga Y, Ohnishi M, Ohtsubo E, Hattori M, Oguma K (2005) The

13

Mol Genet Genomics (2014) 289:1267–1274 genome sequence of Clostridium botulinum type C neurotoxinconverting phage and the molecular mechanisms of unstable lysogeny. Proc Natl Acad Sci USA 102(48):17472–17477 Sebaihia M, Peck MW, Minton NP, Thomson NR, Holden MT, Mitchell WJ, Carter AT, Bentley SD, Mason DR, Crossman L, Paul CJ, Ivens A, Wells-Bennik MH, Davis IJ, Cerdeno-Tarraga AM, Churcher C, Quail MA, Chillingworth T, Feltwell T, Fraser A, Goodhead I, Hance Z, Jagels K, Larke N, Maddison M, Moule S, Mungall K, Norbertczak H, Rabbinowitsch E, Sanders M, Simmonds M, White B, Whithead S, Parkhill J (2007) Genome sequence of a proteolytic (group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. Genome Res 17(7):1082–1092 Smith TJ, Hill KK, Foley BT, Detter JC, Munk AC, Bruce DC, Doggett NA, Smith LA, Marks JD, Xie G, Brettin TS (2007) Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and/B1 clusters are located within plasmids. PLoS One 2(12):e1271 Sugiyama H (1980) Clostridium botulinum neurotoxin. Microbiol Rev 44(3):419–448 Umeda K, Seto Y, Kohda T, Mukamoto M, Kozaki S (2009) Genetic characterization of Clostridium botulinum associated with type B infant botulism in Japan. J Clin Microbiol 47(9):2720–2728. doi: 10.1128/JCM.00077-09 Umeda K, Seto Y, Kohda T, Mukamoto M, Kozaki S (2012) Stability of toxigenicity in proteolytic Clostridium botulinum type B upon serial passage. Microbiol Immunol 56(5):338–341. doi:10.1111/j.1348-0421.2012.00441.x Umeda K, Wada T, Kohda T, Kozaki S (2013) Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: comparison with other isolates and genotyping methods. Infect Genet Evol 16:298–304. doi:10.1016/j.meegid.2013.02.022 Yamaichi Y, Niki H (2000) Active segregation by the Bacillus subtilis partitioning system in Escherichia coli. Proc Natl Acad Sci USA 97(26):14656–14661 Yamakawa K, Karasawa T, Kakinuma H, Maruyama H, Takahashi H, Nakamura S (1997) Emergence of Clostridium botulinum type B-like nontoxigenic organisms in a patient with type B infant botulism. J Clin Microbiol 35(8):2163–2164 Ye Q, Roh Y, Carroll SL, Blair B, Zhou J, Zhang CL, Fields MW (2004) Alkaline anaerobic respiration: isolation and characterization of a novel alkaliphilic and metal-reducing bacterium. Appl Environ Microbiol 70(9):5595–5602. doi:10.1128/ AEM.70.9.5595-5602.2004 Zielenkiewicz U, Ceglowski P (2001) Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems. Acta Biochim Pol 48(4):1003–1023

Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan.

Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a...
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