{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T23:56:59Z","timestamp":1781567819303,"version":"3.54.5"},"reference-count":52,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2021,7,8]],"date-time":"2021-07-08T00:00:00Z","timestamp":1625702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT\/B1 strain CDC 1632, C. argentinense BoNT\/G strain CDC 2741, and Clostridium parabotulinum BoNT\/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont\/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT\/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont\/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.<\/jats:p>","DOI":"10.3390\/toxins13070473","type":"journal-article","created":{"date-parts":[[2021,7,8]],"date-time":"2021-07-08T10:42:17Z","timestamp":1625740937000},"page":"473","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense"],"prefix":"10.3390","volume":"13","author":[{"given":"Theresa J.","family":"Smith","sequence":"first","affiliation":[{"name":"Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ 86011, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Renmao","family":"Tian","sequence":"additional","affiliation":[{"name":"Institute for Food Safety and Health, Illinois Institute of Technology, Bedford Park, IL 60501, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8768-0453","authenticated-orcid":false,"given":"Behzad","family":"Imanian","sequence":"additional","affiliation":[{"name":"Institute for Food Safety and Health, Illinois Institute of Technology, Bedford Park, IL 60501, USA"},{"name":"Food Science and Nutrition, Illinois Institute of Technology, Chicago, IL 60616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charles H. D.","family":"Williamson","sequence":"additional","affiliation":[{"name":"Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ 86011, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3972-9208","authenticated-orcid":false,"given":"Shannon L.","family":"Johnson","sequence":"additional","affiliation":[{"name":"Los Alamos National Laboratory, Los Alamos, NM 87545, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hajnalka E.","family":"Daligault","sequence":"additional","affiliation":[{"name":"Los Alamos National Laboratory, Los Alamos, NM 87545, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9222-3911","authenticated-orcid":false,"given":"Kristin M.","family":"Schill","sequence":"additional","affiliation":[{"name":"Center for Food Safety and Applied Nutrition, Food and Drug Administration, Bedford Park, IL 60501, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Peck, M.W., Smith, T.J., Anniballi, F., Austin, J.W., Bano, L., Bradshaw, M., Cuervo, P., Cheng, L.W., Derman, Y., and Dorner, B.G. (2017). Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature. Toxins, 9.","DOI":"10.3390\/toxins9010038"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Smith, T.J., Williamson, C.H., Hill, K., Sahl, J.W., and Keim, P. (2018). Botulinum neurotoxin -producing bacteria\u2014Isn\u2019t it time we called a species a species?. MBio, 9.","DOI":"10.1128\/mBio.01469-18"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fmicb.2021.566908","article-title":"The distinctive evolution of orfX Clostridium parabotulinum strains and their botulinum neurotoxin type A and F gene clusters is influenced by environmental factors and gene interactions via mobile genetic elements","volume":"12","author":"Smith","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1128\/am.27.1.251-258.1974","article-title":"Interconversion of type C and D strains of Clostridium botulinum by specific bacteriophages","volume":"27","author":"Eklund","year":"1974","journal-title":"Appl. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1128\/iai.13.3.855-860.1976","article-title":"Antigenicity of converting phages obtained from Clostridium botulinum types C and D","volume":"13","author":"Oguma","year":"1976","journal-title":"Infect. Immun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2087","DOI":"10.1128\/iai.63.5.2087-2091.1995","article-title":"The genes for the Clostridium botulinum type G toxin complex are on a plasmid","volume":"63","author":"Zhou","year":"1995","journal-title":"Infect. Immun."},{"key":"ref_7","unstructured":"Montecucco, C. (1995). Ecology of neurotoxigenic strains of clostridia. Clostridial Neurotoxins, The Molecular Pathogenesis of Tetanus and Botulism, Springer."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.bbrc.2007.06.166","article-title":"Plasmid encoded neurotoxin genes in Clostridium botulinum serotype A subtypes","volume":"361","author":"Marshall","year":"2007","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Smith, T.J., Hill, K.K., Foley, B.T., Detter, J.C., Munk, A.C., Bruce, D.C., Doggett, N.A., Smith, L.A., Marks, J.D., and Xie, G. (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.","DOI":"10.1371\/journal.pone.0001271"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Franciosa, G., Maugliani, A., Scalfaro, C., and Aureli, P. (2009). Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0004829"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3856","DOI":"10.1128\/AEM.00080-13","article-title":"Plasmid-borne type E neurotoxin gene clusters in Clostridium botulinum strains","volume":"79","author":"Zhang","year":"2013","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1093\/gbe\/evw017","article-title":"Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts","volume":"8","author":"Carter","year":"2016","journal-title":"Genome Biol. Evol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Hill, K.K., Xie, G., Foley, B.T., Smith, T.J., Munk, A.C., Bruce, D., Smith, L.A., Brettin, T.S., and Detter, J.C. (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.","DOI":"10.1186\/1741-7007-7-66"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Dover, N., Barash, J.R., Hill, K.K., Davenport, K.W., Teshima, H., Xie, G., and Arnon, S.S. (2013). Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: Bont\/A2, bont\/F4 and bont\/F5. PLoS ONE, 8.","DOI":"10.1371\/annotation\/b482f80f-c5b6-4b9c-8e9b-8b7139dc37f1"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3250","DOI":"10.1128\/AEM.00284-14","article-title":"Clostridium botulinum strains producing BoNT\/F4 or BoNT\/F5","volume":"80","author":"Raphael","year":"2014","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.meegid.2014.12.002","article-title":"Genomic sequences of six botulinum neurotoxin-producing strains representing three clostridial species illustrate the mobility and diversity of botulinum neurotoxin genes","volume":"30","author":"Smith","year":"2015","journal-title":"Infect. Genet. Evol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2076","DOI":"10.1093\/gbe\/evu164","article-title":"Three classes of plasmid (47-63 kb) carry the type B neurotoxin gene cluster of group II Clostridium botulinum","volume":"6","author":"Carter","year":"2014","journal-title":"Genome Biol. Evol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Skarin, H., Hafstrom, T., Westerberg, J., and Segerman, B. (2011). Clostridium botulinum group III: A group with dual identity shaped by plasmids, phages and mobile elements. BMC Genom., 12.","DOI":"10.1186\/1471-2164-12-185"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2163","DOI":"10.1128\/jcm.35.8.2163-2164.1997","article-title":"Emergence of Clostridium botulinum type B-like nontoxigenic organisms in a patient with type B infant botulism","volume":"35","author":"Yamakawa","year":"1997","journal-title":"J. Clin. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5420","DOI":"10.1128\/AEM.01159-15","article-title":"Implications of genome-based discrimination between Clostridium botulinum Group I and Clostridium sporogenes strains for bacterial taxonomy","volume":"81","author":"Weigand","year":"2015","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1016\/j.bbrc.2011.01.093","article-title":"Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A","volume":"405","author":"Pellett","year":"2011","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1032","DOI":"10.1093\/gbe\/evt068","article-title":"The type F6 neurotoxin gene cluster locus of group II Clostridium botulinum has evolved by successive disruption of two different ancestral precursors","volume":"5","author":"Carter","year":"2013","journal-title":"Genome Biol. Evol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Williamson, C.H., Sahl, J.W., Smith, T.J., Xie, G., Foley, B.T., Smith, L.A., Fernandez, R.A., Lindstrom, M., Korkeala, H., and Keim, P. (2016). Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia. BMC Genom., 17.","DOI":"10.1186\/s12864-016-2502-z"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Smith, T.J., Xie, G., Williamson, C.H.D., Hill, K.K., Sahl, J.W., Keim, P., and Johnson, S.L. (2020). Genomic characterization of newly completed genomes of botulinum neurotoxin-producing species from Argentina, Australia and Africa. Genome Biol. Evol.","DOI":"10.1093\/gbe\/evaa043"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Brunt, J., van Vliet, A.H.M., Carter, A.T., Stringer, S.C., Amar, C., Grant, K.A., Godbole, G., and Peck, M.W. (2020). Diversity of the Genomes and Neurotoxins of Strains of Clostridium botulinum Group I and Clostridium sporogenes Associated with Foodborne, Infant and Wound Botulism. Toxins, 12.","DOI":"10.3390\/toxins12090586"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Halpin, J.L., Hill, K., Johnson, S.L., Bruce, D.C., Shirey, T.B., Dykes, J.K., and Luquez, C. (2017). Finished Whole-Genome Sequence of Clostridium argentinense Producing Botulinum Neurotoxin Type G. Genome Announc., 5.","DOI":"10.1128\/genomeA.00380-17"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"a003616","DOI":"10.1101\/cshperspect.a003616","article-title":"Group II introns: Mobile ribozymes that invade DNA","volume":"3","author":"Lambowitz","year":"2011","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"247","DOI":"10.4161\/bioe.21004","article-title":"ClosTron-mediated engineering of Clostridium","volume":"3","author":"Kuehne","year":"2012","journal-title":"Bioengineered"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/S0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"W16","DOI":"10.1093\/nar\/gkw387","article-title":"PHASTER: A better, faster version of the PHAST phage search tool","volume":"44","author":"Arndt","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"W347","DOI":"10.1093\/nar\/gkr485","article-title":"PHAST: A fast phage search tool","volume":"39","author":"Zhou","year":"2011","journal-title":"Nucleic Acids Res."},{"key":"ref_32","first-page":"221","article-title":"Another type of Clostridium botulinum","volume":"215","author":"Gimenez","year":"1970","journal-title":"Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. I"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1093\/infdis\/143.1.22","article-title":"Isolation of Clostridium botulinum type G and identification of type G botulinal toxin in humans: Report of five sudden unexpected deaths","volume":"143","author":"Sonnabend","year":"1981","journal-title":"J. Infect. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1099\/00207713-38-4-375","article-title":"Clostridium argentinense sp. nov.: A genetically homogeneous group composed of all strains of Clostridium botulinum toxin type G and some nontoxigenic strains previously identified as Clostridium subterminale or Clostridium hastiforme","volume":"38","author":"Suen","year":"1988","journal-title":"Int. J. Syst. Bacteriol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1128\/aem.54.6.1405-1408.1988","article-title":"Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G","volume":"54","author":"Eklund","year":"1988","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1128\/MMBR.00031-06","article-title":"Insertion sequence diversity in archaea","volume":"71","author":"Filee","year":"2007","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_37","unstructured":"Bowmer, E.J. (1962). Antitoxins of Clostridium botulinum Types A, B, C, D, and E; Preparation and Assay of Proposed International Standards, University of Liverpool."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Guedon, G., Libante, V., Coluzzi, C., Payot, S., and Leblond-Bourget, N. (2017). The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements that Pirate Bacterial Conjugative Systems. Genes, 8.","DOI":"10.3390\/genes8110337"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1517\/14622416.5.4.433","article-title":"Solexa Ltd","volume":"5","author":"Bennett","year":"2004","journal-title":"Pharmacogenomics"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1126\/science.1162986","article-title":"Real-time DNA sequencing from single polymerase molecules","volume":"323","author":"Eid","year":"2009","journal-title":"Science"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1101\/gr.074492.107","article-title":"Velvet: Algorithms for de novo short read assembly using de Bruijn graphs","volume":"18","author":"Zerbino","year":"2008","journal-title":"Genome Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1038\/nmeth.2474","article-title":"Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data","volume":"10","author":"Chin","year":"2013","journal-title":"Nat. Methods"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1101\/gr.8.3.186","article-title":"Base-calling of automated sequencer traces using phred. II. Error probabilities","volume":"8","author":"Ewing","year":"1998","journal-title":"Genome Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1101\/gr.8.3.175","article-title":"Base-calling of automated sequencer traces using phred. I. Accuracy assessment","volume":"8","author":"Ewing","year":"1998","journal-title":"Genome Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2936","DOI":"10.1093\/bioinformatics\/btt515","article-title":"Consed: A graphical editor for next-generation sequencing","volume":"29","author":"Gordon","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1093\/bioinformatics\/btp324","article-title":"Fast and accurate short read alignment with Burrows-Wheeler transform","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1122","DOI":"10.1093\/bioinformatics\/btq090","article-title":"An Ergatis-based prokaryotic genome annotation web server","volume":"26","author":"Hemmerich","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1128\/AEM.02616-15","article-title":"Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing","volume":"82","author":"Schill","year":"2016","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: A flexible trimmer for Illumina sequence data","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing","volume":"19","author":"Bankevich","year":"2012","journal-title":"J. Comput. Biol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast gapped-read alignment with Bowtie 2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat. Methods"}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/7\/473\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:27:56Z","timestamp":1760164076000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/7\/473"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,8]]},"references-count":52,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,7]]}},"alternative-id":["toxins13070473"],"URL":"https:\/\/doi.org\/10.3390\/toxins13070473","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,8]]}}}