Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts

GBE Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts Andrew T. Carter1,*, John W. Austin2, Kelly A. Weedmark3, and Michael W. Peck1 1 Gut Health and Food Safety, Institute of Food Research, Norwich Research Park, Norwich, United Kingdom 2 Bureau of Microbial Hazards, Health Products and Food Branch, Health Canada, Ottawa, ON, Canada 3 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada *Corresponding author: E-mail: . Accepted: January 28, 2016 Abstract Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxinencoding genes were located on plasmids. Seven closely related (134–144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together. Key words: botulism, genome evolution, horizontal gene transfer, CRISPR. Introduction The conventional taxonomic name of Clostridium botulinum masks the fact that it is a physiologically diverse group of anaerobic, spore-forming bacteria defined by the ability to produce the deadly botulinum neurotoxin (BoNT), which when ingested by humans or animals can cause the neuroparalytic disease, botulism. Four groups of C. botulinum are recognized: Groups I and II primarily cause human botulism, Group III is responsible for botulism in birds and animals, and Group IV has yet to be associated with botulism, although it has been associated with sudden unexpected deaths (Sonnabend 1981; Hatheway 1988; Johnson 2007; Peck 2009). Seven different serotypes of BoNT have been described (A to G). Members of the mesophilic (minimum growth temperature 12  C) C. botulinum Group I possess one to three neurotoxin genes. In some bivalent strains one of these genes may be silent, and in others one gene may be expressed at a much lower rate than the other neurotoxin gene; however up to three different neurotoxins may be formed by the same strain (Dover et al. 2013; Kalb et al. 2014). Clostridium botulinum Group I members produce toxin types A, B, and F. Clostridium botulinum Group II is ß The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 540 Genome Biol. Evol. 8(3):540–555. doi:10.1093/gbe/evw017 Advance Access publication February 14, 2016 Data deposition: Plasmid sequences for this study have been deposited at GenBank under the accession KT897275 (plasmid p12/29), KT897276 (plasmid pINGR16-02E1), KT897277 (plasmid pST0210E1), KT897278 (plasmid pFWSKR40E1), KT897279 (plasmid pSWKR38E2), KT897280 (plasmid pFI1111E1), and KT901798 (plasmid pGA0702E1CS). Read data used in this study are listed under NCBI BioProject PRJNA295286 and include 35 newly released data sets. Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters of type E neurotoxin are known, formed by strains that have been isolated from a wide geographical area (Hill and Smith 2013; Hill et al. 2015; Mazuet et al. 2015). Until recently all type E neurotoxin gene clusters were thought to be chromosomally located at the rarA gene locus, including those of subtypes E4 and E5, present in neurotoxigenic strains of Clostridium butyricum (Macdonald et al. 2011; Smith, Hill, Raphael, et al. 2015). However, as a result of using pulsed field gel electrophoresis (PFGE) to separate intact chromosomes from plasmid DNA species, followed by Southern analysis, it was discovered that three Nordic type E1 strains bore their neurotoxin gene on a plasmid of approximately 146 kb (Zhang et al. 2013). One of these strains, CB11/1-1, was subjected to genome sequencing and the data deposited in the GenBank DNA sequence database, but no plasmid-specific sequences were identified. In a recent study of Canadian type E strains derived from food, clinical, and environmental sources, data for 175 genomes were obtained by next-generation sequencing (Weedmark et al. 2014, 2015). We present here the results of screening these data for the presence of type E neurotoxigenic plasmids, as well as three newly sequenced Canadian isolates. Together with a type E1 strain from a Hungarian soil sample, and by further analysis of the publically available data for CB11/1-1, we show for the first time that genes encoding subtypes E1, E3, and E10 may all be carried by a family of closely related 133- to 144-kb neurotoxigenic plasmids. Furthermore, annotation of these plasmids has provided evidence for the route by which the more commonly encountered chromosomal type E neurotoxin gene cluster has been derived. Materials and Methods Culture Conditions, DNA Isolation, and Genome Sequencing These have been described previously for the Canadian C. botulinum type E strains (Weedmark et al. 2014, 2015). Clostridium botulinum type E1 strain IFR 12/29 was isolated at the Institute of Food Research (IFR), Norwich, UK, from a soil sample collected at Kiskunmajsa, Bács-Kiskun county, Hungary, using isolation and culture techniques previously described (Peck et al. 2010). Genomic DNA from a 10-ml TYG (tryptone, yeast extract, glucose) broth culture was prepared using standard Gram positive lysis techniques followed by phenol/chloroform purification (Carter et al. 2014). Genome sequencing was performed using the Illumin (...truncated)