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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 .

Botulinum neurotoxin-producing clostridia are diverse in the types of toxins they produce as well as in their overall genomic composition. They are globally distributed, with prevalent species and toxin types found within distinct geographic regions, but related strains containing the same toxin types may also be located on distinct continents. The mechanisms behind the spread of these bacteria and the independent movements of their bont genes may be understood through examination of their genetic backgrounds. The generation of 15 complete genomic sequences from bacteria isolated in Argentina, Australia, and Africa allows for a thorough examination of genome features, including overall relationships, bont gene cluster locations and arrangements, and plasmid comparisons, in bacteria isolated from various areas in the southern hemisphere. Insights gained from these examinations provide an understanding of the mechanisms behind the independent movements of these elements among distinct species., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2020. This work is written by US Government employees and is in the public domain in the US.)

Botulinum neurotoxins (BoNTs) are produced by a diverse set of seven clostridial species, though alternate naming systems have developed over the last 100 years. Starting in the 1950s, a single-species taxonomy where any bacterium producing BoNT would be designated Clostridium botulinum was introduced. As the extreme diversity of these strains was recognized, a secondary system of taxonomic "groups" evolved. It became clear that these groups also had members that did not produce BoNT, and in some cases, they were given formal species names. Genomic analysis now clearly identifies species affiliations whether an isolate is toxigenic or not. It is clear that C. botulinum group nomenclature is no longer appropriate and that there are recognized species names for each clostridium. We advocate for the use of the scientific binomials and that the single-species group nomenclature be abandoned., (Copyright © 2018 Smith et al.)

Background: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin., Results: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants., Conclusions: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.

The whole genomes for six botulinum neurotoxin-producing clostridial strains were sequenced to provide references for under-represented toxin types, bivalent strains or unusual toxin complexes associated with a bont gene. The strains include three Clostridium botulinum Group I strains (CDC 297, CDC 1436, and Prevot 594), a Group II C. botulinum strain (Eklund 202F), a Group IV Clostridium argentinense strain (CDC 2741), and a Group V Clostridium baratii strain (Sullivan). Comparisons of the Group I genomic sequences revealed close relationships and conservation of toxin gene locations with previously published Group I C. botulinum genomes. The bont/F6 gene of strain Eklund 202F was determined to be a chimeric toxin gene composed of bont/F1 and bont/F2. The serotype G strain CDC 2741 remained unfinished in 20 contigs with the bont/G located within a 1.15Mb contig, indicating a possible chromosomal location for this toxin gene. Within the genome of C. baratii Sullivan strain, direct repeats of IS1182 insertion sequence (IS) elements were identified flanking the bont/F7 toxin complex that may be the mechanism of bont insertion into C. baratii. Highlights of the six strains are described and release of their genomic sequences will allow further study of unusual neurotoxin-producing clostridial strains., (Published by Elsevier B.V.)

Botulinum neurotoxins are a varied group of protein toxins that share similar structures and modes of activity. They include at least seven serotypes and over forty subtypes that are produced by seven different clostridial species. These bacterial species are not limited strictly to BoNT-producers as neuro-toxigenic and non-neuro-toxigenic members have been identified within each species. The nomenclature surrounding these toxins and associated bacteria has been evolving as new isolations and discoveries have arisen, resulting in challenges in diagnostic reporting, epidemiology and food safety studies, and in the application of therapeutic products. An understanding of the intricacies regarding the nomenclature of BoNTs and BoNT-producing clostridia is crucial for communication that allows for accurate reporting of information that is pertinent to each situation. [ABSTRACT FROM AUTHOR]

Of the seven currently known botulinum neurotoxin-producing species of Clostridium , C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes. [ABSTRACT FROM AUTHOR]

Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism’s biological potential, the proteins expressed provide a functional readout of the organism’s response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples. [ABSTRACT FROM AUTHOR]

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters. [ABSTRACT FROM AUTHOR]