Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense

Abstract

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.

Endogenous CRISPR-Cas Systems in Group I Clostridium botulinum and Clostridium sporogenes Do Not Directly Target the Botulinum Neurotoxin Gene Cluster

“…DFPST0006. These chromosomal plasmid integrations have recently been independently reported ( Smith et al, 2021a ). In strains where both type I-B and III-B or III-D CRISPR-Cas systems were present, the gene clusters encoding the two CRISPR-Cas systems were frequently adjacent to each other within the chromosome ( Figure 2D ).…”

“…Analysis of the flanking region showed the bont gene clusters predominantly occupy sites near type I-B CRISPR-Cas systems at site 1, which encompasses brnQ and arsC bont integration sites. Two bont gene clusters occurred within fully or partially chromosomally integrated plasmid sequences near the CRISPR-Cas systems integration regions ( Supplementary Figure 2 ; Dover et al, 2014 ; Hill et al, 2015 ; Smith et al, 2021a ). The only cas gene clusters observed outside of the chromosome were cas1/2/4(−) I-B ** cas gene clusters exclusive to a family of bont (−) plasmids and nuclease(−) cas1/2/4/3 type I-B* cas gene clusters exclusive to bont (+) conjugative plasmids.…”

“…transposons that lack a cas3 nuclease gene and possess cas6, cas7, and a cas5/8 fusion genes and are utilized to achieve CRISPR-mediated site-specific transposition within the genome (Peters et al, 2017;Klompe et al, 2019;Mcdonald et al, 2019). The integrated bont(+) plasmids (Dover et al, 2014;Smith et al, 2021a) observed at integration sites 1 and 4 are intriguing within that context and suggest that much remains to be learned about these still cryptic plasmids (Figure 3B). Direct functional investigation and characterization of this unique, bont(+) plasmid exclusive, type I-B system will provide additional insight into its function and potential relationship to the bont gene cluster.…”

Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China

“…According to the classic view, bont genes confer an advantage to their clostridia hosts because BoNT production ultimately provides a suitable anaerobic substrate for the spread of clostridia [ 1 ]. Recently, a rare chromosomal integration of bont -encoding plasmids has been observed in different clostridia species and has been suggested to facilitate the stable retention of the bont gene in clostridia hosts [ 34 ].…”

Genomic Diversity, Competition, and Toxin Production by Group I and II Clostridium botulinum Strains Used in Food Challenge Studies

“…Whole genome sequencing of an increasing number of C. botulinum strains is changing the landscape of the C. botulinum and BoNT research field. The sequences for over 1000 strains of C. botulinum are currently available, and bioinformatics analyses are revealing previously uncovered insights into aspects of strain relatedness, lateral gene transfer, immune defenses, evolution, and physiology of the strains [ 13 , 14 , 39 , 51 , 59 , 61 , 65 ]. These data are of particular interest for food safety studies, which for decades have relied on assays utilizing select strains to represent the entire species.…”