Dataset Information

A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum.

ABSTRACT: Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental isolate of C. botulinum. Bioinformatic analysis revealed that the genetic structure of the yin element resembles previously reported mobile intervening elements associated with sporulation genes. Within a pure C. botulinum culture, we observed two subpopulations of cells with the yin element either integrated into the yabG locus or excised as a circular DNA molecule. The dynamics between the two observed conformations of the yin element was growth-phase dependent and likely mediated by recombination events. The yin element was not required for sporulation by C. botulinum but triggered an earlier entry into sporulation than in a related isolate lacking this element. So far, the yin element has not been found in any other C. botulinum strains or other endospore-forming species. It remains to be demonstrated what kind of competitive edge it provides for C. botulinum survival and persistence.

SUBMITTER: Douillard FP

PROVIDER: S-EPMC10747680 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum.

Douillard François P FP Portinha Inês Martins IM Derman Yağmur Y Woudstra Cédric C Mäklin Tommi T Dorner Martin B MB Dorner Martin B MB Korkeala Hannu H Henriques Adriano O AO Lindström Miia M

Viruses 20231214 12

Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental ...[more]

PMID: 38140671

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Project description:The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt "bookmark" sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and "bookmark"-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique "watermark" through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

Project description:BackgroundClostridium botulinum is a taxonomic designation for at least four diverse species that are defined by the expression of one (monovalent) or two (bivalent) of seven different C. botulinum neurotoxins (BoNTs, A-G). The four species have been classified as C. botulinum Groups I-IV. The presence of bont genes in strains representing the different Groups is probably the result of horizontal transfer of the toxin operons between the species.ResultsChromosome and plasmid sequences of several C. botulinum strains representing A, B, E and F serotypes and a C. butyricum type E strain were compared to examine their genomic organization, or synteny, and the location of the botulinum toxin complex genes. These comparisons identified synteny among proteolytic (Group I) strains or nonproteolytic (Group II) strains but not between the two Groups. The bont complex genes within the strains examined were not randomly located but found within three regions of the chromosome or in two specific sites within plasmids. A comparison of sequences from a Bf strain revealed homology to the plasmid pCLJ with similar locations for the bont/bv b genes but with the bont/a4 gene replaced by the bont/f gene. An analysis of the toxin cluster genes showed that many recombination events have occurred, including several events within the ntnh gene. One such recombination event resulted in the integration of the bont/a1 gene into the serotype toxin B ha cluster, resulting in a successful lineage commonly associated with food borne botulism outbreaks. In C. botulinum type E and C. butyricum type E strains the location of the bont/e gene cluster appears to be the result of insertion events that split a rarA, recombination-associated gene, independently at the same location in both species.ConclusionThe analysis of the genomic sequences representing different strains reveals the presence of insertion sequence (IS) elements and other transposon-associated proteins such as recombinases that could facilitate the horizontal transfer of the bonts; these events, in addition to recombination among the toxin complex genes, have led to the lineages observed today within the neurotoxin-producing clostridia.

Project description:BackgroundClostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. While studies have characterised variability between strains of Group I (proteolytic) C. botulinum, the genetic and physiological variability and relationships between strains within Group II (non-proteolytic) C. botulinum are not well understood. In this study the genome of Group II strain C. botulinum Eklund 17B (NRP) was sequenced and used to construct a whole genome DNA microarray. This was used in a comparative genomic indexing study to compare the relatedness of 43 strains of Group II C. botulinum (14 type B, 24 type E and 5 type F). These results were compared with characteristics determined from physiological tests.ResultsWhole genome indexing showed that strains of Group II C. botulinum isolated from a wide variety of environments over more than 75 years clustered together indicating the genetic background of Group II C. botulinum is stable. Further analysis showed that strains forming type B or type F toxin are closely related with only toxin cluster genes targets being unique to either type. Strains producing type E toxin formed a separate subset. Carbohydrate fermentation tests supported the observation that type B and F strains form a separate subset to type E strains. All the type F strains and most of type B strains produced acid from amylopectin, amylose and glycogen whereas type E strains did not. However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth. No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains.ConclusionsAlthough Group II C. botulinum form a tight genetic group, genomic and physiological analysis indicates there are two distinct subsets within this group. All type B strains and type F strains are in one subset and all type E strains in the other.

Dataset Information

A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum.

Publications

A Novel Prophage-like Insertion Element within <i>yabG</i> Triggers Early Entry into Sporulation in <i>Clostridium botulinum</i>.

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