Abstract
We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question.
Highlights
The Clostridium butyricum species is one of the six phylogenetic clostridia Groups whose members may possess the ability to produce the botulinum neurotoxin (BoNT), i.e. the powerful protein toxin causing the neuroparalytic disease of botulism in man and animals
Considering that fragment length estimates by pulsed-field gel electrophoresis (PFGE) are not precise [13], the, 150 kb size difference observed between the dp positive NruI restriction bands (,400 kb and, 250 kb, respectively) from the two clusters of C. butyricum type E strains was similar to the estimated, 215 kb size difference between the larger (, 825 kb) and the smaller (, 610 kb) megaplasmids [6]. These results suggested that most of the,150 kb genetic region missing from the genomic DNAs of C. butyricum type E strains ISS-20, ISS-21, ISS-109, and ISS-145/1 was from the megaplasmid
A striking feature of the genetic region common to the megaplasmids is that it includes a number of transcriptional regulators and two-component regulatory systems, the latter recently involved in the regulation of expression of the bont/A gene [23]
Summary
The Clostridium butyricum species is one of the six phylogenetic clostridia Groups whose members may possess the ability to produce the botulinum neurotoxin (BoNT), i.e. the powerful protein toxin causing the neuroparalytic disease of botulism in man and animals. While most C. butyricum strains are nonneurotoxigenic, rare botulinum neurotoxigenic C. butyricum strains have been isolated that produce BoNT type E (BoNT/E), one of the seven (A to G) known BoNT serotypes [1]. The heterogeneity of C. botulinum, along with the first isolations of botulinum neurotoxigenic C. butyricum type E and C. barati type F strains [2,3], and the recognition of a new BoNT/Gproducing species (namely, C. argentinense) [4], led to the present classification of the BoNT-producing clostridia in six phylogenetic Groups. Each Group of highly related clostridia includes strains that are non-neurotoxigenic. According to this classification, botulinum neurotoxigenic C. butyricum type E and nonneurotoxigenic C. butyricum strains constitute clostridia Group VI [5]
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