Structure and Genetic Content of the Megaplasmids of Neurotoxigenic Clostridium butyricum Type E Strains from Italy

Franciosa G (2013) Structure and Genetic Content of the Megaplasmids of Neurotoxigenic Clostridium butyricum Type E Strains from Italy. PLoS ONE 8(8): e71324. doi:10.1371/journal.pone.0071324 Structure and Genetic Content of the Megaplasmids of Neurotoxigenic Clostridium butyricum Type E Strains from Italy Angelo Iacobino 0 Concetta Scalfaro 0 Giovanna Franciosa 0 Holger Bruggemann, Aarhus University, Denmark 0 Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanita` , Rome , Italy 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. - 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. In fact, 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]. Conventionally, the BoNT production ability has been ascribed to the C. botulinum species, which consists of highly heterogenic clostridia. 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 also 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]. We recently showed that each of ten neurotoxigenic C. butyricum type E strains, of which six were from Italy and the remaining four were from China, harbors a differently-sized megaplasmid whose structure was proposed to be linear [6]. A -lactamase gene was detected in the megaplasmid of eight strains associated to human botulism, whereas it was absent from the megaplasmid of the two remaining strains of environmental origin, suggesting that the lactamase encoding megaplasmids may confer a benefit to their microbial hosts in a clinical environment. However, the ubiquitous presence of the megaplasmids in all analyzed neurotoxigenic C. butyricum type E strains, including the two environmental strains that lack the -lactamase gene, would suggest that the megaplasmids have additional functions in their microbial hosts, besides conferring antibiotic resistance. In this study, we focused on the megaplasmids of the six neurotoxigenic C. butyricum type E strains isolated thus far in Italy (Table 1) [2,69]. Following macrorestriction with XhoI and SmaI enzymes and pulsed-field gel electrophoresis (PFGE) analysis, the six strains (ISS-20, ISS-21, ISS-86, ISS-109, ISS-145/1, ISS-190) had previously been grouped in two distinct but closely related PFGE clusters, consistent with the two sizes of the megaplasmid in the strains: in the first cluster (strains ISS-20, ISS-21, ISS-109, ISS145/1) the megaplasmid was about 610 kb, and in the second cluster (strains ISS-86 and ISS-190), the megaplasmid was about 825 kb [6]. The fact that the two PFGE clusters of strains shared a high (.90%) level of similarity [6] suggested a clonal origin of the C. butyricum type E strains and led us to suppose that most of the PFGE pattern diversity between the strains might arise from their differently-sized megaplasmids. Therefore, we sought to more thoroughly analyze and compare the megaplasmids of the six C. butyricum type E strains, to identify any conserved and/or recently acquired genetic regions that may play crucial roles in the biology of these peculiar microorganisms. Results and Discussion Genetic Maps of the Differently-sized Megaplasmids Most attempts to isolate the megaplasmid bands from Seakem or low-melting point PFGE agarose gels for subsequent restriction enzyme mapping were unsuccessful, possibly because of the low DNA yields and/or because of the unusual conformation of the large DNA molecules determined by the PFGE run conditions [10]. Therefore, we subjected the total genomic DNA of the C. butyricum type E strains to restriction analysis and PFGE to select the enzyme(s) yielding the smallest number of clear differences between the PFGE profiles of the two clonal groups of strains. Unexpectedly, BamHI did not digest genomic DNA from any of the C. butyricum type E strains, possibly indicating some methylation at the BamHI restriction sites (59 GGATCC 39). Cytosine methylation at the internal GATC sites was confirmed by the fact that the DNAs were resistant to digestion with Sau3AI, which is unable to cut GATC sequences with methylated cytosine, whereas they were digested by MboI, which cuts the GATC sites regardless of cytosine methylation (data not shown) [11]. Among the other rest (...truncated)