Comparative genomics of Clostridium butyricum reveals a conserved genome architecture and novel virulence-related gene clusters

Bacteria from the species Clostridium butyricum encompass a diverse range of phenotypes. While some strains are used as probiotics, others have been isolated from cases of botulism and necrotizing enterocolitis (NEC) in preterm neonates. We identify a unique genomic feature of this species, namely a highly conserved extrachromosomal element of ~0.8 Mb. This replicon satisfies the three principal criteria used to define a chromid, which include the possession of core genes that are encoded on the main chromosome in other species. Although C. butyricum is the type species of Clostridium, we find that the possession of a chromid is not a typical feature of members of this genus and represents a unique genomic fingerprint of the species C. butyricum. Furthermore, we show that pathogenic C. butyricum strains from the sequenced examples are not monophyletic, which suggests that virulence has evolved multiple times from related non-pathogenic ancestors. However, we were able to identify common genes which are found exclusively in these pathogenic strains. In addition to the botulinum neurotoxin genes, these include a novel set of genes involved in the biosynthesis of a capsular polysaccharide (CPS), and genes that confer the ability to utilize the mucin-derived sugar l-fucose, which may provide a competitive advantage for growth in the colon. Moreover, by identifying NEC strain-associated virulence factors, we are able to further the understanding of these particularly harmful strains.