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

Bacteria from the species <i>Clostridium butyricum</i> 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 <i>C. butyricum</i> is the type species of <i>Clostridium</i>, 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 <i>C. butyricum</i>. Furthermore, we show that pathogenic <i>C. butyricum</i> 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.