Comparative genomic analysis and proposal of Clostridium yunnanense sp. nov., Clostridium rhizosphaerae sp. nov., and Clostridium paridis sp. nov., three novel Clostridium sensu stricto endophytes with diverse capabilities of acetic acid and ethanol production.

Objectives: Genus Clostridium sensu stricto is generally regarded as the true Clostridium genus, which includes important human and animal pathogens and industrially relevant microorganisms. Besides, it is also a prominent member of plant-associated endophytes. However, our knowledge of endophytic Clostridium is limited.

Methods: In this study, the endophytes were isolated under anaerobic condition from the roots of Paris polyphylla Smith var. yunnanensis. Subsequently, a polyphasic taxonomic approach was used to clarify their taxonomic positions. The fermentation products were measured in the isolates with HPLC analysis. Comparative genomics was performed on these new strains and other relatives.

Results: In total, nine endophytic strains belonging to the genus Clostridium sensu stricto were isolated, and three of them were identified as new species. Seven of nine strains could produce acetate, propionate, and butyrate. Only two strains could produce ethanol, although genomics analysis suggested that only two of them were without genes for solventogenesis. Different from the endophytic strains, the phylogenetically closely related non-endophytic strains showed significant enrichment effects on some metabolic pathways involving environmental information processing, carbohydrate, and amino acid metabolisms, etc. It suggests that the genomes of these endophytic strains had undergone subtle changes associated with environmental adaptations.

Conclusion: Consequently, strains YIM B02505, YIM B02515, and YIM B02565 are proposed to represent a new species of the genus Clostridium sensu stricto, for which the names Clostridium yunnanense sp. nov., Clostridium rhizosphaerae sp. nov., and Clostridium paridis sp. nov. are suggested.