Taxonomy and phylogeny of industrial solvent-producing clostridia

We performed a systematic study of 55 solvent-producing clostridial strains, the majority of which are currently classified as Clostridium acetobutylicum strains, by using a combination of biotyping and DNA fingerprint analysis. The biotyping procedures used included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. The 55 strains examined exhibited a good correlation between their biotypes and DNA fingerprints, which allowed us to divide them into nine groups. The DNA fingerprints of the nine groups differed markedly, but within each group the DNA fingerprints exhibited a high level of similarity. To determine the phylogenetic relationships of the nine groups, we performed a 16S rRNA gene sequence analysis. The results of a comparative analysis of the partial sequence corresponding to positions 830 to 1383 (Escherichia coli numbering) of the 16S rRNA gene indicated that the nine biotype groups could be assembled into four taxonomic groups. The complete 16S rRNA sequences of strains representing these groups were determined. Our phylogenetic analysis revealed that the amylolytic type strain C. acetobutylicum ATCC 824 (taxonomic group I) was only distantly related to the saccharolytic strains belonging to taxonomic groups II, III, and IV (levels of sequence similarity, 90 to 90.5%). The strains belonging to taxonomic groups II, III, and IV, represented by C. acetobutylicum NCP 262, "Clostridium saccharoperbutylacetonicum" N1-4, and C. acetobutylicum NCIMB 8052T (T = type strain), respectively, were closely related (levels of sequence similarity, 98.2 to 98.9%). C. acetobutylicum NCIMB 8052T exhibited a level of similarity of 100% with the type strain of Clostridium beijerinckii. Reclassification of the saccharolytic solvent-producing strains is necessary, and possible names for the four taxonomic groups are discussed.