Unravelling the genetic diversity of ruminal bacteria belonging to the CFBphylum

Molecular biology approaches ware employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle. By this means we were able to identify cultured strains t hat represent some of the larger CFB clusters previously identified only by PCR amplification and sequencing. Complete 16S rDNA sequences were obtaine d for 16 previously isolated rumen strains, including the type strains of P revotella ruminicola, P. bryantii, P. brevis and P. albensis to represent a wide range of diversity. Phylogenetic analysis of cultured strains reveale d the existence of three clusters of ruminal CFB: (i) a cluster of Prevotel la strains, which have been found only in the rumen, including the two type strains, P. brevis GA33(T) and P. ruminicola 23(T), (ii) Prevotella spp. t hat cluster with prevotellas from other ecological niches such as the oral cavity and which include the type strains, P. bryantii B(1)4(T) and P. albe nsis M384(T); (iii) two Bacteroides sop. strains clustering with B. forsyth us of oral origin. In order to establish whether the cultivated isolates co ver the whole range of ruminal CFB genetic diversity, 16S rRNA gene sequenc es were amplified and cloned from DNA extracted from the same rumen samples (one cow in Slovenia, one in Scotland and three in Japan). Sequencing and phylogenetic analysis of 16S rRNA genes confirmed the existence of two supe rclusters of ruminal Prevotella, one exclusively ruminal and the other incl uding non-ruminal species. In the case of ruminal Bacteroides spp., however , phylogenetic analysis revealed the existence of three new superclusters, one of which has as yet no cultivable counterpart. Interestingly, these Bac teroides clusters were represented almost exclusively by clone libraries fr om the Japanese cattle and only three sequences were from the European catt le. This study agrees with previous analyses in showing that rumen Prevotel la/Bacteroides strains exhibit a remarkable degree of genetic diversity and suggests that different strain groupings may differ greatly in their recov ery by cultural methods. The most important conclusion, however, is that cu ltured strains can be identified that represent some of the larger clusters previously identified only by PCR amplification and sequencing. (C) 2000 F ederation of European Microbiological Societies. Published by Elsevier Scie nce B.V. All rights reserved.