Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes

Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage/corn silage/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In experiment 1, 31 cloned rDNA sequences were analysed. In experiment 2, DNA extractions were amplified using either 12 or 30 cycles of PCR in order to examine biases introduced during the reactions. A set of 53 sequences were analysed in experiment 2 from DNA amplified using 12 cycles and 49 sequences from PCR using 30 cycles. Sequences from the 5' end of 16S rRNA gene were compared with existing sequences in the Ribosomal Database Project. Clones from experiment 1 produced a data set in which 55% of the sequences were similar to low G+C Gram-positive bacteria related to the genus Clostridia, the majority of which were closely related to bacteria in Cluster XIV. Approximately 30% of the cloned sequences were related to bacteria in the Prevotella-Bacteroides group. Clones from experiment 2 produced a data set in which the majority of sequences were related to the Prevotella-Bacteroides group, regardless of the number of cycles of PCR. The remaining sequences clustered with members of the genus Clostridia. The majority of rDNA sequences analysed in this study represent novel rumen bacteria which have not yet been isolated.