The use of groEL and 16S rRNA gene sequences for classification of Ruminococcus albus and Ruminococcus flavefaciens bacterial species and for identification of unknown bacteria.

摘要

The use of 16S ribosomal RNA as a molecular chronometer has been used to increase our understanding of the phylogeny of microorganisms. This method includes hybridization and sequence analysis of conserved and variable regions of the 16S rRNA. Although the 16S rRNA has been an excellent marker for determining phylogenetic inference, more than one chronometer is essential for the proper identification and classification of microorganisms. This study involves the use of the groEL DNA sequence to determine the phylogeny of Ruminococcus species including Ruminococcus flavefaciens FD-1, JF1, LB4, JM1, B146, C94 and Ruminococcus albus strains 7, 8, B199, R013, and KF1. The groEL protein, a chaperonin, is a member of the heat shock protein family. Like the 16S rRNA, groEL is ubiquitously expressed and exhibits a highly conserved function of mediating protein folding in all organisms. Segments of the groEL gene and the 16S ribosomal DNA were amplified by polymerase chain reaction and cloned into pGEM-T easy vector. The plasmid was extracted from the clones containing the appropriate size groEL and 16S PCR product and the insert was sequenced. Comparisons of these data with other groEL sequences in the National Center for Biotechnology Information database were performed using Basic Local Alignment Search Tool. The sequences were compared to demonstrate the relatedness among phylogenetic loci. Sequence alignments of Ruminococcus flavefaciens FD-1, JF1, LB4, JM1, B146, C94 and Ruminococcus albus strains 7, 8, B199, R013, and KF1 using CLUSTALW showed a high percentage of homology between strains. Intraspecies similarities of the 16S sequences were higher than those of groEL sequences by as much as 20%. Two non-ruminococci bacteria, Butyrivibiro fibrosolvens D1 and Prevotella ruminocola GA33 used as 16S out-groups and showed no similarity to the Ruminococci groups. Phylogenetic trees were constructed using PAUP 4.0 and TreeView PPC. Output trees were the same from each program and there was a strong similarity between the trees generated using the groEL and the 16S rDNA sequences. A combination of groEL and 16S rDNA sequences proved that the two sets of sequences are useful in generating a robust tree. The use of both groEL and 16S sequences served as proficient markers for the identification of unknown cellulolytic bacteria.