Phylogenetic reconstruction of Gram-positive organisms based on comparative sequence analysis of molecular chaperones...

Primers designed on the basis of nucleotide sequences conserved in DnaK and GroEL from Gram-positive organisms were used to PCR amplify internal regions of the cognate genes from the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1. Genome walking was then utilized to elucidate the remainder of the sequences in addition to upstream and downstream regions. The full sequence of the gene encoding the GroES protein (groES) was found directly upstream from groEL. The deduced amino acid sequence of the groEL gene showed the highest homology with the amino acid sequence of the Clostridium thermocellum GroEL protein (72% amino acid identity). Similarly, translation of the groES nucleotide sequence showed highest homology to the C. thermocellum GroES protein (61% amino acid identity). Analysis of the upstream region of this chaperonin operon revealed a CIRCE regulatory element 45 bp upstream from the putative start of the groES ORF. The deduced amino acid sequence of the putative dnaK gene showed the highest homology with the amino acid sequence of the Clostridium acetobutylicum DnaK protein (68% amino acid identity). Phylogenetic analyses based on the translated sequences reiterate this relationship between R. flavefaciens and the Clostridia. However, when the nucleotide sequences of Gram-positive organisms are analyzed, a different topology occurs of the relationship between high- and low-G+C Gram-positive organisms to the 16S rRNA interpretation.