RNA polymerase of Aquifex pyrophilus: Implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria

A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, c ontaining the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA -directed RNA polymerase subunits beta and beta' (rpoBC), alanyl-tRNA synth etase (alaS), and subunit A of proteinase Clp (clpA). Enzymatic activity an d extreme thermostability of purified A. pyrophilus RNA polymerase were ver ified. Transcription initiation on a DNA construct harboring the T7 A1 prom oter was demonstrated by elongation of a P-32-labeled trinucleotide. Phylog enetic analyses of the two largest subunits of bacterial RNA polymerases (b eta and beta') showed overall consistency with the 16S rRNA-based phylogeny , except for the positions of the hyperthermophiles A. pyrophilus and Therm otoga maritima and for the location of the root of the domain Bacteria. In the phylogenies for both RNA polymerase subunits beta and beta', A. pyrophi lus was placed within the Gram-negative bacteria below the epsilon subdivis ion of the Proteobacteria. No support was found for the 165 rRNA-based hypo thesis that A. pyrophilus might be the deepest branch of the Bacteria, but the cell wall-less mycoplasmas were found with a high confidence at the roo t of the Bacteria phylogenies. This raised doubts not only about whether th e original Bacteria were indeed like the hyperthermophiles, but also concer ning the value of single-gene phylogenies for hypotheses about the evolutio n of organisms.