ARRANGEMENT AND NUCLEOTIDE-SEQUENCE OF THE GENE (FUS) ENCODING ELONGATION-FACTOR-G (EF-G) FROM THE HYPERTHERMOPHILIC BACTERIUM AQUIFEX PYROPHILUS - PHYLOGENETIC DEPTH OF HYPERTHERMOPHILIC BACTERIA INFERRED FROM ANALYSIS OF THE EF-G FUS SEQUENCES/

The gene fus (for EF-G) of the hyperthermophilic bacterium Aquifex pyr ophilus was cloned and sequenced. Unlike the other bacteria, which dis play the streptomycin-operon arrangement of EF genes (5'-rps12-rps7-fu s-tuf-3'), the Aquifex fus gene (700 codons) is not preceded by the tw o small ribosomal subunit genes although it is still followed by a tuf gene (for EF-Tu). The opposite strand upstream from the EF-G coding l ocus revealed an open reading frame (ORF) encoding a polypeptide havin g 52.5% identity with an E., coli protein (the pdxJ gene product) invo lved in pyridoxine condensation. The Aquifex EF-G was aligned with ava ilable homologs representative of Deinococci, high G + C Gram positive s, Proteobacteria, cyanobacteria, and several Archaea. Outgroup-rooted phylogenies were constructed from both the amino acid and the DNA seq uences using first and second codon positions in the alignments except sites containing synonymous changes. Both datasets and alternative tr ee-making methods gave a consistent topology, with Aquifex and Thermot oga maritima (a hyperthermophile) as the first and the second deepest offshoots, respectively. However, the robustness of the inferred phylo genies is not impressive. The branching of Aquifex more deeply than Th ermotoga and the branching of Thermotoga more deeply than the other ta xa examined are given at bootstrap values between 65 and 70% in the fi ts-based phylogenies, while the EF-G(2)-based phylogenies do not provi de a statistically significant level of support (less than or equal to 50% bootstrap confirmation) for the emergence of Thermotoga between A quifex and the successive offshoot (Thermus genus). At present, theref ore, the placement of Aquifex at the root of the bacterial tree, albei t reproducible, can be asserted only with reservation, while the emerg ence of Thermotoga between the Aquificales and the Deinococci remains (statistically) indeterminate.