Molecular cloning of the dnaK gene region from Bacillus sphaericus in the context of genomic comparisons

The dnaK gene region of Bacillus sphaericus was cloned as a 3.8 kb HindIII fragment and an overlapping 1.7 kb EcoRI fragment by using an internal B. sphaericus specific dnaK gene probe generated by polymerase chain reaction (PCR). Complete DNA sequencing of the two fragments revealed three complete open reading frames (ORFs). These ORFs exhibited a high degree of identity to the grpE dnaK, and dnaJ heat shock genes from other gram-positive bacteria. The order of the genes was found to be grpE-dnaK-dnaJ. Additionally, the 5'-end and 3'-end contained amino acid sequences that were homologous to the C-terminal sequence of the hrcA gene and the N-terminal sequence of ORF35 (yqeT), respectively, from Bacillus subtilis. The entire hrcA gene from B. sphaericus was then isolated by high-fidelity PCR and completely sequenced. A transcription stop site is located between the dnaK and dnaJ genes but not after the dnaJ gene. Consistent with this observation, the dnaJ gene is immediately followed by an ORF that shows a high degree of identity to ORF35 from B. subtilis, Staphylococcus aureus, and Clostridium acetobutylicum. The presence of ORF35 is not indicated in other genera representing the gram-positive bacteria. The amino acid sequence of ORF35 exhibited nearly 30% identity with the methyltransferase for large subunit ribosomal protein L11 from gram-negative Proteobacteria and the related protein from cyanobacteria, other gram-negative bacteria, and Archaea, suggesting the presence of the gene for this protein in the common ancestor of Bacteria and Archaea. The absence of the ORF35 gene in Mycobacterium tuberculosis and other gram-positive bacteria indicates that the loss of this gene must have occurred in an ancestor of other gram-positive bacteria following their divergence from the ancestor of Bacillus/Clostridium/staphylococcus lineage.