Evidence for the evolution of a single component phenol/cresol hydroxylase from a multicomponent toluene monooxygenase.

We have previously reported on the organization of a unique toluene-3-monooxygenase pathway for the degradation of alkyl-substituted petroleum hydrocarbons including characteristics of the second step in the pathway transforming phenols to catechols. In the present work we have focused on the regulation and unusual genetic organization of this metabolic step. In particular, we have sequenced the 3-kb DNA interval between the region encoding the tbuD gene product (phenol/cresol hydroxylase) and part of the toluene-3-monooxygenase operon of strain PKO1. Then, various regions of this DNA were fused to a LacZ expression system to ascertain the location of the tbuD gene promoter and the binding site for its regulator, TbuT. The 5' end for transcripts for the putative promoter of the tbuD gene was also analyzed using primer extension analysis. Collectively, these results revealed that the promoter was located 2.5-kb upstream of the region encoding the tbuD gene product whose N-terminal region had been previously determined by peptide sequencing. Remarkably, the intervening 2.5-kb region showed sequence identity to results we reported previously for a multi-subunit toluene-2-monooxygenase cloned from a different bacterium, strain JS150, for which phenols are also substrates and effectors. When the DNA sequence for the tbuD gene and its contiguous 2.5-kb upstream region were compared to the entire toluene-2-monooxygenase sequence cloned from strain JS150, a promoter proximal region encoding three reading frames showed 99% identity to subunits for the toluene-2-monooxygenase operon. Within the contiguous tbuD gene region, however, DNA sequence homology was reduced to 64% overall identity and deduced amino acid sequence homology was only 21% similar. Although regions internal to the tbuD gene showed homology to corresponding toluene-2-monooxygenase subunits, domains associated with the putative functions proposed for such subunits were deleted. We believe that these results suggest that through evolution either tbuD was derived from the 2-monooxygenase pathway by deletions and molecular rearrangements, or alternatively the tbuD gene recruited part of the 2-monooxygenase pathway and its regulatory system which is activated by benzene, alkyl-substituted benzenes and phenols.