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

Abstract

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.

Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

“…The other peptides that would be expressed from pRO1966, TbuB, TbuU, and TbuV, would not be expected to be detectable in this analysis since the molecular masses of these components are 12.3, 9.6, and 11.7 kDa, respectively. These results are consistent with the toluene-3-monooxygenase peptide expression patterns that we have previously determined from related work on regulation of the tbu pathway (5,24,40,41), and they support the model that deletion of tbuX impaired the ability of toluene to enter the cell, which in turn negatively affected the expression of the toluene-3-monooxygenase structural genes as a result of the lack of effector binding to the transcriptional activator, TbuT. Therefore, Pseudomonas cells harboring pHYK1000 could not utilize toluene even though the toluene-3-monooxygenase structural genes and the transcriptional regulatory gene were present.…”

“…Comparisons of the nucleotide sequences of both the putative tbuX promoter and the putative TbuT-binding site upstream of tbuX with similar sequences found within the Pu promoter of the upper TOL operon from P. putida mt-2 (47), the Po promoter of the dmp operon from Pseudomonas sp. strain CF600 (57), the Ptbm promoter upstream of the toluene and benzene monooxygenase operon from Burkholderia cepacia JS150 (20), the PtbuD promoter upstream of the phenol/cresol hydroxylase structural gene (40), and the PtbuA1 promoter upstream of the toluene-3-monooxygenase structural genes (6) are shown in Fig. 3B.…”

“…Total RNA was extracted from 2 ml of the cultures using Trizol reagent (Gibco BRL) according to the manufacturer's recommended protocol, except that after the cells were mixed with the Trizol reagent, the suspension was incubated at 68°C for 10 min and then allowed to cool to room temperature. The remainder of the RNA extraction protocol was carried out as previously described (6,27,40). The concentration and purity of the RNA were estimated from the A 260 /A 280 ratio and from visual inspection of RNA bands following electrophoretic analysis.…”

“…Gels were stained with Coomassie brilliant blue, destained in methanol- In vivo analysis of promoter activity. Promoter activity, assessed by in vivo transcomplementation, was determined by assaying ␤-galactosidase activity in cells of P. aeruginosa PAO1c essentially as described previously (6,40). P. aeruginosa PAO1c cells harboring pKRZ1 and its derivatives together with pRO1614 and its derivatives were grown overnight in LB broth containing kanamycin (600 g/ml) and tetracycline (50g/ml) to maintain plasmids.…”

“…The desired tbuX deletion construct was designated pHYK1000 (Table 1). Other DNA restriction digests, ligations, and transformation procedures were performed according to previously published methods (38,40,49).…”