Cascade regulation of the toluene-3-monooxygenase operon (tbuA1UBVA2C) of Burkholderia pickettii PKO1: role of the tbuA1 promoter (PtbuA1) in the expression of its cognate activator, TbuT

Abstract

Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chromosomally encoded toluene-3-monooxygenase pathway. Expression of the toluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regulator, TbuT, in the presence of toluene. We have identified the TbuT coding region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is similar to XylR and DmpR, two members of the NtrC family of transcriptional activators which control toluene-xylene and (methyl)phenol catabolism, respectively, it is significantly different in the domain associated with effector specificity. Using a tbuA1-lacZ fusion reporter system, we determined that TbuT is activated not only by aromatic effectors but also the chlorinated aliphatic hydrocarbon trichloroethylene. Expression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxygenase promoter. As a result, transcription of tbuT is low when the toluene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene. Thus, the toluene-3-monooxygenase promoter drives the cascade expression of both the toluene-3-monooxygenase operon and tbuT, resulting in a positive feedback circuit. Examination of the nucleotide sequence upstream of the toluene-3-monooxygenase operon for promoter-like sequences revealed a -24 TGGC, -12 TTGC sequence, characteristic of sigma54 (rpoN)-dependent promoters. Primer extension and tbuA1-lacZ fusion analyses demonstrated that this -24, -12 promoter sequence, referred to as PtbuA1, was the toluene-3-monooxygenase promoter. Upstream of PtbuA1, a DNA region with dyad symmetry exhibited homology with the XylR-binding site present upstream of the Pu promoter. Deletions within this DNA sequence resulted in complete loss of expression from PtbuA1, suggesting that this region may serve as the TbuT-binding site.