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

Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chro mosomally encoded toluene-3-monooxygenase pathway, Expression of the t oluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regula tor, TbuT, in the presence of toluene. We have identified the TbuT cod ing region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is s imilar to XylR and DmpR, two members of the NtrC family of transcripti onal activators which control toluene-xylene and (methyl)phenol catabo lism, respectively, it is significantly different in the domain associ ated with effector specificity, Using a tbuA1-lacZ fusion reporter sys tem, we determined that TbuT is activated not only by aromatic effecte rs but also the chlorinated aliphatic hydrocarbon trichloroethylene, E xpression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxy genase promoter. As a result, transcription of tbuT is low when the to luene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene, Thus, the toluene-3-monooxygenase p romoter drives the cascade expression of both the toluene-3-monooxygen ase operon and tbuT, resulting in a positive feedback circuit, Examina tion of the nucleotide sequence upstream of the toluene-3-monooxygenas e operon for promoter-like sequences revealed a -24 TGGC, -12 TTGC seq uence, characteristic of sigma(54) (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-monooxy genase promoter. Upstream of PtbuA1, a DNA region with dyad symmetry e xhibited homology with the XylR-binding site present upstream of the P re promoter, Deletions within this DNA sequence resulted in complete l oss of expression from PtbuA1, suggesting that this region may serve a s the TbuT-binding site.