Identification of the Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase regulatory gene (touR) and of its cognate promoter

Toluene-o-xylene monooxygenase is an enzymatic complex, encoded by the touA BCDEF genes, responsible for the early stages of toluene and o-xylene degra dation in Pseudomonas stuizeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysi s and complementation studies were carried out with Pseudomonas putida PaW3 40 as a heterologous host harboring pFB1112, a plasmid that allowed regulat ed expression, inducible by toluene and o-xylene and their corresponding ph enols, of the toluene-o-xylene monooxygenase. A locus encoding a positive r egulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic com pound catabolic pathways belonging to the NtrC family and, in particular, t o DmpR (83% similarity), which controls phenol catabolism. By using a touA- C2,3O fusion reporter system and by primer extension analysis, a TouR cogna te promoter (P-ToMO) was mapped, which showed the typical -24 TGGC, -12 TTG C sequences characteristic of sigma(54)-dependent promoters and putative up stream activating sequences. By using the reporter system described, we fou nd that TouR responds to mono- and dimethylphenols, but not the correspondi ng methylbenzenes. In this respect, the regulation of the P, stutzeri syste m differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effecters. Northern analyses indica ted low transcription levels of tou structural genes in the absence of indu cers. Basal toluene-o-xylene monooxygenase activity may thus transform thes e compounds to phenols, which then trigger the TouR-mediated response.