Regolazione trascrizionale della toluene/o-xilene monossigenasi (ToMO) di pseudomonas stutzeri OX1: studi in vivo e in vitro

Dottorato di ricerca in scienze genetiche. 12. ciclo. A.a. 1996-99. Supervisore Enrica Galli Fossati e Paola BarbieriConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

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 touABCDEF genes, responsible for the early stages of toluene and o-xylene degradation in Pseudomonas stutzeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysis and complementation studies were carried out with Pseudomonas putida PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and o-xylene and their corresponding phenols, of the toluene–o-xylene monooxygenase. A locus encoding a positive regulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using a touA-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (P(ToMO)) was mapped, which showed the typical −24 TGGC, −12 TTGC sequences characteristic of ς(54)-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of the P. stutzeri system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels of tou structural genes in the absence of inducers. Basal toluene–o-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response

TouR-Mediated Effector-Independent Growth Phase-Dependent Activation of the σ(54) Ptou Promoter of Pseudomonas stutzeri OX1

Transcription of the catabolic touABCDEF operon, encoding the toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1, is driven by the σ(54)-dependent Ptou promoter, whose activity is controlled by the phenol-responsive NtrC-like activator TouR. In this paper we describe for the first time a peculiar characteristic of this system, namely, that Ptou transcription is activated in a growth phase-dependent manner in the absence of genuine effectors of the cognate TouR regulator. This phenomenon, which we named gratuitous activation, was observed in the native strain P. stutzeri OX1, as well as in a Pseudomonas putida PaW340 host harboring the reconstructed tou regulatory circuit. Regulator-promoter swapping experiments demonstrated that the presence of TouR is necessary and sufficient for imposing gratuitous activation on the Ptou promoter, as well as on other σ(54)-dependent catabolic promoters, whereas the highly similar phenol-responsive activator DmpR is unable to activate the Ptou promoter in the absence of effectors. We show that this phenomenon is specifically triggered by carbon source exhaustion but not by nitrogen starvation. An updated model of the tou regulatory circuit is presented

New insights into the activation of o-xylene biodegradation in Pseudomonas stutzeri OX1 by pathway substrates

The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o-xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins. The set-up included the prokaryotic enhancer-binding protein TouR, the σ(54)-dependent P(ToMO) promoter and the σ(54)-containing RNA polymerase. With this system we prove that direct binding of 2-methylphenol (o-cresol) to TouR is the only regulatory step for activation of P(ToMO) in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing. In addition, we found that while TouR failed entirely to activate P(ToMO) in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o-cresol addition. The results presented here support the view that TouR-like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation