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

Abstract

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 sigma(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.

FIG. 1

Restriction maps of pFB1112, which allows regulated expression of ToMO, and its derivatives. Only the relevant restriction sites are shown. Under the map of pFB1112, the white arrows indicate the location and the direction of transcription of the previously described ToMO gene cluster (touABCDEF) and orfA, putatively coding for a transposase (4). Shaded arrows indicate the regulatory gene (touR) and two additional ORFs (orf1 and orf2) identified in this work. Black thin arrows represent the touR (P_touR) and ToMO (P_ToMO) promoters. ToMO specific activity was measured in P. putida PaW340 cells carrying the indicated plasmids grown in the absence or in the presence of either toluene or o-cresol supplied as inducers. A minimum of three independent experiments (variation within 10%) were performed for each strain; results from representative assays are shown. ND, not determined.

FIG. 2

Mapping of the 5′-mRNA start of the touR gene (A) and of the tou operon (B). Primer extension analyses were performed as described in Materials and Methods. The primer extension products were run next to the sequence reactions performed on pFP3038 (A) and pPP4062 (B). To the right of each panel, an expanded view of the nucleotide sequence surrounding the transcriptional start site (+1) is shown.

FIG. 3

Deletion analysis of the putative ToMO promoter region. Portions of the putative ToMO promoter region were cloned upstream from the C2,3O reporter gene as described in Materials and Methods. The arrow represents the location of the incomplete touA ORF. C2,3O specific activity was measured in P. putida PaW340 cells carrying the pPP4062 derivatives shown in trans (+) or not (−) with touR, cloned in pFB3028, and grown in the presence (o-cresol) or absence (none) of the effector. A minimum of three independent experiments (variation within 10%) were performed for each strain; results from representative assays are shown.

FIG. 4

Analysis of the ToMO promoter region. (A) DNA sequence alignment of the ς⁵⁴-dependent promoter sequences of the xyl upper operon (Pu) (23), the dmp operon (Po) (40), and the tou operon (P_ToMO). The −24 and −12 sequences within the consensus sequence are boxed and labeled accordingly. The transcription starts are shown in boldface. (B) DNA sequence alignment of the palindromic regions (UASs) upstream of the promoters mentioned above. Nucleotides conserved in all three sequences are shown in boldface. The consensus sequences established from the comparison of the regions within and upstream of the Pu, Po, and P_ToMO operons are displayed below each alignment. Gaps, indicated by dashes, were introduced to maximize homology.

FIG. 5

Northern analysis of the ToMO transcripts. RNA was extracted from P. putida PaW340(pFB1112) cells not exposed (none) or exposed to either toluene or o-cresol. Samples were collected at 30-min intervals (shown at the top) after addition of the inducer (t₀). From each sample, 20 μg of RNA was analyzed by gel electrophoresis and probed with the touA gene to detect the ToMO transcripts.