Organization and regulation of meta cleavage pathway genes for toluene and o-xylene derivative degradation in Pseudomonas stutzeri OX1

Abstract

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.

FIG. 1

Restriction endonuclease maps of the P. stutzeri OX1 chromosomal DNA fragment cloned in pFB3401 and its derivatives (3). pFB3401 allowed the host cells to convert toluene, o-xylene, and their phenolic derivatives into the corresponding ring fission products. The ToMO-encoding operon and the regulatory gene (touR) controlling its expression are indicated (1, 4). The point of the arrows indicates the direction of transcription. The ability (+) or inability (−) of the plasmids to direct the conversion of o-cresol into ring fission products in the absence (−) or in the presence (+) of touR, cloned in pRZ7085 and provided in trans, is indicated to the right of the restriction maps. +/−, mildly positive reaction after prolonged exposure. Abbreviations: A, ApaI; B, BamHI; E, EcoRI; H, HindIII; N.A., not applicable; P, PstI; S, SacI; X, XhoI; Xb, XbaI. Only relevant restriction sites are indicated.

FIG. 2

Restriction endonuclease maps of DNA fragments subcloned from pFB1021 (pJ and pBZ series) and from pFB3411 (pVS series). Only the plasmids containing the minimal region coding for PH, C2,3O, HMSH, and HMSD are reported. The black arrowheads indicate the position of the Plac of the vector. To the right of the restriction maps, the enzymatic activities expressed from the indicated plasmids in uninduced (−) or IPTG-induced (+) E. coli JM109 cells are reported. At the bottom, the map obtained by subcloning, Southern, and sequence analyses (see text for details) displaying the gene arrangement and the putative promoter is shown. The presence of the gene indicated in parentheses can be hypothesized but has not been investigated. Abbreviations: B, BamHI; E, EcoRI; H, HindIII; M, MluI; ND, not detectable; P, PstI; S, SacI; Sm, SmaI; X, XhoI; Xb, XbaI. Only relevant restriction sites are indicated.

FIG. 3

A model for the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1. See text for details. The arrows indicate the direction of transcription. tnp, transposase-like ORF (4).