Genetic characterization of the styrene lower catabolic pathway of Pseudomonas sp. strain Y2

Pseudomonas sp. strain Y2 is a styrene-degrading bacterium, which initiates the catabolism of this compound via its transformation into phenylacetate by the sequential oxidation of the vinyl side chain. The styrene upper catabolic gene cluster (sty genes) had been localized in a 9.2-kb chromosomal region. This report describes the isolation, sequencing and analysis of an adjacent 20.5-kb chromosomal region that contains the genes of the styrene lower degradative pathway (paa genes), which are involved in the transformation of phenylacetate into aliphatic compounds that can enter the Krebs cycle. Hence, Pseudomonas sp. strain Y2 becomes the first microorganism whose entire styrene catabolic cluster has been completely characterized. Analysis of the paa gene cluster has revealed the presence of 17 open reading frames as well as gene duplications and gene reorganizations that are absent in other phenylacetate catabolic clusters described so far. The functionality of these genes has been proved by means of both complementation experiments on Pseudomonas putida mutants and in vitro enzymatic assays. Moreover, a DNA cassette encoding the whole styrene lower pathway has been constructed and has been used to expand the ability of Pseudomonas strains to degrade phenylacetic acid. For the first time, two functional phenylacetate-CoA ligases have been identified in an aerobic phenylacetic acid degradation pathway. Although the upper and lower styrene catabolic clusters are adjacent in the Pseudomonas sp. strain Y2 chromosome, their particular base composition and codon usage suggest a distinct evolutionary history.