High stability and fast recovery of expression of the TOL plasmid-carried toluene catabolism genes of Pseudomonas putida mt-2 under conditions of oxygen limitation and oscillation.

Pseudomonas putida mt-2 harbors the TOL plasmid (pWWO), which contains the genes encoding the enzymes necessary to degrade toluene aerobically. The xyl genes are clustered in the upper operon and encode the enzymes of the upper pathway that degrade toluene to benzoate, while the genes encoding the enzymes of the lower pathway (meta-cleavage pathway) that are necessary for the conversion of benzoate to tricarboxylic acid cycle intermediates, are encoded in a separate operon. In this study, the effects of oxygen availability and oscillation on the expression of catabolic genes for enzymes involved in toluene degradation were studied by using P. putida mt-2 as model bacterium. Quantitative reverse transcription-PCR was used to detect and quantify the expression of the catabolic genes xylM (a key gene of the upper pathway) and xylE (a key gene of the lower pathway) in cultures of P. putida mt-2 that were grown with toluene as a carbon source. Toluene degradation was shown to have a direct dependency on oxygen concentration, where gene expression of xylM and xylE decreased due to oxygen depletion during degradation. Under oscillating oxygen concentrations, P. putida mt-2 induced or downregulated xylM and xylE genes according to the O₂ availability in the media. During anoxic periods, P. putida mt-2 decreased the expression of xylM and xylE genes, while the expression of both xylM and xylE genes was immediately increased after oxygen became available again in the medium. These results suggest that oxygen is not only necessary as a cosubstrate for enzyme activity during the degradation of toluene but also that oxygen modulates the expression of the catabolic genes encoded by the TOL plasmid.