Biodegradation kinetics of toluene, m-xylene, p-xylene and their intermediates through the upper TOL pathway in Pseudomonas putida (pWWO).

Pseudomonas putida mt-2, harbouring TOL plasmid pWWO, is capable of degrading toluene and a range of di- and tri-alkylbenzenes. In this study, chemostat-grown cells (D = 0.05 h-1, toluene or m-xylene limitation) of this strain were used to assess the kinetics of the degradation of toluene, m-xylene, p-xylene, and a number of their pathway intermediates. The conversion kinetics for the three hydrocarbons showed significant differences: the maximal conversion rates were rather similar [11-14 mmol h-1 (g dry wt)-1] but the specific affinity (the slope of the v vs s curve near the origin) of the cells for toluene [1300 I (g dry wt)-1 h-1] was only 5% and 14% of those found for m-xylene and p-xylene, respectively. Consumption kinetics of mixtures of the hydrocarbons confirmed that xylenes are strongly preferred over toluene at low substrate concentrations. The maximum flux rates of pathway intermediates through the various steps of the TOL pathway as far as ring cleavage were also determined. Supply of 0-5 mM 3-methylbenzyl alcohol or 3-methylbenzaidehyde to fully induced cells led to the transient accumulation of 3-methylbenzoate. Accumulation of the corresponding carboxylic acid (benzoate) was also observed after pulses of benzyl alcohol and benzaldehyde, which are intermediates in toluene catabolism. Analysis of consumption and accumulation rates for the various intermediates showed that the maximal rates at which the initial monooxygenation step and the conversion of the carboxylic acids by toluate 1,2-dioxygenase may occur are two- to threefold lower than those measured for the two intermediate dehydrogenation steps.

Effect of growth rate, nutrient limitation and succinate on expression of TOL pathway enzymes in response to m-xylene in chemostat cultures of Pseudomonas putida (pWW0).

Previous studies have shown that expression of the toluene and m- and p-xylene degradation pathway in Pseudomonas putida (pWW0) is subject to catabolite repression by succinate. We report here that the expression level of the upper part of this so-called TOL pathway in cells grown in chemostat culture is strongly influenced by nutrient limitation when m-xylene is the sole carbon and energy source. The benzylalcohol dehydrogenase (BADH) levels in cells that are growth-limited by anabolic processes [sulphate (S)-, phosphate (P)- or nitrogen (N)-limiting conditions] were 3-12% of those in cells growing under oxygen limitation (when catabolism limits growth). BADH levels under S-, P- and N-limitation were further decreased (three- to fivefold) when succinate was supplied in addition to m-xylene. Levels of the meta-cleavage pathway enzyme catechol 2,3-dioxygenase were less affected by the growth conditions but the general pattern was similar. Dilution rate also influenced the expression of the TOL pathway: BADH levels gradually decreased with increasing dilution rates, from 1250 mU (mg protein)-1 at D = 0.05 h-1 under m-xylene limitation to 290 mU (mg protein)-1 at D = 0.58 h-1 (non-limited growth). BADH levels were shown to be proportional to the specific affinity whole cells for m-xylene. It may, therefore, be expected that natural degradation rates are adversely affected by anabolic nutrient limitations, especially at relatively low concentrations of the xenobiotic compound.