Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture

Pseudomonas putida mt-2, harbouring the TOL plasmid pWW0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates. The fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and xylene (TOL- cells), was determined. Genetic analysis revealed that all TOL- cells isolated harboured partially deleted plasmids, lacking the TOL catabolic genes. The growth-rate advantage of the TOL- cells was quantified from the kinetics of their increase as a fraction of the total population. At a dilution rate of 0.1 h-1 no growth-rate advantage of TOL- cells was found when phosphate or ammonium were limiting. Under sulphate-limitation, ingrowth of TOL- cells was evident but did not follow a straightforward pattern. Under succinate-limitation the growth-rate advantage was the highest, particularly at low dilution rates (about 50% at D = 0.05 h-1). In phauxostat culture, at the maximal growth rate, the growth-rate advantage of TOL- cells was less than 1%. The specific activity in TOL+ cells of the plasmid-encoded enzyme catechol 2,3-dioxygenase was relatively high at a low growth rate.