PHENOTYPIC CHARACTERIZATION OF COPPER-RESISTANT MUTANTS OF METHYLOSINUS-TRICHOSPORIUM OB3B

Cultures of Methylosinus trichosporium OB3b grown in the presence of v ery low concentrations of copper synthesize a soluble methane monooxyg enase (sMMO) that efficiently catalyzes the oxidation of trichloroethy lene and other organic pollutants. Recently, we isolated five M. trich osporium OB3b mutants that express sMMO activity when grown in the pre sence of elevated copper concentrations (P. A. Phelps, S. K. Agarwal, G. E. Speitel, Jr., and G. Georgiou, Appl. Environ. Microbiol. 58:3701 -3708, 1992). Here we show that, in contrast to the results for the wi ld-type cells, the addition of copper to mutant cultures grown on meth ane and nitrate as the nitrogen source has no noticeable effect on the growth rate and sMMO expression. In vitro experiments indicated that the copper-resistant phenotype does not arise from an increased stabil ity of sMMO to copper deactivation. Furthermore, the mutant cultures e xhibit altered speciation of copper in the extracellular fluid and hav e substantially decreased levels of cell-associated copper. On the bas is of these results, we propose that the mutant phenotype arises from defects in copper uptake and metabolism rather than from changes in sM MO expression or enzyme stability.