CATABOLITE-MEDIATED MUTATIONS IN ALTERNATE TOLUENE DEGRADATIVE PATHWAYS IN PSEUDOMONAS-PUTIDA

Pseudomonas putida 54g grew on mineral salts with toluene and exhibite d catechol-2,3-dioxygenase (C230) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol(-)) variants approa ched nearly 10% of total CFU. Auxotrophs were not detected among varia nts, suggesting selective loss of catabolic function(s). Variant forma tion was substrate dependent, since Tol(-) cells were observed on neit her ethylbenzene, glucose, nor peptone based media nor when toluene ca tabolism was suppressed by glucose. Unlike wild-type cells, variants d id not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C230 activities were restored to variants via transfer of a 78-mDa TOL-lik e plasmid from a wild-type Tol(+) donor. Tests for reversion of varian ts to Tol(+) were uniformly negative, suggesting possible deletion or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene e ncoding C230. Cells grown on benzoate remained Tol(+) but were C230(-) and contained a plasmid of reduced size or were plasmid free, suggest ing an alternate chromosomal catabolic pathway, also defective in vari ants. Cells exposed to benzyl alcohol, the initial oxidation product o f toluene, accumulated > 13% variants in 5 days, even when cell divisi on was repressed by nitrogen deprivation to abrogate selection process es. No variants formed in identical ethylbenzene-exposed controls. The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pa thway.