Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida

Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU. Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s). Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose. Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor. Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O. Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants. Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes. 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 pathway.