Toxic effects of chlorinated and brominated alkanoic acids on pseudomonas-putida PP3 : selction at high-frequencies of mutations in genes encoding dehalogenases

Mutant strains of Pseudomonas putida PP3 capable of utilizing monochloroacetate (MCA) and dichloroacetate (DCA) as the sole sources of carbon and energy were isolated from chemostat cultures. The mutants differed from the parent strain in that they could grow on products of MCA and DCA dehalogenation (catalyzed by inducible dehalogenases I and II) and were resistant to growth inhibition by the two substrates. The growth inhibition of strain PP3 by MCA, DCA, and other halogenated alkanoic acids was studied. Sensitivity to dehalogenase substrates was related to the expression of the dehalogenase genes. For example, mutants producing elevated levels of one or both of the dehalogenases were sensitive to 2-monochloropropionate and 2-monochlorobutanoate at concentrations which did not affect the growth of strain PP3. P. putida PP1, the parent of strain PP3, was resistant to the inhibitory effects of MCA and DCA. Spontaneous mutants of strain PP3, also resistant to MCA and DCA, were selected at high frequency, and four different classes of these strains were distinguished on the basis of dehalogenase phenotype. All dehalogenase-producing mutants were inducible; no constitutive mutant has yet been isolated. Most of the resistant mutants examined did not produce one or both of the dehalogenase, and over half of those tested failed to revert back to the parental (strain PP3) phenotype, indicating that the observed mutations involved high-frequency deletion of DNA base sequences affecting expression of genes encoding dehalogenases and associated permease(s)