Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating

Abstract

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmol.min-1.mg-1, Km = 114 microM, Kss [the inhibition constant] = 951 microM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmol.min-1.mg-1, Km = 1 microM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 microM).(ABSTRACT TRUNCATED AT 250 WORDS)