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 nmolmin-' mg-', Km = 114 R,M, K.. [the inhibition constant] = 951 ,uM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmolmin-' * mg-', Km = 1 ,uM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 ,uM). In resting-cell assays, recombinant strain CB15 and strain P6 both metabolized 3CB faster than 3,3'-dichlorobiphenyl. However, 3,3'-dichlorobiphenyl could not be utilized as a growth substrate by strain CB15, nor did its presence have any effect on the rate of 3CB mineralization.

Characterization of chlorobenzoate degraders isolated from polychlorinated biphenyl-contaminated soil and sediment in the Czech Republic

“…In our recent experiment focusing on the degradation of commercial PCB mixture Delor 103 by co-metabolizing biphenyl degraders we have noticed the accumulation of high amounts of orthochlorinated benzoates, namely 2,5-DCB, in a reaction mixture. This finding is relevant to the already known inhibition effects of metabolic products from the lower part of the PCB metabolic pathway on the enzymes from the upper region, namely on 3-phenylcatechol 2,3-dioxygenase (Adams et al 1992). Therefore, this study should be followed by more experiments focusing on the degradation activity of CB degraders on PCB-contaminated sites to better understand their bioremediation potentials.…”

“…Plasmid DNA isolation and DNA digestions by restriction endonucleases were performed as described by Sambrook et al (1989) and total DNA was isolated by the method described by Adams et al (1992). A non-radioactive DNA labelling and detection kit (Genius; Boehringer Mannheim, Indianapolis, IN, USA) was used according to the manufacturer's instructions.…”

The use of mutants to discern the degradation pathway of 3,4′-dichlorobiphenyl in Pseudomonas acidovorans M3GY

“…Inhibition of phenylcatechol-1,2-dioxygenase by 3-CC was previously shown with parental strain Pseudomonas sp. CB15 [14].…”

The molecular basis for adaptive evolution in novel extradiol dioxygenases retrieved from the metagenome

“…As for 7E11 and I.2.G enzymes, maximal activities were obtained with 50-80 mM substrate concentrations. Substrate inhibition has also been observed in other EDOs, especially when 2,3-dihydroxybiphenyl was used (Adams et al, 1992;Heiss et al, 1995;Andú jar et al, 2000). We therefore used substrate concentrations at which inhibition would be negligible.…”