Construction of a Novel Polychlorinated Biphenyl-Degrading Bacterium: Utilization of 3,4′-Dichlorobiphenyl by Pseudomonas acidovorans M3GY

Abstract

Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4'-dichlorobiphenyl (3,4'-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Krickel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CCI and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4'-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 ,ug ml-' and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction ofP. acidovorans M3GY, with the novel capability to utilize 3,4'-DCBP, thus involves the complete use ofmeta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.

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

“…P. acidovorans strain M3GY [4] and Escherichia coli C600 pGS9: :Tn5 [6] have been described elsewhere.…”

“…Degradation products from 3,4P-DCBP were identi¢ed by gas chromatography^infrared spectrometry^mass spectrometry (GC^IR^MS) performed with a 5890 Series II gas chromatograph Hewlett-Packard (Palo Alto, CA, USA), which was interfaced with a Hewlett-Packard 5898A mass spectrometer and 5965B infrared detector. The analytical conditions were previously described [4].…”

“…Culture supernatants were extracted as described previously [4]. Catecholic intermediates in culture extracts were converted to their trimethylsilyl derivatives prior to GCÎ R^MS analysis.…”

“…Samples were redissolved in 120 Wl dichloromethane, and 40 Wl of MSTFA was added. Culture supernatants were saturated with NaCl or (NH 4 )SO 4 and acidi¢ed with HCl. The saturated samples were then extracted with 3 volumes of ether.…”

“…The concept of combining genes from biphenyl degraders and chlorobenzoate degraders was ¢rst suggested by [3]. In an earlier communication [4], we described the construction and catabolic abilities of recombinant bacterium Pseudomonas acidovorans strain M3GY, which has the novel ability to utilize 3,4P-dichlorobiphenyl (3,4P-DCBP) as a sole source of carbon and energy. Metabolism of 3,4P-DCBP was shown to proceed through the meta-cleavage pathway by oxidation of the 3P-ring with subsequent rupture and hydrolysis to form 4-chlorobenzoate (4-CBa), which was metabolized through 4-chlorocatechol (4-CC) by the meta-¢ssion pathway.…”