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

Abstract

Pseudomonas acidovorans strain M3GY is a recombinant bacterium with the novel ability to utilize 3,4′-dichlorobiphenyl (3,4′-DCBP) as a growth substrate. This strain was previously shown to oxidize the 3′-ring and produce 4-chlorobenzoate (4-CBa) through the standard biphenyl pathway. Although 4-CBa was metabolized through the meta-fission pathway, the genes encoding the ortho-chlorocatechol pathway were retained. Nevertheless, neither 3-CBa nor 3-chlorocatechol (3-CC) were detected as intermediates during metabolism of 3,4′-DCBP, nor was 4-CBa utilized as a sole carbon source, by this strain. Two mutant strains were produced to resolve these anomalies. Mutant strain M3GY-9 was obtained by Tn 5 insertion and selection for growth on biphenyl, and was unable to grow on 3-CBa. It accumulated 3-CC from 3,4′-DCBP when grown on biphenyl. Thus, M3GY attacks both rings, and the failure to isolate 3-CBa or 3-CC is due to rapid turnover by the enzymes of the ortho-chlorocatechol pathway in the wild-type strain. Mutant strain M3GY-1 grew on 4-CBa, unlike the wild-type strain. Washed cell suspensions of mutant strain MEGY-1 consumed 4-fluorobenzoate, 4-bromobenzoate, and, to a lesser extent 4-iodobenzoate. The mutation that resulted in the ability of mutant strain M3GY-I to effectively utilize 4-CBa as a sole carbon source was associated with a transport mechanism.