The identification and cloning of genes encoding haloaromatic catabolic enzymes and the construction of hybrid pathways for substrate mineralization

Abstract

This paper reviews the genetic basis of haloaromatic biodegradation by bacteria, with a focus on the genetic analysis of Alcaligenes eutrophus JMP134, an organism which can utilize 3-chlorobenzoate, 2,4-dichlorophenoxyacetate (2,4-D) and related compounds as sole carbon and energy sources, and Pseudomonas sp. B13, a chlorobenzoate degrader. The involvement of transmissible plasmids pJP4 and pWR1, isolated from strains JMP134 and B13, respectively, in chloroaromatic mineralization has been examined, and restriction fragments of both plasmids have been cloned on the broad host range plasmid vector pKT231. Transposon Tn5 mutagenesis of these and other soil isolates enriched in and purified from mixed cultures utilizing 2,4,5-trichlorophenoxyacetate (2,4,5-T) as sole carbon and energy source, has been carried out using a "suicide" transposon donor, pLG221 (Co1Ibdrd-1::Tn5). Mapping of Tn5 insertions in mutants which accumulate pathway intermediates has facilitated the identification and cloning of genes encoding chlorocatechol 1,2-dioxygenase, and other key enzymes in haloaromatic catabolism. There are good prospects for the genetic construction of hybrid haloaromatic catabolic pathways by combining genes encoding broad specificity enzymes, capable of transforming halogenated analogues of their natural substrates, with genes for halocatechol degradation.