Cloning and expression of the transposable chlorobenzoate-3,4-dioxygenase genes of Alcaligenes sp. strain BR60.

Growth on 3-chlorobenzoate was found to induce the enzymes of the protocatechuate meta ring fission pathway in Alcaligenes sp. strain BR60. The chlorobenzoate catabolic genes, designated cba, were localized to a 3.7-kb NotI-EcoRI fragment within the nonrepeated region of the composite transposon Tn5271. The cba genes were cloned onto two broad-host-range vectors and expressed in Escherichia coli and Alcaligenes sp. strain BR6024. In E. coli, expression of the cba genes with the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible tac promoter of the IncQ vector pMMB66HE resulted in the production of protocatechuate and chlorodihydroxybenzoate metabolites of 3-chlorobenzoate. Expression of this construct in one orientation resulted in the formation of two polypeptides 51 and 42 kDa in size. This result was confirmed by subcloning into pGEM3Zf and then incorporating L-35S-methionine into newly synthesized proteins, using the thermally regulated T7 polymerase-promoter system. Introduction of the NotI-EcoRI fragment into Alcaligenes sp. strain BR6024 (Cba-P), using the IncP broad-host-range, mobilizable plasmid pBW13, restored the 3-chlorobenzoate-degradative phenotype and resulted in the accumulation of protocatechuate and chlorodihydroxybenzoate intermediates. The data indicate that a two-component dioxygenase specified by Tn5271 oxidizes 3-chlorobenzoate at the 3,4- or 4,5-positions. This activity extends the range of pathways for chloroaromatic compounds known to be functional in the environment. The new pathway avoids the toxicity attributed to the accumulation of chlorocatechol metabolites in bacteria degrading chlorobenzoates.