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The chlorobenzoate dioxygenase genes of Burkholderia sp. strain NK8 involved in the catabolism of chlorobenzoates

Soil General Microbiology Laboratory, National Institute of Agro-Environmental Sciences, 3-1-1 Kannondai, Tsukuba City, Ibaraki 305-8604, Japan¹

Author for correspondence: Kiyotaka Miyashita. Tel: +81 298 38 8256. Fax: +81 298 38 8199. e-mail: kmiyas{at}s.affrc.go.jp

Burkholderia sp. NK8 grows abundantly on 3-chlorobenzoate (3CB),4-chlorobenzoate (4CB) and benzoate. The genes encoding the oxidation of (chloro)benzoates (cbeABCD) and catechol (catA, catBC), the LysR-type regulatory gene cbeR and the gene cbeE with unknown function, all of which form a single cluster in NK8, were cloned and analysed. The protein sequence of chlorobenzoate 1,2-dioxygenase (CbeABC) is 50–65% identical to the benzoate dioxygenase (BenABC) of Acinetobacter sp. ADP1, toluate dioxygenase (XylXYZ) of the TOL plasmid pWW0 and 2-halobenzoate dioxygenase (CbdABC) of Burkholderia cepacia 2CBS. Disruption of the cbeA gene resulted in the simultaneous loss of the ability to grow on benzoate and monochlorobenzoates, indicating the involvement of the cbeABCD genes in the degradation of these aromatics. The cbeABCD genes are preceded by catA, the gene for catechol dioxygenase. lacZ transcriptional fusion studies in Pseudomonas putida showed that catA and cbeA are co-expressed under the positive control of cbeR, a LysR-type transcriptional regulatory gene. The cbeA::lacZ transcriptional fusion studies showed that the inducers of the genes are 3CB, 4CB, benzoate and probably cis,cis-muconate. On the other hand, 2-chlorobenzoate (2CB) did not activate the expression of the genes. The chlorobenzoate dioxygenase was able to transform 2CB, 3CB, 4CB and benzoate at considerable rates. 2CB yielded both catechol and 3-chlorocatechol (3CC), and 3CB gave rise to 4-chlorocatechol and 3CC as the major and minor intermediate products, respectively, indicating that the NK8 dioxygenase lacks absolute regiospecificity. The absence of growth of NK8 on 2CB, despite its considerable degradation activity against 2CB, is apparently due to the inability of CbeR to recognize 2CB as an inducer of the expression of the cbe genes.

Keywords: Burkholderia, chlorobenzoate dioxygenase, cbeABC, cbeR, catA

Abbreviations: 2CB, 2-chlorobenzoate; 3CB, 3-chlorobenzoate; 4CB, 4-chlorobenzoate; 3CC, 3-chlorocatechol; 4CC, 4-chlorocatechol; BSMG, basal synthetic medium + glucose; DA, Davis-adonitol medium; DHB, dihydrodihydroxybenzoate (3,5-cyclohexadiene-1,2-diol-1-carboxylic acid); PCBs, polychlorinated biphenyls

The GenBank accession number for the sequence reported in this paper is AB024746.

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