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

Abstract

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.

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

“…For example, CbeR (11) and CbnR (12) of Burkholderia sp. NK8 and Ralstonia eutropha NH9, respectively, respond to muconate, benzoate, and͞or chloro-substituted versions of these compounds.…”

“…Although multiple effectors sometimes regulate gene expression (11)(12)(13)(14)(15)(16)(17), the combined effect of these compounds on transcriptional control by a single regulator is poorly understood. Synergistic effects on transcriptional regulation have previously been attributed to two distinct contacts made between RNA polymerase and a single DNA-bound activator or between RNA polymerase and two different activators, as reviewed in ref.…”

Analysis of a Bacterial Community in 3-Chlorobenzoate-Contaminated Soil by PCR-DGGE Targeting the 16S rRNA Gene and Benzoate 1,2-Dioxygenase Gene (benA)

“…To date, various bacteria that can degrade CBs have been isolated from different environmental samples 3,[6][7][8]15,25,29) . It is not clear, however, which bacterial groups are the main contributors to the degradation of CBs under environmental conditions, because most of the degraders are obtained by screening based on their ability to degrade CBs on agar plates or in liquid cultures.…”

Isolation of Effective 3-Chlorobenzoate-Degraders in Soil Using Community Analyses by PCR-DGGE

“…Although 3CB-degrading Burkholderia spp. have been isolated from various soils 12,14,15,32) , it had not been confirmed whether this group of bacteria contributes to the degradation of 3CB in the soil environment. Our results suggest that several Burkholderia strains actually function as effective 3CB-degraders in soil.…”

“…Chlorobenzoates can be generated from the biodegradation of polychlorinated biphenyls 11) , and 3CB has been most frequently used to study the bacterial degradation of this group of compounds. Up to now, 3CB-degrading bacteria have been isolated from environmental samples based on their 3CB-degrading ability in culture media 4,6,[12][13][14][15][16]20,29,31) , but there have been few attempts to isolate these degraders based on their ability in the environment. Previously, we showed that several Burkholderia-like bacteria became dominant in a forest soil after 3CB was added, by PCR-DGGE targeting the 16S rRNA gene and benzoate 1,2-dioxygenase alpha subunit gene (benA) 25) .…”