Genes for 2,4,5-trichlorophenoxyacetic acid metabolism in Burkholderia cepacia AC1100: characterization of the tftC and tftD genes and locations of the tft operons on multiple replicons.

Chlorinated phenols and phenoxyacetates are a group of chemical compounds which have been used extensively as pesticides, wood preservatives, and herbicides in the agricultural industry. They are a major group of recalcitrant environmental pollutants (11, 26). Burkholderia cepacia AC1100 is a bacterium capable of using the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy (21). 2,4,5-T is converted to 2,4,5-trichlorophenol (2,4,5-TCP) by 2,4,5-T oxygenase (6, 16, 47). The genes encoding the oxygenase component of this complex, tftA and tftB, have been cloned and sequenced (6). The tftA and tftB genes are clustered (6) and their expression is controlled by a constitutive Escherichia coli ς70-like fusion promoter created by insertion of IS1490 (5, 19). 2,4,5-TCP is then converted to 5-chloro-2-hydroxy-1,4-benzoquinone (5-CHQ) in a two-step hydroxylation catalysis (48). In resting-cell experiments, Karns et al. (20) showed that when AC1100 was grown on glucose, succinate, or lactate as the sole carbon source, these cells could convert 2,4,5-T to 2,4,5-TCP but were unable to dechlorinate 2,4,5-TCP. Growth in the presence of either 2,4,5-T or 2,4,5-TCP allowed induction of tftCD, resulting in dechlorination of 2,4,5-T and 2,4,5-TCP. It was therefore hypothesized that the steps converting 2,4,5-TCP to other pathway intermediates are inducible. Inhibitor studies suggested that a flavin-containing enzyme(s) is responsible for at least one of the two 2,4,5-TCP hydroxylation steps (42). The enzyme responsible for both hydroxylation reactions on 2,4,5-TCP, chlorophenol-4-monooxygenase, was subsequently purified and shown to be composed of a 58-kDa polypeptide and a 22-kDa polypeptide (48). Natural horizontal transfer of genes responsible for biodegradation has been described for several bacterial systems (43, 44) and seems to play a major role in the acclimation of bacterial communities to environmental pollutants. B. cepacia AC1100 acquired its catabolic ability to metabolize 2,4,5-T over a period of several months after being subjected for a prolonged period to strong selective pressure in a continuous culture (3). The evolution of this catabolic capacity occurred most likely through recruitment of genes present in the chemostate consortium and their integration into its genome. The two 2,4,5-T catabolic gene clusters tftAB and tftEFGH have previously been identified and shown to be specific for the upper and the lower portion of the pathway. The presence of copies of the three known insertion sequence (IS) elements in AC1100, namely, IS931, IS932, and IS1490, adjacent to both gene clusters supports their possible role in stimulating rapid evolution through gene acquisition and expression (6, 9, 15, 16, 19, 41). In this paper, the identification and characterization of tftCD provide the missing link in the characterization of the upper portion of the 2,4,5-T pathway. Through the use of pulsed-field gel electrophoresis (PFGE) we demonstrate that the three 2,4,5-T gene clusters are localized on different replicons, which supports the hypothesis that the 2,4,5-T pathway was assembled by a series of independent gene transfers. Comparison of AC1100 with different 2,4,5-T-negative mutants shows that the tft genes are deleted and rearranged at high frequencies.