Substrate diversity and expression of the 2,4,5-trichlorophenoxyacetic acid oxygenase from Burkholderia cepacia AC1100.
Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid as a sole source of carbon and energy. The genes encoding the proteins involved in the first step (tftA and tftB [previously designated tftA1 and tftA2, respectively]) have been cloned and sequenced. The oxygenase, TftAB, is capable of converting not only 2,4,5-trichlorophenoxyacetic acid to 2,4,5-trichlorophenol but also a wide range of chlorinated aromatic phenoxyacetates to their corresponding phenolic derivatives, as shown by whole-cell and cell-free assays. The rate of substrate utilization by TftAB depends upon the extent of chlorination of the substrate, the positions of the chlorines, and the phenoxy group. These results indicate a mechanistic similarity between TftAB and the 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate-dependent dioxygenase, TfdA, from Alcaligenes eutrophus JMP134. The promoter of the oxygenase genes was localized by promoter-probe analysis, and the transcriptional start site was identified by primer extension. The beta-galactosidase activity of the construct containing the promoter region cloned upstream of the beta-galactosidase gene in the promoter-probe vector pKRZ-1 showed that this construct is constitutively expressed in Escherichia coli and in AC1100. The -35 and -10 regions of the oxygenase genes show significant sequence identity to typical Escherichia coli sigma 70 promoters.
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago 60612, USA.
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Danganan
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Shankar
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Ye
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Chakrabarty
D001483:Base Sequence; D016956:Burkholderia cepacia / Q000201:enzymology*; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D010105:Oxygenases; C090875:2,4,5-trichlorophenoxyacetic acid oxygenase