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.
Danganan
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CE
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago 60612, USA.
Shankar
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S
Ye
R W
RW
Chakrabarty
A M
AM
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