tfdA -Like Genes in 2,4-Dichlorophenoxyacetic Acid-Degrading Bacteria Belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia Cluster in α- Proteobacteria

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Abstract
The 2,4-dichlorophenoxyacetate (2,4-D)/α-ketoglutarate dioxygenase gene ( tfdA ) homolog designated tfdAα was cloned and characterized from 2,4-D-degrading bacterial strain RD5-C2. This Japanese upland soil isolate belongs to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in the α subdivision of the class Proteobacteria on the basis of its 16S ribosomal DNA sequence. Sequence analysis showed 56 to 60% identity of tfdAα to representative tfdA genes. A MalE-TfdAα fusion protein expressed in Escherichia coli exhibited about 10 times greater activity for phenoxyacetate than 2,4-D in an α-ketoglutarate- and Fe(II)-dependent reaction. The deduced amino acid sequence of TfdAα revealed a conserved His-X-Asp-X 146 -His-X 14 -Arg motif characteristic of the active site of group II α-ketoglutarate-dependent dioxygenases. The tfdAα genes were also detected in 2,4-D-degrading α- Proteobacteria previously isolated from pristine environments in Hawaii and in Saskatchewan, Canada (Y. Kamagata, R. R. Fulthorpe, K. Tamura, H. Takami, L. J. Forney, and J. M. Tiedje, Appl. Environ. Microbiol. 63: 2266-2272, 1997). These findings indicate that the tfdA genes in β- and γ- Proteobacteria and the tfdAα genes in α- Proteobacteria arose by divergent evolution from a common ancestor.