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Applied and Environmental Microbiology, July 2002, p. 3449-3454, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3449-3454.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Kazuhito Itoh,1,2,3* Rie Kanda,1 Yoko Sumita,4 Hongik Kim,4 Yoichi Kamagata,4 Kousuke Suyama,1 Hiroki Yamamoto,1 Robert P. Hausinger,2,3 and James M. Tiedje2,3

Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504,1 Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan,4 Department of Microbiology and Molecular Genetics,2 Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 488243

Received 29 November 2001/ Accepted 3 April 2002

The 2,4-dichlorophenoxyacetate (2,4-D)/{alpha}-ketoglutarate dioxygenase gene (tfdA) homolog designated tfdA{alpha} 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 {alpha} subdivision of the class Proteobacteria on the basis of its 16S ribosomal DNA sequence. Sequence analysis showed 56 to 60% identity of tfdA{alpha} to representative tfdA genes. A MalE-TfdA{alpha} fusion protein expressed in Escherichia coli exhibited about 10 times greater activity for phenoxyacetate than 2,4-D in an {alpha}-ketoglutarate- and Fe(II)-dependent reaction. The deduced amino acid sequence of TfdA{alpha} revealed a conserved His-X-Asp-X146-His-X14-Arg motif characteristic of the active site of group II {alpha}-ketoglutarate-dependent dioxygenases. The tfdA{alpha} genes were also detected in 2,4-D-degrading {alpha}-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 ß- and {gamma}-Proteobacteria and the tfdA{alpha} genes in {alpha}-Proteobacteria arose by divergent evolution from a common ancestor.

* Corresponding author. Mailing address: Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan. Phone: 81-852-32-6521. Fax: 81-852-32-6597. E-mail: itohkz{at}life.shimane-u.ac.jp.

Applied and Environmental Microbiology, July 2002, p. 3449-3454, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3449-3454.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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