EVIDENCE FOR ACQUISITION IN NATURE OF A CHROMOSOMAL 2,4-DICHLOROPHENOXYACETIC ACID ALPHA-KETOGLUTARATE DIOXYGENASE GENE BY DIFFERENT BURKHOLDERIA SPP

We characterized the gene required to initiate the degradation of 2,4- dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2, 4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage o f the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an alp ha-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumo ri and R P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tf dA gene was identified in a recombinant plasmid which complemented a t fdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5 alph a, as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% s imilarity to a tfdA gene cloned from the chromosome of a different Bur kholderia species (strain RASC) isolated from a widely separated geogr aphical area. This chromosomal gene has 77.2% sequence identity to tfd A from plasmid pJP4 (Y. Suwa, W E. Holben, and L. J. Forney, abstr. Q- 403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 an d RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different ba cterial species suggests that this chromosomal gene can be horizontall y transferred.