Phylogenetic analyses indicate independent recruitment of diverse gene cassettes during assemblage of the 2,4-D catabolic pathway

To investigate the diversify of genes of the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic pathway and to infer phylogenetic relationships within e ach gene family, we compared partial nucleotide sequences of putative tfdA, tfdB and tfdC genes. These sequences were obtained from a range of bacteri al isolates degrading 2,2,4-dichlorophenoxyacetate from disparate origins a s well as from collection strains able to degrade only 2,4-dichlorophenol o r 3-chlorobenzoate. PCR primers targeted to putatively conserved areas of t he tfdB and tfdC genes were used to amplify tfdB and tfdC fragments from 2, 4-D degrading strains known to present various degrees of homology to the c anonical tfdB and tfdC genes from ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134. We observed a large diversity of gene sequences and Gene assemblies. Sequence comparison showed a wide divergence suggesting an anc ient origin of the tfd genes. The tfdB, tfdC phylogenetic trees and the pre viously obtained tfdA phylogenetic tree showed significant discrepancies in their organisations. Particularly, we showed that the Burkholderia sp. str ain TFD2 was harbouring a tfdA gene which was closely related to the RASC ( Burkholderia sp.) IfdA gene while its tfdB gene was grouped with the tfdB g ene from a Sphingomonas sp. (strain TFD26) located on a distinct branch fro m the RASC tfdB gene. The lack of divergence in the partial nucleotide sequ ences of the tfdB genes from Burkholderia sp. TFD2 and Sphingomonas sp. TFD 26 suggested that the gene recruitment was a relatively recent event, Discr epancies were also observed between the tfdB and tfdC phylogenetic trees, g iving additional evidence for the involvement of independent gene recruitme nt of diverse, significantly divergent genes during the assembly of the 2,4 -D catabolic pathway. (C) 1999 Federation of European Microbiological Socie ties. Published by Elsevier Science B.V. All rights reserved.