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

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Abstract
To investigate the diversity of genes of the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic pathway and to infer phylogenetic relationships within each gene family, we compared partial nucleotide sequences of putative tfdA, tfdB and tfdC genes. These sequences were obtained from a range of bacterial isolates degrading 2,4-dichlorophenoxyacetate from disparate origins as well as from collection strains able to degrade only 2,4-dichlorophenol or 3-chlorobenzoate. PCR primers targeted to putatively conserved areas of the 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 canonical 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 ancient origin of the tfd genes. The tfdB, tfdC phylogenetic trees and the previously obtained tfdA phylogenetic tree showed significant discrepancies in their organisations. Particularly, we showed that the Burkholderia sp. strain TFD2 was harbouring a tfdA gene which was closely related to the RASC (Burkholderia sp.) tfdA gene while its tfdB gene was grouped with the tfdB gene from a Sphingomonas sp. (strain TFD26) located on a distinct branch from the RASC tfdB gene. The lack of divergence in the partial nucleotide sequences of the tfdB genes from Burkholderia sp. TFD2 and Sphingomonas sp. TFD26 suggested that the gene recruitment was a relatively recent event. Discrepancies were also observed between the tfdB and tfdC phylogenetic trees, giving additional evidence for the involvement of independent gene recruitment of diverse, significantly divergent genes during the assembly of the 2,4-D catabolic pathway.

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