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

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.

2,4-Dichrolophenoxyacetic Acid-degrading Genes from Bacteria Isolated from Soil in Japan: Spread of Burkholderia cepacia RASC-type Degrading Genes Harbored on Large Plasmids

“…Analysis of the pattern of hybridization of total DNA digested with restriction enzymes Total DNA from strains, whose 2,4-D-degrading gene homologs were highly similar to those in Burkholderia cepacia strain RASC 45,48,51) , was digested with the restriction enzyme EcoRI or HindIII (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions; the digested DNA was electrophoresed in 0.7% agarose gels. Subsequent Southern hybridization was performed as described in the section Southern hybridization for plasmid analysis, except that the hybridized membranes were washed twice in 2×SSC, 0.1% SDS and twice in 0.1×SSC, 0.1% SDS.…”

“…Some 2,4-D-degrading plasmids, such as pJP4 of strain JMP134 4) and pEST4011 of strain EST4002 31) , are conjugative plasmids. In addition, highly similar 2,4-D-degrading genes have often been obtained from diverse genera 3,10,17,18,28,33,51) . These findings suggest a lateral spread of 2,4-D-degrading genes among bacteria.…”

“…Some of the 2,4-D-degrading betaproteobacteria have DNA fragments whose nucleotide sequences have 95% or more similarity to those of the tfdA, tfdBI, and tfdCI genes of strain JMP134 2,3,33,51) . Other Betaproteobacteria, including Achromobacter xylosoxidans strain EST4002 31) and Burkholderia cepacia strain RASC 45) , have tfdA, tfdB, and tfdC genes whose nucleotide sequences have 90% or more similarity to each other, and 60-80% similarity to those of the tfdA, tfdB I , and tfdC I genes of strain JMP134 3,14,22,23,27,33,41,50,51) . Among 2,4-D-degrading alphaproteobacteria (e.g., Sphingomonas spp.…”

Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

“…One litre of this culture was used to inoculate a fermenter (INFORS, ALGU 503) operating at a working volume of 4 l. Cultivation proceeded under aerobic conditions at pH 8 O, 0.5. (R,S)-2,4-DP was supplied as the sole carbon and energy source in a fed batch regime by adapting the feed rate to the substrate consumption capacity of the culture.…”

“…Genes encoding this type of enzyme, i.e. tfdA, were found to be widespread in microbial communities [3][4][5][6][7][8][9]. By contrast, there are only a few examples of axenic strains that are able to productively utilise phenoxypropionate herbicides: Sphingomonas herbicidovorans (Flavobacterium sp.)…”