The metabolic pathway of 2,4-dichlorophenoxyacetic acid degradation involves different families of tfdA and tfdB genes according to PCR-RFLP analysis

Abstract

Twenty‐five 2,4‐dichlorophenoxyacetic acid (2,4‐D) degrading bacteria from geographically diverse locations and presenting various degrees of similarity or no similarity to the tfdA and tfdB genes from Alcaligenes eutrophus JMP134 were analysed by PCR‐RFLP (restriction length fragment polymorphism). Primers for the 2,4‐D etherase gene were derived by sequence alignment of the tfdA genes from A. eutrophus JMP134 and Burkholderia sp. RASC. Primers for the 2,4‐dichlorophenolhydroxylase gene were based on the tfdB gene sequence from A. eutrophus JMP134 by taking codon degeneration and variations in amino acid residue sequences into consideration. PCR amplification using the tfdA primer set produced fragments of 0.3 kb from 17 strains which showed varying degrees of similarity to the tfdA gene probe from A. eutrophus JMP134. Significant variations in the gene sequences were confirmed by PCR‐RFLP analysis. DNA amplification using the tfdB primer set produced a 1.1 kb fragment from 19 strains. Amongst them, two did not show any similarity to the tfdB gene probe. The size and restriction pattern of the products obtained from A. eutrophus JMP134 were in accordance with the expected size calculated from the A. eutrophus tfdA and tfdB gene sequence and their theoretical PCR‐RFLP patterns. Some strains which did not amplify using the tfdA primer set did however amplify with the tfdB primer set. These results suggest the independent evolution of these two genes in the construction of the 2,4‐D metabolic pathway. Our tfdA and tfdB primer sets could be used for the detection of similar sequences in bacteria and soils. Moreover, PCR‐RFLP patterns could also be used to select subsets of strains for sequencing to study the phylogeny of the tfdA and tfdB genes.

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

“…The first step in the degradation pathway is catalyzed by an a-ketoglutarate-dependent dioxygenase encoded by the tfdA gene (Figure 1). Prior work has shown that diversity among the functional tfdA genes encoding this dioxygenase enzyme is high (Vallaeys et al, 1996;Kamagata et al, 1998). McGowan et al (1998) suggested classifying tfdA genes into three classes (I-III) with sequences between classes showing 75-80% homology and Kamagata et al (1998) and Itoh et al (2000) isolated 2,4-D-degrading strains possessing the tfdA-like genes tfdAa and cadA.…”

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

“…Moreover, a molecular weight of 32 kD is found with the canonical TfdA protein from R. eutropha JMP134 [2], cleaving 2,4-D with high specificity. The application of primers, derived from conserved regions of tfdA genes [7], did not result in a specific amplification product in PCR with the genome of strain MC1 [12,16] and strain MH as the template. This might be expected in agreement with the different substrate specificity found with the various strains (i.e.…”

“…This means that a similar herbicide consumption profile in these strains must be provoked by a divergent genetic background. This deviates from the experience with tfdA: this gene determining 2,4-D degradative specificity is distributed in the microbial world in a highly conserved manner [7]. To complete this picture, a third strain, Rhodoferax sp.…”

“…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.)…”

Delftia acidovoransMC1 Resists High Herbicide Concentrations — a Study of Nutristat Growth on (RS)-2-(2,4-Dichlorophenoxy)propionate and 2,4-Dichlorophenoxyacetate

“…They have been shown to be degradable by microbes, as was found for a number of bacterial strains with respect to 2,4-D, [2][3][4][5][6][7] but also holds for the enantiomers of (RS)-2-(2,4-dichlorophenoxy)propionate [(RS)-2,4-DP] with e.g. strains Sphingomonas herbicidovorans MH, 8,9) Rhodoferax sp.…”