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

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.