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 f$dA and tfdB genes from Alcaligenes eurrophus JMP134 were analysed by PCR-RFLP (restriction length fragment polymorphism). Primers for the 2,4-D etherase gene were derived by sequence alignment of the t$dA genes from A. eutrophus JMP134 and Burkholderia sp. RASC. Primers for the 2,4-dichlorophenolhydroxylase gene were based on the t$dB gene sequence from A. eutrophus JMP134 by taking codon degeneration and variations in amino acid residue sequences into consideration. PCR amplification using the rfdA primer set produced fragments of 0.3 kb from 17 strains which showed varying degrees of similarity to the tfdA gene probe from A. eutrophus JMPI 34. Significant variations in the gene sequences were confirmed by PCR-RFLP analysis. DNA amplification using the tfdB primer set produced a 1.1 kb fragment from 19 strains. Amongst them, two did not show any similarity to the rfdB gene probe. The size and restriction pattern of the products obtained from A. eutrophus JMP134 were in accordance with the expected size calculated from the A. eutrophus tjZ.4 and t$dB gene sequence and their theoretical PCR-RFLP patterns. Some strains which did not amplify using the @!A primer set did however amplify with the rfdB primer set. These results suggest the independent evolution of these two genes in the construction of the 2.4-D metabolic pathway. Our .@A 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 $CdB genes.

Diversity of tfdC genes: distribution and polymorphism among 2,4-dichlorophenoxyacetic acid degrading soil bacteria

“…The tfdCII gene of R. eutropha was found to be more closely related to the tfdC gene of P. putida PaW85 than to the canonical tfdC gene of R. eutropha JMP134. This data indicated that the P. putida PaW85 tfdC gene type could be more widely distributed than the canonical tfdC from R. eutropha JMP134 and it could explain why recent studies, using pJP4-based probes [16,18,27], failed to detect tfdC genes in 2,4-D degrading strains isolated from soil. The tfdC gene of strain RASC, isolated in the USA, is closely related to those of P.…”

“…2,4-D degrading isolates from the MSDJ (Microbiologie des Sols Dijon) collection were isolated in 1995 from a grassland soil (Dijon soil) with no previous known application of 2,4-D, and from an agricultural gleyic luvisol of Citeaux in the Dijon area, with no previous history of 2,4-D treatments. An enrichment step of the soil samples in 2,4-D minimal liquid medium (2,4-D MM) [16] was carried out as previously described [18]. Strains were then isolated on 2,4-D MM plates, solidi¢ed by 15 g l 3I agar and supplemented with 200 mg l 3I of 2,4-D. All strains were maintained on 2,4-D minimal medium agar slopes supplemented with 50 mg l 3I of 2,4-D. For long-term storage strains were kept frozen at 380³C in 12.5% glycerol.…”

“…This discrepancy could be explained by sequence divergence in the tfdC regions chosen as PCR priming sites. Three strains (RASC, PDC4, TFD6) failed to utilize the carbon of the ring, although they have been reported to degrade the 2,4-D ring [9,16,26]. These strains gave 460-bp ampli¢cation products which did hybridize with at least one of the two probes.…”

“…Duplication has been reported for di¡erent genes of the 2,4-D degradative pathway on plasmid pJP4 harbored by R. eutropha: tfdA [13], tfdD [14] and tfdC [15]. Moreover, a high degree of interspecies diversity has been described for the 2,4-D dioxygenase gene tfdA and the 2,4-dichlorophenol hydroxylase gene tfdB among di¡erent 2,4-D degrading soil isolates [16]. These observations are in agreement with the assumption that gene duplication and sequence divergence are important for enlarging the range of substrates degraded by soil microorganisms.…”

Expression of tfdA genes in aquatic microbial communities during acclimation to 2,4-dichlorophenoxyacetic acid

“…Of 32 strains analysed, 26 possessed tfdA-homologous genes with either s 90%, 75^90% or 60^75% homology to the tfdA gene of strain JMP134. Vallaeys et al [17] used tfdA-speci¢c PCR ampli¢cation to show that 17 of 25 isolates had tfdA-like sequences. The widespread abundance of tfdA-homologous genes in 2,4-D degrading bacteria implies that levels of tfdA genes and transcripts can be related to the presence and the activity of 2,4-D degrading microbial populations.…”