Development and Application of PCR Primers for the Detection of thetfd Genes inDelftia acidovorans P4a Involved in the Degradation of 2,4-D

Abstract

Primers specific for the genes tfdD, tfdE and tfdF, derived from conserved amino acid sequence motifs of the corresponding homologous enzymes, and primers specific for the genes tfdA and tfdB as well as tfdC taken from the literature were applied in PCR reactions using the genomic DNA of Delftia acidovorans P4a as the template. PCR products were obtained with all primer pairs that were similar in size to those found with the genomic DNA of strains harbouring plasmid pJP4 as the carrier of tfd genes. The nucleotide sequences and the corresponding amino acid sequences of the PCR products obtained with Strain P4a were compared with the sequence databases. According to BLAST analyses, the partial sequences of tfdA and tfdB exhibited a 94-99% degree of identity with the homologous sequences of the 2,4-D-degrading strains Achromobacter xylosoxidans subsp. denitrificans EST4002 (pEST4011), Burkholderia sp. RASC, Variovorax paradoxus TV1 (pTV1) and Burkholderia cepacia 2a (pIJB1), whereas the partial sequences of the tfdC, tfdD, tfdE and tfdF genes revealed a 96-100% degree of identity with the homologous sequences of the chlorobenzene-utilizing strains Ralstonia eutropha NH9 (pENH91), Pseudomonas chlororaphis RW71 and Pseudomonas sp. P51 (pP51).

Uptake Kinetics of 2,4-Dichlorophenoxyacetate byDelftia acidovoransMC1 and Derivative Strains: Complex Characteristics in Response to pH and Growth Substrate

“…The tfdA gene is widely distributed in a range of bacterial strains that belong to the and subdivisions of Proteobacteria. [4][5][6][7][8][9][10][11][12] Strains selected for their capability to utilize racemic phenoxypropionate herbicides like (RS)-2-(2,4-dichlorophenoxy)propionate (2,4-DP, dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy)propionate (MCPP, mecoprop) are more rare. Examples are Sphingobium herbicidovorans MH, 8,[13][14][15] Delftia acidovorans MC1, 16,17) Rhodoferax sp.…”

Localization and Characterization of Two Novel Genes Encoding Stereospecific Dioxygenases Catalyzing 2(2,4-Dichlorophenoxy)propionate Cleavage in Delftia acidovorans MC1

“…The identity of the suggested substrate-binding residues was confirmed by site-directed mutagenesis and inactivation studies (8). The tfdA genes are widespread among bacteria (17,31,33,36,39,42,47,53,54,55,56), also among strains unable to degrade 2,4-D (19). Recently, several ␣-proteobacterial strains were shown to carry tfdA␣ genes, which are tfdA-like, showing between 46 and 60% nucleotide identity to the canonical tfdA genes and forming a phylogenetic clade distinct from tfdA (22,23).…”

“…Amplification of tfdB, tfdC, and tfdD gene fragments was performed as described in reference 35. Fragments of tfdE and tfdF were amplified according to the method of Hoffmann et al (17). Sequencing and sequence analysis.…”

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

“…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.)…”