Abundance and Expression of Enantioselective rdpA and sdpA Dioxygenase Genes during Degradation of the Racemic Herbicide ( R , S )-2-(2,4-Dichlorophenoxy)Propionate in Soil

Abstract

The rdpA and sdpA genes encode two enantioselective ␣-ketoglutarate-dependent dioxygenases catalyzing the initial step of microbial degradation of the chiral herbicide (R,S)-2-(2,4-dichlorophenoxy)propionate (R,Sdichlorprop). Primers were designed to assess abundance and transcription dynamics of rdpA and sdpA genes in a natural agricultural soil. No indigenous rdpA genes were detected, but sdpA genes were present at levels of approximately 10 3 copies g of soil ؊1 . Cloning and sequencing of partial sdpA genes revealed a high diversity within the natural sdpA gene pool that could be divided into four clusters by phylogenetic analysis. BLASTp analysis of deduced amino acids revealed that members of cluster I shared 68 to 69% identity, cluster II shared 78 to 85% identity, cluster III shared 58 to 64% identity, and cluster IV shared 55% identity to their closest SdpA relative in GenBank. Expression of rdpA and sdpA in Delftia acidovorans MC1 inoculated in soil was monitored by reverse transcription quantitative real-time PCR (qPCR) during in situ degradation of 2 and 50 mg kg ؊1 of (R,S)-dichlorprop. (R,S)-Dichlorprop amendment created a clear upregulation of both rdpA and sdpA gene expression during the active phase of 14 C-labeled (R,S)-dichlorprop mineralization, particularly following the second dose of 50 mg kg ؊1 herbicide. Expression of both genes was maintained at a low constitutive level in nonamended soil microcosms. This study is the first to report the presence of indigenous sdpA genes recovered directly from natural soil and also comprises the first investigation into the transcription dynamics of two enantioselective dioxygenase genes during the in situ degradation of the herbicide (R,S)-dichlorprop in soil.

(R,S)‐dichlorprop herbicide in agricultural soil induces proliferation and expression of multiple dioxygenase‐encoding genes in the indigenous microbial community

“…Interestingly, rdpA and sdpA were still transcribed following the initial degradation event which is in agreement with our previous findings where low levels of constitutive gene expression were detected from D. acidovorans MC1 both prior to and after ( R,S )‐dichlorprop degradation but were further induced when re‐exposed to the herbicide (Paulin et al ., 2010). Previous pure culture studies have also hinted towards constitutive expression of both genes through measurement of catalytic enzyme activity (Müller et al ., 2001; 2004).…”

“…This band was exclusively associated with soil DNA extracts and was absent from pure culture DNA extracts. Sequencing and comparison to the GenBank database revealed that clones of this band showed closest match (≥ 90% identity) to uncultured bacterial clones of sdpA previously described in the same soil receiving no herbicide treatment (Paulin et al ., 2010). However, these sequences were not detected in the mRNA samples and were presumably not actively involved in ( R,S )‐dichlorprop degradation.…”

“…Our findings are in agreement to this and are the first to demonstrate the importance of rdpA gene sequences during in situ degradation of phenoxypropionate herbicides in soil. In contrast, the natural sdpA gene pool has recently been reported as highly diverse in soils receiving no herbicides (Paulin et al ., 2010). While this diversity was reflected in the DNA DGGE profiles herein, one band in particular was dominant and consistently detected throughout the course of the experiment regardless of herbicide presence.…”

Establishment of Bacterial Herbicide Degraders in a Rapid Sand Filter for Bioremediation of Phenoxypropionate-Polluted Groundwater

“…The strain was grown under selective conditions in mineral salts solution supplemented with 400 mg/liter (R/S)-DCPP, as previously described (22). Duplicate 10-fold dilutions of the D. acidovorans MC1 culture were prepared in a 0.9% NaCl solution, and 100 l of each dilution was spiked into tubes with 500 mg fresh sand (like that used to prepare the sand filters).…”

“…The numbers of rdpA and sdpA phenoxypropionate degrader genes and mRNA transcripts were quantified by qPCR using a CFX96 real-time system (Bio-Rad Laboratories, USA) or an iCycler thermocycler (Bio-Rad). PCR mixtures were prepared with 1ϫ SsoFast EvaGreen supermix (BioRad), 1 mg/ml BSA (Bioron), 1 l DNA or cDNA template, and primers rdpA_f and rdpA_r (0.4 M each for the rdpA assay) or sdpA_f (0.4 M) and a mixture of sdpA_r1 and sdpA_r2 (0.2 M each for the sdpA assay), as previously described (22). PCR cycling conditions were as follows: 98°C for 2 min, 40 cycles of 98°C for 10 s and 55°C for 20 s (CFX96) or 30 s (iCycler), and 72°C for 2 min.…”