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

Schleinitz, Kathleen M.; Kleinsteuber, Sabine; Vallaeys, Tatiana; Babel, Wolfgang

doi:10.1128/aem.70.11.6948.2004

Cited by 16 publications

(18 citation statements)

References 45 publications

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“…Recently, sequence analyses of cosmid clones from the MC1 plasmid indicated an open reading frame (ORF) which was located upstream to the rdpA gene. This has similarity by 29% to tfdK (Schleinitz et al 2004). The TfdK protein, in turn, is a member of the Major Facilitator Superfamily (MFS) transporter and belongs to the sub-family of aromatic acid:H + symporter (AAHS) (Pao et al 1998).…”

Section: Discussionmentioning

confidence: 86%

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Hoffmann

Müller

2006

Biodegradation

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Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain's degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (mu max) of 0.158 h(-1). The half-maximum rate-associated substrate concentration (Ks) was 45 microM. At pH 8.5 mu max was only 0.05 h(-1) and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that mu max with dichlorprop was around 0.2 h(-1) at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with mu max of 0.147 h(-1) and Ks of 267 microM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 microM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)-2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h(-1) at pH 6.8 and up to D = 0.2 h(-1) at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.

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Section: Discussionmentioning

confidence: 86%

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Hoffmann

Müller

2006

Biodegradation

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“…Since very few studies have targeted rdpA genes, sequence information to date is limited and comes from just a few cultured strains including S. herbicidovorans MH (Müller et al ., 2004), D. acidovorans MC1 and Rhodoferax sp. P230 (Schleinitz et al ., 2004), and a Burkhoderia sp. isolate (Zakaria et al ., 2007).…”

Section: Discussionmentioning

confidence: 99%

“…Nucleic acids were then extracted as described above and 1/10 dilutions of DNA were used as templates for qPCR. The target genes in both organisms are single copy in the cells (Schleinitz et al ., 2004; Pérez‐Pantoja et al ., 2008). Consequently, DNA standard curves were prepared from DNA/RNA extractions of cell dilutions and used to quantify both DNA and mRNA from rdpA , sdpA or tfdA with mRNA being quantified as DNA gene equivalents and presented as ratios of mRNA normalized to DNA as previously described (Nicolaisen et al ., 2008).…”

Section: Methodsmentioning

confidence: 99%

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

Paulin

Nicolaisen

Sørensen

2011

Environmental Microbiology

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We investigated the effect of (R,S)-dichlorprop herbicide addition to soil microcosms on the degrading indigenous microbial community by targeting multiple α-ketoglutarate-dependent (α-KG) dioxygenase-encoding genes (rdpA, sdpA and tfdA group I) at both gene and transcript level. The soil microbial community responded with high growth of potential degraders as measured by the abundance of dioxygenase-encoding genes using quantitative real-time PCR (qPCR). rdpA DNA was not detectable in unamended soil but reached over 10⁶ copies g⁻¹ soil after amendment. sdpA and tfdA were both present prior to amendment at levels of ~5 × 10⁴ and ~ 10² copies g⁻¹ soil, respectively, and both reached over 10⁵copies g⁻¹ soil. While expression of all three target genes was detected during two cycles of herbicide degradation, a time-shift occurred between maximum expression of each gene. Gene diversity by denaturing gradient gel electrophoresis (DGGE) uncovered a diversity of sdpA and tfdA genes at the DNA level while rdpA remained highly conserved. However, mRNA profiles indicated that all transcribed tfdA sequences were class III genes while rdpA transcripts shared 100% identity to rdpA of Delftia acidovorans MC1 and sdpA transcripts shared 100% identity to sdpA from Sphingomonas herbicidovorans MH. This is the first report to describe expression dynamics of multiple α-KG dioxygenase-encoding genes in the indigenous microbial community as related to degradation of a phenoxypropionate herbicide in soil.

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“…(R)-2-(2,4-dichlorophenoxy)propionate/ α-ketoglutarate-dependent dioxygenases (RdpA), which attacks the R-enantiomers of 2-phenoxypropionates, is identical in the primary amino acid sequence in Delftia acidovorans MC1, Sphingobium herbicidovorans MH, and Rhodoferax sp. P230, as follows from the rdpA gene sequences [9]. Nevertheless, significant differences in the kinetic parameters of RdpA synthesized in these three strains and upon heterologous expression in E. coli [10] were found, based on strain-dependent K m (Michaelis-Menten constant) values for 4-chloro-2-methylphenoxyacetate and 2,4-D.…”

Section: Introductionmentioning

confidence: 96%

Posttranslational oxidative modification of (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA) leads to improved degradation of 2,4‐dichlorophenoxyacetate (2,4‐D)

Leibeling

Maess

Centler

et al. 2013

Engineering in Life Sciences

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Microbial activities and the versatility gained through adaptation to xenobiotic compounds are the main biological forces to counteract environmental pollution.The current results present a new adaptive mechanism that is mediated through posttranslational modifications. Strains of Delftia acidovorans incapable of growing autochthonously on 2,4-dichlorophenoxyacetate (2,4-D) were cultivated in a chemostat on 2,4-D in the presence of (R)-2-(2,4-dichlorophenoxy)propionate. Long-term cultivation led to enhanced 2,4-D degradation, as demonstrated by improved values of the Michaelis-Menten constant K m for 2,4-D and the catalytic efficiency k cat /K m of the initial degradative key enzyme (R)-2-(2,4-dichlorophenoxy)propionate/ α-ketoglutarate-dependent dioxygenases (RdpA). Analyses of the rdpA gene did not reveal any mutations, indicating a nongenetic mechanism of adaptation. 2-DE of enzyme preparations, however, showed a series of RdpA forms varying in their pI. During adaptation increased numbers of RdpA variants were observed. Subsequent immunoassays of the RdpA variants showed a specific reaction with 2,4-dinitrophenylhydrazine (DNPH), characteristic of carbonylation modifications. Together these results indicate that posttranslational carbonylation modified the substrate specificity of RdpA. A model was implemented explaining the segregation of clones with improved degradative activity within the chemostat. The process described is capable of quickly responding to environmental conditions by reversibly adapting the degradative potential to various phenoxyalkanoate herbicides.Keywords: 2,4-Dichlorophenoxyacetate (2,4-D) / (R)-2-(2,4-Dichlorophenoxy)propionate / α-ketoglutarate-dependent dioxygenases (RdpA) / Individual-based modeling / Posttranslational carbonylationAdditional supporting information may be found in the online version of this article at the publisher's web-site

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

Cited by 16 publications

References 45 publications

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

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

Posttranslational oxidative modification of (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA) leads to improved degradation of 2,4‐dichlorophenoxyacetate (2,4‐D)

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