Faculty Opinions recommendation of Molecular characterization of a deletion/duplication rearrangement in tfd genes from Ralstonia eutropha JMP134(pJP4) that improves growth on 3-chlorobenzoic acid but abolishes growth on 2,4-dichlorophenoxyacetic acid.

2001 ◽  
Author(s):  
Peter Williams
Keyword(s):  
Molecular Characterization ◽  
Ralstonia Eutropha ◽  
Dichlorophenoxyacetic Acid ◽  
Chlorobenzoic Acid ◽  
2,4 Dichlorophenoxyacetic Acid

scholarly journals Characterization of a Second tfd Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in Ralstonia eutropha JMP134(pJP4)

2000 ◽  
Vol 182 (15) ◽  
pp. 4165-4172 ◽  
Author(s):  
Caroline M. Laemmli ◽  
Johan H. J. Leveau ◽  
Alexander J. B. Zehnder ◽  
Jan Roelof van der Meer
Keyword(s):  
Ralstonia Eutropha ◽  
Open Reading Frames ◽  
Dichlorophenoxyacetic Acid ◽  
Dot Blot ◽  
Catabolic Plasmid ◽  
Pathway Expression ◽  
Dienelactone Hydrolase ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Extension Analysis

ABSTRACT Within the 5.9-kb DNA region between the tfdR andtfdK genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutropha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (tfdCDEF and tfdB) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows:tfdD II C II E II F IIand tfdB II (in short, thetfd II cluster), by analogy totfdCDEF and tfdB (thetfd I cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown R. eutropha JMP134 identified a single transcription start site in front of the first gene of the cluster, tfdD II, suggesting an operon-like organization for the tfd II genes. By expressing each ORF in Escherichia coli, we confirmed that tfdD II coded for a chloromuconate cycloisomerase, tfdC II coded for a chlorocatechol 1,2-dioxygenase, tfdE II coded for a dienelactone hydrolase, tfdF II coded for a maleylacetate reductase, and tfdB II coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that bothtfd I and tfd II genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfd II genes seem to be redundant with respect to those of the tfd I cluster. One reason why the tfd II genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR andtfdS.

scholarly journals A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil

2000 ◽  
Vol 66 (10) ◽  
pp. 4589-4594 ◽  
Author(s):  
Anthony G. Hay ◽  
James F. Rice ◽  
Bruce M. Applegate ◽  
Nathan G. Bright ◽  
Gary S. Sayler
Keyword(s):  
Ralstonia Eutropha ◽  
Dichlorophenoxyacetic Acid ◽  
Chlorobenzoic Acid ◽  
Whole Cell ◽  
Bioluminescent Response ◽  
2,4 Dichlorophenoxyacetic Acid

ABSTRACT A bioreporter was made containing atfdRPDII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R 2 = 0.9825) in the range of 2.0 μM to 1.1 × 102 μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 102μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.

Characterization of the Reproductive Mode in Guayule In Vitro

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 483a-483
Author(s):  
Roy N. Keys ◽  
Dennis T. Ray ◽  
David A. Dierig
Keyword(s):  
Field Experiments ◽  
Reproductive Mode ◽  
Dichlorophenoxyacetic Acid ◽  
Initial Field ◽  
Breeding Lines ◽  
Reproductive Modes ◽  
Desert Southwest ◽  
2,4 Dichlorophenoxyacetic Acid

Guayule (Parthenium argentatum Gray, Asteraceae) is a latex-producing perennial desert shrub that is potentially of economic importance as an industrial crop for the desert Southwest. It is known to possess complex reproductive modes. Diploids are predominantly sexual and self-incompatible, while polyploids show a range of apomictic potential and self-compatibility. This paper describes the development of a relatively rapid and simple technique for characterizing reproductive modes of breeding lines of P. argentatum. Initial field experiments were based on an auxin test used successfully to characterize reproductive mode in the Poaceae. The application of 2,4-dichlorophenoxyacetic acid inhibited embryo formation in P. argentatum, but this was not the case with other auxins tested. Results of field experiments were ambiguous because: 1) the floral structure of P. argentatum is such that auxins might not have penetrated to the ovules, and 2) there was potential self-fertilization by pollen released within isolation bags. Therefore, in vitro culture of flower heads was tested because it provided much better control of environmental conditions, growth regulator application, and pollen release. Auxin alone, or in combination with gibberellic acid or kinetin, inhibited parthenogenesis in vitro. Embryo production did not vary using two substantially different nutrient media. In vitro flower head culture using a (Nitsch and Nitsch) liquid nutrient medium without growth regulators, enabled characterization of the reproductive mode of seven breeding lines, ranging from predominantly sexual to predominantly apomictic. The results of this technique were substantiated using RAPD analyzes of progeny arrays from controlled crosses.

scholarly journals Characterization of Hapten-Protein Conjugates: Antibody Generation and Immunoassay Development for Chlorophenoxyacetic Acid Pesticides

2009 ◽  
Vol 92 (6) ◽  
pp. 1773-1779 ◽  
Author(s):  
Robin C Boro ◽  
K Vikas Singh ◽  
C Raman Suri
Keyword(s):  
Inhibitory Concentration ◽  
Dichlorophenoxyacetic Acid ◽  
Protein Conjugation ◽  
Protein Conjugates ◽  
Highly Sensitive ◽  
Ic50 Values ◽  
Chlorophenoxyacetic Acid ◽  
2,4 Dichlorophenoxyacetic Acid

Abstract The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1500 ng/mL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

scholarly journals Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

2006 ◽  
Vol 72 (7) ◽  
pp. 4853-4861 ◽  
Author(s):  
Tina A. Müller ◽  
Thomas Fleischmann ◽  
Jan Roelof van der Meer ◽  
Hans-Peter E. Kohler
Keyword(s):  
Amino Acids ◽  
Divalent Cations ◽  
Propanoic Acid ◽  
Dichlorophenoxyacetic Acid ◽  
Purification And Characterization ◽  
Subgroup Ii ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Acid Herbicides ◽  
Growth Experiments

ABSTRACT α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His6-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX24TX131HX10R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent Km values were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparent Km values for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAα-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.

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