http://bioinformatica.uab.es/biocomputacio/treballs02-03/rburgos/dades/articulos/engieneria.pdf

Engineering bacteria for bioremediation.

The subject invention provides novel plants that are not only resistant to 2,4-D and other phenoxy auxin herbicides, but also to aryloxyphenoxypropionate herbicides. Heretofore, there was no expectation or suggestion that a plant with both of these advantageous properties could be produced by the introduction of a single gene. The subject invention also includes plants that produce one or more enzymes of the subject invention alone or 'stacked' together with another herbicide resistance gene, preferably a glyphosate. resistance gene, so as to provide broader and more robust weed control, increased treatment flexibility, and improved herbicide resistance management options. More specifically, preferred enzymes and genes for use according to the subject invention are referred to herein as AAD (aryloxyalkanoate dioxygenase) genes and proteins. No α-ketoglutarate-dependent dioxygenase enzyme has previously been reported to have the ability to degrade herbicides of different chemical classes and modes of action. This highly novel discovery is the basis of significant herbicide tolerant crop trait opportunities as well as development of selectable marker technology. The subject invention also includes related methods of controlling weeds. The subject invention enables novel combinations of herbicides to be used in new ways. Furthermore, the subject invention provides novel methods of preventing the formation of, and controlling, weeds that are resistant (or naturally more tolerant) to one or more herbicides such as glyphosate.

Novel herbicide resistance genes

Is biological treatment a viable alternative for micropollutant removal in drinking water treatment processes

This review highlights the analytical chemistry, environmental occurrence, and environmental fate of individual stereoisomers of chiral emerging pollutants, which are modern current-use chemicals of growing environmental concern due to their presence in the environment and potential for deleterious effects. Comparatively little is known about individual stereoisomers of pollutants, which can have differential toxicological effects and can be tracers of biochemical weathering in the environment. Stereoisomers are resolved by gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Separation techniques in environmental analysis are typically coupled to mass spectrometry (MS) and tandem mass spectrometry (MS/MS), as these provide the sensitivity and selectivity needed. The enantiomer composition of phenoxyalkanoic and acetamide herbicides, organophosphorus and pyrethroid pesticides, chiral polychlorinated biphenyl metabolites, synthetic musks, hexabromocyclododecane, and pharmaceuticals in the environment show species-dependent enantioselectivity from biotransformation and other biologically mediated processes affecting enantiomers differentially. These enantiomer compositions are useful in detecting biologically mediated environmental reactions, apportioning sources of pollutants, and gaining insight into the biochemical fate of chiral pollutants in the environment, which are needed for accurate risk assessment of such chemicals.

Environmental fate processes and biochemical transformations of chiral emerging organic pollutants

The subject invention provides novel plants that are not only resistant to 2,4-D and other phenoxy auxin herbicides, but also to aryloxyphenoxypropionate herbicides. Heretofore, there was no expectation or suggestion that a plant with both of these advantageous properties could be produced by the introduction of a single gene. The subject invention also includes plants that produce one or more enzymes of the subject invention alone or “stacked” together with another herbicide resistance gene, preferably a glyphosate resistance gene, so as to provide broader and more robust weed control, increased treatment flexibility, and improved herbicide resistance management options. More specifically, preferred enzymes and genes for use according to the subject invention are referred to herein as AAD (aryloxyalkanoate dioxygenase) genes and proteins. No α-ketoglutarate-dependent dioxygenase enzyme has previously been reported to have the ability to degrade herbicides of different chemical classes and modes of action. This highly novel discovery is the basis of significant herbicide tolerant crop trait opportunities as well as development of selectable marker technology. The subject invention also includes related methods of controlling weeds. The subject invention enables novel combinations of herbicides to be used in new ways. Furthermore, the subject invention provides novel methods of preventing the formation of, and controlling, weeds that are resistant (or naturally more tolerant) to one or more herbicides such as glyphosate.

Herbicide resistance genes

Research on the biodegradation of chiral xenobiotics can provide a better understanding of the processes that govern enantioselectivity in the microbial degradation and environmental fate of stereoisomers. We have examined the microbial degradation of the widely used chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by a pure bacterial strain that was isolated from soil. This strain, Sphingomonas herbicidovorans MH, can utilize both enantiomers of mecoprop as sole carbon and energy sources for growth (24). Growth experiments with the pure enantiomers as well as with racemic mecoprop revealed that the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. Based on these results, we concluded that specific catabolic enzymes must be involved in the degradation of each enantiomer of mecoprop (24). This view was confirmed by the finding that two α-ketoglutarate-dependent dioxygenases are involved in the enantioselective degradation of mecoprop in S. herbicidovorans MH, one that is specific for (R)-mecoprop and one that is specific for (S)-mecoprop (17). The (S) enantiomer-specific dioxygenase activity is constitutively expressed, whereas the (R) enantiomer-specific enzyme activity is present only when cells grow on the (R) enantiomer. Both enzymes are present in cells grown on the racemic mixture. Extracts of cells grown on complex medium and extracts of cells grown on (S)-mecoprop contain equally high activities of the (S) enantiomer-specific dioxygenase (17). However, intact cells grown on complex medium show very low oxygen uptake rates with (S)-mecoprop, whereas (S)-mecoprop-grown cells show high oxygen consumption with (S)-mecoprop (24). These findings suggested the existence of specific uptake systems and led us to examine the uptake of phenoxyalkanoic acids by S. herbicidovorans after growth on various substrates.

Many aromatic compounds are taken up by bacteria through energy-dependent transport systems (4, 5, 10, 11, 15, 16). The uptake of 4-chlorobenzoate by the coryneform bacterium NTB-1 is inducible and coupled to the proton motive force (4). The uptake of 4-toluene sulfonate by Comamonas testosteroni T-2 is also inducible and shows substrate saturation kinetics (11). Transport of 4-toluene sulfonate is inhibited completely by uncouplers but only marginally by ATPase inhibitors, and the authors proposed that 4-toluene sulfonate is taken up by an inducible secondary proton symport system (11). The uptake of 4-hydroxybenzoate by Pseudomonas putida is driven by the proton motive force, and the gene that encodes a 4-hydroxybenzoate transporter was identified, cloned, and expressed in Escherichia coli. Furthermore, it was found that 4-hydroxybenzoate transport is an integral feature of the β-ketoadipate pathway in P. putida (5, 15, 16). The uptake of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) by Ralstonia eutropha JMP134 is inducible, is sensitive to metabolic inhibitors, and shows substrate saturation kinetics (10). Moreover, the authors identified a gene, designated tfdK, whose translation product shows resemblance to transport proteins of the major facilitator superfamily, and they suggested that tfdK encodes the 2,4-D transporter of R. eutropha JMP134.

Our results indicate that S. herbicidovorans MH has two distinct uptake systems for chiral phenoxypropionic acid herbicides, one for the (R) enantiomer and another one for the (S) enantiomer of dichlorprop and mecoprop. Furthermore, evidence was obtained that 2,4-D was taken up by a third transport system.

Enantioselective uptake and degradation of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid] by Sphingomonas herbicidovorans MH.

Mecoprop and dichlorprop, two chiral herbicides, are enantioselectively degraded by Sphingomonas herbicidovorans MH. Experiments on the biochemistry of mecoprop and dichlorprop metabolism in this bacterium show that one reason for enantioselectivity is the existence of two distinct α-ketoglutarate-dependent dioxygenases—one that is specific for the R enantiomers and one that is specific for the S enantiomers of mecoprop and dichlorprop. Uptake experiments with radiolabeled dichlorprop provide evidence that the first step in the degradation of dichlorprop and mecoprop by strain MH is active transport and that two inducible, proton gradient-driven uptake systems exist—one uptake system for (R)-dichlorprop and (R)-mecoprop and another one for (S)-dichlorprop and (S)-mecoprop. This indicates that the enantioselective degradation of mecoprop and dichlorprop by the soil isolate S. herbicidovorans MH is not only due to enantioselective metabolism but also to enantioselective uptake. Because selective enrichment of one of the enantiomers may occur in the environment it is important to assess the environmental impact of stereoisomers separately. A field study of chiral mecoprop in a contaminated aquifer downstream of a former waste disposal site in Switzerland supports this notion as the study clearly shows that the R enantiomer enriches during groundwater passage of the landfill leachate that was polluted with racemic mecoprop. We conclude that enantioselective microbial degradation increases the enantiomeric ratio of (R)- to (S)-mecoprop and suggest that analysis of enantiomeric ratios of chiral contaminants such as mecoprop can provide information about in situ biodegradation processes.

Monika Bunk

Papers

Enantioselective uptake and degradation of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid] by Sphingomonas herbicidovorans MH.

Microbial Transformation of the Chiral Pollutants Mecoprop and Dichlorprop