EP0972074A1 - Procede pour identifier le site d'action d'agents chimiques xenobiotiques - Google Patents

Procede pour identifier le site d'action d'agents chimiques xenobiotiques

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Publication number
EP0972074A1
EP0972074A1 EP98907633A EP98907633A EP0972074A1 EP 0972074 A1 EP0972074 A1 EP 0972074A1 EP 98907633 A EP98907633 A EP 98907633A EP 98907633 A EP98907633 A EP 98907633A EP 0972074 A1 EP0972074 A1 EP 0972074A1
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EP
European Patent Office
Prior art keywords
detector
bacteria
gene
genotoxic
stressor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98907633A
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German (de)
English (en)
Inventor
David Allen Elsemore
Robert Alan Larossa
Dana Robin Smulski
Tina Kangas Van Dyk
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EIDP Inc
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EI Du Pont de Nemours and Co
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Application filed by EI Du Pont de Nemours and Co filed Critical EI Du Pont de Nemours and Co
Publication of EP0972074A1 publication Critical patent/EP0972074A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • TITLE A METHOD FOR IDENTIFYING THE SITE OF ACTION OF XENOBIOTIC CHEMICALS FIELD OF INVENTION The invention relates to field of molecular biology and to methods for screening-compounds for biological activity. More specifically, a method has been developed to rapidly identify the site of action of various xenobiotic compounds in particular, xenobiotic agrochemicals and antimicrobials. BACKGROUND OF THE INVENTION Technological advances within the chemical arts have made possible the synthesis of vast arrays of chemical compounds of interest to the agrochemical, pharmaceutical and environmental industries. These methods of synthesis are capable of producing far more compounds than can be reasonably screened to identify their utility.
  • This method precisely defines the site of sulfonylurea herbicide action in Salmonella typhimurium LT2.
  • the method uses lawns of bacterial cells grown on solid media and exposed to effective concentrations of herbicide. Sulfonylurea herbicides inhibit Salmonella growth by inhibiting the branched chain amino acid biosynthetic pathway. Nutrient supplementation overcome specific amino acid deficiencies imposed by herbicidal action. Results are seen in about 48 hr.
  • the method of LaRossa et al. is useful for determining a site of action for a specific active xenobiotic compound but, like whole plant assays, is time consuming and requires large amounts of test compound to be screened.
  • Genetic titration in a microbial population offers an alternative to nutritional reversal. Genetic titration has been used during the past 40 years. Selection of regulatory mutations that increase the titer of pathway enzymes in response to a challenge with an antimetabollite are well known in the E. coli and S. typhimurium literature. Gene duplications have been selected in both mammalian cell lines (using drugs and transition state analogs) and with antimetabolites in bacteria. These selections are based upon the increased enzyme content of the cell titering out the deleterious effect of the inhibitor.
  • Genetic titration proceeds generally by first preparing DNA libraries of a bacterial genome in multicopy cloning vectors where each portion of the genome is highly amplified. Bacterial hosts are transformed with the library and screened for growth on plates containing effective inhibitory concentrations of the xenobiotic compound to be tested. Colonies are picked and the plasmid isolated and sequenced. The sequence is compared to known sequences to identify genes that could encode a site of action for the compound.
  • Bioluminescent reporters are l ⁇ iown as toxicological detectors in the art.
  • One of the most common are genes encoding the firefly luciferase.
  • Another is a set of five genes, luxCDABE, that has been isolated from the bioluminescent bacteria Vibrio fischeri. Both eukaryotic and prokaryotic genes have been used in recombinant systems to serve as detectors.
  • cDNA encoding firefly luciferase has been expressed in E. coli under the control of the lacZ promoter [Tatsumi et al., Biochem.
  • recombinant bacteria have been developed by fusing the lux gene complex to chemically responsive bacterial promoters and then placing such chimeras in appropriate hosts. These recombinant bacteria are sensor organisms that glow in response to specific stimuli.
  • indicator bacterial strains are provided (by vector-mediated gene transfer) containing a mer promoter, specifically inducible by Hg ions, fused to a bacterial luciferase (lux AB) genes complex which is responsible for bioluminescence.
  • a mer promoter specifically inducible by Hg ions
  • lux AB bacterial luciferase
  • USSN 08/244,376 teaches the use of detector organisms containing a stress promoter-bioluminescent gene fusion to detect various environmental stresses including those sensitive to protein damage (heat shock), DNA damage (genotoxic), oxidative damage, cell membrane damage, amino acid starvation, carbon starvation, and nitrogen starvation.
  • USSN 08/344,428 demonstrates the use of similarly transformed detector cells as lyophilized reagents.
  • the detector bacteria comprising a genotoxic-sensitive promoter operably linked to a luminescent reporter gene complex to form a gene fusion that confers a bioluminescent positive phenotype upon the detector bacteria wherein exposure of the genotoxic- sensitive promoter to genotoxic compounds drives heightened expression of the luminescent reporter gene complex producing an increased bioluminescent signal;
  • step (ii) selecting for genotoxic or non-genotoxic stressors capable of inhibiting the growth of the detector bacteria of step (i) by monitoring the growth and light output of the detector bacteria;
  • step (iii) submitting the growth-inhibiting, genotoxic or non-genotoxic stressor selected in step (ii) to a site of action screen;
  • the present invention further provides detector bacteria strains comprising a recA-LuxCDABE gene fusion as well as non-bioluminescent parent strains possessing a multiplicity of cellular and membrane mutations.
  • the invention further provides methods for determining whether a compound is genotoxic and comprising the steps of: (i) culturing a detector cell comprising a promoter regulated by a
  • SOS bacterial regulatory circuit and a luxCDABE gene complex wherein the luxCDABE gene complex is positioned in the bacterial chromosome downstream of the SOS promoter such that when the SOS promoter is expressed, then the luxCDABE gene complex is also expressed; (ii) contacting the culture with a substance to be tested and; (iii) determining whether the substance is genotoxic by measuring the amount of luminescence in the culture.
  • the invention additionally provides methods of identifying a. structural gene encoding a stressor target comprising:
  • the bacteria comprising a genotoxic-sensitive promoter operably linked to a luminescent reporter gene complex to form a gene fusion that confers a bioluminescent positive phenotype upon the detector bacteria wherein exposure of the genotoxic- sensitive promoter to genotoxic compounds drives heightened expression of the luminescent reporter gene complex producing an increased bioluminescent signal;
  • the invention provides methods for identifying compounds having glyphosate-like activity, thienylalanine-like activity and ALS-inhibitory activity.
  • FIG. 1 (flow chart-strains la and lb) is a diagram showing the genealogies of the detector cells used in the present invention. Solid arrows indicate construct of non-bioluminescent strains. Broken arrows indicate construction of bioluminescent derivatives. The selection and screens used to isolate bacteria are indicated in capitalized and italicized typeface. Relevant genotypes are italicized, igm indicates improved growth on minimal medium.
  • Figure 2 (method chart) is a flow diagram illustrating the method of the present invention of screening a compound for genotoxicity using the instant detector cells and determining the site of action of non-genotoxic compounds by nutritional reversal of genetic titration.
  • Figure 3 is an illustration of the method for in vivo inhibitor identification of specified targets showing the screening of a compound for a known activity.
  • Figure 4a is a plot of RLU vs. time of strain DPD 1715 containing the ilvB, ilvl, ilvH, ilvG relA and spoT mutations and the recA-LuxCDABE and being tolC+, exposed to the DNA damaging agent mitomycin C.
  • Figure 4b is a plot of RLU vs. time of strain DPD1730 containing the recA-LuxCDABE and tolC+ exposed to the DNA damaging agent mitomycin C.
  • Figure 5 is a kinetic plot of RLU vs. mitocycin C concentration comparing the sensitivity of tolC- and tolC+ stains to mitomycin C.
  • Figure 6 is a plot of RLU vs. time for the strain DPD1718 containing the RecA-LuxCDABE gene fusion, exposed to varying concentrations of . 2,4-dichlorophenoxyacetic acid.
  • Figure 7 is a plot of RLU vs. time for the strain DPD1718 containing the recA-LuxCDABE gene fusion, exposed to varying concentrations of sulfometuron methyl.
  • Figures 8(a)and (b) show plots of RLU vs. time for the strain DPD1715 containing the ilvB, ilvl, ilvH, ilvG relA and spo T mutations and the recA-LuxCDABE and being tolC+ exposed to varying concentrations of glyphosate.
  • Figure 9 is a plot of RLU vs. time for the strain DPD1730 containing the recA-LuxCDABE gene fusion, and the ilvB ilvG UvIHrelA spoTtolC+ and spoT mutations exposed to varying concentrations of methyl viologen.
  • Figure 10(a) is a plot of RLU vs. time for the strain DPD1715 containing the ilvB, ilvl, ilvH, ilvG relA and spoT mutations and the recA-LuxCDABE and being tolC+ exposed to varying concentrations of Sulfometuron methyl.
  • Figure 10(b) is a plot of RLU vs. time for the strain DPD1728 containing the igm relA and spoT mutations and the recA-LuxCDABE and being tolC- exposed to varying concentrations of sulfometuron methyl.
  • Figure 10(c) is a kinetic plot of bioluminescent measure at 80min vs. concentration of sulfometuron methyl comparing the responses of strains DPD1715 and DPD1728.
  • Figure 11 (a) is a plot of RLU vs. time for the strain DPD 1718 containing the recA-LuxCDABE gene fusion, exposed to varying concentrations of 3-(2- thienyl)-L-alanine in minimal medium.
  • Figure 11(b) is a plot of RLU vs. time for the strain DPD1718 containing the recA-LuxCDABE gene fusion, exposed to varying concentrations of 3-(2- thienyl)-L-alanine in Rich LB medium.
  • Figure 12(a) is a plot of RLU vs. time for the strain DPD1718 containing the recA-LuxCDABE gene fusion, exposed to varying concentrations of the oxime carbamate compound OC in minimal medium.
  • Figure 12(b) is a plot of RLU vs. time for the strain DPD 1718 containing the recA-LuxCDABE gene fusion, exposed to varying concentrations of the oxime carbamate compound OC in Rich LB medium.
  • Figure 13(a) is aplot of RLU vs. time for the strain DPD1718 containing the recA-LuxCDABE gene fusion, exposed to varying concentrations of glyphosate in minimal medium.
  • Figure 13(b) is a plot of RLU vs. time for the strain DPD1718 containing the recA-LuxCDABE gene fusion, exposed to varying concentrations of glyphosate in Rich LB medium.
  • Figure 14 is a plot of RLU vs. time for the strain DPD1718 containing the recA-LuxCDABE gene fusion, and the relA and ⁇ lvB mutations demonstrating reversal of ' 3-(2-Thienyl)-L-alanine induced inhibition of bioluminescence by cystein.
  • Figure 15(a) is a plot of RLU vs. time for strain DPD 1718 showing light inhibition by sulfometuron methyl.
  • Figure 15(b) is a plot of RLU vs. time for strain DPD1718 showing restoration of light output after inhibition by sulfometuron methyl as a result of nutritional reversal by the pool comprising the three branched chain amino acids, lysine and histidine.
  • Figure 16 is a plot of bioluminescent response ratio vs. concentration of thienylalanie contacted with strains transformed with 0245 ygaH, phenA, and aroH genes.
  • the transformed E. coli strains DPD 1707, DP 1675, and DPD1718 were deposited on 13 February 1997 with the American Type Culture Collection ("ATCC"), international depository located at 12301 Parklawn Drive, Rockville, MD 20852 U.S.A. under the terms of the Budapest Treaty.
  • ATCC American Type Culture Collection
  • the strains are designated as ATCC 98328, ATCC 98329, and ATCC 98330 respectively.
  • the designations refer to the accession number of the deposited material.
  • Applicants are the first to recognize that stress promoter-Li gene fusions will be useful in enhancing traditional methods of screening for compound sites of action and the first to develop a detector organism capable of dual functionality in such an assay where the organism is able to denote genotoxic compounds in a first stage and identify site of action in a second assay.
  • Applicants have developed a method for discovering the site of action of particular xenobiotic compounds by using a bacterial detector cell having a mutation that confers sensitivity to amino acid starvation and containing a gene fusion comprising a genotoxic-sensitive promoter operably linked to a bacterial ⁇ bioluminescent reporter gene complex.
  • Typical xenobiotics include chemicals active as herbicides, anticancer agents, antimicrobials and crop protection chemicals.
  • the method has high utility in the agrochemical and pharmaceutical industries for identifying the site of action of compounds and for designing new compounds of similar structure and/or function.
  • xenobiotic compounds that are genotoxic interact with the genotoxic-sensitive promoter (driving transcription of the bioluminescent reporter gene complex) and produce an increase in light.
  • Higher concentrations of non- genotoxic xenobiotic compounds may result in the interference of metabolic activity and a decrease in light production.
  • the present method rapidly determines the site of action of a particular compound via a two part screen involving a uniquely constructed detector cell.
  • the detector cell may posses a relA mutation (responsible for diminishing the host cell's response to amino acid starvation) and a lux gene fusion comprising a genotoxic-sensitive promoter operably fused to a bacterial lux gene complex.
  • a relA mutation response to diminishing the host cell's response to amino acid starvation
  • a lux gene fusion comprising a genotoxic-sensitive promoter operably fused to a bacterial lux gene complex.
  • stage two Compounds that result in decreased bioluminescent output relative to the mock-treated control and do not elevate cellular bioluminescence at any tested concentration are subjected to stage two.
  • the second stage uses the detector organism in standard nutritional reversal and genetic titration screens, making use of the bioluminescent gene fusion to identify those nutrients or genes whose supplementation results in prevention of the metabolic interference associated with the xenobiotic compound.
  • the present method demonstrates for the first time that auxanographic reversal of chemical stressor action can be signaled by restoration of light production of a detector cell strain and that patterns of reversal by defined nutrient pools can define pathways inhibited by the stressor chemical.
  • Advantages of the method include increased numbers of compound screenings per unit of time, increased speed of the biological response, and ease of automation of data collection and processing, while decreasing by a factor of approximately 300 times the amount of compound required for analysis.
  • CPC crop protection chemical
  • pesticides paraquat (methyl viologen), copper sulfate, metidathion
  • anti-pathogenic compounds such as fungicides (chlorothalonil, 2-thienylalanine) and profungicides (Oxime Carbamates) or compounds, responsible for insect behavior modulation (pheromones, allomones and kairomones), and herbicides referring to compounds having specific or general toxicity to plant species.
  • Typical herbicides include but are not limited to the class of sulfonylurea herbicides and sulfonanilide herbicides (chlorsulfuron, triasulfuron, metsulfuron-methyl), auxin herbicides (e.g., dicamba, 2-methyl-4-chlorophenoxyacetic acid, picloram, quinclorac, quinmerac), pre- emergence herbicides (metribuzin), and post-emergence herbicides (Clethodim Pendimethalin, oryzalin, dithiopyr, oxadiazon, prodiamine, and 2,4-dichlorophenoxyacetic acid).
  • auxin herbicides e.g., dicamba, 2-methyl-4-chlorophenoxyacetic acid, picloram, quinclorac, quinmerac
  • pre- emergence herbicides metribuzin
  • post-emergence herbicides Clethodim Pendimethalin, oryzalin, di
  • Sulfonylurea herbicides are defined as N-(heterocyclicaminocarbonyl)- arylsulfonamide-containing herbicidal compounds that inhibit the enzyme acetolactate synthase, such as sulfometuron methyl.
  • sulfometuron methyl refers to 2-[[[[[(4,6-dimethyl-2- pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoic acid, methyl ester (CAS registry number 74222-97-2), and is abbreviated as "SM”.
  • ALS a key enzyme responsible for branched chain amino acid biosynthesis.
  • Glyphosate will be abbreviated “GP”, has the CAS registry number 1071 -83-6 and is a herbicide whose site of action is 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) which catalyzes the conversion of shikimate into anthranilate, a key transformation in plant amino acid synthesis.
  • EPSPS 5-enolpyruvylshikimate-3- phosphate synthase
  • thienylalanine-like activity means any substance, natural or synthetic that has the fungicidal activity of chlorothalonil, 2-thienylalanine.
  • glycopohosate-like activity means any substance, natural or synthetic that acts the interfere with 5-enolpyruvylshikimate-3-phosphate synthase activity.
  • ALS-inhibiting activity means any substance, natural or synthetic that inhibits the activity of acetolactate synthetase or expression of the gene encoding acetolactate synthetase.
  • a “luminescent reporter gene complex” means any reporter gene(s) the products of which result in light production. Examples include but are not limited to the bacterial lux genes; the luciferase genes (luc), from, for example, the firefly (Photinus pyralis) or click beetle (Pyrophorus plagiophthalamus); or the gene encoding the luciferase from the sea pansy (Renilla reniformis).
  • Site of action refers to the macromolecular target of a particular stressor or xenobiotic compound. Typical sites of action are specific enzymes in a particular biosynthetic pathway.
  • the terms "plasmid”, “vector”, and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and are usually in the form of circular double-stranded DNA molecules.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single-, or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • Integrant refers to a bacterial strain into whose chromosome has been inserted a foreign gene fragment.
  • multiple copies or “multicopy” as it pertains to the presence of expressible genes in an organisms means a number of copies of the gene that exceeds the normal complement of that gene in the cell.
  • transformation and “transfection” refer to the acquisition of new genes in a cell as a result of the incorporation of nucleic acid.
  • the acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences.
  • transformant refers to the product of a transformation.
  • promoter and “promoter region” refer to a sequence of DNA, usually upstream of (5' to) the protein coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at the correct site. Promoter sequences are necessary but not always sufficient to drive the expression of the gene.
  • Genetoxic sensitive promoter refers to a promoter activated by DNA damage.
  • these promoters include but are not limited to recA, uvrA, lexA, umuDC, uvrA, uvrB, uvrC, sulA, recN, uvrD, ruv, alkA, ada, dinA, dinB, dinD, and dinF as well as other promoters which are members of the adaptive response regulon group such as those disclosed by Rupp in E. coli and Salmonella; Cellular and Molecular Biology [Niedhardt et al., Eds., pp 1190-1220, American Society of Microbiology, Washington, D.C.
  • a “fragment” constitutes a fraction of the DNA sequence of the particular region.
  • Regulation and “regulate” refer to the modulation of gene expression controlled by DNA sequence elements located primarily, but not exclusively, upstream of (5' to) the transcription start of a gene. Regulation may result in an "all or none" response to a stimulation, or it may result in variations in the level of gene expression.
  • operably linked refers to the fusion of two fragments of DNA in a proper orientation and reading frame to be transcribed into functional RNA.
  • expression refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.
  • a DNA chain coding for the sequence of gene product is first transcribed to a complimentary RNA which is often a messenger RNA and, then, the thus transcribed messenger RNA is translated into the above-mentioned gene product if the gene product is a protein.
  • Heightened expression refers to a gene expression greater than that seen in a mock-treated culture. In the case of lux gene fusions, heightened expression is indicated by an increase in bioluminescence above background levels that is characterized by a temporal delay that allows for increased transcription and subsequent translation.
  • stressor refers to a chemical agent or physical treatment that results in suboptiminal growth of an organism. Stressors may include, but are not limited to, chemicals (such as herbicides, crop protection chemicals, environmental pollutants, heavy metals), physical treatments such as changes in temperature, changes in pH, agents producing oxidative damage or DNA damage (such as from UV exposure), anaerobiosis, biological insults such as the introduction of other life forms (viruses, bacteria, etc.) into the bacterial culture, or changes in nutrient availability. Additionally, stressors may include naturally-occurring compounds such as L-valine, galactose-phosphate, 2-ketobutyrate.
  • a “stressor target” means a specific macomolecular target inhibited by a specific stressor.
  • xenobiotic compound amd “xenobiotic chemical” refer to any stressor chemical which does not typically occur in nature.
  • Typical xenobiotics of interest in the present invention include those useful as herbicides, pesticides, fungicides or any other xenobiotic capable of interfering with a specific metabolic site of action.
  • bioluminescence refers to the phenomenon of light emission from a living organism.
  • Bioluminescent positive phenotype refers to a phenotype displaying an increase in light production by a detector cell containing a lux gene fusion.
  • baseline luminescence means the amount of light produced by a cell having a bioluminescent positive phenotype in an unstressed metabolic state.
  • lux gene complex refers to the lux structural genes which include luxA, luxB, luxC, luxD and luxE and which are responsible for the phenomenon of bacterial bioluminescence.
  • a lux gene complex might include all of the independent lux genes, acting in concert, or any subset of the lux structural genes so long as luxA and luxB are part of the complex.
  • Gene fusion is a hybrid DNA fragment comprising a regulatory signal essential for transcription (referred to as a promoter) fused to at least one structural gene sequence coding for a specific polypeptide.
  • lux gene fusion means the fusion of the lux gene complex with a suitable stressor-sensitive promoter.
  • recA-LuxCDABE' refers to the specific fusion of the genotoxic sensitive promoter recA fused to the bacterial Lux gene complex.
  • ilvBN refers to the structural genes encoding, respectively, the large and small subunits of the heterotetrameric ALS I-EC 4.1.3.18.
  • tVvGJW refers to the structural genes encoding, respectively, the large and small subunits of the heterotetrameric ALS II-EC 4.1.3.18.
  • ilvGM is cryptic in E. coli K-12 ⁇ ilvB-a mutation that deletes part of the ilvB gene.
  • ilvIH refers to the structural genes encoding, respectively, the large and small subunits of the heterotetrameric ALS III-EC 4.1.3.18. ilvIH is cryptic in laboratory strains of Salmonella typhimurium.
  • pheA refers to the structural gene encoding the bifuctional polypeptide that displays chorismate mutase (EC 5.4.99.5) and prephenate dehydratase (EC 4.2.1.51) activities.
  • the term "relA” refers to the structural gene encoding the ATP:GTP 3'-pyrophosphotransferase I-EC 2.7.6.5.
  • spoT refers to the structural gene encoding the ATP:GTP 3'- pyrophosphotransferase II-EC 2.7.6.5.
  • aromaticA- refers to the structural gene encoding enolpyruvylshikimate phosphate synthase-EC 2.5.1.19.
  • aromatic H- refers to the structural gene encoding DHAP(tryptophan repressible and feed back inhibitable) synthase-EC4.1.2.15.
  • t ⁇ /C+ refers to the structural gene encoding an outer membrane porin needed for the efflux of many xenobiotics pumped out by a variety of membrane translocases.
  • glk- refers to the structural gene encoding glucokinase- EC2.7.1.2.
  • the terms "detector organism”, “detector bacteria”, and “detector cell” refer to an organism which contains a gene fusion consisting of a genotoxic- sensitive promoter fused to a luminescent reporter gene or gene complex.
  • non-bioluminescent parent of the detector cell is a bacterial strain into which a light producing genetic cassette has not been introduced.
  • Genetic titration refers to an alteration of the genetic makeup of a microbe such that the levels of a macromolecular target are elevated to a point whereby they overcome the action of a stressor.
  • genetic titration will involve a process of screening for biochemcal targets of compounds where a host organism is transfected with a suitable genomic library, transformants are screened for growth in the presence of the compound and portions of the library conveying resistance to the compound are isolated and identified.
  • Nutritional reversal refers to the addition of a nutrient to a culture contacted with a stressor such that the biological output of the culture is restored to the unchallenged level.
  • Nutrient refers to an end product of a biochemical pathway or a compound readily converted to a pathway end product. Typical nutrients are amino acids, vitamins, bases or sugars. Vitamins are readily converted to cofactors which are pathway end products; similarly bases are readily transformed in vivo into nucleotide triphosphates.
  • auxanography means the diagnostic and systematic administration of nutrient pools to determine the pathway blocked in a microorganism as described in Davis, R. W., D. Botstein And J. R. Roth. A
  • RLU Relative Light Unit
  • Host cells suitable in the present invention include any cell capable of expression of the lux gene fusion where prokaryotic cells are preferred and where members of the enteric class of bacteria are most preferred.
  • Enteric bacteria are members of the family Enterobacteriaceae and include such members as Escherichia, Salmonella, and Shigella. They are gram-negative straight rods, 0.3-1.0 X 1.0-6.0 mm, motile by peritrichous flagella (except for Tatumell ⁇ ) or nonmotile. They grow in the presence and absence of oxygen and grow well on peptone, meat extract, and (usually) MacConkey's media. Some grow on D-glucose as the sole source of carbon, whereas others require vitamins and/or mineral(s).
  • Host cells of the present invention optionally may contain mutations that will facilitate the screening process.
  • the appropriate bacterial strain with which to test the effects of a chemical is one whose growth is affected by that chemical. Hence, the chemical of interest must be able to enter the cell, be retained in the cell, and interact with target molecules of the cellular machinery.
  • Various mutations of E. coli are known to affect permeation into and accumulation within the cell. Strains carrying mutant alleles of rfa (Ames, B. N., F.D. Lee, and W. E. Durston, Proc. Nat. Acad. Sci. USA, 70(3): p. 782-786, (1973)), envA (Young, K. and L. L. Silver, J.
  • the target macromolecule of a chemical may be intrinsically resistant to the action of that chemical.
  • E. coli has two isozymes of the enzyme acetolactate synthase, one of which has a poor binding affinity for the sulfonylureas herbicides.
  • An appropriate host strain of E. coli or other bacteria may be constructed to carry a known mutation or combinations of mutations.
  • an appropriately sensitive strain may also be found by screening for growth inhibition following mutagenesis by transposon insertion or chemical or physical treatments. Mutations Conferring Stress Sensitivity
  • Detector cells of the present invention optionally may contain mutations that will convey sensitivity to a particular stress to be screened for.
  • the relA mutation for example, prevents the cell from responding to amino acid starvation.
  • Other mutations that will be useful in stress sensitivity include, for example, mutations that result in sensitivity to various anticancer drugs and compounds that cause oxidative stress. It is known that many anticancer drugs interfere with DNA replication while compounds that cause oxidative stress may be useful in controlling fungal pathogens of crops and bacterial pathogens of humans or animals.
  • Mutations in genes conveying sensitivity to stresses are preferred, including but not limited to, mutant genes selected from the group consisting of cya, crp, spoT, arcAB, envZ, ompR, marR, earAB,fur, oxrG,fruR, rpoS, rpoE, creB, creC, glnG, glnL, glnB, glnD, glnF, phoB, phoP, phoQ, phoR, phoU, rpoH, lexA, recA, Irp, soxRS, oxyR,fnr, atbR, ada, and relA where the relA mutation is most preferred.
  • genetic titration may indicate unexpected roles for regulatory genes. Such discovery, coupled with inactivation of the regulatory locus, may optimize the sensor strains.
  • strains generally contained mutations such that only a single ALS (I or III) sensitive to sulfonylurea herbicides was expressed.
  • a sensitive host may either be screened from wild type after standard transposon, chemical (e.g., HNO 2 and NH 2 OH), UV, intercalating dye (e.g., acridine dyes) or other mutagenesis protocols have generated the appropriate hypersensitive mutations or can be constructed by combining mutations that together yield the desired sensitivity.
  • chemical e.g., HNO 2 and NH 2 OH
  • intercalating dye e.g., acridine dyes
  • Other mutagenesis protocols have generated the appropriate hypersensitive mutations or can be constructed by combining mutations that together yield the desired sensitivity.
  • the detector cell of the present invention optionally may also, contain a stress-sensitive reporter for the detection of particularly desirable or undesirable characteristics of the stressor compound to be screened.
  • stress- sensitive reporters are comprised of a stress-sensitive promoter operably linked to a suitable reporter element.
  • the promoter must be chosen so as to be expressible within the specific detector cell desired.
  • the promoters will be chosen from stress-inducible bacterial promoters. Examples of stress-inducible promoters suitable in the present invention are those responsive to chemicals, environmental pollutants, heavy metals, xenobiotics, changes in temperature, changes in pH as well as agents producing oxidative damage, DNA damage, anaerobiosis, changes in nitrate availability or pathogenesis.
  • suitable bacterial stress-promoters include, but are not limited to, those sensitive to protein damage such as the heat shock genes (grpE, dnaK, Ion, rpoD, groESL, lysU, htpE, htpG, htpl, htpK, clpP, clpB, htpN, htpO, and htpX), those sensitive to DNA damage such as those controlled by the SOS regulatory circuit (recA, uvrA, lexA, umuDC, uvrA, uvrB, uvrC, sulA, recN, uvrD, ruv, dinA, dinB, dinD, and dinF), those sensitive to oxidative damage (katG, ahp, micF, sodA, nfo, zwf, and soi), those sensitive to membrane damage (fabA), those sensitive to amino acid starvation and
  • Reporter genes suitable for fusion to the stress inducible promoter are structural genes under the control of such a promoter and able to report a detectable signal.
  • Many bacterial reporters such as lacZ, galK, xylE, luc, luxAB, luxCDABE, phoA, uidA (GUS), cat, npt-II, SUC2 and ubiquitin are known in the art (Miller, J. H., A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 63-67).
  • bioluminescent reporter genes including but not limited to the bacterial lux genes; lucifersae genes (luc) from, for example, the firefly, Photinus pyralis, or click beetle, Pyrophorus plagiophthalamus; or the gene encoding the luciferase from the sea pansy, Renilla reniformis. Because its gene products function well in E. coli under a wide range of temperatures, most preferred is the promoterless Photorhabdus luminescens luxCDABE gene complex obtained from the pCGLSl plasmid containing the lux gene complex. This complex is fully described by Rosson, R. A., in PCT International Application WO 93/03179 (1993).
  • Bacterial bioluminescence as produced by the luxCDABE gene complex is a phenomenon in which the products of 5 structural genes (luxA, luxB, luxC, luxD and luxE) work in concert to produce light.
  • the luxD product generates a 14 carbon fatty acid from a precursor.
  • the 14 carbon fatty acid is activated in an ATP dependent reaction to an acyl-enzyme conjugate through the action of the luxE product which couples bacterial bioluminescence to the cellular energetic state.
  • the acyl-enzyme (luxE product) serves as a transfer agent, donating the acyl group to the luxC product.
  • the acyl-IwxC binary complex is then reduced in a reaction in which NADPH serves as an electron pair and proton donor reducing the acyl conjugate to the C 14 aldehyde.
  • This reaction couples the reducing power of the cell to bacterial light emission.
  • the light production reaction catalyzed by luciferase (the product of luxA and luxB), generates light.
  • the energy for light emission is provided by the conversion of aldehyde to fatty acid and FMNH2 oxidation, providing another link between light production and the cellular energy state.
  • luxCDABE usefulness of luxCDABE are limited by the inherent thermolability of the protein products of these genes. The temperature requirement of this reporter system has limited overlap with the need to grow bacteria rapidly in defined media. Applicants have solved this problem by using luxCDABE that encode protein products capable of functioning in the desired temperature range (28-42°C).
  • a multiplicity of strains were engineered, each having a specific genotype useful for the specific site of action screen desired. Construction of the strains is reviewed in Figure 1 and the genotypes are summarized in Table 1 in the GENERAL METHODS. All strains engineered to contain the recA-Ewx -CD ABE gene fusion functioned as detector cells. Detector cells may contain only the gene fusion, or optionally may possess other mutations affecting membrane permeability or stressor sensitivity.
  • Useful strains possess a variety of genotypes including where the expression or suppression of the ilvB, relA, tolC, igm, spoTan ⁇ ilvG genes were used to give useful detector cells.
  • Methods of strain construction are well known in the art and use the basic elements of molecular biology and microbiology fully discussed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Escherichia coli and Salmonella; Cellular and Molecular Biology (Niedhardt et al. Eds., American Society of Microbiology, Washington, D.C. (1996))].
  • Preferred growth medium in the present invention are common defined media such as Vogel-Bonner medium (Davis et al., Advanced Bacterial Genetics, (1980), Cold Spring Harbor, NY: Cold Spring Harbor Laboratory). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. In some instances rich or complete media such as NB (Nutrient broth) are used. Suitable pH ranges for bacterial growth are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition.
  • Growth of the bacterial cells in liquid medium allows a uniform population of cells to be stressed at various growth phases such as early log phase, mid log phase, late log arithmic phase, or stationary phase.
  • Stress is the condition produced in a cell as the result of exposure to a cellular insult or stressor.
  • This cellular stress may be caused by any substance or change in the cellular environment that results in an alteration of normal cellular metabolism in a bacterial cell or population of cells.
  • chemicals such as herbicides, crop protection chemicals, environmental pollutants, or heavy metals to the growth media can cause such a stress.
  • changes in temperature, changes in pH, agents producing oxidative damage or DNA damage (such as from UV exposure), anaerobiosis, or changes in nitrate availability are insults that may cause stress as well.
  • the present method is useful for identifying the site of action for a variety of xenobiotic chemicals and particularly those compounds useful as crop protection chemicals.
  • the instant method is useful for determining the site of action of compounds selected from but not limited to sulfonylurea herbicides such as sulfometuron methyl, glyphostate, the profungicides such as the oxime carbamates, oxidants such as methyl viologen (paraquat), hormonal herbicides such as 2,4-dichlorophenoxyacetic acid, and fungicides such as 2-thienylalanine. Any chemical that negatively impacts bacterial metabolism can be anlyszed in this manner.
  • Nutritional Reversal The above defined media provides those nutrients (inorganic compounds and carbons sources) necessary and sufficient for the growth of wild type E. coli.
  • E. coli detector cells (containing the recA-Lux gene fusion) grow in these media converting the nutrients into organic chemicals required for cell growth and metabolism. If a stressor chemical interferes with one of the biosynthetic processes, the cellular metabolism and the bioluminescence of a culture will be reduced or eliminated.
  • the present method of nutritional reversal involves first screening compounds for the ability to depress bioluminescence of the detector organism containing the recA-Z(7Xgene fusion. Next, panels of cultures are initiated to test which nutrient will reverse the growth inhibition as indicated by a reversal of bioluminescent depression. Stressor chemicals meeting this criteria are defined as nutritionally reversed. The specific pathway affected by a stressor chemical is then determined by auxanographic analysis. Supplementation with biosynthetic intermediates from the identified pathway to determine which intermediates obviate the depression of bioluminescence may be used to define the target within the pathway. Modulation by non-pathway nutrients (Van Dyk and LaRossa, pp.
  • the genetic titration protocol is predicated upon the fact that chemical stressor action can be overcome by increasing the intracellular concentration of the macromolecular target above that normally found. In this manner the inhibitor action is titrated out and cells grow under conditions that normally inhibit the growth of the wild type.
  • the means of increasing the macromolecular target could be through the selection of genetic duplications (e.g., Anderson and Roth, Ann Rev Microbiol, 31:473-505 (1977); Wahl et al, JBiol Chem, 254: 8679-89 (1979), Alt et al., JBiol Chem, 253: 1357-70 or through the selection of high level constitutive regulatory mutants (Roth et al., J Mol. Biol.
  • a preferred embodiment is that pioneered by Falco [Falco and Dumas, Genetics 109:21-35 (1985)] and Rine [Rine et al., RN4S USA 80:6750-6754 (1983)] in yeast in which gene amplification is created by (a) construction of genomic libraries in high copy number autonomously replicating plasmids and (b) the introduction of such plasmid libraries into a suitable host strain creating a catalog of mero-multipoids [LaRossa, (1996), in Escherichia coli and Salmonella; Cellular and Molecular Biology (Niedhardt et al. Eds., pp 1400-1416, American Society of Microbiology, Washington, D.C. (1996))].
  • present detector cells may be employed to identify compounds on the basis of the prevention of inhibitor action.
  • the present application exemplifies the incorporation of several genomic fragments suspected of or known to confer growth resistance to thienylalanine into a bioluminescent or other tester strains by transformation selecting for ampicillin resistance.
  • the present detector cells may be modified to incorporate disrupted membrane proteins which in turn may be exploited to identify compounds having a specific biological activity. For example, genetic titration with SM identified tolC as a resistance determinant. Similarly titration with GP recognized yhhTS, thienylalanine recognized o245 ygaH, acivicin recognized yedA and mitomycin C recognized mdfA as resistance determinants (Examples 7-9) . Each of these genes encodes a predicted membrane protein. It is axiomatic that disruption of these cloned genes and incorporation of the disruptant into the E. coli chromosome can be achieved by standard techniques without undo experimentation. The utility of such disruptants can be readily assessed by bioluminescent assays that determine the doses that reduce light output by a factor of 2. These disrupted genes will be useful for a plethora of sensitive bioassays.
  • a preferred embodiment of the present method is illustrated in Figure 2.
  • a detector organism (1) having a recA-luxCDABE fusion and a relA mutation (inhibiting the cell's response to amino acid starvation) is exposed to a battery of compounds (stressors) (2) to be screened for crop protection activity.
  • Compounds producing an increase in light from the detector cell are discarded as genotoxic.
  • Compounds that slow cell growth but do not produce an increase in light are subjected to nutritional reversal screens or genetic titration screens to determine the site of action of the compound.
  • detector cells are grown in a minimal medium supplemented with a multiplicity of different nutrient pools, each pool composed of a different mixture of nutrients (3).
  • the pool supplying the amino acid necessary to reverse metabolic inhibition is detected by the recovery of light production by the bioluminescent gene fusion
  • genomics make a number of related embodiments possible.
  • an ordered set of overlapping, high copy number plasmids is placed such that each contain different segments of the E. coli chromosome.
  • This set of resulting strains may be used in a genetic titration screen of a chemical stressor to identify those chromosomal regions that upon amplification restore bioluminescent output to uninhibited levels.
  • genetic titration may be performed in alternative selection and screening modes.
  • detector cells containing the recA-LUX gene fusion were constructed by transformation of suitable hosts with the chimera according to standard methods (Sambrook supra). The transformants possessed a variety of mutations including ilvB, relA and tolC. Detector cells were constructed so as to contain one or more of these mutations. These detector cells emitted a baseline luminescence that was altered by the exposure to various xenobiotics. The sensitivity of the detector cell to genotoxic agents was examined by exposing the cell to mitomycin C. Moderate levels (0.3-20 ug/mL for a tolC + strain, 0.3-1.25 ug/mL for a tolC derivative) of mitomycin C resulted in an increase in baseline luminescence. High levels (>2.5 ug/mL in a tolC strain) of mitomycin C resulted in a decrease in luminescence ( Figures 4 and 5).
  • Detector cells containing a variety of mutations were treated with 4 different herbicides (SM, MV, GP and 2,4-D) to determine the effect of the compounds on bioluminescent output of the detector cell.
  • SM herbicide
  • MV MV
  • GP GP
  • 2,4-D cyclopentadiene
  • kinetic plots indicate a dose-dependent decrease in light emission in response to the herbicides. Data illustrating the effect is seen in Figures 6-9).
  • Detector cells containing the tolC mutation were exposed to varying concentrations of SM and mitomycin C to determine the effect of the mutation on the sensitivity of the assay. As is noted in Tables 2 and 3 and Figures 4 and 10 and 4 the tolC mutation not only enhanced the responsiveness of the detector cell to the lipophillic SM but also to mitomycin C. These tests demonstrated the utility of the tolC mutation as a component of the detector cell.
  • the site of action of two CPC's were analyzed using a detector cell comprising the recA-LuxCDABE fusion.
  • bioluminescent detector cells nutritional reversal was applied to each of the compounds to determine which nutrient would reverse the growth inhibiting effects of the compound.
  • cysteine metabolism was identified as the potential affected site for cells treated with the oxime carbamate compound while phenylalanaine metabolism was the identified site of action for the cells treated with thienylalanine.
  • detector cells harboring ilvB, relA and tolC alleles were used in genetic titration assays to determine the site of action of the SM, 2-thienylalanine and GP.
  • the detector cells were transformed with E coli cDNA libraries and transformants were screened for resistance to SM as indicated by changes in bioluminescence of the transformants. Resistant colonies were picked and the plasmids isolated, sequenced and analyzed for genes encoding for possible targets. In this fashion the ⁇ 7V2JW and ilvIH genes were identified and confirmed as the genes encoding ALS, the target for SM. In similar fashion the aroA, the known target for GP encoding ⁇ PSPS, was selected. The aroH gene was obtained in a selection for 2-thienylalanine resistant clones.
  • the present detector cells may also be utilized in a method to screen for compounds where the site of action is known.
  • a detector organism may be constructed to include not only the stress promoter-/wxCZ ) ⁇ RE gene fusion, but also a plasmid expressing the gene target of the compound to be screened.
  • the gene target for GP is 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoded by the aroA gene, and plasmids have been constructed so as to express the EPSPS gene product.
  • EPSPS 5-enolpyruvylshikimate-3- phosphate synthase
  • E. coli aroA gene may be substituted with plant or other bacterial aroA homologues.
  • chemistry for any target of interest can be identified in this manner.
  • the method of in vivo inhibitor identification of specified targets here exemplified could be used to identify many useful compounds that include, but are not limited to, new crop protection chemistries, antibacterial chemistries, antifungal chemistries, anticancer chemistries, anti-viral chemistries, chemistries preventing biofilm formation and anticorrosives.
  • DPD 1718 could be transformed with pBR322 selecting for ampicillin or tetracycline resistance yielding strain pBR322/DPD1718.
  • DPD1718 may be transformed with an ilvIH containing plasmid selecting for ampicillin resistance yielding strain pIlvIH/DPD1718 having an ALS III phenotype.
  • Non-ALS toxicant 1 1
  • the recA-LUX detector cell is used as a means for screening compounds for mutagencity.
  • the efficacy of the present SOS regulated bioluminescent gene fusions is seen in the comparison of fusion containing detector cells as indicators of compound mutagenicity as compared with the standardized Ames test (Example 10).
  • Compounds chosen at random which tested positive in the standard Ames test were confirmed as mutagenic by giving a 'lights-on" response in bioluminescent detector cells.
  • Crop protection chemicals used in the following examples were sulfometuron methyl [obtained from DuPont Agricultural Products, Wilmington, DE], glyphosate [obtained from Sigma], methyl viologen [obtained from Sigma] 2-Thienylalanine [obtained from Aldrich], compound OC, member of the class of oxime carbamates having profungicide activity was prepared by DuPont Agricultural Products. Mitomycin C and acivicin was obtained from Sigma. Stock solutions were prepared as follows:
  • ATCC American Type Culture Collection
  • DPD 1012 as DPD 1690 but igm (improved growth on minimal medium, an unmapped mutation)
  • DPD1013 as DPD 1690 but igm (improved growth on minimal medium, an unmapped mutation)
  • DPD1680 as RK4988 but to/C::miniTn/0
  • DPD1010 as DPD 1682 but igm (improved growth on minimal medium, an unmapped mutation)
  • DPD1011 as DPD 1682 but igm (improved growth on minimal medium, an unmapped mutation)
  • DPD1728 as DPD1010 but lacZ::[recA ' ⁇ luxCDABE cat]
  • DPD1730 as DPD1682 but lacZ::[recA ' ⁇ luxCDABE cat]
  • Chromosomal DNN isolated from E. coli W3110 [B. Bachmann, in E. coli and Salmonella typhimurium; Cellular and Molecular Biology (Niedhardt et al., Eds., pp 1190-1220, American Society of Microbiology, Washington, D.C. (1987))] was partially digested with restriction enzyme S ⁇ w3Al and size fractionated on agarose gels. Fractions of two size ranges (average sizes of approximately 2.5 and 4.0 Kbp) were ligated to pBR322 (0.11 pmol) or pUC18 (0.11 pmol) that had previously been digested with restriction enzyme BamHl and treated with calf intestinal alkaline phosphatase.
  • the molar ratio of chromosomal DNA to vector in each of the ligation reactions was approximately 0.2: 1.
  • the ligation products were used to transform ultracompetent E. coli XL2Blue (Stratagene) to AmpR. Pooled transformants (>10 5 for each transformation) were used to isolate plasmid DNA.
  • strain DPD 1675 containing tolC and ilvB proceeded by the manipulation of CU847, an E. coli strain, possessing the ilvB mutation and having the genotype [ilvB2101 ara thi ⁇ pro lac] from H.E. Umbarger, Purdue University.
  • a Pl v/ > phage stock was grown on strain DEI 12 [tolC: :miniTnl0; fully described in Van Dyk et al., Applied and Environ. Microbiol. 60:1414-1420 (1994), isogenic with RM443 described in B. Bachmann, in E.
  • DPD 1707 containing the recA-LuxCDABE gene fusion was constructed as follows.
  • Plasmid precALux3 was isolated from strain DPD2794 (fully described in U.S. 5683868), isogenic with RM443 described in B. Bachmann, in E. coli and Salmonella typhimurium; Cellular and Molecular Biology (Niedhardt et al. Eds., pp 1190-1220, American Society of Microbiology, Washington, D.C. (1987))].
  • the promoter region (recA) was amplified by PCR using primers 1,2 (SEQ ID NOS:l and 2 respectively). Primer 1 :
  • the resulting product was digested with BamHl and Sail and was mixed with a similarly digested pJT205 plasmid (formerly called pCGLS205) containing the Photorhabdus luminescens luxCDABE gene complex, fully described by (Rosson, R. A., PCT International Application WO 93/03179 (1993)). After ligation the mixture was transformed into strain DH5 (ATCC) and ampicillin resistant colonies were selected. The colonies were screened for bioluminescence. One such bioluminescent transformant was designated DPD 1657. Plasmid pRecALxxl, isolated from strain DPD1657, was digested with Pstl and Ecorl.
  • This digested plasmid was mixed with similarly digested pBrint.CM. plasmid, [from F. Valle, Insituto de Biotechnologia, UNAM, Cuernavaca, Mexico; Balabas et al., Gene 172:65-69 (1996)].
  • pBrint.CM. plasmid [from F. Valle, Insituto de Biotechnologia, UNAM, Cuernavaca, Mexico; Balabas et al., Gene 172:65-69 (1996)].
  • ligation chloramphenacol resistant transformants were recovered in strain DH5. The resistant colonies were screened for a bioluminescent phenotype. One such transformant was termed DPD 1696.
  • the plasmid in this strain was called pDEW14. Plasmid pDEW14 contained a fusion of the recA promoter to the Photorhabdus luminescens luxCDABE gene complex.
  • Plasmid pDEW14 was isolated from DPD 1696 and the DNA was introduced into strain JC7623 [B. Bachmann, in E.coli and Salmonella typhimurium; Cellular and Molecular Biology (Niedhardt et al. Eds., pp 2466, American Society of Microbiology, Washington, D.C. (1987))] by transformation and chloramphenacol resistant colonies were selected. Colonies that were ampicillin sensitive and lacZ negative (i.e., could not cleave X-gal) designated as DPD1707. It had the recALux fusion integrated into the lacZ locus of the E. coli chromosome and was bioluminescent.
  • a phage stock of Pl v/ > was prepared on strain DPD 1707 (Pl r and the method are fully described in J. H. Miller, Experiments in Molecular Genetics, (1972) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 201-205. Construction of DPD1718 (lacZr.recA ' ⁇ 'luxCDABE) proceeded from the strain DPD 1692. A P 1 v ⁇ phage stock grown on strain DPD 1707 was mixed as described by Miller (supra) with strain DPD 1692; chlorampehnicol resistant recombinants were selected. One such recombinant that also displayed bioluminescence was designated DPD1718. The same transductional methods were used to create strains DPD1715, DPD1730, DPD1728, DPD 1729, DPD1716, DPD1708, DPD1719, DPD1709, and DPD1714's reported in Table 1.
  • strains DPD 1715 and DPD 1730 were grown to mid-logarithmic phase in LB medium. Strain DPD1715 and DPD1730 differ only in that DPD1730 is tolC while DPD1715 is tolC+. To the medium was added varying concentrations of
  • Mitomycin C ranging from 0 to 20 ug/mL.
  • the kinetics of light emission after introduction of the genotoxin was monitored using a microtiter plate format luminometer as described previously (Van Dyk et al., Applied and Environmental Microbiology 60, 1414, (1994)) except that the temperature was controlled at 37 °C.
  • induction is lexA -dependent as indicated by kinetic and dose- response curves of the fusion introduced by transduction into an isogenic pair of strains differing in the lexA gene that controls the SOS response to DNA damage.
  • the LexA repressor encoded by lexA must be cleaved if the SOS response is to be activated [Walker, 1996, in E. coli and Salmonella typhimurium; Cellular and Molecular Biology (Niedhardt et al. Eds., pp 1400-1416, American Society of Microbiology, Washington, D.C.
  • lacZr.recA ' ⁇ 'luxCDABE fusion was introduced by transduction into strains OM800(lexA + ) and DM803 (non inducible lexA due to a non-cleavable repressor gene product [Mount et al., J Bad., 112: 886, (1972)] using a phage stock grown on strain DPD1707.
  • DPD1714 lacZ::recA ' ⁇ 'luxCDABE lexA +
  • DPD1709 lacZ::recA ' ⁇ 'luxCDABE lexA
  • Example 3 illustrates the effect of four different crop protection chemicals on the light output of the detector cells.
  • the detector strains were growth to mid-log arithmic phase as described above treated with sulfometuron methyl, methyl viologen, glyphosate and 2,4-dichlorophenoxyacetic acid (2,4-D) according to the following conditions where all cultures were maintained at 37 °C and tests were run over a time period of 0-90 min. Strain DPD1718 was exposed to 2,4-D over a concentration range of
  • Strain DPD1715 was exposed to glyphosate over a concentration range of 2.5-2500 ug/mL. Modulation in bioluminescence is seen in Figures 8(a) and (b). Strain DPD1718 was exposed to sulfometron methyl over a concentration range of 24-1800 ug/mL. Modulation in bioluminescence is seen in Figure 7. Strain DPD 1730 was exposed to methyl viologen over a concentration range of 0.75-48 ug/mL. Modulation in bioluminescence is seen in Figure 9.
  • Example 4 illustrates the effect of the tolC mutation on the detector cell response to sulfometuron methyl and Mitomycin C.
  • Detector cells were growth to mid-logarithmic phase as described above. Detector cells were exposed to a range of crop protection chemical and bioluminescence was measure at 80 min post- exposure. Strain DPD1715 (tolC+) and strain DPD1728(to/Q were exposed to Sulfometuron methyl over a concentration range of 0.006 to 0.4 ug/mL and responses are compared in Figures l ⁇ (a-c).
  • Example 5 demonstrates the use of a detector cell having to identify the site of action of the oxime carbamate OC, glyphosate and thienylalanine.
  • Strain DPD1718 was grown in minimal medium or LB medium at 37 °C and was exposed to either 3-(2-thienyl)-L-alanine or the oxime carbamate profungicide compound OC over a variety of concentrations. Concentrations of 3-(2-thienyl)-L-alanine ranged between 0 and 100 ug/mL for growth in both minimal and LB medium.
  • Concentrations of compound OC ranged from 0 to 100 ug/mL for cells grown in minimal medium and from 0 to 1000 ug/mL for cells grown in LB medium. Concentrations of glyphosate for cells grown in both minimal and LB Rich medium ranged between 0 and 5000 ug/mL.
  • Strain DPD1718 was grown to mid-logarithmic phase in minimal medium. A total of 11 individual pools are created which represent various bases, vitamins and amino acids (Tables 2, 5). Each pool shares one component with one other pool. Each pool was added singly into a microtiter well in duplicate.
  • the volume added per well was 10 ⁇ L of an individual pool plus 40 ⁇ L of fresh minimal media which has been supplemented with a predetermined inhibitory concentration of a given compound.
  • Fifty microliters of midlog arithmic cells grown in minimal media were added to the microtiter well.
  • the microtiter plate was then placed into a ML2000 luminometer and the bioluminescence of the sample was monitored over time. The data was compared to the sample of cells not challenged with the compound.
  • Table 2 records the data from the interaction of the compound OC with DPD1718.
  • a lights-off response (approximately 1000 fold diminution in light output) is observed on minimal but not rich media.
  • Auxanography (Table 3) indicated that the presence of pool 6, containing cysteine, was able to prevent the lights-off response.
  • the reversing agent is cysteine, the common component between the 2 pools. Cysteine could exert it effects either by reversing a chemical-induced auxotrophy or by forming an adduct with the oxime carbamate or with its hydrolysis product. To attempt to distinguish between these 2 models, the stereoisomers of cysteine were tested as reversing agents:
  • D-cysteine can also reverse compound OC effects. Applicants then tested and found that D-cysteine satisfies the auxotrophy of 2 cysD strains, a cysA mutant, a cysH strain and a cysB mutant. The data suggests that either the D-cysteine is not optically pure or that E. coli has the ability to interconvert the two stereoisomers. Additional experiments, showed that a free sulfhydryl group, present on a variety of compounds including 2-mercaptoethanol, was responsible for quenching the profungicide inhibition of bioluminescence (data not shown).
  • Nutritional reversal of thienylalanine inhibition was accomplished in a similar fashion as with compound OC. Pools were created as described above. Reversal of thienylalanine inhibition by pools 1 and 8 was observed as shown in Figure 14. The reversal can be attributed to phenylalanine, but not to the other components, of the pools. Nutritional Reversal of Glyphosate Action
  • Glyphosate is expected to be inhibitory to E. coli K12.
  • sulfometuron methyl is not inhibitory towards E. coli due to the presence of the refractile ALS I isozyme encoded by ilvBN. Both compounds inhibit an ilvB mutant of E coli.
  • Zone of inhibition assays demonstrate that introduction of a relA null allele which prevents the mounting of the stringent response to amino acid starvation into the ilvB mutant results in hypersensitivity to GP but does not change the response to sulfometuron methyl.
  • Strain DPD 1675 expresses a single SM-sensitive ALS, ALS III, encoded by ilvIH due to the presence of an ilvB mutation that prevents production of the wild type ALS I naturally resistant to SM. It also contains a tolC mutation that prevents an efflux pump mediated expulsion of SM from the cytoplasm. These two mutations together create a strain quite sensitive to SM. Applicants determined the MIC of strain DPD 1675 to be 3 ug/mL in minimal medium containing plates.
  • the genetic titration of the herbicide sulfometuron methyl was performed in the strain DPD 1675 (Table 1).
  • This strain harbors a mutation in the ilvB allele and tolC, which result in rendering the cell sensitive to the herbicide and more permeable to hydrophobic compounds.
  • the MIC of the herbicide in this strain was determined to be 3 ⁇ g/mL following 1 day of growth on minimal plates.
  • Frozen competent cells of DPD 1675 (prepared by the method of Nishimura et al., Nucleic Acids Research 18:6169 (1990)) were transformed with 0.5 ⁇ L of plasmid DNA from two different E. coli libraries, one pBR322-based and one pUCl 8-based.
  • the transformation mix was washed one time and resuspended in 1 X ⁇ [Davis et al, supra, pp. 202-203] before being plated to minimal ⁇ plates supplemented with thiamine, proline, glycine and glucose at standard concentrations that can be found in Davis et al., supra, pp. 201-210, and Miller, 1972, supra, p. 432. .
  • the selection for the desired clones was Ampicillin (100 ⁇ g/mL) and Sulfometuron methyl (9 ⁇ g/mL) which was also included in the media.
  • Strain DPD 1675 was transformed with 2 libraries containing random fragments- of the E. coli chromosome ligated into either pBR322 or pUC18. Plasmids from 19 isolates, when reintroduced into strain DPD 1675, conferred resistance to SM (Table 6).
  • Strain DPD 1675 containing the other 13 plasmids maintained the bile salt sensitive phenotype of the plasmid free host.
  • the nineteen plasmids were also used to transform strain MF2000.
  • MF2000 harbors mutant ilvBN and ilvIH alleles and so is devoid of ALS activity.
  • the strain is an isoleucine-valine auxotroph.
  • Introduction of the tolC + containing plasmids does not alter the observed auxotrophy of strain MF2000.
  • the transformants of strain MF2000 containing the other 19 plasmids were isoleucine- valine prototrophs.
  • Strain DPD 1692 was used for the selection of glyphosate resistant meromultiploids. Applicants determined the MIC of strain DPD 1692 to be 3.0 mM (0.56 mg/mL) glyphosate in minimal media plates.
  • Competent DPD 1692 cells transformed with 0.5 uL (of either library one (pBR22-based; 0.0375 ug DNA) or library three (pUC18-based, 0.15 ug, DNA) yielded 10 4 transformants thus having a frequency of about 3 x 10 5 and 0.7 x 10 5 transformants per microgram DNA, respectively when plated on rich media plus ampicillin.
  • Approximately 2 x 10 3 cells were plated per selective plate which consisted of minimal ⁇ media amended with glucose, glycine, proline, uracil and thiamine as well as the selective agents Ampicillin (100 ug/mL) and glyphosate (3.3 mM 0.56 mg/mL).
  • Glyphosate resistant meromultiploids appeared throughout a 96 h incubation at 37 °C.
  • ORF's identified to date include previously undescribed component IIC and IIB PTS enzymes.
  • the predicted amino acid sequence encoded by these two genes share homology with component IIC and IIB enzymes encoded by a fructose-like PTS operon (Reizer, J. A. et al., Microbiol. 141, 961, (1995)).
  • this insert contains the single E. coli glucokinase gene, glk.
  • EXAMPLE 7 IDENTIFICATION OF MITOMYCIN C - RESISTANCE GENES
  • Example 7 illustrates the identification and isolation of genes having resistance to Mitomycin C that may be used in the construction of a detector cell containing the recA-L UX fusion.
  • Mitomycin C inhibits colony formation of a lexA ind mutant of E. coli ,DM803, with a MIC of 3 ug/ml. This MIC is 5 x lower than that of the lexA + strain DM800.
  • Competent DM 803 cells were transformed with a pBR322 library and pUC18 library. Colonies were selected by plating on LB Amp 150 plates in the presence of 15 ⁇ g/ml of mitomycin C (3X the MIC) at 37°C. After 2 days, 4 colonies appeared on plates where cells were transformed with the pUCl 8 based E. coli library. These 4 colonies were picked; all plates were incubated for another 5 days without the appearance of further colonies.
  • Plasmids were purified from the four colonies and named plexA3.1, 3.2, 3.3 and 3.4. Reintroduction of each plasmid by retransformation of strain DM803 showed linkage between the ampicillin resistance and mitomycin C resistance indicating that the mitomycin C resistance in each case was a plasmid encoded trait.
  • Plasmid purification from DM803 host was difficult - poor yield and degradation of samples upon storage were observed. Thus plasmids were transferred to RFM443 for routine purification of plasmid template for sequencing. Sequences obtained:
  • Lex A3.1 forward- primed sequence maps to region 291 out of 400 (minute 18) as defined by the Blattner E. coli sequencing project - git region, with putative f ⁇ mbrial chaperone gene, and yhcA while the reverse-primed sequence maps to region 76 out of 400 - this region -contains dacC (a penicillin binding protein) and deoR (deoxyribose operon repressor) as well as several open reading frames encoding proteins of unknown function. Since these are two non-continuous regions of the chromosome, the selected plasmid is a chimera of 2 non adjacent fragments presumably fused during the library construction.
  • LexA3.2 forward- and reverse-primed sequences map to region 76 out of 400 in E. coli database. This region contains dacC (a penicillin binding protein) and deoR (deoxyribose operon repressor) among the8.6kb found in the insert.
  • TheLex A3.3 reverse-primed sequence maps to the same region as LexA3.2; while the forward-primed sequence is unavailable at this time.
  • Lex A3.4 reverse-primed sequence also maps to same region as LexA3.2 and appears to contain the same junction as Lex3.3; again forward-primed sequence is unavailable.
  • Strain RFM443 is /e ⁇ + and thus was used for genetic titration with mitomycin C.
  • the MIC for mitomycin C for this strains was determined to be between 1 and 3 ug/ml on rich, solidified LB medium.
  • LB solidified with agar and supplemented with 6 ug/ml of mitomycin C and 150 ug/ml of ampicillin was used to select mitomycin resistant clones after transformation of strain RFM443 with E. coli genomic libraries constructed in either pBR322 or pUC18.
  • Plasmid DNA purified from such resistant isolates was reintroduced into RFM443 to demonstrate if the resistance was a plasmid encoded trait.
  • DNA sequencing of the vector-insert junctions served to define those sequences that conferred resistance to mitomycin C. Such resistances mapped to 3 sites as defined by sequencing of the inserts (see following).
  • Site A isolated 7 times, coincides with the site at minute 18 present in the pLexA3 plasmids (above).
  • This set of plasmids (see Table 7 below) demonstrates that one specific gene, o410 (mdfA), a recently described multidrug transporter, in the minute 18 region is capable of conferring resistance to mitomycin C when present in multiple copies.
  • the inhibitory action of another DNA damaging agent, C0360 is not effected by site A clones.
  • Site B mediated resistance due to high dosage of the minute 43 region, is defined by 16 distinct inserts (see Table 8 below).
  • the only intact gene shared in common by these inserts from site B is sdiA, which encodes a positive activator of fisQAZ operon whose products are essential for cell division. Both the sdiA and rpoS gene products act on distinct promoters of ftsQAZ.
  • the inhibitory action of the DNA damaging agent C0360 is lessened by the presence of several site B clones.
  • Site C inserts were identified by sequencing (see Table 9 below). Arising from the minute 44 region, they have but one common intact gene, sbmC. Expression of this gene is induced by microcin B17, a small peptide antibiotic that causes double strand DNA breaks, other DNA damaging agents and entry into stationary phase. A limited sampling indicates that site C clones do not confer cross- resstance to the DNA damaging agent C0360.
  • Example 8 illustrates the identification and isolation of genes having resistance to Acivicin that may be used in the construction of a detector cell containing the recA -L UX fusion.
  • Acivicin inhibits colony formation of E. coli strain DPD 1675 on minimal medium with a MIC of 1 ug/ml.
  • Competent DPD 1675 cells were transformed with a pBR322 library and pUC18 library each containing random fragments of the E. coli chromosome. Colonies were selected by plating on E plates supplemented with glucose, thiamine, proline, 100 ug/ml ampicillin and 3 ⁇ g/ml of acivicin (3X the MIC) at 37°C. After prolonged incubation, colonies appeared on the plates. Plasmids were purified from the colonies and named. Reintroduction of each plasmid by retransformation of strain DPD 1675 showed linkage between the ampicillin resistance and acivicin resistance indicating that the acivicin resistance in each case was a plasmid encoded trait.
  • Plasmid purification from the resistant clones provided a plasmid template for sequencing. Sequences obtained
  • Class 1 clones come from region 287 at about 43 minutes. 9 in number, their inserts vary from about 2800-5800 bp but all contain an intact yedA. Perusal of Table 10 indicates that no other gene in this region is intact in all class 5clones that confer resistance. There is but a single class 2 clone that maps to region 374 at about
  • Example 9 illustrates the identification and isolation of genes having resistance to Thienylalanine that may be used in the construction of a detector cell containing the recA-LUX fusion. The method proceeded by the isolation of Thienylalanine resistant genes and transformation of an appropriate detector cell. Genetic Titrations of Thienylalanine Action
  • Thienylalanine inhibits colony formation of E. coli strain DPD 1675 on minimal medium with a MIC of 75 ug/ml.
  • Competent DPD 1675 cells were transformed with a pBR322 library and pUC18 library each containing random fragments of the E. coli chromosome. Colonies were selected by plating on E plates supplemented with glucose, thiamine, proline, 100 ug/ml ampicillin and 150 ⁇ g/ml of thienylalanine (2X the MIC) at 37°C. After prolonged incubation, colonies appeared on the plates. Plasmids were purified from the colonies and named. Reintroduction of each plasmid by retransformation of strain DPD 1675 showed linkage between the ampicillin resistance and thienylalanine resistance indicating that the thienylalanine resistance in each case was a plasmid encoded trait.
  • Plasmid purification from the resistant clones provided a plasmid template for sequencing.
  • Plasmid pAHHl a multicopy plasmid obtained from R. Baurele, Univeristy of Virginia, that contains ⁇ roHbut not adjacent genes
  • DPD 1675 results in a thienylalanine resistant phenotype indicating that aroH is the gene responsible for the multicopy mediated resistance.
  • the clones isolated by thienylalanine resistance were of two classes.
  • One resistant class defined by overlapping regions of 12 independently isolated clones contained the genes o245 yagaH which are located at 59.79 minutes of the E. coli chromosome.
  • the other resistant class defined by overlapping regions of 5 independently isolated clones contained a region located at 38.22 minutes on the
  • E. coli chromosome including ydiG(A) aroHydiEf478.
  • DPD1750 (pDEW45) was used as template to subclone either the o245 or ygaH ORF independently.
  • the primers were designed with defined restriction sites flanking the ORF to allow one to PCR amplify each individual ORF and then directionally clone into pBR322.
  • the forward direction primers were constructed such that they contain a BamHI restriction site (5' GGA TCC 3') incorporated into their nucleotide sequence.
  • the reverse primers have an EcoRI restriction site (5' GAA TTC 3') incorporated into their nucleotide sequence.
  • the specific primers for the PCR reaction to isolate the o245 gene were "o245f ' and "o245r", respectively.
  • the specific primers for the PCR reaction to isolate the ygaH gene were "ygaHf and "ygaHr", respectively. Their specific nucleotide sequences are described in below.
  • the PCR reaction was run for 40 cycles of 94°C, 1 min.; 50°C, 1 min.; 72°C, 1 min. and the primer conditions were 100 pmole each.
  • the concentrations and sizes of the PCR products were confirmed by electrophoresis on a 2.0%> agarose gel.
  • the o245 PCR reaction yielded a product of the predicted 1236 bp.
  • the ygaH PCR reaction amplified a product of the predicted size 661 bp.
  • the PCR products were purified by column filtration (Microcon) and then enzyme digested.
  • Sequential BamHI and EcoRI restriction digestions were performed on the o245 and ygaH PCR products and pBR322 vector which would serve as the host vector during the ligation. A fraction of the samples were run on a 0.7% agarose gel to determine their DNA concentrations. Ligation reactions were performed over night at 4°C into the host vector pBR322 with either the ygaH or 0245 digested PCR products as the insert DNA. Alloquots of the ligated pDNA was transformed into the DH5 ⁇ host selecting ampicillin resistance at 150 ⁇ g/ml and screening for tetracycline sensitivity at 20 ⁇ g/ml. Plasmid DNA was isolated from the ampicillin resistant, tetracycline sensitive isolates.
  • the host strain DPD1718 contains a chromosomally integrated recAr.lux p , making the basal level of bioluminescence very high in terms of RLU' s (relative light units).
  • plasmids were transformed into DPD 1718 to serve as positive and negative controls, namely pBR322, AH1 (aroH) (isolated from CB18), and ppheA ⁇ isolated by selecting for a pBR322 clone that complements a phe A auxotroph.
  • the plotted data is shown in figure 16 as ratios (normalized to the unchallenged cells in terms of light production). Only the last time point is shown at all of the concentrations (usually at approximately 60 minutes).
  • Zone assays were also performed on the cells according to the following protocol. Strains were grown overnight at 37°C in LB medium supplemented with 150 ug/ml of ampicillin. Cultures were collected by centrifugation prior to resuspension in an equal volume of E medium. 0.1 ml portions were plated in 2.5 ml of E medium amended with 0.7% agar to effect an even lawn of cells on E agar plates supplemented with glucose, proline, uracil and 100 ug/ml ampicillin. Filter disks containing the indicated quantities of compounds were placed upon the lawns. Plates were incubated overnight at 37°C before zones of clearing were measured.
  • Zone assays confirmed the trend in sensitivity to thienylalanine as illustrated in Table 12.
  • Beta-2-thiazolyl-D,L-alanine 200 nz 15c, 22t mimosine 200 l ie 15c azaleucine 200 14t 23c, 43t thienylalanine 200 nz 60c sulfometuron methyl 40 40c 42c glyphosate 200 18c, 25t 27c rifampicin 000 35c 40c nz nozone
  • EXAMPLE 10 IDENTIFICATION OF MUTAGENIC COMPOUNDS vs. STANDARD AMES REVERTANT ASSAY Example 10 compares the sensitivity and accuracy of a screen for mutagenic compounds using a recA-LUX containing detector cell as opposed to a standard revertant based Ames test.
  • the example evaluated the mutagenic potential of the submitted test substances in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2 uvrA (pKMlOl).
  • the Salmonella strains are unable to synthesize histidine, an essential amino acid, because of mutations in the genes coding for histidine biosynthetic enzymes. Additional mutations in the defective genes can result in individual Salmonella bacteria regaining the ability to synthesize histidine [(Maron, D. M. and B. N. Ames, Mutation Research 113, " " 173-215, (1983)].
  • coli WP2 uvrA (pKMlOl) is unable to synthesize tryptophan due to an ochre mutation in a gene required for tryptophan biosynthesis.
  • E. coli reversion mutants may arise either from further changes at the ochre site or from suppressor mutations at a locus in tRNA genes. [Brusick et al, Mutation Research 76, 169-190, (1980)].
  • a trace of histidine or tryptophan in the top agar permits several generations of auxotrophic cell division to fix pro- mutagenic lesions. This results in the formation of a microscopic "lawn" of bacteria.
  • DMSO Dimethyl sulfoxide
  • Positive indicators included the following: 2-aminoanthracene (2AA), 2-nitrofluorene (2NF), sodium azide (NAAZ), ICR 191 Acridine (ICR 191), and methyl methanesulfonate (MMS).
  • Deionized water was the solvent for NAAZ, ICR 191, and MMS.
  • the solvent for other positive indicators was DMSO.
  • the positive indicators were assumed to be stable in this study and no evidence of instability was observed. Any impurities were not expected to have interfered with the study.
  • Salmonella Tester Strain Characterization S. typhimurium tester strains were obtained from Dr. Bruce Ames,
  • uvrB a gene which codes for DNA excision repair
  • LPS lipopolysaccharide
  • TA97 was the recommended replacement for TA1537 and has been demonstrated to be more sensitive to frameshift mutagens.(l,5)
  • TA97a is now routinely used in place of TA97 due to its improved growth properties (personal communication with Bruce Ames and associates).
  • E. coli WP2 uvrA (pKMlOl) was obtained from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland. Because tryptophan biosynthesis is blocked by an ochre nonsense mutation, revertants arise as a result of base pair substitution. A second class of mutants may arise as a result of nonsense suppressor mutations in genes coding for tRNAs. Frameshift mutagens are not generally expected to be detected by this strain. (Brusick, et al. supra) Salmonella Tester Strain Storage and Culture
  • test substance was classified as positive when: (1) the average number of revertants in any strain at any test substance concentration studied was at least two times greater than the average number of revertants in the negative control; and (2) there was a positive dose-response relationship in that same strain.
  • a test substance was classified as negative when either: (1) there were no test substance concentrations with an average number of revertants which was at least two times greater than the average number of revertants in the negative control; and (2) there was no positive dose-response relationship.
  • Test substances were evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2 uvrA (pKMlOl) without an exogenous metabolic activation system (S9).
  • AA, BB, CC, EE, FF, and GG displayed evidence of mutagenic activity. Due to what was judged as test-substance related toxicity, there were insufficient acceptable concentrations to assess the mutagenicity of DD.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Cette invention se rapporte à un procédé permettant de découvrir le site d'action de composés xénobiotiques. Ce procédé consiste à soumettre une cellule détecteur comprenant un promoteur sensible aux agents génotoxiques liés opérationnellement à un complexe de gènes reporteurs luminescents. L'exposition des bactéries détecteurs à des composés qui sont génotoxiques produit un accroîssement de la luminescence. Ces composés sont groupés et peuvent être analysés pour que leur activité pharmaceutique soit déterminée. L'exposition des bactéries détecteurs à des agents xénobiotiques actifs qui ne sont pas génotoxiques produit une inhibition de la bioluminescence ou de la luminescence stable. En soumettant les bactéries détecteurs à un tri, tel qu'un titrage génétique ou d'inversion nutritionnelle, on dévoile le site d'action du composé en question.
EP98907633A 1997-02-28 1998-02-26 Procede pour identifier le site d'action d'agents chimiques xenobiotiques Withdrawn EP0972074A1 (fr)

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