EP0972074A1 - A method for identifying the site of action of xenobiotic chemicals - Google Patents

A method for identifying the site of action of xenobiotic chemicals

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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)
French (fr)
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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Publication of EP0972074A1 publication Critical patent/EP0972074A1/en
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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.
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Abstract

A method is disclosed for the discovery of the site of action of xenobiotic compounds. The method involves subjecting a detector cell comprising a genotoxic-sensitive promoter operably linked to a luminescent reporter gene complex. Exposure of the detector bacteria to compounds that are genotoxic results in an increase in luminescence. Such compounds are grouped and may be analyzed for pharmaceutical activity. Exposure of the detector bacteria to active xenobiotics that are not genotoxic results in inhibition of bioluminescence or stable luminescence. Subjection of the detector bacteria to a screen such as nutritional reversal or genetic titration reveals the site of action of the compound.

Description

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. The utility of these compounds is modeled on various structure-function relationships and is often confirmed through screening methods that associate these compounds with desired, known activities. Once a compound or class of compounds has been subjected to a screen and a putative activity has been determined, further analysis is needed to determine the mechanism also known as site of action, i.e., the target, of the compound. The effort to understand the precise site of action of a chemical or environmental agent is driven by a concern for environmental safety and a desire to capture the value associated with this understanding for use in the agrochemical and pharmaceutical industries. A need exits, therefore, for methods of determining the site of action of synthetic xenobiotic compounds that are rapid, accurate and able to accommodate the vast numbers of compounds being produced.
Methods for determining the site of action of various compounds have been developed using the complementary methods of nutritional reversal and genetic titration in microbially based systems. LaRossa et al., [JBio Chem., 259 (14), (1984) 8753-8757] teach a pool strategy for nutritional reversal previously used to classify the pathway effected by an auxotrophic mutation, termed auxanography, [Davis, R. W., D. Botstein and J. R. Roth: A Manual For Genetic Engineering: Advanced Bacterial Genetics. X+254p. Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., U.S.A., 1980]. 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. Systematic selection for such titrations using antimetabolites in yeast in conjunction with multicopy recombinant DNA based yeast chromosomal libraries were pioneered by Falco [Falco et al., Genetics 109:21-35 (1985)] and Rine [Rine et al., Proc Natl AcadSci USA 80: 6750-4 (1983)]. More recently, Sternberg and Chattajee PNAS 92:8950-8954 (1995)] have used this recombinant DNA based methodology in E. coli. This art teaches the use of the method of genetic titration in yeast to identify various macromolecular targets of a crop protection chemical and a few pharmaceuticals. It also teaches that recombinant DNA mediated genetic titration may identify a bacterial target.
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.
These methods of nutritional reversal and genetic titration are both limited by the need to place cells of bacteria or yeast on agar plates and subsequently score or monitor for cell growth in response to the presence of the compound. This limitation makes the methods slow and cumbersome. Further, neither of these methods offers a way to determine the relative physiological or environmental toxicity of the compounds being screened. One of the significant disadvantages of any current screening method is that many active compounds may be identified in initial screens that must be later discarded due to poor results in toxicological screens. Incorporation of a toxicological pre-screen into the site of action screen would minimize this costly investment of time and resources.
Bioluminescent reporters are lαiown 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. Biophys Ada., 1131, (2), pp 161-165., (1992)], and the luxAB fusion gene has been expressed in Bacillus at levels comparable to those achievable in E. coli by placing it under the control of the powerful Pxyn promoter (Jacobs et al., Mol Gen. Genet., 230(1-2), pp 251-256, (1991)]. Bacterial genes have also been isolated and expressed. Rychert et al.,
[Environ. Toxic. Water Qual, 6 (4), pp 415-422, (1991)] have shown that recombinant E. coli harboring the plasmid, pJΕ202, that contains the promoterless Vibrio fischeri lux gene complex, was sensitive to Zn2+, ethidium bromide, sodium pentachoropheate, Cu2+ and 2,4-dichloropheoxyacetic acid. Response in this assay was registered by a decrease in baseline light emitted by the transformed E. coli.
In addition to the use of the promoterless bacterial gene complex, foreign promoters have been fused to the bacterial bioluminescent structural genes for use in assays specific to various toxicants. For example, 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. An example of this type of gene fusion is described by Burlage et al., [J Bacteriol, 172 (9) pp 4749-4757 (1990)] where a DNA fragment from plasmid NAH7 containing a promoter for the naphthalene degradation pathway was fused to the lux genes of Vibrio fischeri and used to transform a strain of Pseudomonas. The resulting transformant displayed an increase in light emission in the presence of naphthalene. The induction of bioluminescence was demonstrated to coincide with naphthalene degradation by the transformed organism. Another test system specifically responsive to mercury (Hg) is described by H. Molders (EP456667). Here, 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. The test system of Molders relies on the induction of the mer promoter by the presence of mercury and the subsequent increase in light emission from the recombinant bacteria.
Applicants have previously disclosed the use of various stress promoters, including those sensitive to genotoxic stress, in commonly owned USSN 08/244,376 and USSN 08/344,428. 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. Finally, in commonly owned USSN 08/735,545 Applicants teach the use of bioluminescent reporters fused to bacterial promoters isolated from restriction of genomic DNA for the identification of new, compound sensitive promoters. The problem to be overcome therefore is to develop a rapid, facile method for the identification of the site of action of agrochemically or pharmaceutically active xenobiotic compounds that are non-toxic to animals, including humans, or the environment.
SUMMARY OF THE INVENTION The present invention provides a method for the identification of the site of action of a stressor comprising:
(i) contacting a stressor with a detector bacteria, 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;
(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;
(iii) submitting the growth-inhibiting, genotoxic or non-genotoxic stressor selected in step (ii) to a site of action screen;
(iv) identifying the site of action in the detector bacteria; and (vi) confirming the site of action in a target organism. 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:
- (i) contacting a stressor with a detector bacteria, 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;
(ii) selecting for genotoxic or non-genotoxic stressors capable of inhibiting the growth the detector bacteria of step (i) by monitoring the growth and light output of the detector bacteria; (iii) submitting the growth-inhibiting, genotoxic or non-genotoxic stressor to a site of action screen wherein the stressor target is identified and; (vi) isolating the structural gene encoding the stressor target. In another embodiment, the invention provides methods for identifying compounds having glyphosate-like activity, thienylalanine-like activity and ALS-inhibitory activity.
BRIEF DESCRIPTION OF THE FIGURES. BIOLOGICAL DEPOSITS AND SEQUENCE LISTING Figure 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 ΔϊlvB 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. 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 have provided a sequence listing in conformity with Rules for the Standard Representation of Nucleotide and Amino Acid Sequences in Patent Applications (Annexes I and II to the Decision of the President of the ΕPO, published in Supplement No. 2 to OJ ΕPO, 12/1992) and with 37 C.F.R. 1.821-1.825 and Appendices A and B (Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences). SΕQ ID NO: 1-10 are primer sequneces. DETAILED DESCRIPTION OF THE INVENTION
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.
Inihe method, 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. In the first stage, detector cells are exposed to specific concentrations of a xenobiotic compound to be screened for and cultures are analyzed for growth inhibition or an increase in bioluminescence. Compounds producing an increase in bioluminescence are discarded as genotoxic. 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.
The following definitions are used herein and should be referred to for claim interpretation.
The terms "crop protection chemical" and "CPC" refer to compounds having toxic or repellent effect on insects, plant pathogens, or crop-competing plant species known to damage crop plants. CPC's include 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).
"Sulfonylurea herbicides" are defined as N-(heterocyclicaminocarbonyl)- arylsulfonamide-containing herbicidal compounds that inhibit the enzyme acetolactate synthase, such as sulfometuron methyl.
The term "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".
"Acetolactate synthetase" or "ALS" is 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.
The term "thienylalanine-like activity" means any substance, natural or synthetic that has the fungicidal activity of chlorothalonil, 2-thienylalanine. The term "glypohosate-like activity" means any substance, natural or synthetic that acts the interfere with 5-enolpyruvylshikimate-3-phosphate synthase activity.
The term "ALS-inhibiting activity" means" 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.
The term "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.
The terms "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. The term "transformant" refers to the product of a transformation.
The terms "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.
"Genotoxic sensitive promoter" refers to a promoter activated by DNA damage. Examples of 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. (1996))] and members of the SOS regulon group disclossed by Walker in E. coli and Salmonella typhimurium; Cellular and Molecular Biology [Niedhardt et al, Eds., pp 1400-1416, American Society of Microbiology, Washington, D.C. (1996))].
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.
The. term "operably linked" refers to the fusion of two fragments of DNA in a proper orientation and reading frame to be transcribed into functional RNA.
The term "expression" refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product. In the expression of a gene, 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.
The terms "stressor" or "insult" refer 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. The terms "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. The term "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. The term "baseline luminescence" means the amount of light produced by a cell having a bioluminescent positive phenotype in an unstressed metabolic state.
The term "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.
The term "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.
The term "ilvBN" refers to the structural genes encoding, respectively, the large and small subunits of the heterotetrameric ALS I-EC 4.1.3.18.
The term "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 Δ ilvB-a mutation that deletes part of the ilvB gene.
The term "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.
The term "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.
The term "spoT" refers to the structural gene encoding the ATP:GTP 3'- pyrophosphotransferase II-EC 2.7.6.5.
The term "aroA-" refers to the structural gene encoding enolpyruvylshikimate phosphate synthase-EC 2.5.1.19.
The term "aroH-" refers to the structural gene encoding DHAP(tryptophan repressible and feed back inhibitable) synthase-EC4.1.2.15. The term "tø/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.
The term "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.
The term "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. Within the context of the present invention 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.
The term "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
Manual For Genetic Engineering: Advanced Bacterial Genetics. X+254p. Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., U.S.A., 1980.
The term "Relative Light Unit" is abbreviated "RLU" and refers to a measure of light emission as measured by a luminometer, calibrated against an internal standard unique to the luminometer being used.
DETECTOR CELLS AND NONBIOLUMINESCENT STRAINS
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 Tatumellά) 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). They are chemoorganotrophic with respiratory and fermentative metabolism but are not halophilic. Acid and often visible gas is produced during fermentation of D-glucose, other carbohydrates, and polyhydroxyl alcohols. They are oxidase negative and, with the exception of Shigella dys_enteriae 0 group 1 and Xenorhabdus nematophilus, catalase positive. Nitrate is reduced to nitrite (except by some strains oϊErwinia and Yersina). The G + C content of DNA is 38-60 mol% (Tm, Bd). DNAs from species within most genera are at least 20% related to one another and to Escherichia coli, the type species of the family. Notable exceptions are species of Yersina, Proteus,
Providenica, Hafnia and Edwardsiella, whose DNAs are 10-20% related to those of species from other genera. Except for Erwinia chrysanthemi, all species tested contain the enterobacterial common antigen (Bergy's Manual of Systematic Bacteriology, D. H. Bergy et al., Baltimore: Williams and Wilkins, 1984). Particularly useful in the present invention are E. coli and members of the genus Salmonella and those enterics for which the recA gene and its upstream promoter region has been identified. Membrane Mutations
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. Bacteriol, 173: p. 3609-3614, (1991)), imp (Sampson, B. A., R. Misra, and S. Benson, Genetics, 122: p. 491-501, (1989)), C. Z., S. Hayashi, and H. C. Wu, J. Biol Chem., (259): p. 5601-5605, (1984)) or surA (Tormo, A., M. Almiron, and R. Kolter, J Bacteriol, (172): p. 4339-4347,
(1990)) have increased sensitivity to a variety of chemicals. Destruction of efflux pumps, with mutations such as emr (Ma, D., et al, Mol Microbiol., 16: p. 45-55, (1995)), or acrAB (Paulsen, I.T., et al., Mol. Micro., 19: p. 1167-1175, (1996)), or the channels they use, with mutations such as tolC (Schnaitman et al., J Bacteriol, 172 (9), pp 5511-5513, (1990)), also result in increased chemical sensitivity.
Moreover, it is contemplated that genetic titration' s in conjunction with genomics will permit the development of method for the identification of such sensitizing mutations which can then be integrated in to an appropriate detector cell background. In some instances, the target macromolecule of a chemical may be intrinsically resistant to the action of that chemical. For example, E. coli has two isozymes of the enzyme acetolactate synthase, one of which has a poor binding affinity for the sulfonylureas herbicides. Mutations which destroy the function of ilvBN, encoding the resistant isozyme, result in a strain with greatly increased susceptibility to growth inhibition by sulfonylureas that target acetolactate synthase (LaRossa, R. A. and D. R. Smulski, J Bacteriol., 160: p. 391-394, (1984)). An appropriate host strain of E. coli or other bacteria may be constructed to carry a known mutation or combinations of mutations. Furthermore, 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. Moreover, genetic titration, may indicate unexpected roles for regulatory genes. Such discovery, coupled with inactivation of the regulatory locus, may optimize the sensor strains.
Within the context of the present invention strains generally contained mutations such that only a single ALS (I or III) sensitive to sulfonylurea herbicides was expressed. Such a sensitive host may either be screened from wild type after standard transposon, chemical (e.g., HNO2 and NH2OH), 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. For a review of the methods of mutagenesis, see, for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology. Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. Stress Sensitive Reporter; Escherichia coli and Salmonella; Cellular and Molecular Biology (Niedhardt et al. Eds., American Society of Microbiology, Washington, D.C. (1996))].
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. Typically, 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. Typically in bacterial detector cells, 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. Specific examples of 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 under the control of the relA, or spoT regulatory genesis, z7vRN, ilvGMED, and thr ABC), those sensitive to carbon starvation and under the control of the cya, and crp regulatory genes (lac, mal, and gal), those sensitive to phosphate starvation under the control of the phoB, phoM, phoR,andpho U regulatory genes (phoA, phoBR, phoE, phoS, aphA, himA,pepN, ugpAB, psiD, psiE, psiF, psiK, psiG, psil, psiJ, psiN, psiR, psiH, phiL, phiO or a, tna, dsd, and hut), and those sensitive to nitrogen starvation under the control of the glnB, glnD, glnG, and glnL regulatory genes (glnA, and hut). Preferred within the context of the present invention are those stress-inducible promoters sensitive to genotoxicity or DΝA damage under the control of the SOS regulatory circuit where the recA gene is most preferred.
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). Of particular use in the present invention are 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 C14 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.
The 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, spoTanά 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))]. Culture Conditions:
Typically, cells are grown at 37 °C in an appropriate media. 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. The addition of chemicals such as herbicides, crop protection chemicals, environmental pollutants, or heavy metals to the growth media can cause such a stress. Additionally, 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. STRESSOR XENOBIOTICS AND CROP PROTECTION CHEMICALS 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. Thus, not only antibacterials, but also antivirals, anticancer agents, anticorrosives , disinfectants and crop protection chemicals may be analyzed with this invention since the site of action of many such compounds are often enzymes or cofactors shared by bacteria and target organisms. Moreover, products of normal metabolism whose accumulation is detrimental can be analyzed with these methods. SITE OF ACTION SCREENS
Determination of the site of action of various stressors and xenobiotic compounds by the present method proceeded using the complementary screens of nutritional reversal and genetic titration. 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. 123-130 and LaRossa et al, pp 109-121 both in Biosynthesis of Branched Chain Amino Acids, Barak et al., eds. Balaban Publishers, Weinheim, FRG, 1990) can also help to define the macromolecular target of the stressor chemical. Genetic Titration
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. 22:305-323 (1966); " Hartman, Ames, Anton Yanofsky Calvo et al., J Bacteriol. 97:1272-1282 (1969), Calvo et al., Genetics, 61 :777-787 (1969)). These methods, however, lead to rather laborious analyses to define the overexpressed gene product. 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))]. In the next stage, (c), the meromultiploids conferring resistance to the chemical stressor are identified by selection for colonies that grow above the compound's MIC (minimal inhibitory concentration). Such methodology has been recently extended to E. coli (Chatterjee and Sternberg, RN4SUSA 92:8950-8954 (1995)).
Prevention Of Inhibitor Action By Partial Genome Amplification.
In addition to the techniques of nutritional reversal and genetic titration it is contemplated that present detector cells may be employed to identify compounds on the basis of the prevention of inhibitor action. For example, 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 resultant bioluminescent strains, along with negative controls in which the multicopy plasmid vectors lacking an insert were used for transformation, were exposed to various concentrations of thienylalanine and kinetic plots of bioluminescence versus time were obtained (Example 9) . As demonstrated in Example 9, clone o245 ygaH conferred the greatest degree of protection from thienylalanine while other clones confer intermediate levels of resistance. It is evident from this Example that (a) cross resistance can be discovered by simply replacing thienylalanine with other compounds in the test (indeed disk diffusion assays have revealed cross resistance conferred by o245, ygaH, to several other amino acid analogs including those identified in table 12) and; (b) other chemicals hitting a specific target can be identified by asking if particular target specifying gene in multiple copies confers inhibitor resistance. In this manner new inhibitors of acetolactate synthase have been revealed). Membrane Alterations
In another embodiment it is contemplated that 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.
Discussion of the Preferred Embodiments
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. Where nutritional reversal is used, 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
(4). The skilled person will appreciate that advances in genomics make a number of related embodiments possible. For example, into the detector organism described in the preceding paragraph 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. Thus, genetic titration may be performed in alternative selection and screening modes.
In another embodiment, 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. In each case 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. Using the 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. As illustrated in Tables 2 and 3, 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. Finally, 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 Ϊ7V2JW 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 ΕPSPS, was selected. The aroH gene was obtained in a selection for 2-thienylalanine resistant clones. In another alternate embodiment describing in vivo inhibitor identification of specified targets, the present detector cells may also be utilized in a method to screen for compounds where the site of action is known. Such an embodiment is illustrated in Figure 3. It is contemplated that 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. For example, it is known that 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. Compounds to be screened would be exposed to a standard detector cell (1) and the modified detector cell (2) containing. the expressible gene target (3) at suitable concentrations. No addition of compounds (4) will give a baseline luminescence in both detector cells. Addition of compounds that inhibit growth (5) will produce a decrease in luminescence in both detector cells. However, where exposure of the standard detector cell (1) to a glyphostate-like molecule (6) will produce a decrease in light production, exposure of the same compound to the modified detector cell (2) will give a smaller or no decrease in light production. The compound was genetically titrated by the expression of the gene target in the modified detector cell(2).
It will be appreciated by one skilled in the art that the E. coli aroA gene may be substituted with plant or other bacterial aroA homologues. Moreover, it is contemplated that 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.
The method of in vivo inhibitor identification of specified targets may also be used to detect inhibitors of ALS III, for example. DPD 1718 could be transformed with pBR322 selecting for ampicillin or tetracycline resistance yielding strain pBR322/DPD1718. Similarly DPD1718 may be transformed with an ilvIH containing plasmid selecting for ampicillin resistance yielding strain pIlvIH/DPD1718 having an ALS III phenotype. These two transformed strains may then be used to screen for specific inhibitors of ALS III since the titer of ALS III will be much greater in strain pI H/DPD1718 than in pBR322/DPD1718. Expected results follow:
Chemical Relative MLC* pI H/DPD1718 pBR322/DPD1718
Non-ALS toxicant 1 =1
ALS III inhibitor >1 =\
* Minimal Lumino - Inhibitory Concentration It is contemplated then that one can score other growth related phenotypes or growth itself to identify novel compounds interfering with EPSP, ALS or other desired cellular functions.
The above embodiment is more fully exemplified in Examples 7-9 which demonstrate how the introduction of genes conveying resistance to Mitomycin C, Acivicin and Thienylalanine into a recA-LUX detector cell may be used to screen for compounds having to Mitomycin C, Acivicin and Thienylalanine activity respectively.
In another embodiment 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.
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
EXAMPLES GENERAL METHODS Procedures for phosphorylations, ligations and transformations are well known in the art. Techniques suitable for use in the following examples may be found in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press (1989).
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, DC. (1994) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. Standard bacterial genetic protocols can be found in Miller, J. H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory (1972); Miller, J. H., (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press; Silhavey, T. J., Berman, M. L., and Enquist, L. W., Experiments with Gen Fusions (1984), Cold Spring Harbor Laboratory, and Davis, R. W., Botstein, D. and Roth, J. R. (1980), A Manual for Genetic Engineering-Advanced Bacterial Genetics, Cold Spring Harbor Laboratory. All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis, MO) unless otherwise specified.
The meaning of abbreviations is as follows: "h" means hour(s), "min" means minute(s), "sec" means second(s), "d" means day(s), "mL" means milliliters, "L" means liters.
Bioluminescence was measured in a luminometer, model ML3000 (Dynatech Laboratories, Chantilly, VA)according to the manufacturer's instructions.
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:
2-thienylalanine - (10 mg/mL) in water sulfometuron methyl - (3.6 mg/mL) in 0.01 N NaOH glyphosate - (5 mg/mL) in water compound OC - (10 mg/mL) in DMSO (dimethylsulfoxide) methyl viologen - (2 mg/mL) in water mitomycin C - (1 mg/mL) in water _acivicin 10 mg/ml in water. Strains
The transformed E. coli strains DPD1707, DP1675, and DPD1718 were deposited on 13 February 1997 with the American Type Culture Collection
("ATCC"), international depository. The strains are designated as ATCC 98328, ATCC 98329, and ATCC 98330 respectively.
Additional strains of detector cells useful in the determination of CPC sites of action are given in Table 1, below. TABLE 1
Strain Genotype
CU847 ilvB2101 ara thi JE(pro-lac), [from E. Umbarger, Purdue University]
DPD1678 as CU847 but ArelA251::Kan
DPD 1675 as CU847 but tolC: :miniTn70
DPD1690 - as DPD1675 but Δre/Λ25/::Λ:<2«
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)
RK4988 F' araD139 E(laclPOZYA) U169 strA thi? non gyrA E(ilvB-uhpA)2089 [From
R. Kadner via Ying-Ying Chang, University of Illinois]
DPD 1692 as Kan resistant, lactose non-utilizing
DPD1680 as RK4988 but to/C::miniTn/0
DPD1693 as DPD1680 but ArelA251::Kan
FD1062 Hfr ara-14 ilvI614 ilvH612 λ- glyA18 relAl spoTl ilvB619 bglR20 rbs-5::Tn5 ilvG468 (IlvG+) thi-1, [from Bob Lawther, University of South Carolina, reference: Babczinski, P. and T. Zelinski, (1991) Pestic. Sci. 31 : 305-323]
DPD1682 as FD1062 but to/C::miniTn70
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)
DPD1707 as JC7623 but lacZrfrecA 'φluxCDABE cat]
DPD1708 as JC7623 but lacZrfrecA 'φluxCDABE cat]
DPD 1715 as FD 1062 but lacZ: .[recA 'φluxCDABE cat]
DPD 1716 as CU847 but lacZ: :[recA 'φluxCDABE cat]
DPD1719 as DPD1693 but lacZ::frecA 'φluxCDABE cat]
DPD1728 as DPD1010 but lacZ::[recA 'φluxCDABE cat]
DPD 1729 as DPD 1011 but lacZ: :[recA 'φluxCDABE cat]
DPD1730 as DPD1682 but lacZ::[recA 'φluxCDABE cat]
JC7623 thr-1 ara-14 leuB6 d(gpt-proA)62 lacYl tsx-33 supE44 galK2 I- rac- hisG4
(Oc) rfbDl mgl-51 rpsL31 kdgK51 xyl-5 mtl-1 argE3 (oc) thi-1 qsr' sbcC201 sbcB15 recB21 recC22 [from F. Valle, Insituto de Biotechnologia, UNAM, Cuernavaca, Mexico]
DM800 F-, metA28, lacYl or Z4, thi-1, xyl-5 or -7, galK2, tsx-6 [from Brooks Low,
Yale University, fully described in J. Bacteriol., 12:886, (1972)]
DM803 as DM800 but lexAl [from Brooks Low, Yale University, fully described in
J. Bacteriol., 12:886, (1972)]
DPD1714 as DM800 but lacZ::[recA 'φluxCDABE cat]
DPD1709 as DM800 but lacZ::[recA 'φluxCDABE cat] Construction of libraries
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 Sαw3Al 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 (>105 for each transformation) were used to isolate plasmid DNA.
EXAMPLE 1 CONSTRUCTION OF DETECTOR CELL STRAINS
Construction of stain DPD 1675
Construction of 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 Δpro lac] from H.E. Umbarger, Purdue University. A Plv/> 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. coli and Salmonella typhimurium; Cellular and Molecular Biology (Niedhardt et al., Eds., pp 1190-1220, American Society of Microbiology, Washington, D.C. (1987))] by standard methods as described in J. H. Miller, Experiments in Molecular Genetics, (1972) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 201-205. The phage stock was mixed as described by Miller (supra) with strain CU847, tetracycline resistant recombinants were selected and a recombinant that was unable to grow on MaConkey agar (Miller, supra) and hence displaying a bile salt sensitive phenotype was designated DPD 1675. Construction of DPD 1707
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 :
5' ACT Ttt aagctt AGA GAA GCC TGT CGG CAC 3' recA-upperH (SEQ ID NOT)
Primer 2:
5' AGC TTT ggatcc CGC TTT CTG TTT GTT TT 3' recA-lowerB (SEQ ID NO:2)
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)]. After 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. This fusion is situated within lacZ coding sequences. 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 Plv/> 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 'φ'luxCDABE) proceeded from the strain DPD 1692. A P 1 vι 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.
EXAMPLE 2 ENHANCED BIOLUMINESCENCE IN THE PRESENCE OF THE
DNA DAMAGING AGENT MITOMYCIN C To test the detector cells for sensitivity to genotoxic substances, 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.
The kinetics are presented in Figures 4 (a) and (b), while a dose-response curve is shown in Figure 5. For DPD 1730 [tolC-] (figure 4(b)), high concentrations of mitomycin C, (here above 2.5 ug/mL) will either inhibit or prevent the increase in light production attributable to growth. At low concentrations (below 2.5 ug/mL) the cell does not acknowledge the presence of the chemical. At intermediate concentrations (0.63 and 0.31 ug/mL) the cell responds to the damage by inducing the SOS response as conveniently monitored by the large increase in bioluminescence, above the mock treated control, that develops as a function of time. In contrast, the tolC+ strain, DPD1715 (figure 4(a)) was much less sensitive to mitomycin C (see figure 5). Induction was observed throughout the entire range of mitomycin C concentrations. Inhibitory effects were not observed.
As expected, 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. (1996))] The lacZr.recA ' φ'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. One chlorampehnical transductant from each cross that was bioluminescent was saved and designated as DPD1714 (lacZ::recA ' φ'luxCDABE lexA+) and DPD1709 (lacZ::recA ' φ'luxCDABE lexA). Mitomycin C treatment caused enhanced light production from strain DPD 1714 but not from strain DPD 1709 indicating that the treatment elicited a true SOS response since the inability to cleave the LexA repressor prevented the bioluminescent increase. EXAMPLE 3
DECREASED BIOLUMINESCENCE AFTER TREATMENT WITH 4 CROP PROTECTION CHEMICALS 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
78-5000 ug/mL. Modulation in bioluminescence is seen in Figure 6.
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.
As is evident from the figures illustrating kinetic data, all crop protection chemicals gave a dose-dependent decrease in light emission from the detector strains.
EXAMPLE 4 tolC ENHANCES THE RESPONSE TO SULFOMETURON METHYL
AND MITOMYCIN C 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 lθ(a-c). Similarly tolC+ and tolC strains were exposed to concentrations of 0-20 ug/mL of mitomycin C and responses are compared in Figure 5. From the data it is clear that the tolC mutation has the effect of enhancing sensitivity of the detector strain to the CPC. The bioluminescent assay demonstrating the efficacy of the tolC- strains was confirmed using disk diffusion assays [Stephens et al., RN4S 72, 4389, (1975); LaRossa R.A. and JN. Schloss, J.Biol.Chem., 259, 8753 (1984)] (data not shown). EXAMPLE 5
INDICATIONS OF SITE OF ACTION OF OXIME CARBAMATE OC. GLYPHOSATE AND THIENYLALANINE BY NUTRITIONAL REVERSAL 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.
As seen by the data in Figures 11(a), (b), 12(a), (b) and 13(a), (b), treatment of the detector cell with these compounds caused a decrease in light production in minimal medium, whereas in rich LB medium, light emission was not decreased.
Auxanography was used to determine the nutrient responsible for obviating the light-inhibiting effect of these experimental crop protection chemicals.
Auxano graphic protocol:
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.
Composition of the pools makes metabolic sense as described in Davis, R. W., D. Botstein And J. R. Roth, A Manual For Genetic Engineering: Advanced Bacterial Genetics. X+254p. Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., U.S.A., 1980, see pp. 206-208. Minor changes to the described 11 pools from that of Davis et al. were made to accommodate metabolic differences between Salmonella typhimurium and E. coli. The pool components follow; their concentrations in the pools are as described by Davis et al., supra: pool-components
1-adenosine, histidine, phenylalanine, glutamine, thymine 2-guanosine, leucine, tyrosine, asparagine, serine 3-cysteine, isoleucine, tryptophan, uracil, glutamate 4-methionine, lysine, threonine, aspartate, DAP
5- isoleucine, valine, proline, arginine, gly cine 6-adenosine, guanosine, cysteine, methionine, isoleucine 7-histidine, leucine, isoleucine, lysine, valine
8-phenylalanine, tyrosine, tryptophan, threonine, proline, PABA, DHBA 9-glutamine, asparagine, uracil, aspartate, arginine
10-thymine, serine, glutamate, DAP, glycine 11-pyridoxine, nicotinate, biotin, pantothenate, alanine The volume added per well was 10 μL of an individual pool plus 40 μL 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. The pool that enabled the cell to overcome the inhibitory effects of the compound, as determined by a restoration in bioluminescence, was determined to be the pool that contained the component that reversed the dampening effect of the compound. Finally, individual components of the pool were tested to determine the specific ingredient responsible for restoration of bioluminescence. Nutritional Reversal of Compound OC Action
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.
TABLE 2 Cycle 10: Cmpd OC ADDITION %LUM
=ιoσ
+ Pool 1 0.051
" - + Pool 2 0.13
+ Pool 3 0.051
+ Pool 4 0.042
+ Pool 5 0.022
+ Pool 6 89
+ Pool 7 0.070
+ Pool 8 0.095
+ Pool 9 0.028
+ Pool 10 0.11
+ Pool 11 0.13 + - (U0
* Medium: E+Bl+glucose+glycine+proline+uracil 37 °C/DPD1718/compound OC at 25 ug/mL
A weak prevention of the light-off response was seen at some time points with pool 3 (data not shown). Thus the individual components of pools 3 and 6 were tested for their ability to prevent the lights off response:
TABLE 3 Cmpd. OC ADDITION %LUM
Pool 6 components
=100 + Pool 6 90
+ Ade 0.053
+ Gua 0.038
+ Cys 94
+ Met 0.034
+ He 0.027
+ - 0.031 Cmpd. OC ADDITION %LUM
Pool 3 components
+ Pool 3 0.048
+ Cys 94
He 0.027
+ Trp 0.046 + Glut 0.13
+ Ura 0.027
It is evident that 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:
TABLE 4
%LUM
Inhibitor:
Supplement Cmpd. OC Analog None
.04 .06 =100
L-cys 45 45 D-cys 69 75
It is apparent that 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). The finding indicated that compounds with free SH groups obviate compound OC action, suggesting that L-cysteine biosynthesis is not compromised by compound OC action. Thus, nutritional reversal experiments can suggest agents capable of obviating inhibitors action. Such experimentation must be buttressed with further investigation. Nutritional Reversal of 2-Thienylalanine Action
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
Nutritional reversal of glyphosate inhibition was accomplished in a similar fashion as with compound OC and thienylalanine. Reversal of glyphosate inhibition only by pool 8 was observed as shown in Figure 13(a-b) and Table 5.
TABLE 5
Cycle 10:
Glyphosate ADDITION %LUM
=100
+ Pool l 19
+ Pool 2 19
+ Pool 3 16
+ Pool 4 18
+ Pool 5 29
+ Pool 6 14
+ Pool 7 23
+ Pool 8 105
+ Pool 9 19
+ Pool 10 19
+ Pool 11 19
+ 18
♦Medium: E+Bl+glucose+glycine+proline+uracil 37 °C/DPD1718/Glyphosate at 600 ug/mL
Reversal by pool 8 only is indicative of a block in the common aromatic amino acid pathway (Davis et al, supra, p. 208). Thus the target pathway of glyphosate is accurately identified by this method.
Nutritional Reversal of Sulfometuron Methyl (SM ) Action
The bioluminescence of Strain DPD1718 growing in minimal E medium supplemented with thiamine, glucose and uracil was inhibited by sulfometuron methyl in a dose-dependent manner (see Figure 15(a) in which the concentrations tested were 1800, 900, 450, 225, 113, 61, 31 and 0 ug/mL). Nutritional reversal of SM inhibition caused by administration of 250 ug/mL of the herbicide is depicted in the bottom panel of Figure 15b. The most effective pool at reversing the inhibition was pool 7 composed of the three branched chain amino acids, lysine and histidine. Reversal by this pool is consistent with the known site of SM action. Incomplete reversal to a lesser degree and with greatly delayed kinetics was seen with pools 1 (adenosine, histidine, phenylalanine, glutamine, thymine), 9 (glutamine, asparagine, uracil, aspartate, arginine) and 11 (pyridoxine, nicotinate, biotin, pantothenate, alanine).
EXAMPLE 6 IDENTIFICATION OF SITE OF ACTION OF SULFOMETURON METHYL AND GLYPHOSATE BY GENETIC TITRATION Characterization of GP and SM Sensitivity by Analysis of Their Interactions with Selected Mutants
Glyphosate is expected to be inhibitory to E. coli K12. In contrast, 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. SM resistant meromultiploids
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 μg/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 μL 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 Ε [Davis et al, supra, pp. 202-203] before being plated to minimal Ε 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 μg/mL) and Sulfometuron methyl (9 μg/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).
TABLE 6
Selection Plasmid:
Agent ug/mL Host Vector Name Inserted Genes
SM 9 DPD 1675 pBR322 pDEW21 tolC pDEW18 tolC pDEW22 tolC pDEW123 tolC pDEW24 tolC pDEW25 tolC pDEW20 'emrDivbL ilvBN uhpA uhpB uhpC uhpT' pDEW26 glvA glvB glvGyidL yidK yidJyidl yudH yidG tidF emrD evbL ilvBN uhpA uhpB uhpC uhpT' pDEW27 yidG ' yidF emrD ivbL ilvBN uhpA uhpB ' pDEW28 'yidF emrDivbL ilvBN uhpA uhpB uhpC pDEW19 ilvIH
SM 9 DPD 1675 pUC18 pDEW15 'emrDivbL ilvBN uhpA uhpB' pDEW34 'yidF emrDivbL ilvB ilvN pDEW29 yidG ' yidF emrD ivbL ilvBN uhpA uhpB uhpC pDEW30 ilvBN' pDEW31 yidl ' yidH yidG yidF emrD ivbL ilvB ilvN pDEW32 'emrDivbL ilvBN uhpA uhpB uhpC pDEWT7 ilvIH
GP 560 DPD 1692 pUC18 pDEWHO 'yhhRyhhSyhhTyhhU' pDEW112 yhhQ yhhRyhhSyhhT yhhU'
GP 560 DPD 1692 pBR322 pDEW114 β ' 89 o230 serC aroA pDEW115 β ' 07 o552 criRf461 ybeG cspEybelybeH' pDEW116 f • 415fl08 glk o418 ol08
The response of these transformants to bile acids was scored; bile acid sensitivity indicates a tolC~ phenotype while bile acid resistance indicates a tol JA phenotype. The introduction of 6 plasmids into DPD 1675 resulted in a bile salt resistant isolate suggesting that these strains are tolC+ltolC heterozygotes and that to/C" was dominant. This supposition was confirmed by sequencing both strands of each insert The primers used for sequencing the inserts into pBR322 were:
GCC ACTATC GAC TAC GCG (SEQ ID NO: 3)
CTG TGG CGC CGG TGA TGC (SEQ ID NO: 4)
while those used for sequencing the inserts into pUC18 were:
GTA A A A CGA CGG CCA GT (SEQ ID NO: 5)
AGC GGA TAA CAA TTT CAC ACA GGA (SEQ IDNO: 6).
Informatics methods were used to place the obtained sequences onto the E. coli genome (Genbank Accession number U00096). These data (Table 6) led
Applicants to conclude that each plasmid conferring both SM and bile salt resistance carried tolC .
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. As such, the strain is an isoleucine-valine auxotroph. Introduction of the tolC+ containing plasmids does not alter the observed auxotrophy of strain MF2000. In contrast, the transformants of strain MF2000 containing the other 19 plasmids were isoleucine- valine prototrophs. This indicated that they were expressing either ALS I or ALS III and that the plasmids were likely to harbor ilvBN or ilvIH. These inserts' ends were also sequenced leading to the identification of the genes harbored on the inserts by comparison with the complete primary sequence of the E. coli genome (Genbank Accession number U00096; see Table 6). From the data presented we concluded that there were 9 ilvBN+lilvBN heterozygotes (see Table 6) and 2 ilvIH+/ilvIH+ homozygotes in the original collection of SM-resistant meromultiploids. Selection and characterization of glyphosate resistant meromultiploids 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 104 transformants thus having a frequency of about 3 x 105 and 0.7 x 105 transformants per microgram DNA, respectively when plated on rich media plus ampicillin. Approximately 2 x 103 cells were plated per selective plate which consisted of minimal Ε 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.
Glyphosate selection resulted in the isolation of 7 transformants from the pBR#22 library, and 18 transformants from lthe puC18 library (Table 6). Retransformation of the original host strain DPD 1692 with the 25 plasmids conferred glyphosate resistance, insuring that the plasmid conferred the resistance phenotype. Presence ofaroA, the gene that encodes the known target of glyphosate, was screened by transforming an auxotrophic aroA mutant E. coli strain AB2829 with the plasmids that conferred glyphosate resistance. Prototrophic transformants indicated that the aroA gene was resident on the glyphosate-resistance conferring plasmid. Two of the 25 isolated plasmids restored growth of AB2829 on minimal media. The presence oϊaroA on these two plasmids was confirmed by sequence analysis (see Table 6) using the primers described above. These two plasmids conferred the strongest resistance to glyphosate when the 25 glyphosate resistance plasmids were compared in strain DPD 1692 (data not shown).
Of the remaining 23, non-aroA containing plasmids, three distinct inserts conferring novel glyphosate resistances have been identified by sequence analysis (See Table 6). Group A had 17 members, group B had 4 members and group C had 2 members. Localization of the inserts relative to the E. coli genome map was accomplished by FASTA and BLAST (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), jMadison, Wise.) scans of the databases. The group A inserts, mapping to 77.8 minutes on the E. coli chromosome, contain two previously sequenced ORF's, yhhS . and yhhT . The predicted protein structures for both of these ORF's contain domains predicted to span the membrane and they are thus likely to be permeases. The 4 members of the second group, B, mapping to the 14.2 minute region, contain an uncharacterized region of the E. coli chromosome. The third group of two inserts maps to the 52.3 minute region of the E. coli chromosome. Sequence analysis has revealed genes related to carbohydrate metabolism are contained in this insert. 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)). In addition to new PTS enzyme II homologues, 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.
Genetic Titrations of Mitomycin C Action
Mitomycin C (a DNA damaging agent) inhibits colony formation of a lexAind 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 15μg/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:
(1) The two ends of the LexA3.1 insert were sequened. 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 fϊmbrial 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.
(2) 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.
(3) Similarly the 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.
Thus it appears that a gene or genes within the region 76 of the chromosome of E. coli confers mulitcopy resistance to mitomycin C. It is obvious that division of the segment and retesting will define a novel gene or genes responsible for mitomycin C resistance.
These genes and open reading frames included on pLexA3.2 are: ol27,o371,βlO,dacC = penicillin-binding protein 6 precursor, <ieøR=deoxyribose operon repressor,/; 98, o410=mdfA, J94, f262, f402 & ol 78.
Further experiments were performed with a strain proficient in the SOS response since the non-inducible allele could compromise a subset of potential resistance mechanisms. 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. Thus 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, however, (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.
TABLE 7 Site A Clones.
RESPONSE TO:
ND-not determined; + indicates growth; - indicates no growth
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.
TABLE 8 Site B Clones
RESPONSE TO:
* only one end of these inserts were sequenced; ND-not determined; + indicates growth; - indicates no growth
10 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.
TABLE 9
The clones in the sbmC region, site C.
RESPONSE TO:
ND-not determined; + indicates growth; - indicates no growth EXAMPLE 8 IDENTIFICATION OF ACIVICIN RESISTANCE GENES 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.
Genetic Titrations of Acivicin Action
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 μg/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
69 minutes. It contains a 1831 bp isert that contains the intact iciA and/7(5 genes. Relevant clones are identified in Table 10:
TABLE 10
EXAMPLE 9 IN VIVO INHIBITOR IDENTIFICATION OF THIENYLALANINE LIKE COMPOUNDS
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 μg/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.
Sequences were obtained that yieled the following plasmid insert structures, shown in Table 11 : TABLE 1 1
Introduction of plasmid pAHHl (a multicopy plasmid obtained from R. Baurele, Univeristy of Virginia, that contains αroHbut not adjacent genes) into strains
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.
Subcloning the individual ORFs from DPD 1750 (o245 vasaH) The strain DPD 1750 contains the largest insert containing proX 088 o305
0245 ygaH (pDEW45). PCR amplification utilizing plasmid DNA isolated from
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.
"o245f ' 5' GTT AGC GGA TCC CTA ATT TCA GCC TCA GCC3' [SEQ ID NO: 7]
"o245r" 5'AGC AGT GAA TTC GTG TAT CGT GCA TCA CTT C3' [SEQ ID NO:8]
"ygaHf 5'GTT AGC GGA TCC TAA TAG GGG CAT TCT CCG3' [SEQ ID NO:9]
"ygaHr" 5'AGC AGT GAA TTC CAG CCG ATA TAG TAA CGA CAG3' [SEQ ID NO:l l]
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. Likewise, 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 DH5α host selecting ampicillin resistance at 150 μg/ml and screening for tetracycline sensitivity at 20 μg/ml. Plasmid DNA was isolated from the ampicillin resistant, tetracycline sensitive isolates. Transformations into DPD1718 for Reverse Genetic Titrations The o245 ligated plasmid (pDEW40) or the ygaH ligated plasmid(pDEW39), were transformed into DPD1718 selecting ampicillin 150 μg/ml resistance and screening for bioluminescence, chloramphenical (20 μg/ml) and kanamycin resistance (25 μg/ml). 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). Other 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. Experimental Protocol
Strains were grown at 37°C in minimal E medium supplemented with 0.4%ι thiamine and 0.4%ιglucose with shaking. The cultures were diluted into fresh minimal media the next morning and grown to early log. Serial two fold dilutions of a thienylalanine stock solution (10 mg/ml dissolved in dH20) were performed into minimal media in a microtiter plate with highest concentration @ 100 μg/ml. The bioluminescence was monitored for one hour and the kinetic data collected.
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.
TABLE 12 zone of inhibition (dia. in mm)
DPD 1749
(multicopy DPD3134 compound ug/disk ygαH o245) (vector alone)
D,L-allyl glycine 200 24t 26c
1,2,4-rriazole 200 15t 17c
5-methyl-D,L-trytophan 200 15t, 23vt 14c, 24t
D,L-5-aza-tryptophane hydrate 200 30c, 41t 38c
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
Relevant genotypes of the strains employed are given in Table 13.
TABLE 13 pDEW40 o245 insert in pBR322-PCR amplified from DPD 1750 pDEW 39 ygaH.3 insert in pBR322- PCR amplified from DPD1750 pDE W45 proX 088 o305 0245 ygaH in pBR322 thienylalanine resistant clone
DPD 1748 ydiG(A) aroHydiEf478/237 in pBR322 thienylalanine resistant clone
DPD 1749 0245 ygaH in pBR322 thienylalanine resistant clone
DPD 1750 proX 088 o305 0245 ygaH in pBR322 thienylalanine resistant clone
DPD1770 pDEW 39/DH5α
DPD1771 pDEW 39/ DH5α -second transformant( g(3//.5) of pDEW39
DPD 1772 pDEW40/ DH5α
DPD1773 pDEW 39/ DPD1675
DPD1774 pDEW 39/ DPD1675 -a second transformant (ygaH.5) of pDEW39
DPD 1775 pDEW40/DPD1675
DPD1777 pDEW 39/DPD1718
DPD1778 pDEW 39/ DPD1718-a second transformant (ygaH.5) of pDEW39
DPD1779 pDEW40/DPD1718
DPD 1780 pDEW45 DPD 1675
DPD 1675 HvB tolC
DPD1718 lacZ::Ω(recAE col,::luxP | )
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)]. E. 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. However, only those bacteria that revert to a prototrophic state can continue to divide and form macroscopic colonies. These revertants can be scored by their ability to grow on agar plates deficient in histidine (S. typhimurium strains) or tryptophan (E. coli). By comparing the number of chemically induced revertants to the number of spontaneous revertants, the mutagenicity of the test substance can be assessed. Test Substance and Negative Control Dimethyl sulfoxide (DMSO) was the solvent used for all compounds tested and thus was chosen as the test substance solvent and as the negative control. Dilutions of the test substance were performed at room temperature in DMSO. The negative control was assumed to be stable during this study, and no evidence of instability was observed. Any impurities were not expected to have interfered with the study. Positive Indicators
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,
Berkeley, CA. The phenotypic characteristics and mutational sensitivities of the S. typhimurium strains are summarized in Table 14 as follows: TABLE 14
The deletion in uvrB (a gene which codes for DNA excision repair) increases the bacterial sensitivity to mutagens. [Ames, B. N., F. D. Lee, and W. E. Durston, Proc. Natl Acad. Sci. USA 70, 782-786, (1973)]. The uvrB trait is confirmed by demonstrating an increased bacterial sensitivity to ultraviolet light. Because this deletion also extends through a gene needed for biotin biosynthesis, the bacteria require exogenous biotin for growth. The rfa mutation causes a partial loss in the integrity of the lipopolysaccharide (LPS) cell wall so that permeability to large molecules is increased (Ames et al., 1973). The presence of the R-factor plasmid (plasmid pKMlOl which carries ampicillin resistance as a marker gene) results in an enhancement of an error-prone DNA repair system which is endogenous to these bacteria. [McCann et al., Proc. Natl Acad. Sci. USA 72, 979-983, (1975)]
Although the testing guidelines cited require S. typhimurium strain TA1537, the more recently developed TA97a strain was used. TA97 was the recommended replacement for TA1537 and has been demonstrated to be more sensitive to frameshift mutagens.(l,5) However, the reconstructed strain, 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
All bacterial strains were stored frozen in 8% DMSO in Oxoid nutrient broth at approximately -70°C. Overnight cultures were prepared by inoculating 20 mL of Oxoid nutrient broth with 0.1 mL of thawed bacterial suspension and incubating at 37°C with shaking. Overnight cultures were then stored on ice until used for mutagenesis assays. Bacterial strain phenotypes were confirmed on previous stored isolates and not concurrently with each trial. Results for the frozen permanent stock of strains has demonstrated the appropriate responses. Dose Selection The highest concentration evaluated was a solution or suspension of the test substance at concentrations of 100 μg/plate. Solubility information was confirmed empirically, as needed. Concentrations were calculated with the assumption that addition of the test substance to the solvent did not change the volume of the resulting solution. Stability And Concentration Verification
Solutions of the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions of the study. Treatment and control dosing solutions were not analyzed for concentration, uniformity, or stability. Top agar was not assayed for stability or concentration of the test substance or control articles since this assessment was not considered necessary to achieve the objectives of the study. Bacterial Mutagenicity Assays
This study consisted of one trial without metabolic activation. Three replicates were plated for each tester strain, test concentration, and condition. Positive indicators and negative controls were included in all assays. Treatments with activation were conducted by adding 0.1 mL of negative control or test substance solution, 0.5 mL of S9 mix, and 0.1 mL of an overnight culture containing at least 1 xl08 bacteria to 2 mL of top agar [0.6%) agar (w/v) and 0.6% NaCl (w/v)] supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin for S. typhimurium strains or 0.05 mM L-tryptophan for the E. coli strain. These components were mixed and poured onto a plate containing 25 mL of Davis minimal agar with dextrose (minimal agar plates, purchased from MOLTOX. Revertant colonies were counted after the individually labeled plates were incubated at approximately 37°C for about 48 hours. When necessary, plates were refrigerated prior to counting. Statistical Analysis
For each tester strain, the average number of revertants and the standard deviation at each concentration were calculated. Classification Guidelines A 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, AA, BB, CC, DD, EE, FF, and GG, 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).
Concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 (g/plate were evaluated in comparison to negative (solvent) controls. Under the conditions of these studies, evidence of mutagenic activity was detected in five of the seven test articles assessed. Results are summarized in Table 15 below:
TABLE 15
Lowest cone. in ug/ml: inducing preventing for compound luminescence^) luminescence^3) mutationW for growth inhibitio ")
AA 0.3 1.3 50 100
BB - 5.0 - ....
CC 0.1 0. 6 6 100
DD 0.6 1.3 - 6
EE 0.6 - 6 25
FF 1.3 2.5 100
GG 0.3 1.3 6 13
(1) (2) (3) (4) (5)
(a) Assay volumn of lOOul
(b) Plate volume of 28ml
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.
Five of the six light inducing compounds (col. 2) are also mutagenic (col. 4) while the threshold for inducing light appears equal or lower (col. 2) than that causing mutation (col. 4). Moreover the lights off assay (col. 3) appears superior to the growth inhibition assay (col. 5). SEQUENCE LISTING
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(D) TOPOLOGY: linear
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(2) INFORMATION FOR SEQ ID NO: 2:
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AGCTTTGGAT CCCGCTTTCT GTTTGTTTT 29
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCCACTATCG ACTACGCG 18
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTAAAACGAC GGCCAGT 17
(2) INFORMATION FOR SEQ ID NO: 6:
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(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AGCGGATAAC AATTTCACAC AGGA 24
(2) INFORMATION FOR SEQ ID NO : 7 :
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(B) TYPE: nucleic acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
GTTAGCGGAT CCCTAATTTC AGCCTCAGCC 30
(2) INFORMATION FOR SEQ ID NO: 8:
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(A) LENGTH: 31 base pairs
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
AGCAGTGAAT TCGTGTATCG TGCATCACTT C 31 (2) INFORMATION FOR SEQ ID NO: 9:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GTTAGCGGAT CCTAATAGGG GCATTCTCCG 30
(2) INFORMATION FOR SEQ ID NO: 10:
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(A) LENGTH: 33 base pairs
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(C) STRANDEDNESS: single
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AGCAGTGAAT TCCAGCCGAT ATAGTAACGA CAG 33
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B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [ I
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Claims

CLAIMS:
1. A method for the identification of the site of action of a stressor comprising:
(i) contacting a stressor with a detector bacteria, 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;
(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;
(iii) submitting the growth-inhibiting, genotoxic or non-genotoxic stressor selected in to a site of action screen;
(iv) identifying the site of action in the detector bacteria; and (vi) confirming the site of action in a target organism.
2. The method of Claim 1 wherein the stessor of step (ii) is genotoxic and the stressor of step (iii) is non-genotoxic.
3. The method of Claim 1 wherein the luminescent reporter gene complex is selected from the group consisting of thermostable lux, luxCDABE, thermolabile lux, renella lux, and luc.
4. The method of Claim 1 wherein the detector bacteria is an enteric bacteria and comprises a first mutation conferring the ability to accumulate crop protection chemicals and a second mutation preventing a global cellular response to the active stressor.
5. The method of Claim 4 wherein the second mutation prevents the global cellular response to amino acid starvation.
6. The method of Claim 1 wherein the stressor is selected from the group consisting of crop protection chemicals, antibacterial chemicals, antiviral chemicals, anticancer chemicals, and anticorrosive chemicals.
7. The method of Claim 6 wherein the stressor is selected from the group of crop protection chemicals consisting of herbicides, pesticides, and fungicides.
8. The method of Claim 7 wherein the stressor is selected from the group consisting of sulfonylurea herbicides, profungicides, fungicides, oxidants, and hormonal herbicides.
9. The method of Claim 8 wherein the stressor is selected from the group consisting of Sulfometuron methyl, glyphostate, oxime carbamates, methyl viologen, 2,4-dichlorophenoxyacetic acid, 2-thienylalanine, acivicin, and mitomycin C.
10. The method of Claim 1 wherein the genotoxic-sensitive promoter is selected from the group of genotoxic-sensitive promoters controlled by the SOS regulatory circuit.
11. - . The method of Claim 10 wherein the genotoxic-sensitive promoter is selected from the group of genotoxic-sensitive promoters consisting of recA, uvrA, lexA, umuDC, uvrA, uvrB, uvrC, sulA, recN, uvrD, ruv, dinA, dinB, dinD, and dinF.
12. The method of Claim 11 wherein the genotoxic-sensitive promoter is recA.
13. The method of Claim 1 wherein the site of action screen is a genetic titration screen or a nutritional reversal screen.
14. A detector cell strain comprising a recA-LuxCDABE gene fusion selected from the group consisting of DPD1707, DPD1708, DPD1715, DPD1716, DPD1718, DPD1719 DPD1728, DPD1729, and DPD1730.
15. A non-bioluminescent parent strain selected from the group consisting of DPD1678, DPD1675, DPD1690 DPD1012, DPD1013, DPD1692, DPD1680, DPD1693, DPD1682 DPD1010 and DPD1011.
16. A method for the identification of thienylalanine-like compounds comprising:
(i) contacting a compound suspected of having thienylalanine-like activity with a first detector bacteria, and with a second detector bacteria, the first detector bacteria comprising: a) an expressible luxCDABE gene fusion; and b) the normal cellular complement of a gene selected from the group consisting of pheA and aroH wherein the gene is not overexpressed; and the second detector cell comprising: a) an expressible luxCDABE gene fusion; and b) multiple copies of an expressible gene selected from the group consisting of pheA and aroH wherein the expressible gene is overexpressed; the first and second detector cells being isogenic and wherein the expressible luxCDABE gene fusion generates baseline luminescence;
(ii) monitoring the light output of the first and second detector cells contacted with a compound suspected of having thienylalanine-like whereby a) light output equal to baseline luminescence in both the first and the second detector cells indicates a compound with no biological activity; b) a decrease in light output as compared with baseline luminescence in both the first and the second detector cells indicates a compound with growth inhibitory activity and; c) a decrease in light output in the first detector cell as compared with baseline luminescence and an increase or maintenance of light output in the second detector cell indicates a compound having thienylalanine-like activity.
17. A method for the identification of Glyphosate-like compounds comprising: (i) contacting a compound suspected of having glyphosate-like activity with a first detector bacteria and with a second detector bacteria, the first detector bacteria comprising: a) an expressible luxCDABE gene fusion; and b) the normal cellular complement of an aroA gene wherein the aroA gene is not overexpressed the second detector cell comprising: a) an expressible luxCDABE gene fusion; and b) multiple copies of an expressible aroA gene selected wherein the aroA gene is overexpressed; the first and the second detector cells being isogenic and wherein the expressible luxCDABE gene fusion generates baseline luminescence;
(ii) monitoring the light output of the first and the second detector cells contacted with a compound suspected of having glyphostate-like activity whereby a) light output equal to baseline luminescence in both the first and the second detector cells indicates a compound with no biological activity; b) a decrease in light output as compared with baseline luminescence in both the first and the second detector cells indicates a compound with growth inhibitory activity; and c) a decrease in light output in the first detector cell as compared with baseline luminescence and an increase or maintenance of light output in the second detector cell indicates a compound having glyphosate-like activity.
18. A method for the identification of ALS-inhibiting compounds comprising:
(i) contacting a compound suspected of having ALS-inhibiting activity with a first detector bacteria and with a second detector bacteria, the first detector bacteria comprising: a) an expressible luxCDABE gene fusion; and b) the normal cellular complement of a gene selected from the group consisting of ilvBN and ilvIH wherein the gene is not overexpressed; the second detector cell comprising: a) an expressible luxCDABE gene fusion; and b) multiple copies of an expressible gene selected from the group consisting of ilvBN and ilvIH wherein the expressible gene is overexpressed; the first and the second detector cells being isogenic and wherein the expressible luxCDABE gene fusion generates baseline luminescence;
(ii) monitoring the light output of the first and the second detector cells contacted with a compound suspected of having ALS-inhibiting activity whereby a) light output equal to baseline luminescence in both the first and the second detector cells indicates a compound with no biological activity; b) a decrease in light output as compared with baseline luminescence in both the first and the second detector cells indicates a compound with growth inhibitory activity; and c) a decrease in light output in the first detector cell as compared with baseline luminescence and an increase or maintenance of light output in the second detector cell indicates a compound having ALS-inhibiting activity.
19. A method for identifying a genotoxic substance, 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 of step (i) with a substance to be tested; and
(iii) determining whether the substance is genotoxic by measuring the amount of luminescence in the culture.
20. The method of Claim 19 wherein the promoter regulated by a SOS bacterial regulatory circuit is selected from the group consisting of recA, uvrA, lexA, umuDC, uvrA, uvrB, uvrC, sulA, recN, uvrD, ruv, dinA, dinB, dinD, and dinF.
21. A method of identifying a structural gene encoding a stressor target comprising:
(i) contacting a stressor with a detector bacteria, 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; (ii) selecting for genotoxic or non-genotoxic stressors capable of inhibiting the growth said detector bacteria of step (i) by monitoring the growth and light output of the detector bacteria;
(iii) submitting the growth-inhibiting, genotoxic or non-genotoxic stressor to a site of action screen wherein the stressor target is identified and; (vi) isolating the structural gene encoding the stressor target.
22. A method for the identification of the site of action of a stressor comprising:
(i) contacting a stressor with a detector bacteria, the detector bacteria comprising an expressible luxCDABE gene fusion wherein the expressible luxCDABE gene fusion generates baseline luminescence in the detector bacteria; (ii) selecting for stressors capable of inhibiting the growth of the detector bacteria of step (i) by monitoring the growth and light output of the detector bacteria;
(iii) submitting the growth-inhibiting stressor selected in step (ii) to a site of action screen;
(iv) identifying the site of action in the detector bacteria; and (vi) confirming the site of action in a target organism.
23. The method of Claim 22 wherein the expressible luxCDABE gene fusion further comprises a stressor sensitive promoter selected from the group consisting of grpE, dnaK, Ion, rpoD, groESL, lysU, htpE, htpG, htpl, htpK, clpP, clpB, htpN, htpO, htpX, katG, ahp, micF, sodA, nfo, zwfi soi, fabA, his, ilvBN, ilvGMED, thr ABC, lac, mal, and gal, phoA, phoBR, phoE, phoS, aphA, himA, pepN, ugpAB, psiD, psiE, psiF, psiK, psiG, psil, psiJ, psiN, psiR, psiH, phiL, phiO or a, tna, dsd, glnA, and hut.
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