WO1994001561A9 - Element d'activation de genes - Google Patents

Element d'activation de genes

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Publication number
WO1994001561A9
WO1994001561A9 PCT/US1993/006300 US9306300W WO9401561A9 WO 1994001561 A9 WO1994001561 A9 WO 1994001561A9 US 9306300 W US9306300 W US 9306300W WO 9401561 A9 WO9401561 A9 WO 9401561A9
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WIPO (PCT)
Prior art keywords
sequence
gene
strain
dna sequence
bacterial
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PCT/US1993/006300
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English (en)
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WO1994001561A1 (fr
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Priority to AU46625/93A priority Critical patent/AU679925B2/en
Priority to JP6503435A priority patent/JPH07509605A/ja
Priority to EP93916933A priority patent/EP0649469A1/fr
Publication of WO1994001561A1 publication Critical patent/WO1994001561A1/fr
Publication of WO1994001561A9 publication Critical patent/WO1994001561A9/fr

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  • the present invention relates to the identification, isolation, cloning and use of genetic elements which contribute to the activation of genes in bacterial strains. More specifically, the invention relates to the identification of two classes of genetic elements which interact with each other in the activation of genes in bacteria. Manipulation of either or both types of element can be used to manipulate bacterial phenotype.
  • Rhizoctonia is a particularly problematic plant pathogen for several reasons. First, it is capable of infecting a wide range of crop plants. Second, there are no commercially available chemical fungicides that are effective in controlling the fungus. Because of these circumstances, an inhibitor against R. solani would be of substantial interest as a potential control for this pathogen.
  • the present invention is the first to describe the unexpected finding that certain natural bacterial isolates carry undetected, latent genes which can be activated upon introduction of a bacterial transcription activator derived from a different organism (in this case, either upon introduction of a 2.0 kb Xhol fragment of P. fluorescens strain 915 DNA containing ORF 5 or upon introduction of a cloned E. coli gene homologous to ORF 5, gafA) .
  • DNA sequences and genes are provided that activate genes in bacterial strains. It is a further feature of the present invention that modified DNA sequences and genes be provided which encode modified proteins, which enhance the activation of genes in bacterial strains. Such modifications may improve the efficacy of regulatory genes.
  • biocontrol agents may be produced which are able to inhibit a broad spectrum of plant pathogens.
  • biocontrol agents may be produced which are able to aggressively compete in the plant rhizosphere, which biocontrol agents contain a DNA sequence that activates genes in the bacterial biocontrol agent.
  • the above objectives may be carried out by the isolation and use of genetic elements or gene activating sequences that are able to activate genes that are not normally turned on in bacterial strains.
  • the isolation of these gene activating sequences is important for several reasons.
  • the activated strains produce substances, such as pyrrolnitrin and chitinase, which are able to inhibit plant pathogens, particularly fungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineae and species of the genera Pythium and Fusarium. Therefore, use of bacterial strains transformed with the ORF 5-type or lemA-type genetic element provides an environmentally safe and effective method of control of these pathogens.
  • solani may become a growing problem pathogen of wheat (J. Cook, personal communication) .
  • the gene activating sequence for activation of genes which are effective against R. solani could be introduced into the biocontrol strains currently used to protect wheat from take-all to extend their range of effectiveness to include R. solani.
  • the present invention comprises an isolated DNA sequence consisting essentially of the 2 kb fragment deposited as pCIB 137, or of the ORF 5 sequence shown in SEQUENCE ID No. 1. These DNA sequences are capable of activating latent gene activity in a bacterial strain.
  • the present invention also comprises methods of activating latent gene activity in a host bacterial strain comprising introducing the DNA sequence into the genome of a host bacterial strain.
  • the host bacterial strain may be a pseudcmonad, particularly strains of the species Pseudomonas fluorescens.
  • the present invention further comprises recombinant DNA sequences in which a bacterial regulatory element is operably linked to the DNA sequence of SEQUENCE ID No. 1.
  • the bacterial regulatory element may be a promoter from a gene isolated from Pseudomonas, Bacillus, or E_ coli. In one embodiment of the present invention, the bacterial regulatory element is the native promoter of ORF 5.
  • the bacterial regulatory element may be from a gene which is homologous or heterologous to the host bacterial strain.
  • the present invention also includes methods of activating latent gene activity in a host bacterial strain by transforming the host bacterial strain with the recombinant DNA sequences of the present invention.
  • the transformed host bacterial strain is rendered active against fungal pathogens, such as Rhizoctonia solani, Hehninthosporium qramineae and species of the genera Pythium and Fusari m.
  • the present invention further comprises isolated DNA sequences encoding the lemA gene. These sequences are capable of restoring the production of hydrolytic enzymes such as chitinase and gelatinase and the production of antifungal secondary metabolites such as pyrrolnitrin and cyanide in some mutants lacking these functions.
  • the invention further comprises recombinant DNA sequences in which a bacterial regulatory element is operably linked to the DNA coding sequence of lemA.
  • the bacterial regulatory element may be a promoter from a gene isolated from Pseudomonas, Bacillus, or E coli. In one embodiment of the present invention, the bacterial regulatory element is the native promoter of lemA.
  • the bacterial regulatory element may also be from a gene which is homologous or heterologous to the host bacterial strain.
  • the present invention also includes methods of activating latent gene activity in a host bacterial strain by transforming the host bacterial strain with the recombinant DNA sequences of the present invention.
  • the transformed host bacterial strain is rendered active against fungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineae and species of the genera Pythium and Fusarium.
  • the gene activating sequence includes the deposited DNA sequence as well as fragments thereof. By fragments is intended a DNA sequence which is capable of functioning as a gene activating sequence.
  • Promoter or Regulator DNA sequence An untranslated DNA sequence which assists in, enhances, or otherwise affects the transcription, translation or expression of an associated structural DNA sequence which codes for a protein or other DNA product.
  • the promoter DNA sequence is usually located at the 5' end of a translated DNA sequence, typically between 20 and 100 nucleotides to the 5' end of the starting site for translation.
  • Structural or Coding DNA sequen ⁇ e A DNA sequence that is translated or transcribed in an organism to produce an RNA, a protein or other DNA product.
  • Two DNA sequences which are "associated” or “operably linked” are related physically or functionally.
  • a promoter or regulator DNA sequence is said to be “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are operably linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
  • a first DNA sequence or fragment is said to be "derived from” a second DNA sequence or fragment if the former is physically isolated from the latter, or if the former is isolated by using part or all of the latter as a probe for isolation.
  • a DNA sequence is said to be "homologous" to a host organism, such as a bacterial strain, if that DNA sequence was originally isolated from, or naturally originates in, the genome of an organism of similar biological classification as the host organism.
  • a host organism to be transformed is of the species Pseudomonas fluorescens
  • a DNA sequence is homologous if it originates from a pseudo onad strain, particularly from a strain of the genus Pseudomonas, especially the species Pseudomonas fluorescens.
  • the term "heterologous” is used to indicate a recombinant DNA sequence in which the promoter or regulator DNA sequence and the associated DNA sequence are isolated frqm organisms of different biological classification.
  • Q meric oonstruct/r-ii eric DNA sequence A recombinant DNA sequence in which a regulator or promoter DNA sequence is associated with, or operably linked to, a DNA sequence that codes for an mRNA or which is expressed as a protein, such that the regulator DNA sequence is able to regulate transcription or expression of the associated DNA sequence.
  • the regulator DNA sequence of the chimeric construct is not normally operably linked to the associated DNA sequence as found in nature.
  • Genome refers to the entire native genetic content of an organism.
  • the genome of bacterial organisms may include both the chromosomal and plasmid DNA content of an organism.
  • Gene activating sequences Sequences which, when transformed into a host, have the ability to turn on other genes which are not expressed (i.e. latent) or expressed at low levels in the naturally occurring state of the host. These sequences typically encode proteins which play a role in the pathways which regulate gene expression.
  • Figure 1 This figure shows restriction maps of three cosmid clones, pANT5, pANT9 and pANTIO, that were found to complement the ANT ⁇ phenotype of mutant 2-1.
  • 'B' indicates a BamHI restriction site
  • 'E' an EcoRI restriction site
  • 'H' a Hindlll restriction site.
  • Figure 2 This figure shows the ability of DNA subfragments derived fran the- pANT5 clone of Pseudomonas fluorescens strain 915 to complement the ANT ⁇ phenotype of mutant 2-1 and the wild type strains 914 and 922.
  • the subfragment labelled 3 is the approximately 11 kb region which has been called the Ell fragment.
  • FIG. 3 This figure indicates the organization of the Ell fragment. The figure indicates the location of five identified open reading frames (ORF) and restriction sites for various enzymes.
  • ORF open reading frames
  • Figure 4 This figure shows the ability of DNA subfragments derived from the clone pCIB 146 of Pseudomonas fluorescens strain 915 to complement the mutant CGP 21.
  • Pseudomonas fluorescens strain 915 was isolated from the roots of a cotton plant grown in a Texas cotton field and was identified as an effective biocontrol strain of Pythium ultimum- and Rhizoctonia solani-induced damping off of cotton. We have determined that certain mutant derivatives of the bacterial biological control strain Pseudomonas fluorescens strain 915 are deficient or altered in a variety of functions. Such pleiotropic mutants can be isolated following mutagenesis techniques known to those skilled in the art (e.g. nitrosoguanidine mutagenesis, transposon mutagenesis) or can arise spontaneously.
  • mutant 2-1 One such mutant, obtained after mutagenesis with the chemical mutagen nitrosoguanidine was designated mutant 2-1. Seven further mutants were identified by introducing the transposon TnCIB116 into strain 915. These mutants can be identified on the basis of their inability to inhibit in vitro the growth of the phytopathogenic fungus Rhizoctonia solani. They also fail to synthesize the antifungal metabolite pyrrolnitrin, and no longer produce cyanide or the enzyme chitinase, each of which has the potential to inhibit fungal growth (Voisard et al., EMBO J. 8:351-358 (1989); Jones et al., EMBO J. 5:467-473 (1986)) .
  • the mutants' production of an enzyme with gelatinase activity is significantly reduced, and they have an altered colony morphology.
  • a summary comparing the characteristics of the pleiotropic mutants with the corresponding characteristics of wild-type P. fluorescens strain 915 is presented in Table 1.
  • An 11 kilobase EcoRI restriction fragment (referred to as fragment "Ell") of P. fluorescens strain 915 was identified on the basis of its ability to restore antibiosis to a mutant, designated strain 2-1, and two further mutants (derived from insertion mutagenesis) which were otherwise incapable of inhibiting the growth of the phytopathogen Rhizoctonia solani in vitro or in greenhouse biological control assays.
  • fluorescens strain 915 and a 2.0 kb Xhol subclone of this fragment containing gafA (ORF 5), each restored all of the lost or altered functions listed in Table 1 when introduced by conjugation into one class of pleiotropic mutants derived from strain 915.
  • Introduction of fragment Ell or the 2.0 kb Xhol subclone into the P. fluorescens strains 914 and 922 unexpectedly activate the expression of latent genes involved in the synthesis of pyrrolnitrin, cyanide, and chitinase, and in the case of P.
  • fluorescens strain 914 cause an alteration in colony morphology on minimal medium from large, circular, flat, translucent, with undulate edge to small, circular, convex, opaque white, with entire edge.
  • P. fluorescens strains 914 and 922 to effective biocontrol strains with activity against the phytopathogen Rhizoctonia solani.
  • fluorescens strain 914 activates the expression of genes involved in the synthesis of cyanide, chitinase, and pyrrolnitrin. This result indicates that genes of the gafA class are sufficient for the activation of latent genes in heterologous bacterial strains.
  • ORF 5 coli, at a position equivalent to ORF 5 (gafA) , exists a putative transcriptional activator gene of unknown function (Moolenaar et al., Nucl. Acids Res. 15:4273-4289 (1987)). ORF 5 (gafA) exhibits homology to this putative activator gene. Furthermore, comparison of the fragment Ell sequence with DNA sequences contained in the Genbank database reveals that ORF 5 has substantial homology to a proposed transcriptional activator gene isolated from Pseudomonas fluorescens CHAO by Laville et al., Proc. Natl. Acad. Sci. USA 89:1562-1566 (1992).
  • ORF 2 and ORF 5 share significant homology with numerous sensor and activator co ⁇ ponents, respectively, of bacterial two-component regulatory systems (reviewed in Albright et al., Annu. Rev. Genet. 23:311-336 (1989)).
  • Subcloning experiments are performed with fragment Ell with the aim of determining whether the gene(s) responsible for restoring lost functions to the pleiotropic mutants and for activating latent activities in heterologous Pseudomonas strains could be isolated on a smaller restriction fragment.
  • a 2.0 kb Xhol subclone containing ORF 5 is prepared, as is a 3.7 kb EcoRI-Xbal subclone containing ORF1 and ORF 2.
  • the 2.0 kb Xhol subclone is sufficient to restore the lost functions in the class of pleiotropic mutants originally complemented by fragment Ell and activated the expression of latent genes in P. fluorescens strains 914 and 922.
  • the 3.7 kb EcoRI-Xbal subclone had no measurable effect. Furthermore, when the gafA gene was cloned from the strain 914, transferred to plasmid pLAFR3 and reintroduced into strain 914 the latent genes are activated indicating that strain 914 does contain a gafA gene capable of functioning, but that the expression of the gafA gene in strain 914 is presumably not at levels high enough to activate the latent genes. To determine whether transcriptional activators of the gafA class are generally capable of activating the ej ⁇ ression of latent genes in heterologous bacterial strains, we cloned the putative transcriptional activator gene described by Moolenar et al. and introduced it into P.
  • fluorescens strain 914 The E. coli gene, which encodes a protein which is approximately 60% homologous to that encoded by gafA, activated the ej ⁇ ression of genes involved in the production of cyanide, chitinase, and pyrrolnitrin in P. fluorescens strain 91 .
  • i ⁇ proved biological control strains can be identified following the introduction of transcriptional activators of the gafA class into a variety of environmental isolates. This approach represents a method for the identification of potentially effective biocontrol strains which would otherwise not be selected by any of the screening methods currently available.
  • recombinant DNA sequences are obtained which comprise an approj mately 2 Kb DNA sequence consisting essentially of the DNA sequence of gafA.
  • This DNA sequence demonstrates pleiotropic effects of activating latent gene activity or increasing the efficacy of other genes.
  • the pleiotropic effects of the gafA DNA sequence are the increased ability to inhibit the growth of fungal pathogens, such as Rhizoctonia solani, Hel ⁇ nthosporivim gramineae and species of the genera Pythium and Fusarium
  • This DNA sequence may be derived from bacterial strains which are effective biocontrol agents against Rhizoctonia.
  • the DNA sequence may be derived from the clone pANT5, which was isolated from a strain of Pseudomonas fluorescens.
  • the DNA sequence may comprise the approximately 11 kb Ell fragment of pANT5.
  • the DNA sequence consists essentially of the appro ⁇ mately 2 kb fragment, or the DNA sequence of SEQUENCE ID No. 1.
  • the clone pANT5 has been deposited with ATCC and has been designated ATCC accession number 40868.
  • a plasmid containing the 11 kb Ell fragment of pANT5 has been deposited with ATCC and has been designated ATCC accession number 40869.
  • the approximately 2 kb ORF 5 DNA sequence may be obtained from the Ell fragment of pANT5 as a 2 kb fragment after digestion with Xhol. This fragment has been designated pCIB137 • and has been deposited with the USDA Agricultural Research Service Culture Collection, Northern Regional Research Center (NRRL) .
  • the recombinant DNA sequences of the present invention may be chimeric and may be heterologous or homologous.
  • the recombinant DNA sequences of the present invention may further comprise one or more regulatory DNA sequences operably linked to the structural DNA sequence above.
  • regulatory DNA sequences include promoter sequences, leader sequences, and other DNA sequences which may affect the ej ⁇ ression of the regulatory DNA sequences, as well as those fragments of a regulator DNA sequence that are able to act with such effect.
  • lemA would predictably activate latent gene ej ⁇ ression in strains of Pseudomonas in which lemA ej ⁇ ression is the rate- limiting factor preventing the production of chitinase, gelatinase, pyrrolnitrin and cyanide.
  • Other bacterial genes functionally homologous to lemA would be able to act in the same way as lemA.
  • Such genes comprise a class of sensory component genes capable of phosphorylating and therefore activating the gafA class of activators described above.
  • Members of the class can be identified and isolated by co ⁇ plementation of the appropriate class of pleiotropic mutants as described herein. For eca ⁇ ple, the corresponding E_ coli gene responsible for the phosphorylation of uvrY is one such gene.
  • biocontrol agents are provided which are able to inhibit the growth of fungal pathogens, such as Rhizoctonia solani, Hel ⁇ nthosporium gramineae and species of the genera Pythium and Fusarium.
  • These biocontrol agents may be bacteria, plant cells or animal cells transformed with the recombinant DNA sequences above, but are preferably bacterial strains, and more preferably gram negative bacterial strains, such as the pseudcttonads.
  • Most preferred as the biocontrol agent are strains of the species Pseudomonas fluorescens.
  • Another embodiment of the present invention provides methods of inhibiting the growth of fungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineae and species of the genera Pythium and Fusarium.
  • the gene activating DNA sequences can be introduced into the genome of a bacterial strain which may not ordinarily be effective as an inhibitor of fungal pathogens, resulting in an effective biocontrol strain.
  • DNA in the form of plasmids can be transferred from one bacterium to another by a sejcual process termed conjugation.
  • Plasmids capable of conjugal transfer contain genes that code for the synthesis of sex pili.
  • Sex pili are hollow tubes that join the plasmid-containing bacterium (the donor) with another bacterium (the recipient) and through which replicated copies of the plasmid pass from the donor to the recipient. This procedure occurs naturally in nature and is utilized in the laboratory as a method of transferring genes from one bacterium to another. For some strains of bacteria, such as Pseudomonas, conjugal transfer of DNA is the preferred method since these bacteria are not readily transformed with ejtraneous DNA.
  • kits for producing antibiotic substances which are effective in inhibiting the growth of fungal pathogens, such as Rhizoctonia solani, Helminthosporiim gramineae and species of the genera Pythium and Fusarium.
  • This method comprises introducing the recombinant DNA sequences of the present invention into the genome of a biocontrol agent to form a transformed biocontrol agent, allowing the transformed biocontrol agent to produce antibiotic substances, such as pyrrolnitrin, and e tracting the antibiotic substance from the transformed biocontrol agent.
  • the present invention embraces the preparation of antifungal compositions in which one or more of the transformed biocontrol bacterial strains are used as active ingredient.
  • the present invention further embraces the preparation of antifungal compositions in which the active ingredient is the antifungal metabolite or antibiotic co ⁇ pound produced by the transformed biocontrol agent of the present invention.
  • the active ingredient is a biocontrol bacterial strain
  • the biocontrol preparation may be applied in any manner known for seed and soil treatment with bacterial strains.
  • the bacterial strain may be homogeneously mixed with one or more compounds or groups of compounds described herein, provided such co ⁇ pound is compatible with bacterial strains.
  • the present invention also relates to methods of treating plants, which co ⁇ prise application of the bacterial strain, or antifungal compositions containing the bacterial strain, to plants.
  • the active ingredient of the present invention may also be an antifungal metabolite, such as an antibiotic co ⁇ pound, produced by the biocontrol agents of the present invention.
  • the present invention also relates to methods of treating plants, which comprise application of the antifungal metabolite, such as an antibiotic co ⁇ pound, or antifungal compositions containing the metabolite, to plants.
  • the active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with further co ⁇ pounds.
  • These co ⁇ pounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, mollusicides or ijctures of several of these preparations, if desired together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation.
  • Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
  • a preferred method of applying active ingredients of the present invention or an agrochemical co ⁇ position which contains at least one of the active ingredients is leaf application.
  • the number of applications and the rate of application depend on the intensity of infestation by the corresponding pathogen (type of fungus) .
  • the active ingredients can also penetrate the plant through the roots via the soil (systemic • action) by impregnating the locus of the plant with a liquid composition, or by applying the co ⁇ pounds in solid form to the soil, e.g. in granular form (soil application) .
  • the active ingredients may also be applied to seeds (coating) by impregnating the seeds either with a liquid formulation containing active ingredients, or coating them with a solid formulation. In special cases, further types of application are also possible, for
  • BSTITUTE ShE ⁇ T RULE 26 example, selective treatment of the plant stems or buds.
  • the active ingredients are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for exa ⁇ ple, in polymer substances.
  • the methods of application such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.
  • Advantageous rates of application are normally from 50 g to 5 kg of active ingredient (a.i.) per hectare ("ha", approximately 2.471 acres), preferably from 100 g to 2 kg a.i./ha, most preferably from 200 g to 500 g a.i./ha.
  • compositions or preparations containing the active ingredients and, where appropriate, a solid or liquid adjuvant are prepared in known manner, for ejcample by homogeneously mijing and/or grinding the active ingredients with ectenders, for ejcample solvents, solid carriers and, where appropriate, surface-active co ⁇ pounds (surfactants) .
  • Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for ejcample, jcylene mictures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons'such as cyclohej ⁇ ane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclonejanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfocide or dimethyl formamide, as well as epo dized vegetable oils such as epojidized coconut oil or soybean oil; or water.
  • aromatic hydrocarbons preferably the fractions having 8 to 12 carbon atoms, for ejcample, jcylene mictures or substituted naphthalenes, phthalates such as dibut
  • the solid carriers used e.g. for dusts and dispersible powders are normally natural mineral fillers such as calcite, talcum, kaolin, mont ⁇ orillonite or attapulgite.
  • mineral fillers such as calcite, talcum, kaolin, mont ⁇ orillonite or attapulgite.
  • highly dispersed silicic acid or highly dispersed absorbent polymers In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive
  • ET RULE 26 ⁇ carriers are porous types, for ejca ⁇ ple pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand.
  • suitable nonsorbent carriers are materials such as calcite or sand.
  • pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.
  • suitable surface-active co ⁇ pounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties.
  • surfactants will also be understood as comprising mixtures of surfactants.
  • Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active co ⁇ pounds.
  • Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms) , for ejca ⁇ ple the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mictures which can be obtained for ejca ⁇ ple from coconut oil or tallow oil.
  • the fatty acid methyltaurin salts may also be used.
  • so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.
  • the fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammoniums salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for ejca ⁇ ple, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a m cture of fatty alcohol sulfates obtained from natural fatty acids.
  • These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts.
  • the sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms.
  • Ejcamples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnapthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product.
  • corresponding phosphates e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene ojdde.
  • Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.
  • non-ionic surfactants are the water-soluble adducts of polyethylene ocide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.
  • non-ionic surfactants re nonylphenolpolyethocyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenojcypolyethojcyethanol, polyethylene glycol and octylphenocyethocyethanol.
  • Fatty acid esters of polyojcyethylene sorbitan and polyojcyethylene sorbitan trioleate are also suitable non-ionic surfactants.
  • Cationic surfactants are preferably quaternary ammonium salts which have, as N-substituent, at least one Cg-C ⁇ al yl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydrojcyalkyl radicals.
  • the salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethyla ⁇ onium bromide.
  • the agrochemical compositions usually contain from about 0.1 to about 99 %, preferably about 0.1 to about 95 %, and most prferably from about 3 to about 90 % of the active ingredient, from about 1 to about 99.9 %, preferably from abut 1 to about 99 %, and most preferably from about 5 to about 95 % of a solid or liquid adjuvant, and from about 0 to about 25 %, preferably about 0.1 to about 25 %, and most preferably from about 0.1 to about 20 % of a surfactant.
  • the gene activating sequences of the present invention can be used in a variety of microorganisms to induce the production of gene products and secondary metabolites.
  • the activating sequences are capable of inducing or enhancing the expression of genes which may be latent or natively expressed at low levels.
  • the activating elements find use in the production of antibiotics in microorganisms for the purposes of biocontrol. Such elements are also useful in the production of antibiotics for phar auceutical purposes, particularly in strains of microorganisms which natively e ⁇ ress the biosynthetic genes at low levels or not at all. Ejca ⁇ ples of strains suitable for manipulation in this manner include strains of Streptomyces for the production of tetracycline, erythromycin, chloromycetin, and streptomycin; strains of Bacillus for the production of bacitracin; and strains of Penicillium for the production of penicillin.
  • the activating elements find further use in the production of vitamins, growth factors and hormones in selected strains of microorganisms.
  • Ecamples include the enhanced production of vitamin B2 and B12 from Ashbya and Streptomyces, respectively; the production of
  • Other applications include but are not limited to the enhanced production of butanol, acetone, and ethanol from Clostridium; the production of glycerol from Saccharomyces; the production of lactic acid from Lactobacillus; the production of polysaccharides such as alginate, canthan, dextran from Leuconostoc and other organisms; the production of secreted proteins and enzymes such as analyses, proteases, pectinases, chitinases, cellulases, gelatinases, collagenases, elastases, etc. from Bacillus, Aspergillus and other organisms; and the production of invertase from Saccharomyces.
  • the wild-type Pseudomonas fluorescens strain llc-1-38 (strain 915) was utated by ej ⁇ osure to the mutagen N-Msthyl-N'-nitro-N- nitrosoguanidine. Approximately two dozen antibiotic-affected mutants were identified by screening individual mutants for their ability to inhibit the growth of P. ultimu and R. solani on nutrient agar. Most of these were shown to have reduced, or no, antibiotic activity against one of the two phytopathogenic fungi, but were not affected in their inhibition of the other fungus. However, one mutant, that we have named mutant 2-1, was found to lack antibiotic activity against both test fungi. These results strongly indicated that P. fluorescens strain llc-1-38 produced two distinct antibiotics, one effective against P. ultimum and the other effective against R. solani, rather than one antibiotic that was effective against both fungi.
  • a gene library of total DNA isolated from the parent strain was constructed by partial Sail restriction of the DNA, size fractionation to yield fragments of 20-30 kilobases, and ligation into Xhol-restricted vector pVKlOO. Knauf et al., Plasmid 8:45-54 (1982) .
  • the gene library was transferred to the antibiotic mutant 2-1 by triparental conjugation with EL coli harboring the tra + plasmid pRK2013. Ditta et al., Proc. Natl. Acad. Sci. USA 77:7347-7351 (1980) .
  • Transgenic exconjugants were tested for the production of antibiotic by measuring growth inhibition of P. ultimum and R. solani. Three overlapping clones were identified that restored antibiotic activity against R. solani, but not against P. ultimum, to mutant 2-1. Restriction maps of these clones were determined and are shown in Figure 1.
  • the genetic region necessary for the functional complementation of antibiotic biosynthesis in mutant 2-1 was defined by subcloning portions of the larger region and assessing their ability to co ⁇ plement mutant 2-1 for antibiosis (Figure 2) .
  • the smallest fragment that was demonstrated to co ⁇ plement the mutation was the 4.9 kb Hindlll/EcoRI fragment, which was indicated as subfragment 6 in Figure 2 and was designated subfragment H/E4.9.
  • An active antibiotic co ⁇ pound can be ejetracted from the growth medium of the transformed P. fluorescens strain that produces this antibiotic. This was accomplished by ejctraction of the medium with 80 % acetone followed by removal of the acetone by evaporation and a second extraction with diethyl ether. The diethyl ether was removed by evaporation and the dried ejctract is resuspended in a small volume of water. Small aliquots of the antibiotic ejctract applied to small sterile filter paper discs placed on an agar plate will inhibit the growth of R. solani, indicating the presence of the active antibiotic co ⁇ pound. The antibiotic was determined by NMR and mass spectrometry to be pyrrolnitrin.
  • mutant 2-1 The mutant derivative of P. fluorescens strain 915 designated mutant 2-1 was initially isolated on the basis of its reduced ability to inhibit the growth of the fungus Rhizoctonia solani in vitro. The production of an antibiotic metabolite, subsequently identified as pyrrolnitrin, was lacking in mutant 2-1. While lack of pyrrolnitrin production was the first defect observed in mutant 2-1, additional e ⁇ erimentation, details of which are described below, reveal that mutant 2-1 is a member of one class of pleiotropic mutants with characteristics summarized in Table 1.
  • P. fluorescens strain 915 and mutant 2-1 were tested for pyrrolnitrin production by growing the respective cultures for three days in 50 ml of nutrient broth containing AMBERLITE XAD-4 resin (Rohm and Haas) (5% v/v) at 28 C. The resin was collected in a sieve and washed ectensively with water. The pyrrolnitrin was eluted from the resin by two consecutive ejctractions with isopropanol (0.5X volume) . The two ejctractions were combined and dessicated under vacuum in a rotary evaporator at 40 C.
  • the dessicated material is dissolved in 2 ml of isopropanol and was further analyzed by HPLC chromatography using a Hypersil CBS column (2.1 mm dia. x 10 cm) with a mobile phase consisting of a water/methanol ⁇ ubcture with a starting co ⁇ position of 0/100% and gradually changing to a final co ⁇ position of 100/0%.
  • a Hypersil CBS column 2.1 mm dia. x 10 cm
  • 100 ul of each ejctract was dessicated under vacuum and resuspended in the same volume of water/methanol (50/50) .
  • the material eluting from the column was monitored by B absorbance at 212 nm and at 252 nm, and was fractionated by elution time. P.
  • fluorescens strain 915 ejctracts contained a peak which comigrates with a pyrrolnitrin standard and which was determined to be pyrrolnitrin by NMR spectroscopy and by mass spectrometry. Mutant 2-1 and the other identified pleiotropic mutants lack this peak.
  • P. fluorescens strain 915 and mutant 2-1 and additional pleiotropic mutants were tested for the production of cyanide.
  • Pieces of Whatman paper were impregnated with 5 mg/ml chloroform cupric ethyl acetoacetate and 5 mg/ml chloroform 4,4'-methylene bis-(N,N dimethyl aniline) and chloroform was allowed to evaporate. Papers were then placed under the covers of microtiter plates whose wells have been inoculated with cultures of strain 915 and the various pleiotropic mutants. Plates are wrapped in aluminum foil and incubated overnight at 28 C. The paper turned a blue color above the well of each culture producing cyanide. The results indicated that strain 915 produced cyanide, while the pleiotropic mutants failed to produce cyanide.
  • P. fluorescens strain 915 and various pleiotropic mutants were tested for the presence of chitinase activity by either of two methods.
  • 300 ml L-broth cultures of each strain were incubated at 28 C for 12 hours. Cells were collected by centrifugation and washed once in 20 M phosphate buffer (10 nM Na2HPO4/10 mM KH2P04) . Following centrifugation, the cell pellets were resuspended in 5 ml of the 20 mM phosphate buffer. Cells were lysed by sonication for 60 seconds with the microtip of a Branson sonifier and cell debris is removed from the cell ejctracts by centrifugation.
  • Chitinase activity was assayed by incubation of 100 ul of cell ejctract with 100 ul of tritiated chitin (0.5% w/v; approcimately 0.1 mCi/ml) in a 250 ul total volume of 0.03 M sodium phosphate, pH 6.5 for 1 hour at 37 C.
  • the reaction was stepped by addition of an equal volume of 2 M TCA, followed by centrifugation. 200 ul aliquots were counted in a liquid scintillation counter to determine soluble counts released from the insoluble chitin molecules as a result of chitinase activity.
  • Typical results, presented in Table 2, indicate that a transposon mutant designated #736, which proved to be a member of one class of pleiotropic mutants, lacks the chitinase activity found in P. fluorescens strain 915.
  • Gelatinase activity of P. fluorescens strain 915 and various pleiotropic mutant derivatives was assayed by incubating the bacteria on nutrient agar plates supplemented with 3% w/v Bacto-gelatin (Difco) .
  • a cloudy halo forms in the agar surrounding colonies synthesizing and ecporting a protease capable of hydrolyzing the gelatin.
  • Prominent halos appeared around colonies of strain 915 following 24 hour incubation at 28 C. Such halos failed to appear around colonies of the pleiotropic mutants within the 24 hour time period, appearing instead after approj mately 48 hours.
  • the pleiotropic mutants are not totally devoid of gelatinase activity, they either fail to synthesize the species of protease which appears in strain 915 within 24 hours, or else the synthesis and/or e ⁇ ort of that species is delayed.
  • P. fluorescens strain 915 forms small, circular, convex white opaque colonies with entire edges. All pleiotropic mutants ecamined thus far formed larger, circular, flat, translucent colonies with undulate edges.
  • Figure 3 depicts the genetic organization of the 11 kilobase fragment Ell determined to date from DNA sequence analysis.
  • a variety of subclones of fragment Ell were prepared as double-stranded templates for dideocy sequencing reactions by digestion of fragment Ell with various restriction endonucleases, either singly or in combination, followed by ligation with appropriate cloning vectors such as pBS SK+ (Stratagene) .
  • pBS SK+ Stratagene
  • Ejca ⁇ ples of other bacterial sensor co ⁇ ponent genes include cheA of E. coli, rcsC of E. coli, frzE of Myxococcus xanthus, and bvgS of Bordetella pertussis.
  • the P. fluorescens strain 915 glyW tRNA gene is 100% homologous to the glyW tRNA locus of E. coli.
  • ORF 3 shares substantial homology with the pgsA gene of E. coli which encodes phosphatidyl glycer ⁇ phosphate, an enzyme involved in phospholipid metabolism (Gopalakrishnan et al., J. Biol. Chem. 261:1329-1338 (1986)).
  • ORF 4 shares substantial homology with the uyrC gene of E. coli which encodes a co ⁇ ponent of the ultraviolet light damage repair excinuclease (Sharma et al., Nucl. Acids Res. 14:2301-2318 (1986)).
  • ORF 5 which is present on the gene-activating 2.0 kb Xhol subclone of fragment Ell, shares substantial homology with numerous genes encoding transcription activator co ⁇ ponents of bacterial two-component regulatory sequences.
  • ORF 5 is highly similar to the gacA gene of Pseudomonas fluorescens CHA0 (Laville et al., 1992) and to the uvr-23 gene of E. coli (Moolenar et al., 1987) .
  • Ejca ⁇ ples of some other bacterial transcriptional activator genes with sequence similarity to ORF 5 include sacU of Bacillus subtilis, bvqA of Bordetella pertussis, and algR of Pseudomonas aeruginosa.
  • the entire coding region of ORF 5 is presented as SEQUENCE ID NO. 1.
  • SEQUENCE ID NO. 1 The entire sequence of a a. 5.6 kilobase portion of fragment Ell, bounded by the left-most EcoRI site depicted in Figure 3 and by an internal Hindlll site, is presented as SEQUE ID 2.
  • SEQUE ID 2 The coordinates of open reading frames contained in sequence ID 2 are as follows:
  • ORF 1 210-1688; transcribed left to right
  • ORF 2 1906-3633; transcribed left to right glyW: 4616-4691; transcribed right to left ORF 3: 4731-5318; transcribed right to left
  • Ejcample 6 Cloning of 2 kb fragment.
  • the mutant strain 2-1 was derived from the biocontrol Pseudomonas strain 915 following N-Methyl-N'-nitro-N- nitrosoguanidine treatment. It is initially identified on the basis of its inability to inhibit the growth of the fungi Rhizoctonia solani and Pythium ultimum in vitro. Further characterization revealed that the strain was also defective in the ej ⁇ ression of a number of activities, including pyrrolnitrin, chitinase, and cyanide production. In addition, mutant 2-1 is morphologically distinguishable from strain 915.
  • the wild type parent (strain 915) formed small, circular, convex, white opaque colonies with entire edges.
  • Mutant 2-1 formed larger, circular, flat, translucent colonies with undulate edges.
  • 0RF5 is highly similar to the gacA gene of Pseudomonas fluorescens CHA0 and to the uyr-23 gene of E ⁇ coli. 0RF5 is located entirely within an approjdmately 2kb region defined by Xhol restriction sites. This approximately 2 kb Xhol fragment was cloned into the broad host range plasmid pVKlOO (Knauf et al. (1982), Plasmid 8:45-54) to yield the plasmid pCIB137. pCIB137 was deposited with the USDA Agricultural Research Service Culture Collection, Northern Regional Research Center (NRRL) at 1815 North University Street, Peoria, 111. 61604 on June 24, 1992 and has been accorded the accession number .
  • pCIB137 was introduced into the E. coli host strain S17-1 (Simon et al.(1983). Biotechnology 1:784-791) by transformation. It was then transferred into strain 2-1 by conjugation. Fresh overnight cultures of S17-1(pCIB137) and strain 2-1 were mixed (50 ⁇ l each) on an L agar plate and allowed to incubate overnight at 28C. Loopfuls of bacteria from the mating mijcture were then streaked on IMS agar containing 15 ⁇ g/ml tetracycline and further incubated at 28C. Tetracycline resistant colonies were purified and ejcamined for the presence of pCIB137. Transconjugants of strain 2-1 containing pCIB137 were shown to produce pyrrolnitrin, chitinase, and cyanide. They also display the morphology of strain 915.
  • strain 914 and strain 922 do not produce detectable levels of pyrrolnitrin, chitinase, or cyanide. They formed large, circular, flat, translucent colonies with undulate edges on IMS agar.
  • pCIB137 is introduced into these strains by conjugation as described in ejca ⁇ ple 2. Transconjugants of strain 914 containing pCIB137 were shown to produce pyrrolnitrin, chitinase, and cyanide. They also displayed the morphology of P. fluorescens strain 915.
  • Transconjugants of strain 922 containing pCIB137 were also shown to produce pyrrolnitrin, chitinase, and cyanide, but they did not display a change in morphology.
  • pCIB137 into Pseudomonas fluorescens strains (e.g. strain 914 and strain 922) which are ineffective in the in vitro or in vivo inhibition of Rhizoctonia solani, unej ⁇ ectedly activated expression of previously inactive, undetected chitinase, cyanide, and pyrrolnitrin genes, and converted these strains into effective biocontrol agents in greenhouse assays.
  • strain 914 introduction of fragment Ell unexpectedly caused a conversion in colony morphology to one very similar to that listed for P. fluorescens strain 915 in Table 1.
  • Ejca ⁇ ple 8 Intact CRF 5 is necessary for gene activation.
  • DNA sequence analysis revealed the presence of two Nael recognition sites, located 72 bp apart, within ORF5. Removal of the intervening DNA reduces the predicted gene product by 24 amino acids and may render the gene product nonfunctional.
  • the 2 kb Xhol fragment was cloned into the Xhol site of the plasmid pSP72 (Promega) .
  • the resulting construct, designated pCIB138 contains no other Nael sites besides the two in ORF5.
  • pCIB149 is identical to PCIB137 except for the deletion of the 72fcp of DNA.
  • pCIB149 was introduced into strains 914 and 922 by conjugation as described in exa ⁇ ple 5, pyrrolnitrin, chitinase, or cyanide production was not detected.
  • an intact ORF5 is necessary for gene activation in bacterial strains.
  • Example 9 Gene-replacement e ⁇ eriment.
  • ORF 5 The effect of ORF 5 on gene regulation in strain 915 was demonstrated by the following gene-replacement experiment.
  • the right 6.8kb of fragment Ell (bounded by right-most EcoRI and BamHI sites, see Fig. 3) was cloned into pBR322 (digested with EcoRI and BamHI) to form pBREB6.8.
  • the 2kb Xhol fragment containing ORF 5 was removed from pBREB6.8 by digestion with Xhol, then self-ligation to form pC3 139.
  • a kanamycin resistance marker was introduced into pCIB139 by substituting the Hindlll- Sall kana ⁇ tycin resistance fragment from Tn5 for the tetracycline resistance region bounded by Hindlll and Sail in pCIB139.
  • the resulting plasmid, PCIB154 was used to receive the 2kb Xhol fragment (with the 72bp Nael deletion) from pCIB150 to form pCIB156.
  • This plasmid was transformed into the Escherichia coli strain S17-1 (Simon et al. (1983) BioTechnology 1 :784-791), then introduced into strain 915 by conjugation, selecting for kanamycin resistance.
  • the kanamycin resistant transconjugants contained the plasmid. and its kanamycin resistant determinant, integrated into the chromosome. The most frequent integration events took place by homologous recombination and at the region of homology provided by the 2kb Xhol fragment and its surrounding sequences. Such transconjugants contain two copies of the 2kb Xhol region, one wildtype and one with the 72bp Nael deletion. Such duplications are unstable in bacteria with a proficient homologous recombination system and are lost at detectable frequencies spontaneously when selective conditions favoring their formation are removed.
  • transonjugant is cultured in liquid medium without kanamycin selection and then plated on solid agar medium to obtain individual colonies, again without kanamycin selection. Individual colonies were tested for kana ⁇ tycin sensitivity. Such colonies were obtained at approximately 2% of the total. These kanamycin sensitive colonies fall into two morphological classes, one resembling the wildtype, the other resembling the pleiotropic mutants. Southern hybridization results confirmed that both classes of colonies had lost the integrated plasmid and that the class with the wildtype morphology contained an intact 2kb Xhol region, while the other class contains a smaller Xhol region corresponding to that with the 72bp Nael deletion. Colonies of the former class were identical to strain 915.
  • Colonies of the latter class were identical to strain 915 except for a 72bp Nael deletion in ORF 5. These derivatives of strain 915 no longer produce pyrrolnitrin, chitinase, or cyanide. Thus, an intact ORF 5 is necessary for gene activation in strain 915.
  • Ejca ⁇ ple 10 The native promoter of ORF 5 is conta ned within the 2 kb Xhol fragment.
  • ORF 5 a promoter native to the 2 kb Xhol fragment was directing transcription of ORF 5 when ORF 5 was introduced into pleiotropic mutant derivatives of P. fluorescens 915 or into P. fluorescens strains 914 and 922.
  • ORF 5 class of transcriptional activators in seme bacterial strains, one skilled in the art will recognize that it might be beneficial to operably link the structural ORF 5 class gene with a promoter and/or ribosome binding site which functions more efficiently in the desired host bacterial genus to activate latent genes.
  • the ORF 5 class gene may be operably linked to a Bacillus regulatory region.
  • Ejca ⁇ ple 11 Analysis of the ORF 5 gene.
  • the ORF 5 coding region is capable of encoding a 213 amino acid protein with features of a bacterial transcription activator. For ejca ⁇ ple. there is strong homology between domains 1 and 2 of transcriptional regulators reviewed by Albright et al. (1989) and ccnparable regions in ORF 5.
  • the predicted aspartic acid residue at position 54 of the protein lines up with the conserved aspartic acid residues of other transcriptional activators of this class. It is the aspartic acid at this position which is typically phosphorylated by interaction with a sensor co ⁇ ponent protein. Alignment of ORF 5 with uyr-23 of E. coli and with gacA of P.
  • ORF 5 contains the unusual translation start codon TTG, which is less efficient than either ATG or GTG start codons. It is worth noting that at amino acid position 49 of ORF 5 resides an aspartic acid residue, while a tyrosine residue is present at the equivalent position of gacA.
  • virG an Agrobacterium tumefaciens transcriptional activator, an asparagine to aspartic acid substitution near the conserved phosphorylation site converted virG to a constitutive transcription activator which presumably no longer required phosphorylation by a sensor co ⁇ ponent. It is possible that our ORF 5 is such a constitutive activator by virtue of the substitution of aspartic acid for tyrosine.
  • a promoter directing transcription of ORF 5 likely resides within the 2 kb Xhol fragment. It is possible to map the location of this promoter by, for ejcample, a combination of SI nuclease mapping (Aiba et al., J. Biol. Chem. 256:11905-11910 (1981)) and primer extension mapping (Debarbouille and Raibaud, J. Bacteriol. 153:1221-1227 (1983) ) as was done for a different Pseudomonas promoter by, for ejca ⁇ ple, Gaffney et al., J. Bacteriol. 172:5593-5601 (1990). Once located, a DNA fragment containing this promoter can be operably linked if desired to ORF 5-class activators either by ligation of the appropriate DNA restriction fragments or by the overlap ejctension primer ejctension method of Horton et al.
  • Bacterial regulatory elements can be obtained from various sources including commercially available vectors, bacterial regulatory elements known in the art, and bacterial regulatory elements identified using promoterless marker-containing transposons, or promoter selection vectors such as pKK175-6 and pKK232-8 (Pharmacia, Piscatoway, NJ) .
  • Commercially available bacterial regulatory elements are available from a number of sources such as the plasmid ej ⁇ ression vectors pKK233-2, pDR540, pDR720, pYEJOOl, pPL-la ⁇ ibda (Pharmacia) , or pGEMEX expression vectors (Promega Biotec, Madison, WI) .
  • Bacterial regulatory elements known in the art include any bacterial regulatory element that is known to function as a promoter, enhancer, ribosome binding site, and/or any other regulatory control mechanism of the associated coding DNA sequence.
  • An associated coding DNA sequence is a DNA sequence that is adjacent or adjoining 3' to the regulatory elements and which codes for a protein when transcribed and translated.
  • Appropriate bacterial elements include those of Deretic et al., Bio/Technology 7:1249-1254 (1989); Deuschle et al., EMBO J. 5:2987-2994 (1986); Hawley and McClure, Nucleic Acids Res. 11:2237-2255 (1983); Rosenberg and Court, Annu. Rev. Genet.
  • Bacterial regulatory elements include hybrid regulatory regions co ⁇ prising mijctures of parts of regulatory elements from different sources.
  • trp/lac (trc) promoter of pKK232-2 Pharmacia
  • trc trp/lac
  • pKK232-2 Pharmacia
  • promoters for selectable markers on the broad-host-range plasmid RSF1010 are known to function in at least the following bacterial genera: Acetobacter, Actinobacillus, Aerdbacter, Aercmonas, Agrobacterium, Alcaligenes, Azotobacter, Azospirillum, Cauldbacter, Desulfovibrio, Erwinia, Escherichia, Gluconobacter, Hyphomicrobium, Klebsiella, Methylophilus, Moraxella, Paracoccus, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Serratia, Xanthomonas, Vibrio, Yersinia, and Zymomonas (Morales et al.,1990.
  • Pseudomonas Biotransformations, Pathogenesis
  • Ejca ⁇ ple 12 Biocontrol efficacy of transgenic strains. Biocontrol efficacies of the pCIB137-containing transconjugants of strains 914 and 922 were compared to their natural parents. Bacterial cultures were grown overnight in Luria broth at 28C. Cells were pelleted by centrifugation, then resuspended in sterile water to an optical density
  • Rhizoctonia solani was cultured on autoclaved millet, then dried and ground into powder. Soil was prepared by mijcing equal parts of potting soil
  • the 2.0 kb Xhol fragment containing the gafA gene of P. fluorescens strain 915 was labelled and hybridized with Xhol-digested total genomic DNA from P. fluorescens strain 914.
  • a 2.0 kb Xhol fragment from strain 914 which hybridized to the probe is cloned in pBluescript SK+ and DNA sequencing is performed to verify that the clone contains a gafA hcmologue.
  • the DNA sequence of the gafA ho ⁇ ologue in strain 914 is presented in Table 5.
  • the strain 914 gafA hcmologue differs from the strain 915 gafA at nine nucleotide positions, but only one of these nucleotide differences is predicted to generate an amino acid change difference in the two proteins (amino acid residue 182 is threonine in the strain 915 GafA protein and isoleucine in the 914 GafA protein) .
  • the strain 914 gafA gene was subcloned into the broad-host-range plasmid pVKlOO and the resulting recombinant plasmid was introduced by conjugation into strain 914.
  • strain 914 derivatives containing multiple plasmid copies of the 914 gafA .gene did synthesize pyrrolnitrin, chitinase, and cyanide.
  • EXAMPIE 14 Activation of latent gene ej ⁇ ression with the E. ooli uyr-23 gene.
  • the E. coli uyr-23 gene (also designated uyrY) is likely a member of a class of bacterial transcriptional activators, although no known function has yet been assigned to it in E. coli.
  • a DNA fragment containing the uyr-23 gene was obtained by a ⁇ plifying by the polymerase chain reaction (Mullis and Faloona, Methods in Enzy ⁇ ology 155:335-350 (1987)) a ca. 1.1 kb portion of the widely available E. coli K12 strain AB1157 genome. Primers for the polymerase chain reaction were prepared based upon the published sequence of uyr-23 (Sharma et al.. Nucleic Acids Res. 14:2301-2318 (1986)) .
  • Two PCR primer oligonucleotides 5'-GGCGGAGTATACCATAAG-3' and 5 • ⁇ - AAGCTTACC ⁇ ⁇ GC_A_ ⁇ CGTAC-3 , were used in the amplification of the ca. 1.1 kb DNA fragment at a concentration of 1 uM each in a 50 ul reaction mix containing, in reaction buffer supplied by the PCR kit manufacturer (Perkin Elmer Cetus) , the four deojcyribonucleotides (dATP, dCTP, dGTP, and dTTP; 200 uM with respect to each), approcimately 100 ng of E. coli K12 strain AB1157 gencmic DNA, and 1 unit of Taq DNA polymerase.
  • Typical a ⁇ plification cycle times and temperatures weree 94 C for 1 min, followed by 45 C for 1 min, followed by 72 C for 1 min (30 cycles total) .
  • Amplified DNA fragments were digested with the restriction endonuclease Hindlll, ligated with HindiII-digested pLAFR3, a broad-host-range plasmid capable of replication in Pseudomonas (Staskawicz et al., J. Bacteriol. 169:5789-5794 (1987)), and used to transform E. coli.
  • a pLAFR3 derivative containing the E When a pLAFR3 derivative containing the E.
  • coli uyr-23 gene was mobilized by conjugation into Pseudomonas fluorescens strain 914, the uyr-23 gene activated expression of genes involved in the production of cyanide, chitinase, and pyrrolnitrin.
  • Optimal activation in P. fluorescens strain 914 apparently depends upon ej ⁇ ression of uyr-23 from the lac promoter of pLAFR3.
  • E. coli ORF 5-like transcription activator could activate latent genes in P. fluorescens strain 914 (Ejca ⁇ ple 12), coupled with the fact that disruption of ORF 5 in the 2.0 kb Xhol fragment abolishes the gene-activating ability of this fragment, indicates that it should be possible to define a smaller DNA fragment than the 2 kb fragment with gene-activating ability, provided ORF 5 is intact and expressed.
  • Ej ⁇ ression of ORF 5 can be directed either from its native promoter, from a vector promoter, or from a heterologous promoter operatively joined to the ORF 5 coding region.
  • DNA fragments are prepared from a template consisting of the cloned 2 kb Xhol fragment essentially by the procedure described for isolating the uyr-23 gene from E. coli in Ejca ⁇ ple 5. Pairs of oligonucleotide primers are prepared for use in polymerase chain reaction amplification reactions. A common primer annealing to the template downstream of ORF 5 is present in each primer pair, while the remaining primer of each pair anneals to a sequence at a different distance upstream of ORF 5.
  • DNA fragments fr ⁇ n amplification reactions are cloned in a broad-host-range plasmid such as pLAFR3 for introduction into P. fluorescens. P. fluorescens transconjugants are tested for activation of latent gene activities by assaying production of chitinase, cyanide, and pyrrolnitrin. The smallest fragment activating latent genes is identified.
  • Exa ⁇ ple 16 Formalations of antifungal compositions employing liquid co ⁇ positions of transformed P. fluorescens bacteria which produce antibiotic substance inhibitory to the growth of R. solani as the active ingredient.
  • Emulsions of any required concentration can be produced from such concentrates by dilution with water.
  • the active ingredient is dissolved in methylene chloride, the solution is sprayed onto the carrier, and the solvent is subsequently evaporated off in vacuo.
  • Ready-to-use dusts are obtained by intimately ⁇ u ng the carriers with the active ingredient.
  • the active ingredient is thoroughly mixed with the adjuvants and the mijcture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations.
  • Emulsions of any required concentration can be obtained from this concentrate by dilution with water.
  • Ready-to-use dusts are obtained by mixing the active ingredient with the carriers, and grinding the mijcture in a suitable mill.
  • the active ingredient is mixed and ground with the adjuvants, and the mijcture is subsequently moistened with water.
  • the mijcture is ejctruded and then dried in a stream of air.
  • the finely ground active ingredient is uniformly applied, in a mixer, to the kaolin moistened with polyethylene glycol. Non-dusty coated granulates are obtained in this manner.
  • the finely ground active ingredient is intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desire concentration can be obtained by dilution with water.
  • the transposon TnCIB116 is introduced into strain 915 by conjugation (Lam et al (1990) Plant Soil 129:11-18) .
  • a collection of transposon insertion mutants of strain 915 is obtained and screened for the loss of pyrrolnitrin and chitinase production as described in Ejcample 4. After screening 10,000 transposon mutants, seven pleiotropic mutants which no longer produce pyrrolnitrin, chitinase, or cyanide were obtained.
  • Ejca ⁇ ple 19 Two genetic regions are required for gene activation in strain 915.
  • the seven pleiotropic mutants fall into two genetic classes. pCIB137 restored two of the seven transposon-induced mutants as well as mutant 2-1, to wildtype phenotype, suggesting that the genetic defects in these mutants were in ORF 5. Five mutants are not restored, indicating that at least one other geneic locus is required for gene activation in strain 915. A total gene library of strain 915 was introduced into these mutants by conjugation and transconjugants which has regained wildtype morphology were obtained. These transconjugants also produce pyrrolnitrin, chitinase, and cyanide. The restoring clones were isolated from these transconjugants. Restriction analysis indicates that the clones form an overlapping family of genetic fragments.
  • the clones tested restored all five mutants of the second class to wildtype phenotype and had no effect on the two mutants of the first class. These clones define a second genetic region required for gene activation in strain 915. The smallest clone in the family, pCIB146, is analysed further.
  • Ejca ⁇ ple 20 Functional analysis of the second genetic region.
  • the clone in pCIB146 was flanked by EcoRI sites (Fig. 4) . There is one internal EcoRI site in the clone. The two EcoRI subclones were obtained and tested for restoring ability. Neither one was able to restore mutant CGP 21, one of the five class II mutants, to wildtype phenotype, indicating that the internal EcoRI site defines a site critical to the functioning of the second genetic locus. There are two internal BamHI fragments in the clone. When these internal fragments were removed (pCIB191) , the restoring ability was not affected. Finally, a 6kb subclone containing only the region from the internal Hindlll site to the leftmost internal BamHI site (pCIB168) retained restoring ability. Two clones were deposited which were able to co ⁇ plement the mutant: pCIB 146 (about 25 kb) and pCIB 168 (about 6kb) .
  • Ejca ⁇ ple 21 The second genetic region contains a gene homologous to lemA.
  • Ejca ⁇ ple 22 Introduction of the lemA gene into Pseudomonas strains for the restoration of biocontrol funtions.
  • Clone pCIB146 is introduced into mutant Pseudomonas strains which lack biocontrol functions due to an absence of the lemA gene.
  • pCIB146 is introduced into Pseudomonas strains from E. coli by conjugation and is found to restore biocontrol functions, including chitinase, gelatinase, pyrrolnitrin and cyanide production.
  • Clone pCIB146 is also introduced into Pseudomonas strains which have no apparent defect in either the native lemA or gafA genes.
  • An enhancement of biocontrol function, including production of chitinase, gelatinase, pyrrolnitrin and cyanide is found in the transformed strains by virtue of the increased lemA production in these strains overcoming a limitation in the capacity to phosphorylate the gafA protein.
  • the lemA gene is also introduced into Pseudomonas strains into which the gafA gene has already been introduced by the procedure described in Ejca ⁇ ple 13.
  • the lemA gene is introduced on a plasmid which utilizes an origin of replication different to pLAFR3 to enable both gene constructions to be co ⁇ patable in Pseudomonas.
  • biocontrol function including production of chitinase, gelatinase, pyrrolnitrin and cyanide is found in the strains e ⁇ ressing both transgenes by virtue of the increased lemA production in these strains overcoming a limitation in the capacity to phosphorylate the gafA protein, which in turn arises by virtue of the increased abundance of gafA in the pseudomonad cells.
  • the lemA gene could be expressed behind a heterologous promoter, instead of from its own promoter. Such a promoter would be required to be expressible in Pseudomonas cells and may be expressed either constitutively or in an inducible fashion.
  • Exa ⁇ ple 23 adification of the lemA gene to increase its kinase activity on gafA.
  • lemA functions by interaction of the amino-terminal part of the protein with an unknown signal, autophosphorylation of a histidine located towards the carbojcyterminus of the protein, which thus allows the phosphorylation of the gafA protein.
  • the kinase activity on gafA is increased using two experimental approaches.
  • the amino acid environment flanking the histidine autophosphorylation target is modified using PCR and cloning techniques well known in the art.
  • Introduction of the modified lemA gene into Pseudomonas is achieved using a gene replacement technique (see ejca ⁇ ple 9), and the Pseudomonas strains thus modified are assessed against non- modified strains for chitinase, gelatinase, pyrrolnitrin, and cyanide production.
  • Constructions which have a modified amino acid environment adjacent to the target histidine which render the histidine a better target for autophosphorylation also phosphorylate gafA more efficiently and thus produce elevated levels of chitinase, gelatinase, pyrrolnitrin and cyanide.
  • the amino-terminal sensor part of the lemA gene is modif ed by deletion/substitution of amino acids using PCR and cloning techniques well known in the art.
  • a series of modified constructions thus prepared are introduced into Pseudomonas strains using gene replacement techniques (see ejca ⁇ ple 9), and the Pseudomonas strains thus modified are assessed against non-modified strains for chitinase, gelatinase, pyrrolnitrin, and cyanide production.
  • Constructions which have a modified sensor domain are able to autophosphorylate the target histidine without the necessary interaction of the signal and may therefore phosphorylate gafA more efficiently and thus produce elevated levels of chitinase, gelatinase, pyrrolnitrin and cyanide.
  • Exa ⁇ ple 24 Modification of the gafA gene to increase the efficiency of phosphorylation of the protein.
  • the amino acid environment flanking the presumed receiver domain of the gafA protein (around residue 54) is modified using PCR and cloning techniques.
  • a series of modified constructions thus prepared are introduced into Pseudomonas strains using gene replacement techniques (see ejca ⁇ ple 8b), and the Pseudomonas strains thus modified are assessed against non-modified strains for chitinase, gelatinase, pyrrolnitrin, and cyanide production.
  • Constructions which have a modified receiver domain and which are more readilly phosphorylated produce elevated levels of chitinase, gelatinase, pyrrolnitrin and cyanide.
  • Example 25 Pseudomonas strains carrying improved lemA and gacA genes.
  • lemA and gafA modifications described in Ejca ⁇ ples 19 and 20 which when introduced into Pseudomonas cause a phenotype of elevated production of chitinase, gelatinase, pyrrolnitrin and cyanide are combined into the same Pseudomonas strain by remodifying the improved lemA-carrying strain by repeating the gene replance ent experiment with the improved gafA construction.
  • Ejca ⁇ ple 26 Modification of gafA to render the protein phosphorylation- independent.
  • the gafA gene is modified so as to render the gafA protein phosphorylation independent. Since phosphorylation of the activator co ⁇ ponent of bacterial two-component regulatory systems leads to a conformational change in the DNA-binding domain of the activator, any specific amino acid substitutions, insertions, or deletions which lead to an equivalent conformational change render the activator phosphorylation- independent.
  • the use of a phosphorylation-independent version of gafA in the activation of latent genes in bacterial strains removes the requirement that a strain contain an active version of LemA or an equivalent kinase.
  • gafA The amino acid environment within the N-terminal half of gafA is modified using PCR and cloning techniques well known in the art.
  • Such mutagenized versions of the gafA gene are cloned into broad-host-range plasmids and introduced into a lemA- mutant derivative of strain 915. Since introduction of the unaltered version of gafA into the lemA- mutant fails to restore synthesis of chitinase, pyrrolnitrin, cyanide, and gelatinase (see ejca ⁇ ple 15) , any altered versions of the GafA protein which do restore some level of synthesis of these compounds in the lemA- strain are locked into a constitutively active conformation (i.e. phosphorylation- independent) .
  • MOLECULE TYPE DNA (genomic)
  • CTTAACAGCG CCTGATGCCA GGCGTCCGGC GAGCCGGAAC GGTAGTCACG GTCTTCCAGC 3900
  • CTGCTCGAGA ACGGCCAGGC CGGGACATTG CTCAACGGTC AGCGACCGGA TGGAGCTCGA 4080
  • MOLECULE TYPE DNA (genomic)
  • GCCCAAGACC GCTGCCCTGG TGGGGCGCAT CGCAGATCGA AACCACCATC ACCGCCAACG 960

Abstract

L'invention se rapporte à des séquences d'activation de gènes qui activent l'expression d'autres gènes bactériens latents ou présentant de faibles niveaux d'expression. Les séquences d'activation de gènes confèrent l'aptitude à produire plusieurs métabolites, et peuvent être transférées à des souches bactériennes. Les agents de biorégulation transformés agissent de façon à inhiber la croissance de champignons pathogènes.
PCT/US1993/006300 1992-07-02 1993-07-02 Element d'activation de genes WO1994001561A1 (fr)

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AU46625/93A AU679925B2 (en) 1992-07-02 1993-07-02 Gene activating element
JP6503435A JPH07509605A (ja) 1992-07-02 1993-07-02 遺伝子活性化要素
EP93916933A EP0649469A1 (fr) 1992-07-02 1993-07-02 Element d'activation de genes

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US5639949A (en) * 1990-08-20 1997-06-17 Ciba-Geigy Corporation Genes for the synthesis of antipathogenic substances
US5891688A (en) * 1996-12-06 1999-04-06 Novartis Finance Corporation DNA encoding lemA-independent GacA and its use in activating gene expression
US5955348A (en) * 1997-11-25 1999-09-21 Novartis Ag Genetically modified pseudomonas strains with enhanced biocontrol activity
US5756087A (en) * 1996-12-06 1998-05-26 Novartis Finance Corporation Genetically modified Pseudomonas strains with enhanced biocontrol activity
EP0941350A1 (fr) * 1996-12-06 1999-09-15 Novartis AG SOUCHES DE $i(PSEUDOMONAS) GENETIQUEMENT MODIFIEES AYANT UNE ACTIVITE DE LUTTE BIOLOGIQUE AMELIOREE
ES2178576B1 (es) * 2000-11-22 2004-08-16 Consejo Superior De Investigaciones Cientificas Procedimiento para la construccion de sistemas de expresion de proteinas en la rizosfera de plantas.

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