AU679925B2 - Gene activating element - Google Patents

Gene activating element Download PDF

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AU679925B2
AU679925B2 AU46625/93A AU4662593A AU679925B2 AU 679925 B2 AU679925 B2 AU 679925B2 AU 46625/93 A AU46625/93 A AU 46625/93A AU 4662593 A AU4662593 A AU 4662593A AU 679925 B2 AU679925 B2 AU 679925B2
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gene
sequence
strain
gafa
bacterial
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J. Ole Becker
Thomas D Gaffney
Dwight Steven Hill
Charles R. Howell
Stephen T. Lam
James M. Ligon
Jeffrey I. Stein
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Novartis AG
US Department of Agriculture USDA
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    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

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Description

VEMSION*J
Ipgcs IM7, description, replaced by new pages 1.63; pages 76-79, ITT claims, replaced by now pages 6467: due to late transmittal by ilha I N TfRN A T10N AL A! P reC 'r%1 vol fihc
A
S(SI) International Patent Classlflenllon 5:(11) International Publicatlon Number: C12N 15/31, 15/78, 15/63 Al (3 nentoa ulcto ae C12N 1/21 AOIN 63/00 (3 nentoa ulcto ae /1C2N 1/21,0C2Rf1:39) 0 q Lt Y1, /Ci3 'ION TREATY (PM(1 WVO 94/01561 20OJanuary 1994 (20,01,94) (21) Interrntlonal Application Number: (22) International Ffllng' Date., PCTIJUS9306300 2 July 1993 (01,07.93) Priority data., 07 1908.284 2 July 1992 (02 0.92) Parent Application or Grunt (63) Related by Continuation us Filed on 07'908,284 (CIII) 2 July 1992 (02,07.92) (74) Agent, SP'RUILL, WV, Murray; Ciba-Geigy Corporation, 3054 Cornwallis Road. Research Triangle Park, NC 27709 (US), (81) IDesignated States: AU, 1111 0G, Bit. HY, CA, CZ, Fl, flU, Jr KrX, KRt. KZ, LK. MO. MN, MW, NO. NZ, III., RO, RU, SI), SK. UA, US, VN, Europtan patent (AT, 11E, CII. DE, 1)K. ES. FR, O0, ORt, 10, IT, LU, MC, NL" PT. SE), OAPI patent (1W, BJ, CF. CO. Cf. CM. GA, ON, NIL, MR. NE, SN. TD), TGJ.
P'ulish~ed With intecrnatioal searc'h report, fleffire the exp~iration of th inteo linit for andngfclIU the da,,n and to be re'publishecd in the et'l of the reccipt of el,,f't? UY AG 10lt"C111; Klybeckstrasse 141, ('1002 Basic, (72)1 Inventors,. and linentors/ApplieaniS (fior (18 onk: GAFFNEY, Thiomas, IUS-,usi; 123 Tradescdnt Road, Chapel Hill, NC 275141 LAM. Stephen, T. lUSIUS], 8900 Jeanew Court, Raleigh, NC '27613 HAILL, Dwight. Steven~ IUSIUS]; 311 Melanie Lane, Cary, NC 27511 (US).
STEIN, Jefrcy, 1. IUSIUSI: .3725 Surry Trail, Ilillsbo.
rough, NC 27278 110WELL. Charles, It. IUS/USI;.
805 Avondale, Biryan, TX 77802 BECKER, Ole lDE13,USJ; 6164 Osevego, Riverside, CA 92506 1L1.
GON. James, M. IUS,'USI; 120 Marquette Drive, Cary, NC 27513 (US).
(54) TItle: GENE ACTIVATING ELEMIENT 6Ak7 4
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EcoRI eamHI1 ORF I BaMHI ORF 2 BamHI Hit tORF 3
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I diM Xhol X h ol EcoRi ORF 4 ORF (57) Abstract Gene activating sequences which activate the expression or other bacterial genes, which are latent or expressed at lowv levels, are provided. The gene activating sequences conrer the ability to produce several metabolites and may be transferred to bacterial strains. Thle transrormed biocontrol agents are active to inhibit the growth or thle rungal pathogens, (Referred to in 110 Oattu No 1411994, Setion~ 11) WO 94/01561 PCT/US93/06300 1 GM ACTIrWAnG ELENT Field of the Invention 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.
Background of the Invention It has been recognized that crops grown in some soils are naturally resistant to certain fungal pathogens. Furthermore, soils that are conducive to the velopment of these diseases can be rendered suppressive, or resistant, by aie addition of small quantities of soil from a suppressive field. Scher et al. Phytopatholoqy 70:421 (1980). Conversely, suppressive soils can be made conducive to fungal diseases by autoclaving, indicating that the factors responsible for disease control are biological.
Subsequent research has demonstrated that root colonizing bacteria ire responsible for this phenomenon known as biological disease control (BDC).
Baker et al., Biological control of plant pathogens, (Freeman Press, San Francisco) (1974).
In many cases, the most efficient strains of biological disease controlling bacteria are fluorescent Pseudomonads. Weller et al., Phytopatholo 73:463-469 (1983). These bacteria have also been shown 'to promote plant growth in the absence of a specific fungal pathogen by the suppression of detrimental rhizosphere microflora present in most soils.
Kloepper et al., Phytopatholocy 71:1020-1024 (1981). Important plant pathogens that have been effectively controlled by seed inoculation with these bacteria include Gaemanncmyces gramminis, the causative agent of SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 2 take-all in wheat, Cook et al., Soil Biol. Biochem 8:269-273 (1976) and Pythium and Rhizoctonia, pathogens involved in damping off of cotton.
Howell et al., Phytopathology 69:480-482 (1979). 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 cormmercially 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.
Many biological disease controlling Pseudomonas strains produce antibiotics that inhibit the growth of fungal pathogens. Howell et al., Phytopathology 69:480-482 (1979); Howell et al. Phytopatholoy 70:712-715 (1980). These have been implicated in the control of fungal pathogens in the rhizosphere. Several past studies have focused on the effects of mutations that result in the inability of the disease control bacterium to synthesize these antibiotics. Kloepper et al., Phytopatholoqy 71: 1020-1024 (1981); Howell et al., Can. J. Microbiol. 29:321-324 (1983). In these cases, the ability of the organism to control the pathogen is reduced, but not eliminated. In particular, Howell et al., Phytopatholoqy 69:480-482 (1979) discloses a strain of Pseudomonas fluorescens which was shown to produce an antibiotic substance that is antagonistic to Rhizoctonia solani.
In Baker et al., Biological Control of Plant Pathogens, (American Phytopathological Society, St. Paul, Minn.)( 1982), pages 61-106, it is reported that an inportant factor in biological control is the ability of an organism to compete in a given environment. Thus, it is desirable to obtain strains of biocontrol agents which are effective to control the growth of fungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineae and species of the genera Pythium and Fusarium and are able to aggressively compete with indigenous bacteria and microflora that exist in the rhizosphere of the plant. In order to achieve this objective, it is further desirable to obtain DNA sequences which are useful in conferring resistance to fungal pathogens which may be used to genetically engineer strains of biocontrol agents that combine the ability to control the growth S U.BTTTS .HEET WO 94/01561 PCT/US93/06300 3 of fungal pathogens with the ability to control other plant pathogens and/or the ability to aggressively compete in the rhizosphere.
Bacterial two-coaponent regulatory systems have been extensively reviewed Albright et al., Annu. Rev. Genet. 23:311-336 (1989); Bourret et al., Annu. Rev. Biochem. 60:401-441 (1991); Mekalanos, J.
Bacteriol. 174:1-7 (1992)). In most instances, an environmental signal is received by a sensor protein camponent. Reception of the signal induces an autophosphorylation event and a change in the conformation of the sensor protein. In this new conformation, the sensor is capable of phosphorylating the amino-terminal portion of the activator protein coaponent. This phosphorylation event is thought to alter the conformation of the activator protein such that the DNA binding module of its carboxy-terminal end is capable of interacting with the promoter regions of regulated genes within the network.
Laville et al. PNAS:USA 89:1562-1566 (1992) disclose the isolation from Pser" 'monas fluorescens CHAO, an activator-component gene designated acA which was required for the expression of genes involved in the synthesis of the antifungal secondary metabolites 2,4-diacetylphloroglucinol, cyanide, and pyoluteorin. The strain was indicated to be capable of suppressing black root rot of tobacco caused by the fungal pathogen Thielaviopsis basicola. Disruption of the qacA gene resulted in a mutant unable to synthesize any of these secondary metabolites and significantly reduced in its ability to suppress black root rot. Laville et al. concluded that the qacA gene was involved in regulation of secondary metabolism in P. fluorescens and inferred that extracellular secondary metabolites produced under qcA control are important for the biocontrol of black root rot. They noted the presence of a gene homologous to acA in E. coli and cited preliminary evidence from hybridization experiments that a sequence similar to qacA exists in Pseudomonas aeruginosa.
Our data indicate that antifungal secondary metabolites are likely only a subset of factors under the regulatory control of the ORF 5 gene we have cloned from Pseudamonas fluorescens, since we have discovered that ORF SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 4 (gafA) also affects the production of hydrolytic enzymes such as chitinase and gelatinase which are involved in the catabolism of polymeric carbon sources. Laville et al. did not disclose or suggest that the gcA gene was involved in activation of latent genes by a transcriptional activator or suggest that an activator derived from one bacterial strain would induce expression of genes from a heterologous bacterium. To our knowledge, 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, qafA).
A number of sensory-component genes have been characterized from different microorganisms (for reviews see Bourret et al., 1991 and Stock et al., 1990). In one case the sensory component gene, referred to as lemA, was found to be required for the pathogenicity of Pseudomonas syringae (Hrabak et al. (1992) J. Bacteriol. 174:3011-3020. However, in contradiction to the data presented in this application, Hrabak et al.
proposed that lemA alone may perform sensory and regulatory functions. Our experiments characterize a member of the same gene family cloned from Pseudomonas fluorescens and reveal that it plays a crucial role in gene activation by interacting with the global regulatory element gafA.
Summary of the Invention Accordingly, it is one object of the present invention to provide DNA sequences which are useful in activiating genes in bacterial strains.
It is another object of the present invention to provide genes that can be used to improve the biocontrol capabilities of strains of bacteria used for biocontrol.
It is one feature of the present invention that DNA sequences and genes are provided that activate genes in bacterial strains.
SUBSTITUTE SHEET (RULE 26) WO 94/01561 PC/US93/06300 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.
It in an advantage of the present invention that biocontrol agents may be produced which are able to inhibit a broad spectrum of plant pathogens.
It is another advantage of the present invention that biocontrol agents may be produced which are able to aggressively cTompete in the plant rhizosphere, which biocontrol agents contain a DNA sequence that activates genes in the bacterial biocontrol agent.
According to the present invention, 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. First, the activated strains produce substances, such as pyrrolnitrin and chitinase, which are able to inhibit plant pathogens, particularly fungal pathogens, such as RhizoctonJa 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.
In addition, we have demonstrated that these gene activating sequences can be transferred to other bacterial strains, especially pseudcunonad strains, that otherwise are not effective biocontrol agents for R. solani and thereby transform them into effective biocontrol strains.
The use of the gene activating sequences to improve the biocontrol capabilities of other strains of rhizosphere biocontrol strains is also part of the present invention. For example, United States Patent No.
4,456,684, (Weller et al.) discloses that take-all, a disease of.wheat caused by the fungus Gaermanncnyces qramminis, can be controlled in saome SUBSTITUTE SHEET (RULE 26) 6 cases by the application of bacteria inhibitory to this pathogen to wheat seeds prior to planting. However, where the growth of G. gramminis is effectively under control, R.
solani may become a growing problem pathogen of wheat 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.
According to a first embodiment of this invention there is provided use of a recombinant DNA molecule to activate in an organism transformed with said recombinant DNA molecule expression of at least one gene that is latent or natively expressed at low levels, wherein said recombinant DNA molecule comprising at least one copy of an isolated gene activating element comprising a nucleotide sequence of a gafA class of transcriptional regulators, wherein said gene activating element induces expression of said gene in said bacterial host strain.
According to a second embodiment of this invention there is provided a method of 16 activating in a bacterial host strain expression of at least one gene that is latent or natively expressed at low levels comprising integrating into the genome of said host strain a recombinant DNA molecule comprising at least one copy of an isolated gene activating element comprising a nucleotide sequence of a gafA class of transcriptional regulators, wherein said gene activating element induces expression of said gene in said bacterial host strain.
According to a third embodiment of this invention there is provided a method of inhibiting the growth of a pathogen comprising transforming into a bacterial host strain a recombinant DNA molecule comprising at least one copy of an isolated gene activating element comprising a nucleotide sequence of a gafA class of transcriptional regulators, wherein said gene activating element induces expression of said gene in said bacterial host strain, and applying the transformed host strain to the environment where the pathogen is to be inhibited.
According to a fourth embodiment of this invention there is provided an isolated gene activating element comprising a nucleotide sequence of a gafA class of transcriptional regulators, wherein said gene activating element is capable of inducing expression of at least one gene that is latent or natively expressed at low levels in a bacterial host strain.
According to a fifth embodiment of this invention there is provided the method of the second embodiment, wherein activation of expression leads to production of an antibiotic substance inhibiting a pathogen.
According to another embodiment of this invention, there is provided use of a recombinant DNA molecule to activate in an organism transformed with said recombinant DNA molecule expression of latent genes, wherein said recombinant DNA molecule EN:ULlVV]00676:TCWMI 6A encodes a component of a bacterial two-component regulatory system derived from a different organism.
According to a further embodiment of this invention, there is provided a method of activating in a bacterial host strain expression of latent genes comprising integrating into the genome of said host strain a recombinant DNA molecule which encodes a component of a bacterial two-component regulatory system derived from a different bacterial organism, According to a further embodiment of this invention, there is provided a method of inhibiting the growth of a pathogen comprising transforming into a bacterial host strain a recombinant DNA molecule encoding a component of a bacterial two-component regulatory system derived from a different bacterial organism, and applying dithe transformed host strain to the environment where the pathogen is to be inhibited.
According to another embodiment of this invention, there is provided a bacterial strain producing an antibiotic substance inhibiting a pathogen, wherein said inhibition has been activated by integration of a recombinant DNA molecule encoding a component of a bacterial two-component regulatory system derived from a different bacterial organism.
According to a further embodiment of this invention, there is provided a 20 recombinant DNA molecule encoding the Pseudomionas fluorescens gafA protein having the amino acid sequence given in SEQ TD NO: 2.
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 26 activity in a bacterial strain. Thus, 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. In preferred embodiments of the invention, the host bacterial strain may be a pseudomonad, particularly strains of the species Pseudomonasfluorescens.
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. 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 36 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. In a particular embodiment of the present invention, the transformed host bacterial strain is rendered active against fungal pathogens, such as [N:\LIBVVI00676.VMJ 6B Rlzizoctonia solani, Helinintlzosporiuin granzineae and species of the genera Pythiun and Fusariumt.
The present invention further comprises isolated DNA sequences 00 S04 *ab Go ab 00a [N:\LIIVV]00676:VMJ WO 94/01561 PCT/US93/06300 7 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 ccprises 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 fran a gene isolated from Pseudcmonas, Bacillus, or E. coli. In one embodiment of the present invention, the bacterial regulatory element is the native promoter of le 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. In a particular embodiment 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.
Examples of the gene activating sequences of the present have been deposited. Accordingly, 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.
DEFINITIONS
As used in the present application, the following terms have the meaning set out below: Prcmoter 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 SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 8 between 20 and 100 nucleotides to the 5' end of the starting site for translation.
Structural or Coding DNA sequence: A DNA sequence that is translated or transcribed in an organism to produce an RNA, a protein or other DNA product.
Associated witl/cperably linked: Two DNA sequences which are "associated" or "operably linked" are related physically or functionally.
For example, a promoter or regulator DNA sequence is said to be "associated with", a DNA sequence that codes for an RNA or a nrotein 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.
Derived from: 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.
Hanologous: 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. For example, where a ho, t organism to be transformed is of the species Pseudomonas fluorescens, a DNA s1quence is homologous if it originates from a pseudononad 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 from organisms of different biological classification.
Chimeric construct/chimeric 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 SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 9 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: The term "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 latent) or expressed at low levels in the naturally occurring state of the host. These sequences typically encou proteins which play a role in the pathways which regulate gene expression.
Brief Description of the Figures Figure 1: This figure shows restriction maps of three cosmid clones, pANT5, pANT9 and pANT10, that were found to complement the ANT phenotype of mutant 2-1. In this figure, indicates a BamHI restriction site; an EcoRI restriction site; and a HindIII restriction site.
Figure 2: This figure shows the ability of DNA subfragments deiived from 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.
Figure 3: This figure indicates the organization of the Ell fragment. The figure indicates the location of five identif tad open reading frames (ORF) and restriction sites for various enzymes.
Figure 4: This figure shows the ability of DNA subfragments derived from the clone pCIB 146 of Pseudomonas fluorescens strain 915 to ccmplement the mutant CGP 21.
Detailed Description of the Invention SUBSTITUTE SHEET (RULE 26 WO 94/01561 PCT/US93/06300 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 ultimrm 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 nitrosoguanidine mutagenesis, transposon mutagenesis) or can arise spontaneously. 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., I 30 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 Muced, and they have an altered colony morphology. A summary comparincr the characteristics of the pleiotropic mutants with the corresponding -aracteristics of wild-type P. fluorescens strain 915 is presented in Table 1.
TABLE 1 Character ,stic P. fluorescens 915 Pleiotropic mutants Pyrrolnitrin Cyanide Chitinase Gelatinase reduced Colony rorphology circular, entire, circular, undulate, convex and opaque flat and translucent Inhibition of Rhizoctonia solani SUBSTIT1TE SHEET (RULE 26) WO 94/01561 PCr/US93/0630 11 A total of eight pleiotropic mutants was identified. These all have the phenotype described in Table 1 above and fall into two distinct genetic classes, those which can be restored to 915-phenotype by introduction of the gafA gene (see A below) and those which can be restored to 915-phenotype by the introduction of the lemA gene (see B below).
A. Mutant Complementation with the gafA gene 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. The 11 klobase EcoRI restriction fragment (fragment Ell) of P. fluorescens strain 915, and a 2.0 kb Xhol subclone of this fragment containing qafA (ORF 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 co'ony morphology on minimal medium from large, circular, flat, translucent, with undulate edge to small, circular, convex, opaque white, wi~h entire edge. Accompahying these phenotypic changes associated with the introduction of fragment Ell or the 2.0 kb XhoI subclone is the conversion of P. fluorescens strains 914 and 922 to effective biocontrol strains with activity against the phytopathogen Rhizoctonia solani. We have also demonstrated that introduction of an Escherichia coli gene homologous to ORF 5 (gafA) into P. fluorescens strain 914 activates the expression of genes involved in the synthesis of cyanide, chitinase, and pyrrolnitrin. This result indicates that genes of the qafA class are sufficient for the activation of latent genes in heterologous bacterial strains.
DNA sequence analysis of fragment Ell has, to date, allowed SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 12 identification of five open reading frames, as well as a tRNA gene (gqly).
The organization of these open reading frames within fragment Eli is depicted in Figure 3. The first potential gene regulation element we' identified is ORF 2, which shared homology with numerous sensor components of bacterial two-caponent regulatory systems (reviewed in Albright et al., Annu. Rev. Genet. 23:311-336 (1989)). We determined that the organization of qlyw, ORF 3 (which has homology to the Escherichia coli gene gsA) and ORF 4 (which has homology to the E. coli gene uvrC) is identical to the gene organization found near map position 42 of the E. coli genome. In E.
coli, at a position equivalent to ORF 5 (gafA), exists a putative transcriptionil 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 Eli 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). Thus, two of the open reading frames, ORF 2 and ORF 5, share significant homology with numerous sensor and activator components, 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-XbaI subclone containing ORF1 and ORF 2. The 2.0 kb XhoI 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. fQuorescens 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.
SUBSTITUTE SHEFT (R i! F 9Mi WO 94/01561 PC/US3/06300 13 To determine whether transcr ionil activators of the gafA class are generally capable of activating the expression of latent genes in heterologous bacterial strains, we cloned the putative transcriptional activator gene described by 1bolenar 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 expression of genes involved in the production of cyanide, chitinase, and pyrrolnitrin in P. fluorescens strain 914.
It is an aspect of the present invention that improved biological control strains can be identified following the introduction of transcriptional activators of the qafA 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.
Those skilled in the art will also be aware that it will be possible to improve a biological control strain by placing additional genes under the control of transcriptional activators of the gafA class.
This can be accomplished by identifying the gafA-responsive promoter element(s) and operably linking the desired gene or genes to such element before introducing such genes into the desired strain.
In one embodiment of the present invention, recombinant DNA sequences are obtained which comprise an approximately 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. Among the pleiotropic effects of the gafA DNA sequence are the increased ability to inhibit the growth of fungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineac and species of the genera Pythium and Fusarium This DNA sequence may be derived from bacterial strains which are effective biocontrol agents against Rhizoctonia. Preferably the DNA sequence may be derived from the clone pANT5, which was isolated from a strain of Pseudomonas fluorescens.
SUBSTITUTE SHEE- 'RULE 26) 14 More preferably, the DNA sequence may comprise the approximately 11 kb Ell fragment of pANTS. In particular embodiments of the invention, the DNA sequence consists essentially of the approximately 2 kb fragment, or the DNA sequence of SEQUENCE ID No. 1. The clone pANTS has been deposited with ATCC and has been designated ATCC accession number 40868. A plasmid containing the 11 kb Ell fragment of pANTS 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 pANTS as a 2 .b 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).
Organism Deposit Date Accession No.
Plasmid DNA pANT5 17 August 1990 ATCC 40868 Plasmid DNA pRK-E11B 17 August 1990 ATCC 40869 Bacterial sp. 24 June 1992 NRRL B-18918 CGE 417[S17-1(pCIB 137) 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 rLay further comprise one or more regulatory DNA sequences operably linked 1i to the structural DNA sequence above. Such regulatory DNA sequences include promoter sequences, leader sequences, and other DNA sequences which may affect the expression of the regulatory DNA sequences, as well as those fragments of a regulator DNA sequence that are able to act with such effect.
B: Mutant Complementation with the lemA Gene Of the eight pleiotropic mutants isolated, five are not complemented by plasmids carrying the gafA gene indicating that at least one other genetic locus is required for gene activation. A total gene library of strain 915 was introduced into these mutants CGP 21) by conjugation and transconjugants which had regained wildtype morphology are obtained. These transconjugants also produced pyrrolnitrin, chitinase, and cyanide. The restoring clones are isolated from the transconjugants and characterised. A 6 kb subclone which encodes a gene with high homology to lmA (Hrabak et al., (1992) supra) was found to retain the phenotype restoration ability. This clone was deposited as pCIB 168.
This clone is not required for enablement but was deposited as Bacterial strain (pCIB 168) with the Agricultural Research Service Culture Collection (NRRL) of 1815 North University Street, Peoria, Illinois 61604, United States of America on 29 July 1993 and accorded number NRRL B-21117. Consequently the lemA gene clearly has the ability to restore the biocontrol phenotype in these pleiotropic mutants. However, when the emA gene was introduced into strain 914 it was not capable of activating latent gene expression, [Z:q.LIvV]o0676:TCW WO 94/01561 PCT/US93/06300 corroborating the assertion that strain 914 does not produce chitinase, gelatinase, pyrrolnitrin and cyanide because of inadequate gafA expression.
By way of corollary, lemA would predictably activate latent gene expression in strains of Pseudomonas in which lemA expression is the ratelimiting 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 leiA. Such genes comprise a class of sensory conponent genes capable of phosphorylating and therefore activating the gafA class of activators described above. Members of the class can be identified and isolated by complementation of the appropriate class of pleiotropic mutants as described herein. For example, the corresponding E. coli gene responsible for the phosphorylation of uvrY is one such gene.
Clearly the gene activating sequences aafA and lemA play pivotal roles in the activation of a series of genes involved in the production of enzymes and metabolites important in the biocontrol phenotype of Pseudomonas.
The Use of Gene Activating Sequences for Plant Pathogen Control In another embodiment of the present invention, biocontrol agents are provided which are able to inhibit the growth of fungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineae and species of the genera Pythium and Fusarium. These biocontrol agents may be bacteria, plant cells or animal cells transfoLmed with the recombinant DNA sequences above, but are preferably bacterial strains, and more preferably gram negative bacterial strains, such as the pseudomonads. 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.
In the methods of the present invention, the gene activating DNA sequences can be introduced into the gename of a bacterial strain which may not ordinarily be effective as an inhibitor of fungal pathogens, resulting in ,ql IRqTTI ITr CIrcT /t11 r n.\ WO 94/01561 PCr/US93/06300 16 an effective biocontrol strain.
DNA in the form of plasmids can be transferred from one bacterium to another by a sexual 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 Pseudoronas, conjugal transfer of DNA is the preferred method since these bacteria are not readily transformed with extraneous
DNA.
In yet another embodiment of the present invention, methods are provided for producing antibiotic substances which are effective in inhibiting the growth of fungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineae and species of the genera Pythium and Fusarium.
This method comprises introducing the reccmbinant 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 extracting 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 campound produced by the transformed biocontrol agent of the present invention. Where 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 carpound is compatible with bacterial strains. The present invention also relates to methods of treating plants, which comprise SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 17 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 conpound, 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 compound, 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 compounds. These compounds 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 mixtures 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 composition 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). However, the active ingrndients 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 compounds 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 SUBSTITUTE ShEET (RULE 26) WO 94/01561 PCT/US93/06300 18 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 therefo-e 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 example, in polymer substances. Like the nature of the compositions, 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 per hectare 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.
The formulations, compositions or preparations containing the active ingredients and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, for example by homogeneously mixing and/or grinding the active ingredients with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dinethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil; or water.
The solid carriers used e.g. for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. 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 SUBSTITUT SHEET (RULE 2, WOb 94/01561 PCT/US93/06300 19 carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.
Depending on the nature of the active ingredient to be used in the formulation, suitable surface-active ccpounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "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 compounds.
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 example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained for example from coconut oil or tallow oil. The fatty acid methyltaurin salts may also be used.
More frequently, however, 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 example, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture 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. Examples of alkylarylsulfonates are the SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibut,. lapthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.
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.
Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide 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 propy.ane glycol unit.
Representative examples of non-ionic surfactants re nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylpheni'-vethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.
Cationic surfactants are preferably quaternary ammonium salts which have, as N-substituent, at least one C 8
-C
2 2 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi (2-chloroethyl) ethylammonium bromide.
The surfactants customarily employed in the art of formulation are described, for example, in "McCutcheon's Detergents and EulsifirCs Annual," MC Publishing Corp. Kingwood, New Jersey, 1979, and Sisely and SUBSTITUTE SHEET (RULE 26, WO 94/01561 PCT/US93/06300 21 Wood, "Encyclopedia of Surface Active Agents," Chemical Publishing Co., Inc. New York, 1980.
The agrochemical carpositions usually contain from about 0.1 to about 99 preferably about 0.1 to about 95 and most prferably fran 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 to about 95 of a solid or liquid adjuvant, and from about 0 to about preferably about 0.1 to about 25 and most preferably from about 0.1 to about 20 of a surfactant.
Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations.
Further Uses of Gene Activating Sequences It is recognized that 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.
As discussed above, 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 pharmauceutical purposes, particularly in strains of microorganisms which natively express the biosynthetic genes at low levels or not at all.
Examples of strains suitable for manipulation in this manner include strains of Streptonyces for the production of tetracycline, erythromycin, chlorcmycetin, 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. Examples include the enhanced production of vitamin B2 and B12 from As and Streptcowces, respectively; the production of SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 22 gibberellin from Fusarium and Gibberella; the production of ll-a-hydroxyprogesterone from Rhizopus; and the produciton of corticosterone from Curvularia.
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 product on of polysaccharides such as alginate, xanthan, dextran from Leuconostoc and other organisms; the production of secreted proteins and enzymes such as amalyses, proteases, pectinases, chitinases, cellulases, gelatinases, collagenases, elastases, etc. from Bacillus, Aspergillus and other organisms; and the production of invertase from Saccharomyces.
The following examples are offered by way of illustration and not by way of limitation.
Examples Exanple 1. iitant Isolation and Functional Carplementaticn.
The wild-type Pseudomonas fluorescens strain 11c-1-38 (strain 915) was mutated by exposure to the mutagen N-Methyl-N'-nitro-Nnitrosoguanidine. Approximately two dozen antibiotic-affected mutants were identified by screening individual mutants for their ability to inhibit the growth of P. ultimum and R. solani on nutrient agar. Most of these were shown to have reduced, or no, antibiotic activit.y against ono 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-l-'8 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 SUBSTITUTE SHEET (RULE 26\ WO 94/01561 /I'/US93/063' 23 constructed by partial Sail restriction of the DNA, size fractionation to yield fragments of 20-30 kilobases, and liaation into XhoI-restricted vector pVK100. Knauf et al., Plasmid 8:45-54 (1982). The gene library was transferred to the antibiotic mutant 2-1 by triparental conjugation with E.
coli harboring the tr plasmid pRK2013. Ditta t- 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 antib 4 otic 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.
Example 2. Characterization of the Ell gene region.
The genetic region necessary for the functional complementation of antibiotic biosynthesis in mutant 2-1 was defined by subcloning portions of .ie larger region and assessing their ability to complement mutant 2-1 for antibiosis (Figure The smallest fragment that was demonstrated to complement the mutation was the 4.9 kb HindIII/EcoRI fragment, which was indicated as subfragment 6 in Figure 2 and was designated subfragment H/E4.9. Some of the subcloned DNA fragments derived from the antibiotic gene parent clone, which was designated pANT-5, were transferred to two wild type P. fluotescens strains, llc-1-33 (strain 914) and 11-1-6 (strain 922), that were not ordinarily able to produce antibiotic against R.
solani.
The results, shown in Figure 2, indicated that an 11 -b EcoRI subfragment, which is indicated as subfragment 3 in Figure 2 and was designated subfragment Ell, imparts R. solani-active antibiosis to both strains.
Example 3. Inhibition of Rhizoctonia sou -Li.
An active antibiotic caopound can be extracted from the g' wth ,edium of the transformed P. fluorescens strain that produces this antibiotic. This was accomplished by extraction of the medium with 80 SUBSTITUTE SHEET (RULE 26) .1 WO 94/01561 PCT/US93/06300 24 acetone followed by removal of the acetone by evaporatioi and a second extraction with diethyl ether. The diethyl ether was removed by evaporation and the dried extract is resuspended in a small volume of water. Small aliquots of the antibiotic extract 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 compound. The antibiotic was determined by NMR and mass spectrometry to be pyrrolnitrin.
Exanple 4. Description of the pleiotropic defects restored by fragment Eli and by the 2.0 kb _XoI subfrajment; ability of these fragments to active-e latent genes in other bacterial strains.
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 ide. :ified as pyrrolnitrin, was lacking in mutant 2-1. While lack of pyrrolnitrin production was the first defect observed in mutant 2-1, additional experimentation, 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.
a) Loss of pyrrolnitrin production 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) v/v) at 28 C. The resin was collected in a sieve and washed extensively with water. The pyrrolnitrin was eluted from the resin by two consecutive extractions with isopropanol (0.5X volure). The two extractions 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 ODS column (2.1 mm dia. x 10 ancm) with a mobile phase consisting of a water/methanol mixture with a starting composition of 0/100% and gradually changing to a final composition of 100/0%. Prior to injection into the HPLC, 100 ul of each extract was dessicated under vacuum and SUBSTITUTE SiH-EET (HiuLE 26N W;O 9001561C PCT/US93/06300 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. fluorescen. strain 915 extracts contained a peak which coaigrates 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.
Loss of cyanide production 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 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.
loss of chitinase production P. fluorescens strain 915 and various pleiotropic mutants were tested for the presence of chitinase activity by either of two methods. In the first method, 300 ml L-broth cultures of each strain were incubated at 28 C for 12 hours. Cells were collected by centrifugation an l washed once in 20 mM phosphate buffer (10 mM Na2HP04/10 mM KH2PO4). 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 extracts by centrifugation. Chitinase activity was assayed by incubation of 100 ul of cell extract with 100 ul of tritiated chitin w/v; approximately 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 stopped by addition SUBST I uTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 26 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 rwtant designated #736, which proved to be a member of one class of pleiotropic mutants, lacks the chitinase activity found in P.
fluorescens strain 915.
TABLE 2 EXTRACT COONTS/MINUTE P. fluorescens 915 32205 Transposon mutant 736 4366 No extract 4993 In the second method, 200 ul cultures of each strain were grown overnight at 28 C in L-broth in the wells of a 96-well microtiter plate. The overnight cultures were frozen and allowed to thaw before further use to release some enzyme which might otherwise be cell-bound. 10 ul of 0.5 mM 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside was added to the wells of an opaque black microtiter plate containing 10 ul of each overnight bacterial culture and 80 ul of a solution consisting of 50 mM sodium phosphate, pH 7.0; 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, and 10 mM beta-mercaptoethanol. Incubation was for 3 hours at 37 C. Release of fluorescent methylumbelliferyl groups by chitinase activity was monitored by reading the microtiter plates at an excitation of 355 nm and an emission of 460 nm on a Titertek Fluoroscan II fluorescent spectrophotometer. Typical results, presented in Table 3, indicate that pleiotropic mutants lack the chitinase activity found in P.
fluorescens strain 915.
TABLE 3 STRAIN FIURESCENCE ONITS SUBSTITUTE SHEET (RI ll F 9Mn WO 94/01561 PCT/US93/06300 27 P. fluorescens 915 Pleiotropic mutant Reduction in gelatinase activity 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 exporting 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 approximately 48 hours. Thus, while 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 export of that species is delayed.
Alteration in colony morphology On minimal growth medium, P. fluorescens strain 915 forms small, circular, convex white opaque colonies with entire edges. All pleiotropic mutants examined thus far formed larger, circular, flat, translucent colonies with undulate edges.
Example 5: Analysis of the 11 kilobase fragment (Ell) 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 dideoxy 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). As regions of contiguous DNA sequence were generated, they are compared against sequences contained in the GenBank database for homology with known bacterial gene coding regions. This analysis has thus far led to the SUBSTT[1TF LRH iT iK I1 WO 94/01561 PCT/US93/06300 28 assignment of the genetic organization for fragment Ell depicted in Figure 3. ORF 1 shares substantial homology with the cheR gene of E. coli and the frzF gene of Myxococcus xanthus. cheR in E. coli has a methyl transferase activity which is involved in mediating the chenotaxis response (Springer and Koshland, Proc. Natl. Acad. Sci. USA 74:533-537) and frzF appears to have a similar function in M. xanthus (McCleary et al, J.
Bacteriol. 172:4877-4887 (1990)). ORF 2 shares substantial homology with numerous genes encoding sensor components of so-called bacterial two-component regulatory sequences (Albright et al., 1989; Bourret et al., 1991; and Mekalanos, 1992). Examples of other bacterial sensor component 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 glycerophosphate, an enzyme involved in phospholipid metabolism (Gopalakrishnan et al., J. Biol. Chem. 261:1329-1338 (1986)).
ORF 4 shares substantial homology with the uvrC gene of E. coli which encodes a component 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 XhoI subclone of fragment Ell, shares substantial homology with numerous genes encoding transcription activator components of bacterial two-component regulatory sequences. In particular, ORF 5 is highly similar to the gacA gene of Pseudomonas fluorescens CHAO (Laville et al., 1992) and to the uvr-23 gene of E. coli (Moolenar et al., 1987). Examples of some other bacterial transcriptional activator genes with sequence similarity to ORF 5 include sacU of Bacillus subtilis, bvA of Bordetella pertussis, and algR of Pseudomonas aeruginosa. The entire coding region of ORF 5 is presented as 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 HindIII site, is presented as 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 qlyW: 4616-4691; transcribed right to left SUBSTITUTE SHEET (RULE 26: WO 94/01561 PCT/US93/06300 29 ORF 3: 4731-5318; transcribed right to left The combined results that the 2.0 kb XhoI subclone of fragment Ell containing ORF 5 activates latent gene expression in Pseudmonas strains and that the E. coli uvr-23 gene, which is homologous to ORF 5, is capable of activating latent Pseudomonas gene expression indicate that transcriptional activators of the ORF 5 class have the unexpected capability of activating the expression of latent bacterial genes.
Example 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 expression of a number of activities, including pyrrolnitrin, chitinase, and cyanide production. In addition, mutant 2-1 is morphologically distinguishable from strain 915. On agar plates containing the defined medium LM KH 2
PO
4 0.1% Na 2
HPO
4 0.1% NaCI, 0.4% (NH 4 2
SO
4 0.02% glucose and 0.66% MgSO4, and 1.6% agar) 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.
The approximately 2 kb Xhol fragment containing ORF5 was cloned into the broad host range plasmid pVK100 (Knauf et al. (1982), Plasmid 8:45-54) to yield the plasmid pCIB137.
Example 7: Analysis of the 2 kilcbase XhoI fragment Comparison of the ORF5 sequence to DNA sequences in the GenBank database revealed substantial hanology with numerous transcription activator genes in known bacterial two-component regulatory systems. In particular, ORF5 is highly similar to the gacA gene of Pseudamonas fluorescens CHAO and to the uvr-23 gene of E. coli. ORF5 is located entirely within an approximately 2kb region defined by Xhol restriction ,l IRSTITI iTF HFFT (ill F 9R) WO 94/01561 PCT/US93/06300 sites. This approximately 2 kb XhoI fragment was cloned into the broad host range plasmid pVK100 (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, Ill. 61604 on June 24, 1992 and has been accorded the accession number N gfl.--3- lqciSg 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 (50pl each) on an L agar plate and allowed to incubate overnight at 28C. Loopfuls of bacteria from the mating mixture were then streaked on LM3 agar containing 15 pg/ml tetracycline and further incubated at 28C. Tetracycline resistant colonies were purified and examined 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.
The natural Pseudomonas isolates 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 LM3 agar. pCIB137 is introduced into these strains by conjugation as described in example 2. T::ansconjugants 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.
Thus, it was shown that the introduction of pCIB137 into Pseudomonas fluorescens strains strain 914 and strain 922) which are ineffective in the in vitro or in vivo inhibition of Rhizc tonia solani, unexpectedly activated e.xpression of previously inactive, undetected chitinase, cyanide, and pyrrolnitrin genes, and converted these strains into effective biocontrol agents in greenhouse assays. Also, in the case of 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.
SUBSTITUTE SHEET (RULE 26)
LBI
WO 94/01561 PCT/US93/06300 31 Example 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 XhoI fragment was cloned into the XhoI site of the plasmid pSP72 (Promnga). The resulting construct, designated pCIB138, contains no other NaeI sites besides the two in Digestion of pCIB138 DNA with NaeI followed by ligation at a concentration of 30 ng/pl and transformation into E. coli strain S17-1 yielded pCIB150, which contains the desired 72 bp deletion. The presence of the deletion was confirmed by DNA sequencing. The ORF5-containing XhoI fragment from pCIB150 was cloned into PVK100 to yield pCIB149. pCIB149 is identical to pCIB137 except for the deletion of the 72bp of DNA. When pCIB149 was introduced into strains 914 and 922 by conjugation as described in example pyrrolnitrin, chitinase, or cyanide production was not detected. Thus, an intact ORF5 is necessary for gene activation in bacterial strains.
Example 9: Gene-replacement experiment.
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 XhoI fragment containing ORF 5 was removed from pBREB6.8 by digestion with Xhol, then self-ligation to form pCIB139. A kanamycin resistance marker was introduced into pCIB139 by substituting the HindIII- Sall kanamycin resistance fragment from Tn5 for the tetracycline resistance region bounded by HindIII and SalI in pCIB139. The resulting plasmid, pCIB154, was used to receive the 2kb XhoI fragment (with the 72bp NaeI deletion) from pCI3150 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. Since pCIB156 cannot be maintained autonomously in strain 915, the kanamycin resistant transconjugants contained the plasmid, SUBSTITUTE SHEET (RULE 26) II I I' I II III l WO 94/01561 PCT/US93/06300 32 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 XhoI 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. One such 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 kanamycin 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 XhoI region, while the other class contains a smaller XhoI region corresponding to that with the 72bp NaeI 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 NaeI deletion in ORF 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.
Exarple 10: The native promoter of CORF 5 is contained within the 2 kb XboI fragment.
The following evidence indicates a promoter element directing transcription of ORF 5 is likely to be found within the 2 kilobase Xhol fragment. A version of the broad host range plasmid pVKl00 containing the 2 kb Xhol fragmnent was isolated with the 2 kb insert in the opposite orientation from that found in pCIB137. This plasmid was designated pCIB151. Introduction of pCIB151 into 31eiotropic mutants and into P.
fluorescens strain 914 activated gene e)pression. The ability of the 2 kb Xhol fragment to activate gene expression in each orientation, and the SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 33 previously described requirement of functional ORF 5 gene product for gene activation, made it likely that transcription of ORF 5 relies on the presence of a prcmoter located within the 2 kb insert. Furthermore, Moolenar et al., (1987) have identified a promoter directing transcription of the O}F 5-related uvr-23 gene of E. coli. This promoter was contained within the first 100 bas pairs upstream of the uvr-23 structural gene. At a nearly identical position upstream of ORF 5, we have identified the sequence TTGTCA-17bp-TITTTTT which is similar to the sigma-70 promoter consensus sequence described by Rosenberg and Court, Annu. Rev. Genet.
13:319-353 (1979). The sequence of a 983 base pair region containing the ORF 5 structural gene, the possible promoter region, and the 5' end of ORF 4 (which is homologous to uvrC of E. coli) is presented in SEQUENCE ID 3.
The coordinates of these elements in sequence ID 3 are as follows: Sequence with promoter homology: 23-51 ORF 5 structural gene: 99-740 end of ORF 4 (uvrC) 743-983 It is likely that a promoter native to the 2 kb XholI 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. However, to express the ORF 5 class of transcriptional activators in some 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 praomoter and/or ribosome binding site which functions more efficiently in the desired host bacterial genus to activate latent genes. For example, to activate latent genes in Bacillus species with ORF 5 class genes, the ORF class gene may be operably linked to a Bacillus regulatory region. Such regulatory regions are readily available to those skilled in the art. One possible method for accaomplishing this fusion between bacterial regulatory sequences and ORF 5-class structural genes involves use of the overlap extension polymerase chain reaction strategy (Horton et al., Gene 77:61).
Exanple 11: Analysis of the CEF 5 gene.
a. The ORF 5 coding region is capable of encoding a 213 amino acid protein with features of a bacterial transcription activator. For example, SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 34 there is strong homology between domains 1 and 2 of transcriptional regulators reviewed by Albright et al. (1989) and ccnparable regions in ORF 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 whi-h is typically phosphorylated by interaction with a sensor component protein.
Alignment of ORF 5 with uvr-23 of E. coli and with qacA of P. florescens CHAO leads to the conclusion that ORF 5 contains the unusual translation start codon TIG, 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 qacA. In virG, an Agrobacterium tumefaciens transcriptional activator, an asparagine to aspartic acid substitutiqn near the conserved phosphorylation site converted virG to a constitutive transcription activator which presumably no longer required phosphorylation by a sensor component. It i: possible that our ORF 5 is such a constitutive activator by virtue of the substitution of aspartic acid for tyrosine.
b. As noted above, a promoter directing transcription of ORF likely resides within the 2 kb XhoI fragment. It is possible to map the location of this promoter by, for example, a combination of Sl 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 example, 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 activators either by ligation of the appropriate DNA restriction fragments or by the overlap extension primer extension method of Horton et al.
c. 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 SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCT/US93/06300 number of sources such as the plasmid expression vectors pKK233-2, pDR540, pDR720, pYEJ001, pPL-lambda (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. 13:319-353 (1979), and references cited therein. Likewise, promoters for use in gram positive microorganisms such as Bacillus species are readily accessible to those skilled in the art. Any of the above can be synthesized using standard DNA synthesis techniques. Bacterial regulatory elements include hybrid regulatory regions coprising mixtures of parts of regulatory elements from different sources. For example, the trg/lac (trc) promoter of pKK232-2 (Pharmacia) which cambines the -35 region of the E. coli tryptophan operon promoter with the -10 region of the E. coli lac operon promoter functions effectivexy in Pseudomonas (Bagdasarian et al., Gene 26:273-282 (1983).
Certain bacterial promoters have the capability of functioning efficiently in a variety of bacterial genera. For example, promoters for selectable markers on the broad-?'ost-range plasmid RSF1010 are known to function in at least the following bacterial genera: Acetobacter, Actinobacillus, Aerobacter, Aeromonas, Agrobacterium, Azotobacter, Azospirillum, Caulobacter, Desulfovibrlo, Erwinia, Escherichia, Gluconobacter, Hyphomicrobium, Klebsiell-, Methylophilus, Moraxella, Paracoccus, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Serratia, Xanthomonas, Vibrio, Yersinia, and Zymomonas (Morales et al.,1990. In: Pseudomonas: Biotransformations, Pathogenesis and Evolving Biotechnology, (Silver, Chakrabarty, Iglewski, and Kaplan, eds.) pp.
229-241.) Example 12: Biocontrol efficacy of transgenic strains.
I I PZTJT IT E: QLJ fl i WO 94/01561 PCT/US93/06300 36 Biocontrol efficacies of the pCIB137-containing transconjug;nts of strains 914 and 922 were conaared to their natural parents. Bacterial cultures were grown overnight in Luria broth at 28C. Cells were pelleted by centriiugation, then resuspended in sterile water to an optical density of 2.5 at 600nm (approximately 2x109 colony forming units per ml.).
Rhizoctonia solani was cultured on autoclaved millet, then dried and ground into powder. Soil was prepared by mixing equal parts of potting soil (Metro-mix 360), sand, and vermiculite. This was used to fill diameter pots. A 2cm deep circular furrow with a total length of 30cm is formed at the perimeter of each pot. Ten cotton seeds (stoneville 506) were placed in each furrow. R. solani-infested millet powder was sprinkled evenly over the seeds in the furrows at the rate of 100mg/pct, followed by the application of 20mi of bacterial suspension for each pot. Water was added in place of bacterial suspension in the unbacterized control. Each treatment consisted of four replicate pot, for a total of 40 seeds per treatment. The plants were grown in an environmentally controlled chamber with a day/night temperature regime of 26/21C. The plants were rated for disease severity after 10 days. The results (Table 4) clearly ind :ate that strains 914 and 922 provide no disease control, whereas their pCIB137-containing transconjugants provided good control of R. solani in cotton.
TABLE 4 (Stand 10 DAPa) Treatment Repl Rep2 Rep3 Rep4 Mean %Biocontrol NP.NTb 9 10 9 9 9.25 100.0 P.NTc 0 2 1 1 1.00 0.0 914 3 0 3 1 1.75 9.1 914(pCIB149) 0 1 0 2 0.75 914(pCIB137) 5 4 5 7 5.25 51.5 922 1 1 1 3 1.50 6.1 922(pCIB137) 8 10 7 6 7.75 81.8 1 Uninfested control designated 100% biocontrol; Infested control designated 0% biocontrol a Days After Planting SUBSTITUTE SHEET (RULE 26) WO 94/01561 WO 9401561PCT/US93/06300 37 b No Pathogen, No Treatment (Uninfested control) c Pathogen, Wo TT,?atment (Infest-d control) Rxanple 13: M~itiple copies of a gafA gene isolated frcm the Pseuct1xcnas fluorescens strain 914 activate expressic'i of latent genes in strain 914.
The 2.0 kb XhoI fragwvnt containing the gafA gene of P. fluorescens strain 915 was labelled and hybridized with X(hoI-digested total gent-mic DNA from P. fluorescens strain 914. A 2.0 Kb~ Xhol fragnent fromn strain 914~ which hybridized to the probe is cloned n pBluescript SK+ and DNA sequencing is performed to verify that the c~lone contains a gafA 1.=nlogue.
Tne DNA sequence of the gafA homologue in strain 914 J s p~rese-nted in Table The strain 914 gafA hraologue differs frczn the strain 915 gafA at nine nucleotide positions, but o.rly one of these nucleotide differences is predicted to generate an amino acid change difference in the two proteins (amino acid residuie 182 is threonine in the strain 915 GafA protein and isoleucine in the 914 GafA protein). I.,e strain 914 gafA gene was subcloned into the broad-1host-range plasmid pVKlOO and the resulting recomibinant plasmid was introduced by conjugation into strain 914. Wh~a expression of the single chrmsomal gafA gene in strain 914 was not capable of activating expression of genes required fox. tl~e synthesis of pyrrol-nitrin, chitinase, and cyanide, strain 914 derivatives containing Multiple plas.MnId copies cf the 914 gafA gene did synthesize pyrrolnitrin, chitinase, and cyanide.
I
51 101 151 201 251 301 351 '01
TMGATTAGGG
TACCCr4AAG
AGTCAGGGGA
GTCCTCATGG
CAAATI"gMG
GTAGAAGA
TAI'GN=C
GGMGTFIGM
TGT1'CAAGTC Table 2g open reading frame frmn pCIB 3341 TC1AGTGI CGM'GACCAT GATCCGF,., GTC'G&7.RT CSGGGC2GACA TcGA!'GGOCT GCAA(-"= C-4GGVIXGCCG GGAG2CCZ1'IG CTCAAGGCCC GGAG'ITGhk X./GA~rG ACGTCAAGAT GCYZCGGGATC GGCGGT=1~ AAGCC.ACI3OG CGCAGT'C CGGATATM AMGTIGGCC &1'CACOGIT CCCG1=CCcG ACCCGC TGCAAGCCGG TGCGGCGGGT AAGGTGCGGG CCAATGAA A!TGGIGCAGG CCATTMGCCT GGCCAGCGTT ALAJYIGCCC GCAAA=C CAGCAGTTGG ATTCCAGCCT TCCAGIGAM. CACCGTI'CGA TGCITT=C Q1 IPCOT Ill T CZQI4 (Pil PI 01 WO 94/01561 PCT/US93/06300 38 451 GAGCGGGAAA TCCAGATCGC GCTGATGA7T GTCGGC CC AGAAAGTGCA 501 GATCATCTCC GACAAGCIG GCCITGTCICC GAAAACCGIT AATATCTACC 551 GTTACCGCAT CTICGAAAAG CICTCGATCA GCAGCGATGT TGAACTGACA 601 TIGCIGGCGG TICGCCACGG CATGGTCGAT GCCAGIGCCT GA EX7WFE 14: Activation of latent gene expression with the E. coli uvr-23 gene.
The E. coli uvr-23 gene (also designated uvrY) is likely a member of a class of bacterial transcriptional activators, although no known function has yet been asaigned to it in E. coli. A DNA fragment containing the uvr-23 gene was obtained by amplifying by the polymerase chain reaction (Mullis andc Faloona, Methods in Enzymology 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 uvr-23 (Sharma t al., Nucleic Acids Res. 14:2301-2318 (1986)).
Two PCR primer oligonucleotides, 5' -GGCGGAGTATACCATRAG-3' and -ATAAGCTACCCACAGCATCGTAC-3' were iised in the anplification 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 deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP; 200 uM with respect to each), approximately 100 ng of E. coli K12 strain AB1157 genomic DNA, and 1 unit of Taq DNA polymerase. Typical amplification cycle times and temperatures weree 94 C for 1 min, followed by '0 C for 1 min, followed by 72 C for 1 min (30 cycles total) Amplified DNA fragments were digested with the restriction endonuclease HindII, ligated with Hindll-digested pLAFR3, a broad-host-range plasmid capable of replication in Pseudacmonas (Staskawicz et al., J. Bacteriol. 169:5789-5794 (1987)), and used to transform E. coli. When a pLAFR3 derivative containing the E. coli uvr-23 gene was mobilized by conjugation into Pspudomvnas fluorescens strain 914, the u'r 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 expression of uvr-23 from the lac promoter of pLAFR3.
Exanple 15: Further delimitation of the 2.0 kb XhoI fragment to identify oSUBSTITUTE SHEET (RULE 28)
W
WO 94/01561 PCT/US93/06300 39 the smallest intact subunit that is functional.
The fact that an E. coli ORF 5-like transcription activator could activate 'atent genes in P. fluorescens strain 914 (Example 12), coupled with the fact that disruption of ORF 5 in the 2.0 kb XhoI 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.
Expression 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. Smaller DNA fragments are prepared from a template consisting of the cloned 2 kb Xhol fragment essentially by the procedure described for isolating the uvr-23 gene from E. coli in Example 5. Pairs of oligonucleotide primers are prepared for use in polymerase chain reaction amplification reactions. A ccmmon 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 from 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.
Exanple 16: Formulations of antifungal compositions employing liquid coupositions of transformed P. fluorescens bacteria which produce antibiotic substance inhibitory to the growth of R. solani as the active ingredient.
In the following examples, percentages of comaposition are given by weight: 1. Emulsifiable concentrates: a b c Active ingredient 20 40 50 Calcium dodecylbenzenesulfonate 5 8 6 Cast oil polyethlene glycol 5 ether (36 moles of ethylene oxide) SUBSTITUTE SHEET (RULE 26 WO 94/01561 I'CT/US93/06300 Tributylphenol polyethylene glycol ether (30 moles of ethylene oxide) Cyclchexanone Xylene mixture 12 15 70 25 4% 20 20 Emulsions of any required concentration can be produced from such concentrates by dilution with water.
2. Solutions: Active ingredient Ethylene glycol monomethyl ether Polyethylene glycol 400 N-methyl-2-pyrrolidone Epoxidised coconut oil Petroleum distillate (boiling range 160-1900) a 80 20 b 10
C
5% d 95 70 20 1 94 These solutions are suitable for application in the form of microdrops.
3. Granulates: Active ingredient Kaolin Highly dispersed silicic acid Attapulgite a b 5 10 94 1% 90 The active ingredient is dissolved in solution is sprayed onto the carrier, and the evaporated off in vacuo.
methylene chloride, the solvent is subsequently 4. Dusts: Active ingredient Highly dispersed silicic acid Talcum a b 2 1% 37 SI RITITJ JTF. HFFT IRJ II P WO 94/01561 WO94/ 1561PC'r/US93/06300 Kaolin 90 Ready-to-use dusts are obtained by intimiately mixing the carriers with the active ingredient.
Exanpie 17: Foniulaticri of antifungjal cczupositions caxpositicis of transfonxed P. fluorescens bacteria antibiotic substance inhibitory to the growth of R.
ingredient.
euployincg sol-id which produce solani as the active In the following exarrples, percentages of conqositions are by weight.
1. Wttable powders: a A..tive ingredient 20 SodiL-.m lignosulfonate 5 Sodium lauryl sulfate 3 Sodium diisobutylnaphthalene- sulfonate Octyiphenol polyethylene glycol ether (7-8 nmles of ethylene oxide) Highly dispersed silicic acid 5 Kaolin 67 b c 60 75 5 6 10 2 27 10 The active ingredient is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations.
2. Emulsifiable concentrate: Active ingredient Octylphenol, polyethylene glycol ether mles of ethylene oxide) Calcium dodecylbenzenesulfonate Castor oil polyglycol ether (36 moles of ethylene oxide) WO 94/01561 PCT/US93/06300 42 Cyclohexanone 30 Xylene mixture 50 Smulsions of any required concentration can be obtained from this concentrate by dilution with water.
3. Dusts: a b Active ingredient 5 8 Talcum 95 Kaolin 92 Ready-to-use dusts are obtained by mixing the active ingredient with the carriers, and grinding the mixture in a suitable mill.
4. Extruder granulate: Active ingredient 10 Sodium lignosulfonate 2 Carboxymethylcellulose 1 Kaolin 87 The active ingredient is mixed and ground with the adjuvants, and the mixture is subsequently moistened with water. The mixture is extruded and then dried in a stream of air.
Coated granulate: Active ingredient 3 Polyethylene glycol 200 3 Kaolin 94 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.
6. Suspension concentrate: Active ingredient 40 Ethylene glycol 10 Sft I I I 11*0: Qc~ ,r~if r7,- WO 94/01561 PCT/US93/06300 43 Nonylphenol polyethylene glycol 6 moles of ethylene oxide) Sodium lignosulfonate 10 Carboxymethylcellulose 1 37 aqueous formaldehyde solution 0.2 Silicone oil in 75 aqueous emulsion 0.8 Water 32 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.
Example 18: Isolation of further pleiotropic mutants.
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 Example 4.
After screening 10,000 transposon mutants, seven pleiotropic mutants which no longer produce pyrrolnitrin, chitinase, or cyanide were obtained.
Example 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 SUBSTITUTE SHEET (RULE 26) 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 fu:ther. This clone is not required for enablement but was deposited as Bacterial strain 517-1 (pCIB 146) with the Agricultural Research Service Culture Collection (NRRL) of 1815 North University Street, Peoria, Illinois 61604, United States of America on 29 July 1993 and accorded number NRRL B-21118.
Example 20: Functional analysis of the second genetic region.
The clone in pCIB146 was flanked by EcoRI sites (Fig. 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 HindIUl site to the leftmost internal BamHI site (pCIB168) retained restoring ability. Two clones were deposited which were able to complement the mutant: pCIB 146 (about 25 kb) and pCIB 168 (about 6kb).
Example 21: The second genetic region contains a gene homologous to lemA.
DNA sequences surrounding the internal EcoRI site in pCIB146 were obtained.
Comparison against sequences contained in the GenBank database revealed significant homology with the lemA gene of Pseudomonas syringae pv. syringae strain B728a (Hrabak and Willis, 1992), a gene in the sensor family of two-component regulatory systems. See Table 6.
Table 6 Comparison of pCIB168 with the published lemA sequence by Hrabak et al. (1992) J. Bacteriol, 174:3011-3020 lemA sequence homology coding start GTG 788 788-1063 72% 1101-1886 77% 1616-1886 87% 2182-2308 78% [N:\LIBVV]00676:TCW WO 94/01561 PCT/US93/06300 72% Example 22: Introduction of the lemA gene into Pseudoamnas strains for the restoration of biocontrol funtions.
Clone pCIB146 is introduced into mutant Pseudaronas 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 Example 13. In this case the lemA gene is introduced on a plasmid which utilizes an origin of replication different to pLAFR3 to enable both gene constructions to be compatable in Pseudomonas. An enhancement of biocontrol function, including production of chitinase, gelatinase, pyrrolnitrin and cyanide is found in the strains expressing 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.
In any of the experimental approaches described above 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.
SUBSTiTUTJ SHrFT (Pi IIP9 R WO 94/01561 PCT/US93/06300 46 Exarple 23: Modification of the lemA gene to increase its kinase activity n gafA.
By corollary with other sensor components it is assumed that lemA functions by interaction of the amino-terminal part of the protein with an unknown signal, autophosphorylation of a histidine located towards the carboxyterminus of the protein, which thus allows the phosphorylation of the gafA protein. The kinase activity on gafA is increased using two experimental approaches.
First, 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 example and the Pseudomonas strains thus modified are assessed against nonmodified 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.
Second, the amino-terminal sensor part of the lemA gene is modified 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 example 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.
Exaple 24: Modification of the gafA gene to increase the efficiency of phosphorylation of the protein.
SUBSTITUTE SHEET (RULE 26) WO 94/01561 PCI/US93/06300 47 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 Pseudononas strains using gene replacement techniques (see example 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: Pseudcmonas strains carrying improved lemA and gacA genes.
lemA and gafA modifications described in Examples 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 Pseudononas strain by renodifying the improved lemA-carrying strain by repeating the gene replancement experiment with the improved gafA construction.
Example 26: Modification of gafA to render the protein phosphorylationindependent.
The gafA gene is modified so as to render the gafA protein phosphorylation independent. Since phosphorylation of the activator component 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 phosphorylationindependent. The use of a phosphorylation-independent version of gafA in the activation of latent genes in bacterial strains renoves the requirdment that a strain contain an active version of LemA or an equivalent kinase.
The amino acid environment within the N-terminal half of qafA 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.
C01 IFQTITt I TC QCLCT /DI II C1 OR\ WO 94/01561 PC/US93/06300 48 Since introduction of the unaltered version of gafA into the lemA- mutant fails to restore synthesis of chitinase, pyrrolnitrin, cyanide, and gelatinase (see example 15), any altered versions of the GafA protein which do restore some level of synthesis of these compounds in the lemA- strain are locked .ito a constitutively active conformation phosphorylationindependent).
While the present invention has been described with reference to specific embodinents thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications and embodiments are to be regarded as being within the spirit and scope of the present invention.
SUBSTITUTE SHEET (RULE 26) WO 94/01561 WO 9401561PCr/ US93/06300 49 SEQUENCE LISTING GENERAL. INFORMATION: APPTICA2NT: Gaffney, Thomnas D.
Lam, Stephen T.
Ligon, Jam~s M.
Hill, Dwight S.
Stein, Jeffrey I.
Howell, Charles R.
Becker, J. Ole (ii) TITLE OF INVENTION: Gene Activating Eleme~nt (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: CIBA-GEIGY Corporation STREET: 7 Skyline Drive CITY: Hawthorne STATE: New York COUNTRY: USA ZIP: 10532 COMPUYTER READABLE FORM: MEDIUM TYPE: Floppy disk Mt4PTER: IBM PC Compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTW7ARE: Patentln Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US93/06300 FILING DATE: 02-JUL-19 93
CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: S I IRRTITI ITP Q1.4:T in;;'i r n WO 94/01561 WO 9401561P Cri US93/06300 APPLI0CTION NUMBER: US 07/908, 284 FILING DATE: 02-JUJL-1992 (viii) ATIOBNEY/AGENT INFRTION: NAMEZ: Spruill, W. Murray REGISTRATION NUMBER: 32, 943 PEFEREN(JE/DCCKET NUMBER: S-18210/A/CGCl5O 6/PC (ix) TEIECOC4M~JICATION INFORMATION: TELEPHONE: (919) 541-8615 TELEFAX: (919)541-8689 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 642 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY~: linear (ii) bMDLFCULE TYPE: DNA (genanic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) OFCCGINAL SOURCE: ORGANISM: Pseudomnas fluorescens STRAZ-4: CGA267356 INDIVIDAL ISOLATE: ORF (vii) fl4AEDIATE SOURCE: CLONE: pCIB137 (ix) FEATURE: WO 94/01561 /6rUS9?/06300 51 NYE/KEY: CDS L()ATION: 1..639 OTHER INFORMATION: /tanslexcept= (pos: 3, aa: Mt) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATT AGG GIG CTA GTA GC GhT GAO CAT GAT CIC GTT CGT ACA GGI Ile Arg Val Leu Val Val Asp Asp His Asp Leu Val Arg Thr Gly 5 10
TIG
Met 1 ATT ACA CGA ATG CTG GCT GAC AC GAT GGC Tr, CA". GTG GT(_ GGC CAG Ile Thr Arg Meet Leu Ala Asp Ile Asp Gly Leu JLf Val Val Gly Gin GCC GAG TCA Ala Giu Ser GCG GAG GAA Gly Giu Glu TOC CTG C1'C AAG GCC CGG GAG T1G AAM CCC Ser Leu Leu Lys Ala Arg Giu Leu Lys Pro 40 GAT GTG Asp Val GTC CT ATG GAO GTC AAG Val Leu Viet Asp Val Lys 55 ATG CCC GGG ATC GGC CGT CTT GAA Mt Pro Gly Ile Giy Giy Leu Glu
GC
Ala ACG CGC AAA IG TIG CGC Thr Arg Lys Leu Leu Arg 70 AGT CAC CS GAT ATC AM G1 Ser His Pro Asp Ile Lys Val 75 G1IG GCC Val Ala CAA GCC Gin Ala 240 GTC ACC GIG TGT GAA GM GAAW Val Thr Val Cys Giu Giu Asp CCG TTC CCG Pro Phe Pro 90 ACC CGC 'ffrG CTG Thr Arg Leu Leu GGC CG GCOG GGT TAO CIG ACC AAG GGC Gly Ala Ala Giy Tyr Leu Thr Lys Gly CCG GGC CTC AA GAA G G G Ala Gly Leu A.n Giu Met Val 110 SUBS-,-, rr E S) WO 94/01561 WO 9401561PCTI US93/0 6300 GAG GCC ATI CGC CI'G GIG TIT GCC GGC CAG CGT TAC ATC AGO COG CAM Gin Ala Ile Arg Leu Val Phe 115 Ala 120 Gly Gin Arg Tyr le Ser Pro Gin 125 AW1 GOC CAG le Ala Gin 130 GAG TTG GTG 71C AAG Gin Leu Val Phe Lys TCA TTC Ser Phe CAG CCI TCC AGT GAT TCA Gin Pro 140 Ser Ser Asp Ser
COG
Pro 145 TIC GAT GOT TIG TOOC Phe Asp Ala Leu Ser 150 GAG CGG GMA ATC CAG Glu Arg Giu le Gin 155 ATC GOG GIG ATG ATI' Ile Ala Leu Met Ile 160 G 1C GGC TGG CAG A Val Gly Cys Gin Lys 165 GIG GAG ATC ATC 'ICC Val Gin Ile le Ser 170 ACC TAO CGT TAO CGC Thr Tyr Arg Tyr Arg 185 GAO MAG Asp Lys GIG TGC CTG TCT Leu Cys Lieu Ser 175 CCG M ACC Pro Lys Th~r ATC AGC AGO Ile Ser Ser 195 Ar. TIC GMA MG aCT TCG Ile Phe Giu Lys Leu Ser 190 GAT GT1' Asp Vai GMA CIG ACA Giu Leu Thr 200 MI GIG GCG M'r =G GA GGC ATG Leu tleu Ala Val Arg 205 his Gly Met G=G GAT GOC AGT GOC 'rcjA 642 Val Asp Ala Ser Ala 210 INORMlIO FOR SEQ ID NO:2: SEQUENCE CHAACTERISTICS: LENGTH: 213 amino acids TYPE: amino acid TOPOLMG: linear SUBSTITUTE SHEET (RULE 26)
I
WO 94/01561 WO 9401561PCr/0S93/06300 53 (ii) MVLECULE TYPE: protein (xi) SEQUENCE LJESCRIPTION: SEQ ID NO:2: 'A l P L v )1i I Ld V Met Ile Arg VaiLeu Val Val Asp Asp His Asp Lau Val Arg Thr Gly Ile Thr Arg Yet Leu Ala Asp Ile Asp Gly Le.-u Gin Val Val Gly Gin Leu Lys Pro Ala Giu Ser Gly Giu Glu Ser Leu Leu Lys Ala Arg Asp Val Val. Leu Met Asp Val Lys Met Pro Gly Gly Gly Leu Glu Ala Thr Arg Lys Leu Arg Ser His Pro le Lys Val. Val Ala Val Val Cys Glu Asp Pro Phe Pro Thr Arg Leu Leu Gin Ala Gly Ala ita Gly 100 Tyr Leu Thr Lys Gly 105 Ala Gly Leu Asn Giu Met Val.
110 Gin Ala Ile 115 Arg Leu Val. Phe Gly Gin Arg Tyr Ile 125 Ser Pro Gin le Ala 130 Pro Phe 145 Gin Gin Leu Val Lys Ser Phe Gin Pro 140 Ser Ser Asp Ser Asp Ala M~u Ser Giu 150 Arg Glu Ile Gin Ile Ala Leu Met Ile 155 160 Ser Asp Lys Leu Cys Loeu Ser 170 175 Val. Gly Cys Gin Val Gin Ile Ile
IS!"T(FUE
WO 94/01561 WO 9401561PCr/US93/06300 Pro Lys Thr Val Asn 180 Ile Ser Ser Asp, Val.
195'-.
Th~r Tyr Arg Arg le Phe Glu Lys Leu Ser 190 Glu ILeu Thr 200 Leu Leu Ala Val Arg His Gly Met 205 Val Asp 210 Ala Ser Ala INFORMATION FOR SEQ ID NO:3: Wi SEQUENCE CHARACERISTICS: LENGTH: 5559 base pairs T1~YPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) WLIECULE TIYPE: DNA (genomic) (iii) HYPOTHIETICAL: NO (iv) lANTI-SENSE: NO (vi) ORIGINAL SOURCE:, ORGANISM: Pseudomonas fluorescens STRAIN: CGA267356 INDIVIDUAL ISOLATE: 5.6 kb EcoRI-HindIII restriction f ragmeant (vii) IMI4ATNE SOURCE:.
CLONE: &IB137 (iX) FEAaTURE: NAME/KEY: misc feature LOCAION: 210..1688 SUBSTITUTE SHFt (r J 12 WO 94/01561 WO 94/ 1561PCr/US93/06300 OTHER IZNFORMATION: /note= "ORF 1, transcribed left to right" (ix) FEATURE: NX E/KEY: misc feature LOCATION: 1906. .3633 OTHER INFRMATION: /note= "1OR 2f transcribed left to right" (ix) FEATURE: NAME/KEY: misc feature LOATION: 4616..469.
OTHER 110 RMATION: /notex- "glyW, transcribed right to left" (ix) FEATrURE: NA1E/KEY: misc feature WOCATION:'4731. .318 OTHER INFORMATION: /note= "ORE' 3, transcribed right to left" (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:3: GAA'rTCGATG ACATGCCGCG CGCCGGCATC GACACGCAAA TGG
T
CGACCr GTGGWCGAAA TGCCGCAGAA GCTCTGGAG C7GTGGCGCA. ACTC1'CAGCT CCGACCGCCA ACGATCCGCA AATCAAGGTC TCGGCGCCGG TGTCCAMACG GCGGCGAACA GCAGCTGCAA, GACATCIGA TGCITGCG CACCGGCACC TCMAGCATTIA CAAGCGCGCC ACGGTGCTGC GGCGGATCGA GCGCCCGCTG CCCAGCCGGA cCCTCGCCGCC TM'CACGATT ACCTGCAGAT GCACCCTGAA
GGTGCTGCCG
CATCACCCTG
CGATGCCGCG
GGCCATGACT
CAGG'ICACCG
GAAACCAAGG
SUBSTiTUTE SHEET (RULE 26) WO 94/01561 WO 9401561PCr/US93/06300 56 CGC~TGCTGGG CGACAC ACGGCGTGA CCATI= TCGCGACCGC GTAGGCCTIC AAGCCCTGGA GCGCAXIGTC ATICCTG=~ TGGTGAAGTC CTIGCAGGAC ZASCCAACCGC ACCGTGAAGA CGTGCGCAIC TC-GCCGCCG GCTIGCTCCAC GGGTGAAGAG GC=1ATAGCC TGCAA= C GCCAGCGAG CAGATGGCOC 'IGGAGGCCTG CAACGCCMAG C1'GCAGGThI' 'ICGcOACCGA TATCGACGAT CGGCC CCCAGGGACG CAAGGGGGTC TATCCCGAAG 420 '480 540 600 660 CGATCG'rTAC
ATTACCGGGT
CGGATCCGCC
ACCGCGACGT
ACCGTfCCT
ACAAGCGCAA
CCATGCCGCG
WWTGCGCAA
CGAGCATGAT
TCCTGCGCTA
TGCGCCTA
CCGCCGGGTG
CGATGTGCCT
GCGCMAGGAG
ATITGCAG
GCAACGGGAG
CGGTTCCTCC
~CGCATFI
ACGGCGTACA
AACCTCGX
CG7CGATGCC
TGTGCGGGG
ACTGCGCACC
CGC-ATCGAGC
CCGCAGCGCA
ANTICGAMA
ATCGACTTGA
TGCGCCAGTA
ACG7CTGT
TCGTC'IGCCG
CI=CGG
CGCCAAGCAC
TAACCTGCT
GAAMACCAGC
AGCCTGCTGG
ATCTACCTGG
A='CTGCAGA TGTTCr-ACI CGCCCTGOGT CCIGGAGGCT
GAATCCGCGG
CGGGTACGGC
TGCGCACCAT
GCCGACA'ICC
AACGCCGACA
ACGGTCCA
CCAkCICGGC
CGCICOMCCC
ACC=IGCGC
'ICCTGCACAT
GGATCG=
CACGGTI=G
CACCCCGTGG
CCTGGAAAMG
GAGCGAAGGC
GACCCTGATC
GTCCGGTGT
ATCGACCTCA
GTCCGGCG
CGCGCGCCCA
AAACCAAT
TGCGCGCCGC
GCCGGCCGGI'
CAGCCCGAGC
GCGGTGACCG
CAGCCCGCCC
720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 GAAAWTC GCAACCT ACGFICC AGTGCAACA GGGMAAAGAA GCC1GTIC CF1CAAGGAC GAGGAAMCOG ACAACGAATIA W.LTGCIGG GI=TIGAGG AGACCGAGGC S LI3SITU THE (RULE 26) WO 94/01561 WO 9401561PCI'1US93/06300
CGACCCACGG
CCTCGAGCGG
AGIVCrAGC
GCGCTCGGCC
GCTGCTGACG
CTACCI,.AC
GCGCATCCGC
CGACGCICA
GCGACCT
TGGTACATCG
CTGACCI=C
GAACGCATGC
CAGGGCCICA
GAGTCTGG
CCGGAAAGCG
ATACGCMAGG
AGcCCTGCAAG
GAGCAGAACC
GAGC1'GcG
GAGTIGCAGC
GAGGAGCTCA
ACCGAAGAGC
GTCAATTACG
MACCT'GATCG
TGGTTCACCC
TACTGGACAT
TCGAGWG1'
CACGCCTGTT
TCGATATCAC
GCCITGCGC
TCACCGACTG
GCGCC1'ATrA
AGCTCC=
ACGGCGAGCG
GCTAZGIGAA
AGAAGCTGAT
AGACCAGCGC CAGCCAGACG GGACCAAATI GCACCTGCAG AGGCGTCGAA CGAAGAAAG
GAAAACCAGA
GACACCATICG
CAGGCGCTCA
TGCTG-GCCAA
AGCAA'ICGGA
ATGAAGAGCI'
TGGAAACCAG
AGCTGAAAAC
CCTCCACCGA
CGCGCGCCAC
CACCCACCGC
GAGCATfmA= GCCCrAT=G
CAAGCGCCGG
CGAAAGCACC
GAACACCGGG
CGACCTGAT
CGCCCGCGAA
cFFFFIrTGC
AATIAGCCCGC
GGAGACCCAG.
CAAGGAAGAG TTGCAGTCGA TCAAGAAGA CAAGGICGAG GAAACCGACA AGATCAACGA CArICGCCACG
CGACX
T
IT
CTGAACTACC
GAGCGTCAA
TCCAGCGAAG
CTGGCCGAGG
CATGA2G
GCGCAACTGA
GIG'TCGTCG
AGCATGCTGC
CGGAAATGGC
'ICAACAGCGA
ACCATATCGA
AGGAACTGCG
CCATCATCAT
TCTICGGCTA
ACCGCAACAT
CGGTGACAC
CGAGGACGCC
CGATCAGCGC
CGGCACCGTG
CC'TGGGCGAA
CCTCGACAAC
TACCAAGGAC
CGGCGGCGrTG
ACGCTGGCAT
CAAGGGTGAC
CAIXGCtGGAC
TGAGFI'T=
1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 TTCGCGCCTG AGGACCGCGC CACGGCCGCA GCGACGAIGA AGCGGCGAAG 'ICACGCGGCr GACCM'ACG GCCACAAACG ACCCACGCC AcCCCAAGGA SUBSTITUTE S"HEET (RULE 26) WO 94/01561 WO 9401561PCr/US93/06300 58 GCGGTGATGT CCCTIGAACT CMGCATCCG CICAACCIGA TCCPAGCTCAA CGCCGAGTIG
CTIGCGTCGCC
TGCGAGGCITG
CGCACCGGCA
ATCCATACCG
CCACCGCAAC
AACCTGGTGA
CAGCGGGTCG
GAAGACC1ASA
CAACGCGACG
GGCTCGGTICA
CCCCTGAGCC
CICCGGCA
CTGCTGCTGG
GGCAATGCCA
TGCCGACGAC
TCTCCAGCCA
AGCTCAAGCT
GGCICAG
CGCCGTI'AAT
ACAACGCCCT
AGGATAAGGC
ACAAGG=I'
GGCI'GGGCAT
GCGTGCAGTC
ACCCCAACGA
TCAAGGTGV=
AGATGGAGGG
GGAACAACAG
CMAGGCGGOC
GGC-GCGGATC
GAAGAAACAG
CGAAGGGCAT
GATCGATGCC
GAAA2
T
I'CACC
GCACGTGGAT
CGACCIMC
GMAGGGGCTG
CAGCGCTCCC
GCTGGTGGAC
CGCGCAA=
TTACGACCTG
GCCCCT1GCCC TCAAGGCGGT CAATACCATI'
ATCGACGACC
CCGG'GATC
TCGGATGT GCGGCGT=I TIGGCCGGAT CCTGCAGGAC CGCTGCAGG TGACGC1'GCA
GAGOACGC
CCGGCCAATG
GMVATCGACA
GGCCAGGCGG
CTGG1'GCGCC
GGCCAGGGAT
AAACAGCCCG
GACTCGCGGG
GAGGCC
ATCATITCAG
GGCTGAGCA
GC'IUCGGTCA
GCGGCG'IGGG
CCAACCAGCA
ACCTGG" )GGA
GCACTTAC
CGTCGCGGGG
AAGTCATGGA
ACGACCCGCT
ACATCGG(,AT
AGTGCCIM
GGTGAW
G'fDGAaCGCC
CCI'GGCCGAG
CGGCACTCAT
AGCCCACGGC
CGTGCI'C=I
TGTGAACGC
AGTCGCAA
GCAGGCCII=
GCCGATTATG
2580 28~40 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 AACGGCTACG ATZATGCA GAACCTGCGC CAGATCGCTC ACCTGCACCA TACGCCAGCG ATTIGCGCIGA CCGG7TACGG GATCGGCATG TGTAGCAAAC CGCCAGCAGC GACCGAGA AGTCCAGCA TGCGGGATIC CG~GTCICAG GACCGC7GA TCGACCTGA CAGGGAGCTIG 3540 3600 SU B ST ITU T 2E (R UL r 2 6) WO 94/01561 W094/1561PCT/US93/06300 TCAGCCAGG GCITGCGCTC GGCIGAGCAC TGATC=A GACCCGGCGA ACCCACCI'CG TCGGCC71GA GCGCGGCGAG CGCCATIGCC TGCTGGGCAG CTAT1CACGC TTIGCGGATCG
TCGCGCTGC
TI 3AGCTGA CTrAACJGCG AGGCC ITGCC ~Tr'ArT CTGC ICGAGA
GCGCCATGCC
TGAATACCGG
AGCCAATGGT
TAAAGGGCTC
TCGATTIATC
T IrcAr
CGCATTGCGC
GGCCACGGCA
TATE=r'
CGGGAAACGA
GGGCCACCGC
GCCAAWTC
TCCA
AGGCGTICCAG
GCCGATAAAT
ACGGCCAGGC
AWCGACCAGC
TTCGCCGTCC
CG ICAGG
CACCCGCI'CG
GAGCA=G
CCTITITGAT
GGCGMATGGG
ATACGGGCT
CGCAMAAAA
GACTCGAACT
CCIU)AT1G
CCAATCGATC
GGCGICCGGC
=ICGGTGT
CGGCGAGTGG
CGGGACATVIG
GTCAAGG7CA
GGATCCAACT
WCGGCCCCA
CGATICTCAA
TCGAT=TCC
GCCCGCCCGA
CGACTGAA
GcCTCCATAGGCGGCGrIT GGCCTGGTCC ACGCCGTIG GCAGCAACTC CICGGCCGCG GAGCCGGAAC GGTAGICACG
TT'GCGTICAT
GTGGIGGGF1' C1'CAACGGIC
GITCTCGAT
CATCGTAGAA
GGTAATACTT
TGACATAACG
AGCAGAGAA
CGCTGGCGAG
TCCGATXC'G
TGACCATGGC
T=TGGATAT
AGCGACCGGA
CGTGCCGGCG
AAAGCGCGTG
GCCGICAAGG
TA'FICAGCG
TCCGGTAGAC
CCIGCI'CGC
GTTCCAGC
AGOCACGC
GCGCCCrGIC
TGGAGCTCGA
ATCCGGTCCT
CCTICGAGCC
AAAAACCCGG
2T3CATACCTG
CGCGTGCTI
GCGTCTGC
3660 31720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 AAGTGCCAG CTPIGCGGCCC CAGGTACGGC TTAGAP AGAA GAATGAOGAT TGGCTCGACA AATGGACCTC TITMAGAGG CGCGACCG ACFIGGCAA TCCATITIA ATAMTTGGAG GGTCGIGCICr TACCAAeI'GA SUBSTT~w.~j.*T RLE E26) WO 94/01561 W094/1561PCT/US93/06300 GCTAT CCG CGTGGTG GATTCAAAAA GTTICT G1'GTGCTI' TATAGAAAT1' CGAAACTGCG TCAACCCCTT TITIr.ACCA, TCGGC= GGTGCGGCCA GGCAGCGCGC AGGTACTGCA ACATCGACCA CAGGGTCAGC CCTCCGGCGAT CAGCAGGAA GGCATAACC AGCAGCACCC GGCCGGCGGA TTWCC~jCA GGATICACCAG CGCCAGCATC AGAAGG7CGAA TGCGCGGCAG TITI'CCGATT TGCCCATGTT GGACACCGGC CACCTGGGCG CGTGCGNCCG AGCICGGCCA TCCACICGC GCCGGCAGGG TCAGCCACAG
AAGGGCGGAC
GTTGCCGTGC
CGGATCGAGG
AAGCCAGTCG
GTAAGGCAGG
ACCACGA TT CACGCCCGAT TI'IG(ACCA. GCAGCACCAG AAGGCCCCGA ACGGCGTGCT GTWGCCGCGG CGAACGCA TAAMACAGCA AAATGAAT ACCA2CAACT
CGCCGCGCCA
GCGGCCATGT
AGAACGCGTA
TGTCGGCCAC
GGTAGCCATC
AGCCCA I
GAACGGTGAT
GAWCACCGCT1
GGCCACCGCC
CTGCTCCAGA
GACGGAACTG
CGGGATGAGC
CTACGAGGTG
ACGGGCI'ICT
AACTTCAGAA
GATCGGGATC
4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5559 AGTG~GCAATC TACTCGGAAA CAGA'FTAGGG ATAICATCG GCACMACTGG AGACAGCAGA TGGGrAGCA. CGGCCATTCT G2WICCAAGAC 2TCGGGCCGC TCGAAAGGC GAC~ATTCAGT TCTITGTAA GTACCGTGTA
GCCICAGCGT
GGTCTACA.CG
GTCTAACACT
CGCAAAATAA
TM'CAGCGGT GCCCCGCCAA, AAAGGAAGCC TIGAAGCTT INFORM@AION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 983 base pairs TYPE: nucleic acid SrRANDEDNESS: single SUB, S T IT U SH IE ET (R U L E 26) WO 94/01561 PTU9/60 PCT/US93/06300 61 TIOPOLGY: linear (ii) MO)LECUJLE TYPE: DNA (genanic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURE: ORGANISM: Pseudomonas fluorescens STRAIN: CGA267356 (vii) I TDIATE SO;,TCE: CLONE: pCIB137 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 23. .51 OTHER INFORMATION: /note= "sequence with promoter homology" (ix) FEATURE: NAmE/KEY: misc feature LOCATION: 99. .740 OTHER INFORMATION: /note= (ix) FEATURE: NAME/KY: misc feature LOCATION: 743..983 OTHER INFORMATION: /note= "1ORF 5 structural gene" end of ORF 4 (uvrC)"1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CCAAGTGCTT TITATATGGT GM1ITG=CTT AGGTCAGCAC GC1'GCTrTT TGCI'AAG'I'G S1 IRST1TI ITF P-W-PT 11 PZ 26 WO 94/01561 WO 9401561PCr/US93/0(r300 TCCGGCAACC TATAAGACCC AAATCGCGAG GTGTCTGCIT GA2
T
TAGGGTG CTAGTAGTCG ATGACCAGA TCTCGTT'CGT ACAGGTAITA CACGAATGCT GGCTGACATC GATGGCCTGC
AAGIGGTCGG
CCGATG=G
AATTGTTIGCG
CGITCCCGAC
TCAATGAAAT
AAAT1'GCCCA urriWGTCGA
TCATCCCGA
TCGAAAAGC1'
TGGTGATGC
ClrzCAGTGGC
CCAGGCCGAG
CCTCATGGAC
CAGTCACCCG
CCGCTTGCTG
GGTGCAGGCC
GCAGTTGGTG
'ICAGGGGAGG AATCC1CTr G1'CAAGATGC
GATATCAAAG
CAAGCCGGCG
AT1'CGCC1'GG
CCGGGATCGG
TCGTGCCGT
CGGCGGGTTA
TGrIGCCGG
CAAGGCCCGG
CGGTCTTGAA
CACCGTG=G
CCTGACCAAG
GAGTIG~AAAC
GCCACGCGCA
GAAGAAGATC
GGGGCGGGCC
CCAGCGTTAC ATGAGCCCGC 120 180 240 300 360 420 480 540 600 660 720 780 840 900 TICAAGTCAT TCCAGCC2TC CAGTGATICA CCGTTGATG GCGGGAAATC CAGATCGCGC CAAG' TC
CTCGATCAGC
CAGTGCCTGA
CCTCCTGGCG
CTG=C2CGA
AGCGAT
CAATGACCGA
TGTATCGCAT
IGATGAT
AAACCG'ITAA
AACIGA7T'
CCCGITGAT
CGGCTGCCAG
TACCTACCGT
GC4-LG T
CCCAGTGC=
AAAGTGCAGA
TACCGCAT
CGCCACGGCA
TCTC-CAC
GTTCGACAGC GATACGCT TGC1'GTACGT CGGTAAAGCC AAGAACCTGA AGAGCCGCCT GGCCAGCTAC 'TI=CCGA CCGGCCI'cGC GCCCAALGACC GCTGCCCTGG LGGGGCGCAT AGACCGAAGC CCTGCTGCTC GAG INFORMATION FOR SEQ ID SEQUENCE CHARACERISTICS LENGTH: 642 base pa CGCAkGATCGA AACCAGCATC ACCGCCAACG SUB3S T17 71 SHEET RUE 26) WO 94/01561 WO 9401561PCr/US93/06300 63 TYPE: nucleic acid STRANDEDNESS: single 'IOPOLCGY: linear (ii) MODLECULE TYPE: DNA (gencmnic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TIGATTAGGG TCTAGTG= CGATGACCAT GMC1LGTI' GTACAGGTAT TACCCGAATG C1'GGC1'GACA T CGATGGCCT GCAAGTGGTC CTCAAGGCCC GGGAGTISMA ACCCGATGTG GGCGGTCMTG AAGCCACGCC CAAATIX= GTCA.CCGGT GTAAGAAGA CCCGTTCCCG TACCTGACCA AAGGTGCGGG CCTCAATGAA GGCCAGCGTT ACATCAGCCC GCAAATI'GCC TCCAGI3A T CACCGT1CGA ITGTT C &I'CGGCTXC AGAAAGTGCA GATCAWIC AATAWCTACC GITAhCGCAT CTICGAAAA3
GGTCAGGCCG
GTCCTTGG
CGCAGTC
ACCCGCTGC
ATGIGGCAG
CALCAGTTGG
GAGCGGGAAA
GACAAGCTGT
CTCTCGATCA
AGTCAGGGGA GGAGTCCCTG ACGTCAAGAT GCCCGGGATC CGGATATC.AA AGICGTCGCC TGCAAGCCGG TGCGGCGGGT CCATI7CGCCT GGTG'TGC TGTICAAGTC ATTCCAGCCT TCCAGATICGC GC1XGATGAT GCCTGWCTCC GAAAACCGTT GCAGCGATGT TGAACTGACA
GA
120 180 240 300 360 420 480 540 600 642 T1'GCTGGCGG 71ICGCCACGG CATGGTCGAT GCCAGCCT

Claims (27)

1. Use of a recombinant DNA molecule to activate in an organism transformed with said recombinant DNA molecule expression of at least one gene that is latent or natively expressed at low levels, wherein said recombinant DNA molecule comprising at least one copy of an isolated gene activating element comprising a nucleotide sequence of a gafA c ass of transcriptional regulators, wherein said gene activating element induces expression of said gene in said bacterial host strain.
2. The use according to claim 1, wherein said nucleotide sequence is a Pseudomcnas gafA sequence.
3. The use according to claim 2, wherein said nucleotide sequence is a Pseudomonas fluorescens gafA sequence.
4. The use according to claim 3, wherein the said gafA sequence encodes a protein having the amino acid sequence given in SEO ID NO: 2. The use according to any one of claims to 4, further comprising introducing into said bacterial host strain at least one copy of a Pseudomonas lemA sequence.
6. The use according to claim 5, wherein the lemA sequence is defined by the DNA fragment deposited as pCIB168.
7. The use of claim 1, wherein activated expression leads to production of an antibiotic substance inhibiting a pathogen.
8. The use of claim 7, wherein the pathogen is Rhizoctonia solani.
9. A method of activating in a bacterial host strain expression of at least one gene that is latent or natively expressed at low levels comprising integrating into the genome of said host strain a recombinant DNA molecule comprising at least one copy of an isolated gene activating element comprising a nucleotide sequence of a gafA class of transcriptional regulators, wherein said gene activating element induces expression of said gene in said bacterial host strain. The method of claim 9, wherein said nucleotide sequence is a Pseudomonas gafA sequence.
11. The method of claim 10, whet in said nucleotide sequence is a Pseudomonas fluorescens gafA sequence.
12. The method according to claim 11, wherein the said gafA sequence encodes a protein having the amino acid sequence given in SEQ ID NO: 2.
13. The method according to claim 12, wherein the said gafA sequence encodes a protein having the amino acid sequence given in SEQ ID NO: 2, except that amino acid residue 182 may be either isoleucine or threonine.
14. The method of any one of claims 9 to 13, further comprising introducing into said bacterial host strain at least one copy of a Pseudomonas lemA sequence. The method of claim 14, wherein the lemA sequence is defined by the DNA fragment deposited as pCIB168. S [N:\LIBxx]00884:VMJ
16. The method of claim 9, wherein activation of expression leads to production of an antibiotic substance inhibiting pathogen.
17. The method of claim 1b, wherein the pathogen inhibited is Rhizocionia solani.
18. The method of claim 9, wherein the recombinant DNA molecule is operably linked to a bacterial regulatory element.
19. The method of claim 9, wherein said host strain is from .he genus Pseudomonas Bacillus, or E. coh. A method of inhibiting the growth of a pathogen comprisilg transforming into a bacterial host strain a recombinant DNA molecule comprising at least one copy of an isolated gene activating element comprising a nucleot:de sequence of a gafA class of transcriptional regulators, wherein said gene activating element induces expression of said gene a said bacteri di 'ost strain, and applying the transformed host strain to the environment where the pathogen is to be inhibited.
21. An isolated gene activating element comprising a nucleotide sequence of a gafA class of transcriptional regulators, wherein said gene activating element is capable of inducing expression of at least one gene that is latent or natively expressed at low levels in a bacterial host strain.
22. An isolated gene activating element to claim 21, wherein said nucleotide sequence is a Pseudomonas gafA sequence.
23. An isolated gene activating element according to claim 22, wherein said nucleotide seauence is a Pseudomonasfluorescens gafA sequence.
24. An isolated gene activating eleme.t according to claim 23, wherein thr. said gafA sequence encdJes a protein having the amino acid sequence given in SEQ ID NO: 2. An isolated gene activating element according to claim 24, wherein the said gafA sequence encodes a protein having the amino acid sequence given il SEQ ID NO: 2, except Chat amino acid residue 182 may be either isoleucine or threonine.
26. An isolated gene activating element according to claim 23, wherein the said C 30 gafA sequence has the nucleotide sequence set forth in SEQ ID NO: 1.
27. An isolated gene activating element according to claim 23, wherein the said gafA sequence has the nucleotide sequence set forth in SEQ ID NO:
28. A chimeric expression construct comprising the gene activating element of any one of claims 21 to 27. S 35 29. A pla-mid vector comprising the gene activating element of any one of claims 21 to 27. A bacterial strain producing an antibiotic substance inhibiting a pathogen, wherein said inhibition has been activated by integration of a recombinant DNA molecuic according to any one of claims 21 to 27. [N:\LIBxx]00884:VMJ 1 A.2 66
31. A transgenic bacterial host strain into which the gen' activating element according to any one of claims 21 to 27 has been introduced.
32. A transgenic host strain according to claims 30 and 31, which is a strain of Pseudomonasfluorescens.
33. A bacterial strain producing an antibiotic substance inhibiting a pathogen, which strain is defined in any one of claims 30 to 32 and substantially as herein described with reference to the Examples.
34. Use of a recombinant DNA molecule as defined in claim 1 and substantially as herein described with reference to the Examples. Dated 5 May, 1997 Ciba-Geigy AG United States Department of Agriculture Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON t 0IIW884:VMX I II ii
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US5639949A (en) * 1990-08-20 1997-06-17 Ciba-Geigy Corporation Genes for the synthesis of antipathogenic substances
US5756087A (en) * 1996-12-06 1998-05-26 Novartis Finance Corporation Genetically modified Pseudomonas strains with enhanced biocontrol activity
US5955348A (en) * 1997-11-25 1999-09-21 Novartis Ag Genetically modified pseudomonas strains with enhanced biocontrol activity
US5891688A (en) * 1996-12-06 1999-04-06 Novartis Finance Corporation DNA encoding lemA-independent GacA and its use in activating gene expression
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