AU4662593A - Gene activating element - Google Patents

Description

GENE ACTIVATING ELEMENT

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 development of these diseases can be rendered suppressive, or resistant, by the addition of small quantities of soil from a suppressive field. Scher et al. Phytopathology 70:421 (1980) . Conversely, suppressive soils can be made conducive to fungal diseases by a toclaving, indicating that the factors responsible for disease control are biological. Subsequent research has demonstrated that root colonizing bacteria are 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. , Phytopathology, 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., Phytopathology 71:1020-1024 (1981). Important plant pathogens that have been effectively controlled by seed inoculation with these bacteria include Gaemannomyces gramminis, the causative agent of take-all in wheat, Cook et al., Soil Biol. Biochem 8:269-273 (1976) and Pvthium 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 commercially available chemical fungicides that are effective in controlling the fungus. Because of these circumstances, an inhibitor against R. solani would be of substantial interest as a potential control for this pathogen.

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. Phytopathology 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., Phytopathology 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., Phytopathology 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 important 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 of fungal pathogens with the ability to control other plant pathogens and/or the ability to aggressively compete in the rhizosphere. Bacterial two-component regulatory systems have been extensively reviewed (e.g. 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 component. 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 component. 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 Pseudomonas fluorescens CHAO, an activator-component gene designated gacA which was required for the expression of genes involved in the synthesis of the antifungal secondary metabolites 2,4-diacetylρhloroglucinol, 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 gacA 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 gacA gene was involved in regulation of secondary metabolism in P_. fluorescens and inferred that extracellular secondary metabolites produced under gacA control are important for the biocontrol of black root rot. They noted the presence of a gene homologous to gacA in E. coli and cited preliminary evidence from hybridization experiments that a sequence similar to gacA 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 Pseudomonas fluorescens, since we have discovered that ORF 5, (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 gacA 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 Xhoi fragment of P_. fluorescens strain 915 DNA containing ORF 5 or upon introduction of a cloned E. coli gene homologous to ORF 5, gafA) .

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 syrinqae (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.

It is a further feature of the present inventicfn 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 is 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 compete 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 Rhizoctonia solani, Helminthosporium gramineae and species of the genera Pythium and Fusarium. Therefore, use of bacterial strains transformed with the ORF 5-type or lemA-type genetic element provides an environmentally safe and effective method of control of these pathogens.

In addition, we have demonstrated that these gene activating sequences can be transferred to other bacterial strains, especially pseudomonad 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 Gaemannomyces gramminis, can be controlled in some 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 (J. Cook, personal communication) . The gene activating sequence for activation of genes which are effective against R. solani could be introduced into the biocontrol strains currently used to protect wheat from take-all to extend their range of effectiveness to include R. solani.

The present invention comprises an isolated DNA sequence consisting essentially of the 2 kb fragment deposited as pCIB 137, or of the ORF 5 sequence shown in SEQUENCE ID No. 1. These DNA sequences are capable of activating latent gene activity in a bacterial strain. 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 Pseudomonas fluorescens.

The present invention further comprises recombinant DNA sequences in which a bacterial regulatory element is operably linked to the DNA sequence of SEQUENCE ID No. 1. The bacterial regulatory element may be a promoter from a gene isolated from Pseudomonas, Bacillus, or E^ coli. In one embodiment of the present invention, the bacterial regulatory element is the native promoter of ORF 5. The bacterial regulatory element may be from a gene which is homologous or heterologous to the host bacterial strain.

The present invention also includes methods of activating latent gene activity in a host bacterial strain by transforming the host bacterial strain with the recombinant DNA sequences of the present invention. 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.

The present invention further comprises isolated DNA sequences encoding the lemA gene. These sequences are capable of restoring the production of hydrolytic enzymes such as chitinase and gelatinase and the production of antifungal secondary metabolites such as pyrrolnitrin and cyanide in some mutants lacking these functions. The invention further comprises recombinant DNA sequences in which a bacterial regulatory element is operably linked to the DNA coding sequence of lemA. The bacterial regulatory element may be a promoter from a gene isolated from Pseudomonas, Bacillus, or E. coli. In one embodiment of the present invention, the bacterial regulatory element is the native promoter of lemA. The bacterial regulatory element may also be from a gene which is homologous or heterologous to the host bacterial strain.

The present invention also includes methods of activating latent gene activity in a host bacterial strain by transforming the host bacterial strain with the recombinant DNA sequences of the present invention. 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:

Promoter or Regulator DNA sequence: An untranslated DNA sequence which assists in, enhances, or otherwise affects the transcription, translation or expression of an associated structural DNA sequence which codes for a protein or other DNA product. The promoter DNA sequence is usually located at the 5' end of a translated DNA sequence, typically between 20 and 100 nucleotides to the 5' end of the starting site for translation.

Structural or Coding DNA sequence: A DNA sequence that is translated or transcribed in an organism to produce an RNA, a protein or other DNA product. Associated with/operably 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 protein if the two sequences are operably linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.

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.

Homologous: 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 host organism to be transformed is of the species Pseudomonas fluorescens, a DNA sequence is homologous if it originates from a pseudomonad 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 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 (i.e. latent) or expressed at low levels in the naturally occurring state of the host. These sequences typically encode proteins which play a role in the pathways which regulate gene expression.

Brief Description of the Figures

Figure 1: This figure shows restriction maps of three cosmid clones, pANT5, ρANT9 and pANTlO, that were found to complement the ANT- phenotype of mutant 2-1. In this figure, 'B' indicates a BamHI restriction site; Ε" an EcoRI restriction site; and 'H' a Hindlll restriction site.

Figure 2: This figure shows the ability of DNA subfragments derived 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 identified 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 complement the mutant CGP 21. Detailed Description of the Invention

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 Pythiu ultimum- and Rhizoctonia solani-induced damping off of cotton. We have determined that certain mutant derivatives of the bacterial biological control strain Pseudomonas fluorescens strain 915 are deficient or altered in a variety of functions. Such pleiotropic mutants can be isolated following mutagenesis techniques known to those skilled in the art (e.g. nitrosoguanidine mutagenesis, transposon mutagenesis) or can arise spontaneously. One such mutant, obtained after mutagenesis with the chemical mutagen nitrosoguanidine was designated mutant 2-1. Seven further mutants were identified by introducing the transposon TnCIB116 into strain 915. These mutants can be identified on the basis of their inability to inhibit _in vitro the growth of the phytopathogenic fungus Rhizoctonia solani. They also fail to synthesize the antifungal metabolite pyrrolnitrin, and no longer produce cyanide or the enzyme chitinase, each of which has the potential to inhibit fungal growth (Voisard et al., EMBO J. 8:351-358 (1989); Jones et al., EMBO J. 5:467-473 (1986)). The mutants' production of an enzyme with gelatinase activity is significantly reduced, and they have an altered colony morphology. A summary comparing the characteristics of the pleiotropic mutants with the corresponding characteristics of wild-type P_. fluorescens strain 915 is presented in Table 1.

TABLE 1

Characteristic P. fluorescens 915 Pleiotropic mutants

Pyrrolnitrin + Cyanide + Chitinase + Gelatinase reduced Colony morphology circular, entire, circular, undulate, convex and opaque flat and translucent

Inhibition of Rhizoctonia solani

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 kilobase EcoRI restriction fragment (fragment Ell) of P_. fluorescens strain 915, and a 2.0 kb Xhoi subclone of this fragment containing gafA (ORF 5) , each restored all of the lost or altered functions listed in Table 1 when introduced by conjugation into one class of pleiotropic mutants derived from strain 915. Introduction of fragment Ell or the 2.0 kb Xhoi subclone into the P_. fluorescens strains 914 and 922 unexpectedly activate the expression of latent genes involved in the synthesis of pyrrolnitrin, cyanide, and chitinase, and in the case of P_. fluorescens strain 914, cause an alteration in colony morphology on minimal medium from large, circular, flat, translucent, with undulate edge to small, circular, convex, opaque white, with entire edge. Accompanying these phenotypic changes associated with the introduction of fragment Ell or the 2.0 kb Xhoi subclone is the conversion of £. 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 gafA class are sufficient for the activation of latent genes in heterologous bacterial strains.

DNA sequence analysis of fragment Ell has, to date, allowed identification of five open reading frames, as well as a tRNA gene (glyW) . The organization of these open reading frames within fragment Ell 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-component regulatory systems (reviewed in Albright et al., Annu. Rev. Genet. 23:311-336 (1989)). We determined that the organization of glyW, ORF 3 (which has homology to the Escherichia coli gene pgsA) 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 transcriptional activator gene of unknown function (Moolenaar et al., Nucl. Acids Res. 15:4273-4289 (1987)). ORF 5 (gafA) exhibits homology to this putative activator gene. Furthermore, comparison of the fragment Ell sequence with DNA sequences contained in the Genbank database reveals that ORF 5 has substantial homology to a proposed transcriptional activator gene isolated from Pseudomonas fluorescens CHAO by Laville et al., Proc. Natl. Acad. Sci. USA 89:1562-1566 (1992). 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 Xhoi subclone containing ORF 5 is prepared, as is a 3.7 kb EcoRI-Xbal 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_. fluorescens strains 914 and 922. The 3.7 kb EcoRI-Xbal subclone had no measurable effect. Furthermore, when the gafA gene was cloned from the strain 914, transferred to plasmid pLAFR3 and reintroduced into strain 914 the latent genes are activated indicating that strain 914 does contain a gafA gene capable of functioning, but that the expression of the gafA gene in strain 914 is presumably not at levels high enough to activate the latent genes.

To determine whether transcriptional activators of the gafA class are generally capable of activating the expression of latent genes in heterologous bacterial strains, we cloned the putative transcriptional activator gene described by Moolenar et al. and introduced it into P.. fluorescens strain 914. The E. coli gene, which encodes a protein which is approximately 60% homologous to that encoded by gafA, activated the 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 gafA class into a variety of environmental isolates. This approach represents a method for the identification of potentially effective biocontrol strains which would otherwise not be selected by any of the screening methods currently available.

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 (s) 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 gramineae and species of the genera Pythium and Fusarium This DNA sequence may be derived from bacterial strains which are effective biocontrol agents against Rhizoctonia. Preferably the DNA sequence may be derived from the clone pANT5, which was isolated from a strain of Pseudomonas fluorescens. More preferably, the DNA sequence may comprise the approximately 11 kb Ell fragment of pANT5. 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 pANT5 has been deposited with ATCC and has been designated ATCC accession number 40868. A plasmid containing the 11 kb Ell fragment of pANT5 has been deposited • with ATCC and has been designated ATCC accession number 40869. The approximately 2 kb ORF 5 DNA sequence may be obtained from the Ell fragment of pANT5 as a 2 kb fragment after digestion with Xhoi. This fragment has been designated pCIB137 and has been deposited with the USDA Agricultural Research Service Culture Collection, Northern Regional Research Center (NRRL) .

The recombinant DNA sequences of the present invention may be chimeric and may be heterologous or homologous. The recombinant DNA sequences of the present invention may further comprise one or more regulatory DNA sequences operably linked to the structural DNA sequence above. 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 (e.g. 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 characterized. A 6 kb subclone which encodes a gene with high homology to lemA (Hrabak et al., (1992) supra) was found to retain the phenotype restoration ability. This clone was deposited as pCIB 168. Consequently the lemA gene clearly has the ability to restore the biocontrol phenotype in these pleiotropic mutants. However, when the lemA gene was introduced into strain 914 it was not capable of activating latent gene expression, 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 rate-limiting factor preventing the production of chitinase, gelatinase, pyrrolnitrin and cyanide. Other bacterial genes functionally homologous to lemA would be able to act in the same way as lemA. Such genes comprise a class of sensory component genes capable of phosphorylating and therefore activating the gafA class of activators described above. Members of the class can be identified and isolated by 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 seguences gafA 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 transformed 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 genome of a bacterial strain which may not ordinarily be effective as an inhibitor of fungal pathogens, resulting in an effective biocontrol strain.

DNA in the form of plasmids can be transferred from one bacterium to another by a 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 Pseudomonas, 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 recombinant DNA sequences of the present invention into the genome of a biocontrol agent to form a transformed biocontrol agent, allowing the transformed biocontrol agent to produce antibiotic substances, such as pyrrolnitrin, and 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 compound 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 compound is compatible with bacterial strains. The present invention also relates to methods of treating plants, which comprise 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 compound, 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 ingredients can also penetrate the plant through the roots via the soil (systemic action) by impregnating the locus of the plant with a liquid composition, or by applying the 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 example, selective treatment of the plant stems or buds.

The active ingredients are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for 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 (a.i.) per hectare ("ha", approximately 2.471 acres), preferably from 100 g to 2 kg a.i./ha, most preferably from 200 g to 500 g a.i./ha.

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 dimethyl 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 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 compounds 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 sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnapthalenesulfonic 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 propylene glycol unit.

Representative examples of non-ionic surfactants re nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol. 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 C₈-C_j alky***- 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 Emulsifiers Annual," MC Publishing Corp. Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopedia of Surface Active Agents," Chemical Publishing Co., Inc. New York, 1980.

The agrochemical compositions usually contain from about 0.1 to about 99 %, preferably about 0.1 to about 95 %, and most prferably from about 3 to about 90 % of the active ingredient, from about 1 to about 99.9 %, preferably from abut 1 to about 99 %, and most preferably from about 5 to about 95 % of a solid or liquid adjuvant, and from about 0 to about 25 %, preferably about 0.1 to about 25 %, and most preferably from about 0.1 to about 20 % of a surfactant.

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 Streptomyces for the production of tetracycline, erythromycin, chloromycetin, and streptomycin; strains of Bacillus for the production of bacitracin; and strains of Penicillium for the production of penicillin.

The activating elements find further use in the production of vitamins, growth factors and hormones in selected strains of microorganisms. Examples include the enhanced production of vitamin B2 and B12 from Ashbya and Streptomyces, respectively; the production of gibberellin from Fusarium and Gibberella; the production of 11-α-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 production 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 Example 1. Mutant Isolation and Functional Complementation.

The wild-type Pseudomonas fluorescens strain llc-1-38 (strain 915) was mutated by exposure to the mutagen N-Methyl-N'-nitro-N- nitrosoguanidine. Approximately two dozen antibiotic-affected mutants were identified by screening individual mutants for their ability to inhibit the growth of P_. ultimum and R. solani on nutrient agar. Most of these were shown to have reduced, or no, antibiotic activity against one of the two phytopathogenic fungi, but were not affected in their inhibition of the other fungus.

However, one mutant, that we have named mutant 2-1, was found to lack antibiotic activity against both test fungi. These results strongly indicated that P. fluorescens strain llc-1-38 produced two distinct antibiotics, one effective against P.. ultimum and the other effective against R. solani, rather than one antibiotic that was effective against both fungi.

A gene library of total DNA isolated from the parent strain was constructed by partial Sail restriction of the DNA, size fractionation to yield fragments of 20-30 kilobases, and ligation into Xhol-restricted vector pVKlOO. Knauf et al., Plasmid 8:45-54 (1982) . The gene library was transferred to the antibiotic mutant 2-1 by triparental conjugation with E. coli harboring the tra plasmid pRK2013. Ditta et al., Proc. Natl. Acad. Sci. USA 77:7347-7351 (1980) . Transgenic exconjugants were tested for the production of antibiotic by measuring growth inhibition of P_. ultimum and R. solani. Three overlapping clones were identified that restored antibiotic activity against R. solani, but not against P_. ultimum, to mutant 2-1. Restriction maps of these clones were determined and are shown in Figure 1. 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 the larger region and assessing their ability to complement mutant 2-1 for antibiosis (Figure 2) . The smallest fragment that was demonstrated to complement the mutation was the 4.9 kb Hindlll/EcoRI fragment, which was indicated as subfragment 6 in Figure 2 and was designated subfragment H/E4.9. Some of the subcloned DNA fragments derived from the antibiotic gene parent clone, which was designated pANT-5, were transferred to two wild type £. fluorescens 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 kb 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 solani.

An active antibiotic compound can be extracted from the growth medium of the transformed P. fluorescens strain that produces this antibiotic. This was accomplished by extraction of the medium with 80 % acetone followed by removal of the acetone by evaporation and a second extraction with diethyl ether. The diethyl ether was removed by evaporation and the dried 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.

Example 4. Description of the pleiotropic defects restored by fragment Ell and by the 2.0 kb Xhoi subfragment; ability of these fragments to activate 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 identified 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) (5% 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 volume). 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 cm) 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 resuspended in the same volume of water/methanol (50/50) . The material eluting from the column was monitored by B absorbance at 212 nm and at 252 nm, and was fractionated by elution time. P_. fluorescens strain 915 extracts contained a peak which comigrates with a pyrrolnitrin standard and which was determined to be pyrrolnitrin by NMR spectroscopy and by mass spectrometry. Mutant 2-1 and the other identified pleiotropic mutants lack this peak. b.) 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 5 mg/ml chloroform 4,4'-methylene bis-(N,N dimethyl aniline) and chloroform was allowed to evaporate. Papers were then placed under the covers of microtiter plates whose wells have been inoculated with cultures of strain 915 and the various pleiotropic mutants. Plates are wrapped in aluminum foil and incubated overnight at 28 C. The paper turned a blue color above the well of each culture producing cyanide. The results indicated that strain 915 produced cyanide, while the pleiotropic mutants failed to produce cyanide. c.) 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 and washed once in 20 mM phosphate buffer (10 mM Na2HPO4/10 mM KH2P04) . Following centrifugation, the cell pellets were resuspended in 5 ml of the 20 mM phosphate buffer. Cells were lysed by sonication for 60 seconds with the microtip of a Branson sonifier and cell * debris is removed from the cell extracts by centrifugation. Chitinase activity was assayed by incubation of 100 ul of cell extract with 100 ul of tritiated chitin (0.5% 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 of an equal volume of 2 M TCA, followed by centrifugation. 200 ul aliquots were counted in a liquid scintillation counter to determine soluble counts released from the insoluble chitin molecules as a result of chitinase activity. Typical results, presented in Table 2, indicate that a transposon mutant designated #736, which proved to be a member of one class of pleiotropic mutants, lacks the chitinase activity found in P_. fluorescens strain 915.

TABLE 2 EXTRACT COUNTS /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 FLUORESCENCE UNITS

P. fluorescens 915 13.2

Pleiotropic mutant 0.687 d.) Reduction in gelatinase activity

Gelatinase activity of £. 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. e.) 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 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 chemotaxis 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 seguences (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 bvqS 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 uyrC 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, bvgA 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 Hindlll 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 glyW: 4616-4691; transcribed right to left 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 Pseudomonas strains and that the E. coli uyr-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 LMG (0.1% KH₂P0₄, 0.1% Na₂HP0₄, 0.1% NaCl, 0.4% (NH₄>₂S0₄, 0.02% glucose and 0.66% MgS0₄, 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 Xhoi fragment containing ORF5 was cloned into the broad host range plasmid pVKlOO (Knauf et al.(1982), Plasmid 8:45-54) to yield the plasmid pCIB137. Example 7: Analysis of the 2 kilobase Xhoi fragment

Comparison of the ORF5 sequence to DNA sequences in the GenBank database revealed substantial homology with numerous transcription activator genes in known bacterial two-component regulatory systems. In particular, ORF5 is highly similar to the gacA gene of Pseudomonas fluorescens CHAO and to the uyr-23 gene of E^ coli. ORF5 is located entirely within an approximately 2kb region defined by Xhoi restriction sites. This approximately 2 kb Xhoi fragment was cloned into the broad host range plasmid pVKlOO (Knauf et al.(1982), Plasmid 8:45-54) to yield the plasmid pCIB137. pCIB137 was deposited with the USDA Agricultural Research Service Culture Collection, Northern Regional Research Center (NRRL) at 1815 North University Street, Peoria, 111. 61604 on June 24, 1992 and has been accorded the accession number .

PCIB137 was introduced into the E. coli host strain S17-1 (Simon et al.(1983), Biotechnology 1:784-791) by transformation. It was then transferred into strain 2-1 by conjugation. Fresh overnight cultures of S17-1 (pCIB137) and strain 2-1 were mixed (50μl each) on an L agar plate and allowed to incubate overnight at 28C. Loopfuls of bacteria from the mating mixture were then streaked on LMG agar containing 15 μg/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 LMG agar. pCIBl37 is introduced into these strains by conjugation as described in example 2. Transconjugants of strain 914 containing pCIB137 were shown to produce pyrrolnitrin, chitinase, and cyanide. They also displayed the morphology of P. fluorescens strain 915. Transconjugants of strain 922 containing pCIB137 were also shown to produce pyrrolnitrin, chitinase, and cyanide, but they did not display a change in morphology. Thus, it was shown that the introduction of pCIB137 into Pseudomonas fluorescens strains (e.g. strain 914 and strain 922) which are ineffective in the _in vitro or _in vivo inhibition of Rhizoctonia solani, unexpectedly activated expression 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. Example 8: Intact ORF 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 (Promega) . The resulting construct, designated pCIB138, contains no other Nael sites besides the two in ORF5. Digestion of pCIB138 DNA with Nael followed by ligation at a concentration of 30 ng/μl 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 5, 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 ρBREB6.8 by digestion with Xhoi, then self-ligation to form pCIB139. A kanamycin resistance marker was introduced into pCIB139 by substituting the Hindlll-Sall kanamycin resistance fragment from Tn5 for the tetracycline resistance region bounded by Hindlll and Sail in pCIB139. The resulting plasmid, pCIB154, was used to receive the 2kb Xhoi fragment (with the 72bp Nael deletion) from pCIB150 to form pCIB156. This plasmid was transformed into the Escherichia coli strain S17-1 (Simon et al. (1983) BioTechnology JL:784-791), then introduced into strain 915 by conjugation, selecting for kanamycin resistance. Since ρCIB156 cannot be maintained autonomously in strain 915, the kanamycin resistant transconjugants contained the plasmid, and its kanamycin resistant determinant, integrated into the chromosome. The most frequent integration events took place by homologous recombination and at the region of homology provided by the 2kb Xhoi fragment and its surrounding sequences. Such transconjugants contain two copies of the 2kb Xhoi region, one wildtype and one with the 72bρ 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 Nael deletion. Colonies of the former class were identical to strain 915. Colonies of the latter class were identical to strain 915 except for a 72bp Nael deletion in ORF 5. These derivatives of strain 915 no longer produce pyrrolnitrin, chitinase, or cyanide. Thus, an intact ORF 5 is necessary for gene activation in strain 915. Example 10: The native promoter of ORF 5 is contained within the 2 kb Xhoi fragment.

The following evidence indicates a promoter element directing transcription of ORF 5 is likely to be found within the 2 kilobase Xhoi fragment. A version of the broad host range plasmid pVKlOO containing the 2 kb Xhoi fragment was isolated with the 2 kb insert in the opposite orientation from that found in ρCIB137. This plasmid was designated pCIB151. Introduction of pCIB151 into pleiotropic mutants and into P_. fluorescens strain 914 activated gene expression. The ability of the 2 kb Xhoi fragment to activate gene expression in each orientation, and the 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 promoter located within the 2 kb insert. Furthermore, Moolenar et al., (1987) have identified a promoter directing transcription of the ORF 5-related uyr-23 gene of E. coli. This promoter was contained within the first 100 base pairs upstream of the uyr-23 structural gene. At a nearly identical position upstream of ORF 5, we have identified the sequence TTGTCA-17bp-TTTTTT 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 uyrC 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

5' end of ORF 4 (uyrC) 743-983

It is likely that a promoter native to the 2 kb Xhoi 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 promoter 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 5 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 accomplishing 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). Example 11: Analysis of the ORF 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, there is strong homology between domains 1 and 2 of transcriptional regulators reviewed by Albright et al. (1989) and comparable regions in ORF 5. The predicted aspartic acid residue at position 54 of the protein lines up with the conserved aspartic acid residues of other transcriptional activators of this class. It is the aspartic acid at this position which is typically phosphorylated by interaction with a sensor component protein. Alignment of ORF 5 with uyr-23 of E. coli and with gacA of . fluorescens CHAO leads to the conclusion that ORF 5 contains the unusual translation start codon TTG, which is less efficient than either ATG or GTG start codons. It is worth noting that at amino acid position 49 of ORF 5 resides an aspartic acid residue, while a tyrosine residue is present at the equivalent position of gacA. In virG, an Agrobacterium tumefaciens transcriptional activator, an asparagine to aspartic acid substitution near the conserved phosphorylation site converted virG to a constitutive transcription activator which presumably no longer required phosphorylation by a sensor component. It is 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 5 likely resides within the 2 kb Xhoi fragment. It is possible to map the location of this promoter by, for example, a combination of SI nuclease mapping (Aiba et al., J. Biol. Chem. 256:11905-11910 (1981)) and primer extension mapping (Debarbouille and Raibaud, J. Bacteriol. 153:1221-1227 (1983)) as was done for a different Pseudomonas promoter by, for 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 5-class 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 number of sources such as the plasmid expression vectors pKK233-2, pDR540, pDR720, pYEJOOl, 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 comprising mixtures of parts of regulatory elements from different sources. For example, the trp/lac (trc) promoter of pKK232-2 (Pharmacia) which combines the -35 region of the E. coli tryptophan operon promoter with the -10 region of the E. coli lac operon promoter functions effectively 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-host-range plasmid RSF1010 are known to function in at least the following bacterial genera: Acetobacter, Actinobacillus, Aerobacter, Aeromonas, Agrobacterium, Alcaligenes, Azotobacter, Azospirillum, Caulobacter, Desulfovibrio, Erwinia, Escherichia, Gluconobacter, Hyphomicrobium, Klebsiella, 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.

Biocontrol efficacies of the pCIB137-containing transconjugants of strains 914 and 922 were compared to their natural parents. Bacterial cultures were grown overnight in Luria broth at 28C. Cells were pelleted by centrifugation, then resuspended in sterile water to an optical density of 2.5

9 at 600nm (approximately 2x10 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 15cm diameter pots. A 2cm deep circular furrow with a total length of 30cm was 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 lOOmg/pot, followed by the application of 20ml of bacterial suspension for each pot. Water was added in place of bacterial suspension in the unbacterized control. Each treatment consisted of four replicate pots 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 indicate that strains 914 and 922 provide no disease control, whereas their pCIB137-containing transconjugants provided good control of R. solani in cotton.

Treatment NP.NTb P.NTc 914 914(pCIB149) 0 1 0 2 0.75 -3.0

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 b = No Pathogen, No Treatment (Uninfested control) c = Pathogen, No Treatment (Infested control)

Example 13: Multiple copies of a gafA gene isolated from the Pseudomonas fluorescens strain 914 activate expression of latent genes in strain 914.

The 2.0 kb Xhoi fragment containing the gafA gene of P. fluorescens strain 915 was labelled and hybridized with Xhol- digested total genomic DNA from P. fluorescens strain 914. A 2.0 kb Xhoi fragment from strain 914 which hybridized to the probe is cloned in pBluescript SK+ and DNA sequencing is performed to verify that the clone contains a gafA homologue. The DNA sequence of the gafA homologue in strain 914 is presented in Table 5. The strain 914 gafA homologue differs from the strain 915 gafA at nine nucleotide positions, but only one of these nucleotide differences is predicted to generate an amino acid change difference in the two proteins (amino acid residue 182 is threonine in the strain 915 GafA protein and isoleucine in the 914 GafA protein) . The strain 914 gafA gene was subcloned into the broad-host-range plasmid pVKlOO and the resulting recombinant plasmid was introduced by conjugation into strain 914. Whereas expression of the single chromosomal gafA gene in strain 914 was not capable of activating expression of genes required for the synthesis of pyrrolnitrin, chitinase, and cyanide, strain 914 derivatives containing multiple plasmid copies of the 914 gafA gene did synthesize pyrrolnitrin, chitinase, and cyanide.

Table 5 gafA open reading frame from pCIB 3341

1 TTGATTAGGG TGCTAGTGGT CGATGACCAT GATCTCGTTC GTACAGGTAT

51 TACCCGAATG CTGGCTGACA TCGATGGCCT GCAAGTGGTC GGTCAGGCCG

101 AGTCAGGGGA GGAGTCCCTG CTCAAGGCCC GGGAGTTGAA ACCCGATGTG

151 GTCCTCATGG ACGTCAAGAT GCCCGGGATC GGCGGTCTTG AAGCCACGCG

201 CAAATTGTTG CGCAGTCACC CGGATATCAA AGTCGTGGCC GTCACCGTGT

251 GTGAAGAAGA CCCGTTCCCG ACCCGCTTGC TGCAAGCCGG TGCGGCGGGT

301 TACCTGACCA AAGGTGCGGG CCTCAATGAA ATGGTGCAGG CCATTCGCCT

351 GGTGTTTGCC GGCCAGCGTT ACATCAGCCC GCAAATTGCC CAGCAGTTGG

401 TGTTCAAGTC ATTCCAGCCT TCCAGTGATT CACCGTTCGA TGCTTTGTCC

451 GAGCGGGAAA TCCAGATCGC GCTGATGATT GTCGGCTGCC AGAAAGTGCA

501 GATCATCTCC GACAAGCTGT GCCTGTCTCC GAAAACCGTT AATATCTACC

551 GTTACCGCAT CTTCGAAAAG CTCTCGATCA GCAGCGATGT TGAACTGACA

601 TTGCTGGCGG TTCGCCACGG CATGGTCGAT GCCAGTGCCT GA

EXAMPLE 14: Activation of latent gene expression with the E. coli uyr-23 gene.

The E. coli uyr-23 gene (also designated uyrY) is likely a member of a class of bacterial transcriptional activators, although no known function has yet been assigned to it in E. coli. A DNA fragment containing the uyr-23 gene was obtained by amplifying by the polymerase chain reaction (Mullis and 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 uyr-23 (Sharma et al., Nucleic Acids Res. 14:2301-2318 (1986) ) . Two PCR primer oligonucleotides,

5'-GGCGGAGTATACCATAAG-3' and 5'-ATAAGCTTACCACCAGCATCGTAC-3' were used in the amplification of the ca. 1.1 kb DNA fragment at a concentration of 1 uM each in a 50 ul reaction mix containing, in reaction buffer supplied by the PCR kit manufacturer (Perkin Elmer Cetus) , the four 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 45 C for 1 min, followed by 72 C for 1 min (30 cycles total) . Amplified DNA fragments were digested with the restriction endonuclease Hindlll, ligated with HindiII-digested pLAFR3, a broad-host-range plasmid capable of replication in Pseudomonas (Staskawicz et al., J. Bacteriol. 169:5789-5794 (1987)), and used to transform E. coli. When a pLAFR3 derivative containing the E. coli uyr-23 gene was mobilized by conjugation into Pseudomonas fluorescens strain 914, the uyr-23 gene activated expression of genes involved in the production of cyanide, chitinase, and pyrrolnitrin.

Optimal activation in £. fluorescens strain 914 apparently depends upon expression of uyr-23 from the lac promoter of pLAFR3.

Example 15: Further delimitation of the 2.0 kb Xhol fragment to identify the smallest intact subunit that is functional.

The fact that an E_. coli ORF 5-like transcription activator could activate latent genes in P_. fluorescens strain

914 (Example 12) , coupled with the fact that disruption of ORF

5 in the 2.0 kb Xhol fragment abolishes the gene-activating ability of this fragment, indicates that it should be possible to define a smaller DNA fragment than the 2 kb fragment with gene-activating ability, provided ORF 5 is intact and expressed. 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 uyr-23 gene from E. coli in Example

5. Pairs of oligonucleotide primers are prepared for use in polymerase chain reaction amplification reactions. A common primer annealing to the template downstream of ORF 5 is present in each primer pair, while the remaining primer of each pair anneals to a sequence at a different distance upstream of ORF

5. DNA fragments from amplification reactions are cloned in a broad-host-range plasmid such as pLAFR3 for introduction into

£. 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.

Example 16: Formulations of antifungal compositions employing liquid compositions 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 composition are given by weight:

1. Emulsifiable concentrates: a_ b £

Active ingredient 20 % 40 % 50 %

Calcium dodecylbenzenesulfonate 5 % 8 % 6 %

Castor oil polyethlene glycol 5 % ether (36 moles of ethylene oxide)

Tributylphenol polyethylene glycol - 12 % 4 % ether (30 moles of ethylene oxide)

Cyclohexanone - 15 % 20 %

Xylene mixture 70 % 25 % 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 - 70 % -

N-methyl-2-pyrrolidone - 20 % -

Epoxidised coconut oil - - 1 % 5 %

Petroleum distillate - 94 %

(boiling range 160-190 )

These solutions are suitable for application in the form of microdrops.

3. Granulates: a b

Active ingredient 5 % 10 %

Kaolin 94 %

Highly dispersed silicic acid 1 %

Attapulgite - 90 %

The active ingredient is dissolved in methylene chloride, the solution is sprayed onto the carrier, and the solvent is subsequently evaporated off In vacuo.

4. Dusts: a b Active ingredient 2 % 5 % Highly dispersed silicic acid 1 % 5 % Talcum 97 % Kaolin - 90 %

Ready-to-use dusts are obtained by intimately mixing the carriers with the active ingredient.

Example 17: Formulation of antifungal compositions employing solid compositions 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 compositions are by weight.

1. ettable powders:

Active ingredient

Sodium lignosulfonate

Sodium lauryl sulfate

Sodium diisobutylnaphthalene- sulfonate Octylphenol polyethylene glycol ether - 2 % (7-8 moles of ethylene oxide)

Highly dispersed silicic acid 5 % 27 % 10 %

Kaolin 67 %

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 10 %

Octylphenol polyethylene glycol ether 3 % (4-5 moles of ethylene oxide)

Calcium dodecylbenzenesulfonate 3 %

Castor oil polyglycol ether 4 %

(36 moles of ethylene oxide)

Cyclohexanone 30 %

Xylene mixture 50 %

Emulsions 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.

5. 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 %

Nonylphenol polyethylene glycol 6 % (15 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 the two mutants of the first class. These clones define a second genetic region required for gene activation in strain 915. The smallest clone in the family, pCIB146, is analysed further.

Example 20: Functional analysis of the second genetic region.

The clone in pCIB146 was flanked by EcoRI sites (Fig. 4) . There is one internal EcoRI site in the clone. The two EcoRI subclones were obtained and tested for restoring ability. Neither one was able to restore mutant CGP 21, one of the five class II mutants, to wildtype phenotype, indicating that the internal EcoRI site defines a site critical to the functioning of the second genetic locus. There are two internal BamHI fragments in the clone. When these internal fragments were removed (pCIB191), the restoring ability was not affected. Finally, a 6kb subclone containing only the region from the internal HindiII 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 syrinqae 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%

2528-2843 72%

Example 22: Introduction of the lemA gene into Pseudomonas strains for the restoration of biocontrol funtions.

Clone pCIB146 is introduced into mutant Pseudomonas strains which lack biocontrol functions due to an absence of the lemA gene. pCIB146 is introduced into Pseudomonas strains from E. coli by conjugation and is found to restore biocontrol functions, including chitinase, gelatinase, pyrrolnitrin and cyanide production.

Clone pCIB146 is also introduced into Pseudomonas strains which have no apparent defect in either the native lemA or gafA genes. An enhancement of biocontrol function, including production of chitinase, gelatinase, pyrrolnitrin and cyanide is found in the transformed strains by virtue of the increased lemA production in these strains overcoming a limitation in the capacity to phosphorylate the gafA protein.

The lemA gene is also introduced into Pseudomonas strains into which the gafA gene has already been introduced by the procedure described in 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.

Example 23: Modification of the lemA gene to increase its kinase activity on 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 9) , and the Pseudomonas strains thus modified are assessed against non- modified strains for chitinase, gelatinase, pyrrolnitrin, and cyanide production. Constructions which have a modified amino acid environment adjacent to the target histidine which render the histidine a better target for autophosphorylation also phosphorylate gafA more efficiently and thus produce elevated levels of chitinase, gelatinase, pyrrolnitrin and cyanide.

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 9) , and the Pseudomonas strains thus modified are assessed against non-modified strains for chitinase, gelatinase, pyrrolnitrin, and cyanide production. Constructions which have a modified sensor domain are able to autophosphorylate the target histidine without the necessary interaction of the signal and may therefore phosphorylate gafA more efficiently and thus produce elevated levels of chitinase, gelatinase, pyrrolnitrin and cyanide.

Example 24: Modification of the gafA gene to increase the efficiency of phosphorylation of the protein.

The amino acid environment flanking the presumed receiver domain of the gafA protein (around residue 54) is modified using PCR and cloning techniques. A series of modified constructions thus prepared are introduced into Pseudomonas strains using gene replacement techniques (see 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: Pseudomonas 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 Pseudomonas strain by remodifying 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 phosphorylation-independent.

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 phosphorylation-independent. The use of a phosphorylation- independent version of gafA in the activation of latent genes in bacterial strains removes the requirement that a strain contain an active version of LemA or an equivalent kinase.

The amino acid environment within the N-terminal half of gafA is modified using PCR and cloning techniques well known in the art. Such mutagenized versions of the gafA gene are cloned into broad-host-range plasmids and introduced into a lemA- mutant derivative of strain 915. Since introduction of the unaltered version of gafA into the lemA- mutant fails to restore synthesis of chitinase, pyrrolnitrin, cyanide, and gelatinase (see 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 into a constitutively active conformation (i.e. phosphorylation-independent).

* * * * * * * * * * * *

While the present invention has been described with reference to specific embodiments 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.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Gaffney, Thomas D. Lam, Stephen T. Ligon, James M. Hill, Dwight Steven Stein, Jeffrey I. Howell, Charles R. Becker, J. Ole

(ii) TITLE OF INVENTION: Gene Activating Element

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CIBA-GEIGY Corporation

(B) STREET: 7 Skyline Drive

(C) CITY: Hawthorne

(D) STATE: New York

(E) COUNTRY: USA

(F) ZIP: 10532

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE: 02-JUL-1992

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/570,184

(B) FILING DATE: 20-AUG-1990

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Lazar, Steven R.

(B) REGISTRATION NUMBER: 32,618

(C) REFERENCE/DOCKET NUMBER: CGC 1506/CIP

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (919)541-8615

(B) TELEFAX: (919)541-8689

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 642 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pseudomonas fluorescens

(B) STRAIN: CGA267356

(C) INDIVIDUAL ISOLATE: ORF 5

(vii) IMMEDIATE SOURCE:

(B) CLONE: pCIB137

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..642

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TTG ATT AGG GTG CTA GTA GTC GAT GAC CAT GAT CTC GTT CGT ACA GGT 4 Leu lie Arg Val Leu Val Val Asp Asp His Asp Leu Val Arg Thr Gly 1 5 10 15

ATT ACA CGA ATG CTG GCT GAC ATC GAT GGC CTG CAA GTG GTC GGC CAG 9 lie Thr Arg Met Leu Ala Asp He Asp Gly Leu Gin Val Val Gly Gin 20 25 30

GCC GAG TCA GGG GAG GAA TCC CTG CTC AAG GCC CGG GAG TTG AAA CCC 14 Ala Glu Ser Gly Glu Glu Ser Leu Leu Lys Ala Arg Glu Leu Lys Pro 35 40 45

GAT GTG GTC CTC ATG GAC GTC AAG ATG CCC GGG ATC GGC GGT CTT GAA 19 Asp Val Val Leu Met Asp Val Lys Met Pro Gly He Gly Gly Leu Glu 50 55 60

GCC ACG CGC AAA TTG TTG CGC AGT CAC CCG GAT ATC AAA GTC GTG GCC 24 Ala Thr Arg Lys Leu Leu Arg Ser His Pro Asp He Lys Val Val Ala 65 70 75 80

GTC ACC GTG TGT GAA GAA GAT CCG TTC CCG ACC CGC TTG CTG CAA GCC 28 Val Thr Val Cys Glu Glu Asp Pro Phe Pro Thr Arg Leu Leu Gin Ala

85 90 95

GGC GCG GCG GGT TAC CTG ACC AAG GGG GCG GGC CTC AAT GAA ATG GTG 33 Gly Ala Ala Gly Tyr Leu Thr Lys Gly Ala Gly Leu Asn Glu Met Val 100 105 110

CAG GCC ATT CGC CTG GTG TTT GCC GGC CAG CGT TAC ATC AGC CCG CAA 38 Gin Ala He Arg Leu Val Phe Ala Gly Gin Arg Tyr He Ser Pro Gin 115 120 125 ATT GCC CAG CAG TTG GTG TTC AAG TCA TTC CAG CCT TCC AGT GAT TCA 4 He Ala Gin Gin Leu Val Phe Lys Ser Phe Gin Pro Ser Ser Asp Ser 130 135 140

CCG TTC GAT GCT TTG TCC GAG CGG GAA ATC CAG ATC GCG CTG ATG ATT 4 Pro Phe Asp Ala Leu Ser Glu Arg Glu He Gin He Ala Leu Met He 145 150 155 160

GTC GGC TGC CAG AAA GTG CAG ATC ATC TCC GAC AAG CTG TGC CTG TCT 5 Val Gly Cys Gin Lys Val Gin He He Ser Asp Lys Leu Cys Leu Ser

165 170 175

CCG AAA ACC GTT AAT ACC TAC CGT TAC CGC ATC TTC GAA AAG CTC TCG 5 Pro Lys Thr Val Asn Thr Tyr Arg Tyr Arg He Phe Glu Lys Leu Ser 180 185 190

ATC AGC AGC GAT GTT GAA CTG ACA TTG CTG GCG GTT CGC CAC GGC ATG 6 He Ser Ser Asp Val Glu Leu Thr Leu Leu Ala Val Arg His Gly Met 195 200 205

GTC GAT GCC AGT GCC TG 6

Val Asp Ala Ser Ala 210

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 213 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Leu He Arg Val Leu Val Val Asp Asp His Asp Leu Val Arg Thr Gly 1 5 10 15

He Thr Arg Met Leu Ala Asp He Asp Gly Leu Gin Val Val Gly Gin 20 25 30

Ala Glu Ser Gly Glu Glu Ser Leu Leu Lys Ala Arg Glu Leu Lys Pro 35 40 45

Asp Val Val Leu Met Asp Val Lys Met Pro Gly He Gly Gly Leu Glu 50 55 60

Ala Thr Arg Lys Leu Leu Arg Ser His Pro Asp He Lys Val Val Ala 65 70 75 80

Val Thr Val Cys Glu Glu Asp Pro Phe Pro Thr Arg Leu Leu Gin Ala

85 90 95 Gly Ala Ala Gly Tyr Leu Thr Lys Gly Ala Gly Leu Asn Glu Met Val 100 105 110

Gin Ala He Arg Leu Val Phe Ala Gly Gin Arg Tyr He Ser Pro Gin 115 120 125

He Ala Gin Gin Leu Val Phe Lys Ser Phe Gin Pro Ser Ser Asp Ser 130 135 140

Pro Phe Asp Ala Leu Ser Glu Arg Glu He Gin He Ala Leu Met He 145 150 155 160

Val Gly Cys Gin Lys Val Gin He He Ser Asp Lys Leu Cys Leu Ser

165 170 175

Pro Lys Thr Val Asn Thr Tyr Arg Tyr Arg He Phe Glu Lys Leu Ser 180 185 190

He Ser Ser Asp Val Glu Leu Thr Leu Leu Ala Val Arg His Gly Met 195 200 205

Val Asp Ala Ser Ala 210

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5559 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pseudomonas fluorescens

(B) STRAIN: CGA267356

(C) INDIVIDUAL ISOLATE : 5 . 6 kb EcoRI-HindlH restriction fragment

(vii) IMMEDIATE SOURCE:

(B) CLONE: pCIB137

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 210..1688

(D) OTHER INFORMATION: /note= "ORF 1, transcribed left to right"

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 1906..3633

(D) OTHER INFORMATION: /note= "ORF 2, transcribed left to right"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 4616..4691

(D) OTHER INFORMATION : /note= "glyW, transcribed right to left"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 4731..5318

(D) OTHER INFORMATION : /note= "ORF 3 , transcribed right to left"

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 3 :

GAATTCGATG ACATGCCGCG CGCCGGCATC GACACGCAAA TGGTCGACCT GGTGCTGCCG

GTGGTCGAAA TGCCGCAGAA GCTGCTGGAG CTGTGGCGCA ACTCTCAGCT CATCACCCTG

CCGACCGCCA ACGATCCGCA AATCAAGGTC TCGGCGCCGG TGTCCAAACG CGATGCCGCG

GCGGCGAACA GCAGCTGCAA GACATCCTGA TGCTGTTGCG CACCGGCACC GGCCATGACT

TCAAGCATTA CAAGCGCGCC ACGGTGCTGC GGCGGATCGA GCGCCCGCTG CAGGTCACCG

CCCAGCCGGA CCTCGCCGCC TATCACGATT ACCTGCAGAT GCACCCTGAA GAAACCAAGG

CGCTGCTGGG CGACATGCTG ATCGGCGTGA CCAATTTCTT TCGCGACCGC GAGGCCTTCG

AAGCCCTGGA GCGCAATGTC ATTCCTGCCC TGGTGAAGTC CTTGCAGGAC AGCCAACCGC

ACCGTGAAGA CGTGCGCATC TGGTCCGCCG GCTGCTCCAC GGGTGAAGAG GCCTATAGCC

TGGCAATCGT CGCCAGCGAG CAGATGGCCC TGGAGGCCTG CAACGCCAAG CTGCAGGTAT

TCGCGACCGA TATCGACGAT CGTGCCATCG CCCAGGGACG CAAGGGGGTC TATCCCGAAG

CGATCGTTAC CGATGTGCCT CCGCAGCGCA TGCGCCAGTA CTTTTCCCGG GAAAACCAGC

ATTACCGGGT GCGCAAGGAG ATTCGCGAAA AGGTGCTGTT CGCCAAGCAC AGCCTGCTGG

CGGATCCGCC ATTTTCGCAG ATCGACTTGA TCGTCTGCCG TAACCTGCTG ATCTACCTGG

ACCGCGACGT GCAACGGGAG ATCCTGCAGA TGTTCCACTT CGCCCTGCGT CCTGGAGGCT

ACCTGTTCCT CGGTTCCTCC GAATCCGCGG ACGGCTGCCA GGATCTGTTC GTGCCGGTCG

ACAAGCGCAA CCGCATTTTC CGGGTACGGC CCAACTCGGC CACGGTTCGC CGCGCGCCCA 1

CCATGCCGCG ACGGCGTACA TGCGCACCAT CGGCAGCCCC CACCCCGTGG AAACCAAGTG 1

TCTCGCGCAA AACCTCGTTC GCCGACATCC ACCTTCGCGC CCTGGAAAAG TGCGCGCCGC 1 CGAGCATGAT CGTCGATGCC AACGCCGACA TCCTGCACAT GAGCGAAGGC GCCGGCCGGT

TCCTGCGCTA TGTCGCGGGG GAAATCACCC GCAACCTGCT GACCCTGATC CAGCCCGAGC

TGCGCCTTGA ACTGCGCACC ACGCTGTTCC AGGTGCAACA GTCCGGTGTT GCGGTGACCG

CCGCCGGGTG CGCATCGAGC GGGAAAAGAA GCCTTGTTTC ATCGACCTCA CAGCCCGCCC

CTTCAAGGAC GAGGAAACCG ACAACGAATA TGTGCTGGTG GTGTTCGAGG AGACCGAGGC

CGACCCACGG GAGCTGCGCG AGACCAGCGC CAGCCAGACG GAAAACCAGA TGCTGGCCAA

CCTCGAGCGG GAGTTGCAGC GGACCAAATT GCACCTGCAG GACACCATCG AGCAATCGGA

AGTCTCCAGC GAGGAGCTCA AGGCGTCGAA CGAAGAAATG CAGGCGCTCA ATGAAGAGCT 1

GCGCTCGGCC ACCGAAGAGC TGGAAACCAG CAAGGAAGAG TTGCAGTCGA TCAATGAAGA 1

GCTGCTGACG GTCAATTACG AGCTGAAAAC CAAGGTCGAG GAAACCGACA AGATCAACGA 1

CTACCTGACC AACCTGATCG CCTCCACCGA CATCGCCACG GTGTTCGTCG ACCGCAACAT 1

GCGCATCCGC TGGTTCACCC CGCGCGCCAC CGACATTTTC AGCATGCTGC CGGTGGACAC 1

CGACGCTCAT TACTGGACAT CACCCACCGC CTGAACTACC CGGAAATGGC CGAGGACGCC 1

GCGACCGTGT TCGAGTCGTT GAGCATGATC GAGCGTGAAG TCAACAGCGA CGATCAGCGC 1

TGGTACATCG CACGCCTGTT GCCCTATCGC TCCAGCGAAG ACCATATCGA CGGCACCGTG 2

CTGACCTTCA TCGATATCAC CAAGCGCCGG CTGGCCGAGG AGGAACTGCG CCTGGGCGAA 2

GAACGCATGC GCCTGGTCGC CGAAAGCACC CATGATTTCG CCATCATCAT CCTCGACAAC 2

CAGGGCCTCA TCACCGACTG GAACACCGGG GCGCAACTGA TCTTCGGCTA TACCAAGGAC 2

GAAGTGCTGG GCGCCTATTA CGACCTGATT TTCGCGCCTG AGGACCGCGC CGGCGGCGTG 2

CCGGAAAGCG AGCTGCTCAC CGCCCGCGAA CACGGCCGCA GCGACGATGA ACGCTGGCAT 2

ATACGCAAGG ACGGCGAGCG CTTTTTCTGC AGCGGCGAAG TCACGCGGCT CAAGGGTGAC 2

AGCCTGCAAG GCTACGTGAA AATAGCCCGC GACCTGACGG GCCACAAACG CATGCAGGAC 2

GAGCAGAACC AGAAGCTGAT GGAGACCCAG ACCCACAGCC ACCTCAAGGA TGAGTTTTTC 2

GCGGTGATGT CCCATGAACT CAAGCATCCG CTCAACCTGA TCCAGCTCAA CGCCGAGTTG 2

CTGCGTCGCC TGCCGACGAC CAAGGCGGCC GCCCCTGCCC TCAAGGCGGT CAATACCATT 2

TGCGAGGCTG TCTCCAGCCA GGCGCGGATC ATCGACGACC TGCTGGATGT GCGGCGTTTG 2

CGCACCGGCA AGCTCAAGCT GAAGAAACAG CCGGTGGATC TTGGCCGGAT CCTGCAGGAC 2

ATCCATACCG TGGTGCTCAG CGAAGGGCAT CGCTGCCAGG TGACGCTGCA AGTGCCGTTG 2 CCACCGCAAC CGCCGTTAAT GATCGATGCC GATGCGACGC GGCTGGAGCA GGTGATCTGG 2

AACCTGGTGA ACAACGCCCT GAAATTCACC CCGGCCAATG GCTTGGTCCA GTTGATCGCC 2

CAGCGGGTCG AGGATAAGGC GCACGTGGAT GTCATCGACA GCGGCGTGGG CCTGGCCGAG 3

GAAGACCAGA ACAAGGTGTT CGACCTTTTC GGCCAGGCGG CCAACCAGCA CGGCACTCAT 3

CAACGCGACG GGCTGGGCAT CGGCCTGTCA CTGGTGCGCC AGCTGGTGGA AGCCCACGGC 3

GGCTCGGTCA GCGTGCAGTC GAAGGGGCTG GGCCAGGGAT GCACCTTTAC CGTGCTCTTG 3

CCCCTGAGCC ACCCCAACGA CAGCGCTCCC AAACAGCCCG CGTCGCGGGG TGTCGAACGC 3

CTTGCCGGCA TCAAGGTGCT GCTGGTGGAC GACTCGCGGG AAGTCATGGA AGTCCTGCAA 3

CTGCTGCTGG AGATGGAGGG CGCGCAAGTC GAGGCCTTCC ACGACCCGCT GCAGGCCTTG 3

GGCAATGCCA GGAACAACAG TTACGACCTG ATCATTTCAG ACATCGGCAT GCCGATTATG 3

AACGGCTACG AACTGATGCA GAACCTGCGC CAGATCGCTC ACCTGCACCA TACGCCAGCG 3

ATTGCGCTGA CCGGTTACGG CGCCAGCAGC GACCAGAAGA AGTCCCAGCA TGCGGGATTC 3

GATCGGCATG TGAGCAAACC CGTGGCTCAG GACCCGCTGA TCGACCTGAT CAGGGAGCTG 3

TGCAGCCAGG GCTTGCGCTC GGCTGAGCAC TGATGGTCTA GACCCGGCGA ACCCACCTCG 3

TCGGCCTTGA GCGCGGCGAG CGCCATTGCC TGCTGGGCAG CTATTCACGC TTGCGGATCG 3

TCGCGCCTGC GGGCCACCGC CTCTTTGATG GCTTGCTCAT AGGCGGCGTT GGCCTGGTCC 3

TTGAGCTTGA GCCAATCGTC CCAATCGATC ACGCCGTTGC GCAGCAACTC CTCGGCCGCG 3

CTTAACAGCG CCTGATGCCA GGCGTCCGGC GAGCCGGAAC GGTAGTCACG GTCTTCCAGC 3

AGGCCTTGCC AGGCGTCCAG TTCCGGTGTC TTGCGTTCAT TGACCATGGC AGCCACGGCC 3

TTTGTTCATT GCCGATAAAT CGGCGAGTGG GTGGTGGGTT TCTCGGATAT GCGCCCTGTC 4

CTGCTCGAGA ACGGCCAGGC CGGGACATTG CTCAACGGTC AGCGACCGGA TGGAGCTCGA 4

GCGGCATGCC ATCGACCAGC GTCAAGGTCA GGTTCTCGAT GGTGCCGGCG ATCCGGTCCT 4

TGAATACCGG TTCGCCGTCC GGATCCAACT CATCGTAGAA AAAGCGCGTG CCTTCGAGCC 4

AGCCAATGGT CGTTTGCAGG TCCGGCCCCA GGTAATACTT GCCGTCAAGG AAAAACCCGG 4

TAAAGGGCTC CACCCGCTCG CGATTCTCAA TGACATAACG TATTCCAGCG TGCATACCTG 4

TCGATTTATC GAGCATGGCG TCGATCTCCC AGCAGATGAA TCCGGTAGAC CGCGTGGCTT 4

TTTCACTGTT CCTTTTGATT GCCCGCCCGA CGCTGGCGAG CCTTGCTCGC GCGTCCTGGC 4

CGCATTGCGC GGCGAATGGG CGACGTCGAA TCCGATCTGC AAGTGCCCAG CTAGCGGCCC 4 GGCCACGGCA ATACGGGCTT CAGGTACGGC TTAGAAAGAA GAATGACGAT TGGCTCGACA

TATTTTTTGG CGCAAAAAAA AATGGACCTC TTTTCAGAGG TCCATTTTTA ATATTTGGAG

CGGGAAACGA GACTCGAACT CGCGACCCCG ACCTTGGCAA GGTCGTGCTC TACCAACTGA

GCTATTCCCG CGTCTTGGTG GTGTGCATTT TATAGAAATT CGAAACTGCG TCAACCCCTT

GATTCAAAAA GTTTTATTTC TTTTCTACCA TCGGTCTTCA GGTGCGGCCA GGCAGCGCGC

AGGTACTGCA ACATCGACCA CAGGGTCAGC CCTCCGGCGA TCAGCAGGAA GGCATAACCC

AGCAGCACCC AGAAGGTGAA GGCCGGCGGA TTGGCCAGCA GGATCACCAG CGCCAGCATC

TGCGCGGCAG TTTTCCGATT TGCCCATGTT GGACACCGGC CACCTGGGCG CGTGCGNCCG

AGCTCGGCCA TCCACTCGCG AAGGGCGGAC ACCACGATTT CACGCCCGAT GATCACCGCT

GCCGGCAGGG TCAGCCACAG GTTGCCGTGC TCTTGCACCA GCAGCACCAG GGCCACCGCC

ACCATCAACT TGTCGGCCAC CGGATCGAGG AAGGCCCCGA ACGGCGTGCT CTGCTCCAGA

CGCCGCGCCA GGTAGCCATC AAGCCAGTCG GTGGCCGCGG CGAACGCAAA GACGGAACTG

GCGGCCATGT AGCTCCAGTT GTAAGGCAGG TAAAACAGCA AAATGAAGAT CGGGATGAGC

AGAACGCGTA GAACGGTGAT CAGATTAGGG ATATTCATCG GCACAACTGG CTACGAGGTG

AGTGGCAATC TACTCGGAAA AGACAGCAGA TGAGGTAGCA CGGCCATTCT ACGGGCTTCT

GCCACAGCGT GTCTAACACT GTTCCAAGAC TTCGGGCCGC TCGAAAGAGC AACTTCAGAA

GGTCTACACG CGCAAAATAA GACATTCAGT TCTTCTGTAA GTACCGTGTA GATCGGGATC

TATCAGCGGT GCCCCGCCAA AAAGGAAGCC TTGAAGCTT (2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 983 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(ii) MOLECULE TYPE : DNA (genomic) (iii) HYPOTHETICAL : NO (iv) ANTI-SENSE : NO

(vi) ORIGINAL SOURCE :

(A) ORGANISM : Pseudomonas fluorescens

(B) STRAIN : CGA267356

(vii) IMMEDIATE SOURCE : (B) CLONE : pCIB137

(ix) FEATURE :

(A) NAME/KEY : misc_feature

(B) LOCATION : 23 . .51

(D) OTHER INFORMATION: /note= "sequence with promoter homology"

(ix) FEATURE :

(A) NAME/KEY : misc_feature

(B) LOCATION: 99 . .740

(D) OTHER INFORMATION: /note= "ORF 5 structural gene"

(ix) FEATURE :

(A) NAME/KEY : misc_feature

(B) LOCATION : 743 . . 983

(D) OTHER INFORMATION: /note= "5' end of ORF 4 (uvrC) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CCAAGTGCTT TTTATATGGT GTTTGTCATT AGGTCAGCAC GCTGCTTTTT TGCTAAGGTG

TCCGGCAACC TATAAGACCC AAATCGCGAG GTGTCTGCTT GATTAGGGTG CTAGTAGTCG

ATGACCATGA TCTCGTTCGT ACAGGTATTA CACGAATGCT GGCTGACATC GATGGCCTGC

AAGTGGTCGG CCAGGCCGAG TCAGGGGAGG AATCCCTGCT CAAGGCCCGG GAGTTGAAAC

CCGATGTGGT CCTCATGGAC GTCAAGATGC CCGGGATCGG CGGTCTTGAA GCCACGCGCA

AATTGTTGCG CAGTCACCCG GATATCAAAG TCGTGGCCGT CACCGTGTGT GAAGAAGATC

CGTTCCCGAC CCGCTTGCTG CAAGCCGGCG CGGCGGGTTA CCTGACCAAG GGGGCGGGCC

TCAATGAAAT GGTGCAGGCC ATTCGCCTGG TGTTTGCCGG CCAGCGTTAC ATCAGCCCGC

AAATTGCCCA GCAGTTGGTG TTCAAGTCAT TCCAGCCTTC CAGTGATTCA CCGTTCGATG

CTTTGTCCGA GCGGGAAATC CAGATCGCGC TGATGATTGT CGGCTGCCAG AAAGTGCAGA

TCATCTCCGA CAAGCTGTGC CTGTCTCCGA AAACCGTTAA TACCTACCGT TACCGCATCT

TCGAAAAGCT CTCGATCAGC AGCGATGTTG AACTGACATT GCTGGCGGTT CGCCACGGCA

TGGTCGATGC CAGTGCCTGA CAATGACCGA CCCGTTTGAT CCCAGTGCTT TTCTTTCCAC

CTGCAGTGGC CGTCCTGGCG TGTATCGCAT GTTCGACAGC GATACGCGTC TGCTGTACGT

CGGTAAAGCC AAGAACCTGA AGAGCCGCCT GGCCAGCTAC TTTCGCAAGA CCGGCCTGGC

GCCCAAGACC GCTGCCCTGG TGGGGCGCAT CGCAGATCGA AACCACCATC ACCGCCAACG

AGACCGAAGC CCTGCTGCTC GAG

Claims (35)

What is claimed is: CLAIMS:

1. A gene activating sequence capable of inducing the expression of at least one gene, wherein said gene is latent or expressed at low levels in a target organism.

2. The gene activating sequence of claim 1, wherein said target organism is a strain other than a strain from which said gene activating sequence was derived.

3. The gene activating sequence of claim 1, wherein said sequence is a gafA sequence.

4. The gene activating sequence of claim 1, wherein said sequence is a lemA sequence.

5. The gene activating sequence of claim 3, wherein said gafA sequence consists essentially of the 2 kb fragment deposited as pCIB137.

6. An isolated DNA sequence derived from the DNA sequence of claim 5.

7. The gene activating sequence of claim 4, wherein said lemA sequence consists essentially of the 6 kb fragment deposited as pCIB168.

8. An isolated DNA sequence derived from the DNA sequence of claim 7.

9. The DNA sequence of claim 8, wherein said DNA sequence encodes lemA.

10. A method of activating a gene which may be latent or natively expressed at low levels in a host strain, said method comprising introducing at least one gene activating sequence of claim 1 into said host strain.

11. The method of claim 10, wherein said host strain is a strain other than a strain from which said gene activating sequence was derived.

12. The method of claim 10, wherein said gene activating sequence is a lemA or gafA sequence.

13. The method of claim 12, wherein said gafA sequence consists essentially of the 2 kb fragment deposited as pCIB137 or a fragment thereof.

14. The method of claim 12, wherein said lemA sequence consists essentially of the 6 kb fragment deposited as PCIB168.

15. The method of claim 10, wherein the host strain is a bacterial strain.

16. The method of claim 15, wherein the host strain is of the genus Pseudomonas.

17. An isolated DNA sequence consisting essentially of the gafA sequence as shown in SEQUENCE ID NO 1.

18. An isolated DNA sequence derived from the DNA sequence of claim 17.

19. A recombinant DNA sequence comprising a bacterial regulatory element operably linked to the gene activating sequence of claim 1.

20. A recombinant DNA sequence comprising a bacterial regulatory element operably linked to the DNA sequence of claim 3.

21. A recombinant DNA sequence comprising a bacterial regulatory element operably linked to the DNA sequence of claim 4.

22. The recombinant DNA sequence of claim 20, wherein the bacterial regulatory element is a promoter from a gene isolated from a bacterial strain selected from the group consisting of Pseudomonas genus and Bacillus genus and E_^ coli species.

23. The recombinant DNA sequence of claim 22, wherein the bacterial regulatory element is homologous to the host bacterial strain.

24. A transgenic bacterial strain into which the recombinant DNA sequence of claim 20 has been introduced.

25. A transgenic bacterial strain into which the recombinant DNA sequence of claim 22 has been introcduced, wherein the bacterial regulatory element of the recombinant DNA sequence is from a gene which is homologous to the transgenic bacterial strain.

26. A method for activating the expression of at least one gene in a host strain wherein said gene is latent or natively expressed at low levels, said method comprising: introducing into said host strain at least one gene activating sequence.

27. The method of claim 26, wherein the gene activating sequence is a DNA sequence.

28. The method of claim 27, wherein the DNA sequence is operably linked to a bacterial regulatory element.

29. The method of claim 28, wherein said regulatory element is a promoter from a gene isolated form a bacterial strain selected from the group consisting of Pseudomonas genus and Bacillus genus and E^ coli species.

30. The method of claim 26, wherein said gene activating sequence is a gafA sequence.

31. The method of claim 30, wherein the gafA sequence is Sequence ID No. 1.

32. The method of claim 26, wherein said gene activating sequence is a lemA sequence.

33. The method of claim 32, wherein the lemA sequence is the lemA contained in pCIB168.

34. A method of rendering a bacterial strain effective against fungal pathogens, said method comprising introducing into the bacterial strain the DNA sequence of claim 1.

35. A method of rendering a bacterial strain effective against fungal pathogens, said method comprising introducing into the bacterial strain the DNA sequence of claim 19.