EP1163347A2 - Zwittermicin a biosynthetic gene from bacillus cereus - Google Patents

Abstract

Disclosed herein is an isolated DNA construct encoding enzymes necessary for the biological synthesis of zwittermicin A, a unique aminopolyol antibiotic produced by Bacillus cereus that is active against diverse bacteria and lower eucaryotes. Also disclosed herein are hosts transformed to contain and express the DNA construct, and a method to produce zwittermicin A using the construct and transformed hosts.

Description

ZWITTERMICIN A BIOSYNTHETIC GENE FROM BACILLUS CEREUS

Priority is hereby claimed to provisional application Serial No. 60/125,769, filed March 23, 1999, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Plants routinely become infected by disease-causing fungi and bacteria (referred to herein-as "phytopathogens"). Many microbial phytopathogen species have evolved to utilize different modes of infection, such as introduction of air-born phytopathogens through foliar surfaces, introduction from plant-to-plant contact, or introduction by various vectors. Other phytopathogens are soil-borne and preferentially infect roots and newly germinated seedlings. In addition to infection by fungi and bacteria, many plant diseases are caused by soil-borne nematodes that infect roots, typically causing serious damage when the same crop species is cultivated for successive years on the same area of ground.

Plant diseases cause considerable crop loss from year to year, which causes economic hardship to farmers and nutritional deprivation for local populations in many parts of the world. The severity of the destructive process of disease depends on the aggressiveness of the phytopathogen and the response of the host. Various attempts have been made to inhibit or decrease the distribution of phytopathogens. One aim of most plant breeding programs is to increase the resistance of host plants to disease. For example, the development of plants resistant to disease has typically had only a limited period of success in many crop-pathogen systems due to the rapid evolution of phytopathogens to overcome resistance genes. In addition, there are several documented cases of the evolution of fungal strains that are resistant to particular fungicides. Variation in the sensitivity of fungi to fungicides has been documented for powdery mildew (to triadimenol), wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Pseudocercosporella (to MBC-type fungicides), and Mycosphaerella fψensis (to triazoles).

Diseases caused by nematodes have been controlled successfully by pesticide application. However, in contrast to most fungicides which are relatively harmless to mammals, nematicides have a relatively high toxicity to mammals. Most nematicides used to control soil nematodes are of the carbamate, organochlorine, or organophosphorous groups and must be applied to the soil with particular care.

In some crop species, the use of biocontrol organisms has been developed as a further alternative to protect crops. Biocontrol organisms, i.e., an organism that is capable of affecting the growth of a pathogen such that the ability of the pathogen to cause a disease is reduced, have the advantage of being able to colonize and protect parts of the plant inaccessible to conventional fungicides. This practice developed from the recognition that crops grown in some soils are naturally resistant to certain fungal phytopathogens and that the suppressive nature of these soils is lost by autoclaving. Furthermore, it was recognized that soils that are conducive to the development of certain diseases could be rendered suppressive by the addition of small quantities of soil from a suppressive field.

Subsequent research demonstrated that root colonizing bacteria were responsible for this phenomenon, now known as "biological disease control" (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 of the species Pseudomonas fluorescens (Weller et al. Phytopathology 73 : 463-469 (1983); Kloepper et al. Phytopathology 71 : 1020-1024 (1981)). Important plant pathogens that have been effectively controlled by seed inoculation with these bacteria include Gaeumannomyces graminis, the causative agent of take-all in wheat (Cook et al. Soil Biol. Biochem 8: 269-273 (1976)) and the Pythiurn and Rhizoctonia phytopathogens involved in damping-off of cotton (Howell et al. Phytopathology 69: 480-482 (1979)). Several biological disease controlling Pseudomonas strains produce antibiotics that inhibit the growth of fungal phytopathogens (Howell et al. Phytopathology 69: 480-482 (1979); Howell et al. Phytopathology 70: 712-715 (1980)) and these have been implicated in the control of fungal phytopathogens in the rhizosphere. Although biocontrol was initially believed to have considerable promise as a method of widespread application for disease control, it has found application mainly in the environment of greenhouse crops where its utility in controlling soil-borne phytopathogens is best suited for success. Large-scale field application of naturally-occurring microorganisms has not proven possible due to constraints of microorganism production (they are often slow growing), distribution (they are often short lived), and high cost (the result of the slow growth and short lifetimes of these organisms). In addition, the success of biocontrol approaches is also largely limited by the identification of naturally-occurring strains which may have a limited spectrum of efficacy. Some initial approaches have also been taken to control nematode phytopathogens using biocontrol organisms. Although these approaches are still exploratory, some Streptomyces species have been reported to control the root knot nematode (Meloidogyne spp.) (WO 93/118135 to Research Technology Corporation), and toxins from some Bacillus thuringiensis strains (such as B. israeliensis) have been shown to have broad anti-nematode activity. Spore or bacillus preparations may thus provide suitable biocontrol opportunities (EP 0 352 052 to Mycogen, WO 93/19604 to Research Technology Corporation).

SUMMARY OF THE INVENTION Traditional methods of protecting crops against disease, including plant breeding for disease resistance, the continued development of fungicides, and more recently, the identification of biocontrol organisms, have all met with limited success. Thus, there is a continued need for the development of new methods with which to protect crops against disease.

Definitions

"Nucleic acid fragment" as used herein refers to a linear polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A nucleic acid fragment may include both coding and non-coding regions that can be obtained directly from a natural source (e.g., a microorganism), or can be prepared with the aid of recombinant or synthetic techniques.

A "coding region" is a linear form of nucleotides that encodes a polypeptide, usually via mRNN when placed under the control of appropriate regulatory sequences. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A "non-coding region" is a linear form of nucleotides that does not encode a polypeptide.

"Polypeptide" as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.

The term "complement" and "complementary" as used herein, refers to the ability of two single stranded nucleic acid fragments to base pair with each other, where an adenine on one nucleic acid fragment will base pair to a thymine on a second nucleic acid fragment and a cytosine on one nucleic acid fragment will base pair to a gaunine on a second nucleic acid fragment. Two nucleic acid fragments are complementary to each other when a nucleotide sequence in one nucleic acid fragment can base pair with a nucleotide sequence in a second nucleic acid fragment. For instance, 5'-ATGC and 5'-GCAT are complementary, as are 5'- ATGrC and 5'-GCAT. The term complement and complementary also encompasses two nucleic acid fragments where one nucleic acid fragment contains at least one nucleotide that will not base pair to at least one nucleotide present on a second nucleic acid fragment. For instance the third nucleotide of each of the two nucleic acid fragments 5'-ATTGC and 5¹- GrCTAT will not base pair, but these two nucleic acid fragments are complementary as defined herein. Typically two nucleic acid fragments are complementary if they hybridize under the standard conditions referred to herein. Typically two nucleic acid fragments are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity. As used herein, "hybridizes," "hybridizing," and "hybridization" means that a single stranded nucleic acid fragment forms a noncovalent interaction with a second nucleic acid fragment under certain conditions, as described herein.

As used herein, the term "isolated" means that a nucleic acid fragment or polypeptide is either removed from its natural environment or synthetically derived. Preferably, the nucleic acid fragment or polypeptide is purified, i.e., essentially free from any other nucleic acid fragment and associated cellular products or other impurities.

"Host cells" refer to, for example, microorganisms including prokaryotic (e.g., bacteria), and eukaryotic (e.g., fungi) cells, and plant cells that can be used as recipients for introduction of a recombinant vector. An "antibody" is an immunoglobulin produced by the immune system of a vertebrate in response to exposure to an antigen and able to bind to that antigen. Typically, an antigen is a polypeptide. The epitope of an antigen is the portion of the antigen to which an antibody binds. The epitope can be present on other antigens.

"Transgenic" as used herein refers to any cell, cell line, tissue plant part or plant the genotype of which has been altered by the presence of a foreign coding region. Typically, the foreign coding region was introduced into the genotype by a process of genetic engineering, or was introduced into the genotype of a parent cell or plant by such a process and is subsequently transferred to later generations by sexual crosses or asexual propagation. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a map of the B. cereus UW85 genome. Arrows indicate the direction of transcription of the genes. Restriction sites are abbreviated as follows: RI = EcoRI, Sp = Sphl, Ba = Barri , Bl = Blpl, Bg = BgHl, RV = EcoRV, and Sa = Sail. FIG. 2 is a map of pΔzmaR showing insertional inactivation of zmaR. Dotted lines indicate deleted DNA. Spr designates that the plasmid confers resistance to spectinomycin. All other abbreviations as in Fig. 1.

FIG. 3 is a map of pΔor 2 showing insertional inactivation of or/ Dotted lines indicate deleted DNA. All abbreviations as in Figs. 1 and 2. FIG. 4 is a gel demonstrating expression of zmaR in B. cereus UW85 and

UW85Δ_zmαi?. Cultures of UW85 and UW85Δ_z/wα^ _were grown in 50% TSB. Time points consisting of 3.0 mL culture were taken at 6, 12, 24, 48, and 72 hours. SDS-PAGΕ gels containing 20 μg total protein per lane were run, and protein was transferred to PVDF membrane, with subsequent western blot analysis using the ΕCL kit. Production of zwittermicin A is first detected after 48 hours. The limit of detection for zwittermicin A is 0.33 μg mL (Milner et al., Appl. Microbiol. Biotechnol. 43 :685-691 (1995)).

BRIEF DESCRD?TION OF THE SEQUENCE LISTING

SEQ. ID. NO: 1 is the nucleotide sequence of a 3.9-kb DNA region containing zmaR, orfl, orf2, and orf3. The locations of zmaR, orfl, orf2, and orf3 are noted in the "features" fields, as are various restriction enzyme recognition sites, the putative ribosome binding sites, and the acyltransferase (AT) active site motif in orfl.

The deduced amino acid sequences of orfl, orfl, orf3 and zmaR are presented in SEQ. LD. NOS: 42 & 43 (orfl gene and Orfl protein, respectively), SEQ. LD. NOS: 44 & 45 ((orfi gene and Orf2 protein, respectively), SEQ. ID. NOS: 46 & 47 (orf3 gene and Orf3 protein, respectively), and SEQ. ID. NOS: 48 & 49 (zmaR gene and zmaR protein, respectively).

SEQ. ID. NOS: 2-25 are the PCR primers presented in Table 2.

SEQ. ID. NO: 26 is the R. cereus mutant designated 11.35

SEQ. ID. NO: 27 is the R. cereus mutant designated 32.18 SEQ. ID. NO: 28 is the B. cereus mutant designated 52.6

SEQ. ID. NO: 29 is the R. cereus mutant designated 56.34

SEQ. ID. NO: 30 is the R. cereus mutant designated 64.27

SEQ. ED. NO: 31 is the R. cereus mutant designated 78.24 SEQ. ID. NO: 32 is the R. cereus mutant designated 89.3 SEQ. ID. NO: 33 is the B. cereus mutant designated 96.3 SEQ. ED. NO: 34 is the R. cereus mutant designated 101.19 SEQ. ID. NO: 35 is the R. cereus mutant designated 120.4 SEQ. ED. NO: 36 is a consensus transcription initiation sequence for plants. SEQ. ED. NO: 37 is another consensus transcription initiation sequence for plants. SEQ. ED. NOS: 38-41 are the PCR sequencing primers used to sequence the R. cereus mutants.

DETAILED DESCRIPTION OF THE INVENTION

Zwittermicin A is the only known aminopolyol antibiotic:

It also has structural features in common with peptide and polyketide antibiotics. The alternating hydroxyl groups on the carbon backbone are similar to a partially reduced polyketide structure; the nitrogen-rich end of zwittermicin A may be derived from an amino acid, possibly citrulline, similar to a polypeptide antibiotic. Several polypeptide antibiotic biosynthetic pathways have been described from Bacillus spp. Although the polyketidic antibiotics dif icidin and aurantinin have been identified from species of Bacillus, nothing is known about the genes necessary for their biosynthesis. In fact, the vast majority of genetic studies of polyketide antibiotic biosynthesis have been conducted in Streptomyces spp.

Therefore, elucidation of the pathway for biosynthesis of zwittermicin A will likely increase understanding of this novel, bioactive molecule and the organisms that produce it, and contribute to fundamental knowledge of the diversity of antibiotic biosynthetic pathways. Polyketide antibiotics share a common pattern of biosynthesis. The molecules are built up from two carbon building blocks, the β-carbon of which always carries a keto group, thus the name polyketide. Polyketides include many important antibiotics, immunosuppressants, and other compounds possessing a broad range of biological properties. The tremendous structural diversity of polyketides derives from the different lengths of the polyketide chain and the different side-chains introduced, either as part of the two carbon building blocks, or after the polyketide backbone is formed. The keto groups may also be reduced to hydroxyls or removed altogether. Each round of two carbon addition is carried out by a complex of enzymes called the polyketide synthases (PKS) in a manner similar to fatty acid biosynthesis.

The biosynthetic genes for an increasing number of polyketide antibiotics have been isolated and sequenced and it is believed that the PKS genes are structurally conserved. The encoded proteins generally fall into two types: type I proteins are polyfunctional, with several catalytic domains carrying out different enzymatic steps covalently linked together (e.g. PKS for eiythromycin, soraphen, and avermectin; whereas type II proteins are monofunctional.

For the simpler polyketide antibiotics such as actinorhodin (produced by Streptomyces coelicolor), the several rounds of two carbon additions are carried out iteratively on PKS enzymes encoded by one set of PKS coding regions. In contrast, synthesis of the more complicated compounds such as eiythromycin and soraphen involves sets of PKS coding regions organized into modules, with each module carrying out one round of two-carbon addition (for review see Hopwood et al. in: Industrial Microorganisms: Basic and Applied Molecular Genetics, (ed.: Baltz et al.), American Society for Microbiology, Washington D.C. pp. 267-275 (1993)).

It is known from earlier investigations on actinomycetes, for example, that coding regions involved in the individual steps of secondary metabolite biosynthesis are usually organized in the form of coding region clusters on the bacterial chromosome. It is believed that large multi-functional peptide synthetase enzymes catalyze the sequential condensation of amino acids through a thiotemplate. It is further believed that the sequence of events may be primarily determined by the spatial arrangement of catalytic domains of the protein, which include activities of the activation of the amino acid as an acyladenylate, the attachment of the amino acid as a thioester to a site on the enzyme, and the transpeptidation and formation of the peptide bond. Once completed, it is believed that the peptide chain is released by cyclization or the action of a thioesterase. See, e.g.. Kleinkauf, H., et al., Europ. J. Biochem., 236, 335-351 (1996); Marahiel, M.N, FEBS Letters, 307, 40-43 (1992); and Turgay, K, et al., Mol. Micro., 6, 529-546 (1992).

There are few examples of bioactive molecules synthesized via a hybrid polyketide/polypeptide biosynthetic pathway. A locus encoding putative polypeptide/polyketide functions is mutated in a strain of Proteus mirabilis defective in swarming (Gaisser, S., et al., Mol. Gen. Genet. 253, 415-427 (1997)). A polypeptide with homology to polypeptide synthetase enzymes, which is believed to function in the incorporation of L-pipecolate into and cyclization of rapamycin, cooperates with the PKS largely responsible for the biosynthesis of rapamycin (Schwecke, T., et al., Proc. Νatl. Acad. Sci. USN 92:7839-7843 (1995)). Probably the best example of a hybrid polyketide/polypeptide biosynthetic mechanism is the plant phytotoxin coronatine, which contains a polyketide-derived component (Rangaswamy, V., et al, J. Bacteriol. 180, 3330- 3338 (1998)) and a cyclized amino acid component (Ullrich, M.,et al., J. Bacteriol. 176, 7574-7586 (1994)) coupled by an amide bond. The present invention is directed to the isolation of nucleic acid molecules including, and at least consisting of, nucleic acid fragments and expression thereof which encode polypeptides involved in the biosynthesis of aminopolyol antibiotics. Nucleic acid molecules in accordance with the present invention can be cloned using a variety of techniques. To that end, the invention provides isolated nucleic acid fragments encoding at least one polypeptide necessary for the biosynthesis of an aminopolyol antibiotic wherein the nucleic acid fragment includes at least the following: orfl, orfl, and or/3. Preferably, the nucleic acid fragment encodes at least one polypeptide necessary for the biosynthesis of zwittermicin A. More preferably, the nucleic acid fragment has the sequence of SEQ. ID. NO: 1. Most preferably, a nucleic acid fragment complementary to the nucleic acid fragment encoding at least one polypeptide necessary for the biosynthesis of an aminopolyol antibiotic hybridizes to SEQ. ED. NO: 1.

The term "or " refers to a coding region. "Orf ' refers to the polypeptide encoded by the orf. Both orf and Orf are acronyms for open reading frame. SEQ. ED. NO: 1 contains three coding regions of the present invention, referred to as or/1, orfl, and or/3. Each individual or/is shown in isolation in the Sequence Listing at SEQ. ED. NOS: 42 (orfl), 44 (orfl), and 46 (orf 3). The polypeptides encoded by these coding regions are Orfl, Orf2, and Orf3, respectively. The amino acid sequences of Orfl, Orf2, and Oιf3 are depicted in SEQ. ED. NOS: 43, 45, and 47, respectively. These polypeptides display similarity with acyl-CoA dehydrogenases, polyketide synthases, and polypeptide synthetases, respectively.

One method of cloning nucleic acid fragment of the invention involves a standard technique that can be used to clone nucleic acid fragments encoding polypeptides involved in the biosynthesis of aminopolyol antibiotics is the use of transposon mutagenesis to generate "knockout" mutants; that is, an aminopolyol antibiotic-producing organism which, after mutagenesis, fails to produce the aminopolyol antibiotic. For example, one preferred transposon can be classified as a class II transposon isolated from Bacillus ssp. One more preferred transposon has been designated Tn5401 and described by Baum, J., J. of Bacter., 176, 2835-2845 (1994). Generally, the region of the genome responsible for aminopolyol antibiotic production is tagged by the transposon and can be easily recovered and used as a probe to isolate the coding regions from an unmutagenized strain.

Individual wild-type microorganisms can be screened for the presence of nucleotide sequences that are similar to the coding regions of the present invention. Screening methods include, for instance, hybridization of a detectably labeled probe with a nucleic acid fragment. Hybridizing conditions are appreciated by those with skill in the art, such as those described by Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 9.47-9.55 (2ⁿ ed, Cold Spring Harbor Laboratory Press 1989). Generally the probe does not have to be complementary to all the nucleotides of the nucleic acid fragment as long as there is hybridization under the above-stated conditions. Preferred probes are nucleic acid fragments complementary to a coding region or another nucleotide sequence which plays a part in the synthesis of an aminopolyol antibiotic. For instance, a probe can comprise a consecutive series of nucleotides complementary to a portion of SEQ. ED. NO: 1. Methods of detectably labeling a probe are well known to the art. The nucleic acid fragment that is identified by the probe is further analyzed to determine if it encodes a polypeptide involved in the biosynthesis of an aminopolyol antibiotic by, for example, polymerase chain reaction (PCR) technology.

Individual wild-type microorganisms containing nucleic acid fragments encoding polypeptides involved in the biosynthesis of aminopolyol antibiotics can also be identified using monoclonal or polyclonal antibodies. Preferably the antibody is directed to a polypeptide involved in the biosynthesis of zwittermicin N where the polypeptide is encoded by a nucleic acid fragment, the complement of which hybridizes to SEQ. ED. NO: 1. Preferably the antibody is directed to a polypeptide involved in the biosynthesis of zwittermicin A where the polypeptide is Ofrl (SEQ. ID. NO: 43), Orf2 (SEQ. DD. NO: 45), or Orf3 (SEQ. ID. NO: 47).

Hybridization of a probe to a coding region present in individual wild-type microorganisms can be used as a method to identify a coding region identical or similar to a coding region present in SEQ. ID. NO: 1. The coding region can then be isolated and ligated into a vector as described below. Two nucleic acid sequences are "similar" if the two nucleic acid sequences can be aligned so that a percentage of corresponding residues are identical. Preferably, two nucleotide acid sequences have at least about 60%ι, more preferably at least about 70%, most preferably at least about 80% identity when no gaps are permitted in aligning the sequences. A "gap" refers to a space inserted in a nucleic acid sequence to permit better alignment of the two nucleic acid sequences being compared. Preferably, two nucleotide acid sequences have at least about 80%, more preferably at least about 90%, most preferably at least about 95% identity when gaps are permitted in aligning the sequences.

As mentioned above, a nucleic acid fragment of the invention can be inserted in a vector. Construction of vectors containing a nucleic acid fragment of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) or Ausubel, R.M., ed. Current Protocols in Molecular Biology (1994). A vector can provide for further cloning (amplification of the nucleic acid fragment), i.e., a cloning vector, or for expression of the polypeptide encoded by the coding region, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, or artificial chromosome vectors. Typically, a vector is a vector capable of replication in a bacterial host, for instance E. coli or B. cereus. Preferably the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. Suitable plasmids for expression in a bacterial host include, for example, pUC(X), pKK223-3, pKK233-2, pTrc99N and pET-(X) wherein (X) denotes a vector family in which numerous constructs are available. pUC(X) vectors can be obtained from Pharmacia Biotech (Piscataway, ΝH) or Sigma Chemical Co. (St. Louis, MO). pKK223-3, pKK233-2 and pTrc99A can be obtained from Pharmacia Biotech. pET-(X) vectors can be obtained from Promega (Madison, WI), Stratagene (La Jolla, CA), and Νovagen (Madison, WI). To facilitate replication inside a host cell, the vector preferably includes an origin of replication (known as an "ori") or replicon. For example, ColEl and P15A replicons are commonly used in plasmids that are to be propagated in E. coli. An expression vector optionally includes regulatory regions operably linked to the coding region. "Regulatory region" refers to a nucleic acid fragment that regulates expression of a coding region to which a regulatory region is operably linked. Non-limiting examples of regulatory regions include promoters, transcription initiation sites, translation start sites, translation stop sites, and terminators. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory element is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory region. The invention is not limited by the use of any particular promoter, and a wide variety are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region. The promoter used in the invention can be a constitutive or an inducible promoter. It can be, but need not be, heterologous with respect to the host cell. Preferred promoters for bacterial transformation include lac, lacUV5, tac, trc, T7, SP6, and ara. An expression vector can optionally include a Shine Dalgarno site (e.g., a ribosome binding site), and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the enzyme. It can also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNN thus ending polypeptide synthesis. The nucleic acid fragment used to transform the host cell can optionally further include a transcription termination sequence. The rrnB terminator, which is a stretch of DΝA that contains two terminators, TI and T2, is an often-used terminator that is incorporated into bacterial expression systems (J. Brosius et al., J. Mol. Biol, 148: 107-127 (1981)).

The nucleic acid fragment used to transform the host cell optionally includes one or more marker sequences, which typically encode a polypeptide that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence can render the transformed cell resistant to an antibiotic, or it can confer compound-specific metabolism on the transformed cell. Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, and tetracycline. For the expression of operons encoding multiple coding regions involved in aminopolyol antibiotic biosynthesis, the operon can be inserted into a vector such as pKK223-3 in transcriptional fusion, allowing the wild-type ribosome binding site of the heterologous coding regions to be used. Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al., In: Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz et al. (eds.), American Society for Microbiology, Washington (1993)). Alternate systems for overexpression rely on yeast vectors and include the use of Pichia, Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrial microorganisms: basic and applied molecular genetics, Baltz, et al. (eds.), American Society for Microbiology, Washington (1993); Dequin & Barre, Biotechnology 12: 173-177 (1994); van den Berg et al., Biotechnology 8: 135-139 (1990)).

The present invention also provides for polypeptides involved in the biosynthesis of an aminopolyol antibiotic. Preferably the antibiotic is zwittermicin A. Preferably, a polypeptide includes an amino acid sequence wherein at least a portion of the amino acid sequence is encoded by a nucleic acid fragment, such that a complement of the nucleic acid fragment hybridizes to SEQ. ED. NO: 1. Most preferably, the polypeptides are selected from the group consisting of SEQ. ED. NO: 43, SEQ. ED. NO: 45, and SEQ. ED. NO: 47.

Generation of Novel Molecules Through Combinatorial Biochemistry.

Much work in the field of polyketide antibiotics focuses on the generation of new polyketides through domain swapping (Katz, L., et al., Annu. Rev. Microbiol. 47:875-912 (1993); Khosla, C, et al., Trends Biotechnol. 14, 335-341 (1996); Ruan, X., et al., J. Bacteriol. 179, 6416-6425 (1997)); similar experiments have begun in the synthesis of new polypeptide antibiotics (Stachelhaus, T., et al., Science 269:69-72 (1995)). The fusion of two structurally distinct biosynthetic pathways, as may be offered by the zwittermicin A biosynthetic pathway, could markedly increase the potential number of molecules generated through combinatorial biochemistry. Isolated nucleic acid fragments of the invention can be expressed in heterologous bacterial or fungal hosts to enable the production of the aminopolyol antibiotic with greater efficiency than might be possible from native hosts, which may increase the efficacy of a biocontrol aspect of the microorganism. "Heterologous bacterial or fungal hosts" are host cells containing a nucleic acid fragment encoding polypeptides involved in the biosynthesis of aminopolyol antibiotics, where the host cell is not the organism from which the nucleic acid fragment was isolated.

Isolated aminopolyol biosynthesis coding regions can also be expressed in heterologous bacterial and fungal hosts with the aim of increasing the efficacy of biocontrol strains of such bacterial and fungal hosts. A "biocontrol strain," "biocontrol agent," or "biocontrol organism" is an organism that is capable of affecting the growth of a pathogen such that the ability of the pathogen to cause a disease is reduced. A "pathogen" is an organism that causes a deleterious effect on a second organism under appropriate conditions. When the second organism is a plant, the pathogen is typically referred to as a

"phytopathogen." Within the scope of this invention, the term pathogen is intended to include fungi, bacteria, nematodes, viruses, viroids, and insects. Biocontrol agents for plants include microorganisms which are capable of colonizing plants or the rhizosphere. Organisms may act as biocontrol agents in their native state or when they are modified to express an aminopolyol antibiotic, preferably zwittermicin A.

Biocontrol agents can be modified to express an aminopolyol antibiotic, wherein the biocontrol agent is a microorganism which is capable of colonizing plants or the rhizosphere. Such microorganisms can be brought into contact with phytopathogenic fungi, phytopathogenic bacteria and phytopathogenic nematodes. Preferably, contact with a biocontrol agent inhibits the growth of a phytopathogen. Suitable biocontrol agents that can be modified include gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia, the gram-positive microorganism Bacillus and the fungi Trichoderma and Gliocladium.

For example, Bacillus ssp. containing a mutation, preferably a single insertion mutation, within an aminopolyol antibiotic pathway can be applied directly to an area to be cultivated can act as a biocontrol agent. Preferably, the Bacillus ssp. contains a mutation such that the mutant Bacillus ssp. produces the aminopolyol in an amount greater than the wild type Bacillus ssp.(i.e., the same Bacillus ssp. that does not contain the mutation), as determined by a zone of inhibition assay based on Erwinia herbicola (defined in detail in the Examples). Preferably, the microorganism produces the aminopolyol in an amount greater than about 1.1 times that of the wild type microorganism, and more preferably in an amount of about 2 times greater than the wild type microorganism. More preferably, the mutant Bacillus ssp. is R. cereus containing a single insertion mutation within a portion of the genome encoding a transcriptional regulator, preferably including a nucleic acid fragment comprising SEQ. ED. NO: 29. In this instance, the biocontol agent is a homologous host with respect to the nucleic acid fragment encoding the desired affect, i.e., the nucleic acid fragment is derived from Bacillus. For example, the nucleic acid fragment containing the mutation might be derived from Bacillus cereus and the biocontol agent might be Bacillus subtilis or Bacillus cereus including the mutant nucleic acid fragment.

However, heterologous hosts can also be used and suitable hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesens, Trichoderma viride, Trichoderma harzianum, and Gliocladium virens. In a preferred embodiment of the invention, the nucleic acid fragments of the present invention are transferred to a preferred heterologous host listed above. In a particularly preferred embodiment, the biosynthetic coding regions for zwittermicin A are transferred to and expressed in Pseudomonas fluorescens strain CGA267356 (described in U.S. Pat. No. 5,348,742), which has biocontrol utility due to its production of pyrrolnitrin. In another preferred embodiment, the nucleic acid fragments are transferred to Pseudomonas aureofaciens strain 30-84, which has biocontrol characteristics due to its production of phenazine. Expression in heterologous bacterial or fungal hosts requires the selection of vectors appropriate for replication in the chosen host and a suitable choice of promoter. Techniques are well known in the art for expression in gram-negative and gram-positive bacteria and fungi and are described herein.

Nucleic acid fragments of this invention can be expressed in plants, preferably transgenic plants, thus causing the biosynthesis of an aminopolyol antibiotic, such as zwittermicin A. In this way, transgenic plants with enhanced resistance to phytopathogenic fiingi, phytopathogenic bacteria and phytopathogenic nematodes are generated. The nucleic acid fragments of the present invention may be modified in ways known to one of skill in the art to optimize their expression in transgenic plants. For instance, it is known in the art that all organisms have specific preferences for codon usage, and the codons of the coding regions of the invention can be changed to conform with plant preferences while maintaining the amino acid sequence of the encoded polypeptide. High expression of transgenic coding regions in plants is best achieved from coding regions having at least 35%> GC content, and preferably more than 45%>. Changes to nucleic acid fragments and coding regions described herein can be made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction. See, e.g., U.S. Patent 5,716,849 (Ligon et al.), which describes that preferred nucleotide sequences may be modified to account for the specific codon preferences and/or GC content preferences of monocotyledons or dicotyledons to increase coding region expression.

Dicot and monocot plants that can be genetically manipulated can be used in the present invention. A plant that can be genetically manipulated is a plant into which foreign coding regions can be introduced, expressed, stably maintained, and transmitted to subsequent generations of progeny.

Transgenic plants may be obtained from transgenic seeds set by parental transgenic plants. Methods of making a transgenic plant of the invention typically involve the transformation of a cell of a plant with a nucleic acid fragment comprising a coding region encoding a polypeptide involved in aminopolyol biosynthesis. The nucleic acid fragment is typically present on a vector. In a plant cell, the vector can replicate autonomously, i.e., extrachromosomally, which can allow for high numbers of the vector to be maintained and potentially result in higher polypeptide production, or can be integrated into the genomic DNA. Preferably the vector is integrated into the genomic DNA of a plant cell. Vectors are preferably circular, but can be linear.

A coding region present in a nucleic acid fragment of the invention is typically flanked by operably linked regulatory regions that regulate expression of a coding region in a transformed plant cell. A typical regulatory region operably linked to the coding region includes a promoter. The invention is not limited by the use of any particular promoter, and a wide variety are known. Plant-specific promoters are preferred. These include, but are not limited to, constitutive promoters, inducible promoters, and tissue-specific promoters. It can be, but need not be, heterologous with respect to the host. Promoters may be obtained from Ti- or Ri-plasmids, from plant cells, plant viruses or other hosts where the promoters are found to be functional in plants. Illustrative promoters include the octopine synthetase promoter, the nopaline synthase promoter, the manopine synthetase promoter, etc., as illustrative of promoters of bacterial origin functional in plants. Viral promoters include the cauliflower mosaic virus full length (CaMV35S) and region VI promoters, etc. Endogenous plant promoters include the ribulose-l,6-biphosphate (RUBP) carboxylase small subunit (ssu) promoter, the b -conglycinin promoter, the phaseolin promoter, the ADH promoter, GPAL2 promoter, GPAL3 promoter, heat-shock promoters, tissue specific promoters, e.g., promoters associated with fruit ripening, etc.

The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. For the protection of plants against foliar pathogens, expression in leaves is preferred; for the protection of plants against ear pathogens, expression in inflorescences (e.g. spikes, particles, cobs etc.) is preferred; for protection of plants against root pathogens, expression in roots is preferred; for protection of seedlings against soil-borne pathogens, expression in roots and/or seedlings is preferred. In many cases, however, protection against more than one type of phytopathogen will be sought, and thus expression in multiple tissues will be desirable, as described in U.S. Patent No. 5,716,849 (Ligon et al.). Preferably, a suitable promoter is selected from the group consisting of a wound inducible promoter, a green tissue specific promoter, a root specific promoter, a stem specific promoter, and a flower specific promoters.

Another typical regulatory region operably linked to a coding region includes a terminator, i.e., a nucleic acid fragment that can cause the termination of transcription of the exogenous coding region, present 3' of the exogenous coding region. The invention is not limited by the use of any particular terminator, and a wide variety are known. Plant-specific terminators are preferred. These include, but are not limited to, a nopaline synthase terminator derived from the Agrobacterium tumefaciens Ti plasmid (nos ter).

For efficient initiation of translation, nucleotide sequences 5' to the initiating coding of the polypeptide encoded by the coding region can be modified to increase expression. Modifications include the inclusion of sequences known to be effective in plants. For instance the sequence GTCGACCATGGTC (SEQ. ID. NO: 36) (Joshi, Nuc. Acids Res., 15:6643- 6653 (1987)) has been suggested as a consensus translation initiator for the expression of the E. coli-uidA coding region in plants. Another potential translation initiation sequence that can be used adjacent to the initiating codon is AAACAATGGCT (SEQ. ED. NO: 37) (Joshi, Nuc. Acid Res., 15:6643-6653 (1987)). These translation initiation sequences may be used with the coding regions of the invention. The sequences are incorporated into the nucleic acid fragment upstream of the initiating codon.

Synthesis of a aminopolyol antibiotic, such as zwittermicin N in a transgenic plant will frequently require the simultaneous overexpression of multiple genes encoding the aminopolyol antibiotic biosynthetic enzymes. This can be achieved by transforming the individual aminopolyol biosynthetic coding regions into different plant lines individually, and then crossing the resultant lines. Selection and maintenance of lines carrying multiple coding regions is facilitated if each the various transformation constructions utilize different selectable markers. A line in which all the required aminopolyol biosynthetic coding regions are present will synthesize the aminopolyol antibiotic, whereas other lines will not. This approach may be suitable for hybrid crops such as maize in which the final hybrid is necessarily a cross between two parents.

A variety of techniques are available for the introduction of the nucleic acid fragment into a plant cell. However, the particular manner of introduction of the nucleic acid fragment into the host cell is not critical to the practice of the present invention, and any method which provides for efficient transformation may be employed. For the introduction of the nucleic acid fragment into a plant cell, in addition to transformation using plant transformation vectors derived form the tumor-inducing (Ti) or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used. Such conventional methods may include, for example, the use of liposomes, transformation using viruses or pollen, chemicals that increase the direct uptake of DNN microinjection, electroporation, or high-velocity microprojectiles.

The choice of plant tissue source or cultured plant cells for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is regenerable, in that it will retain the ability to regenerate whole, fertile plants following transformation.

The transformation is carried out under conditions directed to the plant tissue of choice. Buffers and media used will also vary with the plant tissue source and transformation protocol.

Following exposure to the nucleic acid fragment under the appropriate conditions, the plant cells or tissue may be cultivated for varying lengths of time prior to selection, or may be immediately exposed to a selective agent such as those described hereinabove. Protocols involving exposure to Agrobacterium may also include an agent inhibitory to the growth of the Agrobacterium cells. Commonly used compounds are antibiotics such as cefotaxime and carbenicillin. The media used in the selection may be formulated to maintain transformed callus or suspension culture cells in an undifferentiated state, or to allow production of shoots from callus, leaf or stem segments, tuber disks, and the like. Cells or callus observed to be growing in the presence of normally inhibitory concentrations of the selective agents are presumed to be transformed and may be subcultured several additional times on the same medium to remove non-resistant sections. The cells or calli can then be assayed for the presence of the nucleic acid fragment, or may be subjected to known plant regeneration protocols. In protocols involving the direct production of shoots, those shoots appearing on the selective media are presumed to be transformed and may be excised and rooted, either on the selective medium suitable for the production of roots, or by simply dipping the excised shoot in a root-inducing compound and directly planting it in vermiculite. Production of Aminopolyol Antibiotics.

The present invention also provides methods for obtaining aminopolyol antibiotics such as zwittermicin A from heterologous hosts transformed with the appropriate aminopolyol biosynthetic coding regions. These aminopolyol antibiotics may be effective in the inhibition of growth of microbes, particularly phytopathogenic microbes. The aminopolyol antibiotics can be produced from organisms in which the aminopolyol biosynthetic coding regions have been overexpressed, and suitable organisms for this include gram-negative and gram-positive bacteria and yeast, as well as plants. For the purposes of aminopolyol antibiotic production, the significant criteria in the choice of host organism are its ease of manipulation, rapidity of growth (i.e. fermentation in the case of microorganisms), and its lack of susceptibility to the antibiotic being overproduced. These methods of aminopolyol antibiotic production have significant advantages over the chemical synthesis technology usually used in the preparation of antibiotics. These advantages are the cheaper cost of production, and the ability to synthesize compounds of a preferred biological enantiomer, as opposed to the racemic mixtures inevitably generated by organic synthesis. The ability to produce stereochemically appropriate compounds is particularly important for molecules with many chirally active carbon atoms. Antibiotics produced by heterologous hosts can be used in medical (i.e. control of pathogens and/or infectious disease) as well as agricultural applications. The present invention also provides for an antipathogen composition in which the active ingredient is the aminopolyol antibiotic. The aminopolyol antibiotic is typically produced by the biocontrol agent of the present invention. Alternatively, the composition is a suspension or concentrate of the biocontrol agent. The active ingredient is homogeneously mixed with one or more compounds or groups of compounds described herein. The present invention also relates to methods of treating plants, which comprise application of the composition 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 include fertilizers or micronutrient donors, selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations. The compositions can further include carriers, surfactants, and adjuvants. A description of the formulation of antipathogenic compositions can be found in U.S. Patent 5,716,849. EXAMPLES

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. The Examples below demonstrate the identification of a genetic region required for zwittermicin A synthesis. DNA sequence analysis identified open reading frames with sequence similarity to enzymes involved in polyketide and polypeptide antibiotic biosynthesis. Coding region inactivation studies in R. cereus UW85 identified coding regions necessary for zwittermicin A production and resistance, suggesting that the synthesis of zwittermicin A may be directed by a hybrid of polypeptide and polyketide biosynthetic enzymes.

Example 1:

Nucleotide sequence and deduced functions of orfl, orfl, and orf3.

Bacterial strains, plasmids, and culture conditions. The strains and plasmids used in this study are described in Table 1. Unless otherwise indicated, E. coli strains were grown at 37°C on Luria-Bertani (LB) broth or agar. Unless otherwise indicated, Bacillus strains were grown at 28°C on 50% trypticase soy broth (TSB) or agar (TSA) (Difco Laboratories, Detroit, MI). The media 100MH8.1 was prepared by amending Mueller-Hinton medium (Difco) with 40 mM 3-(morpholino)propane-sulfonic acid (MOPS) and 40 mM tris(hydroxymethyl)aminomethane (Tris) and adjusting the pH to 8.1 with NaOH. Antibiotics were added at the following levels for E. coli: ampicillin, 50 mg/L; spectinomycin, 100 mg/L; chloramphenicol, 12 mg/L, and at the following levels for Bacillus: spectinomycin, 150 mg/L or 200 mg/L; eiythromycin, 10 mg/L; chloramphenicol 5 mg/L. Table 1. Bacterial strains and plasmids used in this study Strain or plasmid Description Source or reference

Escherichia coli DH5α 80d/αcZΔM15 ΔQacZYA-argF) J\69deoR supE44 Hanahan, 1983 hsdRll recAl endAl gyrA95 thi-1 relAl

DH5αF'IQ DH5αF' proAB^* /αc^qZΔM15 (Km^r) Gibco-BRL

Bacillus B. cereus UW85 Wild type Handelsman et al., B cereus UW030 UW85 zma-030. lacks zmaR Milner et al . 1996,

B cereus UW85 AzmaR UW85 denvauve with zmaR deleted Sp^r B cereus \JWS5A₀rfl UW85 denvauve with orfl deleted Sp^r B thuringiensis EG10368 Highly transformable strain Ecogen Corp

Plasmids pGEM-3Zf(+) Cloning and sequencing vector, Ap^r Promega Corp pGEMdSH pGEM-3Zf(+) with Smal-HtndUl of MCS deleted. Ap * pZMG6 pGEM with 1 2-kb Sphl-BamΑ subclone of zmaR, Ap^r Milner et al . 1996 pDG1726 Sp^r cassette from Enterococcus faecalis in plasnud Guerout-Fleury et al . pSB119, Ap^r Sp^r 1995 pZMG6S+ pZMG6 with internal deletion of zmaR and Sp^r cassette * inserted, Ap^r Sp^r pZME5 5 pGEMdSH with 5 5-kb EcoRl fragment from HM11 containing zmaR and orfl, Ap^r pZME5 5Aorf2 pZME5 5 with internal deletion of orfl and Sp^r cassette inserted, Ap^r Sp^r pZME5 SAzmaR pZME5 5 with internal deletion of zmaR and Sp^r cassette inserted, Ap^r Sp^r pl4B' Bactllus-E coli vector with T^* oπgin of replication for marker exchange mutagenesis, Ap^r Cm^r pΔzmaR pl4B' with EcoRl fragment from pZME5 5A_zmaR,

Ap^r Cm^r pΔorfi pl4B' with EcoRl fragment from pZME5 S orfl,

Ap^r Cm^r pORF2 pHT304 with 1 7-kb subclone of orfl, Ap^r Em^r pZMS7 pHT304 with 1 2-kb Sphl-BamHl subclone of zmaR, Milner et al , 1996

Ap^r Em^r pZME6R pHT304 with 6-kb EcoRl fragment containing zmaR, Milner et al , 1996 orfl, and orfl, Ap^r Em^r pZMES17/pZMES18 pHT304 with 1 2-kb EcόBI-Sphl fragment from Milner et al , 1996 pZME6R, Ap^r Em^r pZMB14/pZMB20 pHT304 with 3 1-kb Bamm fragment Milner et al , 1996 from pZME6R, Ap^r Em^r pZMR6 pLA2917 with z/wα/?-contammg insert Milner et al , 1996

HM11 pBeloBAC with 14-kb insert containing zmaR, Cm^r * T^s ongin of replication

Ap^r Plasmid confers resistance to ampicilhn

Cm^r Plasmid confers resistance to chloramphenicol

Em^r Plasmid confers resistance to erythromycm

Sp^r Plasmid confers resistance to spectinomycin

* Constructed as described herein

DNA manipulations and analysis. Plasmid DNA was isolated from strains of E. coli using Wizard minipreps (Promega Corp , Madison, WI) or Qiagen (Chatsworth, CA) plasmid kits Plasmid DNA was isolated from strains of Bacillus using a modified alkaline lysis extraction described elsewhere (Milner et al , 1996) Total genomic DNA was isolated from R. cereus using the Easy-DNA kit (Invitrogen Corp , Carlsbad, CA) Restriction and modification enzymes were used according to manufacturers' directions (Promega Corp , New England Biolabs, Beverly, MA, Gibco BRL, Richmond, CA) To change buffer conditions between different enzymatic reactions and to isolate DNA fragments from agarose gels, DNA was purified using QIAquick kits (Promega Corp ) Southern blot analysis was carried out with the Genius Kit (Boehringer Mannheim Biochemicals, Indianapolis, EN) using the conditions described in the manufacture's specifications However, for Southern blots in which B. cereus genomic DNA was the target, probes often failed to hybridize to the target under high-stringency conditions, presumably because B. cereus DNA is AT rich (36%> GrC) For those blots, reduced stringency hybridization conditions the hybridization temperature was reduced from 42°C to 40°C, and the excess probe was washed from blots with 0 5X SSC (IX SSC is 0 15 M NaCl, 0 015 M sodium citrate) at 65°C instead of 0 IX SSC at 68°C Plasmids were introduced into E. coli and Bacillus by electroporation as described previously (Milner et al , 1996) To increase the transformation efficiency of B. cereus, we found it necessary to first introduce and purify plasmid DNA from B. thuringiensis EG 10368 (Table 1), a highly-transformable strain, prior to electroporation ofR. cereus

DNA sequencing and analysis. B. cereus DNA fragments cloned into pLA2917, pHT304, or pGEM (Table 1), were isolated from E. coli and used as templates for sequencing Both strands of a 2 7-kb region were sequenced by primer walking and with

M13/pUC forward and reverse primers (Table 2), numbered according to the DNA sequence in SEQ YD NO 1 Sequencing reactions were carried out using the PPJZM cycle sequencing kit (Applied Biosystems, Inc , Foster City, CA) Primer synthesis and separation of DNA sequencing products was performed by the University of Wisconsin-Madison Biotechnology Center (Madison, WI)

Table 2 Primers used for sequencing

Primer name SEP. ID. NO: Primer sequence (5' to 3') Position of primer Reference forward 2 GTTTTCCCAGTCACGAC vector Promega

Corp reverse CAGGAAACAGCTATGAC vector Promega

Corp

9522 ATAGTAGAATTTGGACGGCG 209- 190 upstream of nucleotide 1

8838 CTGCGACTCAGGAGAAAGAA 94-75 upstream of nucleotide 1

4367 6 GAAAGTAATGAAGAGCTTCCC 416-436

4486 7 CTAACTGAGCCAGAGGTTGG 695-714

4553 8 TTGTTAGGACCAATTGGGGG 977-996

4552 9 AGCAGCTCGTTCGTTATGCC 1189-1208

1721 10 GCACTAGATCTAGGATGG 2491-2508 Milner

(1996)

3556 11 AAAACTGAAGAGGGACGAGC 2557-2576

4476 12 TGTGCTTCTGGAGCAATTGC 2927-2946

4551 13 GAAACAGGCATTAGTACCTG 3292-3311 *

4646 14 CAGAAAAAGGTAGTACGCCC 3653-3672 *

8328 15 GCAACAATAGTTTCCTCTGC 154-135 *

4556 16 CAAACTCATCAACTACTCCC 380-361 *

4555 17 CTCTTTTGCTCTTCAGTCCC 645-626 *

4554 18 AATCTCAGCTATATGCGAGG 943-924 *

4477 19 TCTTGGCATAACGAACGAGC 1212-1193 *

3555 20 AATCAACGAGAAGTGGAG 1546-1529 *

677 21 TAAAGCTCGTCCCTCTTCAG 2580-2561 Milner

(1996)

4963 22 CATAGAGCTGTATATTCTCCC 2928-2908

5054 23 CTATGACAGGTACTAATGGC 3317-3298

4965 24 CAACTACAGTTAAACACCCTG 3614-3594

4966 25 AAGGATCTGATGGCGCAGG downstream of

Sau3 A junction

" Position of pnmer based on nucleotide sequence in SEQ DD NO. 1 * This study The partial DNA sequences were aligned and compiled, open reading frames were identified, and codon usage was analyzed with Seaman and EditSeq software (DNAStar, Inc.), and Codon Use 3.3 (Conrad Hailing, University of Chicago). DNA and protein comparisons were carried out using the BLAST algorithms (Altschul et al., 1990) via the NCBI BLAST E- mail server and the GCG Wisconsin Sequence Analysis Software Package (Genetics Computer Group, Inc.).

Sequence analysis of zmaR-flanking region. SEQ. ID. NO: 1 shows the 3,934- nucleotide sequence of the zm R-flanking region, which includes the previously published nucleotide sequence of a 1.2-kb Sphl-BamHl fragment (bases 1438 to 2665). Analysis of the sequence, applying codon usage typical of Bacillus spp., identified three open reading frames (orfs) having the same orientation of transcription as zmaR. Two orfs were identified upstream of zmaR, and one orf was identified downstream. The three open reading frames, or/7 (bases 338 to 1486 of SEQ. ED. NO: 1), orfl, (bases 2630 to 3847 of SEQ. ED. NO: 1), and orf 3, (bases 78 to 341 of SEQ. ED. NO: 1) all have putative ribosome binding sites 7 to 9 bp upstream of the potential ATG translational start sites. Orfl, orf 3, and zmaR terminate with a TGA stop codon. Orfl terminates with a TAG stop codon. The TGA stop codon of orf 3 overlaps the ATG start codon of orfl, and is shifted one reading frame in relation to orfl. A similar overlapping structure is apparent for the TGA stop codon of orfl and ATG start codon of zmaR. The ATG start codon of orfl is 20 bases downstream of the TGA stop codon of zmaR. The lack of a promoter sequence, the overlapping structure of the start and stop codons, and the shared transcriptional orientation suggest that orfs 1, 1, and 3, and zmaR are organized in an operon. The data suggest that all of these orfs are coding regions that encode proteins. Therefore we designated the open reading frames themselves as or/7, orfl, and or/3 and the deduced proteins encoded by the open reading frames as Orfl, Orf2, and Orf3, respectively.

Deduced functions of Orfs 1, 1, and 3. Comparison of the deduced amino acid sequences of the three predicted proteins using the BLAST algorithm (Altschul et al., 1990) revealed that each of the Orfs has homology to proteins with known function. Orfl (SEQ. ID. NO: 43) is a protein with a predicted molecular weight of 42.1 kDa and a predicted isoelectric point of 6.7. Orfl has a high degree of sequence similarity to acyl-CoA dehydrogenase enzymes from diverse organisms, including B. subtilis (54% amino acid similarity, 34% amino acid identity) and Rattus norvegicus (53% amino acid similarity, 31% amino acid identity) over the full length of the 382-residue protein. Acyl-CoA dehydrogenases are typically involved in the oxidative breakdown of fatty acids. Non-polyketide synthase- associated enzymic activities, such as dehydrogenases (Brown et al., 1996) and methyltransferases (Molnar et al, 1996) have also been implicated in the production of polyketides. Therefore Orfl, which has sequence similarity to acyl-CoA-dehydrogenases, is likely also involved in the biosynthesis of zwittermicin A.

Orf2 is predicted to encode a 45.6 kDa protein (SEQ. YD. NO: 45) with a predicted isoelectric point of 5.3. Orf 2 has sequence similarity to both polyketide and fatty acid synthase enzymes over parts of the predicted protein sequence. Orf2 has 36% to 60% nucleotide identity 51% to 75% amino acid similarity to polyketide synthase enzymes (Schwecke et al, 1995), and 37% to 48% nucleotide identity, 55% to 74% amino acid similarity to fatty acid synthase enzymes, specifically the acyltransferase portion of polyketide synthases and the transacylase of fatty acid synthases. The region of homology includes active site sequences (bases 2897 to 291 1 of SEQ. ED. NO: 1, which correspond to residues 90-94 of the amino acid sequence shown in SEQ. ED. NO: 45) identified for acyltransferases (AT), transacylases, and thioesterases (Cortes et al., 1990). Unexpectedly, although the Orf2 protein has high sequence similarity to the acyltransferase domain of only type I PKSs, the orfl gene appears to encode a single polypeptide, suggesting that it may have more in common with the monofunctional type II PKS enzymes. Orf3 is predicted to encode a 10.2 kDa protein (SEQ. ED. NO: 47) with a predicted isoelectric point of 4.3. Orfl has regions of sequence similarity to polypeptide synthetase enzymes, including gramicidin S synthetase (Turgay et al, 1992) and surfactin synthetase (Cosmina et al, 1993). The known active sites of these enzymes are not included in the region of homology. Polypeptide synthetase enzymes activate and catalyze the formation of peptide bonds between successive amino acids via a thiotemplate mechanism (Marahiel, 1992). As polypeptide synthetase enzymes are typically large, it was surprising that orf 3 is predicted to encode a small protein. It is possible that the Orf3 protein may be a unique sort of polypeptide synthetase enzyme (similar to what is observed with the type I versus II polyketide synthetases, where the peptide synthetases are generally large, multifunctional enzymes). Example 2:

Effect of insertional inactivation of zmaR and orf 2 in B. cereus UW85.

Construction of plasmids for insertional inactivation o ^'zmaR and orf2. pGEMdSH was constructed by digesting pGEM-3Zf(+) (Promega Corp.) with Smαl and Hindlll, creating blunt ends with Klenow enzyme, and self-ligating the DNA. A 5.5-kb EcoRl fragment from HM11 containing orfl, zmaR, and orfl was subcloned into pGEMdSH, yielding pZMΕ5.5. It is believed that HMl 1 does not likely contain the entire biosynthetic pathway because sequence analysis indicated that the very end of the insert appears to contain biosynthetic pathway sequence. The strategy for creating a plasmid construct for insertional inactivation of zmaR was to delete 754 bp of DNA internal to zmaR and insert a 1.2-kb spectinomycin resistance (Sp^r) cassette from plasmid pDG1726 (Table 1) in the same transcriptional orientation as zmaR at the site of the deletion. pZMG6 was digested with Bglil and Blpl, blunt-ended with Klenow, and ligated to a 1.2-kb, blunt-ended PstY fragment containing the (Sp^r) cassette, yielding pZMG6S+. Clones were sequenced with primer 677 to identify constructs with the Sp^r cassette in the same transcriptional orientation as zmaR. The 1.7-kb Sphl-BamYS. fragment from pZMG6S+ was cloned into pZME5.5 which had been digested with Sphϊ and RαmHI, treated with CEP, and gel-purified away from the zmαR-containing 1.2-kb Sphl-Ba HY fragment, yielding pZME5.5AzmaR. The EcoRl fragment from pZME5.5 AzmaR was cloned into pl4B', yielding p AzmaR (Fig. 2). pl4B' (Table 1) is anE. coli-B. cereus shuttle vector, which has with a temperature-sensitive origin of replication, used for marker exchange mutagenesis in B. cereus.

The plasmid pAorfl was constructed by digesting pZMΕ6R with Bglll and Hindlll, gel-purifying the 1.7-kb fragment containing orfl, and ligating this to RαmHI/Hw ΛII-digested and CEP-treated vector pΗT304 (Table 1). The strategy for creating a plasmid construct for insertional inactivation of orfl was to delete 881 bp of DNA internal to orfl, and insert a Sp^r cassette in the same transcriptional orientation as orfl at the site of the deletion. pZME5.5 was digested with RαmHI and EcoKV and ligated to the Sp^r cassette prepared as described above, yielding pZME5.5Δor/2. Clones were sequenced with primer 1721 to identify constructs with the Sp^r cassette in the same predicted transcriptional orientation as orfl. The EcoRl fragment from pZMΕ5.5Δor/2 was ligated into pl4B', yielding pΔor/2 (Fig. 3).

Insertional inactivation o ^'zmaR and orf2. Plasmid p AzmaR or pΔor/2 was transformed into B. cereus UW85. Cultures were grown without antibiotic selection at 42°C, a non-permissive growth temperature for pl4B'-derived plasmids. Putative single integrants were identified by plating at 42°C on media containing chloramphenicol and spectinomycin, and integration was confirmed by Southern blot analysis. Single integrants were grown without antibiotic selection for 48 hours at 42°C with repeated subculturing to promote excision and loss of the plasmid, and plated on non-selective media. Colonies were subsequently screened for spectinomycin resistance and chloramphenicol sensitivity on TSA at 28°C. One-fourth to one-third of spectinomycin-resistant colonies were sensitive to chloramphenicol, indicating they had lost the vector and wild-type allele, but maintained the mutant allele. Southern blot analysis of genomic DNA from the chloramphenicol sensitive, spectinomycin resistant clones was performed to establish the genomic structure of the region. When S/? /RαmHI-digested genomic DNAs from UW85 and UWS5 AzmaR were probed with a fragment internal to the zm R-coding region, a 1.2-kb band was detected from UW85, and a 1.7-kb band was detected from UW85ΔzmαR, confirming the presence of a disrupted copy of the zmaR coding region. When SαΛ/EcoRV-digested DNAs from UW85 and UW85Δor/2 were probed with the same DNA fragment a band of approximately 4.5 kb was detected in UW85, whereas a slightly larger band was detected in UW85Δor/2, confirming the presence of a disrupted copy of the orfl coding region.

Effect of insertional inactivation o ^'zmaR and orf2 in B. cereus UW85. To investigate further the roles of orfl and zmaR in zwittermicin A biosynthesis and zwittermicin A resistance, each wild-type coding region was replaced with a mutant allele carrying a deletion and an antibiotic resistance coding region through homologous recombination of plasmid DNA into the UW85 genome. Southern blot analysis confirmed the genomic structure of the AzmaR and Aorfl mutants, and the mutants appeared to grow and sporulate normally. UW85ΔzmαR was sensitive to zwittermicin A; UW85 Aorfl was resistant to zwitteπnicin N as determined by radial streak assay. UW85Δor/2 did not produce detectable zwittermicin N as shown by a plate assay for inhibition of Erwinia herbicola and direct isolation of zwittermicin A from culture supernatants, whereas UW85Δzm R did produce zwittermicin A (Table 3). These results suggest the presence of a second mechanism of antibiotic self-resistance in UW85, perhaps an efflux pump to quickly remove zwittermicin A from the interior of the cell. and mutants

Strain Zwittermicin A resistance" Zwittermicin A production

UW85 R +

UW85Δ_zmαR S + UW85Δ_z/wαR + pZMS7^c R +

"Zwittermicin A resistance to 300 μg of zwittermicin A was measured by radial streak assay on at least three independent cultures. R (resistant) indicates no zone of inhibition; S

(sensitive) indicates a 3 -mm zone of inhibition. ^Zwittermicin A production was measured by bioassay against a lawn of Erwinia herbicola and by biochemical purification of zwittermicin A using Sep-Pak cation exchange columns and subsequent high-voltage paper electrophoresis. The results reported are representative of at least three independent cultures. ^cpZMS7 contains zmaR in plasmid pHT304. ^dpORF2 contains orfl in plasmid pHT304. When zmaR was introduced in trans on plasmid pZMS7, it restored zwittermicin A resistance to UW85Δz7wαR (Table 3). pHT304, the plasmid vector, does not confer zwittermicin A resistance on sensitive strains of B. cereus (Milner et al., 1996). When orfl was introduced in trans on plasmid pORF2, it restored zwittermicin A production to UW85Δor/2. pHT304 did not restore zwittermicin A production to UW85Δor/2 (Table 3). Together the data indicate that zmaR is necessary for zwittermicin A resistance, and that orfl is necessary for zwittermicin A production.

Example 3:

Expression of zmaR in B. cereus UW85 and UW85 AzmaR Recombinant ZmaR protein visualization, purification, and concentration. A 500-mL culture of E. coli DH5< FTQ carrying pZMG4 was grown to an optical density (at 600 nm) of 0.5 to 0.6 in LB with ampicillin and kanamycin. EPTG (1 mM) was added and cells were allowed to grow for 3 hours. Cells were collected by centrifugation and resuspended in 50 mL of 50 mM Tris (pH 8.0), 2 mM EDTA buffer. The sample was incubated at 30°C for 15 minutes after the addition of 0.6 mg lysozyme and 2.5 mL 1% Triton X-100 to the sample. Cells were disrupted by sonication and inclusion bodies were purified initially as described previously (Milner et al., 1996). Inclusion bodies were further purified by repeated washing with increasing concentrations of urea, and were subsequently solubilized in 5 mL 8 M urea, 1%) SDS, 20 mM Tris-acetate pH 8.0. After addition of 5x SDS sample buffer, the sample was stored at -20°C. Inclusion bodies were further purified on preparative 12% polyacrylamide gels, prepared and run by standard methods (Laemmli, 1970). Protein was visualized by staining the gel with cupric chloride (Deutscher, 1990), and the 43.5-kDa protein band was excised from the gel and stored at 4°C. Protein was electroeluted from the gel using a undirectional electroelutor as directed by the operating manual (EBI, New Haven, CT). Protein concentration and buffer exchange to 20 mM Tris (pH 8.0) was achieved through ultrafiltration using Centriplus MWCO10 and Centricon 10 columns (Amicon, Inc., Beverly, MA). Protein concentration was determined by the bicinchoninic acid assay (Pierce Chemicals, Rockford, IL) (Smith et al., 1985).

Generation of anti-ZmaR polyclonal antiserum. Purified ZmaR protein (350 mg) in 20 mM Tris pH 8.0 (0.5 mL) was mixed with Freund's complete adjuvant and injected intradermally into a rabbit. The rabbit was boosted with 350 mg of purified protein monthly, and was bled from the marginal ear vein two weeks after each boost. Serum was spun in centrifuge (9,800 x g, 10 min) at 4°C to remove blood cells, and frozen in 1-mL aliquots at - 20°C (Harlow and Lane, 1988). Immune serum was pre-adsorbed to acetone powders prepared from cell extracts of E. coli DH5rχFTQ (Stratagene, La Jolla. CA) carrying pGEM- 3Zf(+) and cell extracts of B. cereus UW030 prepared by standard methods (Harlow and Lane, 1988). Isolation of total protein from B. cereus. Three milliliters of culture was pelleted by centrifugation (16,000 xg, 5 min), and the supernatant was removed. Pellets were resuspended in 200 mL protein extraction buffer (10 mM Tris-Cl (pH 8.0), 100 mM EDTN 100 mM DTT, 1% SDS), 50 mL 0.1 mm silica beads were added, and samples were agitated for 4 min at room temperature. Samples were centrifuged (14,000 g, 5 min) at 4°C, 5x SDS protein sample buffer was added to 100 mL sample supernatant, and the sample was stored at -20°C. A 10-mL sample of the supernatant was used for determining protein concentration using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Western blot analysis. Overnight cultures of R. cereus UW85 and UW85ΔzmαR were inoculated 1/100 into fresh media and grown at 28°C with shaking. Three milliliters of culture were removed and pelleted 6, 12, 24, 48, and 72 hours after inoculation. Total protein was isolated, and the protein concentration determined as described above. Twenty milligrams of total B. cereus protein was loaded on each lane of a 12% protein gel prepared and run by standard techniques (Laemmli, 1970). Prestained markers (Bio-Rad Laboratories) were included on all gels. Before transfer, gels were allowed to equilibrate for 10 min in transfer buffer (0.025 M Tris, 0.192 M glycine, 10% methanol) with 0.1% SDS added. Gels were blotted to a 0.45 mm polyvinylidene difluoride (PVDF) membrane (Millipore, Millford, MA) using a Bio-Rad transfer cell (Richmond, CA) at 30 V overnight at 4°C. Western blots were developed according to the ECL Western blotting protocol (Amersham Life Technologies, Arlington Heights, EL) with the following modifications. Blots were blocked for one hour at 34°C in 25 mL of a 5% solution of Carnation powdered milk (Nestle Food Company, Glendale, CA) dissolved in blocking solution (Tris-buffered saline (pH 7.6), 0.1% Tween-20). Primary anti-ZmaR antibodies and secondary anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Sigma, St. Louis, MO) were diluted 1:5,000 in blocking solution and incubated at 34°C for 1 h. All washes were performed at room temperature.

Assays for production of zwittermicin A and zwittermicin A resistance. Zwittermicin A was identified in culture supernatants of sporulated cultures of R. cereus UW85 and UW85- derived mutant constructs by cation exchange chromatography using CM SEP-PAK cartridges (Millipore) followed by high voltage paper electrophoresis (HVPE) as described elsewhere (Milner et al., Appl. Microbiol. Biotechnol. 43:685-691 (1995)). Zwittermicin A production was also tested in a plate bioassay for inhibition of Erwinia herbicola LS005 (Silo-Suh et al, 1994) with the modifications that 0.1% TSA plates were used, and plates were incubated for 24 h at 28°C before scoring for the presence or absence of a zone of inhibition. The zwittermicin A-resistance phenotype of strains of E. coli and R. cereus was determined by radial streak assay on 100MH8.1 agar using 100 mg and 300 mg of zwittermicin A for each organism, respectively, as described elsewhere (Milner et al., 1996).

Expression of ZmaR in B. cereus UW85 and UW85 AzmaR. To determine the pattern of expression of ZmaR in B. cereus UW85 and UW85 Δzm R, we performed a western blot analysis. From the nucleotide sequence of zmαR and the expression of ZmaR inE. coli (Milner et al., 1996), ZmaR was predicted to migrate as a 43.5 kDa protein. A 43.5 kDa band was detected in cell extracts of UW85, but not in UW85ΔzmαR, the mutant in which zmαR had been deleted. No band was detected in cell extracts of UW030 (Table 1), a mutant containing a large genomic deletion that spans zmaR. The data supports the conclusion that the 43.5 kDa band is due to ZmaR. Expression of ZmaR was observed in UW85 after 12 hours, at the end of logarithmic growth phase. The expression of this band increased over time, showing maximal expression after 48-72 hours (Fig. 4). Interestingly, although addition of phosphate and iron to cultures of UW85 suppressed and increased zwittermicin A production, respectively (Milner et al., Appl. Microbiol. Biotechnol. 43:685-691 (1995)), they did not appear to have an effect on ZmaR expression suggesting that there may be multiple levels of regulation that differ between the antibiotic biosynthetic coding regions and antibiotic self-resistance coding regions.

Example 4:

Construction of B. cereus mutants

Generation ofB. cereus mutants using Bacillus transposible element. The plasmid pEG922 (Baum, J., J. Bact., 176, 2835-2845 (1994)) was transformed into Bacillus cereus 101C using the method of Silo-Suh et al., Appl. Environ. Micro., 60, 2023-2030 (1994). The plasmid pEG922 is a shuttle vector containing a Gram-positive origin of replication and carries the Tn5401 transposable element. Bacillus cereus 10 IC is a derivative ofR. cereus UW85 that has been subjected to Tn917 mutagenesis and is lacking the native plasmid, pBC85. See, Silo-Suh, L.N, Ph.D. thesis (1994).

Transformants were selected on half-strength tryptic soy agar (TSA) containing 10 μg/ml tetracycline. Plasmid preparations were performed to confirm the presence of pEG922 in the 101C selected transformants. To construct Tn5401 mutants in 101C, individual colonies of 101C carrying pEG922 were inoculated into half-strength tryptic soy broth (TSB) containing 10 μg ml tetracycline and the cultures were grown overnight at 28°C.

These overnight cultures were then sub-cultured at a dilution of 1 : 100 in TSB and grown six to eight hours with shaking at 42°C. These cultures were then diluted 100-fold in water and 100 μl aliquots of the dilute culture were then plated on half-strength TSA containing 10 μg/ml tetracycline. These plates were incubated overnight at 43°C. Large and small sized colonies were observed after the overnight incubation. Individual large colonies were patched onto a plate with half-strength TSA containing 10 μg/ml tetracycline and a plate with half-strength TSA containing 10 μg/ml chloramphenicol. Patch plates were incubated overnight at 28°C. The tetracycline plate was then placed at 4°C to retard growth and the chloramphenicol plate was incubated another 24 hours at room temperature Chloramphenicol-sensitive, tetracycline-resistant colonies contained Tn5401 insertions as confirmed by Southern blot analysis From each culture subjected to this growth regime, 24 or 48 Tn5401 mutants were generated Southern blot analysis of 18 mutants taken from the same culture indicated that each mutant contained only one insertion and the insertion in each mutant was in a different genomic location Using this protocol, 4800 Tn5401 mutants in B. cereus 10 IC were generated.

Screening ofB. cereus mutants for zwittermicin production. Each mutant was screened for the ability to inhibit Erwinia herbicola, which is an indicator of zwittermicin production. Each mutant was grown in 1 ml of half-strength TSB containing 10 μg/ml tetracycline for three days at 28°C Approximately 10 cells of Erwinia herbicola LS005 were plated on one-thousandth strength TSA Six 8 mm wells were punched out of each dilute TSA plate using a cork borer Into eacji well, a 100 μl aliquot of mutant culture was pipetted One well per plate received B. cereus UW85 as a control The plates were placed at 28°C for two days, after which the E. herbicola zone of inhibition for each well was scored. The following table summarizes the results, which includes a mutant number identifier and the zone score.

From the data above, nine mutants produced a slight inhibition or no inhibition of

Erwinia growth, i.e., the zones of inhibition of these mutants were less than the zone of inhibition of the control (UW85). However, and surprisingly, one mutant was obtained (no. 56.34) inhibited Erwinia to a greater extent, about 2 times greater, than the control, thus indicating an over-production of zwittermicin.

Sequence analysis of the B. cereus mutants. Southern blot analysis of the ten mutants above indicated that each mutant contained one insertion and each insertion was in a different genomic location. In an attempt to clone out the transposon insertion and flanking DNA from each mutant, the plasmid pUC18 was employed. Genomic DNA from each mutant was prepared according to the protocol accompanying the kit commercially available under the trade designation EASY-DNA kit, from Invitrogen, Carlsbad, CA. Prepared genomic DNA was then digested with Pstl restriction enzyme which is believed not to digest within the transposon sequence. Pstl-digested DNA was ligated into the Pstl site of pUClδ. The ligation mixture was electroporated into competent E. coli DH5 cells and transformants were selected on LB agar containing 5 μg/ml tetracycline. Using this method, the transposon and flanking DNA was cloned out for the five mutants 11.35, 32.18, 56.34, 64.27, and 89.3. PCR sequencing was performed using primers synthesized by the University of Wisconsin

Biotechnology Center designed to each end of the transposon Tn5401 and reading into the unique Hpal site of Tn5401 and used in the following pairs:

P 1 GGTCTTCTGANFCGANGAACC (SEQ. ED. NO: 38) with P3 GGAGTAACCTTTTGATGCC (SEQ. ID. NO: 39) and

P2 CCCAGAAGAAGTAAAAGATGGG (SEQ. ED. NO: 40) with P4 CCACCTGCGAGTACAAACTGG (SEQ. ID. NO: 41) PI and P2 were complementary to the ends of Tn5401 and P3 and P4 were complementary to a region surrounding the unique, internal Hpal site. Genomic DNA from each mutant was used as a template for PCR amplification using Taq Plus Long polymerase from Stratagene, La Jolla, CN PCR conditions were as follows: initial hot melt at 95°C for 3 min., followed by 30 cycles of 30 sec. At 95°C, 30 sec. At 55°C, and 5 min. at 72°C, and a final extension at 72°C for 7 min.). For each of the five mutants, at least one PCR product was obtained. For mutants 52.6 and 78.24, only one PCR product was obtained with the P2 - P4 primer set. For mutants 96.3, 101.19, and 120.4, PCR products were obtained with primer sets PI - P3 and P2 - P4. The PCR products were purified from the reaction mix using a purification kit commercially available under the trade designation QIAQUICK PCR Purification Kit, from Qiagen Inc., Valencia, CA. Cycle sequencing was then performed using these purified PCR products as a template to generate sequence information, that was subsequently compared to non-redundant GenBank sequences, and the BLAST results are shown herein for the mutants 11.35, 32.18, 52.6, 56.34, 64.27, 78.24, 89.3, 96.3, 101.19, and 120.4. The following table summarizes the homology for each mutant identified by BLAST.

Evaluation of the biocontrol effect ofB. cereus mutants. The effect of the R. cereus mutants on the inhibition of the growth of a phytopathogen in a simulated plant growth environment was evaluated. Plant seeds and a phytopathogen are applied to a cultivation environment, e.g., soil, vermiculite, or any other medium that will support plant germination from seed. Biocontrol agents are applied to the cultivation medium and % emergence from the medium was evaluated. This is typically calculated as a percent of total seed counfper biocontrol agent that emerged from the medium surface, indicating a viable plant is produced. The controls that are typically included are no biocontrol agent (i.e., seeds plus phytopathogen), B. cereus UW85 (positive control), and B. cereus 101 C (derived from UW85 that has been cured of pBC85). As shown in the accompanying graph, the percent emergence for the treatment of mutant designated 56.34 was comparable to that of the positive control (labeled UW85).

The complete disclosures of all patents, patent applications, publications, and nucleic acid and protein database entries, including for example GenBank accession numbers and EMBL accession numbers, that are cited herein are hereby incorporated by reference as if individually incorporated. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention, and it should be understood that this invention is not to be unduly limited to the illustrative embodiments set forth herein.

Claims

CLAIMSWhat is claimed is:

1. An isolated nucleic acid molecule comprising a nucleic acid fragment encoding at least one polypeptide necessary for the biosynthesis of zwittermicin N wherein the nucleic acid fragment encodes a polypeptide sequence selected from the group consisting of SEQ. YD. NO: 43, SEQ. ID. NO: 45, and SEQ. ID. NO: 47.

2. The isolated nucleic acid molecule of claim 1 comprising a nucleic acid selected from the group consisting of SEQ. ED. NO: 1, SEQ. ID. NO: 42, SEQ. ED. NO: 44, and SEQ. ED. NO: 46.

3. The isolated nucleic acid molecule of claim 1 consisting essentially of a nucleic acid selected from the group consisting of SEQ. YD. NO: 1, SEQ. ED. NO: 42, SEQ. ED. NO: 44, and SEQ. ED. NO: 46.

4. The isolated nucleic acid molecule of claim 1 wherein a nucleic acid probe complementary to the nucleic acid fragment of claim 1 hybridizes to SEQ. ED. NO: 1 under hybridization conditions at 40°C, then washing with 0.5X SSC (IX SSC is 0.15 M NaCl, 0.015 M sodium citrate) at 65°C.

5. The isolated nucleic acid molecule of any one of claims 1, 2, or 3, wherein the nucleic acid fragment is isolated from a Bacillus cereus genome.

6. The isolated nucleic acid molecule of any one of claims 1, 2, or 3 wherein the molecule is a vector.

7. The vector of claim 6, wherein the vector is selected from the group consisting of plasmid vector, viral vector, cosmid vector, and artificial chromosome vector.

8. A host organism transformed to contain and express the isolated nucleic acid molecule of any one of claims 1 through 7.

9. The host cell of claim 8, wherein the host organism is a bacterial cell or a fungal cell.

10. A genetically-engineered microorganism comprising a mutated nucleic acid fragment wherein the genetically-engineered microorganism produces an aminopolyol antibiotic in an amount greater than that shown by a zone of inhibition with Erwinia herbicola for a corresponding wild-type microorganism.

11. The genetically-engineered microorganism of claim 10, wherein the aminopolyol antibiotic is produced by the microorganism in an amount of about 2 times greater than the corresponding wild-type microoganism.

12. The genetically-engineered microorganism of claim 10, wherein the aminopolyol antibiotic is zwittermicin A.

13. The genetically-engineered microorganism of claim 10, wherein the microorganism is Bacillus ssp.

14. The genetically-engineered microorganism of claim 10, wherein the mutated nucleic acid fragment comprises SEQ. ED. NO: 29.

15. A transgenic plant comprising a transgenic nucleic acid fragment which comprises a coding region encoding at least one polypeptide necessary for the biosynthesis of an aminopolyol antibiotic, and wherein the transgenic nucleic acid molecule directs expression the aminopolyol antibiotic in the transgenic plant.

16. The transgenic plant of claim 15, wherein the aminopolyol antibiotic is zwittermicin A.

17. The transgenic plant of claim 15, wherein the transgenic nucleic acid fragment encodes a polypeptide sequence selected from the group consisting of SEQ. ID. NO: 43, SEQ. ED. NO: 45, and SEQ. ED. NO: 47.

18. The transgenic plant of any one of claims 15, 16, or 17, wherein a wound-inducible promoter is operably linked to the transgenic nucleic acid fragment.

19. The transgenic plant of any one of claims 15 through 18, wherein the transgenic nucleic acid fragment comprises a nucleic acid fragment selected from the group consisting of SEQ. YD. NO: 1, SEQ. ED. NO: 42, SEQ. ED. NO: 44, SEQ. ED. NO: 46, and combinations thereof.

20. A polypeptide involved in the biosynthesis of an aminopolyol antibiotic comprising an amino acid sequence wherein at least a portion of the amino acid sequence is encoded by a nucleic acid fragment, such that a complement of the nucleic acid fragment hybridizes to SEQ. YD. NO: 1 under conditions at 40°C, and then washed from with 0.5X SSC (IX SSC is 0.15 M NaCl, 0.015 M sodium citrate) at 65°C.

21. The polypeptide of claim 20 wherein the aminopolyol antibiotic is zwittermicin A.

22. A method for controlling phytopathogens comprising applying a composition comprising a microoganism according to any one of claims 8-14 to soil.

23. A method for producing zwittermicin A comprising cultivating a transgenic host organism according to any one of claims 8-14.

24. An antibody that binds to a polypeptide involved in the biosynthesis of an aminopolyol antibiotic, wherein the polypeptide comprises at least a portion of an amino acid sequence encoded by a nucleic acid fragment, such that a complement of the nucleic acid fragment hybridizes to SEQ. YD. NO: 1 under conditions of 40°C, and then washed with 0.5X SSC (IX SSC is 0.15 M NaCl, 0.015 M sodium citrate) at 65°C.

25. The antibody of claim 24 wherein the antibody is selected form the group consisting of a polyclonal antibody, a monoclonal antibody, and an antibody fragment.

26. The antibody of claim 24 or 25, wherein the antibody is obtained from an animal selected from the group consisting of a rabbit, a mouse, a goat, and a rat.