# Patent

## Title
Uses of antimicrobial genes from microbial genome

## Abstract

We describe a method for mining microbial genomes to discover antimicrobial genes and proteins having broad spectrum of activity. Also described are antimicrobial genes and their expression products from various microbial genomes that were found using this method. The products of such genes can be used as antimicrobial agents or as tools for molecular biology.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage application under 35 U.S.C. 371 of International Patent Application No. PCT/US2007/087691, filed on Dec. 15, 2007, which claims priority to U.S. Provisional patent application Ser. No. 60/870,322, filed on Dec. 15, 2006, the contents of both of which is are hereby incorporated by reference in its entirety for all purposes


STATEMENT OF GOVERNMENTAL SUPPORT
This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by U.S. Department of Energy. The government has certain rights in this invention.


REFERENCE TO SEQUENCE LISTING AND TABLE APPENDIX
The attached sequence listing found in paper form is hereby incorporated by reference. The Sequence Listing describes the sequences of SEQ ID NOS: 1-197, antimicrobial genes and their expression products.
The attached Table 2, hereby incorporated by reference, describes the sequences in greater detail. For each gene, the following details were provided: a) The genome of origin; b) Coordinates on that genome; c) Annotation, with Genbank Accession number if one exists; d) An indication whether the gene is predicted to have a signal sequence for secretion; e) The number of covering small clones and fosmids, and an indication whether this number is regarded as statistically significant low coverage; f) Indication whether this gene was found to have low coverage in other genomes as well—for each comparison genome, the following details are given: (1) BLAST e-value between the gene described and the gene in the comparison genome; (2) GC-content of the other genome; (3) Number of small clones covering the gene in the other genome; (4) Number of fosmid clones covering the gene in the other genome; g) Nucleotide sequence of the gene; and h) Polypeptide sequence of the gene.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for mining microbial genomes for genes that kill, or inhibit the growth of, various bacteria. The present invention also relates to genes and gene products that can be used as antibiotics or as tools for molecular biology.
2. Related Art
Microbes (bacteria, archaea, fungi and viruses) frequently produce and secrete compounds aimed at killing other microbes which help them in their continuous struggle for survival in their ecological niche. Such compounds can be small molecule antibiotics, such as the ones produced by various Streptomyces species [Watve, Arch Microbiol. 2001 November; 176(5):386-90], or proteinacious antibiotics, often known as bacteriocins [Riley & Wertz, Annu Rev Microbiol. 2002; 56: 117-37]. Microbes also produce non-secreted defense molecules that help them escape predation by viruses. For example, “abortive infection” genes are suicidal genes that are activated in some bacteria once they sensed they were infected by a virus. These genes lead to the death of the infected bacterium and, hence, to the survival of the surrounding bacterial community [Chopin, Curr Opin Microbiol. 2005 August; 8(4):473-9]. Viruses also frequently produce molecules that inhibit the growth of, or kill, microbial cells by various mechanisms such as degradation of RNA [Sanson, FEMS Microbiol Rev. 1995 August; 17(1-2):141-50], cell lysis [Schuch, Nature. 2002 Aug. 22; 418(6900):884-9] etc.
Proteins that target bacteria have a broad medical and biotechnological application spectrum. They can be used as direct antibiotics for human and veterinary medicine [Gillor 2005, Curr Pharm Des. 2005; 11(8):1067-75], as growth enhancers in livestock [Brashears, 2003. J. Food Prot. 66, 748-754], as food preservatives [Delves-Broughton, Antonie Van Leeuwenhoek. 1996 February; 69(2): 193-202], as genes engineered into probiotic bacteria [Gillor 2005, Curr Pharm Des. 2005; 11(8):1067-75], as killers of phytopathogenic bacteria for crop management [Penyalver 2000, Eur. J. Plant Pathol. 106, 801-810], etc.
One of the popular methods to study the function of a given gene is to clone it into a model bacterial species (with Escherichia coli (E. coli) being the most popularly used model) and to study the expressed product. However, gene products that are toxic to bacteria will usually be unclonable in E. coli due to their negative effect on the bacterial growth. As described below, the present invention provides a method for identifying regions from microbial genomes that are unclonable into E. coli, retrieve antimicrobial genes that reside in these regions, and demonstrate their toxicity to E. coli and other pathogenic microbes. The method relies on an improvement of the microbial genome sequencing process described in URL:<http://www.jgi.doe.gov/sequencing/strategy.html> and links therein.
One aspect of the present invention involves mapping of sequencing clones onto a “finished” (fully sequenced) microbial genomic sequence. Such mapping was noted to be beneficial for detection of toxic proteins also by Roberts (U.S. Patent Application Publication No. US 2006/0014179 A1), who searched for gaps in clone start sites on either sides of open reading frames, and inferred that such open reading frames can encode for toxic genes or endonucleases.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method for identifying antimicrobial genes in a genome comprising the steps of: (a) Read mapping, wherein sequence reads are mapped back on the genomic sequence and clone positions are identified; (b) Clone coverage calculation, wherein, for each position of the analyzed genome sequence, the number of covering clones (clones that span this position) is calculated; (c) Genomic regions identification, wherein regions having no clone coverage (“uncaptured gaps”), and regions having a statistically significant reduction in coverage, are identified; (d) Toxicity level determination, wherein regions having a statistically significant reduction in fosmid-only coverage (in addition to overall reduction on small [2-4 kb] clone coverage) are marked as containing highly toxic genes; and (e) Gene selection and experimental validation, wherein genes residing in each low- or zero-covered region are identified as antimicrobial. The gene products are then experimentally tested for antimicrobial, more specifically bactericidal or bacteriostatic, effect on the growth of various microorganisms.
Antimicrobial genes were found using the present method. The compositions are nucleotide and amino acid sequences for antimicrobial polypeptides and proteins, the uses of which are further described. Specifically, the present invention provides antimicrobial nucleic acids and polypeptides having a sequence set forth in SEQ ID NOs: 1-172 and 179-197, and variants and fragments thereof. The present invention further provides compositions and methods directed to inducing plant pathogen resistance, particularly bacterial resistance.
In one aspect of the invention, an isolated nucleic acid molecule isolated using the prescribed method for identifying antimicrobial genes in a genome, wherein the nucleic acid molecule encodes a protein or RNA molecule having antimicrobial activity.
In one embodiment, the antimicrobial gene isolated from a microbe and found in Table 1. The isolated nucleic acid, wherein the sequence is selected from the group consisting of sequences, SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, and 159-172.
In another aspect, the encoded protein or RNA molecule having antimicrobial activity. In one embodiment, an antimicrobial protein expressed in vitro from the isolated gene of claim 2 and found in Table 1. In another embodiment, the isolated protein or RNA molecule having antimicrobial activity, comprising a sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 179-197.
The isolated protein or RNA molecule having antimicrobial activity, wherein the protein or RNA molecule has antimicrobial activity against a microbe selected from the group consisting of Staphylococcus aureus, microorganisms of the genus Staphylococcus, Escherichia coli, microorganisms of the genus Escherichia, microorganisms of the genus Aspergillus, microorganisms of the genus Candida, microorganisms of the genus Mucor, microorganisms of the genus Absidia, microorganisms of the genus Cryptococcus, microorganisms of the genus Blastomyces, microorganisms of the genus Paracoccidioides, microorganisms of the genus Coccidioides, microorganisms of the genus Sporothrix, microorganisms of the genus Phialophora, microorganisms of the genus Histoplasma, microorganisms of the genus Trichophyton, microorganisms of the genus Microsporum, microorganisms of the genus Epidermophyton, microorganisms of the genus Bacillus, and microorganisms of the genus Yersinia, microorganisms of the genus Salmonella, and microorganisms of the genus Francisella. 
In another aspect, the nucleic acid molecule encoding antimicrobial expression products, and isolated according to the prescribed method for identifying antimicrobial genes in a genome, wherein said nucleotide sequence is optimized for expression in a plant. An expression cassette comprising the nucleotide sequence operably linked to a promoter that drives expression in a plant. The expression cassette further comprising an operably linked polynucleotide encoding a signal peptide.
In another aspect, a plant comprising in its genome at least one stably incorporated expression cassette, said expression cassette comprising a heterologous nucleotide sequence isolated according to the method of identifying antimicrobial genes from a genome, operably linked to a promoter that drives expression in the plant, wherein the plant displays increased resistance to a plant pathogen. The plant is resistant to a microbe, such as Agrobacterium tumefaciens, Burkholderia cenocepacia, Clavibacter michiganensis, Erwinia carotovora, Erwinia chrysanthemi, Leifsonia xyli, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas campestris, Xylella fastidiosa, Spiroplasma kunkelii, and Onion yellows phytoplasma. The promoter is preferably a pathogen-inducible promoter. In another embodiment, a transformed seed of the plant displaying increased resistance to a plant pathogen.
In another aspect, a cell comprising in its genome at least one stably incorporated expression cassette, said expression cassette comprising a heterologous nucleotide sequence isolated according to the method of identifying antimicrobial genes from a genome, operably linked to a promoter that drives expression in the cell.
In another aspect, a method for inducing pathogen resistance in an organism, said method comprising introducing into an organism at least one expression cassette, said expression cassette comprising a heterologous nucleotide sequence isolated according to the method of identifying antimicrobial genes from a genome operably linked to a promoter that drives expression in the organism. In one embodiment, an expression cassette comprising a nucleotide sequence sequence isolated according to the method of identifying antimicrobial genes from a genome operably linked to a promoter that drives expression in a microorganism. In another embodiment, transformed microorganism comprising at least one expression cassette.
It is an object of the invention to provide an antipathogenic composition comprising at least one protein or RNA molecule isolated according to the method of identifying antimicrobial genes from a genome. The composition further comprising a carrier.
Thus, it is provided a method for protecting a vertebrate from a pathogen comprising applying the antipathogenic composition to the environment of a pathogen. In one embodiment, the antipathogenic composition comprising at least one transformed microorganism, comprising at least one expression cassette comprising a nucleotide sequence isolated according to the method of identifying antimicrobial genes from a genome.
It is further provided, a method for preventing infection in a patient comprising applying the antipathogenic composition in a topical preparation to the patient. The antimicrobial composition can be prepared as in an ointment, cream or lotion for topical application.
In another embodiment, a method for preventing food spoilage comprising applying the antipathogenic composition to a food surface. In one embodiment, the antipathogenic composition is in a preparation form for surface administration such as injections, sprays, liquid solutions, liquid coating agents, gel, ointments, or aerosol.
In yet another embodiment, a method for protecting a plant from a pathogen comprising applying the antipathogenic composition to the environment of a plant pathogen. The method wherein the carrier is a surface-active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protective, a buffer, a flow agent or fertilizers, micronutrient donors, or other preparations that may be used to inhibit or control microbial or bacterial infection or growth. The antipathogenic composition further comprising one or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaracides, plant growth regulators, harvest aids, and fertilizers, can be combined with carriers, surfactants or adjuvants customarily employed in the art of formulation. The antimicrobial agent for preparations according to the present invention useful in exhibiting an antimicrobial effect on plant pathogenic microorganisms such as microorganisms of the genus Agrobacterium, microorganisms of the genus Burkholderia, microorganisms of the genus Clavibacter, microorganisms of the genus Erwinia, microorganisms of the genus Ralstonia, microorganisms of the genus Xanthomonas, microorganisms of the genus Pseudomonas, and microorganisms of the genus Leifsonia, microorganisms of the genus Xylella, microorganisms of the genus Spiroplasma. 



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Growth inhibiting genes will have lower clone coverage. Genomic DNA is in black; Clones are in grey. Genes are marked as arrows. Clones spanning the full length of a growth inhibiting gene (marked by red ‘X’) will cause death or growth inhibition and will therefore be eliminated, leading to lower coverage of the gene.
FIG. 2: Measuring clone coverage. For each clone, forward (dark gray) and reverse (light gray) clone mates are aligned to the genomic sequence (black). Coverage of the clone is measured as the genomic coordinates between the beginning of the forward clone mate to the end of the reverse clone mate (including the space in between, marked by dotted line). Therefore, the coverage in position A is 3; the coverage in position B is 2; and the coverage in position C is 1.
FIG. 3: Recurrence of low coverage in homologous regions in different genomes. Shown is the coverage profile of a  ̃95 kb region in three closely related Shewanella genomes. X-axis, genomic position in each genome (in kilobase). Y-axis, clone coverage. Black curve represents coverage of small clones (sized up to 10 kb); red curve represents coverage of fosmids (sized 10 kb-50 kb). Zero coverage of small clones is observed in three homologous regions (A,B and C).
FIG. 4: Coverage of Geobacter gene YP—386193 (gmet—3255; Gene No: 48; SEQ ID NOS: 96-97). Shown is a snapshot from Artemis genome browser [Rutherford, 2000. Bioinformatics 16 (10) 944-945] of a  ̃20 kb region between positions 3,654,200-3,674,000 of the Geobacter metallireducens GS-15 genome. Clone coverage is in the above curves (red curve—small clones [up to 10 kb]; green curve—large [fosmid] clones [10-50 kb]). On the bottom of the screen in the browser (but not shown in FIG. 4) is the consensus sequence and the predicted proteins from each open reading frame. The gene and protein under the minimum coverage is YP—386193. Partial sequences of SEQ ID NOS:96 and 97 are shown and marked by a black box and highlighted)
FIG. 5: Multiple alignment of YP—386193 homologs. Multiple sequence alignment of SEQ ID NOS: 187-196 was performed using clustalW. Invariant cysteine residues are marked by black arrows. The bracket above the alignment shows the conserved signal peptide region.
FIG. 6: Secondary structure prediction of YP—386193. Shown is the YP—386193 protein without the N-terminal signal sequence (SEQ ID NO: 197). Conserved cysteine residues are marked red. Predicted helical sequences are in blue. Secondary structure was predicted using the NNPREDICT software [Kneller, J. Mol. Biol. (214) 171-182], where only predicted helices longer than 5 residues were taken into account. A helical wheel of the first (N-terminal) helix is also presented. Hydrophobic face is marked by parenthesis; the remaining face is composed of hydrophilic residues.
FIG. 7: Positive and negative controls. RegB, a virally encoded protein that is known to be toxic to E. coli, is used here as positive control for the assay, and E. coli beta-galactosidase, known to be non-toxic to E. coli, is the negative control. Two colonies containing the full length RegB gene grow in a medium without IPTG (right plate) but fail to grow in plates containing concentrations of 200 uM or 1000 uM IPTG, where expression of RegB is induced (middle and left plates). Conversely, colonies containing beta-galactosidase under the regulation of IPTG grow even in high concentrations (1000 uM) of IPTG, The genes were engineered into pET11a vector downstream to the T7 promoter. Vectors were transformed into BL21 (DE)pLys cells (Invitrogen) that contain a choromosomal copy of the T7 polymerase under the control of lac promoter. Correct sequence in the inserts was verified by direct sequencing.
FIG. 8: The protein product of YP—386193 inhibits E coli growth following induction of its expression. Ten colonies containing the full length YP—386193 gene grow in a medium without IPTG (left plate) but fail to grow in the presence of IPTG, where expression of YP—386193 is induced (right plate). The gene was amplified from the Geobacter metallireducens genome and engineered into pET11a vector downstream to the T7 promoter. Vectors were transformed into BL21(DE)pLys cells (Invitrogen) that contain a choromosomal copy of the T7 polymerase under the control of lac promoter. Correct sequence in the inserts was verified by direct sequencing.
FIG. 9: Negative control: Mutated YP—386193 insertions do not kill. Experiments were done as described in FIG. 8. Colonies 1C10, 4C4, 4B4 and 4A11 contain mutated YP—386193 inserts containing various deletions in the coding sequence, as detected by direct sequencing. The remaining four colonies contain unmutated YP—386193 inserts. Bacterial growth is inhibited only upon expression induction of unmutated inserts.
FIG. 10: Experimental results on additional genes. Seventy-eight genes that were predicted using the method described in the present invention were tested using the same system described in FIGS. 7 and 8, and found to be toxic to E. coli upon expression induction. FIG. 10 shows photographs of induction of three different genes and the effect on growth of bacteria. Shown are plating results of bacteria containing each gene on plates containing no IPTG, 250 uM IPTG and 800 uM IPTG. Experiment was performed in a 48-well plate format, with each well containing LB-agar-amp+IPTG in the concentration indicated; pictures of individual wells are shown.
FIG. 11: Positive and negative controls to the experiment presented in FIG. 10. RegB (positive control; see FIG. 7) and beta-galactosidase (negative control; see FIG. 7) were cloned and tested in the same system described for FIG. 10 and the experiments in Example 1 (data not shown). RegB causes growth-reduction (toxic) upon expression induction; beta-galactosidase expression does not affect bacterial growth.
FIG. 12: Antimicrobial genes inhibit bacterial growth when present in the growth media. In-vitro transcription/translation system (Roche RTS100 E. coli HY) was used to produce cell-free protein products of toxic genes. Candidate toxic proteins were mixed with E. coli BL21 bacteria growing in liquid LB medium, and growth was monitored for 5.5 hours by measuring Optical Density (OD) in wavelength 600 nm every 10 minutes. Dark-blue curve shows growth of control bacteria without toxic protein addition (average and standard deviation of 9 repetitions presented); yellow, cyan and pink curves represent 3 repetitions of this growth kinetics experiment for a single toxic protein mixed with the medium. FIG. 12(A) shows antimicrobial activity of protein ABB43836 (SEQ ID NO: 30); and FIG. 12B, shows antimicrobial activity of protein ABB43762 (SEQ ID NO: 46).
FIG. 13: Coverage deficiency in intergenic region in the Burkholderia cenocepacia HI2424 genome. Shown is a snapshot from Artemis genome browser [Rutherford, 2000. Bioinformatics 16 (10) 944-945] of a  ̃22 kb region between positions 748,000-770,000 of chromosome I of the Burkholderia cenocepacia HI2424 genome. Clone coverage is in the above curves (red curve—small clones [up to 10 kb]; green curve—large clones [10-50 kb]). The arrow indicates that the region that has the minimum clone coverage is an intergenic region.
FIG. 14: Multiple sequence alignment of DNA found in homologous low covered intergenic regions from 4 Burkholderia and 2 Cupriavidus (also herein referred to as Ralstonia) species. Red circle marks the conserved sequence core. Blue circle marks a conserved secondary structure predicted to be a rho independent transcriptional terminator. Compensatory mutations in the stem, where a G:C pair is changed to an A:T pair to maintain the stem are marked by blue arrows. An illustration of a typical a rho independent terminator is shown in the lower right side for comparison.
FIG. 15: Structural alignment of the toxic small RNA from 4 Burkholderia and 2 Cupriavidus (Ralstonia) species (SEQ ID NOS:173-178). Multiple sequence alignment of predicted small RNAs is presented. The last row in each alignment block represent the conserved RNA secondary structure in the Infernal format [URL:<http://selabjanelia.org/software.html>], where concentric “<” and “>” signs represent base-pairing within a stem, ‘_’ signs represent loops, “,” represent unstructured parts of the RNA and “.” signs represent gaps. An insertion within the first stem is apparent in the Cupriavidus sequences. The conserved −10 TATA box of the predicted promoter is highlighted in red.
FIG. 16: Toxic small RNA gene structure in Burkholderia (SEQ ID NO:182). Secondary structure calculation was preformed using the RNAfold server [URL:<http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi>]. The transcriptional terminator is marked by blue circle; the conserved core is marked by red circle.
FIG. 17: Toxic small RNA genes structure in Cupriavidus (Ralstonia) genomes. Secondary structure calculation was preformed using the RNAfold server [URL:<http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi>]. The transcriptional terminator is marked by blue circles; the conserved core is marked by red circles. An obvious extension of the first stem structure relative to the Burkholderia RNA is apparent. The small RNA structure in the top panel is SEQ ID NO:179, and the bottom structure is SEQ ID NO:180.
FIG. 18: Two additional RNAs in Burkholderia. Secondary structure calculation was preformed using the RNAfold server [URL:<http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi>]. The transcriptional terminator is marked by blue circles; the conserved core is marked by red circles. Structures of these two RNAs are similar to the above described one, except for the missing first stem. The top structure is SEQ ID NO:185 and the bottom structure is SEQ ID NO:186.
FIG. 19: Low coverage in RNA #2 (SEQ ID NO: 185). Shown is a snapshot from Artemis genome browser [Rutherford, 2000. Bioinformatics 16 (10) 944-945] of a  ̃22 kb region between positions 3,462,000-3,484,000 of chromosome I of the Burkholderia cenocepacia HI2424 genome. Clone coverage is in the above curves (red curve—small clones [up to 10 kb]; green curve—large clones [10-50 kb]). RNA #2 is marked as ‘scRNA’.
FIG. 20: Low coverage in RNA #3 (SEQ ID NO: 186). Shown is a snapshot from Artemis genome browser [Rutherford, 2000. Bioinformatics 16 (10) 944-945] of a  ̃22 kb region between positions 341,000-363,000 of chromosome I of the Burkholderia cenocepacia HI2424 genome. Clone coverage is in the above curves (red curve—small clones [up to 10 kb]; green curve—large clones [10-50 kb]). RNA #3 is marked as ‘scRNA’.
FIG. 21: Small RNAs inhibit E. coli growth following induction of their expression. Colonies containing four small RNA genes grow in a medium without IPTG (left plate) but fail to grow in the presence of IPTG, where expression of small RNAs is induced (right plates). The three small RNAs identified in Burkholderia cenocepacia HI2424 and one of the orthologous small RNA in Ralstonia metallidurans (SEQ ID NOS: 182, 185, 186 and 179) were amplified from their genome of origin and engineered into pcrSmart vector (Lucigen) directly downstream from the T7 promoter. Vectors were transformed into BL21 (DE)pLys cells (Invitrogen) that contain a choromosomal copy of the T7 polymerase under the control of lac promoter. Correct sequence in the inserts was verified by direct sequencing.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Introduction
In one embodiment, the present invention provides a method for identifying regions from microbial genomes that are unclonable into E. coli, wherein the method relies on a derivative of the microbial genome sequencing process.
Typically, the process of microbial genome sequencing is comprised of the following components: First, the genome of the microbe to be sequenced is isolated in multiple copies. Then, the genome is sheared into fragments typically sized 2-4 kb. These fragments are inserted into a carrying plasmid (vector) and each insert-containing vector is transformed into an E. coli bacterium. In some genome sequencing projects fragments of larger size, typically 30-40 kb, are also included in larger vectors called “fosmids”. Vectors carrying small inserts (2-4 kb) are usually found in multiple copies (100-500) in each E. coli host, while fosmids are typically found in a single copy.
Each vector-containing E. coli bacterium is allowed to replicate into a clone, thus creating multiple copies of each insert. Inserts are then sequenced from both their ends using primers matching each end of the vector. Sequence reads that correspond to both ends of the same insert (“clone”) are often called “sister-reads”, “clone-mates”, or “mate-pair”. In the next stage, called genome-assembly, overlaps between resulting reads are used to assemble longer contiguous sequences, called “contigs”. These overlaps are enabled because the amount of DNA to be sequenced is taken such that, on average, each base in the assembled genome is covered by multiple reads (typically at least 8 reads). Despite this fact, genome sequencing projects often need a final stage called “finishing”, in which gaps between assembled contigs are filled [Carraro, Biotechniques. 2003 March; 34(3):626-8, 630-2].
Gaps in the assembly can be classified into two categories: “captured” and “uncaptured”. Captured gaps are gaps for which there is evidence that the missing DNA was successfully cloned but was not successfully sequenced. For example, two sister reads of the same clone can exist at the edges of two different contigs, indicating that the missing DNA lies in the clone from which the sister reads originated. Self-folding, or low complexity, DNA is often the cause for such gaps, and they are typically filled by using different sequencing chemistry that allows the melting of folded DNA.
Uncaptured gaps, on the other hand, are gaps in which the missing DNA was not successfully cloned at all. Cloning independent methods, such as PCR and 454 sequencing can be used to sequence the missing DNA in these gaps [Carraro, Biotechniques. 2003 March; 34(3):626-8, 630-2].
Gaps in or areas of low clone coverage were investigated to determine that genes that target bacteria or inhibit its growth likely exist in these regions, making them an unclonable. In some cases, growth inhibiting genes will not cause uncaptured gaps. This would happen if two (or more) overlapping clones contain parts of the gene but none of them contain the full gene sequence (FIG. 1A). Another possibility is that an immunity gene lies in the genomic vicinity of the growth inhibiting gene. Such immunity genes often protect toxin-producing bacteria from being affected by their own toxins [Sablon, Adv Biochem Eng Biotechnol. 2000; 68:21-60]. In this case, DNA fragments containing the toxic gene will be clonable if they contain the immunity gene as well (FIG. 1B). However, in both these cases the overall clonability of the region will be lower, as some clones will be lethal to the host E coli (for example, clones that contain the lethal gene without the full immunity gene; see FIG. 1).
Thus, the present invention provides a method to discover these unclonable regions in sequenced (and “finished”) microbial genomes, retrieve the killer genes residing in these regions, and demonstrate their toxicity to E. coli and other pathogenic bacteria. The present method also detects genes lying in genomic regions that have a statistically significant reduction in clone coverage.
The present invention also provides the identity and characterizes several genes in microbial genomes investigated and describes the use of the genes and gene products as anti-microbial agents.
DEFINITIONS
As used herein, the term, “host cell,” refers to any cell that can be transformed by foreign DNA where the foreign DNA may be a plasmid or vector containing a gene and the gene can be expressed in the cell. The host cell can be a cell from an organism, for example, microbial, including bacterial, fungal, and viral, plant, animal, or mammalian.
As used herein, the term, “library,” “clone library” or “genomic library” refers to a set of clones containing DNA fragments randomly generated by fragmentation of a genome or large DNA fragment, inserted into a suitable plasmid vector and cloned into a suitable host organism, such as E. coli. Sequencing of clones in a library involves carrying out sequence reactions to sequence the beginning and the end of the DNA fragment inserted into each sequenced clone, also referred to as “end sequences”, or “reads”. The genome or large DNA fragments may be from any eukaryote, including human, mammal, plant or fungus, or prokaryote, including bacteria, virus or archaea.
As used herein, the term, “read,” refers to a sequence corresponding to stretches of nucleotide sequence of on average 200-1000 bp in length, acquired from a single end-sequencing event of a clone in a genomic library.
As used herein, the term “sister reads” or “clone mates” refers to two reads that come from the beginning (forward read) and the end (reverse read) of the same cloned DNA fragment.
As used herein, the term, “mapping,” refers to finding the correct position of a read on an already assembled genomic sequence. The term “clone mapping” refers to finding the correct position of two sister reads on an already assembled genomic sequence.
As used herein, the term, “shotgun sequencing” or “sequencing,” refers the sequencing strategy whereby an entire genomic library is sequenced and assembled as described by the methods found at URL:<http://wwwjgi.doe.gov/sequencing/strategy.html>. The advantage of shotgun sequencing is that a majority, if not all, of the genomic sequence will be represented by random clones about 5-20 times, depending on the number and the sizes of clones in the library.
As used herein, the term, “clone coverage” refers to the number of clones in a library that span a particular position in a genome. A position in the genome is considered as covered by a clone if it is found between the first position of the forward-strand clone mate and the last position of the reverse strand clone mate (see FIG. 2). “Low clone coverage” refers to a particular position or a region having statistically significant under-representation of clones than expected by chance.
As used herein, the term, “gap” refers to a region of the genome or the large DNA fragment where there is an absence or low coverage.
As used herein, the term, “finished” when used referring to a genome, or large DNA fragment, refers to when all or most gaps in the sequence have been closed following additional specific sequencing reactions, and assembly of the final consensus sequence is completed.
As used herein, the term “toxic” when used to define a gene, refers to a gene whose expression product inhibits the growth of a microorganisms, such as bacteria and archae. For example, a toxic gene can be a gene which when expressed in a host cell, causes the host cell to become nonviable or causes cell death, and is thus “toxic” to the cell.
As used herein, the terms, “antipathogenic” and “antimicrobial,” are used interchangeably, e.g., as used in “antipathogenic compositions,” “antimicrobial agents” or “antimicrobial genes or proteins,” and are intended to mean that the compositions have antimicrobial activity and thus are capable of suppressing, controlling, inhibiting and/or killing microorganisms, such as bacteria and archae. An antimicrobial polypeptide of the invention will, for example, reduce the disease symptoms resulting from microbial invasion or challenge by at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80%, or at least about 50% to about 90% or greater.
As used herein, the term “nucleic acid” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.
As used herein, the terms “polypeptide” and “protein” are used interchangeably and are intended to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides of the invention can be produced either from a nucleic acid disclosed herein, or by the use of standard molecular biology techniques. For example, a truncated protein of the invention can be produced by expression of a recombinant nucleic acid of the invention in an appropriate host cell, or alternatively by a combination of ex vivo procedures, such as protease digestion and purification, or in-vitro peptide synthesis.
As used herein, “variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. One of skill in the art will recognize that variants of the nucleic acids of the invention will be constructed such that the open reading frame is maintained. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the antimicrobial polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotide, such as those generated, for example, by using site-directed mutagenesis but which still encode an antimicrobial protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs.
Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, an isolated polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 97 is disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
“Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, antimicrobial activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native antimicrobial protein of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the antimicrobial proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.
Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences and their variants as well as mutant forms. Likewise, the proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired antimicrobial activity.
In nature, some polypeptides are produced as complex precursors which, in addition to targeting labels such as the signal peptides discussed elsewhere in this application, also contain other fragments of peptides which are removed (processed) at some point during protein maturation, resulting in a mature form of the polypeptide that is different from the primary translation product (aside from the removal of the signal peptide). “Mature protein” refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor protein” or “prepropeptide” or “preproprotein” all refer to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may include, but are not limited to, intracellular or extracellular localization signals. “Pre” in this nomenclature generally refers to the signal peptide. The form of the translation product with only the signal peptide removed but no further processing yet is called a “propeptide” or “proprotein.” The fragments or segments to be removed may themselves also be referred to as “propeptides.” A proprotein or propeptide thus has had the signal peptide removed, but contains propeptides (here referring to propeptide segments) and the portions that will make up the mature protein. The skilled artisan is able to determine, depending on the species in which the proteins are being expressed and the desired intracellular location, if higher expression levels or higher antimicrobial activity might be obtained by using a gene construct encoding just the mature form of the protein, the mature form with a signal peptide, or the proprotein (i.e., a form including propeptides) with a signal peptide. For optimal expression in plants or fungi, the pre- and propeptide sequences may be needed. The propeptide segments may play a role in aiding correct peptide folding.
Method for Investigation of Gaps or Areas of Low Clone Coverage
For each finished genome, the present method to select for antipathogenic and antimicrobial genes is applied comprising the following steps: (a) Read mapping, wherein sequence reads are mapped back on the genomic sequence and clone positions are identified; (b) Clone coverage calculation, wherein, for each position of the analyzed genome sequence, the number of covering clones (clones that span this position) is calculated; (c) Genomic regions identification, wherein regions having no clone coverage (“uncaptured gaps”), and regions having a statistically significant reduction in coverage, are identified; (d) Toxicity level determination, wherein regions having a statistically significant reduction in fosmid-only coverage (in addition to overall reduction on small [2-4 kb] clone coverage) are marked as containing highly toxic genes; and (e) Gene selection and experimental validation, wherein genes residing in each low- or zero-covered region are identified, and their products are experimentally tested for a bactericidal or bacteriostatic effect on the growth of various bacteria.
In Read mapping, for each microbial genome that was sequenced and finished, map the original reads back on the finished, assembled genomic sequence. This mapping could be done by a sequence alignment tool, such as BLAST [Altschul, J Mol. Biol. 1990 Oct. 5; 215(3):403-10] or mummer [Delcher, Nucleic Acids Res. 2002 Jun. 1; 30(11):2478-83]. In case that a read aligns to several positions on the genomic sequence, take the region where the alignment has the highest score or a score above a certain threshold.
For each read, identify the position of its clone mate. In case a read has two or more similarly scored positions on the genomic sequence, resolve the correct position by the location of its mate. The two mates should be positioned such that the distance between them is approximately the relevant insert size (usually 2-4 kb or 30-40 kb in case of fosmid-carried inserts). The two mates should also be positioned such that one of them lies on the forward strand and the other on the reverse strand. Clones for which both mates have unambiguous positioning on the genomic sequence are deemed “mapped clones” and taken into further analysis.
In Clone coverage calculation, for each position in the genomic sequence, the number of covering clones is counted. A position in the genome is considered as covered by a clone if it is found between the first position of the forward-strand clone mate and the last position of the reverse strand mate (see FIG. 2). It is possible to calculate, for each genomic position, also fosmid-only- and small-clones-only-coverage parameter. This is useful for determining the level of toxicity of a protein in a low-coverage region (described below).
In Genomic regions identification, for each analyzed genome, the average clone coverage per genomic position and standard deviation are calculated. Regions not covered by any clone, or regions covered by statistically significant less clones than expected by chance, are further analyzed. Genomic regions identification could be done for fosmid-only-clones and small-clones-only-clones, such that regions in which only small-clones have low coverage, regions in which only fosmids have low coverage, and regions in which both library types have low coverage, could be identified.
Regions that are mapped to repetitive genomic sequences (i.e., long sequence stretches that occur two or more times in the genome with high similarity between occurrences) are excluded from the analysis. This is because repetitive genomic regions frequently cause false read-mapping, and hence can appear low covered even though they are not. The identification of repetitive genomic sequences can be done by aligning the genome to itself (using standard alignment software such as Blast), or by following reads that map to two or more regions in the genome, or by other means.
In Gene selection and experimental validation, for each selected genomic region, genes (protein coding and non-protein coding) are identified. If the region is large enough to cover several genes, multiple genes can be selected from a single region.
An optional Toxicity level determination stage could be added, in which genes are predicted to have high or low toxicity according to their fosmid-clones-only coverage. The small clones (2-4 kb) exist in the cell in high-copy number (100-500 copies), and thus the toxicity of genes that are only mildly toxic is enhanced 100-500 fold if cloned into a small-clone library. Large (fosmid) clones, on the other hand, appear only in a single-copy per cell, and thus, if a region has a statistically significant low (or zero) fosmid coverage (as well as low (or zero) small-clone coverage), it means that the gene is highly toxic, because it confers growth inhibition even when introduced into E coli in a single copy.
To test if the protein products of the selected genes inhibit bacterial growth, cell-free protein synthesis could be used to translate the DNA sequence of each gene into protein. Proteins can be applied on various pathogenic and non-pathogenic bacteria to determine the spectrum of activity and whether they have a bactericidal or bacteristatic effect. Minimal inhibitory concentration (MIC) can be determined by testing the growth inhibition activity of serial dilutions of the protein.
To test if the products of the selected genes inhibit bacterial growth when introduced from inside the cell, selected genes can be cloned into a vector that contains a tightly regulated inducible promoter, such that the expression of the gene is induced only after a specific molecule (“inducer”) was added to the growth media. Such vectors could be inserted into E coli without killing it, as the gene product will only be expressed in the cell following induction. Growth of E. coli can be tested before and after induction to determine if the gene has a growth inhibition effect.
Detection of Candidate Genes
The present method was applied to over 30 microbial genomes. To exclude the possibility that the observed low coverage is the result of random fluctuations in clone distribution, we examined the coverage in the genomes of three closely related Shewanella species. As exemplified in FIG. 3, we found that similar (homologous) sequences have a similar coverage profile across all three genomes. This shows that low clone coverage is caused by specific sequences and not by chance.
We then collected the proteins found in the zero- and low-covered regions. For each of the proteins, we looked for homologs in the genomes we analyzed, and tested the coverage of the homologs in those genomes. We prioritized genes according to the following parameters:
a) Genes that had zero- or low-coverage in more than one genome.
b) Genes that displayed coverage deficiency of both small clones and fosmid clones. As vectors carrying small inserts are usually found in a high copy number (100-500) in each E coli cell, the effect of a toxic gene carried on a small insert vector is amplified 100-500 fold. However, if a gene also has low fosmid coverage it is probably highly toxic, because fosmids are maintained in a single copy per cell only.
c) Genes that had homologs in only few (or zero) other genomes, as genes generating antimicrobial agents are known to evolve very fast [Riley, Annu Rev Microbiol. 2002; 56:117-37].
A list of 125 selected genes was compiled as Set 1. The 125 potentially antimicrobial genes discovered using the present invention were detailed.
dWe noted that among the proteins predicted by the present method to be toxic to E. coli, ribosomal proteins are frequently found. This is probably due to the fact that these proteins are parts in the large complex of the ribosome, and their expression should be strictly coordinated to maintain their relative concentrations. An ectopic addition of another copy of a ribosomal protein disrupts this balance and may cause ribosomal misassembly. Indeed, ribosomal proteins are found in a single copy in nearly every microbial genome sequenced so far. Massive ribosomal misassembly or malfunction would reduce translation of cellular proteins leading to overall growth inhibition.
Once compiled, we tested the 125 proteins from Set 1 using an inducible expression system. It was found that 78 of these proteins are indeed toxic when expressed in E. coli. These 78 genes and proteins are described in the attached Sequence Listing and have sequence identifiers of SEQ ID NOS: 1-158.
Table 2 is also attached and provides details for each gene and its expression products. For each gene, the following details were provided: a) The genome of origin; b) Coordinates on that genome; c) Annotation, with Genbank Accession number if one exists; d) An indication whether the gene is predicted to have a signal sequence for secretion; e) The number of covering small clones and fosmids, and an indication whether this number is regarded as statistically significant low coverage; f) Indication whether this gene was found to have low coverage in other genomes as well—for each comparison genome, the following details are given: (1) BLAST e-value between the gene described and the gene in the comparison genome; (2) GC-content of the other genome; (3) Number of small clones covering the gene in the other genome; (4) Number of fosmid clones covering the gene in the other genome; g) Nucleotide sequence of the gene; and h) Polypeptide sequence of the gene.
Exemplary data from several examples are shown in FIG. 10. Table 1 below shows the gene number, internal reference number and the corresponding sequence identifier for the nucleotide and protein sequences in the first four columns. Results are shown below in Table 1 for each protein found to have antimicrobial activity, showing the lowest concentration (uM) of IPTG needed to induce gene expression and inhibition of bacterial growth was observed. “Inhibition of growth” is defined as no more than 10 colonies observed in a well of a 48-well growth plate.







TABLE 1







The gene, Internal Reference number, sequence identifiers of antimicrobial genes


and proteins in the Sequence Listing which were tested and shown to have antimicrobial


activity and the lowest IPTG concentration needed for antimicrobial activity.















Concentration of






IPTG resulting in



Internal


growth elimination


Gene No.
Reference
Nucletoide
Protein
(μM)





Gene No. 1
ABB44040
SEQ ID NO: 1
SEQ ID NO: 2
250


Gene No. 2
ABB44032
SEQ ID NO: 3
SEQ ID NO: 4
250


Gene No. 3
ABB44015
SEQ ID NO: 5
SEQ ID NO: 6
800


Gene No. 4
ABB44001
SEQ ID NO: 7
SEQ ID NO: 8
800


Gene No. 5
ABB43993
SEQ ID NO: 9
SEQ ID NO: 10
800


Gene No. 6
ABB43996
SEQ ID NO: 11
SEQ ID NO: 12
800


Gene No. 7
ABB43996_1
SEQ ID NO: 13
SEQ ID NO: 14
800


Gene No. 8
ABB43899
SEQ ID NO: 15
SEQ ID NO: 16
250


Gene No. 9
ABB43889
SEQ ID NO: 17
SEQ ID NO: 18
100


Gene No. 10
ABB43891
SEQ ID NO: 19
SEQ ID NO: 20
250


Gene No. 11
ABB43856
SEQ ID NO: 21
SEQ ID NO: 22
800


Gene No. 12
ABB43853
SEQ ID NO: 23
SEQ ID NO: 24
600


Gene No. 13
ABB43839
SEQ ID NO: 25
SEQ ID NO: 26
600


Gene No. 14
ABB43838
SEQ ID NO: 27
SEQ ID NO: 28
400


Gene No. 15
ABB43836
SEQ ID NO: 29
SEQ ID NO: 30
600


Gene No. 16
ABB43820
SEQ ID NO: 31
SEQ ID NO: 32
250


Gene No. 17
ABB43819
SEQ ID NO: 33
SEQ ID NO: 34
250


Gene No. 18
ABB43823
SEQ ID NO: 35
SEQ ID NO: 36
600


Gene No. 19
ABB43822
SEQ ID NO: 37
SEQ ID NO: 38
600


Gene No. 20
3634490_gene_1
SEQ ID NO: 39
SEQ ID NO: 40
100


Gene No. 21
ABB43761
SEQ ID NO: 41
SEQ ID NO: 42
600


Gene No. 22
ABB43760
SEQ ID NO: 43
SEQ ID NO: 44
800


Gene No. 23
ABB43762
SEQ ID NO: 45
SEQ ID NO: 46
800


Gene No. 24
ABB43749
SEQ ID NO: 47
SEQ ID NO: 48
400


Gene No. 25
ABB43724
SEQ ID NO: 49
SEQ ID NO: 50
600


Gene No. 26
ABB43725
SEQ ID NO: 51
SEQ ID NO: 52
600


Gene No. 27
ABB43718
SEQ ID NO: 53
SEQ ID NO: 54
600


Gene No. 28
ABB43700
SEQ ID NO: 55
SEQ ID NO: 56
800


Gene No. 29
ABB43688
SEQ ID NO: 57
SEQ ID NO: 58
600


Gene No. 30
ABB43689
SEQ ID NO: 59
SEQ ID NO: 60
250


Gene No. 31
ABB43694
SEQ ID NO: 61
SEQ ID NO: 62
400


Gene No. 32
ABB43693
SEQ ID NO: 63
SEQ ID NO: 64
800


Gene No. 33
YP_516003
SEQ ID NO: 65
SEQ ID NO: 66
250


Gene No. 34
ABB43675_1
SEQ ID NO: 67
SEQ ID NO: 68
400


Gene No. 35
ABB43641
SEQ ID NO: 69
SEQ ID NO: 70
800


Gene No. 36
ABB43625
SEQ ID NO: 71
SEQ ID NO: 72
400


Gene No. 37
ABB43630
SEQ ID NO: 73
SEQ ID NO: 74
800


Gene No. 38
ABB43475
SEQ ID NO: 75
SEQ ID NO: 76
600


Gene No. 39
ABB43462_1
SEQ ID NO: 77
SEQ ID NOs: 78, 79
400


Gene No. 40
ABB43753
SEQ ID NO: 80
SEQ ID NO: 81
600


Gene No. 41
ABB43755
SEQ ID NO: 82
SEQ ID NO: 83
800


Gene No. 42
ABB44224
SEQ ID NO: 84
SEQ ID NO: 85
400


Gene No. 43
ABB45229
SEQ ID NO: 86
SEQ ID NO: 87
100


Gene No. 44
ABB45230
SEQ ID NO: 88
SEQ ID NO: 89
600


Gene No. 45
ABB45231
SEQ ID NO: 90
SEQ ID NO: 91
400


Gene No. 46
ABB45232
SEQ ID NO: 92
SEQ ID NO: 93
400


Gene No. 47
ABB43866
SEQ ID NO: 94
SEQ ID NO: 95
600


Gene No. 48
ABB43867
SEQ ID NO: 96
SEQ ID NO: 97
400


Gene No. 49
ABB43869
SEQ ID NO: 98
SEQ ID NO: 99
400


Gene No. 50
ABB43678_1
SEQ ID NO: 100
SEQ ID NO: 101
800


Gene No. 51
ABB43998
SEQ ID NO: 102
SEQ ID NO: 103
100


Gene No. 52
ABB43913
SEQ ID NO: 104
SEQ ID NOs: 105, 106
600


Gene No. 53
ABB43902
SEQ ID NO: 107
SEQ ID NO: 108
600


Gene No. 54
ABB43892
SEQ ID NO: 109
SEQ ID NO: 110
100


Gene No. 55
ABB43890
SEQ ID NO: 111
SEQ ID NO: 112
400


Gene No. 56
ABB43841
SEQ ID NO: 113
SEQ ID NO: 114
400


Gene No. 57
ABB43827
SEQ ID NO: 115
SEQ ID NO: 116
400


Gene No. 58
ABB43821
SEQ ID NO: 117
SEQ ID NO: 118
400


Gene No. 59
ABB43768
SEQ ID NO: 119
SEQ ID NO: 120
400


Gene No. 60
ABB43764
SEQ ID NO: 121
SEQ ID NO: 122
400


Gene No. 61
ABB43748
SEQ ID NO: 123
SEQ ID NO: 124
400


Gene No. 62
ABB43726
SEQ ID NO: 125
SEQ ID NO: 126
400


Gene No. 63
ABB43722
SEQ ID NO: 127
SEQ ID NO: 128
600


Gene No. 64
ABB43712
SEQ ID NO: 129
SEQ ID NO: 130
400


Gene No. 65
ABB43702
SEQ ID NO: 131
SEQ ID NO: 132
800


Gene No. 66
ABB43704
SEQ ID NO: 133
SEQ ID NO: 134
100


Gene No. 67
ABB43690
SEQ ID NO: 135
SEQ ID NO: 136
250


Gene No. 68
ABB43674
SEQ ID NO: 137
SEQ ID NO: 138
400


Gene No. 69
ABB43678
SEQ ID NO: 139
SEQ ID NO: 140
800


Gene No. 70
ABB43658
SEQ ID NO: 141
SEQ ID NO: 142
400


Gene No. 71
ABB43659
SEQ ID NO: 143
SEQ ID NO: 144
600


Gene No. 72
ABB43655
SEQ ID NO: 145
SEQ ID NO: 146
250


Gene No. 73
ABB43638
SEQ ID NO: 147
SEQ ID NO: 148
800


Gene No. 74
ABB43474
SEQ ID NO: 149
SEQ ID NO: 150
800


Gene No. 75
ABB43461_1
SEQ ID NO: 151
SEQ ID NO: 152
600


Gene No. 76
ABB43457
SEQ ID NO: 153
SEQ ID NO: 154
800


Gene No. 77
ABB43446
SEQ ID NO: 155
SEQ ID NO: 156
600


Gene No. 78
ABB43750
SEQ ID NO: 157
SEQ ID NO: 158
600









To verify that the 78 proteins identified by our method are bona fide antimicrobial proteins, antimicrobial activity can be determined in various ways. In one embodiment, as also described in Example 2, an in-vitro transcription/translation system (e.g., Roche RTS 100 E. coli HY) can be used to produce cell-free protein products of the 78 toxic genes. Candidate toxic proteins are mixed with E. coli strain BL21 bacteria growing in liquid LB medium, and growth is then monitored for several hours by measuring Optical Density (OD) in wavelength 600 nm every 10 minutes. For each protein tested, the OD is measured for growth of control bacteria without toxic protein addition to the medium containing an in-vitro transcription/translation system and compared with growth curves after toxic protein addition. An empirical determination can be made for each protein to determine that they are antimicrobial, such as if the endpoint is at least 0.1 OD lower than the control, or that there is an obvious decrease in the slope of the three curves showing growth is inhibited, then it is concluded that the protein has antimicrobial activity.
For two of the genes and proteins shown in Table 1, growth of bacteria was inhibited in the presence of the translated toxic protein, as compared to growth without toxic proteins in the medium (FIGS. 12A and 12B). Data is not shown for all genes tested. FIGS. 12A and 12B show examples of the OD results obtained for two proteins, ABB43836 (SEQ ID NO: 30) and ABB43762 (SEQ ID NO: 46). It is anticipated that many if not all of the proteins identified in Table 1 have antimicrobial activity and inhibit growth of bacteria and other microbes. These results are significant in showing that not only internal expression of these proteins is toxic, but that the proteins exhibit antimicrobial activity and inhibit growth even if externally applied to a microbe or its environment. This aspect thus enables many of the applications described in the following sections.
These results in totality validate the presently described method in the present invention to identify toxic genes. In another embodiment, the presently described method can be used to identify other genomic elements having antimicrobial activity. Such genomic elements can include but are not limited to, genomic sequence which code for proteins such as genes, various types of RNA, small RNA, etc.
Thus, in another embodiment, the present invention further provides the genes and proteins, SEQ ID NOS:1-158 and identified in Table 1 as having antimicrobial activity. In another embodiment, the present invention provides for small toxic RNA isolated from other organisms. In one embodiment, antimicrobial genes comprise sequences SEQ ID NOS: 159-172, which encode small toxic RNA having antimicrobial activity. In another embodiment, the small toxic RNA found to have antimicrobial activity comprise SEQ ID NOS:179-186, and are encoded by antimicrobial genes having sequences SEQ ID NOS:165-172.
Antimicrobial Polypeptides and Uses Thereof
In one embodiment, to determine whether a protein identified by the described method is indeed antimicrobial, the gene encoding the protein is cloned into an appropriate plasmid under a polymerase promoter, inserted into vector, and used to transform cells, such as E. coli. This system maintains the expression of the inserted gene silent unless an inducer molecule (e.g., IPTG) is added to the medium. As a negative control a non-toxic gene, such as beta-galactosidase (beta-gal) is similarly cloned. FIG. 7 shows that beta-galactosidase does not kill, or inhibit the growth of, E coli following induction of its expression.
Bacterial colonies are allowed to grow after induction of gene expression. The lack of colonies indicates that the protein expressed by the inserted gene is toxic to the cells, showing that the protein has a growth inhibition effect on cells such as E coli, and is thus an antimicrobial agent.
Additionally, in vitro antimicrobial assays that can be used include, for example, the addition of varying concentrations of the antimicrobial composition to paper disks and placing the disks on agar containing a suspension of the pathogen of interest. Following incubation, clear inhibition zones develop around the discs that contain an effective concentration of the antimicrobial polypeptide (Liu et al. (1994) Plant Biology 91:1888-1892, herein incorporated by reference). Additionally, microspectrophotometrical analysis can be used to measure the in vitro antimicrobial properties of a composition (Hu et al. (1997) Plant Mol. Biol. 34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233, both of which are herein incorporated by reference). Assays that specifically measure antibacterial activity are also well known in the art. See, for example, Clinical and Laboratory Standards Institute, Guideline M7-A6, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, herein incorporated by reference.
In one embodiment, the presently described genes, proteins and/or RNA described in SEQ ID NOS: 1-172, and 179-197, and herein referred to as generally antimicrobial compositions or antimicrobial agents, are contemplated for use in any of the antimicrobial applications herein described.
In another embodiment, an antipathogenic polypeptide, selected from any of the polypeptide sequences in SEQ ID NOS:1-159, is localized in cellular compartments by addition of suitable targeting information. This can be accomplished, for example, by adding an endoplasmic reticulum retention signal encoding sequence to the sequence of the gene, or by adding a signal peptide sequence to the antipathogenic polypeptide.
In some embodiments, expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence of the invention, i.e., any nucleotide sequence in SEQ ID NOS:1-172, and 179-186, that encodes an antimicrobial RNA or polypeptide are further provided. The expression cassettes of the invention find use in generating transformed plants, plant cells, and microorganisms and in practicing the methods for inducing plant pathogen resistance disclosed herein. The expression cassette will include 5′ and 3′ regulatory sequences operably linked to a polynucleotide of the invention. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotide that encodes an antimicrobial RNA or polypeptide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional initiation region (i.e., a promoter), translational initiation region, a polynucleotide of the invention, a translational termination region and, optionally, a transcriptional termination region functional in the host organism. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the polynucleotide of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the polynucleotide of the invention may be heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
Where appropriate, the polynucleotides may be optimized for increased expression in the transformed organism. For example, the polynucleotides can be synthesized using preferred codons for improved expression.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFPTM from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
Generally, it will be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen.
In one embodiment, a plant, transformed by the antimicrobial RNA or polypeptides of the present invention is a plant (or an offspring thereof) rendered resistant to a plant-pathogenic microorganism etc., which is regenerated on the basis of host plant cells transformed with the gene of the present invention located under the control of a suitable promoter capable of functioning in plant cells, or with the gene of the present invention integrated in a suitable vector. The transformed plant of the present invention can express, in its body, the protein having an antimicrobial activity according to the present invention.
The expression vector usable in the method of transforming plant cells with the gene of the present invention include pUC vectors (for example pUC118, pUC119), pBR vectors (for example pBR322), pBI vectors (for example pBI112, pBI221), pGA vectors (pGA492, pGAH), pNC (manufactured by Nissan Chemical Industries, Ltd.). In addition, virus vectors can also be mentioned. The terminator gene to be ligated includes 35S terminator gene and Nos terminator gene.
The method of introducing the constructed expression vector into a plant includes an indirect introduction method and a direct introduction method. The indirect introduction includes, for example, a method using Agrobacterium. The direct introduction method includes, for example, an electroporation method, a particle gun method, a polyethylene glycol method, a microinjection method, a silicon carbide method etc.
The method of regenerating a plant individual from the transformed plant cells is not particularly limited, and may make use of techniques known in the art.
In another embodiment, the antimicrobial proteins of the present invention can be produced by methods used conventionally for protein purification and isolation by a suitable combination of various kinds of column chromatography (C18, C8, gel filtration, ion-exchange), prepared by a chemical synthesis method using a peptide synthesizer (for example, peptide synthesizer 430A manufactured by Perkin Elmer Japan) or by a recombination method using a suitable host cell selected from prokaryotes and eukaryotes.
In another embodiment, an expression vector having any one of the nucleic acid sequences in SEQ ID NOS: 1 to 172 and amplifiable in a desired host cells is used to transform bacteria, yeasts, insects or animal cells, and the transformed cells are cultured under suitable culture conditions, whereby a large amount of the protein can be obtained as a recombinant. Culture of the transformant can be carried out by general methods.
The method used in purifying the protein of the present invention from a culture mixture can be suitably selected from methods used usually in protein purification. That is, a proper method can be selected suitably from usually used methods such as salting-out, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, ion-exchange chromatography, hydrophobic chromatography, various kinds of affinity chromatography such as antibody chromatography, chromatofocusing, adsorption chromatography and reverse phase chromatography, using a HPLC system etc. if necessary, and these techniques may be used in purification in a suitable order.
Further, the antimicrobial proteins of the present invention can also be expressed as a fusion protein with another protein or a tag (for example, glutathione S transferase, protein A, hexahistidine tag, FLAG tag, etc.). The expressed fusion protein can be cleaved off with a suitable protease (for example, thrombin etc.), and preparation of the protein can be carried out more advantageously in some cases. Purification of the protein of the present invention may be carried out by using a suitable combination of general techniques familiar to those skilled in the art, and particularly upon expression of the protein in the form of a fusion protein, a purification method characteristic of the form is preferably adopted. Further, a method of obtaining the protein by using the recombinant DNA molecule in a cell-free synthesis method (J. Sambrook, et al.: Molecular Cloning 2nd ed. (1989)) is one of the methods for producing the protein by genetic engineering techniques.
A protein of the present invention can be prepared as it is, or in the form of a fusion protein with another protein, but the protein of the present invention can be changed into various forms without limitation to the fusion protein. For example, the processing of the protein by various techniques known to those skilled in the art, such as various chemical modifications of the protein, binding thereof to a polymer such as polyethylene glycol, and binding thereof to an insoluble carrier, may be conducted. The presence or absence of addition of sugar chains or a difference in the degree of addition of sugar chains can be recognized depending on the host used. The proteins in such cases are also construed to be under the concept of the present invention insofar as they function as proteins having an antimicrobial activity.
Methods are provided for protecting a organism from a pathogen comprising applying an effective amount of an antimicrobial gene expression product, RNA, protein or other composition of the invention to the environment of the pathogen. “Effective amount” is intended to mean an amount of a protein or composition sufficient to control a pathogen. The antimicrobial proteins and compositions can be applied to the environment of the pathogen by methods known to those of ordinary skill in the art.
In one embodiment, an in-vitro transcription/translation system (e.g., Roche RTS 100 E. coli HY) can be used to produce cell-free antimicrobial protein or expression products of the current invention. In another embodiment, an effective amount of any of the 78 toxic proteins comprising a sequence selected from the group consisting of sequences, SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, and/or 158, is applied to a pathogenic microorganism and/or its environment. In another embodiment, the antimicrobial protein is any of SEQ ID NOS: 187-196. In one embodiment, the antimicrobial protein is ABB43836 (SEQ_ID 30) or protein ABB43762 (SEQ_ID 46).
In another embodiment, an effective amount of any of the antimicrobial small RNA products, such as SEQ ID NOS: 179-186, or any small RNA expressed by SEQ ID NOS: 159-172, is applied to a pathogenic microorganism and/or its environment.
In some embodiments, it is preferred that the antimicrobial agents or anticmicrobial compositions, comprising the antimicrobial nucleic acids, proteins or polypeptides of the present invention described above, should demonstrate antipathogenic and antimicrobial activity, but however, be non-toxic or have low toxicity levels to humans, animals and plants or other organisms that are not the target pathogen.
The antimicrobial RNAs, proteins and expression products are preferably used as an agricultural and horticultural fungicide, an antimicrobial agent for pharmaceutical preparations and an antimicrobial agent in industrial, food, and home use.
In many cases, the antimicrobial protein of the present invention can be used alone, or may be used in combination with a suitable excipient or if necessary with other known agrochemicals, pharmaceutical preparations, industrial antimicrobial components, insecticidal components etc. Further, the protein having an antimicrobial activity used as the active ingredient in the present invention may be composed of a single protein or a mixture of several kinds of proteins.
The antimicrobial compositions of the invention may be obtained by the addition of a surface-active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protective, a buffer, a flow agent or fertilizers, micronutrient donors, or other preparations that may be used to inhibit or control microbial or bacterial infection or growth. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaracides, plant growth regulators, harvest aids, and fertilizers, can be combined with carriers, surfactants or adjuvants customarily employed in the art of formulation or other components to facilitate product handling and application for particular target pathogens. 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. The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. For example, the compositions of the present invention may be applied to grain in preparation for or during storage in a grain bin or silo, etc. The compositions of the present invention may be applied simultaneously or in succession with other compounds. Methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention that contains at least one of the antimicrobial proteins, more particularly antibacterial proteins, of the present invention include, but are not limited to, agrochemical composition foliar application, seed coating, and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest or pathogen.
Suitable surface-active agents include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g., the sulfonated condensation product of oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g., the sodium sulfonate or dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate or oleate; or oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt. Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.
The antimicrobial compositions of the present invention can be in a suitable form for direct application or as a concentrate of primary composition that requires dilution with a suitable quantity of water or other diluant before application. The concentration of the antimicrobial polypeptide will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition contains 1 to 98% of a solid or liquid inert carrier, and 0 to 50%, optimally 0.1 to 50% of a surfactant. These compositions will be administered at the labeled rate for the commercial product.
In a further embodiment, the compositions, as well as the transformed microorganisms and antimicrobial proteins, of the invention can be treated prior to formulation to prolong the antimicrobial, particularly antibacterial, activity when applied to the environment of a target pathogen as long as the pretreatment is not deleterious to the activity. Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously affect the properties of the composition(s). Examples of chemical reagents include but are not limited to halogenating agents; aldehydes such a formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride; alcohols, such as isopropanol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixative (see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freeman and Co.).
The antimicrobial agent preparations according to the present invention may be useful in exhibiting an antimicrobial effect on pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, microorganisms of the genus Staphylococcus, microorganisms of the genus Escherichia, microorganisms of the genus Aspergillus, microorganisms of the genus Candida, microorganisms of the genus Mucor, microorganisms of the genus Absidia, microorganisms of the genus Cryptococcus, microorganisms of the genus Blastomyces, microorganisms of the genus Paracoccidioides, microorganisms of the genus Coccidioides, microorganisms of the genus Sporothrix, microorganisms of the genus Phialophora, microorganisms of the genus Histoplasma, microorganisms of the genus Trichophyton, microorganisms of the genus Microsporum, microorganisms of the genus Epidermophyton, microorganism of the genus Bacillus, and microorganisms of the genus Yersinia, microorganisms of the genus Salmonella, microorganisms of the genus Francisella, etc.
In another embodiment, the antimicrobial agent preparations according to the present invention may be useful in exhibiting an antimicrobial effect on plant pathogenic microorganisms such as microorganisms of the genus Agrobacterium, microorganisms of the genus Burkholderia, microorganisms of the genus Clavibacter, microorganisms of the genus Erwinia, microorganisms of the genus Ralstonia, microorganisms of the genus Xanthomonas, microorganisms of the genus Pseudomonas, and microorganisms of the genus Leifsonia, microorganisms of the genus Xylella, microorganisms of the genus Spiroplasma. In one embodiment, the antimicrobial agent exhibits an antimicrobial effect on microorganisms Agrobacterium tumefaciens, Burkholderia cenocepacia, Clavibacter michiganensis, Erwinia carotovora, Erwinia chrysanthemi, Leifsonia xyli, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas campestris, Xylella fastidiosa, Spiroplasma kunkelii, and Onion yellows phytoplasma.
The protein having an antimicrobial activity used as the active ingredient for an industrial antimicrobial agent of the present invention may be blended and mixed with a suitable excipient and adjuvant, for example a binder, a stabilizer etc. to prepare a pharmaceutical preparation in a suitable form in a usual manner, such as a liquid, a hydrate, an emulsion, sol (flowable agent), etc.
In one embodiment for use in agriculture, the antimicrobial compositions of the invention can be applied to the environment of a plant pathogen by, for example, spraying, atomizing, dusting, scattering, coating or pouring, introducing into or on the soil, introducing into irrigation water, by seed treatment or general application or dusting at the time when the pathogen has begun to appear or before the appearance of pathogens as a protective measure. For example, the antimicrobial protein and/or transformed microorganisms of the invention may be mixed with grain to protect the grain during storage. It is generally important to obtain good control of pathogens in the early stages of plant growth, as this is the time when the plant can be most severely damaged. The compositions of the invention can conveniently contain an insecticide if this is thought necessary. In one embodiment of the invention, the composition is applied directly to the soil, at a time of planting, in granular form of a composition of a. Another embodiment is a granular form of a composition comprising an agrochemical such as, for example, a herbicide, an insecticide, a fertilizer, or an inert carrier.
Compositions of the invention find use in protecting plants, seeds, and plant products in a variety of ways. For example, the compositions can be used in a method that involves placing an effective amount of the antimicrobial, composition in the environment of the pathogen by a procedure selected from the group consisting of spraying, dusting, broadcasting, or seed coating.
The industrial antimicrobial agent containing the protein having an antimicrobial activity as the active ingredient according to the present invention can be used in suppression of the growth of microorganisms and fungi in emulsion products such as aqueous paints, adhesive materials, latex, acryl etc., slurry products such as starch, pigments, calcium carbonate etc., and joint cement; preservation of wood such as building materials (construction materials, civil engineering building materials etc.); sterilization and prevention of slime formation in production facilities in factories, cooling towers in building air conditioning, and pulp- and paper-making factories, etc.; antimicrobial treatment by spraying or dipping of fibers, fabrics and hides; protection from the attack of microorganisms and fungi occurring during exposure of a paint coating, particularly a paint coating of an exterior paint, to wind and rain; antimicrobial treatment of interior/exterior materials (for housing, medical facilities), building materials (construction materials, civil engineering materials etc.), home appliances, domestic sundries, sporting goods etc. made of resin such as vinyl chloride, polyurethane, polyethylene, polypropylene, silicone, modified silicone, nylon, epoxy resin etc.; protection from accumulation of slime on devices for producing sugar cane and beet sugar; prevention of accumulation and sedimentation of microorganisms in an air washer and scrubber system and an industrial freshwater-feeding system; maintenance of sanitary environments in food factories etc.; deodorization and sterilization at the time of washing production facilities and in sewage disposal plants etc.; prevention of growth of microorganisms and fungi in paper-coating materials and coating processing; prevention of contamination of cosmetics and toiletries with microorganisms; prevention of microbial growth in a pool, cooling water etc.; prevention of microbial contamination of agricultural blends, an electrodeposition system, and diagnostic and chemical products, medical instruments etc.; and prevention of accumulation of microorganisms in photographic treatment.
In another embodiment for household use as an antimicrobial agent, the present antimicrobial polypeptides are formed into a pharmaceutical preparation and used as it is, or used according to a wide variety of conventional methods of using industrial antimicrobial agents, which include, but are not limited to, a method that involves diluting it with water or a suitable organic solvent and mixing the dilution with various household materials or products, a method of applying or spraying it onto the surfaces of various household and industrial materials or products, and a method that involves dipping various industrial materials or products in a dilution of the household antimicrobial agent of the present invention, etc. The household antimicrobial agent of the present invention described above may be in the form of preparations prepared by mixing the protein with a solution, a suspension, an emulsion etc., for example in the form of tablets, powder, granules, liquids, hydrates, emulsions, injections, aerosol, sprays, sol (flowable agent), etc. The household antimicrobial agents may be used in cleaners, solvents, soaps, disposable cleaning materials, plastics, containers, etc.
In another embodiment, for pharmaceutical use, the antimicrobial polypeptides of the present invention can be prepared from the protein of the present invention alone or by compounding the protein with pharmaceutically acceptable excipients, active ingredients, fillers etc. if necessary. The preparation form includes parenteral administration forms such as injections (subcutaneous, intravenous, intramuscular or intraperitoneal injections), liquid coating agents, gel, ointments, suppositories and aerosol, and oral administration forms such as tablets, capsules, granules, pills, syrups, liquids, emulsions, suspensions etc.
For preparation of the tablets, capsules, granules and pills for oral administration, use is made of excipients such as white sugar, lactose, glucose, starch and mannitol, binders such as syrup, gum arabic, gelatin, sorbitol, tragacanth, methylcellulose and polyvinyl pyrrolidone, disintegrating agents such as starch, carboxymethyl cellulose or a calcium salt thereof, fine crystalline cellulose and polyethylene glycol, lustering agents such as talc, magnesium stearate, calcium stearate and silica, and lubricants such as sodium laurate and glycerol.
For preparation of the injections, liquids, emulsions, suspensions, syrups and aerosol, use is made of solvents for the active ingredient, such as water, ethyl alcohol, isopropyl alcohol, propylene glycol, 1,3-butylene glycol and polyethylene glycol, surfactants such as sorbitan fatty ester, polyoxyethylene sorbitan fatty ester, polyoxyethylene fatty ester, hydrogenated castor oil polyoxyethylene ether, and lecithin, cellulose derivatives such as carboxymethylcellulose sodium salt and methylcellulose, suspending agents such as natural gum such as tragacanth and gum arabic, preservatives such as paraoxybenzoate, benzalkonium chloride and sorbitate.
The clinical dose of the antimicrobial agent is varied depending on the therapeutic effect on the age, body weight, sensitivity and symptom of the patient, but usually the effective daily dose is 0.003 to 1.5 g/kg, preferably 0.01 to 0.6 g/kg. However, a dose outside of the above range can also be used if necessary.
In another embodiment, the antimicrobial agent prepared as in a cream or lotion for topical application. Typically such compositions are prepared with purified water, preservatives, emollient and emulsion stabilizers and humectants used in many cosmetic preparations including, but not limited to, propylene glycol, parabens, cetearyl alcohol, also known as cetostearyl alcohol, polysorbate 60 (polyoxyethylene sorbitan monostearate), cetyl alcohol, and glycerine. For example, a patient can topically apply an antibiotic ointment containing one or more of the antimicrobial agents of the present invention as active ingredients for fighting infection during wound repair, and thereby increasing rate of healing while reducing risk of infection.
In another embodiment of the invention, the bacteria originally producing the antimicrobial agent, or engineered bacteria that express the antimicrobial agent and are also resistant to its effect, may be used as probiotic bacteria for food preservation, livestock growth enhancement, and human health.
In another embodiment, for food processing and preservation use, antimicrobial agents of the present invention can be prepared from the genes, RNA or proteins of the present invention alone or by compounding the agent with commercially acceptable other antibacterial agents, such as chitosan or its derivatives, and/or suitable chelating agents including, but are not limited to, ethylene diaminetetraacetic acid and its salts, cyclodextrins, hydrocarboxylic acids, such as citric acid, acetic acid, lactic acid, tartaric acid, and their salts, alone or in combination. In one embodiment, the antimicrobial agents of the present invention may be combined with other antimicrobial decontaminants, such as hydrogen peroxide, citric acid, lactic acid, or acetic acid, alone, or in combination.
The antimicrobial agents can be used for treatment of whole animals and may be applied to the food animal after stunning, before the animal is bled, during the de-hairing, de-feathering, and skinning processes, and may be applied to decontaminate heads, organs, offal and other carcass parts. The antimicrobial composition of the present invention may be used in connection with any food product which is susceptible to microbial degradation. These include, but are not limited to fruits and vegetables including derived products, grain and grain derived products, dairy foods, meat, poultry, and seafood. In particularly preferred embodiments, the composition is used in connection with meat, poultry and/or seafood, more particularly with fat containing cooked meats such as hotdogs, sausages, roast beef, turkey, corned beef and deli meats. The concentration of the anti-microbial solution, contact time, temperature and other application parameters are controlled to optimize the effectiveness of the treatment.
In another embodiment, the antimicrobial agents are applied to a food surface for the purpose of food processing, preservation or to prevent food spoilage or degradation. The use of the term “food surface” is defined to include any and all internal or external surfaces of the food product being treated. The preparation form includes surface administration forms such as injections, sprays, liquid solutions, liquid coating agents, gel, ointments, and aerosol.
The composition according to the present invention can be used by applying it on the exterior surface of a blended food product, such as a hot dog or bologna, or of a solid food, such as a piece of roasted beef, so as to minimize loss of activity in the fat phase of the food. The composition may alternatively be included in the emulsion or raw ingredients of a food such as sauces or salsas, before or after cooking, or to the interior of solid products, such as hams, by injection or tumbling. In still other embodiments, the composition may be applied as a marinade, breading, seasoning rub, glaze, colorant mixture, and the like, the key criteria being that the antimicrobial composition be available to the surface subject to bacterial degradation. In a preferred embodiment, the composition may be indirectly placed into contact with the food surface by applying the composition to food packaging materials or casings and thereafter applying the packaging to the food surface. The use of surface treatment strategies, whether direct or indirect, benefits from the minimization of loss into the fat phase of the fat containing food product. The optimum effective amount to be used will depend on the composition of the particular food product to be treated and the method used for applying the composition to the food surface, but can be determined by simple experimentation.
EXAMPLE 1
Characterization of a Toxic Gene Found Using the Method
We selected one of the proteins found in our set as an example. This protein, Genbank Accession No. YP—386193 which is hereby incorporated by reference, is found in the Geobacter metallireducens GS-15 genome, and is identified as Gene No. 48 in the attached Sequence Listing. The nucleic acid sequence is SEQ ID NO: 96 and the protein sequence is SEQ ID NO: 97. FIG. 4 demonstrates the deficiency in clone coverage of this gene. The gene encodes a small protein (93 amino-acids long) that has a signal sequence (as predicted by the signalP software [Nielsen, Protein Eng. 1999 January; 12(1):3-9]) and is hence probably secreted. It has an excess of basic residues over acidic ones, and is hence predicted to be positively charged in neutral pH. Homologs of this gene are found only in Geobacter sulfurreducens and Geobacter uraniumreducens, but not outside the Geobacter genus.
FIG. 5 presents a multiple alignment of YP—386193 homologs. All homologs are short (90-100aa), positively charged proteins, with a predicted signal peptide. Two invariant cysteine residues probably indicate on a disulfide bridge formation (FIG. 5). Protein secondary structure prediction indicates that the protein contains two amphiphilic alpha helices, each containing distinct hydrophobic and hydrophilic faces (FIG. 6). From these data we predict that protein YP—386193 might be a cationic antimicrobial peptide, as these peptides are defined as short, positively charged, amphiphilic, secreted proteins that perforate bacterial membranes [Hancock, Trends Microbiol. 2000 September; 8(9):402-10]
To check whether protein YP—386193 indeed inhibits bacterial growth we cloned it into the pET11a plasmid (Stratagene) under a T7 polymerase promoter, and transformed the vector into BL21(DE)pLYS cells. This system maintains the expression of the inserted gene silent unless an inducer molecule (IPTG) is added to the medium. As a positive control we similarly cloned the RegB gene, which is known to be highly toxic to E coli [Sanson, FEMS Microbiol Rev. 1995 August; 17(1-2):141-50]. As a negative control we similarly cloned the E. coli beta-galactosidase gene. FIG. 7 shows that RegB indeed kills E coli following induction of its expression as shown by the lack of colony growth in the top half of the petri dish in the second and third panels, while beta-galactosidase does not affect bacterial growth, as shown by colony growth in the bottom of the petri dishes. FIG. 8 shows that similar to the RegB positive control, colonies containing YP—386193 cannot grow if its expression of YP—386193 is induced, demonstrating its toxicity to E coli. Colonies containing mutated inserts, however, were not growth inhibited following expression induction (FIG. 9). These results show that YP—386193 has a growth inhibition effect on E coli, and is thus an antimicrobial agent.
We tested 77 additional proteins from the set described in the attached Sequence Listing using the same experimental system as described above for protein YP—386193. It was found that each of these 77 proteins are indeed toxic when expressed in E. coli. Results are not shown for each protein in the set, but exemplary data (e.g., photographs of individual wells showing bacteria growth and/or inhibition as a result of induced expression of the antimicrobial protein) from several examples are found in FIG. 10 Photos were taken and the colonies were counted. Table 1 shows the gene number, internal reference number and the corresponding sequence identifier for the nucleotide and protein sequences in the first four columns. In the last column of Table 1, shown is the lowest concentration (uM) of IPTG needed to induce gene expression and inhibition of bacterial growth was observed. A protein was said to inhibit growth of bacteria if a result of no more than 10 colonies was observed in a well of a 48-well growth plate after inducing expression. In this system, IPTG influences the level of activity of the T7 promoter in the pET11a plasmid, thereby inducing expression of the toxic gene. We induced expression at varying concentrations of 100 μM, 250 μM, 400 μM, 600 μM, 800 μM and 1000 μM of IPTG. The manufacturer of the expression system recommends to use 1 mM (1000 uM) IPTG for maximum efficiency and it is generally accepted that IPTG concentrations lower than that cause partial activity. Thus, with even very low IPTG concentrations used to induce expression, it is shown that small amounts of expression of some of the disclosed genes have significant antimicrobial activity. For example, Gene No. 9, ABB43889, having a polynucleotide sequence of SEQ ID NO:17, and when expressed is SEQ ID NO:18, required only 100 μM of IPTG to express ABB43889, and inhibit growth.
To verify that the 78 proteins identified by our method are bona fide antimicrobial proteins, we used an in-vitro transcription/translation system (Roche RTS 100 E. coli HY) to produce cell-free protein products of the 78 toxic genes. Candidate toxic proteins were mixed with E. coli strain BL21 bacteria growing in liquid LB medium, and growth was then monitored for 5.5 hours by measuring Optical Density (OD) in wavelength 600 nm every 10 minutes. For each protein tested, the OD was measured for growth of control bacteria without toxic protein addition to the medium containing an in-vitro transcription/translation system. An average and standard deviation of at least 9 repetitions were performed for each control. At least three repetitions of this growth kinetics experiment for each single toxic protein mixed with the medium containing the in-vitro transcription/translation system was carried out. The OD was taken every 10 minutes and the resulting growth curves were compared to the control curve.
For each of the genes and proteins shown in Table 1, growth of bacteria may be inhibited in the presence of the translated toxic protein, as compared to growth without toxic proteins in the medium. Data not shown for all genes tested, however, FIGS. 12A and 12B show examples of the OD results obtained for two proteins identified using the method in the present invention. FIG. 12(A) shows antimicrobial activity of protein ABB43836 (SEQ_ID 30); and FIG. 12B, shows antimicrobial activity of protein ABB43762 (SEQ_ID 46). These proteins were determined empirically to have antimicrobial activity with the ability to inhibit bacteria growth by external application of the protein to bacteria or its environment. Looking at the graphs in FIGS. 12A and 12B, antimicrobial activity is evident as multiple points including the endpoint of the growth curve of the bacteria exposed to the proteins was at least 0.1 OD lower than the control, and there was an obvious deviation in the slope of the three growth curves as compared to the control. Thus, it was concluded that the two proteins had antimicrobial activity.
EXAMPLE 2
Identification of a Small Toxic RNAs Using the Method
The present method also enables the discovery of genome-encoded antibacterial agents that are not proteins. To demonstrate this, we looked for regions that are not predicted to contain open reading frames (ORFs), but display consistent clone coverage deficiency. One such region, found in the Burkholderia cenocepacia HI2424 genome displays radical reduction of clone coverage as calculated using our method (FIG. 13). This low covered region lies in the intergenic region between the genes encoding to OsmC and to ribosomal protein L13. Examining the coverage of this region in the closely related genomes of Burkholderia cenocepacia AU 1054, Burkholderia sp. strain 383, and Burkholderia—ambifaria_AMMD showed that it has detectable low coverage in all these genomes. The same low coverage pattern is seen in the more distantly related genomes of Ralstonia (Cupriavidus) necator and Cupriavidus metallidurans (not shown).
FIG. 14 shows a multiple sequence alignment of the DNA found in the discussed intergenic region from 4 Burkholderia species (Burkholderia cenocepacia AU 1054 (SEQ ID NO: 159), Burkholderia sp. strain HI2424 (SEQ ID NO: 160), Burkholderia sp. strain 383 (SEQ ID NO: 161), and Burkholderia—ambifaria_AMMD (SEQ ID NO: 162) and 2 Ralstonia species (Ralstonia necator (SEQ ID NO: 163), and Ralstonia metallidurans (SEQ ID NO: 164)). As seen in the figure, the sequences vary significantly, with a conserved core of 20 consecutive invariant nuclecotides in the middle of the alignment, and a conserved stretch of Thymines at the end. Deletions having sizes not divisible by 3 exclude the possibility that this region encodes a protein. Despite the lack of extensive sequence conservation, however, a strongly conserved RNA secondary structure is apparent from compensatory mutations in stem regions (FIG. 14). Therefore, this region is probably populated by an RNA gene which is toxic to E coli. A strongly predicted rho independent terminator defined the 3′ end of the gene (FIG. 14). Scanning the region upstream to the conserved core reveals a strongly predicted promoter that defined the 5′ boundary of the gene (FIG. 15, TATA box is highlighted in red).
The RNA secondary structure of this gene (as predicted by RNAfold [URL:<http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi>]) is shown in FIG. 16. The structure is comprised of 3 stems, of which the middle one hosts the conserved sequence core and the 3′ one forms the transcriptional terminator. The first (most 5′) stem is longer and more complex in the Cupriavidus genomes (FIG. 17). A scan by RFAM (database of known functional RNAs families [URL:<http://www.sanger.ac.uk/Software/Rfam/>]) shows that this RNA gene is not similar to any known functional RNA gene.
To search for more RNA genes having similar structure, we used the cmalign and cmsearch software modules from the infernal software package [URL:<http://infernaljanelia.org/>]. Scanning the Burkholderia genome revealed two more such small predicted RNAs. In both cases, these small RNAs (SEQ ID NOS: 179, 182, 185, and 186) (as shown and identified in FIG. 21) were found in an intergenic region and had an upstream promoter and a rho-independent terminator. Two RNAs have similar structure as the above described small RNA shown in FIG. 16, but lack the first stem (FIG. 18). The clone coverage of both these additional RNAs was extremely low in both cases (FIGS. 19-20).
To check whether the identified small RNAs indeed inhibit bacterial growth we cloned 4 such RNAs (SEQ ID NOS: 165, 168, 171 and 172) into the pcrSmart plasmid (Lucigen) under a T7 polymerase promoter, and transformed the vector into BL21(DE)pLYS cells, as described above for the protein in Example 1. FIG. 21 shows that colonies containing the small RNAs cannot grow if its expression of these RNAs is induced by the addition of IPTG to the medium, demonstrating their toxicity to E coli. These results show that the small RNAs indeed have a growth inhibition effect (i.e., antimicrobial effect) on E coli. 
In summary, we used our method to discover a new family of small RNAs that are toxic to E. coli. These RNAs have detectable homologs only in the small group of betaproteobacteria, to which the Burkholderia and Cupriavidus (Ralstonia) species belong. In all cases we noted these RNAs have extreme clone coverage deficiency (frequently causing zero coverage), indicating their toxicity to the E coli host. This demonstrates the power of our method to detect antimicrobial agents that are not necessarily proteins.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, and patents cited herein are hereby incorporated by reference for all purposes.

## Claims

1. A plant comprising in its genome at least one stably incorporated expression cassette, said expression cassette expressing a protein sequence having at least 85% homology to SEQ ID NO: 97, wherein upon expression of said protein in the plant, said plant displays increased resistance to a plant pathogen.

2. The plant of claim 1, wherein said expression cassette comprises a heterologous nucleotide sequence that is at least 85% homologous to SEQ ID NO: 96 and operably linked to a promoter.

3. The plant of claim 2, wherein said plant pathogen is a microbe.

4. The plant of claim 3, wherein said microbe is selected from the group consisting of Agrobacterium tumefaciens, Burkholderia cenocepacia, Clavibacter michiganensis, Erwinia carotovora, Erwinia chrysanthemi, Leifsonia xyli, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas campestris, Xylella fastidiosa, Spiroplasma kunkelii, and Onion yellows phytoplasma.

5. The plant of claim 2, wherein said promoter is a pathogen-inducible promoter.

6. A transformed seed of the plant of claim 1.

7. A method for inducing pathogen resistance in a plant, said method comprising introducing into a plant at least one expression cassette, said expression cassette comprising a heterologous nucleotide sequence that expresses a protein having at least 85% homology to SEQ ID NO: 97 and operably linked to an inducible promoter that drives expression in the plant; wherein the expressed protein provides pathogen resistance.

8. The method of claim 7, wherein said heterologous nucleotide sequence is at least 90% homologous to SEQ ID NO: 96.

9. The method of claim 8, wherein said heterologous nucleotide sequence is at least 90% homologous to SEQ ID NO: 96.

10. The method of claim 7, wherein said microbe is selected from the group consisting of Agrobacterium tumefaciens, Burkholderia cenocepacia, Clavibacter michiganensis, Erwinia carotovora, Erwinia chrysanthemi, Leifsonia xyli, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas campestris, Xylella fastidiosa, Spiroplasma kunkelii, and Onion yellows phytoplasma.