US20160287677A1 - Bacterial anti-phage defense systems - Google Patents

Bacterial anti-phage defense systems Download PDF

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US20160287677A1
US20160287677A1 US15/131,102 US201615131102A US2016287677A1 US 20160287677 A1 US20160287677 A1 US 20160287677A1 US 201615131102 A US201615131102 A US 201615131102A US 2016287677 A1 US2016287677 A1 US 2016287677A1
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toxin
antitoxin
seq
amino acid
group
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Rotem Sorek
Hila Sberro
Azita Leavitt
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Yeda Research and Development Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention in some embodiments thereof, relates to bacterial polypeptides that comprise toxin or antitoxin activity and, more particularly, but not exclusively, to toxin-antitoxin pairs that may be used as bacterial anti-phage defense systems.
  • a broad array of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various substrates. Because enormous amounts of bacteria are being cultivated each day in large fermentation vats, bacteriophage contamination can rapidly bring fermentations to a halt and cause economic setbacks, and is therefore considered a serious threat in these industries.
  • the dairy fermentation industry has openly acknowledged the problem of phage and has been working with academia and starter culture companies to develop defense strategies and systems to curtail the propagation and evolution of phages for decades.
  • bacteria have developed a variety of anti-phage defense systems (Labrie et al., 2010; Stern and Sorek, 2011). These systems include restriction enzymes that recognize and cleave foreign DNA (King and Murray, 1994), abortive infection (Abi) mechanisms that lead the bacterial cell, upon phage invasion, to commit “suicide”, thus protecting the colony against phage spread (Chopin et al., 2005); and the recently identified adaptive defense system called CRISPR/Cas, which uses small RNAs to target invading phage DNA (Deveau et al., 2010; Horvath and Barrangou, 2010; Sorek et al., 2008; van der Oost et al., 2009).
  • CRISPR/Cas which uses small RNAs to target invading phage DNA
  • TA toxin-antitoxin
  • TA modules play pivotal roles in prokaryotic cellular biology including programmed cell death (Hazan et al., 2004), stress response (Christensen et al., 2001), generation of persister cells (Schumacher et al., 2009), biofilm formation (Kim et al., 2009) and phage defense via abortive infection (Fineran et al., 2009; Hazan and Engelberg-Kulka, 2004; Koga et al., 2011; Pecota and Wood, 1996).
  • TA systems The most prevalent kind of TA systems is type II systems, where both toxin and antitoxin are proteins (as opposed to types I and III where the antitoxin is a non-coding RNA (Fineran et al., 2009; Fozo et al.)).
  • the two genes which reside on the same operon, code for small proteins and inhibition of the toxin is carried out through protein-protein interaction.
  • the toxin is a stable protein and the antitoxin is unstable and degrades rapidly by one of the housekeeping bacterial proteases, usually Lon or ClpP (Aizenman et al., 1996; Cherny and Gazit, 2004; Christensen et al., 2004; Christensen et al., 2001; Christensen et al., 2003; Lehnherr and Yarmolinsky, 1995; Roberts et al., 1994; Van Melderen et al., 1996).
  • Lon or ClpP housekeeping bacterial proteases
  • an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094, wherein the polypeptide has antimicrobial activity.
  • an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094, wherein the polypeptide has antimicrobial activity.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin.
  • an anti-microbial composition comprising a carrier and as an active ingredient an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094.
  • an anti-microbial composition comprising a carrier and as an active ingredient an isolated polynucleotide comprising a nucleic acid sequence which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094.
  • the method comprising administering to the subject a therapeutically effective amount of the anti-microbial composition described herein, thereby treating the infection.
  • a solid support coated with an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544.
  • a method of killing a microbe comprising contacting the microbe with an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544, thereby killing the microbe.
  • a method of protecting a microbe from a toxin comprising expressing in the microbe the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin.
  • an isolated bacterial population genetically modified to express a toxin and a cognate antitoxin thereof, the bacterial population being resistant to a lytic activity of a bacteriophage wherein the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-3117, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2773-2804, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8317-8348, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2805-2871, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8349-8415, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2872-2935, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID
  • a method of protecting a bacterial population from phage attack comprising expressing in the bacterial population a toxin and a cognate antitoxin thereof, wherein the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-3117, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2773-2804, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8317-8348, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2805-2871, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8349-8415, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2872-2935, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8416
  • the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 2773-3117 and 11089-11094.
  • the amino acid sequence consists of the sequences selected from the group as set forth in SEQ ID NOs: 2773-5544.
  • the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 8317-8661.
  • the amino acid sequence consists of the sequences selected from the group as set forth in SEQ ID NOs: 8317-11088.
  • the isolated peptide comprises at least one naturally occurring amino acid.
  • the isolated peptide comprises a synthetic amino acid.
  • the isolated peptide is attached to a cell penetrating agent.
  • the attached is covalently attached.
  • the cell penetrating agent is a peptide agent.
  • the isolated peptide is attached to a sustained-release enhancing agent.
  • the sustained-release enhancing agent is selected from the group consisting of hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene glycol (PEG), glyme and polyisopropylacrylamide.
  • the isolated polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-2772.
  • the isolated polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5545-8316.
  • the carrier is a pharmaceutically acceptable carrier.
  • the anti-microbial composition is formulated for topical application.
  • the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-2772 and 11095-11100.
  • the carrier is a pharmaceutically acceptable carrier.
  • the method further comprises administering to the subject an antibiotic.
  • the contacting is effected in vivo.
  • the contacting is effected ex vivo.
  • the microbe comprises a bacteria.
  • the isolated polynucleotide is operably linked to a promoter.
  • the bacterial population is a lactic acid bacterial population.
  • the bacterial population is of a species selected from the group consisting of Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus species, Pediococcus species and Bifidobacterium.
  • the bacteriophage comprises WT T7 strain.
  • the first isolated polynucleotide encodes a toxin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-3117; and the second isolated polynucleotide encodes an antitoxin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-8661.
  • the first isolated polynucleotide and the second isolated polynucleotide are comprised in a non-identical expression vector.
  • the anti-microbial composition is for treating an infection.
  • FIGS. 1A-1D illustrate that data derived from whole-genome shotgun sequencing exposes toxin-antitoxin pairs.
  • A The “Sanger” based process of DNA sequencing involves random genome fragmentation and transformation of DNA fragments into E. coli .
  • B In a DNA locus spanning a toxin/antitoxin (TA) gene pair, random fragmentation leaving the toxin detached from its cognate antitoxin leads to E. coli growth arrest, whereas a fragment containing both genes, or the antitoxin alone, will be propagated and sequenced.
  • C A known family of toxin/antitoxin gene pairs, of the VapBC type, was found in 11 of the analyzed genomes.
  • FIG. 2 is a diagram of the workflow for systematic discovery of families of toxin/antitoxin associated with anti-phage defense.
  • FIGS. 3A-3C are graphs and diagrams illustrating properties of novel, experimentally verified TA systems
  • A growth of bacteria when only antitoxin is induced (left), only toxin is induced (middle) and both are induced together (right).
  • Toxin and antitoxin were cloned on pRSF (IPTG inducible promoter) and pBAD (arabinose inducible), respectively, in E. coli BL21 (DE3) pLysS.
  • C control bacteria with empty plasmids; 1-pmen system; 2-sana system; 3-psyr system; 4-rleg system; 5-sden system.
  • B Kinetics of E.
  • C Viability assays for cells following exposure to toxin. Transcription of toxin was induced by 100 ⁇ M IPTG. At increasing time points following toxin induction (30, 60, 120, 180, 240 and 300 mins) cells were plated on LB-plates containing 0.3% arabinose and no IPTG, to activate antitoxin expression. Colony forming units (CFUs) were determined by colony counting.
  • FIG. 4A illustrates operon and domain organization of the validated families. Representative pair of each family is shown. For each pair, red and blue genes denote toxin and antitoxin, respectively.
  • FIGS. 4B-4C are tables showing distribution of novel TA system among (B) different bacterial phyla and (C) human associated bacteria. Number of instances of each system within a phylum/bacterial species is indicated, with darker colors indicating higher number of instances.
  • FIGS. 5A-5F illustrate that T7 Gp 4.5 antagonizes abortive infection by interacting with Lon.
  • PFU plaque forming unit
  • EOP Efficiency of plating
  • E Co-immunoprecipitation of Lon and 4.5.
  • the E. coli Lon protease was Flag-tagged at the N-terminus and expressed within E. coli BL21 (DE3) with or without co-expression of gene 4.5. Samples were analyzed by 15% SDS-PAGE. Three left lanes, total soluble proteins; three lanes on the right, following immunoprecipitation with anti-Flag antibody.
  • FIG. 6 is a bar graph demonstrating that expression of gene 4.5 within E. coli results in reduced number of persister bacteria. Bacteria were exposed to ampicillin for 5 hours and % formation of persister bacteria was measured. Experiments were performed in triplicates and error bars represent standard error.
  • FIG. 7 illustrates the statistical assessment of a family of gene pairs as possibly encoding a toxin-antitoxin module.
  • the sequencing clones are re-distributed randomly (maintaining the number of clones and their sizes, but shuffling their positions on the genome).
  • the “TA cloning pattern” for each member is then evaluated based on the randomly distributed clones, and the fraction of members obeying the “TA cloning pattern” by chance is recorded (in the illustration: red, clones that span only the toxin; blue, clones that span only the antitoxin; green, clones that span both genes).
  • This procedure is repeated 1000 times, generating a distribution of fractions.
  • the p-value for a family is determined by comparing the actual fraction of pairs that obey the “TA cloning pattern” to the distribution of fractions obtained using the random clone shuffling.
  • FIG. 8 is a diagram illustrating that predicted new TA systems associate with complex genomic environments. Shown are the schematic environments of 4 systems that are associated with other genes and hence suspected as participating in multi-gene defense systems. Blue and red genes denote putative antitoxin and toxin, respectively; gray genes denote genes associated with the TA system.
  • FIGS. 9A-9B are bar graphs illustrating properties of known TA families.
  • the 21,417 analyzed families of gene pairs were divided into two sets: a positive set which contains all families in which at least one pair is a known TA system (‘Known systems’) and a negative which contains the remaining families (‘Negative set’).
  • A The family diversity measurement for known TA systems (black) as compared to the negative set (light gray)
  • B Mean number of defense island (DI) genes localized +/ ⁇ 5 genes away from known TA families (black) as compared to families in the negative set (light gray).
  • DI Mean number of defense island
  • the present invention in some embodiments thereof, relates to bacterial polypeptides that comprise toxin and/or antitoxin activity and, more particularly, but not exclusively, to toxin-antitoxin pairs that may be used as bacterial anti-phage defense systems.
  • the toxin-antitoxin (TA) complex of bacteria includes a pair of polypeptides that is encoded by bacterial plasmids or chromosomes. It is postulated that in bacteria these polypeptides function to induce programmed cell death or growth inhibition in response to starvation, phage infection or other adverse conditions.
  • the antitoxins neutralize the cognate toxins by forming tight complexes therewith or by other means.
  • the antitoxins are unstable due to degradation by cellular proteases (e.g., Lon or Clp), whereas toxins are stable polypeptides.
  • Toxin-antitoxin pair examples include the pemI-pemK genes of plasmid R100, the phd-doc genes of phage P1, and the ccdA-ccdB genes, of plasmid F.
  • Several toxin-antitoxin encoding gene analogues have been identified on the E. coli K-12 chromosome, such as mazE-mazF.
  • the present inventors took advantage of this typical biased cloning pattern to systematically detect toxin-antitoxin gene pairs within hundreds of microbial genomes.
  • the present inventors showed that the toxin-antitoxin pairs can protect the engineered bacteria from phage attacks ( FIGS. 5A-B ).
  • Such engineered bacteria can be utilized in the dairy industry, where phages cause serious annual losses, as well as in other industries that rely on large-scale bacterial fermentation for biotechnological production.
  • FIGS. 5C-F Infection experiments with T7 phage and multiple T7 phage deletion mutants further showed that two of these new TA pairs provide resistance against T7, and also revealed a general anti-TA mechanism encoded by the phage ( FIGS. 5C-F ). Specifically, the present inventors discovered a phage peptide that inhibits toxin-antitoxin systems by inhibiting Lon protease. This peptide was further shown to inhibit the formation of bacterial persistence, thus active as a mechanism to reduce antibiotics resistance among bacteria.
  • an isolated polypeptide comprising an amino acid sequence at least 90% homologous to a sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094, wherein the polypeptide has antimicrobial activity.
  • antimicrobial activity refers to an ability to suppress, control, inhibit or kill microorganisms, such as bacteria, archaea and fungi.
  • the antimicrobial activity may comprise bactericidal or bacteriostatic activity, or both.
  • the isolated polypeptide comprises an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to SEQ ID NOs: 2773-5544.
  • the isolated polypeptide comprises an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to SEQ ID NOs: 2773-3117.
  • the isolated polypeptide comprises an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to SEQ ID NOs: 11089-11094.
  • Such polypeptides are capable of inhibiting (decreasing activity by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) a member of the Lon protease family.
  • Lon proteases are ATP-dependent serine peptidases belonging to the MEROPS peptidase family S16 (lon protease family, clan SF).
  • an isolated polypeptide comprising an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to any one of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin.
  • Such polypeptides may be expressed in microbial populations thereby protecting the microbial population from the toxic effect of the toxin. Methods of expressing polypeptides are described herein below.
  • Polypeptides which protect microbes from an activity of a toxin may have a Ki of 1 ⁇ 10 ( ⁇ 5) M-1 ⁇ 10 ( ⁇ 10) M for the toxin.
  • sequence selected from the group consisting of SEQ ID NOs. 8317-8661.
  • the peptides of the present invention consist of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2773-5544 or SEQ ID NO:8317-11088 or SEQ ID NOs: 11089-11094.
  • Homology may be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters).
  • the homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.
  • peptide encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds (—C( ⁇ O)—O—), ketomethylene bonds (—CO—CH2-), sulfinylmethylene bonds (—S( ⁇ O)—CH2-), ⁇ -aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g., methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds (—CH2-CH2-), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH ⁇ CH—), fluorinated olefinic double bonds (—CF ⁇ CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally present
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted by non-natural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.
  • Tic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
  • naphthylalanine naphthylalanine
  • ring-methylated derivatives of Phe ring-methylated derivatives of Phe
  • halogenated derivatives of Phe or O-methyl-Tyr.
  • the peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc.).
  • modified amino acids e.g. fatty acids, complex carbohydrates etc.
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • Tables 1 and 2 below list naturally occurring amino acids (Table 1), and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with some embodiments of the invention.
  • peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • the present peptides are preferably utilized in therapeutics or diagnostics which require the peptides to be in soluble form
  • the peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • amino acids of the peptides of the present invention may be substituted either conservatively or non-conservatively.
  • conservative substitution refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties.
  • side-chain of the native amino acid to be replaced is either polar or hydrophobic
  • the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
  • amino acid analogs synthetic amino acids
  • a peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
  • the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
  • non-conservative substitutions refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties.
  • the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
  • Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or —NH—CH[(—CH 2 ) 5 —COOH]—CO— for aspartic acid.
  • Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide having anti-bacterial properties.
  • N and C termini of the peptides of the present invention may be protected by function groups.
  • Suitable functional groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference.
  • Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.
  • the peptides of the present invention may be attached (either covalently or non-covalently) to a penetrating agent.
  • penetrating agent refers to an agent which enhances translocation of any of the attached peptide across a cell membrane.
  • the penetrating agent is a peptide and is attached to the antimicrobial peptide (either directly or non-directly) via a peptide bond.
  • peptide penetrating agents typically have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • the peptides of the present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
  • Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing.
  • Recombinant techniques may also be used to generate the peptides of the present invention.
  • a polynucleotide encoding the peptide of the present invention is ligated into a nucleic acid expression vector, which comprises the polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., promoter sequence) suitable for directing constitutive, tissue specific or inducible transcription of the polypeptides of the present invention in the host cells.
  • a cis-regulatory sequence e.g., promoter sequence
  • prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of some embodiments of the invention.
  • yeast transformed with recombinant yeast expression vectors containing the coding sequence include, but are not limited to; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence.
  • virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • Mammalian expression systems can also be used to express the polypeptides of some embodiments of the invention.
  • bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
  • the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed antimicrobial peptide.
  • the expression of a fusion protein or a cleavable fusion protein comprising the antimicrobial peptides of some embodiments of the invention and a heterologous protein can be engineered.
  • Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein.
  • the antimicrobial can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].
  • polypeptides of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the peptides of the present invention can also be synthesized using in vitro expression systems. These methods are well known in the art and the components of the system are commercially available.
  • the peptides disclosed herein, which comprise anti-microbial properties may be used to kill microbes.
  • a method of killing a microbe comprising contacting the microbe with the isolated peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094.
  • the peptide used for killing a microbe comprises the sequence 11089-11094.
  • the microbe may be for example a gram-positive or gram negative bacteria.
  • Gram-positive bacteria refers to bacteria characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids and are characterized by their blue-violet color reaction in the Gram-staining procedure.
  • Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scro
  • Gram-negative bacteria refer to bacteria characterized by the presence of a double membrane surrounding each bacterial cell.
  • Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus
  • the term “contacting” refers to the positioning of the peptides of the present invention such that they are in direct or indirect contact with the bacterial cells.
  • the present invention contemplates both applying the peptides of the present invention to a desirable surface and/or directly to the bacterial cells.
  • the surface is comprised in a biological tissue, such as for example, mammalian tissues e.g. the skin.
  • the microbes may be comprised inside a particular organism, (e.g. intracellularly or extracellularly) for example inside a mammalian body or inside a plant.
  • the contacting may be effected by administering the peptides per se or by transfecting the cells of the organism with a nucleic acid construct which comprises a nucleic acid sequence which encodes the peptides of the present invention.
  • the present invention contemplates polynucleotide sequences encoding the antimicrobial polypeptides disclosed herein. Such polynucleotide sequences are set forth in SEQ ID NOs: 1-2772.
  • Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
  • Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • Inducible promoters suitable for use with some embodiments of the invention include for example the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804) or pathogen-inducible promoters.
  • Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen.
  • the nucleic acid construct (also referred to herein as an “expression vector”) of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • a typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
  • such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof.
  • the nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
  • the nucleic acid constructs described herein will have a plurality of restriction sites for insertion of the sequence of the invention so that it is under transcriptional regulation of the regulatory regions.
  • Selectable marker genes that ensure maintenance of the vector in the cell can also be included in the expression vector.
  • Preferred selectable markers include those which confer resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469).
  • Selectable markers can also allow a cell to grow on minimal medium, or in the presence of toxic metabolite and can include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
  • the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed.
  • cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
  • neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Recombinant viral vectors are useful for in vivo expression of the antimicrobial peptides of the invention since they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • Exemplary methods of introducing expression vectors into bacterial cells include for example conventional transformation or transfection techniques, or by phage-mediated infection.
  • transformation transformation
  • transduction conjuggation
  • protoplast fusion are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • the polynucleotides may be optimized for increased expression in the transformed organism.
  • the polynucleotides can be synthesized using preferred codons for improved expression.
  • the pre- and propeptide sequences may be needed.
  • the propeptide segments may play a role in aiding correct peptide folding.
  • polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094 have antimicrobial activity
  • the present invention contemplates use thereof for treating infection in a mammalian subject.
  • the peptides are used to treat a topical infection (i.e. infection of the skin) and are provided in a topical formulation.
  • the peptides are used to treat an infection inside the body.
  • the peptides (or polynucleotides encoding same) may be provided ex vivo or in vivo.
  • the present invention contemplates contacting cells with the polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094 (or with expression constructs that encode the peptides) per se or as part of a pharmaceutical composition.
  • the peptide used in the pharmaceutical composition comprises the sequence 11089-11094.
  • the peptides may be used alone or together with additional antimicrobial agents (e.g. antibiotic and/or additional antimicrobial peptides).
  • additional antimicrobial agents e.g. antibiotic and/or additional antimicrobial peptides.
  • antibiotics include, but are not limited to aminoglycoside antibiotics, cephalosporins, quinolone antibiotics, macrolide antibiotics, penicillins, sulfonamides, tetracyclines and carbapenems. It will be appreciated that since the peptides of embodiments of this invention enhance the antibacterial effect of the antibiotic, doses of the antibiotic may be lower (e.g. 20% lower, 30% lower, 40% lower, 50% lower, 60% lower, 70% lower, 80% lower or even 90% lower than those currently in use.
  • compositions of the present invention are administered to a subject in need thereof in order to prevent or treat a bacterial infection.
  • the term “subject in need thereof” refers to a mammal, preferably a human subject.
  • treating refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathogen infection.
  • pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to peptides of the present invention accountable for the intended biological effect. It will be appreciated that a polynucleotide encoding a peptide of the present invention may be administered directly into a subject (as is, or part of a pharmaceutical composition) where it is translated in the target cells i.e. by gene therapy. Accordingly, the phrase “active ingredient” also includes such polynucleotides.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier,” which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • peptides of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
  • antibiotics e.g. rifampicin, chloramphenicol and spectinomycin.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • the preparation of the present invention may also be formulated as a topical composition, such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste and a gel.
  • a topical composition such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste and a gel.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1].
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • the peptides described herein which comprise antitoxin activity may be expressed in microbial populations, thereby protecting the microbial population from the toxic effect of a toxin.
  • the present invention further contemplates polynucleotide sequences encoding the antitoxin polypeptides disclosed herein.
  • polynucleotide sequences are set forth in SEQ ID NOs: 5548-8316.
  • the present inventors have further discovered novel toxin antitoxin pairs that may be expressed in bacterial populations. Expression therein may serve to protect the bacterial population from the lytic effect of a bacteriophage (i.e. impart resistance to the bacteriophage).
  • Antitoxin polypeptide sequences SEQ ID NO: designation SEQ ID NO: designation 2773 >Toxin: Lferr_1303 8317 >Antitoxin: Lferr_1302 2774 >Toxin: Adeg_2177 8318 >Antitoxin: Adeg_2178 2775 >Toxin: BLD_0338 8319 >Antitoxin: BLD_0339 2776 >Toxin: HMPREF0175_1783 8320 >Antitoxin: HMPREF0175_1782 2777 >Toxin: BIL_08340 8321 >Antitoxin: BIL_08350 2778 >Toxin: BL1460 8322 >Antitoxin: BL1461 2779 >Toxin: HMPREF0177_01212 8323 >Antitoxin: HMPREF0177_01213 2780 >Toxin: CJA_3634 8324 >Antitoxin: CJA
  • the present inventors have found that when the toxin comprises an amino acid sequence as set forth in SEQ ID NOs: 3031-3078, the bacterial population in which the toxin antitoxin system is expressed is protected from the lytic activity of the T7 ⁇ 4.3 ⁇ 4.5 ⁇ 4.7 bacteriophage.
  • the toxin comprises an amino acid sequence as set forth in SEQ ID NO: 2805-2871, the bacterial population in which the toxin antitoxin system is expressed is protected from the lytic activity of the WT T7 strain.
  • a bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence into mRNA.
  • a promoter can have a transcription initiation region, which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a bacterial promoter can also have a second domain called an operator, which can overlap an adjacent RNA polymerase binding site at which RNA synthesis begins.
  • the operator permits negative regulated (inducible) transcription, as a gene repressor protein can bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator.
  • positive regulation can be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence.
  • a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression can therefore be either positive or negative, thereby either enhancing or reducing transcription. Other examples of positive and negative regulatory elements are well known in the art.
  • Various promoters that can be included in the protein expression system include, but are not limited to, a T7/LacO hybrid promoter, a trp promoter, a T7 promoter, a lac promoter, and a bacteriophage lambda promoter.
  • Any suitable promoter can be used to carry out the present invention, including the native promoter or a heterologous promoter.
  • Heterologous promoters can be constitutively active or inducible.
  • a non-limiting example of a heterologous promoter is given in U.S. Pat. No. 6,242,194 to Kullen and Klaenhammer.
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1987) Nature 198:1056), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; Yelverton et al. (1981) Nucleic Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publication Nos. 36,776 and 121,775).
  • trp tryptophan
  • the beta-lactamase (bla) promoter system (Weissmann, (1981) “The Cloning of Interferon and Other Mistakes,” in Interferon 3 (ed. I. Gresser); bacteriophage lambda PL (Shimatake et al. (1981) Nature 292:128); the arabinose-inducible araB promoter (U.S. Pat. No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406) promoter systems also provide useful promoter sequences. See also Balbas (2001) Mol. Biotech. 19:251-267, where E. coli expression systems are discussed.
  • synthetic promoters that do not occur in nature also function as bacterial promoters.
  • transcription activation sequences of one bacterial or bacteriophage promoter can be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433).
  • the tac Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21
  • trc Brosius et al. (1985) J. Biol. Chem.
  • promoters are hybrid trp-lac promoters comprised of both trp promoter and lac operon sequences that are regulated by the lac repressor.
  • the tac promoter has the additional feature of being an inducible regulatory sequence.
  • expression of a coding sequence operably linked to the tac promoter can be induced in a cell culture by adding isopropyl-1-thio-.beta.-D-galactoside (IPTG).
  • IPTG isopropyl-1-thio-.beta.-D-galactoside
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
  • a naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.
  • the bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074).
  • a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO Publication No. 267,851).
  • the vector can additionally contain a nucleotide sequence encoding the repressor (or inducer) for that promoter.
  • an inducible vector of the present invention can regulate transcription from the Lac operator (LacO) by expressing the nucleotide sequence encoding the Lad repressor protein.
  • Other examples include the use of the lexA gene to regulate expression of pRecA, and the use of trpO to regulate ptrp.
  • Alleles of such genes that increase the extent of repression (e.g., lacIq) or that modify the manner of induction (e.g., lambda CI857, rendering lambda pL thermo-inducible, or lambda CI+, rendering lambda pL chemo-inducible) can be employed.
  • the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine et al. (1975) Nature 254:34).
  • SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ end of bacterial 16S rRNA (Steitz et al. (1979) “Genetic Signals and Nucleotide Sequences in Messenger RNA,” in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger, Plenum Press, NY).
  • the bacterial protecting peptides can also be secreted from the cell by creating chimeric DNA molecules that encode a protein comprising a signal peptide sequence fragment that provides for secretion of the two-component regulatory polypeptides in bacteria (U.S. Pat. No. 4,336,336).
  • the signal sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids that direct the secretion of the protein from the cell.
  • the protein is either secreted into the growth medium (Gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (Gram-negative bacteria).
  • processing sites which can be cleaved either in vivo or in vitro, encoded between the signal peptide fragment and the protein of the invention.
  • DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb et al. (1984) EMBO J. 3:2437-2442) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212).
  • Other prokaryotic signals include, for example, the signal sequence from penicillinase, Ipp, or heat stable enterotoxin II leaders.
  • transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter, flank the coding sequence. These sequences direct the transcription of an mRNA that can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences (of about 50 nucleotides) that are capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.
  • Bacteria such as Lactobacillus acidophilus generally utilize the translation start codon ATG, which specifies the amino acid methionine (which is modified to N-formylmethionine in prokaryotic organisms). Bacteria also recognize alternative translation start codons, such as the codons GTG and TTG, which code for valine and leucine, respectively. However, when these alternative translation start codons are used as the initiation codon, these codons direct the incorporation of methionine rather than of the amino acid that they normally encode. Lactobacillus acidophilus NCFM recognizes these alternative translation start sites and incorporates methionine as the first amino acid.
  • the polynucleotide sequence encoding the toxin may be comprised in the same expression vector as that comprising the antitoxin or may be comprised on a separate expression vector from that of the antitoxin.
  • Exemplary microbial populations that may be protected from toxins and/or the lytic effect of bacteriophages according to this aspect of the present invention include those bacteria useful in dairy and fermentation processing.
  • microorganisms in which the peptides described herein may be expressed are those that are useful in the manufacture of milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk.
  • the microorganisms may be probiotic organisms.
  • the bacteria is a lactic acid bacteria.
  • lactic acid bacteria refers to a genus selected from the following: Aerococcus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Melissococcus, Alloiococcus, Dolosigranulum, Lactosphaera, Tetragenococcus, Vagococcus, and Weissella (Holzapfel et al. (2001) Am. J. Clin. Nutr. 73:365 S-373S; Bergey's Manual of Systematic Bacteriology, Vol. 2 (Williams and Wilkins, Baltimore (1986)) pp. 1075-1079).
  • the production of bacteria comprising the toxins and/or antitoxins of the present invention can be carried out in accordance with known techniques, including but not limited to those described in Mayra-Makinen and Bigret (1993) Lactic Acid Bacteria. Salminen and vonWright eds. Marcel Dekker, Inc. New York. 65-96; Sandine (1996) Dairy Starter Cultures Cogan and Accolas eds. VCH Publishers, New York. 191-206; Gilliland (1985) Bacterial Starter Cultures for Food. CRC Press, Boca Raton, Fla.
  • fermenting refers to the energy-yielding, metabolic breakdown of organic compounds by microorganisms that generally proceeds under anaerobic conditions and with the evolution of gas.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Clustering of individual genes based on sequence homology was retrieved from IMG (worldwideweb(dot)img(dot)jgi(dot)doe(dot)gov/cgi-bin/w/main(dot)cgi) on November 2010 (based on the “IMG cluster” field). Cluster IDs were recorded for every consecutive pair of genes analyzed. Pairs containing a gene that lacked a cluster ID were discarded. All pairs having the same two cluster IDs (regardless of the order and the strand) were aggregated into a single “family of pairs”. Families containing less than 7 pairs were ignored, to ensure statistical power in next steps of the analysis. This resulted in 21,417 families, containing at least 7 pairs of consecutive genes sharing the same two cluster IDs, which were further analyzed. The following “TA cloning fraction” (F) parameters were calculated for each family:
  • Genus names of all containing organisms were extracted.
  • F sim simulated “TA cloning fraction”
  • DI val ‘mean number of defense island genes in proximity’
  • the locus tags of all genes from the 24 final families were checked against a previously compiled list of known TA systems (Makarova et al., 2009). For a given family, if at least one pair was found in the list of known TA systems, the family was declared as ‘known’. Families that were not declared as ‘known’ were further similarly checked against a list of predicted TA genes downloaded from TADB (http://bioinfo-mml(dot)sjtu(dot)edu(dot)cn/TADB/) (Shao et al., 2011). Families in which at least one pair was found in this TADB list were declared as ‘predicted’. Families found in neither list were declared as ‘novel’.
  • Toxin and antitoxin were amplified from their genomes of origin (in the case of sanaAT, psyrAT and sdenAT) or synthesized (GenScript) for the families pmenAT and rlegAT.
  • the toxin was then directionally ligated into the pRSFDuet-1 vector (EMD Chemicals Inc.) and the antitoxin ligated into the pBAD/HisA plasmid (Invitrogen). Since transformation of the toxin gene alone resulted in mutations in the toxin due to toxicity, the two plasmids (carrying the toxin and antitoxin) were co-transformed into E.
  • clones were cultured in LB medium with 100 ⁇ g/ml ampicillin, 50 ⁇ g/ml kanamycin, 34 ⁇ g/ml chloramphenicol and 0.3% arabinose overnight. The next day, a portion of each overnight culture was inoculated into fresh medium (10-fold dilution) and 100 were spotted on LB plates supplemented with 100 ⁇ g/ml ampicillin, 50 ⁇ g/ml kanamycin and 34 ⁇ g/ml chloramphenicol. Toxin, antitoxin or both were induced by 100 ⁇ M IPTG and 0.3% arabinose, respectively.
  • the viability assay ( FIG. 3C ) was performed as described by Pedersen et al (2002). Briefly, each strain was grown overnight at 37° C. in LB medium containing 100 ⁇ g/ml ampicillin, 50 ⁇ g/ml kanamycin, and 34 ⁇ g/ml chloramphenicol. In the next morning, cells were then diluted 1:1000 in the same medium as above and grown for 3 hours. At time zero cells were then washed once with LB, and then transcription of toxin was induced by 100 ⁇ M IPTG.
  • E. coli strains harboring the antitoxin only or the toxin-antitoxin of psyrAT, sanaAT, pmenAT, rlegAT, and sdenAT binary toxin-antitoxin systems were grown overnight in LB liquid medium supplemented with 35 ⁇ g/ml chloramphenicol, 100 ⁇ g/ml ampicillin and 0.3% L-arabinose with or without 50 ⁇ g/ml kanamycin, respectively. Overnight cultures were diluted 1:100 in fresh LB medium supplemented with inducers and antibiotics as above and aerated with shaking at 37° C. until reaching O.D 600 ⁇ 0.5.
  • EOP efficiency of plating
  • the 4.5 gene (3′ His-tagged) and full-length Lon protease gene (5′ Flag-tagged) were cloned into the 1 st and 2 nd expression cassettes, respectively, of the vector pRSFDuet-1 (EMD Chemicals, Inc.). DNA cloning was performed using the Restriction Free (RF) cloning procedure (Unger et al., 2010). Induction experiments were performed using E. coli BL21 (DE3) cells expressing only the Lon or the 4.5 proteins and cells expressing both Lon and 4.5 proteins. Induction was carried out at 37° C. for 3 hr by addition of 200 ⁇ M of IPTG.
  • RF Restriction Free
  • Cell pellets were lysed by sonication in a buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 ⁇ l/mL protease inhibitor cocktail (Set IV, EMD Chemicals, Inc.). Cell debris were removed by centrifugation at 4° C. for 15 minutes at 18,000 g. Clear supernatants were transferred to 1.5 ml tubes and incubated on a rotator shaker at 4° C.
  • the present inventors To detect families of gene pairs in which one of the genes (putative toxin) is absent from clones unless the adjacent gene (putative antitoxin) is also present, the present inventors first searched for homologous gene pairs that repeatedly appear adjacent to each other in multiple genomes (at least 7 appearances) and aggregated them into families of pairs (Methods). To avoid the analysis of housekeeping genes that appear in conserved operons (e.g., ribosomal protein genes), the present inventors focused on families showing high tendency to undergo horizontal gene transfer (Methods).
  • Each pair of genes (X and Y) in each family was considered as following the “TA cloning pattern” if the number of clones covering gene X (toxin) but not Y (antitoxin) was 0, the number of clones covering gene Y but not X was >0 and the number of clones covering both X and Y was >0 ( FIGS. 1B-C ).
  • the identified families may include genes that follow the TA cloning pattern as a by-product of their functions, and not for reasons associated with classical TA systems.
  • a metabolic enzyme whose expression results in accumulation of toxic intermediates might be neutralized by an accompanying transcriptional repressor. This may be the case for the argininosuccinate synthase and the ArgR repressor of the arginine regulon that obey the TA cloning patterns in 4 out of 12 homologous pairs.
  • the present inventors focused on those families that had high tendency to appear within bacterial “defense islands”.
  • the present analysis retrieved 8 putative novel families of TA systems (Table 5). Six of these families, which appeared as “stand-alone” toxin-antitoxin pairs (see below), were selected for further experimental characterization (Table 6, herein below). A representative pair was selected from each family and co-transformed into E. coli BL21 (DE3) on a compatible two-vector system, so that the putative toxin was under the control of an IPTG-induced promoter, and the antitoxin was under the control of an arabinose-induced promoter ( FIG. 3A ). E. coli bacteria carrying these two plasmids were plated on agar plates containing IPTG, arabinose, or both IPTG and arabinose.
  • the 6 novel validated families were named based on the species from which the validated system was taken: pmenAT ( P. mendocina ); sanaAT (S. sp. ana-3); rlegAT ( R. leguminosarum ); psyrAT ( P. syringae ); sdenAT ( S. denitrificans ); and hhalTA ( H. halophila ).
  • pmenAT P. mendocina
  • sanaAT S. sp. ana-3
  • rlegAT R. leguminosarum
  • psyrAT P. syringae
  • sdenAT S. denitrificans
  • hhalTA H. halophila
  • the toxins in these new systems were further tested in order to analyze whether they have a bacteriocidic (cell-killing) or bacteriostatic (growth-inhibiting) effect.
  • IPTG was used to induce toxin expression for different time intervals (ranging from
  • TA modules described to date share several typical characteristics: the antitoxin appears upstream of the toxin; both the toxin and antitoxin are small proteins (typically ⁇ 100aa); and the antitoxin contains a DNA binding domain (Makarova et al., 2009). Since the present approach does not rely on such attributes for TA modules discovery it has the potential to expand the premises of TA modules properties. Indeed, some of the new families that were experimentally validated deviate significantly from the previously described characteristics.
  • the sizes of many new toxins and antitoxins are significantly larger than 100 aa, with a maximum of 698 aa in the toxin of the psyrAT system; in two families the toxin is located upstream of the antitoxin ( FIG. 4A ); and several antitoxins do not contain a known DNA binding domain (although the possibility cannot be ruled out that some antitoxins code for yet uncharacterized such domains) ( FIG. 4A ).
  • the toxin is a ribonuclease
  • diverse domains within the new toxins detected, including DNA helicase, phosphoribosyl-transferase and nucleotidyl-transferase suggest novel mechanisms of toxicity ( FIG. 4A ).
  • the presence of an ADP-ribose-binding, S-adenosyl-homocysteine hydrolase and nucleotide-binding domains in some of the antitoxins suggest that these antitoxins perform a more complex function than simply masking the activity of the toxin by protein-protein interactions ( FIG. 4A ; Table 5).
  • the TA system is part of a larger operon that is co-horizontally transferred between genomes in the context of defense islands, suggesting their involvement in more complex defense mechanisms (Table 5; FIG. 8 ).
  • toxin-antitoxin systems can be modular, such that members of one toxin family may be associated with several different types of antitoxin, and vice versa (Leplae et al., 2011; Makarova et al., 2009). Indeed, the toxins of three of the present new families carry the same domain, DUF1814, with a different antitoxin associated with this domain in each of the three families (Table 2; FIG. 4A ).
  • rlegAT where the DUF1814 toxin is accompanied by COG5340 as an antitoxin, was previously shown to be enriched in defense islands in bacterial genomes and was suggested as a new TA system based on its two-gene nature (Makarova et al., 2011).
  • the DUF1814 domain was recently classified as a nucleotidyl-transferase domain based on structural information, but its specific substrates are yet unknown (Kuchta et al., 2009).
  • DUF1814 domain was also documented in AbiG, a two-gene system involved in abortive infection in Lactococcus lactis via an unknown mechanism (Makarova et al., 2011; O'Connor et al., 1996), although there is no direct homology between the AbiG system and any of the genes in the new TA systems that were presently detected. Therefore, the results point to DUF1814 domain-containing proteins as a widespread superfamily of toxins that might be involved in anti-phage defense (see below).
  • PCC 6506 648860075 648860076 Parvibaculum lavamentivorans DS-1 640878511 640878512 Pectobacterium carotovorum carotovorum 644864144 644864143 PC1 Pedobacter sp.
  • BAL39 641137361 641137360 Prevotella timonensis CRIS 5C-B1 647314799 647314798 Providencia rustigianii DSM 4541 643141873 643141874 Pseudomonas entomophila L48 637999408 637999409 Pseudomonas mendocina ymp 640501778 640501779 Rhodopseudomonas palustris TIE-1 642711985 642711984 Roseiflexus sp.
  • MZ1T 643700535 643700534 Thermus aquaticus Y51MC23 645188421 645188422 Thiobacillus denitrificans ATCC 25259 637710175 637710174 Zymomonas mobilis subsp. mobilis ZM4 648935037 648935036 ZM4 plasmid pZZM405
  • PCC 8801 643474605 643474606 Dehalogenimonas lykanthroporepellens 648069289 648069288 BL-DC-9 Desulfitobacterium hafniense DCB-2 643560590 643560591 Desulfitobacterium hafniense Y51 637910440 637910441 Desulfotalea psychrophila LSv54 637527656 637527657 Desulfotomaculum acetoxidans 5575, 645033659 645033660 DSM 771 Escherichia coli 83972 644299713 644299712 Escherichia coli ABU 83972 648235458 648235457 Escherichia coli B088 647955723 647955722 Escherichia coli B185 647960289 647960288 Escherichia coli E22 (EPEC) 638663931 638663932 Escherichia
  • PCC 7120 637233840 637233841 Oscillochloris trichoides DG6 650114297 650114298
  • Pelodictyon phaeoclathratiforme BU-1 642727669 642727668 Photorhabdus luminescens laumondii TTO1 637466171 637466172 Polaromonas naphthalenivorans CJ2 639835976 639835975
  • Providencia stuartii ATCC 25827 642341526 642341525 Pseudomonas aeruginosa 39016 650201418 650201417 Pseudomonas syringae pv.
  • NGR234 (ANU265) 643821930 643821929 Rhodomicrobium vannielii ATCC 17100 649745264 649745263 Rhodopseudomonas palustris DX-1 649838192 649838193 Rhodopseudomonas palustris DX-1 649839250 649839249 Slackia heliotrinireducens RHS 1, 644987249 644987248 DSM 20476 Synechococcus sp.
  • MLMS-1 639158034 639158033 Desulfurivibrio alkaliphilus AHT2 646845548 646845547 Eggerthella lenta VPI 0255, DSM 2243 645023812 645023813 Helicobacter canadensis MIT 98-5491, 643921049 643921048 ATCC 700968 Helicobacter canadensis MIT 98-5491, 647542139 647542138 ATCC 700968 Marinobacter sp. ELB17 640637051 640637050 Marinomonas sp. MWYL1 640805575 640805574 Pantoea sp.
  • the present inventors next set out to explore whether any of the TA systems detected can provide defense against phage. For this, efficiency of plating assays of T7 phage on E. coli hosts were performed ( FIG. 5A ). Since these new TA systems are widespread in E. coli strains (but are not found in the lab strains E. coli K-12 and E. coli BL21), it was hypothesized that a successful coliphage, such as T7, might hold anti-defense mechanisms that mitigate the defense conferred by the TA systems. The present inventors therefore tested, in addition to the wild-type (WT) T7 phage, 12 additional T7 mutants lacking genes that are not-essential for infection of E. coli K-12 (Table 12).
  • WT wild-type
  • T7 mutants were used to infect E. coli BL21 or K-12 expressing the five verified new TA systems, as well as control clones expressing only the antitoxin of each system ( FIG. 5A ).
  • One of the tested systems, sanaAT was found to provide E. coli with resistance against T7 ⁇ 4.3 ⁇ 4.5 ⁇ 4.7, reducing sensitivity to this phage strain by almost 3 orders of magnitude ( FIG. 5B ).
  • a second system, rlegAT resulted in opaque plaques with plaque diameters reduced more than fourfold for the WT T7 strain (diameters of 0.47 mm ⁇ 0.06 for E. coli expressing both the toxin and the antitoxin, as compared to 1.77 mm ⁇ 0.03 for bacteria expressing the antitoxin only).
  • the present inventors asked whether the defense provided by the sanaTA system against the phage is Lon-dependent.
  • the Lon protease is one of the major proteolytic machineries in the bacterial cell (Gottesman, 2003), and was implicated in degradation (“destabilization”) of many types of antitoxins in E. coli , thus enabling toxin activity (Christensen et al., 2001; Van Melderen et al., 1996; Wang et al., 2011).
  • T7 mutant growth on E. coli containing the sanaTA system was restored by two orders of magnitude when the E. coli also lacked lon ( FIG. 5D ), suggesting that the sanaTA protection from T7 phage depends on Lon activity.
  • Toxin-antitoxin systems were shown to mediate bacterial resistance to antibiotics through promotion of persister cells (Maisonneuve et al., 2011; Rotem et al., 2010).
  • Persisters are non-growing bacterial cells found in a dormant state and are thus resistant to many antibiotics and other stress conditions (Smith and Romesberg, 2007).
  • Such persisters are stochastically found in bacterial populations as a small fraction of the population. It was recently shown that persistence in E. coli depends on resident type II TA systems as well as on the Lon protease. Indeed, deletion of Lon in E. coli was shown to mitigate TA-system-mediated persistence and resulted in higher sensitivity to antibiotics (Maisonneuve et al., 2011).
  • phage genes that may function as Lon inhibitors
  • the present inventors looked for all phage genomes for genes showing homology to the N-terminus of Lon, roughly where the 4.5 aligns ( FIG. 5D ). 6 such genes were found (including gene 4.5) in a diverse set of phages.
  • phage proteins inhibit Lon protease and can thus function as inhibitors of persistence.

Abstract

An isolated polypeptide is disclosed comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094, wherein the polypeptide has antimicrobial activity. Uses thereof for treating microbial infections are also disclosed.

Description

    RELATED APPLICATION/S
  • This application is a division of U.S. patent application Ser. No. 14/140,793, filed Dec. 26, 2013, which claims the benefit of priority under 35 USC §119(e) of U.S. Provisional Patent Application No. 61/745,838 filed Dec. 26, 2012.
  • The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
    • GOVERNMENT INTERESTS Federally Sponsored Research
  • This invention was made with government support under AI082376 awarded by NIH. The government has certain rights in the invention.
    • SEQUENCE LISTING STATEMENT
  • The ASCII file, entitled 65707SequenceListing.txt, created on Apr. 18, 2016, comprising 20,497,456 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
    • FIELD AND BACKGROUND OF THE INVENTION
  • The present invention, in some embodiments thereof, relates to bacterial polypeptides that comprise toxin or antitoxin activity and, more particularly, but not exclusively, to toxin-antitoxin pairs that may be used as bacterial anti-phage defense systems.
  • A broad array of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various substrates. Because enormous amounts of bacteria are being cultivated each day in large fermentation vats, bacteriophage contamination can rapidly bring fermentations to a halt and cause economic setbacks, and is therefore considered a serious threat in these industries. The dairy fermentation industry has openly acknowledged the problem of phage and has been working with academia and starter culture companies to develop defense strategies and systems to curtail the propagation and evolution of phages for decades.
  • To survive in the face of perpetual phage attacks, bacteria have developed a variety of anti-phage defense systems (Labrie et al., 2010; Stern and Sorek, 2011). These systems include restriction enzymes that recognize and cleave foreign DNA (King and Murray, 1994), abortive infection (Abi) mechanisms that lead the bacterial cell, upon phage invasion, to commit “suicide”, thus protecting the colony against phage spread (Chopin et al., 2005); and the recently identified adaptive defense system called CRISPR/Cas, which uses small RNAs to target invading phage DNA (Deveau et al., 2010; Horvath and Barrangou, 2010; Sorek et al., 2008; van der Oost et al., 2009). Due to the rapid evolution and elaborated biological novelty associated with the bacteria-phage arms race, it is estimated that many additional, yet uncharacterized anti-phage defense systems are encoded by bacteria and archaea (Makarova et al., 2011; Stern and Sorek, 2011). As part of this continuous arms race, successful phages had also developed numerous counter-resistance mechanisms to overcome bacterial defense (Labrie et al., 2010; Stern and Sorek, 2011).
  • The growing availability of genomic sequences has elucidated the vast dispersion of toxin-antitoxin (TA) systems in prokaryotic genomes (Shao et al., 2011). These systems (also called TA modules), composed of a toxic gene and a neutralizing gene, were first suggested to function as plasmid ‘addiction molecules’ (Van Melderen and Saavedra De Bast, 2009; Wozniak and Waldor, 2009), but their prevalent existence on chromosomes (Aizenman et al., 1996; Makarova et al., 2009; Shao et al., 2011) has led to the understanding that this is unlikely their major role. Accumulating evidence suggest that TA modules play pivotal roles in prokaryotic cellular biology including programmed cell death (Hazan et al., 2004), stress response (Christensen et al., 2001), generation of persister cells (Schumacher et al., 2009), biofilm formation (Kim et al., 2009) and phage defense via abortive infection (Fineran et al., 2009; Hazan and Engelberg-Kulka, 2004; Koga et al., 2011; Pecota and Wood, 1996).
  • The most prevalent kind of TA systems is type II systems, where both toxin and antitoxin are proteins (as opposed to types I and III where the antitoxin is a non-coding RNA (Fineran et al., 2009; Fozo et al.)). The two genes, which reside on the same operon, code for small proteins and inhibition of the toxin is carried out through protein-protein interaction. As a rule, the toxin is a stable protein and the antitoxin is unstable and degrades rapidly by one of the housekeeping bacterial proteases, usually Lon or ClpP (Aizenman et al., 1996; Cherny and Gazit, 2004; Christensen et al., 2004; Christensen et al., 2001; Christensen et al., 2003; Lehnherr and Yarmolinsky, 1995; Roberts et al., 1994; Van Melderen et al., 1996). As a result, continuous production of the antitoxin is required to prevent the toxin's deleterious effects (Van Melderen and Saavedra De Bast, 2009).
  • A number of reports demonstrate a role for type II TA systems in phage resistance via an Abi mechanism: the extensively studied MazEF system was shown to eliminate P1 phage infection (Hazan and Engelberg-Kulka, 2004), and the recently identified Rn1A toxin which is activated following T4 phage infection degrades phage mRNAs (Koga et al., 2011). Type I and Type III TA systems (hok/sok and ToxIN, respectively) were also shown to provide resistance against phages (Fineran et al., 2009; Pecota and Wood, 1996). However, based on their presence in bacterial ‘defense islands’, it was hypothesized that TA systems play a much wider role in phage defense (Makarova et al., 2011).
  • The usage of Abi systems for biotech purposes, especially in protecting dairy industry bacteria from bacteriophages is disclosed in U.S. Pat. Nos. 7,754,868, 7,550,576, 7,169,911 and 5,994,118.
  • To date, at least 12 families of type II TA systems have been described (Masuda et al., 2012; Shao et al., 2011). Members of these families are widespread in bacterial and archaeal genomes and undergo extensive horizontal gene transfer (Guglielmini et al., 2008; Makarova et al., 2009; Pandey and Gerdes, 2005; Shao et al., 2011). Computational studies based on the ‘guilt by association’ principle (gene neighbors of known toxins/antitoxins are suspected as antitoxins/toxins themselves) and on other characteristics of TA systems have recently predicted several novel TA families (Leplae et al., 2011; Makarova et al., 2009), which are yet to be validated experimentally.
    • SUMMARY OF THE INVENTION
  • According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094, wherein the polypeptide has antimicrobial activity.
  • According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin.
  • According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094, wherein the polypeptide has antimicrobial activity.
  • According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin.
  • According to an aspect of some embodiments of the present invention there is provided an anti-microbial composition, comprising a carrier and as an active ingredient an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094.
  • According to an aspect of some embodiments of the present invention there is provided an anti-microbial composition, comprising a carrier and as an active ingredient an isolated polynucleotide comprising a nucleic acid sequence which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094.
  • According to an aspect of some embodiments of the present invention there is provided method of treating an infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the anti-microbial composition described herein, thereby treating the infection.
  • According to an aspect of some embodiments of the present invention there is provided a solid support coated with an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544.
  • According to an aspect of some embodiments of the present invention there is provided a method of killing a microbe, the method comprising contacting the microbe with an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544, thereby killing the microbe.
  • According to an aspect of some embodiments of the present invention there is provided a method of protecting a microbe from a toxin comprising expressing in the microbe the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin.
  • According to an aspect of some embodiments of the present invention there is provided an isolated bacterial population genetically modified to express a toxin and a cognate antitoxin thereof, the bacterial population being resistant to a lytic activity of a bacteriophage, wherein the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-3117, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2773-2804, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8317-8348, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2805-2871, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8349-8415, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2872-2935, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8416-8479, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2936-3030, the antitoxin comprises an amino acid selected from the group consisting of SEQ ID NO:8480-8574, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3031-3078, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8575-8622, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3079-3087, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8623-8631, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3088-3094, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8632-8638, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3095-3109, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8639-8653, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3110-3117, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8654-8661.
  • According to an aspect of some embodiments of the present invention there is provided a method of protecting a bacterial population from phage attack, the method comprising expressing in the bacterial population a toxin and a cognate antitoxin thereof, wherein the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-3117, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2773-2804, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8317-8348, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2805-2871, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8349-8415, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2872-2935, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8416-8479, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2936-3030, the antitoxin comprises an amino acid selected from the group consisting of SEQ ID NO:8480-8574, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3031-3078, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8575-8622, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3079-3087, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8623-8631, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3088-3094, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8632-8638, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3095-3109, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8639-8653, wherein when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3110-3117, the antitoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8654-8661.
  • According to an aspect of some embodiments of the present invention there is provided a nucleic acid system comprising:
  • (i) a first isolated polynucleotide which encodes a toxin, the toxin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544; and
  • (ii) a second isolated polynucleotide which encodes a cognate antitoxin to the toxin, the antitoxin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-11088.
  • According to some embodiments of the invention, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2773-3117 and 11089-11094.
  • According to some embodiments of the invention, the amino acid sequence consists of the sequences selected from the group as set forth in SEQ ID NOs: 2773-5544.
  • According to some embodiments of the invention, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 8317-8661.
  • According to some embodiments of the invention, the amino acid sequence consists of the sequences selected from the group as set forth in SEQ ID NOs: 8317-11088.
  • According to some embodiments of the invention, the isolated peptide comprises at least one naturally occurring amino acid.
  • According to some embodiments of the invention, the isolated peptide comprises a synthetic amino acid.
  • According to some embodiments of the invention, the isolated peptide is attached to a cell penetrating agent.
  • According to some embodiments of the invention, the attached is covalently attached.
  • According to some embodiments of the invention, the cell penetrating agent is a peptide agent.
  • According to some embodiments of the invention, the isolated peptide is attached to a sustained-release enhancing agent.
  • According to some embodiments of the invention, the sustained-release enhancing agent is selected from the group consisting of hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene glycol (PEG), glyme and polyisopropylacrylamide.
  • According to some embodiments of the invention, the isolated polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-2772.
  • According to some embodiments of the invention, the isolated polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5545-8316.
  • According to some embodiments of the invention, the carrier is a pharmaceutically acceptable carrier.
  • According to some embodiments of the invention, the anti-microbial composition is formulated for topical application.
  • According to some embodiments of the invention, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-2772 and 11095-11100.
  • According to some embodiments of the invention, the carrier is a pharmaceutically acceptable carrier.
  • According to some embodiments of the invention, the method further comprises administering to the subject an antibiotic.
  • According to some embodiments of the invention, the contacting is effected in vivo.
  • According to some embodiments of the invention, the contacting is effected ex vivo.
  • According to some embodiments of the invention, the microbe comprises a bacteria.
  • According to some embodiments of the invention, the isolated polynucleotide is operably linked to a promoter.
  • According to some embodiments of the invention, the bacterial population is a lactic acid bacterial population.
  • According to some embodiments of the invention, the bacterial population is of a species selected from the group consisting of Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus species, Pediococcus species and Bifidobacterium.
  • According to some embodiments of the invention, when the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2805-2871, the bacteriophage comprises WT T7 strain.
  • According to some embodiments of the invention, the first isolated polynucleotide encodes a toxin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-3117; and the second isolated polynucleotide encodes an antitoxin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8317-8661.
  • According to some embodiments of the invention, the first isolated polynucleotide and the second isolated polynucleotide are comprised in a non-identical expression vector.
  • According to some embodiments of the invention, the anti-microbial composition is for treating an infection.
  • Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
    • BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
  • The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
  • Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
  • In the drawings:
  • FIGS. 1A-1D illustrate that data derived from whole-genome shotgun sequencing exposes toxin-antitoxin pairs. (A) The “Sanger” based process of DNA sequencing involves random genome fragmentation and transformation of DNA fragments into E. coli. (B) In a DNA locus spanning a toxin/antitoxin (TA) gene pair, random fragmentation leaving the toxin detached from its cognate antitoxin leads to E. coli growth arrest, whereas a fragment containing both genes, or the antitoxin alone, will be propagated and sequenced. (C) A known family of toxin/antitoxin gene pairs, of the VapBC type, was found in 11 of the analyzed genomes. In 7/11 cases the gene pair follows the “TA cloning pattern”, significantly higher than the number expected by chance (p=4×10−3). (D) The VapBC locus in Haemophilus somnus 129T (accession NC_008309, locus tags HS_1769-HS_1770). Shown are clones (brown) mapped to the reference genome at that locus. Clones covering the antitoxin but not the toxin are numbered. Only five of the 22 clones covering both toxin and antitoxin are shown.
  • FIG. 2 is a diagram of the workflow for systematic discovery of families of toxin/antitoxin associated with anti-phage defense.
  • FIGS. 3A-3C are graphs and diagrams illustrating properties of novel, experimentally verified TA systems (A) growth of bacteria when only antitoxin is induced (left), only toxin is induced (middle) and both are induced together (right). Toxin and antitoxin were cloned on pRSF (IPTG inducible promoter) and pBAD (arabinose inducible), respectively, in E. coli BL21 (DE3) pLysS. C, control bacteria with empty plasmids; 1-pmen system; 2-sana system; 3-psyr system; 4-rleg system; 5-sden system. (B) Kinetics of E. coli BL21 (DE3) pLysS growth when toxin and antitoxin are co-expressed simultaneously (purple), alone (red and blue for toxin and antitoxin, respectively) or when antitoxin is induced 2.5 hrs following toxin induction (green). Kinetic measurements were performed on biological triplicates in technical duplicates. (C) Viability assays for cells following exposure to toxin. Transcription of toxin was induced by 100 μM IPTG. At increasing time points following toxin induction (30, 60, 120, 180, 240 and 300 mins) cells were plated on LB-plates containing 0.3% arabinose and no IPTG, to activate antitoxin expression. Colony forming units (CFUs) were determined by colony counting.
  • FIG. 4A illustrates operon and domain organization of the validated families. Representative pair of each family is shown. For each pair, red and blue genes denote toxin and antitoxin, respectively.
  • FIGS. 4B-4C are tables showing distribution of novel TA system among (B) different bacterial phyla and (C) human associated bacteria. Number of instances of each system within a phylum/bacterial species is indicated, with darker colors indicating higher number of instances.
  • FIGS. 5A-5F illustrate that T7 Gp 4.5 antagonizes abortive infection by interacting with Lon. (A) Illustration of the plaque forming unit (PFU) assays on E. coli harboring toxin/antitoxin systems. Efficiency of plating (EOP) was calculated by dividing the number of PFUs obtained for a bacterial lawn expressing the toxin+antitoxin by the number of PFUs obtained on a lawn expressing the antitoxin alone. Bars in panels B, C and E represent average±SD of three independent EOP experiments. (B) EOP experiments with E. coli harboring the sanaAT system when infected by WT T7 (left) or by T7Δ4.3Δ4.5Δ4.7 (right). (C) EOP experiments with WT E. coli (left) and E. coli expressing Gp4.5 (right), when infected by T7Δ4.3Δ4.5Δ4.7. (D) EOP experiments with WT E. coli (left) and E. coli lacking Ion (right), when infected by T7Δ4.3Δ4.5Δ4.7. (E) Co-immunoprecipitation of Lon and 4.5. The E. coli Lon protease was Flag-tagged at the N-terminus and expressed within E. coli BL21 (DE3) with or without co-expression of gene 4.5. Samples were analyzed by 15% SDS-PAGE. Three left lanes, total soluble proteins; three lanes on the right, following immunoprecipitation with anti-Flag antibody. Co-immunoprecipitation of Lon and 4.5 was validated by mass spectrometry analysis. Numbers on the left denote protein marker sizes in kDa. (F) Western blot analysis on reciprocal co-IP with flag-tagged Gp4.5 and his-tagged Lon. Immunoprecipitation was done with anti-Flag antibody.
  • FIG. 6 is a bar graph demonstrating that expression of gene 4.5 within E. coli results in reduced number of persister bacteria. Bacteria were exposed to ampicillin for 5 hours and % formation of persister bacteria was measured. Experiments were performed in triplicates and error bars represent standard error.
  • FIG. 7 illustrates the statistical assessment of a family of gene pairs as possibly encoding a toxin-antitoxin module. In each genome where a member of the family exist, the sequencing clones are re-distributed randomly (maintaining the number of clones and their sizes, but shuffling their positions on the genome). The “TA cloning pattern” for each member is then evaluated based on the randomly distributed clones, and the fraction of members obeying the “TA cloning pattern” by chance is recorded (in the illustration: red, clones that span only the toxin; blue, clones that span only the antitoxin; green, clones that span both genes). This procedure is repeated 1000 times, generating a distribution of fractions. The p-value for a family is determined by comparing the actual fraction of pairs that obey the “TA cloning pattern” to the distribution of fractions obtained using the random clone shuffling.
  • FIG. 8 is a diagram illustrating that predicted new TA systems associate with complex genomic environments. Shown are the schematic environments of 4 systems that are associated with other genes and hence suspected as participating in multi-gene defense systems. Blue and red genes denote putative antitoxin and toxin, respectively; gray genes denote genes associated with the TA system.
  • FIGS. 9A-9B are bar graphs illustrating properties of known TA families. The 21,417 analyzed families of gene pairs were divided into two sets: a positive set which contains all families in which at least one pair is a known TA system (‘Known systems’) and a negative which contains the remaining families (‘Negative set’). (A) The family diversity measurement for known TA systems (black) as compared to the negative set (light gray) (B) Mean number of defense island (DI) genes localized +/−5 genes away from known TA families (black) as compared to families in the negative set (light gray).
    •  DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
  • The present invention, in some embodiments thereof, relates to bacterial polypeptides that comprise toxin and/or antitoxin activity and, more particularly, but not exclusively, to toxin-antitoxin pairs that may be used as bacterial anti-phage defense systems.
  • Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
  • The toxin-antitoxin (TA) complex of bacteria includes a pair of polypeptides that is encoded by bacterial plasmids or chromosomes. It is postulated that in bacteria these polypeptides function to induce programmed cell death or growth inhibition in response to starvation, phage infection or other adverse conditions. The antitoxins neutralize the cognate toxins by forming tight complexes therewith or by other means. The antitoxins are unstable due to degradation by cellular proteases (e.g., Lon or Clp), whereas toxins are stable polypeptides. Toxin-antitoxin pair examples include the pemI-pemK genes of plasmid R100, the phd-doc genes of phage P1, and the ccdA-ccdB genes, of plasmid F. Several toxin-antitoxin encoding gene analogues have been identified on the E. coli K-12 chromosome, such as mazE-mazF.
  • In numerous cases where TA systems were studied experimentally, cloning of the toxin was nearly impossible in the absence of the cognate antitoxin. Based on this concept the present inventors reasoned that data derived from Sanger-based whole genome shotgun sequencing can experimentally and systematically reveal active TA pairs. In such a genome sequencing process, randomly fragmented DNA pieces of the genome are serially cloned and propagated within E. coli prior to sequencing. The ends of the cloned fragments are then sequenced, and overlapping sequences are used for genome assembly (FIG. 1A). It has been shown that analysis of clone distribution patterns can reveal genes toxic to bacteria, which are uncloneable and cause gaps (Kimelman et al., 2012; Sorek et al., 2007). However, the toxin in a TA pair is not expected to cause a gap, since the adjacent antitoxin will sometimes be found on the same clone, neutralizing the toxic effect. Nevertheless, random fragments that contain the toxin but not the antitoxin will cause cell death and will be absent from the set of clones covering the genome (FIG. 1B).
  • The present inventors took advantage of this typical biased cloning pattern to systematically detect toxin-antitoxin gene pairs within hundreds of microbial genomes. The analyses, while retrieving many known TA systems, also exposed 9 novel families of TA modules widespread in numerous bacterial species. These systems were subsequently experimentally validated as TA systems (FIGS. 3A-C). By engineering these systems into new bacteria, the present inventors showed that the toxin-antitoxin pairs can protect the engineered bacteria from phage attacks (FIGS. 5A-B). Such engineered bacteria can be utilized in the dairy industry, where phages cause serious annual losses, as well as in other industries that rely on large-scale bacterial fermentation for biotechnological production. Infection experiments with T7 phage and multiple T7 phage deletion mutants further showed that two of these new TA pairs provide resistance against T7, and also revealed a general anti-TA mechanism encoded by the phage (FIGS. 5C-F). Specifically, the present inventors discovered a phage peptide that inhibits toxin-antitoxin systems by inhibiting Lon protease. This peptide was further shown to inhibit the formation of bacterial persistence, thus active as a mechanism to reduce antibiotics resistance among bacteria.
  • Thus, according to one aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 90% homologous to a sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094, wherein the polypeptide has antimicrobial activity.
  • The phrase “antimicrobial activity” as used herein, refers to an ability to suppress, control, inhibit or kill microorganisms, such as bacteria, archaea and fungi. Thus for example the antimicrobial activity may comprise bactericidal or bacteriostatic activity, or both.
  • According to a preferred embodiment, the isolated polypeptide comprises an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to SEQ ID NOs: 2773-5544.
  • According to a preferred embodiment, the isolated polypeptide comprises an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to SEQ ID NOs: 2773-3117.
  • According to a preferred embodiment, the isolated polypeptide comprises an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to SEQ ID NOs: 11089-11094. Such polypeptides are capable of inhibiting (decreasing activity by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) a member of the Lon protease family. Lon proteases are ATP-dependent serine peptidases belonging to the MEROPS peptidase family S16 (lon protease family, clan SF).
  • According to another aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 60%, at least 70%, at least 80%, at least 90% or at least 95% homologous to any one of SEQ ID NOs: 8317-11088, wherein the polypeptide protects a microbe from an activity of a toxin. Such polypeptides may be expressed in microbial populations thereby protecting the microbial population from the toxic effect of the toxin. Methods of expressing polypeptides are described herein below.
  • Polypeptides which protect microbes from an activity of a toxin (i.e. antitoxin) may have a Ki of 1×10(−5) M-1×10(−10) M for the toxin.
  • According to another embodiment, the sequence selected from the group consisting of SEQ ID NOs. 8317-8661.
  • According to still another embodiment, the peptides of the present invention consist of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2773-5544 or SEQ ID NO:8317-11088 or SEQ ID NOs: 11089-11094.
  • Homology may be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters). The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.
  • The term “peptide” as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds (—C(═O)—O—), ketomethylene bonds (—CO—CH2-), sulfinylmethylene bonds (—S(═O)—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g., methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds (—CH2-CH2-), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), fluorinated olefinic double bonds (—CF═CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally present on the carbon atom.
  • These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) bonds at the same time.
  • Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted by non-natural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.
  • The peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc.).
  • The term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.
  • Tables 1 and 2 below list naturally occurring amino acids (Table 1), and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with some embodiments of the invention.
  • TABLE 1
    Three-Letter One-letter
    Amino Acid Abbreviation Symbol
    Alanine Ala A
    Arginine Arg R
    Asparagine Asn N
    Aspartic acid Asp D
    Cysteine Cys C
    Glutamine Gln Q
    Glutamic Acid Glu E
    Glycine Gly G
    Histidine His H
    Isoleucine Ile I
    Leucine Leu L
    Lysine Lys K
    Methionine Met M
    Phenylalanine Phe F
    Proline Pro P
    Serine Ser S
    Threonine Thr T
    Tryptophan Trp W
    Tyrosine Tyr Y
    Valine Val V
    Any amino acid as above Xaa X
  • TABLE 2
    Non-conventional amino Non-conventional amino
    acid Code acid Code
    ornithine Orn hydroxyproline Hyp
    α-aminobutyric acid Abu aminonorbornyl- Norb
    carboxylate
    D-alanine Dala aminocyclopropane- Cpro
    carboxylate
    D-arginine Darg N-(3- Narg
    guanidinopropyl)glycine
    D-asparagine Dasn N-(carbamylmethyl)glycine Nasn
    D-aspartic acid Dasp N-(carboxymethyl)glycine Nasp
    D-cysteine Dcys N-(thiomethyl)glycine Ncys
    D-glutamine Dgln N-(2-carbamylethyl)glycine Ngln
    D-glutamic acid Dglu N-(2-carboxyethyl)glycine Nglu
    D-histidine Dhis N-(imidazolylethyl)glycine Nhis
    D-isoleucine Dile N-(1-methylpropyl)glycine Nile
    D-leucine Dleu N-(2-methylpropyl)glycine Nleu
    D-lysine Dlys N-(4-aminobutyl)glycine Nlys
    D-methionine Dmet N-(2-methylthioethyl)glycine Nmet
    D-ornithine Dorn N-(3-aminopropyl)glycine Norn
    D-phenylalanine Dphe N-benzylglycine Nphe
    D-proline Dpro N-(hydroxymethyl)glycine Nser
    D-serine Dser N-(1-hydroxyethyl)glycine Nthr
    D-threonine Dthr N-(3-indolylethyl) glycine Nhtrp
    D-tryptophan Dtrp N-(p-hydroxyphenyl)glycine Ntyr
    D-tyrosine Dtyr N-(1-methylethyl)glycine Nval
    D-valine Dval N-methylglycine Nmgly
    D-N-methylalanine Dnmala L-N-methylalanine Nmala
    D-N-methylarginine Dnmarg L-N-methylarginine Nmarg
    D-N-methylasparagine Dnmasn L-N-methylasparagine Nmasn
    D-N-methylasparatate Dnmasp L-N-methylaspartic acid Nmasp
    D-N-methylcysteine Dnmcys L-N-methylcysteine Nmcys
    D-N-methylglutamine Dnmgln L-N-methylglutamine Nmgln
    D-N-methylglutamate Dnmglu L-N-methylglutamic acid Nmglu
    D-N-methylhistidine Dnmhis L-N-methylhistidine Nmhis
    D-N-methylisoleucine Dnmile L-N-methylisolleucine Nmile
    D-N-methylleucine Dnmleu L-N-methylleucine Nmleu
    D-N-methyllysine Dnmlys L-N-methyllysine Nmlys
    D-N-methylmethionine Dnmmet L-N-methylmethionine Nmmet
    D-N-methylornithine Dnmorn L-N-methylornithine Nmorn
    D-N-methylphenylalanine Dnmphe L-N-methylphenylalanine Nmphe
    D-N-methylproline Dnmpro L-N-methylproline Nmpro
    D-N-methylserine Dnmser L-N-methylserine Nmser
    D-N-methylthreonine Dnmthr L-N-methylthreonine Nmthr
    D-N-methyltryptophan Dnmtrp L-N-methyltryptophan Nmtrp
    D-N-methyltyrosine Dnmtyr L-N-methyltyrosine Nmtyr
    D-N-methylvaline Dnmval L-N-methylvaline Nmval
    L-norleucine Nle L-N-methylnorleucine Nmnle
    L-norvaline Nva L-N-methylnorvaline Nmnva
    L-ethylglycine Etg L-N-methyl-ethylglycine Nmetg
    L-t-butylglycine Tbug L-N-methyl-t-butylglycine Nmtbug
    L-homophenylalanine Hphe L-N-methyl- Nmhphe
    homophenylalanine
    α-naphthylalanine Anap N-methyl-α-naphthylalanine Nmanap
    penicillamine Pen N-methylpenicillamine Nmpen
    γ-aminobutyric acid Gabu N-methyl-γ-aminobutyrate Nmgabu
    cyclohexylalanine Chexa N-methyl-cyclohexylalanine Nmchexa
    cyclopentylalanine Cpen N-methyl-cyclopentylalanine Nmcpen
    α-amino-α-methylbutyrate Aabu N-methyl-α-amino-α- Nmaabu
    methylbutyrate
    α-aminoisobutyric acid Aib N-methyl-α- Nmaib
    aminoisobutyrate
    D-α-methylarginine Dmarg L-α-methylarginine Marg
    D-α-methylasparagine Dmasn L-α-methylasparagine Masn
    D-α-methylaspartate Dmasp L-α-methylaspartate Masp
    D-α-methylcysteine Dmcys L-α-methylcysteine Mcys
    D-α-methylglutamine Dmgln L-α-methylglutamine Mgln
    D-α-methyl glutamic acid Dmglu L-α-methylglutamate Mglu
    D-α-methylhistidine Dmhis L-α-methylhistidine Mhis
    D-α-methylisoleucine Dmile L-α-methylisoleucine Mile
    D-α-methylleucine Dmleu L-α-methylleucine Mleu
    D-α-methyllysine Dmlys L-α-methyllysine Mlys
    D-α-methylmethionine Dmmet L-α-methylmethionine Mmet
    D-α-methylornithine Dmorn L-α-methylornithine Morn
    D-α-methylphenylalanine Dmphe L-α-methylphenylalanine Mphe
    D-α-methylproline Dmpro L-α-methylproline Mpro
    D-α-methylserine Dmser L-α-methylserine Mser
    D-α-methylthreonine Dmthr L-α-methylthreonine Mthr
    D-α-methyltryptophan Dmtrp L-α-methyltryptophan Mtrp
    D-α-methyltyrosine Dmtyr L-α-methyltyrosine Mtyr
    D-α-methylvaline Dmval L-α-methylvaline Mval
    N-cyclobutylglycine Ncbut L-α-methylnorvaline Mnva
    N-cycloheptylglycine Nchep L-α-methylethylglycine Metg
    N-cyclohexylglycine Nchex L-α-methyl-t-butylglycine Mtbug
    N-cyclodecylglycine Ncdec L-α-methyl- Mhphe
    homophenylalanine
    N-cyclododecylglycine Ncdod α-methyl-α-naphthylalanine Manap
    N-cyclooctylglycine Ncoct α-methylpenicillamine Mpen
    N-cyclopropylglycine Ncpro α-methyl-γ-aminobutyrate Mgabu
    N-cycloundecylglycine Ncund α-methyl-cyclohexylalanine Mchexa
    N-(2-aminoethyl)glycine Naeg α-methyl-cyclopentylalanine Mcpen
    N-(2,2- Nbhm N-(N-(2,2-diphenylethyl) Nnbhm
    diphenylethyl)glycine carbamylmethyl-glycine
    N-(3,3- Nbhe N-(N-(3,3-diphenylpropyl) Nnbhe
    diphenylpropyl)glycine carbamylmethyl-glycine
    1-carboxy-1-(2,2-diphenyl Nmbc 1,2,3,4- Tic
    ethylamino)cyclopropane tetrahydroisoquinoline-3-
    carboxylic acid
    phosphoserine pSer phosphothreonine pThr
    phosphotyrosine pTyr O-methyl-tyrosine
    2-aminoadipic acid hydroxylysine
  • The peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • Since the present peptides are preferably utilized in therapeutics or diagnostics which require the peptides to be in soluble form, the peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • The amino acids of the peptides of the present invention may be substituted either conservatively or non-conservatively.
  • The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
  • As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.
  • For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
  • When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
  • The phrase “non-conservative substitutions” as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or —NH—CH[(—CH2)5—COOH]—CO— for aspartic acid. Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide having anti-bacterial properties.
  • The N and C termini of the peptides of the present invention may be protected by function groups. Suitable functional groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.
  • The peptides of the present invention may be attached (either covalently or non-covalently) to a penetrating agent.
  • As used herein the phrase “penetrating agent” refers to an agent which enhances translocation of any of the attached peptide across a cell membrane.
  • According to one embodiment, the penetrating agent is a peptide and is attached to the antimicrobial peptide (either directly or non-directly) via a peptide bond.
  • Typically, peptide penetrating agents have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • The peptides of the present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
  • Large scale peptide synthesis is described by Andersson Biopolymers 2000; 55(3):227-50.
  • Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing.
  • Recombinant techniques may also be used to generate the peptides of the present invention. To produce a peptide of the present invention using recombinant technology, a polynucleotide encoding the peptide of the present invention is ligated into a nucleic acid expression vector, which comprises the polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., promoter sequence) suitable for directing constitutive, tissue specific or inducible transcription of the polypeptides of the present invention in the host cells.
  • A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of some embodiments of the invention. These include, but are not limited to; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence. Mammalian expression systems can also be used to express the polypeptides of some embodiments of the invention.
  • Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
  • Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed antimicrobial peptide. For example, the expression of a fusion protein or a cleavable fusion protein comprising the antimicrobial peptides of some embodiments of the invention and a heterologous protein can be engineered. Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein. Where a cleavage site is engineered between the antimicrobial and the heterologous protein, the antimicrobial can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].
  • Recovery of the recombinant peptide is effected following an appropriate time in culture. The phrase “recovering the recombinant peptide” refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. Notwithstanding the above, polypeptides of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • In addition to being synthesizable in host cells, the peptides of the present invention can also be synthesized using in vitro expression systems. These methods are well known in the art and the components of the system are commercially available.
  • The peptides disclosed herein, which comprise anti-microbial properties may be used to kill microbes.
  • Thus, according to another aspect of the present invention there is provided a method of killing a microbe, the method comprising contacting the microbe with the isolated peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094.
  • According to a particular embodiment, the peptide used for killing a microbe comprises the sequence 11089-11094.
  • The microbe may be for example a gram-positive or gram negative bacteria.
  • The term “Gram-positive bacteria” as used herein refers to bacteria characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids and are characterized by their blue-violet color reaction in the Gram-staining procedure. Representative Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, Streptococcus sanguis.
  • The term “Gram-negative bacteria” as used herein refer to bacteria characterized by the presence of a double membrane surrounding each bacterial cell. Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestis.
  • As used herein the term “contacting” refers to the positioning of the peptides of the present invention such that they are in direct or indirect contact with the bacterial cells. Thus, the present invention contemplates both applying the peptides of the present invention to a desirable surface and/or directly to the bacterial cells.
  • According to another embodiment the surface is comprised in a biological tissue, such as for example, mammalian tissues e.g. the skin.
  • It will be appreciated that the microbes may be comprised inside a particular organism, (e.g. intracellularly or extracellularly) for example inside a mammalian body or inside a plant. In this case, the contacting may be effected by administering the peptides per se or by transfecting the cells of the organism with a nucleic acid construct which comprises a nucleic acid sequence which encodes the peptides of the present invention.
  • Thus, the present invention contemplates polynucleotide sequences encoding the antimicrobial polypeptides disclosed herein. Such polynucleotide sequences are set forth in SEQ ID NOs: 1-2772.
  • Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
  • Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use with some embodiments of the invention include for example the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804) or pathogen-inducible promoters. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen.
  • The nucleic acid construct (also referred to herein as an “expression vector”) of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof.
  • The nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
  • The nucleic acid constructs described herein will have a plurality of restriction sites for insertion of the sequence of the invention so that it is under transcriptional regulation of the regulatory regions. Selectable marker genes that ensure maintenance of the vector in the cell can also be included in the expression vector. Preferred selectable markers include those which confer resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers can also allow a cell to grow on minimal medium, or in the presence of toxic metabolite and can include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
  • Preferably, the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed. Examples of cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
  • In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Recombinant viral vectors are useful for in vivo expression of the antimicrobial peptides of the invention since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • Various methods can be used to introduce the expression vector of some embodiments of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
  • Exemplary methods of introducing expression vectors into bacterial cells include for example conventional transformation or transfection techniques, or by phage-mediated infection. As used herein, the terms “transformation,” “transduction,” “conjugation,” and “protoplast fusion” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
  • Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • 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. 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.
  • Since the polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094 have antimicrobial activity, the present invention contemplates use thereof for treating infection in a mammalian subject.
  • According to one embodiment, the peptides are used to treat a topical infection (i.e. infection of the skin) and are provided in a topical formulation.
  • According to another embodiment, the peptides are used to treat an infection inside the body. In this case, the peptides (or polynucleotides encoding same) may be provided ex vivo or in vivo.
  • Accordingly, the present invention contemplates contacting cells with the polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2773-5544 and 11089-11094 (or with expression constructs that encode the peptides) per se or as part of a pharmaceutical composition.
  • According to a particular embodiment, the peptide used in the pharmaceutical composition comprises the sequence 11089-11094.
  • The peptides may be used alone or together with additional antimicrobial agents (e.g. antibiotic and/or additional antimicrobial peptides).
  • Exemplary antibiotics include, but are not limited to aminoglycoside antibiotics, cephalosporins, quinolone antibiotics, macrolide antibiotics, penicillins, sulfonamides, tetracyclines and carbapenems. It will be appreciated that since the peptides of embodiments of this invention enhance the antibacterial effect of the antibiotic, doses of the antibiotic may be lower (e.g. 20% lower, 30% lower, 40% lower, 50% lower, 60% lower, 70% lower, 80% lower or even 90% lower than those currently in use.
  • The pharmaceutical compositions of the present invention are administered to a subject in need thereof in order to prevent or treat a bacterial infection.
  • As used herein, the term “subject in need thereof” refers to a mammal, preferably a human subject.
  • As used herein, the term “treating” refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathogen infection.
  • The phrase “pharmaceutical composition”, as used herein refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • As used herein the term “active ingredient” refers to peptides of the present invention accountable for the intended biological effect. It will be appreciated that a polynucleotide encoding a peptide of the present invention may be administered directly into a subject (as is, or part of a pharmaceutical composition) where it is translated in the target cells i.e. by gene therapy. Accordingly, the phrase “active ingredient” also includes such polynucleotides.
  • Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier,” which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
  • Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • Techniques for formulation and administration of drugs may be found in the latest edition of “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., which is herein fully incorporated by reference and are further described herein below.
  • It will be appreciated that the peptides of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
  • Exemplary additional agents include antibiotics (e.g. rifampicin, chloramphenicol and spectinomycin).
  • Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • The preparation of the present invention may also be formulated as a topical composition, such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste and a gel.
  • Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1].
  • Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • As mentioned, the peptides described herein which comprise antitoxin activity may be expressed in microbial populations, thereby protecting the microbial population from the toxic effect of a toxin.
  • Thus, the present invention further contemplates polynucleotide sequences encoding the antitoxin polypeptides disclosed herein. Such polynucleotide sequences are set forth in SEQ ID NOs: 5548-8316.
  • The present inventors have further discovered novel toxin antitoxin pairs that may be expressed in bacterial populations. Expression therein may serve to protect the bacterial population from the lytic effect of a bacteriophage (i.e. impart resistance to the bacteriophage).
  • Such pairs are summarized in Table 3 herein below.
  • TABLE 3
    Toxin polypeptide sequences Antitoxin polypeptide sequences
    SEQ ID NO: designation SEQ ID NO: designation
    2773 >Toxin: Lferr_1303 8317 >Antitoxin: Lferr_1302
    2774 >Toxin: Adeg_2177 8318 >Antitoxin: Adeg_2178
    2775 >Toxin: BLD_0338 8319 >Antitoxin: BLD_0339
    2776 >Toxin: HMPREF0175_1783 8320 >Antitoxin: HMPREF0175_1782
    2777 >Toxin: BIL_08340 8321 >Antitoxin: BIL_08350
    2778 >Toxin: BL1460 8322 >Antitoxin: BL1461
    2779 >Toxin: HMPREF0177_01212 8323 >Antitoxin: HMPREF0177_01213
    2780 >Toxin: CJA_3634 8324 >Antitoxin: CJA_3633
    2781 >Toxin: MldDRAFT_1473 8325 >Antitoxin: MldDRAFT_1471
    2782 >Toxin: MldDRAFT_0420 8326 >Antitoxin: MldDRAFT_0418
    2783 >Toxin: MldDRAFT_4398 8327 >Antitoxin: MldDRAFT_4397
    2784 >Toxin: MldDRAFT_1876 8328 >Antitoxin: MldDRAFT_1875
    2785 >Toxin: DaAHT2_0326 8329 >Antitoxin: DaAHT2_0325
    2786 >Toxin: Elen_0846 8330 >Antitoxin: Elen_0847
    2787 >Toxin: HCAN_1210 8331 >Antitoxin: HCAN_1209
    2788 >Toxin: MELB17_24172 8332 >Antitoxin: MELB17_24167
    2789 >Toxin: Mmwyl1_3205 8333 >Antitoxin: Mmwyl1_3204
    2790 >Toxin: Pat9b_4701 8334 >Antitoxin: Pat9b_4700
    2791 >Toxin: HMPREF0620_1504 8335 >Antitoxin: HMPREF0620_1505
    2792 >Toxin: Pecwa_0953 8336 >Antitoxin: Pecwa_0952
    2793 >Toxin: plu0453 8337 >Antitoxin: plu0454
    2794 >Toxin: plu3932 8338 >Antitoxin: plu3931
    2795 >Toxin: HMPREF0693_2530 8339 >Antitoxin: HMPREF0693_2531
    2796 >Toxin: PMI2316 8340 >Antitoxin: PMI2317
    2797 >Toxin: PMI2493 8341 >Antitoxin: PMI2492
    2798 >Toxin: Sden_0300 8342 >Antitoxin: Sden_0299
    2799 >Toxin: Veis_2844 8343 >Antitoxin: Veis_2845
    2800 >Toxin: XNC1_4222 8344 >Antitoxin: XNC1_4221
    2801 >Toxin: BloniC5_010100004089 8345 >Antitoxin: BloniC5_010100004094
    2802 >Toxin: BloniC5_010100007713 8346 >Antitoxin: BloniC5_010100007718
    2803 >Toxin: HcanM9_010100006460 8347 >Antitoxin: PROVRUST_02882
    2804 >Toxin: PROVRUST_02881 8348 >Antitoxin: HcanM9_010100006455
    2805 >Toxin: AcdelDRAFT_0026 8349 >Antitoxin: AcdelDRAFT_0025
    2806 >Toxin: Ajs_0911 8350 >Antitoxin: Ajs_0912
    2807 >Toxin: A20C1_10775 8351 >Antitoxin: A20C1_10770
    2808 >Toxin: Astex_0661 8352 >Antitoxin: Astex_0662
    2809 >Toxin: bll1927 8353 >Antitoxin: bll1928
    2810 >Toxin: HMPREF0183_2010 8354 >Antitoxin: HMPREF0183_2009
    2811 >Toxin: CAP2UW1_0236 8355 >Antitoxin: CAP2UW1_0235
    2812 >Toxin: pc2001 8356 >Antitoxin: pc2002
    2813 >Toxin: Clim_1931 8357 >Antitoxin: Clim_1932
    2814 >Toxin: Cpha266_0654 8358 >Antitoxin: Cpha266_0653
    2815 >Toxin: Cvib_0668 8359 >Antitoxin: Cvib_0667
    2816 >Toxin: Cwoe_5578 8360 >Antitoxin: Cwoe_5577
    2817 >Toxin: cgR_0545 8361 >Antitoxin: cgR_0546
    2818 >Toxin: Daci_0082 8362 >Antitoxin: Daci_0083
    2819 >Toxin: Dole_3139 8363 >Antitoxin: Dole_3138
    2820 >Toxin: Dbac_1303 8364 >Antitoxin: Dbac_1304
    2821 >Toxin: Elen_3086 8365 >Antitoxin: Elen_3085
    2822 >Toxin: HMPREF1023_01420 8366 >Antitoxin: HMPREF1023_01419
    2823 >Toxin: Smed_6434 8367 >Antitoxin: Smed_6433
    2824 >Toxin: Smed_5454 8368 >Antitoxin: Smed_5453
    2825 >Toxin: Smed_6434 8369 >Antitoxin: Smed_6433
    2826 >Toxin: GobsU_010100024456 8370 >Antitoxin: GobsU_010100024461
    2827 >Toxin: gsr2662 8371 >Antitoxin: glr2661
    2828 >Toxin: Gbro_4918 8372 >Antitoxin: Gbro_4917
    2829 >Toxin: GPA_34760 8373 >Antitoxin: GPA_34770
    2830 >Toxin: Intca_2903 8374 >Antitoxin: Intca_2902
    2831 >Toxin: LLO_0649 8375 >Antitoxin: LLO_0650
    2832 >Toxin: LLO_2898 8376 >Antitoxin: LLO_2899
    2833 >Toxin: LNTAR_09414 8377 >Antitoxin: LNTAR_09419
    2834 >Toxin: LNTAR_09681 8378 >Antitoxin: LNTAR_09686
    2835 >Toxin: SPV1_04688 8379 >Antitoxin: SPV1_04693
    2836 >Toxin: MexAM1_META2p1274 8380 >Antitoxin: MexAM1_META2p1275
    2837 >Toxin: HMPREF0580_0585 8381 >Antitoxin: HMPREF0580_0584
    2838 >Toxin: HMPREF0577_2238 8382 >Antitoxin: HMPREF0577_2237
    2839 >Toxin: HMPREF9278_0956 8383 >Antitoxin: HMPREF9278_0957
    2840 >Toxin: MLBr_02284 8384 >Antitoxin: MLBr_02283
    2841 >Toxin: ML2284 8385 >Antitoxin: ML2283
    2842 >Toxin: MMAR_3595 8386 >Antitoxin: MMAR_3594
    2843 >Toxin: Namu_4516 8387 >Antitoxin: Namu_4515
    2844 >Toxin: Nhal_3749 8388 >Antitoxin: Nhal_3750
    2845 >Toxin: Olsu_1425 8389 >Antitoxin: Olsu_1426
    2846 >Toxin: Plut_0508 8390 >Antitoxin: Plut_0507
    2847 >Toxin: Ppha_0787 8391 >Antitoxin: Ppha_0786
    2848 >Toxin: PHZ_p0168 8392 >Antitoxin: PHZ_p0169
    2849 >Toxin: Pnap_4573 8393 >Antitoxin: Pnap_4574
    2850 >Toxin: Bpro_1527 8394 >Antitoxin: Bpro_1528
    2851 >Toxin: PFREUD_01930 8395 >Antitoxin: PFREUD_01920
    2852 >Toxin: CRD_00786 8396 >Antitoxin: CRD_00785
    2853 >Toxin: Rleg_6339 8397 >Antitoxin: Rleg_6340
    2854 >Toxin: Rleg_6554 8398 >Antitoxin: Rleg_6555
    2855 >Toxin: pRL100221 8399 >Antitoxin: pRL100220
    2856 >Toxin: Arad_0082 8400 >Antitoxin: Arad_0081
    2857 >Toxin: NGR_b00520 8401 >Antitoxin: NGR_b00510
    2858 >Toxin: Rvan_1799 8402 >Antitoxin: Rvan_1798
    2859 >Toxin: Rpdx1_0873 8403 >Antitoxin: Rpdx1_0874
    2860 >Toxin: Rpdx1_1938 8404 >Antitoxin: Rpdx1_1937
    2861 >Toxin: Shel_25040 8405 >Antitoxin: Shel_25030
    2862 >Toxin: RS9917_04250 8406 >Antitoxin: RS9917_04255
    2863 >Toxin: Sfum_2054 8407 >Antitoxin: Sfum_2055
    2864 >Toxin: Vapar_5649 8408 >Antitoxin: Vapar_5648
    2865 >Toxin: VDG1235_790 8409 >Antitoxin: VDG1235_4870
    2866 >Toxin: XOO_2997 8410 >Antitoxin: XOO_2998
    2867 >Toxin: Xoryp_010100009970 8411 >Antitoxin: Xoryp_010100009965
    2868 >Toxin: XOO3154 8412 >Antitoxin: XOO3155
    2869 >Toxin: PXO_01415 8413 >Antitoxin: PXO_01414
    2870 >Toxin: Xcel_2876 8414 >Antitoxin: Xcel_2875
    2871 >Toxin: ObacDRAFT_3823 8415 >Antitoxin: ObacDRAFT_3824
    2872 >Toxin: HMPREF0016_02714 8416 >Antitoxin: HMPREF0016_02713
    2873 >Toxin: NIES39_A02090 8417 >Antitoxin: NIES39_A02080
    2874 >Toxin: Cpha266_1126 8418 >Antitoxin: Cpha266_1127
    2875 >Toxin: PCC8801_1532 8419 >Antitoxin: PCC8801_1533
    2876 >Toxin: Dehly_0318 8420 >Antitoxin: Dehly_0317
    2877 >Toxin: Dhaf_0673 8421 >Antitoxin: Dhaf_0674
    2878 >Toxin: DSY0713 8422 >Antitoxin: DSY0714
    2879 >Toxin: DP0406 8423 >Antitoxin: DP0407
    2880 >Toxin: Dtox_1318 8424 >Antitoxin: Dtox_1319
    2881 >Toxin: HMPREF0358_0171 8425 >Antitoxin: HMPREF0358_0170
    2882 >Toxin: ECABU_c38650 8426 >Antitoxin: ECABU_c38640
    2883 >Toxin: ECCG_03832 8427 >Antitoxin: ECCG_03831
    2884 >Toxin: ECDG_04005 8428 >Antitoxin: ECDG_04004
    2885 >Toxin: ECIAI39_3917 8429 >Antitoxin: ECIAI39_3916
    2886 >Toxin: HMPREF9549_04273 8430 >Antitoxin: HMPREF9549_04274
    2887 >Toxin: HMPREF9553_03184 8431 >Antitoxin: HMPREF9553_03183
    2888 >Toxin: HMPREF9530_02209 8432 >Antitoxin: HMPREF9530_02210
    2889 >Toxin: HMPREF9531_03250 8433 >Antitoxin: HMPREF9531_03251
    2890 >Toxin: HMPREF9534_00693 8434 >Antitoxin: HMPREF9534_00692
    2891 >Toxin: ECO103_4157 8435 >Antitoxin: ECO103_4156
    2892 >Toxin: ECSF_3257 8436 >Antitoxin: ECSF_3256
    2893 >Toxin: EscoliO157_010100006036 8437 >Antitoxin: EscoliO157_010100006031
    2894 >Toxin: EsccoliO157_010100001300 8438 >Antitoxin: EsccoliO157_010100001305
    2895 >Toxin: EschcoliO157_010100002315 8439 >Antitoxin: EschcoliO157_010100002320
    2896 >Toxin: EcoliO157_010100000650 8440 >Antitoxin: EcoliO157_010100000645
    2897 >Toxin: EcolO157_010100000480 8441 >Antitoxin: EcolO157_010100000475
    2898 >Toxin: EcolO15_010100000460 8442 >Antitoxin: EcolO15_010100000455
    2899 >Toxin: EcolO_010100000490 8443 >Antitoxin: EcolO_010100000485
    2900 >Toxin: EcolO1_010100003356 8444 >Antitoxin: EcolO1_010100003351
    2901 >Toxin: Z4801 8445 >Antitoxin: Z4799
    2902 >Toxin: ESCCO14588_0146 8446 >Antitoxin: ESCCO14588_0145
    2903 >Toxin: ECP_3530 8447 >Antitoxin: ECP_3529
    2904 >Toxin: c4223 8448 >Antitoxin: c4222
    2905 >Toxin: EcSMS35_3718 8449 >Antitoxin: EcSMS35_3717
    2906 >Toxin: HMPREF9098_1123 8450 >Antitoxin: HMPREF9098_1124
    2907 >Toxin: L8106_27027 8451 >Antitoxin: L8106_27022
    2908 >Toxin: FB2170_11841 8452 >Antitoxin: FB2170_11846
    2909 >Toxin: Maqu_0559 8453 >Antitoxin: Maqu_0560
    2910 >Toxin: MC7420_773 8454 >Antitoxin: MC7420_724
    2911 >Toxin: NB231_14358 8455 >Antitoxin: NB231_14363
    2912 >Toxin: NE2520 8456 >Antitoxin: NE2521
    2913 >Toxin: alr3451 8457 >Antitoxin: alr3452
    2914 >Toxin: OSCT_2233 8458 >Antitoxin: OSCT_2234
    2915 >Toxin: Ppha_1436 8459 >Antitoxin: Ppha_1435
    2916 >Toxin: plu4452 8460 >Antitoxin: plu4453
    2917 >Toxin: Pnap_2963 8461 >Antitoxin: Pnap_2962
    2918 >Toxin: PstuA_020100002503 8462 >Antitoxin: PstuA_020100002498
    2919 >Toxin: PA39016_001140007 8463 >Antitoxin: PA39016_001140006
    2920 >Toxin: Psyr_3804 8464 >Antitoxin: Psyr_3805
    2921 >Toxin: Rcas_2468 8465 >Antitoxin: Rcas_2467
    2922 >Toxin: Sbal_4347 8466 >Antitoxin: Sbal_4346
    2923 >Toxin: SD1617_2224 8467 >Antitoxin: SD1617_2223
    2924 >Toxin: SDY_3582 8468 >Antitoxin: SDY_3581
    2925 >Toxin: TERTU_3911 8469 >Antitoxin: TERTU_3910
    2926 >Toxin: Tery_1571 8470 >Antitoxin: Tery_1570
    2927 >Toxin: VFA_000662 8471 >Antitoxin: VFA_000663
    2928 >Toxin: EcolE2_01000162 8472 >Antitoxin: EcolE2_01000163
    2929 >Toxin: EcolM6_010100020382 8473 >Antitoxin: EcolM6_010100020377
    2930 >Toxin: EcolM_010100020768 8474 >Antitoxin: EcolM_010100020763
    2931 >Toxin: EcolB_01001160 8475 >Antitoxin: EcolB_01001159
    2932 >Toxin: EcolF_01001475 8476 >Antitoxin: EcolF_01001476
    2933 >Toxin: 8477 >Antitoxin:
    EschercoliO157_010100002920 EschercoliO157_010100002925
    2934 >Toxin: 8478 >Antitoxin:
    EschericoliO157_010100015347 EschericoliO157_010100015342
    2935 >Toxin: 8479 >Antitoxin:
    EschericcoliO157_010100003780 EschericcoliO157_010100003785
    2936 >Toxin: Aave_0160 8480 >Antitoxin: Aave_0161
    2937 >Toxin: Amir_6975 8481 >Antitoxin: Amir_6976
    2938 >Toxin: VSAL_I0392 8482 >Antitoxin: VSAL_I0391
    2939 >Toxin: Mlg_0502 8483 >Antitoxin: Mlg_0503
    2940 >Toxin: AmaxDRAFT_5193 8484 >Antitoxin: AmaxDRAFT_5192
    2941 >Toxin: AmaxDRAFT_5205 8485 >Antitoxin: AmaxDRAFT_5204
    2942 >Toxin: NIES39_K04470 8486 >Antitoxin: NIES39_K04480
    2943 >Toxin: AplaP_010100002817 8487 >Antitoxin: AplaP_010100002812
    2944 >Toxin: SI859A1_01336 8488 >Antitoxin: SI859A1_01337
    2945 >Toxin: bthur0013_57000 8489 >Antitoxin: bthur0013_57010
    2946 >Toxin: BGP_2335 8490 >Antitoxin: BGP_2336
    2947 >Toxin: BHWA1_01320 8491 >Antitoxin: BHWA1_01319
    2948 >Toxin: CAP2UW1_4295 8492 >Antitoxin: CAP2UW1_4296
    2949 >Toxin: Cyan7822_4726 8493 >Antitoxin: Cyan7822_4725
    2950 >Toxin: HMPREF0321_1867 8494 >Antitoxin: HMPREF0321_1868
    2951 >Toxin: HMPREF0326_02542 8495 >Antitoxin: HMPREF0326_02543
    2952 >Toxin: Dd1591_0935 8496 >Antitoxin: Dd1591_0936
    2953 >Toxin: NT01EI_3442 8497 >Antitoxin: NT01EI_3441
    2954 >Toxin: ETAE_3072 8498 >Antitoxin: ETAE_3073
    2955 >Toxin: ETAF_2777 8499 >Antitoxin: ETAF_2776
    2956 >Toxin: ENHAE0001_1187 8500 >Antitoxin: ENHAE0001_1188
    2957 >Toxin: E2348C_1093 8501 >Antitoxin: E2348C_1092
    2958 >Toxin: E4_010100008678 8502 >Antitoxin: E4_010100008683
    2959 >Toxin: HMPREF0381_2362 8503 >Antitoxin: HMPREF0381_2361
    2960 >Toxin: Glov_2199 8504 >Antitoxin: Glov_2198
    2961 >Toxin: glr3370 8505 >Antitoxin: glr3371
    2962 >Toxin: GDI0970 8506 >Antitoxin: GDI0971
    2963 >Toxin: KPK_4974 8507 >Antitoxin: KPK_4973
    2964 >Toxin: HMPREF0554_2331 8508 >Antitoxin: HMPREF0554_2332
    2965 >Toxin: L8106_04306 8509 >Antitoxin: L8106_04311
    2966 >Toxin: MELB17_05794 8510 >Antitoxin: MELB17_05789
    2967 >Toxin: Micau_0029 8511 >Antitoxin: Micau_0028
    2968 >Toxin: Mb0060 8512 >Antitoxin: Mb0061
    2969 >Toxin: BCG_0090 8513 >Antitoxin: BCG_0091
    2970 >Toxin: JTY_0060 8514 >Antitoxin: JTY_0061
    2971 >Toxin: Mtub2_010100014508 8515 >Antitoxin: Mtub2_010100014513
    2972 >Toxin: Mtube_010100000751 8516 >Antitoxin: Mtube_010100000756
    2973 >Toxin: MT0065 8517 >Antitoxin: MT0066
    2974 >Toxin: TBFG_10058 8518 >Antitoxin: TBFG_10059
    2975 >Toxin: MRA_0061 8519 >Antitoxin: MRA_0062
    2976 >Toxin: Rv0059 8520 >Antitoxin: Rv0060
    2977 >Toxin: TBMG_00058 8521 >Antitoxin: TBMG_00059
    2978 >Toxin: MtubK4_020200000305 8522 >Antitoxin: MtubK4_020200000310
    2979 >Toxin: MtubKR_020200000315 8523 >Antitoxin: MtubKR_020200000320
    2980 >Toxin: MtubKV_020200000310 8524 >Antitoxin: MtubKV_020200000315
    2981 >Toxin: TMAG_00729 8525 >Antitoxin: TMAG_00730
    2982 >Toxin: MtubS_010100002549 8526 >Antitoxin: MtubS_010100002554
    2983 >Toxin: MtubSUM_010100000319 8527 >Antitoxin: MtubSUM_010100000324
    2984 >Toxin: MtubSUMu_010100000950 8528 >Antitoxin: MtubSUMu_010100000955
    2985 >Toxin: MtubSUMu0_010100002539 8529 >Antitoxin: MtubSUMu0_010100002544
    2986 >Toxin: MtubSUMu00_010100001797 8530 >Antitoxin: MtubSUMu00_010100001802
    2987 >Toxin: MtubSUMu007_010100002479 8531 >Antitoxin:
    MtubSUMu007_010100002484
    2988 >Toxin: MtubSUMu008_010100002534 8532 >Antitoxin:
    MtubSUMu008_010100002539
    2989 >Toxin: MtubSUMu009_010100020343 8533 >Antitoxin:
    MtubSUMu009_010100020348
    2990 >Toxin: MtubSUMu01_010100020402 8534 >Antitoxin: MtubSUMu01_010100020407
    2991 >Toxin: MtubSUMu011_010100020688 8535 >Antitoxin:
    MtubSUMu011_010100020693
    2992 >Toxin: MtubSUMu012_010100000252 8536 >Antitoxin:
    MtubSUMu012_010100000257
    2993 >Toxin: Nhal_3405 8537 >Antitoxin: Nhal_3406
    2994 >Toxin: NE1363 8538 >Antitoxin: NE1364
    2995 >Toxin: OSCI_3800101 8539 >Antitoxin: OSCI_3800102
    2996 >Toxin: Plav_2928 8540 >Antitoxin: Plav_2929
    2997 >Toxin: PC1_0863 8541 >Antitoxin: PC1_0862
    2998 >Toxin: PBAL39_20435 8542 >Antitoxin: PBAL39_20430
    2999 >Toxin: HMPREF9019_0398 8543 >Antitoxin: HMPREF9019_0397
    3000 >Toxin: PSEEN0280 8544 >Antitoxin: PSEEN0281
    3001 >Toxin: Pmen_0565 8545 >Antitoxin: Pmen_0566
    3002 >Toxin: Rpal_2100 8546 >Antitoxin: Rpal_2099
    3003 >Toxin: RoseRS_0296 8547 >Antitoxin: RoseRS_0297
    3004 >Toxin: ISM_01070 8548 >Antitoxin: ISM_01065
    3005 >Toxin: RflaF_010100007781 8549 >Antitoxin: RflaF_010100007776
    3006 >Toxin: Sputcn32_3528 8550 >Antitoxin: Sputcn32_3529
    3007 >Toxin: STAUR_1046 8551 >Antitoxin: STAUR_1047
    3008 >Toxin: sll8002 8552 >Antitoxin: sll8001
    3009 >Toxin: Slip_1960 8553 >Antitoxin: Slip_1959
    3010 >Toxin: Tmz1t_3595 8554 >Antitoxin: Tmz1t_3594
    3011 >Toxin: TaqDRAFT_4249 8555 >Antitoxin: TaqDRAFT_4250
    3012 >Toxin: Tbd_1490 8556 >Antitoxin: Tbd_1489
    3013 >Toxin: ZZM4_0145 8557 >Antitoxin: ZZM4_0144
    3014 >Toxin: APCC8_010100024013 8558 >Antitoxin: APCC8_010100024018
    3015 >Toxin: APCC8_010100024078 8559 >Antitoxin: APCC8_010100024083
    3016 >Toxin: DthioDRAFT_0920 8560 >Antitoxin: DthioDRAFT_0919
    3017 >Toxin: MettuDRAFT_1183 8561 >Antitoxin: MettuDRAFT_1184
    3018 >Toxin: Mtub0_010100020641 8562 >Antitoxin: Mtub0_010100020646
    3019 >Toxin: Mtub9_010100021293 8563 >Antitoxin: Mtub9_010100021298
    3020 >Toxin: MtubC_01003315 8564 >Antitoxin: MtubC_01003316
    3021 >Toxin: MtubCP_010100002512 8565 >Antitoxin: MtubCP_010100002517
    3022 >Toxin: MtubE_010100020484 8566 >Antitoxin: MtubE_010100020489
    3023 >Toxin: MtubG1_010100021149 8567 >Antitoxin: MtubG1_010100021154
    3024 >Toxin: MtubH_010100021035 8568 >Antitoxin: MtubH_010100021040
    3025 >Toxin: MtubK8_010100002630 8569 >Antitoxin: MtubK8_010100002635
    3026 >Toxin: MtubKZN_010100019149 8570 >Antitoxin: MtubKZN_010100019144
    3027 >Toxin: MtubT1_010100000475 8571 >Antitoxin: MtubT1_010100000480
    3028 >Toxin: MtubT_010100021213 8572 >Antitoxin: MtubT_010100021218
    3029 >Toxin: MtubT9_010100002236 8573 >Antitoxin: MtubT9_010100002241
    3030 >Toxin: PROVRUST_01477 8574 >Antitoxin: PROVRUST_01478
    3031 >Toxin: AcdelDRAFT_0253 8575 >Antitoxin: AcdelDRAFT_0254
    3032 >Toxin: Ajs_1569 8576 >Antitoxin: Ajs_1570
    3033 >Toxin: BURPS1106A_2452 8577 >Antitoxin: BURPS1106A_2451
    3034 >Toxin: Bpse14_010100013779 8578 >Antitoxin: Bpse14_010100013774
    3035 >Toxin: Bpseu9_010100040554 8579 >Antitoxin: Bpseu9_010100040549
    3036 >Toxin: BpseB_010100013291 8580 >Antitoxin: BpseB_010100013286
    3037 >Toxin: BUH_2443 8581 >Antitoxin: BUH_2442
    3038 >Toxin: Clim_0213 8582 >Antitoxin: Clim_0214
    3039 >Toxin: MldDRAFT_0262 8583 >Antitoxin: MldDRAFT_0261
    3040 >Toxin: Dalk_1149 8584 >Antitoxin: Dalk_1150
    3041 >Toxin: HMPREF0358_4229 8585 >Antitoxin: HMPREF0358_4228
    3042 >Toxin: ECABU_c33410 8586 >Antitoxin: ECABU_c33400
    3043 >Toxin: ECFG_02048 8587 >Antitoxin: ECFG_02047
    3044 >Toxin: ECGG_01859 8588 >Antitoxin: ECGG_01858
    3045 >Toxin: HMPREF9346_01205 8589 >Antitoxin: HMPREF9346_01204
    3046 >Toxin: HMPREF9552_04316 8590 >Antitoxin: HMPREF9552_04315
    3047 >Toxin: HMPREF9531_00622 8591 >Antitoxin: HMPREF9531_00623
    3048 >Toxin: HMPREF9534_00156 8592 >Antitoxin: HMPREF9534_00157
    3049 >Toxin: HMPREF9534_02242 8593 >Antitoxin: HMPREF9534_02241
    3050 >Toxin: ECUMN_3416 8594 >Antitoxin: ECUMN_3415
    3051 >Toxin: c3682 8595 >Antitoxin: c3681
    3052 >Toxin: EschWDRAFT_2732 8596 >Antitoxin: EschWDRAFT_2733
    3053 >Toxin: ECW_m4651 8597 >Antitoxin: ECW_m4650
    3054 >Toxin: ESCG_00445 8598 >Antitoxin: ESCG_00444
    3055 >Toxin: LDG_1702 8599 >Antitoxin: LDG_1703
    3056 >Toxin: LPC_1887 8600 >Antitoxin: LPC_1888
    3057 >Toxin: lpl0200 8601 >Antitoxin: lpl0199
    3058 >Toxin: lpp2408 8602 >Antitoxin: lpp2409
    3059 >Toxin: Msip34_2080 8603 >Antitoxin: Msip34_2081
    3060 >Toxin: Plut_0717 8604 >Antitoxin: Plut_0716
    3061 >Toxin: Ppha_1969 8605 >Antitoxin: Ppha_1968
    3062 >Toxin: SKA34_21022 8606 >Antitoxin: SKA34_21027
    3063 >Toxin: PAU_03952 8607 >Antitoxin: PAU_03953
    3064 >Toxin: plu4270 8608 >Antitoxin: plu4271
    3065 >Toxin: PSHAb0102 8609 >Antitoxin: PSHAb0101
    3066 >Toxin: RC1_3156 8610 >Antitoxin: RC1_3157
    3067 >Toxin: Sbal_4457 8611 >Antitoxin: Sbal_4456
    3068 >Toxin: Shewana3_4161 8612 >Antitoxin: Shewana3_4160
    3069 >Toxin: slr5102 8613 >Antitoxin: slr5101
    3070 >Toxin: Veis_3691 8614 >Antitoxin: Veis_3690
    3071 >Toxin: VIBHAR_06811 8615 >Antitoxin: VIBHAR_06812
    3072 >Toxin: pVT1_52 8616 >Antitoxin: pVT1_51
    3073 >Toxin: VV20181 8617 >Antitoxin: VV20180
    3074 >Toxin: VVM_01405 8618 >Antitoxin: VVM_01404
    3075 >Toxin: VSWAT3_18743 8619 >Antitoxin: VSWAT3_18738
    3076 >Toxin: yaldo0001_40550 8620 >Antitoxin: yaldo0001_40560
    3077 >Toxin: YE4109 8621 >Antitoxin: YE4110
    3078 >Toxin: EcolH5_010100022792 8622 >Antitoxin: EcolH5_010100022787
    3079 >Toxin: Nham_0582 8623 >Antitoxin: Nham_0581
    3080 >Toxin: Swol_1548 8624 >Antitoxin: Swol_1549
    3081 >Toxin: Ppro_0237 8625 >Antitoxin: Ppro_0238
    3082 >Toxin: Aave_0159 8626 >Antitoxin: Aave_0158
    3083 >Toxin: Cthe_0519 8627 >Antitoxin: Cthe_0520
    3084 >Toxin: Daci_4205 8628 >Antitoxin: Daci_4206
    3085 >Toxin: Nther_0364 8629 >Antitoxin: Nther_0363
    3086 >Toxin: Ppha_1128 8630 >Antitoxin: Ppha_1127
    3087 >Toxin: Afer_1394 8631 >Antitoxin: Afer_1395
    3088 >Toxin: Mmar10_3055 8632 >Antitoxin: Mmar10_3056
    3089 >Toxin: Hhal_0412 8633 >Antitoxin: Hhal_0413
    3090 >Toxin: Rsph17029_3386 8634 >Antitoxin: Rsph17029_3385
    3091 >Toxin: Acry_3581 8635 >Antitoxin: Acry_3582
    3092 >Toxin: Spro_0571 8636 >Antitoxin: Spro_0572
    3093 >Toxin: PputGB1_4746 8637 >Antitoxin: PputGB1_4747
    3094 >Toxin: Glov_0294 8638 >Antitoxin: Glov_0295
    3095 >Toxin: Psyr_1426 8639 >Antitoxin: Psyr_1427
    3096 >Toxin: Bxe_A1177 8640 >Antitoxin: Bxe_A1178
    3097 >Toxin: Rmet_2360 8641 >Antitoxin: Rmet_2359
    3098 >Toxin: Ajs_1249 8642 >Antitoxin: Ajs_1250
    3099 >Toxin: Ajs_1391 8643 >Antitoxin: Ajs_1392
    3100 >Toxin: Ajs_2179 8644 >Antitoxin: Ajs_2180
    3101 >Toxin: Mpe_A2401 8645 >Antitoxin: Mpe_A2400
    3102 >Toxin: Plav_3431 8646 >Antitoxin: Plav_3430
    3103 >Toxin: Spro_4161 8647 >Antitoxin: Spro_4162
    3104 >Toxin: Daci_4186 8648 >Antitoxin: Daci_4185
    3105 >Toxin: Tmz1t_0941 8649 >Antitoxin: Tmz1t_0942
    3106 >Toxin: Tgr7_2866 8650 >Antitoxin: Tgr7_2865
    3107 >Toxin: Avin_36380 8651 >Antitoxin: Avin_36370
    3108 >Toxin: Rpic12D_0690 8652 >Antitoxin: Rpic12D_0689
    3109 >Toxin: Dd1591_0428 8653 >Antitoxin: Dd1591_0429
    3110 >Toxin: Mbar_A1358 8654 >Antitoxin: Mbar_A1357
    3111 >Toxin: RoseRS_2587 8655 >Antitoxin: RoseRS_2588
    3112 >Toxin: Rcas_4238 8656 >Antitoxin: Rcas_4239
    3113 >Toxin: TRQ2_0318 8657 >Antitoxin: TRQ2_0317
    3114 >Toxin: Clim_0935 8658 >Antitoxin: Clim_0934
    3115 >Toxin: Paes_1991 8659 >Antitoxin: Paes_1990
    3116 >Toxin: Cagg_3810 8660 >Antitoxin: Cagg_3811
    3117 >Toxin: Cagg_3824 8661 >Antitoxin: Cagg_3825
    3118 >Toxin: Ava_B0311 8662 >Antitoxin: Ava_B0312
    3119 >Toxin: Sfri_2090 8663 >Antitoxin: Sfri_2089
    3120 >Toxin: Pnap_4584 8664 >Antitoxin: Pnap_4583
    3121 >Toxin: Mpe_B0172 8665 >Antitoxin: Mpe_B0171
    3122 >Toxin: Fjoh_3012 8666 >Antitoxin: Fjoh_3013
    3123 >Toxin: Swit_5328 8667 >Antitoxin: Swit_5327
    3124 >Toxin: MADE_00121 8668 >Antitoxin: MADE_00120
    3125 >Toxin: Tmz1t_2113 8669 >Antitoxin: Tmz1t_2114
    3126 >Toxin: Mnod_0641 8670 >Antitoxin: Mnod_0642
    3127 >Toxin: Amir_0591 8671 >Antitoxin: Amir_0592
    3128 >Toxin: Mfl052 8672 >Antitoxin: Mfl051
    3129 >Toxin: Bcep18194_B0245 8673 >Antitoxin: Bcep18194_B0246
    3130 >Toxin: RPC_2242 8674 >Antitoxin: RPC_2243
    3131 >Toxin: Pden_4165 8675 >Antitoxin: Pden_4164
    3132 >Toxin: Swit_2282 8676 >Antitoxin: Swit_2281
    3133 >Toxin: Mext_2035 8677 >Antitoxin: Mext_2036
    3134 >Toxin: Mpop_1996 8678 >Antitoxin: Mpop_1997
    3135 >Toxin: Gdia_2649 8679 >Antitoxin: Gdia_2650
    3136 >Toxin: Msil_0447 8680 >Antitoxin: Msil_0446
    3137 >Toxin: Mchl_2310 8681 >Antitoxin: Mchl_2311
    3138 >Toxin: Avi_1240 8682 >Antitoxin: Avi_1242
    3139 >Toxin: Caci_6778 8683 >Antitoxin: Caci_6779
    3140 >Toxin: Tfu_0002 8684 >Antitoxin: Tfu_0003
    3141 >Toxin: Mmcs_0002 8685 >Antitoxin: Mmcs_0003
    3142 >Toxin: Arth_0002 8686 >Antitoxin: Arth_0003
    3143 >Toxin: Noca_0002 8687 >Antitoxin: Noca_0003
    3144 >Toxin: Mkms_0010 8688 >Antitoxin: Mkms_0011
    3145 >Toxin: Mvan_0002 8689 >Antitoxin: Mvan_0003
    3146 >Toxin: Mjls_0002 8690 >Antitoxin: Mjls_0003
    3147 >Toxin: Mflv_0826 8691 >Antitoxin: Mflv_0825
    3148 >Toxin: Strop_0003 8692 >Antitoxin: Strop_0004
    3149 >Toxin: Sare_0002 8693 >Antitoxin: Sare_0003
    3150 >Toxin: Achl_0002 8694 >Antitoxin: Achl_0003
    3151 >Toxin: Amir_0002 8695 >Antitoxin: Amir_0003
    3152 >Toxin: Bcav_0002 8696 >Antitoxin: Bcav_0003
    3153 >Toxin: Ksed_00020 8697 >Antitoxin: Ksed_00030
    3154 >Toxin: Caci_0002 8698 >Antitoxin: Caci_0003
    3155 >Toxin: Svir_00020 8699 >Antitoxin: Svir_00030
    3156 >Toxin: Namu_0002 8700 >Antitoxin: Namu_0003
    3157 >Toxin: B21_04046 8701 >Antitoxin: B21_04047
    3158 >Toxin: RS05352 8702 >Antitoxin: RS05353
    3159 >Toxin: PSPTO_2507 8703 >Antitoxin: PSPTO_2508
    3160 >Toxin: Psyr_2311 8704 >Antitoxin: Psyr_2312
    3161 >Toxin: RPB_2618 8705 >Antitoxin: RPB_2619
    3162 >Toxin: Rfer_0277 8706 >Antitoxin: Rfer_0278
    3163 >Toxin: Pden_4294 8707 >Antitoxin: Pden_4293
    3164 >Toxin: Noca_0421 8708 >Antitoxin: Noca_0420
    3165 >Toxin: Aave_1295 8709 >Antitoxin: Aave_1296
    3166 >Toxin: Pnap_0622 8710 >Antitoxin: Pnap_0621
    3167 >Toxin: Ent638_0383 8711 >Antitoxin: Ent638_0384
    3168 >Toxin: BBta_1616 8712 >Antitoxin: BBta_1615
    3169 >Toxin: Smed_1972 8713 >Antitoxin: Smed_1971
    3170 >Toxin: Mmwyl1_0005 8714 >Antitoxin: Mmwyl1_0006
    3171 >Toxin: YpsIP31758_1619 8715 >Antitoxin: YpsIP31758_1620
    3172 >Toxin: Xaut_0312 8716 >Antitoxin: Xaut_0313
    3173 >Toxin: EcHS_A4466 8717 >Antitoxin: EcHS_A4465
    3174 >Toxin: EcE24377A_4780 8718 >Antitoxin: EcE24377A_4782
    3175 >Toxin: Franean1_3622 8719 >Antitoxin: Franean1_3621
    3176 >Toxin: YpAngola_A2844 8720 >Antitoxin: YpAngola_A2843
    3177 >Toxin: Mext_4022 8721 >Antitoxin: Mext_4023
    3178 >Toxin: EcolC_3795 8722 >Antitoxin: EcolC_3794
    3179 >Toxin: YPK_1727 8723 >Antitoxin: YPK_1728
    3180 >Toxin: Xfasm12_0209 8724 >Antitoxin: Xfasm12_0208
    3181 >Toxin: EcSMS35_4690 8725 >Antitoxin: EcSMS35_4691
    3182 >Toxin: Lcho_1439 8726 >Antitoxin: Lcho_1440
    3183 >Toxin: Mrad2831_0164 8727 >Antitoxin: Mrad2831_0163
    3184 >Toxin: Mrad2831_5255 8728 >Antitoxin: Mrad2831_5256
    3185 >Toxin: XfasM23_0185 8729 >Antitoxin: XfasM23_0184
    3186 >Toxin: PXO_03749 8730 >Antitoxin: PXO_03748
    3187 >Toxin: SbBS512_E4752 8731 >Antitoxin: SbBS512_E4753
    3188 >Toxin: Rpal_2976 8732 >Antitoxin: Rpal_2977
    3189 >Toxin: Gbem_2877 8733 >Antitoxin: Gbem_2876
    3190 >Toxin: M446_0617 8734 >Antitoxin: M446_0618
    3191 >Toxin: ECH74115_5728 8735 >Antitoxin: ECH74115_5729
    3192 >Toxin: Rleg2_2511 8736 >Antitoxin: Rleg2_2510
    3193 >Toxin: Rleg2_6179 8737 >Antitoxin: Rleg2_6178
    3194 >Toxin: Mchl_4391 8738 >Antitoxin: Mchl_4392
    3195 >Toxin: Achl_0581 8739 >Antitoxin: Achl_0580
    3196 >Toxin: A2cp1_3290 8740 >Antitoxin: A2cp1_3289
    3197 >Toxin: Avi_2793 8741 >Antitoxin: Avi_2792
    3198 >Toxin: Amir_0551 8742 >Antitoxin: Amir_0552
    3199 >Toxin: Rleg_2766 8743 >Antitoxin: Rleg_2765
    3200 >Toxin: PC1_4290 8744 >Antitoxin: PC1_4289
    3201 >Toxin: Cpin_0640 8745 >Antitoxin: Cpin_0639
    3202 >Toxin: Caci_0025 8746 >Antitoxin: Caci_0026
    3203 >Toxin: CPR_1661 8747 >Antitoxin: CPR_1660
    3204 >Toxin: SAG0378 8748 >Antitoxin: SAG0379
    3205 >Toxin: BCE_3854 8749 >Antitoxin: BCE_3853
    3206 >Toxin: LMOf2365_1339 8750 >Antitoxin: LMOf2365_1340
    3207 >Toxin: GBAA_3953 8751 >Antitoxin: GBAA_3952
    3208 >Toxin: BAS3667 8752 >Antitoxin: BAS3666
    3209 >Toxin: BT9727_3557 8753 >Antitoxin: BT9727_3556
    3210 >Toxin: Mfl292 8754 >Antitoxin: Mfl293
    3211 >Toxin: BCZK3575 8755 >Antitoxin: BCZK3574
    3212 >Toxin: DET0985 8756 >Antitoxin: DET0984
    3213 >Toxin: SACOL1285 8757 >Antitoxin: SACOL1286
    3214 >Toxin: SERP0833 8758 >Antitoxin: SERP0834
    3215 >Toxin: PMN2A_1023 8759 >Antitoxin: PMN2A_1024
    3216 >Toxin: Ava_1870 8760 >Antitoxin: Ava_1869
    3217 >Toxin: Syncc9902_0594 8761 >Antitoxin: Syncc9902_0593
    3218 >Toxin: Syncc9605_2079 8762 >Antitoxin: Syncc9605_2080
    3219 >Toxin: PMT9312_1585 8763 >Antitoxin: PMT9312_1586
    3220 >Toxin: Synpcc7942_2022 8764 >Antitoxin: Synpcc7942_2021
    3221 >Toxin: Dgeo_1234 8765 >Antitoxin: Dgeo_1235
    3222 >Toxin: Moth_1047 8766 >Antitoxin: Moth_1048
    3223 >Toxin: Tery_3978 8767 >Antitoxin: Tery_3977
    3224 >Toxin: CPF_1943 8768 >Antitoxin: CPF_1942
    3225 >Toxin: Swol_0897 8769 >Antitoxin: Swol_0898
    3226 >Toxin: OEOE_0430 8770 >Antitoxin: OEOE_0431
    3227 >Toxin: LEUM_1358 8771 >Antitoxin: LEUM_1357
    3228 >Toxin: LGAS_0811 8772 >Antitoxin: LGAS_0812
    3229 >Toxin: LACR_0814 8773 >Antitoxin: LACR_0815
    3230 >Toxin: STER_0380 8774 >Antitoxin: STER_0381
    3231 >Toxin: Dvul_2431 8775 >Antitoxin: Dvul_2432
    3232 >Toxin: P9515_16721 8776 >Antitoxin: P9515_16731
    3233 >Toxin: A9601_16961 8777 >Antitoxin: A9601_16971
    3234 >Toxin: P9303_04151 8778 >Antitoxin: P9303_04141
    3235 >Toxin: NATL1_18931 8779 >Antitoxin: NATL1_18941
    3236 >Toxin: Cthe_0994 8780 >Antitoxin: Cthe_0993
    3237 >Toxin: P9301_16831 8781 >Antitoxin: P9301_16841
    3238 >Toxin: Dred_1960 8782 >Antitoxin: Dred_1959
    3239 >Toxin: DehaBAV1_0876 8783 >Antitoxin: DehaBAV1_0875
    3240 >Toxin: SaurJH9_1326 8784 >Antitoxin: SaurJH9_1327
    3241 >Toxin: Lreu_0696 8785 >Antitoxin: Lreu_0697
    3242 >Toxin: RoseRS_3749 8786 >Antitoxin: RoseRS_3750
    3243 >Toxin: SaurJH1_1352 8787 >Antitoxin: SaurJH1_1353
    3244 >Toxin: Bcer98_2468 8788 >Antitoxin: Bcer98_2467
    3245 >Toxin: Rcas_1134 8789 >Antitoxin: Rcas_1133
    3246 >Toxin: Haur_0473 8790 >Antitoxin: Haur_0474
    3247 >Toxin: P9211_16141 8791 >Antitoxin: P9211_16151
    3248 >Toxin: Cphy_2777 8792 >Antitoxin: Cphy_2776
    3249 >Toxin: BcerKBAB4_3638 8793 >Antitoxin: BcerKBAB4_3637
    3250 >Toxin: Teth514_1648 8794 >Antitoxin: Teth514_1647
    3251 >Toxin: Daud_0923 8795 >Antitoxin: Daud_0924
    3252 >Toxin: Nther_1441 8796 >Antitoxin: Nther_1442
    3253 >Toxin: MARTH_orf784 8797 >Antitoxin: MARTH_orf786
    3254 >Toxin: BCAH187_A3863 8798 >Antitoxin: BCAH187_A3862
    3255 >Toxin: Dtur_1206 8799 >Antitoxin: Dtur_1207
    3256 >Toxin: BCB4264_A3914 8800 >Antitoxin: BCB4264_A3913
    3257 >Toxin: PCC7424_2359 8801 >Antitoxin: PCC7424_2358
    3258 >Toxin: PCC8801_2712 8802 >Antitoxin: PCC8801_2711
    3259 >Toxin: BCG9842_B1330 8803 >Antitoxin: BCG9842_B1331
    3260 >Toxin: DvMF_2515 8804 >Antitoxin: DvMF_2514
    3261 >Toxin: BCAH820_3828 8805 >Antitoxin: BCAH820_3827
    3262 >Toxin: Dhaf_3679 8806 >Antitoxin: Dhaf_3678
    3263 >Toxin: Hore_07820 8807 >Antitoxin: Hore_07830
    3264 >Toxin: Ccel_0454 8808 >Antitoxin: Ccel_0455
    3265 >Toxin: Ddes_0061 8809 >Antitoxin: Ddes_0060
    3266 >Toxin: Cyan7425_2089 8810 >Antitoxin: Cyan7425_2090
    3267 >Toxin: Athe_1049 8811 >Antitoxin: Athe_1050
    3268 >Toxin: Afer_0636 8812 >Antitoxin: Afer_0637
    3269 >Toxin: Dbac_2412 8813 >Antitoxin: Dbac_2413
    3270 >Toxin: GWCH70_1152 8814 >Antitoxin: GWCH70_1153
    3271 >Toxin: Elen_1812 8815 >Antitoxin: Elen_1811
    3272 >Toxin: Cagg_3683 8816 >Antitoxin: Cagg_3682
    3273 >Toxin: Ccur_08060 8817 >Antitoxin: Ccur_08050
    3274 >Toxin: Apre_0607 8818 >Antitoxin: Apre_0608
    3275 >Toxin: Cyan8802_3391 8819 >Antitoxin: Cyan8802_3392
    3276 >Toxin: Dtox_3191 8820 >Antitoxin: Dtox_3190
    3277 >Toxin: Aaci_1432 8821 >Antitoxin: Aaci_1433
    3278 >Toxin: Dret_0497 8822 >Antitoxin: Dret_0496
    3279 >Toxin: Ent638_3663 8823 >Antitoxin: Ent638_3662
    3280 >Toxin: SbBS512_E3548 8824 >Antitoxin: SbBS512_E3550
    3281 >Toxin: SNSL254_A3604 8825 >Antitoxin: SNSL254_A3603
    3282 >Toxin: SeHA_C3639 8826 >Antitoxin: SeHA_C3638
    3283 >Toxin: SeSA_A3534 8827 >Antitoxin: SeSA_A3533
    3284 >Toxin: SeAg_B3532 8828 >Antitoxin: SeAg_B3531
    3285 >Toxin: SeD_A3701 8829 >Antitoxin: SeD_A3700
    3286 >Toxin: ECH74115_4545 8830 >Antitoxin: ECH74115_4543
    3287 >Toxin: Patl_0700 8831 >Antitoxin: Patl_0699
    3288 >Toxin: Shewmr7_0726 8832 >Antitoxin: Shewmr7_0725
    3289 >Toxin: Dvul_2762 8833 >Antitoxin: Dvul_2763
    3290 >Toxin: Sbal_1288 8834 >Antitoxin: Sbal_1287
    3291 >Toxin: VC0395_1052 8835 >Antitoxin: VC0395_1053
    3292 >Toxin: PSPA7_0699 8836 >Antitoxin: PSPA7_0700
    3293 >Toxin: PSPA7_5057 8837 >Antitoxin: PSPA7_5056
    3294 >Toxin: Mmwyl1_0582 8838 >Antitoxin: Mmwyl1_0583
    3295 >Toxin: VIBHAR_05030 8839 >Antitoxin: VIBHAR_05029
    3296 >Toxin: YPK_0902 8840 >Antitoxin: YPK_0901
    3297 >Toxin: SNSL254_A2931 8841 >Antitoxin: SNSL254_A2932
    3298 >Toxin: SeHA_C3487 8842 >Antitoxin: SeHA_C3488
    3299 >Toxin: SeAg_B2795 8843 >Antitoxin: SeAg_B2796
    3300 >Toxin: VSAL_I1032 8844 >Antitoxin: VSAL_I1031
    3301 >Toxin: Ddes_0244 8845 >Antitoxin: Ddes_0243
    3302 >Toxin: RSc2303 8846 >Antitoxin: RSc2304
    3303 >Toxin: RS02977 8847 >Antitoxin: RS02976
    3304 >Toxin: Tbd_1300 8848 >Antitoxin: Tbd_1299
    3305 >Toxin: Tbd_1439 8849 >Antitoxin: Tbd_1438
    3306 >Toxin: Nmul_A2583 8850 >Antitoxin: Nmul_A2584
    3307 >Toxin: Mfla_1336 8851 >Antitoxin: Mfla_1337
    3308 >Toxin: Rmet_4706 8852 >Antitoxin: Rmet_4707
    3309 >Toxin: Pput_2293 8853 >Antitoxin: Pput_2292
    3310 >Toxin: PputGB1_2459 8854 >Antitoxin: PputGB1_2458
    3311 >Toxin: PputW619_2468 8855 >Antitoxin: PputW619_2467
    3312 >Toxin: Lcho_3884 8856 >Antitoxin: Lcho_3883
    3313 >Toxin: Avin_29100 8857 >Antitoxin: Avin_29110
    3314 >Toxin: GBAA_2803 8858 >Antitoxin: GBAA_2804
    3315 >Toxin: BAS2613 8859 >Antitoxin: BAS2614
    3316 >Toxin: BCZK2530 8860 >Antitoxin: BCZK2531
    3317 >Toxin: Moth_1254 8861 >Antitoxin: Moth_1255
    3318 >Toxin: Cthe_2752 8862 >Antitoxin: Cthe_2753
    3319 >Toxin: Haur_2771 8863 >Antitoxin: Haur_2772
    3320 >Toxin: Cphy_3555 8864 >Antitoxin: Cphy_3554
    3321 >Toxin: Teth514_2351 8865 >Antitoxin: Teth514_2352
    3322 >Toxin: BCAH187_A2853 8866 >Antitoxin: BCAH187_A2854
    3323 >Toxin: BCAH820_2811 8867 >Antitoxin: BCAH820_2812
    3324 >Toxin: Dhaf_1135 8868 >Antitoxin: Dhaf_1136
    3325 >Toxin: Hore_14040 8869 >Antitoxin: Hore_14050
    3326 >Toxin: Apar_1232 8870 >Antitoxin: Apar_1233
    3327 >Toxin: Dtox_2072 8871 >Antitoxin: Dtox_2071
    3328 >Toxin: SO_1986 8872 >Antitoxin: SO_1985
    3329 >Toxin: PSPTO_1043 8873 >Antitoxin: PSPTO_1042
    3330 >Toxin: Psyr_0892 8874 >Antitoxin: Psyr_0891
    3331 >Toxin: CPS_1377 8875 >Antitoxin: CPS_1378
    3332 >Toxin: RPB_0486 8876 >Antitoxin: RPB_0487
    3333 >Toxin: Jann_4021 8877 >Antitoxin: Jann_4022
    3334 >Toxin: Sde_2471 8878 >Antitoxin: Sde_2472
    3335 >Toxin: Sden_3374 8879 >Antitoxin: Sden_3373
    3336 >Toxin: RPD_0340 8880 >Antitoxin: RPD_0339
    3337 >Toxin: TM1040_0415 8881 >Antitoxin: TM1040_0416
    3338 >Toxin: Patl_1332 8882 >Antitoxin: Patl_1333
    3339 >Toxin: Rru_A0722 8883 >Antitoxin: Rru_A0721
    3340 >Toxin: Shewmr7_2347 8884 >Antitoxin: Shewmr7_2348
    3341 >Toxin: RSP_1092 8885 >Antitoxin: RSP_1093
    3342 >Toxin: Shewmr4_2275 8886 >Antitoxin: Shewmr4_2276
    3343 >Toxin: Mmar10_0274 8887 >Antitoxin: Mmar10_0275
    3344 >Toxin: Sfri_2318 8888 >Antitoxin: Sfri_2319
    3345 >Toxin: Shewana3_2465 8889 >Antitoxin: Shewana3_2466
    3346 >Toxin: Sama_2036 8890 >Antitoxin: Sama_2037
    3347 >Toxin: Ping_0995 8891 >Antitoxin: Ping_0996
    3348 >Toxin: Aave_3570 8892 >Antitoxin: Aave_3569
    3349 >Toxin: Maqu_0518 8893 >Antitoxin: Maqu_0517
    3350 >Toxin: Sbal_2597 8894 >Antitoxin: Sbal_2598
    3351 >Toxin: Rsph17029_2755 8895 >Antitoxin: Rsph17029_2756
    3352 >Toxin: Shew_1476 8896 >Antitoxin: Shew_1477
    3353 >Toxin: Sputcn32_2322 8897 >Antitoxin: Sputcn32_2323
    3354 >Toxin: Rsph17025_2936 8898 >Antitoxin: Rsph17025_2935
    3355 >Toxin: VC0395_A1891 8899 >Antitoxin: VC0395_A1890
    3356 >Toxin: BBta_2959 8900 >Antitoxin: BBta_2958
    3357 >Toxin: Mmwyl1_4075 8901 >Antitoxin: Mmwyl1_4076
    3358 >Toxin: Shew185_2636 8902 >Antitoxin: Shew185_2637
    3359 >Toxin: Plav_1868 8903 >Antitoxin: Plav_1867
    3360 >Toxin: Xaut_1629 8904 >Antitoxin: Xaut_1630
    3361 >Toxin: VIBHAR_03284 8905 >Antitoxin: VIBHAR_03283
    3362 >Toxin: Dshi_0366 8906 >Antitoxin: Dshi_0365
    3363 >Toxin: Sbal195_2711 8907 >Antitoxin: Sbal195_2712
    3364 >Toxin: Caul_1554 8908 >Antitoxin: Caul_1553
    3365 >Toxin: Swoo_2780 8909 >Antitoxin: Swoo_2779
    3366 >Toxin: Rpal_0551 8910 >Antitoxin: Rpal_0550
    3367 >Toxin: Smal_1959 8911 >Antitoxin: Smal_1960
    3368 >Toxin: MADE_01437 8912 >Antitoxin: MADE_01438
    3369 >Toxin: Sbal223_1749 8913 >Antitoxin: Sbal223_1748
    3370 >Toxin: Avi_0089 8914 >Antitoxin: Avi_0090
    3371 >Toxin: CPR_1140 8915 >Antitoxin: CPR_1139
    3372 >Toxin: SAG0350 8916 >Antitoxin: SAG0349
    3373 >Toxin: CPF_1328 8917 >Antitoxin: CPF_1327
    3374 >Toxin: OEOE_1587 8918 >Antitoxin: OEOE_1588
    3375 >Toxin: LEUM_0315 8919 >Antitoxin: LEUM_0314
    3376 >Toxin: LACR_0826 8920 >Antitoxin: LACR_0825
    3377 >Toxin: STER_0435 8921 >Antitoxin: STER_0434
    3378 >Toxin: Lreu_0986 8922 >Antitoxin: Lreu_0987
    3379 >Toxin: Cphy_0519 8923 >Antitoxin: Cphy_0518
    3380 >Toxin: Shel_01090 8924 >Antitoxin: Shel_01080
    3381 >Toxin: Jann_0512 8925 >Antitoxin: Jann_0513
    3382 >Toxin: TM1040_2567 8926 >Antitoxin: TM1040_2566
    3383 >Toxin: Rru_A1858 8927 >Antitoxin: Rru_A1857
    3384 >Toxin: RSP_1668 8928 >Antitoxin: RSP_1669
    3385 >Toxin: Mmar10_1577 8929 >Antitoxin: Mmar10_1576
    3386 >Toxin: Pden_1402 8930 >Antitoxin: Pden_1401
    3387 >Toxin: Rsph17029_0301 8931 >Antitoxin: Rsph17029_0302
    3388 >Toxin: Rsph17025_2578 8932 >Antitoxin: Rsph17025_2577
    3389 >Toxin: Acry_0529 8933 >Antitoxin: Acry_0530
    3390 >Toxin: Plav_2866 8934 >Antitoxin: Plav_2867
    3391 >Toxin: Dshi_0192 8935 >Antitoxin: Dshi_0193
    3392 >Toxin: Gdia_0378 8936 >Antitoxin: Gdia_0377
    3393 >Toxin: B21_03041 8937 >Antitoxin: B21_03040
    3394 >Toxin: PSPTO_4425 8938 >Antitoxin: PSPTO_4424
    3395 >Toxin: Psyr_4119 8939 >Antitoxin: Psyr_4118
    3396 >Toxin: Csal_2206 8940 >Antitoxin: Csal_2205
    3397 >Toxin: HS_0753 8941 >Antitoxin: HS_0754
    3398 >Toxin: Ent638_3665 8942 >Antitoxin: Ent638_3664
    3399 >Toxin: Mmwyl1_2399 8943 >Antitoxin: Mmwyl1_2398
    3400 >Toxin: EcHS_A3419 8944 >Antitoxin: EcHS_A3418
    3401 >Toxin: EcE24377A_3713 8945 >Antitoxin: EcE24377A_3712
    3402 >Toxin: Spro_4348 8946 >Antitoxin: Spro_4347
    3403 >Toxin: COXBURSA331_A1940 8947 >Antitoxin: COXBURSA331_A1939
    3404 >Toxin: YpAngola_A1133 8948 >Antitoxin: YpAngola_A1132
    3405 >Toxin: EcolC_0476 8949 >Antitoxin: EcolC_0477
    3406 >Toxin: YPK_0525 8950 >Antitoxin: YPK_0526
    3407 >Toxin: SbBS512_E3546 8951 >Antitoxin: SbBS512_E3547
    3408 >Toxin: SNSL254_A3607 8952 >Antitoxin: SNSL254_A3605
    3409 >Toxin: SeSA_A3536 8953 >Antitoxin: SeSA_A3535
    3410 >Toxin: SeD_A3704 8954 >Antitoxin: SeD_A3702
    3411 >Toxin: ECH74115_4547 8955 >Antitoxin: ECH74115_4546
    3412 >Toxin: Dd703_3638 8956 >Antitoxin: Dd703_3637
    3413 >Toxin: Dd1591_3796 8957 >Antitoxin: Dd1591_3795
    3414 >Toxin: PC1_0293 8958 >Antitoxin: PC1_0294
    3415 >Toxin: B21_03060 8959 >Antitoxin: B21_03059
    3416 >Toxin: SO_4097 8960 >Antitoxin: SO_4096
    3417 >Toxin: PP_0934 8961 >Antitoxin: PP_0935
    3418 >Toxin: PSPTO_4471 8962 >Antitoxin: PSPTO_4470
    3419 >Toxin: MCA0101 8963 >Antitoxin: MCA0102
    3420 >Toxin: Psyr_4162 8964 >Antitoxin: Psyr_4161
    3421 >Toxin: Psyc_0484 8965 >Antitoxin: Psyc_0485
    3422 >Toxin: CPS_4559 8966 >Antitoxin: CPS_4558
    3423 >Toxin: Daro_0115 8967 >Antitoxin: Daro_0114
    3424 >Toxin: Tbd_0261 8968 >Antitoxin: Tbd_0262
    3425 >Toxin: Tcr_1631 8969 >Antitoxin: Tcr_1632
    3426 >Toxin: Nmul_A0319 8970 >Antitoxin: Nmul_A0318
    3427 >Toxin: Sde_3191 8971 >Antitoxin: Sde_3190
    3428 >Toxin: Mfla_2492 8972 >Antitoxin: Mfla_2493
    3429 >Toxin: Sden_3333 8973 >Antitoxin: Sden_3332
    3430 >Toxin: Csal_2239 8974 >Antitoxin: Csal_2238
    3431 >Toxin: Pcryo_0479 8975 >Antitoxin: Pcryo_0480
    3432 >Toxin: Rmet_0052 8976 >Antitoxin: Rmet_0053
    3433 >Toxin: BCI_0048 8977 >Antitoxin: BCI_0047
    3434 >Toxin: Patl_0184 8978 >Antitoxin: Patl_0185
    3435 >Toxin: Rru_A0471 8979 >Antitoxin: Rru_A0472
    3436 >Toxin: HS_1126 8980 >Antitoxin: HS_1125
    3437 >Toxin: Shewmr7_0480 8981 >Antitoxin: Shewmr7_0481
    3438 >Toxin: Shewmr4_3471 8982 >Antitoxin: Shewmr4_3470
    3439 >Toxin: Mlg_0170 8983 >Antitoxin: Mlg_0171
    3440 >Toxin: Sfri_3737 8984 >Antitoxin: Sfri_3736
    3441 >Toxin: PA14_58130 8985 >Antitoxin: PA14_58120
    3442 >Toxin: Mmc1_1413 8986 >Antitoxin: Mmc1_1414
    3443 >Toxin: Shewana3_3647 8987 >Antitoxin: Shewana3_3646
    3444 >Toxin: Sama_0468 8988 >Antitoxin: Sama_0469
    3445 >Toxin: Ping_1123 8989 >Antitoxin: Ping_1124
    3446 >Toxin: Aave_0295 8990 >Antitoxin: Aave_0296
    3447 >Toxin: Ajs_0240 8991 >Antitoxin: Ajs_0241
    3448 >Toxin: Veis_1609 8992 >Antitoxin: Veis_1608
    3449 >Toxin: Hhal_1006 8993 >Antitoxin: Hhal_1007
    3450 >Toxin: Maqu_2727 8994 >Antitoxin: Maqu_2726
    3451 >Toxin: Sbal_3821 8995 >Antitoxin: Sbal_3820
    3452 >Toxin: Shew_0404 8996 >Antitoxin: Shew_0405
    3453 >Toxin: Pnuc_2022 8997 >Antitoxin: Pnuc_2023
    3454 >Toxin: Ent638_3686 8998 >Antitoxin: Ent638_3685
    3455 >Toxin: Pmen_0857 8999 >Antitoxin: Pmen_0858
    3456 >Toxin: Sputcn32_0575 9000 >Antitoxin: Sputcn32_0576
    3457 >Toxin: VC0395_A2835 9001 >Antitoxin: VC0395_A2836
    3458 >Toxin: Pput_0974 9002 >Antitoxin: Pput_0975
    3459 >Toxin: PsycPRwf_0421 9003 >Antitoxin: PsycPRwf_0422
    3460 >Toxin: PSPA7_5094 9004 >Antitoxin: PSPA7_5093
    3461 >Toxin: Mmwyl1_1938 9005 >Antitoxin: Mmwyl1_1939
    3462 >Toxin: Shew185_0498 9006 >Antitoxin: Shew185_0499
    3463 >Toxin: YpsIP31758_0399 9007 >Antitoxin: YpsIP31758_0400
    3464 >Toxin: VIBHAR_03684 9008 >Antitoxin: VIBHAR_03683
    3465 >Toxin: EcHS_A3439 9009 >Antitoxin: EcHS_A3438
    3466 >Toxin: EcE24377A_3733 9010 >Antitoxin: EcE24377A_3731
    3467 >Toxin: Ssed_0559 9011 >Antitoxin: Ssed_0560
    3468 >Toxin: Spro_4410 9012 >Antitoxin: Spro_4409
    3469 >Toxin: Spea_3753 9013 >Antitoxin: Spea_3752
    3470 >Toxin: Sbal195_0519 9014 >Antitoxin: Sbal195_0520
    3471 >Toxin: COXBURSA331_A1647 9015 >Antitoxin: COXBURSA331_A1646
    3472 >Toxin: YpAngola_A1197 9016 >Antitoxin: YpAngola_A1196
    3473 >Toxin: PputGB1_0941 9017 >Antitoxin: PputGB1_0942
    3474 >Toxin: EcolC_0457 9018 >Antitoxin: EcolC_0458
    3475 >Toxin: PputW619_4281 9019 >Antitoxin: PputW619_4280
    3476 >Toxin: YPK_0466 9020 >Antitoxin: YPK_0467
    3477 >Toxin: Swoo_4401 9021 >Antitoxin: Swoo_4400
    3478 >Toxin: Xfasm12_0669 9022 >Antitoxin: Xfasm12_0670
    3479 >Toxin: EcSMS35_3545 9023 >Antitoxin: EcSMS35_3544
    3480 >Toxin: Lcho_0503 9024 >Antitoxin: Lcho_0502
    3481 >Toxin: Pnec_1739 9025 >Antitoxin: Pnec_1740
    3482 >Toxin: XfasM23_0588 9026 >Antitoxin: XfasM23_0589
    3483 >Toxin: SbBS512_E3281 9027 >Antitoxin: SbBS512_E3280
    3484 >Toxin: Smal_3453 9028 >Antitoxin: Smal_3452
    3485 >Toxin: SNSL254_A3636 9029 >Antitoxin: SNSL254_A3635
    3486 >Toxin: SeHA_C3671 9030 >Antitoxin: SeHA_C3670
    3487 >Toxin: SeSA_A3565 9031 >Antitoxin: SeSA_A3564
    3488 >Toxin: MADE_00265 9032 >Antitoxin: MADE_00266
    3489 >Toxin: SeAg_B3564 9033 >Antitoxin: SeAg_B3563
    3490 >Toxin: Lferr_2193 9034 >Antitoxin: Lferr_2194
    3491 >Toxin: SeD_A3733 9035 >Antitoxin: SeD_A3732
    3492 >Toxin: VSAL_I0484 9036 >Antitoxin: VSAL_I0485
    3493 >Toxin: ECH74115_4566 9037 >Antitoxin: ECH74115_4565
    3494 >Toxin: Tmz1t_0237 9038 >Antitoxin: Tmz1t_0238
    3495 >Toxin: Sbal223_0523 9039 >Antitoxin: Sbal223_0524
    3496 >Toxin: AFE_2563 9040 >Antitoxin: AFE_2564
    3497 >Toxin: Tgr7_0513 9041 >Antitoxin: Tgr7_0512
    3498 >Toxin: Dtpsy_0234 9042 >Antitoxin: Dtpsy_0235
    3499 >Toxin: Avin_12660 9043 >Antitoxin: Avin_12670
    3500 >Toxin: Dd703_3676 9044 >Antitoxin: Dd703_3675
    3501 >Toxin: Dd1591_3834 9045 >Antitoxin: Dd1591_3833
    3502 >Toxin: PC1_0253 9046 >Antitoxin: PC1_0254
    3503 >Toxin: Nwi_1700 9047 >Antitoxin: Nwi_1701
    3504 >Toxin: Noc_2148 9048 >Antitoxin: Noc_2149
    3505 >Toxin: RPB_2392 9049 >Antitoxin: RPB_2391
    3506 >Toxin: RPC_3329 9050 >Antitoxin: RPC_3330
    3507 >Toxin: RPD_3059 9051 >Antitoxin: RPD_3060
    3508 >Toxin: Nham_2421 9052 >Antitoxin: Nham_2422
    3509 >Toxin: Meso_1370 9053 >Antitoxin: Meso_1369
    3510 >Toxin: Ping_2499 9054 >Antitoxin: Ping_2498
    3511 >Toxin: BBta_4778 9055 >Antitoxin: BBta_4779
    3512 >Toxin: Xaut_4540 9056 >Antitoxin: Xaut_4541
    3513 >Toxin: Rpal_3560 9057 >Antitoxin: Rpal_3561
    3514 >Toxin: M446_2256 9058 >Antitoxin: M446_2255
    3515 >Toxin: Msil_0751 9059 >Antitoxin: Msil_0752
    3516 >Toxin: Mnod_0353 9060 >Antitoxin: Mnod_0354
    3517 >Toxin: Francci3_0679 9061 >Antitoxin: Francci3_0680
    3518 >Toxin: Dgeo_0887 9062 >Antitoxin: Dgeo_0888
    3519 >Toxin: Mmcs_1228 9063 >Antitoxin: Mmcs_1229
    3520 >Toxin: Arth_1129 9064 >Antitoxin: Arth_1130
    3521 >Toxin: Mkms_1245 9065 >Antitoxin: Mkms_1246
    3522 >Toxin: Mvan_1587 9066 >Antitoxin: Mvan_1588
    3523 >Toxin: Mjls_1254 9067 >Antitoxin: Mjls_1255
    3524 >Toxin: Mflv_4846 9068 >Antitoxin: Mflv_4845
    3525 >Toxin: TBFG_13349 9069 >Antitoxin: TBFG_13348
    3526 >Toxin: Cphamn1_2514 9070 >Antitoxin: Cphamn1_2515
    3527 >Toxin: Paes_2255 9071 >Antitoxin: Paes_2256
    3528 >Toxin: Achl_1202 9072 >Antitoxin: Achl_1203
    3529 >Toxin: Amir_6464 9073 >Antitoxin: Amir_6463
    3530 >Toxin: Bcav_2952 9074 >Antitoxin: Bcav_2951
    3531 >Toxin: Bfae_21330 9075 >Antitoxin: Bfae_21320
    3532 >Toxin: Mlut_04830 9076 >Antitoxin: Mlut_04840
    3533 >Toxin: Caci_0770 9077 >Antitoxin: Caci_0771
    3534 >Toxin: Svir_05360 9078 >Antitoxin: Svir_05370
    3535 >Toxin: Jden_0819 9079 >Antitoxin: Jden_0820
    3536 >Toxin: Namu_1309 9080 >Antitoxin: Namu_1310
    3537 >Toxin: SERP2170 9081 >Antitoxin: SERP2169
    3538 >Toxin: Moth_0607 9082 >Antitoxin: Moth_0606
    3539 >Toxin: Swol_1512 9083 >Antitoxin: Swol_1513
    3540 >Toxin: Dred_2469 9084 >Antitoxin: Dred_2470
    3541 >Toxin: Gdia_2026 9085 >Antitoxin: Gdia_2027
    3542 >Toxin: Ksed_17560 9086 >Antitoxin: Ksed_17550
    3543 >Toxin: Elen_1387 9087 >Antitoxin: Elen_1386
    3544 >Toxin: Ccur_00390 9088 >Antitoxin: Ccur_00380
    3545 >Toxin: Apre_1243 9089 >Antitoxin: Apre_1242
    3546 >Toxin: Shel_11050 9090 >Antitoxin: Shel_11040
    3547 >Toxin: Apar_0696 9091 >Antitoxin: Apar_0695
    3548 >Toxin: Dtox_3044 9092 >Antitoxin: Dtox_3045
    3549 >Toxin: PG0071 9093 >Antitoxin: PG0070
    3550 >Toxin: CHU_1037 9094 >Antitoxin: CHU_1036
    3551 >Toxin: Fjoh_2903 9095 >Antitoxin: Fjoh_2902
    3552 >Toxin: Oter_2576 9096 >Antitoxin: Oter_2577
    3553 >Toxin: Aasi_0289 9097 >Antitoxin: Aasi_0288
    3554 >Toxin: Coch_1756 9098 >Antitoxin: Coch_1757
    3555 >Toxin: Phep_4279 9099 >Antitoxin: Phep_4278
    3556 >Toxin: Dfer_3015 9100 >Antitoxin: Dfer_3014
    3557 >Toxin: Cpin_1279 9101 >Antitoxin: Cpin_1278
    3558 >Toxin: CPR_2094 9102 >Antitoxin: CPR_2093
    3559 >Toxin: CPF_2382 9103 >Antitoxin: CPF_2381
    3560 >Toxin: Cthe_0163 9104 >Antitoxin: Cthe_0164
    3561 >Toxin: Cphy_2548 9105 >Antitoxin: Cphy_2547
    3562 >Toxin: Dhaf_4334 9106 >Antitoxin: Dhaf_4333
    3563 >Toxin: Hore_14100 9107 >Antitoxin: Hore_14090
    3564 >Toxin: Ccel_1323 9108 >Antitoxin: Ccel_1324
    3565 >Toxin: Apre_0929 9109 >Antitoxin: Apre_0930
    3566 >Toxin: PSPTO_5284 9110 >Antitoxin: PSPTO_5283
    3567 >Toxin: Psyr_4842 9111 >Antitoxin: Psyr_4841
    3568 >Toxin: Sde_0348 9112 >Antitoxin: Sde_0349
    3569 >Toxin: Spro_3823 9113 >Antitoxin: Spro_3822
    3570 >Toxin: COXBURSA331_A1734 9114 >Antitoxin: COXBURSA331_A1733
    3571 >Toxin: YpAngola_A3240 9115 >Antitoxin: YpAngola_A3239
    3572 >Toxin: YPK_1034 9116 >Antitoxin: YPK_1035
    3573 >Toxin: SeHA_C3216 9117 >Antitoxin: SeHA_C3215
    3574 >Toxin: SeSA_A3167 9118 >Antitoxin: SeSA_A3166
    3575 >Toxin: SeAg_B3150 9119 >Antitoxin: SeAg_B3149
    3576 >Toxin: SeD_A3330 9120 >Antitoxin: SeD_A3329
    3577 >Toxin: Dd703_3016 9121 >Antitoxin: Dd703_3015
    3578 >Toxin: PC1_0905 9122 >Antitoxin: PC1_0906
    3579 >Toxin: Rfer_3786 9123 >Antitoxin: Rfer_3785
    3580 >Toxin: Bpro_0265 9124 >Antitoxin: Bpro_0266
    3581 >Toxin: Aave_0347 9125 >Antitoxin: Aave_0348
    3582 >Toxin: Ajs_0287 9126 >Antitoxin: Ajs_0288
    3583 >Toxin: Pnap_0212 9127 >Antitoxin: Pnap_0213
    3584 >Toxin: Veis_1273 9128 >Antitoxin: Veis_1274
    3585 >Toxin: Mpe_A3434 9129 >Antitoxin: Mpe_A3433
    3586 >Toxin: Daci_0400 9130 >Antitoxin: Daci_0401
    3587 >Toxin: Lcho_3990 9131 >Antitoxin: Lcho_3989
    3588 >Toxin: Dtpsy_0282 9132 >Antitoxin: Dtpsy_0283
    3589 >Toxin: Vapar_4904 9133 >Antitoxin: Vapar_4903
    3590 >Toxin: Csal_1644 9134 >Antitoxin: Csal_1645
    3591 >Toxin: Patl_1767 9135 >Antitoxin: Patl_1766
    3592 >Toxin: CHU_1081 9136 >Antitoxin: CHU_1080
    3593 >Toxin: Sfri_1851 9137 >Antitoxin: Sfri_1850
    3594 >Toxin: Fjoh_2211 9138 >Antitoxin: Fjoh_2212
    3595 >Toxin: Spro_3043 9139 >Antitoxin: Spro_3042
    3596 >Toxin: Caul_4167 9140 >Antitoxin: Caul_4168
    3597 >Toxin: MADE_01800 9141 >Antitoxin: MADE_01799
    3598 >Toxin: Phep_2288 9142 >Antitoxin: Phep_2287
    3599 >Toxin: Dfer_5124 9143 >Antitoxin: Dfer_5125
    3600 >Toxin: Mpal_0372 9144 >Antitoxin: Mpal_0371
    3601 >Toxin: Shewmr7_0701 9145 >Antitoxin: Shewmr7_0702
    3602 >Toxin: Sbal_1270 9146 >Antitoxin: Sbal_1271
    3603 >Toxin: Ent638_1343 9147 >Antitoxin: Ent638_1344
    3604 >Toxin: VC0395_1075 9148 >Antitoxin: VC0395_1074
    3605 >Toxin: EcHS_A0912 9149 >Antitoxin: EcHS_A0913
    3606 >Toxin: EcolC_2792 9150 >Antitoxin: EcolC_2791
    3607 >Toxin: YPK_0884 9151 >Antitoxin: YPK_0885
    3608 >Toxin: Mbar_A3105 9152 >Antitoxin: Mbar_A3104
    3609 >Toxin: BURPS1710b_A1055 9153 >Antitoxin: BURPS1710b_A1054
    3610 >Toxin: Pcar_2990 9154 >Antitoxin: Pcar_2991
    3611 >Toxin: Plut_1064 9155 >Antitoxin: Plut_1065
    3612 >Toxin: Nmul_A1660 9156 >Antitoxin: Nmul_A1659
    3613 >Toxin: BTH_II0420 9157 >Antitoxin: BTH_II0421
    3614 >Toxin: Rfer_1162 9158 >Antitoxin: Rfer_1163
    3615 >Toxin: Bxe_A1880 9159 >Antitoxin: Bxe_A1881
    3616 >Toxin: Bxe_C0048 9160 >Antitoxin: Bxe_C0047
    3617 >Toxin: Patl_2669 9161 >Antitoxin: Patl_2670
    3618 >Toxin: RSP_3930 9162 >Antitoxin: RSP_3931
    3619 >Toxin: Sfri_3050 9163 >Antitoxin: Sfri_3051
    3620 >Toxin: Ping_0464 9164 >Antitoxin: Ping_0465
    3621 >Toxin: Pnap_2335 9165 >Antitoxin: Pnap_2334
    3622 >Toxin: Rsph17029_4098 9166 >Antitoxin: Rsph17029_4099
    3623 >Toxin: BMA10247_A0148 9167 >Antitoxin: BMA10247_A0149
    3624 >Toxin: BURPS668_A2793 9168 >Antitoxin: BURPS668_A2792
    3625 >Toxin: BURPS1106A_A2651 9169 >Antitoxin: BURPS1106A_A2650
    3626 >Toxin: BBta_1439 9170 >Antitoxin: BBta_1438
    3627 >Toxin: Mmwyl1_1955 9171 >Antitoxin: Mmwyl1_1956
    3628 >Toxin: Dshi_0436 9172 >Antitoxin: Dshi_0437
    3629 >Toxin: Bind_2774 9173 >Antitoxin: Bind_2775
    3630 >Toxin: Bphy_1128 9174 >Antitoxin: Bphy_1127
    3631 >Toxin: Bphyt_5559 9175 >Antitoxin: Bphyt_5558
    3632 >Toxin: Rpal_1056 9176 >Antitoxin: Rpal_1055
    3633 >Toxin: Paes_0889 9177 >Antitoxin: Paes_0890
    3634 >Toxin: Avin_19680 9178 >Antitoxin: Avin_19690
    3635 >Toxin: Dbac_0951 9179 >Antitoxin: Dbac_0950
    3636 >Toxin: Fjoh_2893 9180 >Antitoxin: Fjoh_2892
    3637 >Toxin: Coch_1073 9181 >Antitoxin: Coch_1072
    3638 >Toxin: Phep_0306 9182 >Antitoxin: Phep_0307
    3639 >Toxin: Dfer_4782 9183 >Antitoxin: Dfer_4783
    3640 >Toxin: Cpin_1735 9184 >Antitoxin: Cpin_1736
    3641 >Toxin: Cpin_2192 9185 >Antitoxin: Cpin_2193
    3642 >Toxin: Cpin_3748 9186 >Antitoxin: Cpin_3747
    3643 >Toxin: DET1408 9187 >Antitoxin: DET1409
    3644 >Toxin: Dgeo_1103 9188 >Antitoxin: Dgeo_1102
    3645 >Toxin: Moth_2402 9189 >Antitoxin: Moth_2403
    3646 >Toxin: Dred_3174 9190 >Antitoxin: Dred_3175
    3647 >Toxin: DehaBAV1_1215 9191 >Antitoxin: DehaBAV1_1216
    3648 >Toxin: Daud_2173 9192 >Antitoxin: Daud_2174
    3649 >Toxin: Elen_1088 9193 >Antitoxin: Elen_1087
    3650 >Toxin: Ccur_09300 9194 >Antitoxin: Ccur_09310
    3651 >Toxin: Shel_23670 9195 >Antitoxin: Shel_23680
    3652 >Toxin: Dtox_0059 9196 >Antitoxin: Dtox_0058
    3653 >Toxin: Aaci_2782 9197 >Antitoxin: Aaci_2783
    3654 >Toxin: RSc0621 9198 >Antitoxin: RSc0620
    3655 >Toxin: Daro_2593 9199 >Antitoxin: Daro_2592
    3656 >Toxin: Bpro_0540 9200 >Antitoxin: Bpro_0541
    3657 >Toxin: Bpro_2731 9201 >Antitoxin: Bpro_2730
    3658 >Toxin: Spro_1511 9202 >Antitoxin: Spro_1510
    3659 >Toxin: Daci_3133 9203 >Antitoxin: Daci_3134
    3660 >Toxin: Bphy_5021 9204 >Antitoxin: Bphy_5020
    3661 >Toxin: Gdia_1698 9205 >Antitoxin: Gdia_1697
    3662 >Toxin: Gdia_2752 9206 >Antitoxin: Gdia_2751
    3663 >Toxin: Vapar_2991 9207 >Antitoxin: Vapar_2990
    3664 >Toxin: Ajs_3305 9208 >Antitoxin: Ajs_3304
    3665 >Toxin: TBFG_12339 9209 >Antitoxin: TBFG_12340
    3666 >Toxin: Oter_4149 9210 >Antitoxin: Oter_4150
    3667 >Toxin: M446_6662 9211 >Antitoxin: M446_6663
    3668 >Toxin: Dtpsy_2658 9212 >Antitoxin: Dtpsy_2657
    3669 >Toxin: Bcav_0146 9213 >Antitoxin: Bcav_0145
    3670 >Toxin: Bfae_28370 9214 >Antitoxin: Bfae_28380
    3671 >Toxin: RSc1117 9215 >Antitoxin: RSc1118
    3672 >Toxin: Reut_A1076 9216 >Antitoxin: Reut_A1077
    3673 >Toxin: Rmet_1040 9217 >Antitoxin: Rmet_1041
    3674 >Toxin: TM1040_1846 9218 >Antitoxin: TM1040_1845
    3675 >Toxin: RSP_0777 9219 >Antitoxin: RSP_0776
    3676 >Toxin: Pden_1840 9220 >Antitoxin: Pden_1839
    3677 >Toxin: BMASAVP1_A2201 9221 >Antitoxin: BMASAVP1_A2200
    3678 >Toxin: Rsph17029_2433 9222 >Antitoxin: Rsph17029_2432
    3679 >Toxin: BMA10247_1474 9223 >Antitoxin: BMA10247_1473
    3680 >Toxin: BURPS668_2584 9224 >Antitoxin: BURPS668_2583
    3681 >Toxin: BURPS1106A_2639 9225 >Antitoxin: BURPS1106A_2638
    3682 >Toxin: Rsph17025_0402 9226 >Antitoxin: Rsph17025_0403
    3683 >Toxin: Glov_0758 9227 >Antitoxin: Glov_0759
    3684 >Toxin: PSPTO_0935 9228 >Antitoxin: PSPTO_0936
    3685 >Toxin: Psyr_0803 9229 >Antitoxin: Psyr_0804
    3686 >Toxin: Psyc_0758 9230 >Antitoxin: Psyc_0757
    3687 >Toxin: Noc_1003 9231 >Antitoxin: Noc_1004
    3688 >Toxin: Bcep18194_B0278 9232 >Antitoxin: Bcep18194_B0277
    3689 >Toxin: Rru_A0901 9233 >Antitoxin: Rru_A0900
    3690 >Toxin: Ping_1268 9234 >Antitoxin: Ping_1269
    3691 >Toxin: Pnap_4659 9235 >Antitoxin: Pnap_4660
    3692 >Toxin: PsycPRwf_0082 9236 >Antitoxin: PsycPRwf_0083
    3693 >Toxin: YpsIP31758_3791 9237 >Antitoxin: YpsIP31758_3792
    3694 >Toxin: YpAngola_A0660 9238 >Antitoxin: YpAngola_A0659
    3695 >Toxin: YPK_3879 9239 >Antitoxin: YPK_3880
    3696 >Toxin: ECH74115_1348 9240 >Antitoxin: ECH74115_1347
    3697 >Toxin: Tbd_0567 9241 >Antitoxin: Tbd_0566
    3698 >Toxin: Mmc1_3717 9242 >Antitoxin: Mmc1_3716
    3699 >Toxin: Pden_4150 9243 >Antitoxin: Pden_4151
    3700 >Toxin: Pnuc_1157 9244 >Antitoxin: Pnuc_1156
    3701 >Toxin: Rsph17025_3918 9245 >Antitoxin: Rsph17025_3917
    3702 >Toxin: BBta_3671 9246 >Antitoxin: BBta_3672
    3703 >Toxin: Dshi_2807 9247 >Antitoxin: Dshi_2806
    3704 >Toxin: Mpop_4156 9248 >Antitoxin: Mpop_4157
    3705 >Toxin: Mnod_7668 9249 >Antitoxin: Mnod_7669
    3706 >Toxin: YpsIP31758_1279 9250 >Antitoxin: YpsIP31758_1280
    3707 >Toxin: Spro_3465 9251 >Antitoxin: Spro_3464
    3708 >Toxin: YpAngola_A2785 9252 >Antitoxin: YpAngola_A2784
    3709 >Toxin: YPK_1392 9253 >Antitoxin: YPK_1393
    3710 >Toxin: Dd703_3212 9254 >Antitoxin: Dd703_3211
    3711 >Toxin: Dd1591_3291 9255 >Antitoxin: Dd1591_3290
    3712 >Toxin: PC1_0756 9256 >Antitoxin: PC1_0757
    3713 >Toxin: Dde_0128 9257 >Antitoxin: Dde_0127
    3714 >Toxin: Rfer_3566 9258 >Antitoxin: Rfer_3565
    3715 >Toxin: RPC_4427 9259 >Antitoxin: RPC_4426
    3716 >Toxin: Rmet_2589 9260 >Antitoxin: Rmet_2590
    3717 >Toxin: Bcen_1535 9261 >Antitoxin: Bcen_1534
    3718 >Toxin: Rxyl_2989 9262 >Antitoxin: Rxyl_2988
    3719 >Toxin: Moth_0726 9263 >Antitoxin: Moth_0727
    3720 >Toxin: Bcen2424_6294 9264 >Antitoxin: Bcen2424_6295
    3721 >Toxin: Bmul_6106 9265 >Antitoxin: Bmul_6107
    3722 >Toxin: Bcenmc03_6953 9266 >Antitoxin: Bcenmc03_6954
    3723 >Toxin: BamMC406_5964 9267 >Antitoxin: BamMC406_5963
    3724 >Toxin: Oter_2151 9268 >Antitoxin: Oter_2150
    3725 >Toxin: Bphy_5836 9269 >Antitoxin: Bphy_5837
    3726 >Toxin: SNSL254_A0178 9270 >Antitoxin: SNSL254_A0179
    3727 >Toxin: SeSA_A0181 9271 >Antitoxin: SeSA_A0182
    3728 >Toxin: SeAg_B0194 9272 >Antitoxin: SeAg_B0195
    3729 >Toxin: SeD_A0177 9273 >Antitoxin: SeD_A0178
    3730 >Toxin: Mnod_8221 9274 >Antitoxin: Mnod_8220
    3731 >Toxin: Phep_0524 9275 >Antitoxin: Phep_0523
    3732 >Toxin: Dd703_0691 9276 >Antitoxin: Dd703_0692
    3733 >Toxin: Dd1591_0648 9277 >Antitoxin: Dd1591_0649
    3734 >Toxin: PC1_3536 9278 >Antitoxin: PC1_3535
    3735 >Toxin: SO_0666 9279 >Antitoxin: SO_0665
    3736 >Toxin: Mfla_2701 9280 >Antitoxin: Mfla_2702
    3737 >Toxin: Bpro_3782 9281 >Antitoxin: Bpro_3781
    3738 >Toxin: VC0395_A0764 9282 >Antitoxin: VC0395_A0765
    3739 >Toxin: Shew185_2088 9283 >Antitoxin: Shew185_2087
    3740 >Toxin: Sbal195_2135 9284 >Antitoxin: Sbal195_2134
    3741 >Toxin: Tgr7_1642 9285 >Antitoxin: Tgr7_1643
    3742 >Toxin: Avi_3008 9286 >Antitoxin: Avi_3009
    3743 >Toxin: Dde_0309 9287 >Antitoxin: Dde_0308
    3744 >Toxin: YpsIP31758_2058 9288 >Antitoxin: YpsIP31758_2057
    3745 >Toxin: YpAngola_A2450 9289 >Antitoxin: YpAngola_A2449
    3746 >Toxin: YPK_2170 9290 >Antitoxin: YPK_2169
    3747 >Toxin: DvMF_2349 9291 >Antitoxin: DvMF_2348
    3748 >Toxin: Avi_2178 9292 >Antitoxin: Avi_2179
    3749 >Toxin: Dd703_1873 9293 >Antitoxin: Dd703_1872
    3750 >Toxin: Dd1591_2093 9294 >Antitoxin: Dd1591_2092
    3751 >Toxin: Dret_2124 9295 >Antitoxin: Dret_2123
    3752 >Toxin: Ent638_0215 9296 >Antitoxin: Ent638_0214
    3753 >Toxin: YpsIP31758_3844 9297 >Antitoxin: YpsIP31758_3845
    3754 >Toxin: Spro_0292 9298 >Antitoxin: Spro_0291
    3755 >Toxin: YpAngola_A0464 9299 >Antitoxin: YpAngola_A0463
    3756 >Toxin: YPK_0356 9300 >Antitoxin: YPK_0355
    3757 >Toxin: Dd703_3708 9301 >Antitoxin: Dd703_3709
    3758 >Toxin: Dd1591_3862 9302 >Antitoxin: Dd1591_3863
    3759 >Toxin: PC1_0224 9303 >Antitoxin: PC1_0223
    3760 >Toxin: CPS_3479 9304 >Antitoxin: CPS_3478
    3761 >Toxin: Sden_1676 9305 >Antitoxin: Sden_1677
    3762 >Toxin: BCI_0426 9306 >Antitoxin: BCI_0425
    3763 >Toxin: Sbal_2404 9307 >Antitoxin: Sbal_2403
    3764 >Toxin: Ent638_1631 9308 >Antitoxin: Ent638_1632
    3765 >Toxin: Sputcn32_2156 9309 >Antitoxin: Sputcn32_2155
    3766 >Toxin: Shew185_2393 9310 >Antitoxin: Shew185_2392
    3767 >Toxin: Spea_1545 9311 >Antitoxin: Spea_1546
    3768 >Toxin: Sbal195_2509 9312 >Antitoxin: Sbal195_2508
    3769 >Toxin: Sbal223_1954 9313 >Antitoxin: Sbal223_1955
    3770 >Toxin: Dd1591_1648 9314 >Antitoxin: Dd1591_1649
    3771 >Toxin: PC1_2477 9315 >Antitoxin: PC1_2476
    3772 >Toxin: B21_02512 9316 >Antitoxin: B21_02513
    3773 >Toxin: SO_3428 9317 >Antitoxin: SO_3429
    3774 >Toxin: GSU0148 9318 >Antitoxin: GSU0147
    3775 >Toxin: MCA0389 9319 >Antitoxin: MCA0388
    3776 >Toxin: Noc_0926 9320 >Antitoxin: Noc_0925
    3777 >Toxin: Pcar_2408 9321 >Antitoxin: Pcar_2407
    3778 >Toxin: Gmet_0201 9322 >Antitoxin: Gmet_0200
    3779 >Toxin: Tcr_1590 9323 >Antitoxin: Tcr_1591
    3780 >Toxin: Mfla_0568 9324 >Antitoxin: Mfla_0569
    3781 >Toxin: Csal_0625 9325 >Antitoxin: Csal_0624
    3782 >Toxin: Patl_3015 9326 >Antitoxin: Patl_3016
    3783 >Toxin: Shewmr7_1198 9327 >Antitoxin: Shewmr7_1197
    3784 >Toxin: Shewmr4_1127 9328 >Antitoxin: Shewmr4_1126
    3785 >Toxin: Mlg_1481 9329 >Antitoxin: Mlg_1482
    3786 >Toxin: Mmc1_0013 9330 >Antitoxin: Mmc1_0012
    3787 >Toxin: Shewana3_1128 9331 >Antitoxin: Shewana3_1127
    3788 >Toxin: Ppro_3176 9332 >Antitoxin: Ppro_3177
    3789 >Toxin: Sama_1048 9333 >Antitoxin: Sama_1047
    3790 >Toxin: Hhal_1653 9334 >Antitoxin: Hhal_1654
    3791 >Toxin: Sbal_3115 9335 >Antitoxin: Sbal_3116
    3792 >Toxin: Shew_1217 9336 >Antitoxin: Shew_1216
    3793 >Toxin: Ent638_3172 9337 >Antitoxin: Ent638_3173
    3794 >Toxin: Sputcn32_2745 9338 >Antitoxin: Sputcn32_2746
    3795 >Toxin: VC0395_A0072 9339 >Antitoxin: VC0395_A0071
    3796 >Toxin: Gura_0222 9340 >Antitoxin: Gura_0221
    3797 >Toxin: Shew185_3124 9341 >Antitoxin: Shew185_3125
    3798 >Toxin: YpsIP31758_3238 9342 >Antitoxin: YpsIP31758_3239
    3799 >Toxin: VIBHAR_03511 9343 >Antitoxin: VIBHAR_03512
    3800 >Toxin: EcHS_A2833 9344 >Antitoxin: EcHS_A2834
    3801 >Toxin: Spro_0843 9345 >Antitoxin: Spro_0842
    3802 >Toxin: Sbal195_3267 9346 >Antitoxin: Sbal195_3268
    3803 >Toxin: COXBURSA331_A0883 9347 >Antitoxin: COXBURSA331_A0882
    3804 >Toxin: EcolC_1015 9348 >Antitoxin: EcolC_1014
    3805 >Toxin: YPK_3373 9349 >Antitoxin: YPK_3374
    3806 >Toxin: EcSMS35_2820 9350 >Antitoxin: EcSMS35_2821
    3807 >Toxin: PXO_00147 9351 >Antitoxin: PXO_00148
    3808 >Toxin: SbBS512_E3180 9352 >Antitoxin: SbBS512_E3179
    3809 >Toxin: Smal_1478 9353 >Antitoxin: Smal_1477
    3810 >Toxin: SNSL254_A3029 9354 >Antitoxin: SNSL254_A3030
    3811 >Toxin: SeHA_C3013 9355 >Antitoxin: SeHA_C3014
    3812 >Toxin: SeSA_A2978 9356 >Antitoxin: SeSA_A2979
    3813 >Toxin: MADE_02846 9357 >Antitoxin: MADE_02847
    3814 >Toxin: Gbem_3633 9358 >Antitoxin: Gbem_3632
    3815 >Toxin: SeAg_B2946 9359 >Antitoxin: SeAg_B2947
    3816 >Toxin: Lferr_1052 9360 >Antitoxin: Lferr_1051
    3817 >Toxin: SeD_A3136 9361 >Antitoxin: SeD_A3137
    3818 >Toxin: ECH74115_3944 9362 >Antitoxin: ECH74115_3945
    3819 >Toxin: Sbal223_1250 9363 >Antitoxin: Sbal223_1249
    3820 >Toxin: AFE_0934 9364 >Antitoxin: AFE_0933
    3821 >Toxin: Tgr7_1289 9365 >Antitoxin: Tgr7_1288
    3822 >Toxin: Dd703_0998 9366 >Antitoxin: Dd703_0997
    3823 >Toxin: Dd1591_0983 9367 >Antitoxin: Dd1591_0982
    3824 >Toxin: PC1_3211 9368 >Antitoxin: PC1_3212
    3825 >Toxin: GM21_3738 9369 >Antitoxin: GM21_3737
    3826 >Toxin: Bpro_1133 9370 >Antitoxin: Bpro_1134
    3827 >Toxin: Aave_0898 9371 >Antitoxin: Aave_0899
    3828 >Toxin: Ajs_3581 9372 >Antitoxin: Ajs_3580
    3829 >Toxin: Pnap_3357 9373 >Antitoxin: Pnap_3356
    3830 >Toxin: Veis_3024 9374 >Antitoxin: Veis_3023
    3831 >Toxin: Mpe_A3193 9375 >Antitoxin: Mpe_A3192
    3832 >Toxin: Daci_1569 9376 >Antitoxin: Daci_1570
    3833 >Toxin: Lcho_3974 9377 >Antitoxin: Lcho_3973
    3834 >Toxin: Dtpsy_2904 9378 >Antitoxin: Dtpsy_2903
    3835 >Toxin: Vapar_4629 9379 >Antitoxin: Vapar_4628
    3836 >Toxin: RSc0670 9380 >Antitoxin: RSc0669
    3837 >Toxin: PP_4990 9381 >Antitoxin: PP_4991
    3838 >Toxin: PSPTO_5032 9382 >Antitoxin: PSPTO_5033
    3839 >Toxin: Psyr_0490 9383 >Antitoxin: Psyr_0489
    3840 >Toxin: Psyc_1816 9384 >Antitoxin: Psyc_1817
    3841 >Toxin: Daro_3897 9385 >Antitoxin: Daro_3898
    3842 >Toxin: Reut_A2621 9386 >Antitoxin: Reut_A2622
    3843 >Toxin: Tbd_2550 9387 >Antitoxin: Tbd_2551
    3844 >Toxin: Noc_0129 9388 >Antitoxin: Noc_0130
    3845 >Toxin: Rfer_1373 9389 >Antitoxin: Rfer_1372
    3846 >Toxin: Sde_3632 9390 >Antitoxin: Sde_3633
    3847 >Toxin: Mfla_1244 9391 >Antitoxin: Mfla_1243
    3848 >Toxin: Bpro_1138 9392 >Antitoxin: Bpro_1137
    3849 >Toxin: Pcryo_2099 9393 >Antitoxin: Pcryo_2100
    3850 >Toxin: Rmet_0671 9394 >Antitoxin: Rmet_0670
    3851 >Toxin: Mlg_0357 9395 >Antitoxin: Mlg_0356
    3852 >Toxin: PA14_05340 9396 >Antitoxin: PA14_05330
    3853 >Toxin: Aave_0903 9397 >Antitoxin: Aave_0902
    3854 >Toxin: Ajs_3576 9398 >Antitoxin: Ajs_3577
    3855 >Toxin: Pnap_3352 9399 >Antitoxin: Pnap_3353
    3856 >Toxin: Veis_3019 9400 >Antitoxin: Veis_3020
    3857 >Toxin: Mpe_A3188 9401 >Antitoxin: Mpe_A3189
    3858 >Toxin: Maqu_3769 9402 >Antitoxin: Maqu_3768
    3859 >Toxin: Pmen_0405 9403 >Antitoxin: Pmen_0404
    3860 >Toxin: Pput_4864 9404 >Antitoxin: Pput_4865
    3861 >Toxin: PsycPRwf_2157 9405 >Antitoxin: PsycPRwf_2158
    3862 >Toxin: PSPA7_0510 9406 >Antitoxin: PSPA7_0509
    3863 >Toxin: Daci_1574 9407 >Antitoxin: Daci_1573
    3864 >Toxin: PputGB1_5040 9408 >Antitoxin: PputGB1_5041
    3865 >Toxin: PputW619_0474 9409 >Antitoxin: PputW619_0473
    3866 >Toxin: Lcho_3969 9410 >Antitoxin: Lcho_3970
    3867 >Toxin: PXO_01604 9411 >Antitoxin: PXO_01603
    3868 >Toxin: Rpic_0614 9412 >Antitoxin: Rpic_0613
    3869 >Toxin: Smal_3087 9413 >Antitoxin: Smal_3088
    3870 >Toxin: Tmz1t_3690 9414 >Antitoxin: Tmz1t_3689
    3871 >Toxin: Tgr7_2902 9415 >Antitoxin: Tgr7_2903
    3872 >Toxin: Dtpsy_2899 9416 >Antitoxin: Dtpsy_2900
    3873 >Toxin: Vapar_4624 9417 >Antitoxin: Vapar_4625
    3874 >Toxin: Rpic12D_0562 9418 >Antitoxin: Rpic12D_0561
    3875 >Toxin: Jann_2718 9419 >Antitoxin: Jann_2719
    3876 >Toxin: TM1040_1520 9420 >Antitoxin: TM1040_1521
    3877 >Toxin: Rru_A2437 9421 >Antitoxin: Rru_A2438
    3878 >Toxin: RSP_0193 9422 >Antitoxin: RSP_0194
    3879 >Toxin: Pden_1269 9423 >Antitoxin: Pden_1268
    3880 >Toxin: Rsph17029_1826 9424 >Antitoxin: Rsph17029_1827
    3881 >Toxin: Rsph17025_1452 9425 >Antitoxin: Rsph17025_1451
    3882 >Toxin: Acry_0172 9426 >Antitoxin: Acry_0171
    3883 >Toxin: Swit_2553 9427 >Antitoxin: Swit_2554
    3884 >Toxin: Plav_3295 9428 >Antitoxin: Plav_3296
    3885 >Toxin: Dshi_1392 9429 >Antitoxin: Dshi_1391
    3886 >Toxin: RSc2588 9430 >Antitoxin: RSc2587
    3887 >Toxin: BTH_I0808 9431 >Antitoxin: BTH_I0809
    3888 >Toxin: Rmet_1336 9432 >Antitoxin: Rmet_1337
    3889 >Toxin: Rmet_1546 9433 >Antitoxin: Rmet_1547
    3890 >Toxin: Bamb_2000 9434 >Antitoxin: Bamb_2001
    3891 >Toxin: Shewana3_1278 9435 >Antitoxin: Shewana3_1279
    3892 >Toxin: Aave_0722 9436 >Antitoxin: Aave_0723
    3893 >Toxin: Pnap_4191 9437 >Antitoxin: Pnap_4192
    3894 >Toxin: PSPA7_3710 9438 >Antitoxin: PSPA7_3709
    3895 >Toxin: PSPA7_3723 9439 >Antitoxin: PSPA7_3722
    3896 >Toxin: Daci_0442 9440 >Antitoxin: Daci_0441
    3897 >Toxin: Bmul_2346 9441 >Antitoxin: Bmul_2347
    3898 >Toxin: Rpic_2641 9442 >Antitoxin: Rpic_2642
    3899 >Toxin: Tmz1t_2064 9443 >Antitoxin: Tmz1t_2063
    3900 >Toxin: Tgr7_1891 9444 >Antitoxin: Tgr7_1892
    3901 >Toxin: Dtpsy_1314 9445 >Antitoxin: Dtpsy_1315
    3902 >Toxin: Dtpsy_3510 9446 >Antitoxin: Dtpsy_3509
    3903 >Toxin: Avin_35760 9447 >Antitoxin: Avin_35750
    3904 >Toxin: Avin_35850 9448 >Antitoxin: Avin_35840
    3905 >Toxin: CPR_1601 9449 >Antitoxin: CPR_1602
    3906 >Toxin: Ent638_4115 9450 >Antitoxin: Ent638_4116
    3907 >Toxin: VC0395_0010 9451 >Antitoxin: VC0395_0011
    3908 >Toxin: EcHS_A3965 9452 >Antitoxin: EcHS_A3964
    3909 >Toxin: EcE24377A_4265 9453 >Antitoxin: EcE24377A_4264
    3910 >Toxin: Spro_4900 9454 >Antitoxin: Spro_4901
    3911 >Toxin: EcolC_4245 9455 >Antitoxin: EcolC_4246
    3912 >Toxin: EcSMS35_4117 9456 >Antitoxin: EcSMS35_4116
    3913 >Toxin: Nther_2687 9457 >Antitoxin: Nther_2688
    3914 >Toxin: SNSL254_A4165 9458 >Antitoxin: SNSL254_A4164
    3915 >Toxin: SeSA_A4094 9459 >Antitoxin: SeSA_A4093
    3916 >Toxin: SeAg_B4109 9460 >Antitoxin: SeAg_B4108
    3917 >Toxin: SeD_A4274 9461 >Antitoxin: SeD_A4273
    3918 >Toxin: ECH74115_5185 9462 >Antitoxin: ECH74115_5184
    3919 >Toxin: Aaci_2279 9463 >Antitoxin: Aaci_2280
    3920 >Toxin: Adeh_0013 9464 >Antitoxin: Adeh_0014
    3921 >Toxin: Anae109_0014 9465 >Antitoxin: Anae109_0015
    3922 >Toxin: Dole_1190 9466 >Antitoxin: Dole_1189
    3923 >Toxin: AnaeK_0013 9467 >Antitoxin: AnaeK_0014
    3924 >Toxin: Dtur_0950 9468 >Antitoxin: Dtur_0951
    3925 >Toxin: A2cp1_0013 9469 >Antitoxin: A2cp1_0014
    3926 >Toxin: Dbac_0487 9470 >Antitoxin: Dbac_0486
    3927 >Toxin: Dret_1117 9471 >Antitoxin: Dret_1116
    3928 >Toxin: Reut_B4429 9472 >Antitoxin: Reut_B4430
    3929 >Toxin: Noc_1239 9473 >Antitoxin: Noc_1240
    3930 >Toxin: Pcar_2552 9474 >Antitoxin: Pcar_2553
    3931 >Toxin: Adeh_0797 9475 >Antitoxin: Adeh_0798
    3932 >Toxin: Nham_4186 9476 >Antitoxin: Nham_4185
    3933 >Toxin: Acid345_3002 9477 >Antitoxin: Acid345_3001
    3934 >Toxin: CHU_2211 9478 >Antitoxin: CHU_2210
    3935 >Toxin: Fjoh_1636 9479 >Antitoxin: Fjoh_1637
    3936 >Toxin: RoseRS_4141 9480 >Antitoxin: RoseRS_4142
    3937 >Toxin: Anae109_0842 9481 >Antitoxin: Anae109_0843
    3938 >Toxin: Rcas_1464 9482 >Antitoxin: Rcas_1465
    3939 >Toxin: Mext_0217 9483 >Antitoxin: Mext_0218
    3940 >Toxin: Mrad2831_6224 9484 >Antitoxin: Mrad2831_6225
    3941 >Toxin: Oter_3933 9485 >Antitoxin: Oter_3932
    3942 >Toxin: Mpop_0019 9486 >Antitoxin: Mpop_0020
    3943 >Toxin: Amuc_1872 9487 >Antitoxin: Amuc_1873
    3944 >Toxin: AnaeK_0845 9488 >Antitoxin: AnaeK_0846
    3945 >Toxin: M446_5823 9489 >Antitoxin: M446_5822
    3946 >Toxin: Gdia_0834 9490 >Antitoxin: Gdia_0833
    3947 >Toxin: Mchl_0161 9491 >Antitoxin: Mchl_0162
    3948 >Toxin: A2cp1_0849 9492 >Antitoxin: A2cp1_0850
    3949 >Toxin: Mnod_6892 9493 >Antitoxin: Mnod_6891
    3950 >Toxin: Rleg_5954 9494 >Antitoxin: Rleg_5953
    3951 >Toxin: Phep_0290 9495 >Antitoxin: Phep_0291
    3952 >Toxin: Dfer_3475 9496 >Antitoxin: Dfer_3474
    3953 >Toxin: Cagg_3385 9497 >Antitoxin: Cagg_3384
    3954 >Toxin: Cpin_0527 9498 >Antitoxin: Cpin_0526
    3955 >Toxin: Arth_2706 9499 >Antitoxin: Arth_2705
    3956 >Toxin: Noca_1440 9500 >Antitoxin: Noca_1441
    3957 >Toxin: Strop_0973 9501 >Antitoxin: Strop_0974
    3958 >Toxin: Franean1_5838 9502 >Antitoxin: Franean1_5837
    3959 >Toxin: Sare_0910 9503 >Antitoxin: Sare_0911
    3960 >Toxin: Achl_2437 9504 >Antitoxin: Achl_2436
    3961 >Toxin: Bfae_20600 9505 >Antitoxin: Bfae_20590
    3962 >Toxin: Mlut_14700 9506 >Antitoxin: Mlut_14690
    3963 >Toxin: Ksed_08660 9507 >Antitoxin: Ksed_08670
    3964 >Toxin: Caci_7585 9508 >Antitoxin: Caci_7584
    3965 >Toxin: Svir_30890 9509 >Antitoxin: Svir_30880
    3966 >Toxin: Jden_1822 9510 >Antitoxin: Jden_1821
    3967 >Toxin: Rfer_2071 9511 >Antitoxin: Rfer_2070
    3968 >Toxin: Bpro_2703 9512 >Antitoxin: Bpro_2704
    3969 >Toxin: Aave_1588 9513 >Antitoxin: Aave_1587
    3970 >Toxin: Ajs_3149 9514 >Antitoxin: Ajs_3150
    3971 >Toxin: Pnap_2537 9515 >Antitoxin: Pnap_2538
    3972 >Toxin: Veis_4366 9516 >Antitoxin: Veis_4365
    3973 >Toxin: Mpe_A1578 9517 >Antitoxin: Mpe_A1577
    3974 >Toxin: Pnuc_0828 9518 >Antitoxin: Pnuc_0827
    3975 >Toxin: Daci_2856 9519 >Antitoxin: Daci_2855
    3976 >Toxin: Lcho_1899 9520 >Antitoxin: Lcho_1898
    3977 >Toxin: Pnec_1011 9521 >Antitoxin: Pnec_1012
    3978 >Toxin: Dtpsy_2496 9522 >Antitoxin: Dtpsy_2497
    3979 >Toxin: Vapar_2587 9523 >Antitoxin: Vapar_2586
    3980 >Toxin: Daro_0008 9524 >Antitoxin: Daro_0007
    3981 >Toxin: Nmul_A2694 9525 >Antitoxin: Nmul_A2693
    3982 >Toxin: Sama_2952 9526 >Antitoxin: Sama_2951
    3983 >Toxin: Hhal_0366 9527 >Antitoxin: Hhal_0365
    3984 >Toxin: Swoo_3314 9528 >Antitoxin: Swoo_3313
    3985 >Toxin: DvMF_1096 9529 >Antitoxin: DvMF_1097
    3986 >Toxin: Tgr7_0082 9530 >Antitoxin: Tgr7_0081
    3987 >Toxin: Avin_50630 9531 >Antitoxin: Avin_50640
    3988 >Toxin: BR0817 9532 >Antitoxin: BR0818
    3989 >Toxin: Nwi_1872 9533 >Antitoxin: Nwi_1871
    3990 >Toxin: RPB_2589 9534 >Antitoxin: RPB_2590
    3991 >Toxin: RPC_2419 9535 >Antitoxin: RPC_2420
    3992 >Toxin: RPD_2870 9536 >Antitoxin: RPD_2869
    3993 >Toxin: Nham_2205 9537 >Antitoxin: Nham_2204
    3994 >Toxin: Rru_A1571 9538 >Antitoxin: Rru_A1572
    3995 >Toxin: Meso_1037 9539 >Antitoxin: Meso_1038
    3996 >Toxin: Mmar10_1371 9540 >Antitoxin: Mmar10_1372
    3997 >Toxin: BBta_4546 9541 >Antitoxin: BBta_4545
    3998 >Toxin: BOV_0812 9542 >Antitoxin: BOV_0813
    3999 >Toxin: Smed_0905 9543 >Antitoxin: Smed_0906
    4000 >Toxin: Oant_2408 9544 >Antitoxin: Oant_2407
    4001 >Toxin: Plav_3211 9545 >Antitoxin: Plav_3210
    4002 >Toxin: Xaut_4618 9546 >Antitoxin: Xaut_4617
    4003 >Toxin: Mext_1070 9547 >Antitoxin: Mext_1069
    4004 >Toxin: Mrad2831_2044 9548 >Antitoxin: Mrad2831_2043
    4005 >Toxin: Mpop_1004 9549 >Antitoxin: Mpop_1003
    4006 >Toxin: Rpal_3283 9550 >Antitoxin: Rpal_3282
    4007 >Toxin: M446_4405 9551 >Antitoxin: M446_4406
    4008 >Toxin: Rleg2_1276 9552 >Antitoxin: Rleg2_1277
    4009 >Toxin: Msil_2933 9553 >Antitoxin: Msil_2934
    4010 >Toxin: Mchl_1199 9554 >Antitoxin: Mchl_1198
    4011 >Toxin: Mnod_4141 9555 >Antitoxin: Mnod_4142
    4012 >Toxin: Avi_1731 9556 >Antitoxin: Avi_1733
    4013 >Toxin: Rleg_1368 9557 >Antitoxin: Rleg_1369
    4014 >Toxin: PMN2A_1141 9558 >Antitoxin: PMN2A_1140
    4015 >Toxin: Ava_0994 9559 >Antitoxin: Ava_0993
    4016 >Toxin: Noc_0362 9560 >Antitoxin: Noc_0361
    4017 >Toxin: Syncc9902_1940 9561 >Antitoxin: Syncc9902_1941
    4018 >Toxin: Syncc9605_0390 9562 >Antitoxin: Syncc9605_0389
    4019 >Toxin: Gmet_1568 9563 >Antitoxin: Gmet_1569
    4020 >Toxin: Synpcc7942_0158 9564 >Antitoxin: Synpcc7942_0159
    4021 >Toxin: Nmul_A0972 9565 >Antitoxin: Nmul_A0971
    4022 >Toxin: Tery_1108 9566 >Antitoxin: Tery_1109
    4023 >Toxin: Mlg_1160 9567 >Antitoxin: Mlg_1159
    4024 >Toxin: P9303_22891 9568 >Antitoxin: P9303_22901
    4025 >Toxin: NATL1_20151 9569 >Antitoxin: NATL1_20141
    4026 >Toxin: Cthe_2539 9570 >Antitoxin: Cthe_2540
    4027 >Toxin: Gura_3943 9571 >Antitoxin: Gura_3942
    4028 >Toxin: P9211_16891 9572 >Antitoxin: P9211_16881
    4029 >Toxin: Nther_1092 9573 >Antitoxin: Nther_1093
    4030 >Toxin: Cphamn1_0645 9574 >Antitoxin: Cphamn1_0646
    4031 >Toxin: Gbem_1980 9575 >Antitoxin: Gbem_1979
    4032 >Toxin: Dhaf_4109 9576 >Antitoxin: Dhaf_4108
    4033 >Toxin: Athe_1186 9577 >Antitoxin: Athe_1187
    4034 >Toxin: GM21_2240 9578 >Antitoxin: GM21_2241
    4035 >Toxin: Mpal_1141 9579 >Antitoxin: Mpal_1140
    4036 >Toxin: BCE_3851 9580 >Antitoxin: BCE_3850
    4037 >Toxin: LMOf2365_1342 9581 >Antitoxin: LMOf2365_1343
    4038 >Toxin: GBAA_3950 9582 >Antitoxin: GBAA_3949
    4039 >Toxin: BAS3664 9583 >Antitoxin: BAS3663
    4040 >Toxin: BT9727_3554 9584 >Antitoxin: BT9727_3553
    4041 >Toxin: BCZK3572 9585 >Antitoxin: BCZK3571
    4042 >Toxin: Tfu_0778 9586 >Antitoxin: Tfu_0779
    4043 >Toxin: Pcar_1555 9587 >Antitoxin: Pcar_1556
    4044 >Toxin: Adeh_1102 9588 >Antitoxin: Adeh_1103
    4045 >Toxin: Francci3_3562 9589 >Antitoxin: Francci3_3561
    4046 >Toxin: Acid345_4217 9590 >Antitoxin: Acid345_4216
    4047 >Toxin: Rxyl_1411 9591 >Antitoxin: Rxyl_1412
    4048 >Toxin: Sfum_1228 9592 >Antitoxin: Sfum_1229
    4049 >Toxin: Acel_1515 9593 >Antitoxin: Acel_1514
    4050 >Toxin: Dvul_2433 9594 >Antitoxin: Dvul_2434
    4051 >Toxin: Bcer98_2465 9595 >Antitoxin: Bcer98_2464
    4052 >Toxin: Franean1_1182 9596 >Antitoxin: Franean1_1183
    4053 >Toxin: Dole_3035 9597 >Antitoxin: Dole_3034
    4054 >Toxin: Sare_1327 9598 >Antitoxin: Sare_1328
    4055 >Toxin: BcerKBAB4_3635 9599 >Antitoxin: BcerKBAB4_3634
    4056 >Toxin: BCAH187_A3860 9600 >Antitoxin: BCAH187_A3859
    4057 >Toxin: Dtur_1208 9601 >Antitoxin: Dtur_1209
    4058 >Toxin: BCB4264_A3911 9602 >Antitoxin: BCB4264_A3910
    4059 >Toxin: BCG9842_B1333 9603 >Antitoxin: BCG9842_B1334
    4060 >Toxin: DvMF_2513 9604 >Antitoxin: DvMF_2512
    4061 >Toxin: A2cp1_1230 9605 >Antitoxin: A2cp1_1231
    4062 >Toxin: Amir_5834 9606 >Antitoxin: Amir_5833
    4063 >Toxin: Dbac_2414 9607 >Antitoxin: Dbac_2415
    4064 >Toxin: GWCH70_1155 9608 >Antitoxin: GWCH70_1156
    4065 >Toxin: Caci_7742 9609 >Antitoxin: Caci_7741
    4066 >Toxin: Svir_14570 9610 >Antitoxin: Svir_14580
    4067 >Toxin: Dret_0495 9611 >Antitoxin: Dret_0494
    4068 >Toxin: Namu_2207 9612 >Antitoxin: Namu_2208
    4069 >Toxin: Nwi_0373 9613 >Antitoxin: Nwi_0372
    4070 >Toxin: RPB_0069 9614 >Antitoxin: RPB_0068
    4071 >Toxin: RPC_0508 9615 >Antitoxin: RPC_0507
    4072 >Toxin: RPD_0093 9616 >Antitoxin: RPD_0092
    4073 >Toxin: Nham_0466 9617 >Antitoxin: Nham_0465
    4074 >Toxin: Mmar10_2381 9618 >Antitoxin: Mmar10_2382
    4075 >Toxin: BBta_7609 9619 >Antitoxin: BBta_7610
    4076 >Toxin: Smed_2552 9620 >Antitoxin: Smed_2553
    4077 >Toxin: Plav_1578 9621 >Antitoxin: Plav_1577
    4078 >Toxin: Xaut_2019 9622 >Antitoxin: Xaut_2020
    4079 >Toxin: Caul_0378 9623 >Antitoxin: Caul_0377
    4080 >Toxin: Bind_0430 9624 >Antitoxin: Bind_0429
    4081 >Toxin: Rpal_0614 9625 >Antitoxin: Rpal_0615
    4082 >Toxin: M446_2063 9626 >Antitoxin: M446_2062
    4083 >Toxin: Rleg2_3582 9627 >Antitoxin: Rleg2_3583
    4084 >Toxin: Msil_1898 9628 >Antitoxin: Msil_1897
    4085 >Toxin: Mnod_0448 9629 >Antitoxin: Mnod_0449
    4086 >Toxin: Avi_4036 9630 >Antitoxin: Avi_4037
    4087 >Toxin: Rleg_3875 9631 >Antitoxin: Rleg_3876
    4088 >Toxin: Arth_1982 9632 >Antitoxin: Arth_1981
    4089 >Toxin: Shew185_0950 9633 >Antitoxin: Shew185_0949
    4090 >Toxin: Bcer98_2848 9634 >Antitoxin: Bcer98_2847
    4091 >Toxin: CHAB381_0432 9635 >Antitoxin: CHAB381_0433
    4092 >Toxin: Sbal195_0985 9636 >Antitoxin: Sbal195_0984
    4093 >Toxin: Cphy_0848 9637 >Antitoxin: Cphy_0849
    4094 >Toxin: Sbal223_0971 9638 >Antitoxin: Sbal223_0970
    4095 >Toxin: Caci_2776 9639 >Antitoxin: Caci_2777
    4096 >Toxin: Rfer_0220 9640 >Antitoxin: Rfer_0219
    4097 >Toxin: Bxe_A1422 9641 >Antitoxin: Bxe_A1421
    4098 >Toxin: Bpro_1628 9642 >Antitoxin: Bpro_1627
    4099 >Toxin: Pnap_2944 9643 >Antitoxin: Pnap_2945
    4100 >Toxin: Veis_0732 9644 >Antitoxin: Veis_0731
    4101 >Toxin: BBta_6640 9645 >Antitoxin: BBta_6641
    4102 >Toxin: Daci_0074 9646 >Antitoxin: Daci_0075
    4103 >Toxin: Bphy_1540 9647 >Antitoxin: Bphy_1541
    4104 >Toxin: Bphyt_2696 9648 >Antitoxin: Bphyt_2697
    4105 >Toxin: Vapar_0093 9649 >Antitoxin: Vapar_0092
    4106 >Toxin: Jann_2733 9650 >Antitoxin: Jann_2732
    4107 >Toxin: TM1040_2312 9651 >Antitoxin: TM1040_2311
    4108 >Toxin: RSP_1856 9652 >Antitoxin: RSP_1857
    4109 >Toxin: Pden_0538 9653 >Antitoxin: Pden_0539
    4110 >Toxin: Rsph17029_0505 9654 >Antitoxin: Rsph17029_0506
    4111 >Toxin: Rsph17025_0642 9655 >Antitoxin: Rsph17025_0643
    4112 >Toxin: Dshi_2404 9656 >Antitoxin: Dshi_2403
    4113 >Toxin: Bpro_3279 9657 >Antitoxin: Bpro_3280
    4114 >Toxin: Aave_2105 9658 >Antitoxin: Aave_2104
    4115 >Toxin: Ajs_2841 9659 >Antitoxin: Ajs_2842
    4116 >Toxin: Pnap_1404 9660 >Antitoxin: Pnap_1403
    4117 >Toxin: Mpe_A2319 9661 >Antitoxin: Mpe_A2320
    4118 >Toxin: BMA10247_2917 9662 >Antitoxin: BMA10247_2916
    4119 >Toxin: BURPS668_0556 9663 >Antitoxin: BURPS668_0555
    4120 >Toxin: BURPS1106A_0571 9664 >Antitoxin: BURPS1106A_0570
    4121 >Toxin: Pnuc_1002 9665 >Antitoxin: Pnuc_1003
    4122 >Toxin: Dshi_1669 9666 >Antitoxin: Dshi_1670
    4123 >Toxin: Daci_2370 9667 >Antitoxin: Daci_2369
    4124 >Toxin: Dtpsy_2330 9668 >Antitoxin: Dtpsy_2331
    4125 >Toxin: Vapar_3530 9669 >Antitoxin: Vapar_3531
    4126 >Toxin: BR0659 9670 >Antitoxin: BR0658
    4127 >Toxin: Meso_0948 9671 >Antitoxin: Meso_0947
    4128 >Toxin: BARBAKC583_0471 9672 >Antitoxin: BARBAKC583_0470
    4129 >Toxin: BOV_0652 9673 >Antitoxin: BOV_0651
    4130 >Toxin: Smed_0680 9674 >Antitoxin: Smed_0679
    4131 >Toxin: Oant_2629 9675 >Antitoxin: Oant_2630
    4132 >Toxin: Rleg2_0996 9676 >Antitoxin: Rleg2_0995
    4133 >Toxin: Rleg_1149 9677 >Antitoxin: Rleg_1148
    4134 >Toxin: Reut_A3273 9678 >Antitoxin: Reut_A3274
    4135 >Toxin: Bcep18194_B3076 9679 >Antitoxin: Bcep18194_B3075
    4136 >Toxin: Rmet_3438 9680 >Antitoxin: Rmet_3439
    4137 >Toxin: PputW619_3105 9681 >Antitoxin: PputW619_3106
    4138 >Toxin: Rpic_3958 9682 >Antitoxin: Rpic_3957
    4139 >Toxin: Bphyt_4351 9683 >Antitoxin: Bphyt_4352
    4140 >Toxin: Rpic12D_4071 9684 >Antitoxin: Rpic12D_4070
    4141 >Toxin: Ent638_2196 9685 >Antitoxin: Ent638_2197
    4142 >Toxin: VC0395_A1332 9686 >Antitoxin: VC0395_A1333
    4143 >Toxin: VIBHAR_01574 9687 >Antitoxin: VIBHAR_01573
    4144 >Toxin: Spro_2657 9688 >Antitoxin: Spro_2658
    4145 >Toxin: YpAngola_A2304 9689 >Antitoxin: YpAngola_A2305
    4146 >Toxin: SNSL254_A1841 9690 >Antitoxin: SNSL254_A1842
    4147 >Toxin: SeHA_C1904 9691 >Antitoxin: SeHA_C1905
    4148 >Toxin: SeSA_A1846 9692 >Antitoxin: SeSA_A1847
    4149 >Toxin: SeAg_B1432 9693 >Antitoxin: SeAg_B1431
    4150 >Toxin: SeD_A1614 9694 >Antitoxin: SeD_A1613
    4151 >Toxin: Dd703_1924 9695 >Antitoxin: Dd703_1925
    4152 >Toxin: Dd1591_2162 9696 >Antitoxin: Dd1591_2163
    4153 >Toxin: PC1_2015 9697 >Antitoxin: PC1_2014
    4154 >Toxin: B21_03000 9698 >Antitoxin: B21_02999
    4155 >Toxin: HS_0247 9699 >Antitoxin: HS_0248
    4156 >Toxin: Ent638_3620 9700 >Antitoxin: Ent638_3619
    4157 >Toxin: YpsIP31758_3610 9701 >Antitoxin: YpsIP31758_3609
    4158 >Toxin: EcHS_A3377 9702 >Antitoxin: EcHS_A3376
    4159 >Toxin: EcE24377A_3669 9703 >Antitoxin: EcE24377A_3668
    4160 >Toxin: Spro_0475 9704 >Antitoxin: Spro_0476
    4161 >Toxin: EcolC_0516 9705 >Antitoxin: EcolC_0517
    4162 >Toxin: EcSMS35_3480 9706 >Antitoxin: EcSMS35_3479
    4163 >Toxin: SbBS512_E3586 9707 >Antitoxin: SbBS512_E3587
    4164 >Toxin: SNSL254_A3563 9708 >Antitoxin: SNSL254_A3562
    4165 >Toxin: SeHA_C3599 9709 >Antitoxin: SeHA_C3598
    4166 >Toxin: SeSA_A3494 9710 >Antitoxin: SeSA_A3493
    4167 >Toxin: SeAg_B3492 9711 >Antitoxin: SeAg_B3491
    4168 >Toxin: ECH74115_4506 9712 >Antitoxin: ECH74115_4505
    4169 >Toxin: Dd703_3358 9713 >Antitoxin: Dd703_3357
    4170 >Toxin: Dd1591_3477 9714 >Antitoxin: Dd1591_3476
    4171 >Toxin: PC1_0560 9715 >Antitoxin: PC1_0561
    4172 >Toxin: B21_03043 9716 >Antitoxin: B21_03044
    4173 >Toxin: CPS_4344 9717 >Antitoxin: CPS_4345
    4174 >Toxin: Sde_3165 9718 >Antitoxin: Sde_3166
    4175 >Toxin: Csal_2208 9719 >Antitoxin: Csal_2209
    4176 >Toxin: Maqu_2697 9720 >Antitoxin: Maqu_2698
    4177 >Toxin: Ent638_3667 9721 >Antitoxin: Ent638_3668
    4178 >Toxin: VC0395_A0102 9722 >Antitoxin: VC0395_A0101
    4179 >Toxin: Mmwyl1_2401 9723 >Antitoxin: Mmwyl1_2402
    4180 >Toxin: YpsIP31758_0458 9724 >Antitoxin: YpsIP31758_0457
    4181 >Toxin: VIBHAR_00880 9725 >Antitoxin: VIBHAR_00879
    4182 >Toxin: EcHS_A3421 9726 >Antitoxin: EcHS_A3422
    4183 >Toxin: EcE24377A_3715 9727 >Antitoxin: EcE24377A_3716
    4184 >Toxin: Spro_4350 9728 >Antitoxin: Spro_4351
    4185 >Toxin: YpAngola_A1135 9729 >Antitoxin: YpAngola_A1136
    4186 >Toxin: EcolC_0474 9730 >Antitoxin: EcolC_0473
    4187 >Toxin: EcSMS35_3528 9731 >Antitoxin: EcSMS35_3529
    4188 >Toxin: SbBS512_E3544 9732 >Antitoxin: SbBS512_E3543
    4189 >Toxin: SNSL254_A3609 9733 >Antitoxin: SNSL254_A3610
    4190 >Toxin: SeHA_C3644 9734 >Antitoxin: SeHA_C3645
    4191 >Toxin: SeSA_A3538 9735 >Antitoxin: SeSA_A3539
    4192 >Toxin: SeAg_B3537 9736 >Antitoxin: SeAg_B3538
    4193 >Toxin: SeD_A3706 9737 >Antitoxin: SeD_A3707
    4194 >Toxin: VSAL_I2665 9738 >Antitoxin: VSAL_I2666
    4195 >Toxin: ECH74115_4549 9739 >Antitoxin: ECH74115_4550
    4196 >Toxin: Dd703_3640 9740 >Antitoxin: Dd703_3641
    4197 >Toxin: Dd1591_3798 9741 >Antitoxin: Dd1591_3799
    4198 >Toxin: PC1_0291 9742 >Antitoxin: PC1_0290
    4199 >Toxin: YpsIP31758_3940 9743 >Antitoxin: YpsIP31758_3939
    4200 >Toxin: Spro_4576 9744 >Antitoxin: Spro_4575
    4201 >Toxin: YpAngola_A3697 9745 >Antitoxin: YpAngola_A3696
    4202 >Toxin: YPK_0256 9746 >Antitoxin: YPK_0257
    4203 >Toxin: Dd703_0373 9747 >Antitoxin: Dd703_0374
    4204 >Toxin: Dd1591_0297 9748 >Antitoxin: Dd1591_0298
    4205 >Toxin: PC1_3849 9749 >Antitoxin: PC1_3848
    4206 >Toxin: GBAA_2146 9750 >Antitoxin: GBAA_2145
    4207 >Toxin: BAS1997 9751 >Antitoxin: BAS1996
    4208 >Toxin: BT9727_1970 9752 >Antitoxin: BT9727_1969
    4209 >Toxin: BCZK1949 9753 >Antitoxin: BCZK1948
    4210 >Toxin: Daro_0816 9754 >Antitoxin: Daro_0817
    4211 >Toxin: Ajs_2839 9755 >Antitoxin: Ajs_2840
    4212 >Toxin: Veis_2593 9756 >Antitoxin: Veis_2594
    4213 >Toxin: GSU2198 9757 >Antitoxin: GSU2197
    4214 >Toxin: Gmet_2296 9758 >Antitoxin: Gmet_2295
    4215 >Toxin: Dde_2378 9759 >Antitoxin: Dde_2379
    4216 >Toxin: Acid345_1874 9760 >Antitoxin: Acid345_1875
    4217 >Toxin: Moth_1112 9761 >Antitoxin: Moth_1111
    4218 >Toxin: Sfum_0419 9762 >Antitoxin: Sfum_0418
    4219 >Toxin: Dvul_2004 9763 >Antitoxin: Dvul_2005
    4220 >Toxin: Gura_3127 9764 >Antitoxin: Gura_3126
    4221 >Toxin: Dole_2355 9765 >Antitoxin: Dole_2354
    4222 >Toxin: SYO3AOP1_0820 9766 >Antitoxin: SYO3AOP1_0821
    4223 >Toxin: Gbem_0761 9767 >Antitoxin: Gbem_0762
    4224 >Toxin: DvMF_1330 9768 >Antitoxin: DvMF_1331
    4225 >Toxin: Ddes_1273 9769 >Antitoxin: Ddes_1274
    4226 >Toxin: Dbac_0582 9770 >Antitoxin: Dbac_0583
    4227 >Toxin: GM21_0777 9771 >Antitoxin: GM21_0778
    4228 >Toxin: Dret_1591 9772 >Antitoxin: Dret_1590
    4229 >Toxin: BR1724 9773 >Antitoxin: BR1725
    4230 >Toxin: Nwi_2732 9774 >Antitoxin: Nwi_2733
    4231 >Toxin: RPB_4464 9775 >Antitoxin: RPB_4465
    4232 >Toxin: RPC_4764 9776 >Antitoxin: RPC_4765
    4233 >Toxin: RPD_4310 9777 >Antitoxin: RPD_4311
    4234 >Toxin: Nham_3529 9778 >Antitoxin: Nham_3530
    4235 >Toxin: Meso_3442 9779 >Antitoxin: Meso_3441
    4236 >Toxin: Mmar10_2444 9780 >Antitoxin: Mmar10_2445
    4237 >Toxin: BARBAKC583_0135 9781 >Antitoxin: BARBAKC583_0136
    4238 >Toxin: BBta_6921 9782 >Antitoxin: BBta_6922
    4239 >Toxin: BOV_1667 9783 >Antitoxin: BOV_1668
    4240 >Toxin: Smed_2648 9784 >Antitoxin: Smed_2649
    4241 >Toxin: Oant_1192 9785 >Antitoxin: Oant_1191
    4242 >Toxin: Plav_2110 9786 >Antitoxin: Plav_2109
    4243 >Toxin: Mext_2364 9787 >Antitoxin: Mext_2365
    4244 >Toxin: Mrad2831_0162 9788 >Antitoxin: Mrad2831_0161
    4245 >Toxin: Bind_3470 9789 >Antitoxin: Bind_3469
    4246 >Toxin: Mpop_2320 9790 >Antitoxin: Mpop_2321
    4247 >Toxin: Rpal_1017 9791 >Antitoxin: Rpal_1016
    4248 >Toxin: M446_5999 9792 >Antitoxin: M446_6000
    4249 >Toxin: Rleg2_3234 9793 >Antitoxin: Rleg2_3235
    4250 >Toxin: Msil_2280 9794 >Antitoxin: Msil_2281
    4251 >Toxin: Mchl_2641 9795 >Antitoxin: Mchl_2642
    4252 >Toxin: Mnod_7288 9796 >Antitoxin: Mnod_7287
    4253 >Toxin: Avi_3642 9797 >Antitoxin: Avi_3640
    4254 >Toxin: Rleg_3530 9798 >Antitoxin: Rleg_3531
    4255 >Toxin: Sala_1670 9799 >Antitoxin: Sala_1671
    4256 >Toxin: Rru_A3324 9800 >Antitoxin: Rru_A3325
    4257 >Toxin: RSP_0690 9801 >Antitoxin: RSP_0691
    4258 >Toxin: Rsph17029_2345 9802 >Antitoxin: Rsph17029_2346
    4259 >Toxin: Smed_5929 9803 >Antitoxin: Smed_5930
    4260 >Toxin: Oant_0463 9804 >Antitoxin: Oant_0464
    4261 >Toxin: Xaut_0465 9805 >Antitoxin: Xaut_0464
    4262 >Toxin: Dshi_0667 9806 >Antitoxin: Dshi_0666
    4263 >Toxin: Rpal_0014 9807 >Antitoxin: Rpal_0015
    4264 >Toxin: M446_6713 9808 >Antitoxin: M446_6712
    4265 >Toxin: Msil_3665 9809 >Antitoxin: Msil_3664
    4266 >Toxin: Avi_2393 9810 >Antitoxin: Avi_2391
    4267 >Toxin: Rleg_4956 9811 >Antitoxin: Rleg_4957
    4268 >Toxin: Mbar_A0098 9812 >Antitoxin: Mbar_A0099
    4269 >Toxin: Mhun_2241 9813 >Antitoxin: Mhun_2242
    4270 >Toxin: Mbur_0013 9814 >Antitoxin: Mbur_0012
    4271 >Toxin: Mthe_1716 9815 >Antitoxin: Mthe_1717
    4272 >Toxin: Tpen_0243 9816 >Antitoxin: Tpen_0244
    4273 >Toxin: Pisl_0560 9817 >Antitoxin: Pisl_0559
    4274 >Toxin: Mlab_0093 9818 >Antitoxin: Mlab_0092
    4275 >Toxin: Memar_0576 9819 >Antitoxin: Memar_0575
    4276 >Toxin: Pcal_1747 9820 >Antitoxin: Pcal_1748
    4277 >Toxin: MmarC5_0167 9821 >Antitoxin: MmarC5_0168
    4278 >Toxin: Pars_1730 9822 >Antitoxin: Pars_1729
    4279 >Toxin: Msed_0103 9823 >Antitoxin: Msed_0102
    4280 >Toxin: Mevan_0722 9824 >Antitoxin: Mevan_0721
    4281 >Toxin: Maeo_1396 9825 >Antitoxin: Maeo_1395
    4282 >Toxin: MmarC7_0656 9826 >Antitoxin: MmarC7_0655
    4283 >Toxin: Mboo_0544 9827 >Antitoxin: Mboo_0543
    4284 >Toxin: Cmaq_1832 9828 >Antitoxin: Cmaq_1833
    4285 >Toxin: MmarC6_1262 9829 >Antitoxin: MmarC6_1263
    4286 >Toxin: Nmar_0798 9830 >Antitoxin: Nmar_0799
    4287 >Toxin: Tneu_1550 9831 >Antitoxin: Tneu_1549
    4288 >Toxin: Hlac_2437 9832 >Antitoxin: Hlac_2438
    4289 >Toxin: Huta_2304 9833 >Antitoxin: Huta_2303
    4290 >Toxin: Mpal_0454 9834 >Antitoxin: Mpal_0453
    4291 >Toxin: Hmuk_1840 9835 >Antitoxin: Hmuk_1839
    4292 >Toxin: Moth_1750 9836 >Antitoxin: Moth_1749
    4293 >Toxin: Daud_1377 9837 >Antitoxin: Daud_1376
    4294 >Toxin: Nther_1816 9838 >Antitoxin: Nther_1815
    4295 >Toxin: Dhaf_0220 9839 >Antitoxin: Dhaf_0221
    4296 >Toxin: Hore_05640 9840 >Antitoxin: Hore_05650
    4297 >Toxin: Dtox_2395 9841 >Antitoxin: Dtox_2394
    4298 >Toxin: Aaci_2129 9842 >Antitoxin: Aaci_2128
    4299 >Toxin: Swol_0491 9843 >Antitoxin: Swol_0492
    4300 >Toxin: Sfum_0261 9844 >Antitoxin: Sfum_0262
    4301 >Toxin: Franean1_1571 9845 >Antitoxin: Franean1_1572
    4302 >Toxin: Glov_0185 9846 >Antitoxin: Glov_0186
    4303 >Toxin: Mlut_00580 9847 >Antitoxin: Mlut_00590
    4304 >Toxin: Caci_6429 9848 >Antitoxin: Caci_6430
    4305 >Toxin: Namu_3589 9849 >Antitoxin: Namu_3588
    4306 >Toxin: Swol_0979 9850 >Antitoxin: Swol_0978
    4307 >Toxin: Dred_1882 9851 >Antitoxin: Dred_1883
    4308 >Toxin: Tpet_0396 9852 >Antitoxin: Tpet_0397
    4309 >Toxin: Pmob_1380 9853 >Antitoxin: Pmob_1379
    4310 >Toxin: Daud_0556 9854 >Antitoxin: Daud_0555
    4311 >Toxin: TRQ2_0412 9855 >Antitoxin: TRQ2_0413
    4312 >Toxin: Dhaf_2730 9856 >Antitoxin: Dhaf_2731
    4313 >Toxin: Dtox_2130 9857 >Antitoxin: Dtox_2129
    4314 >Toxin: Aaci_1489 9858 >Antitoxin: Aaci_1488
    4315 >Toxin: Rfer_2867 9859 >Antitoxin: Rfer_2868
    4316 >Toxin: Meso_2435 9860 >Antitoxin: Meso_2436
    4317 >Toxin: Mlg_2738 9861 >Antitoxin: Mlg_2739
    4318 >Toxin: Ajs_3127 9862 >Antitoxin: Ajs_3128
    4319 >Toxin: Mpe_A2221 9863 >Antitoxin: Mpe_A2222
    4320 >Toxin: Smed_5505 9864 >Antitoxin: Smed_5504
    4321 >Toxin: Dshi_0628 9865 >Antitoxin: Dshi_0627
    4322 >Toxin: Mext_2852 9866 >Antitoxin: Mext_2851
    4323 >Toxin: Mrad2831_6165 9867 >Antitoxin: Mrad2831_6166
    4324 >Toxin: Mpop_2970 9868 >Antitoxin: Mpop_2969
    4325 >Toxin: M446_3944 9869 >Antitoxin: M446_3943
    4326 >Toxin: Mchl_3078 9870 >Antitoxin: Mchl_3077
    4327 >Toxin: Mnod_3623 9871 >Antitoxin: Mnod_3624
    4328 >Toxin: Dtpsy_2474 9872 >Antitoxin: Dtpsy_2475
    4329 >Toxin: Vapar_1590 9873 >Antitoxin: Vapar_1589
    4330 >Toxin: Mmcs_2235 9874 >Antitoxin: Mmcs_2236
    4331 >Toxin: Noca_0933 9875 >Antitoxin: Noca_0932
    4332 >Toxin: Mkms_2282 9876 >Antitoxin: Mkms_2283
    4333 >Toxin: Mvan_0335 9877 >Antitoxin: Mvan_0334
    4334 >Toxin: Mjls_2274 9878 >Antitoxin: Mjls_2275
    4335 >Toxin: Strop_1773 9879 >Antitoxin: Strop_1772
    4336 >Toxin: Sare_1760 9880 >Antitoxin: Sare_1759
    4337 >Toxin: Amir_1992 9881 >Antitoxin: Amir_1993
    4338 >Toxin: Bfae_27580 9882 >Antitoxin: Bfae_27570
    4339 >Toxin: Caci_4842 9883 >Antitoxin: Caci_4843
    4340 >Toxin: Svir_11380 9884 >Antitoxin: Svir_11370
    4341 >Toxin: B21_02992 9885 >Antitoxin: B21_02991
    4342 >Toxin: BCI_0636 9886 >Antitoxin: BCI_0635
    4343 >Toxin: Ping_0813 9887 >Antitoxin: Ping_0814
    4344 >Toxin: Ent638_3610 9888 >Antitoxin: Ent638_3609
    4345 >Toxin: VC0395_A0168 9889 >Antitoxin: VC0395_A0169
    4346 >Toxin: YpsIP31758_3600 9890 >Antitoxin: YpsIP31758_3599
    4347 >Toxin: VIBHAR_03402 9891 >Antitoxin: VIBHAR_03401
    4348 >Toxin: EcHS_A3368 9892 >Antitoxin: EcHS_A3367
    4349 >Toxin: EcE24377A_3661 9893 >Antitoxin: EcE24377A_3660
    4350 >Toxin: Spro_0485 9894 >Antitoxin: Spro_0486
    4351 >Toxin: YpAngola_A3987 9895 >Antitoxin: YpAngola_A3988
    4352 >Toxin: EcolC_0524 9896 >Antitoxin: EcolC_0525
    4353 >Toxin: YPK_3734 9897 >Antitoxin: YPK_3733
    4354 >Toxin: EcSMS35_3472 9898 >Antitoxin: EcSMS35_3471
    4355 >Toxin: SbBS512_E3594 9899 >Antitoxin: SbBS512_E3595
    4356 >Toxin: SNSL254_A3553 9900 >Antitoxin: SNSL254_A3552
    4357 >Toxin: SeHA_C3589 9901 >Antitoxin: SeHA_C3588
    4358 >Toxin: SeSA_A3483 9902 >Antitoxin: SeSA_A3482
    4359 >Toxin: SeAg_B3483 9903 >Antitoxin: SeAg_B3482
    4360 >Toxin: SeD_A3651 9904 >Antitoxin: SeD_A3650
    4361 >Toxin: VSAL_I0594 9905 >Antitoxin: VSAL_I0595
    4362 >Toxin: ECH74115_4498 9906 >Antitoxin: ECH74115_4497
    4363 >Toxin: Avin_42860 9907 >Antitoxin: Avin_42850
    4364 >Toxin: Dd703_3350 9908 >Antitoxin: Dd703_3349
    4365 >Toxin: Dd1591_3469 9909 >Antitoxin: Dd1591_3468
    4366 >Toxin: PC1_0568 9910 >Antitoxin: PC1_0569
    4367 >Toxin: BCE_0330 9911 >Antitoxin: BCE_0329
    4368 >Toxin: GBAA_0301 9912 >Antitoxin: GBAA_0300
    4369 >Toxin: BAS0288 9913 >Antitoxin: BAS0287
    4370 >Toxin: CPS_3456 9914 >Antitoxin: CPS_3455
    4371 >Toxin: Tfu_1437 9915 >Antitoxin: Tfu_1438
    4372 >Toxin: Saro_3197 9916 >Antitoxin: Saro_3196
    4373 >Toxin: Dgeo_2423 9917 >Antitoxin: Dgeo_2422
    4374 >Toxin: Sala_0217 9918 >Antitoxin: Sala_0218
    4375 >Toxin: Sama_2874 9919 >Antitoxin: Sama_2873
    4376 >Toxin: Swit_2131 9920 >Antitoxin: Swit_2130
    4377 >Toxin: Bcer98_0282 9921 >Antitoxin: Bcer98_0281
    4378 >Toxin: Franean1_0106 9922 >Antitoxin: Franean1_0105
    4379 >Toxin: BcerKBAB4_0282 9923 >Antitoxin: BcerKBAB4_0281
    4380 >Toxin: Caul_2841 9924 >Antitoxin: Caul_2840
    4381 >Toxin: Oter_2237 9925 >Antitoxin: Oter_2236
    4382 >Toxin: Smal_1106 9926 >Antitoxin: Smal_1105
    4383 >Toxin: MADE_01581 9927 >Antitoxin: MADE_01582
    4384 >Toxin: BCAH187_A0374 9928 >Antitoxin: BCAH187_A0373
    4385 >Toxin: BCB4264_A0347 9929 >Antitoxin: BCB4264_A0346
    4386 >Toxin: BCG9842_B4973 9930 >Antitoxin: BCG9842_B4974
    4387 >Toxin: BCAH820_0333 9931 >Antitoxin: BCAH820_0332
    4388 >Toxin: Dhaf_0786 9932 >Antitoxin: Dhaf_0785
    4389 >Toxin: Ccel_1361 9933 >Antitoxin: Ccel_1362
    4390 >Toxin: Amir_1211 9934 >Antitoxin: Amir_1210
    4391 >Toxin: GWCH70_0387 9935 >Antitoxin: GWCH70_0386
    4392 >Toxin: Elen_2127 9936 >Antitoxin: Elen_2126
    4393 >Toxin: Dfer_3657 9937 >Antitoxin: Dfer_3656
    4394 >Toxin: Cpin_1882 9938 >Antitoxin: Cpin_1881
    4395 >Toxin: Caci_1922 9939 >Antitoxin: Caci_1923
    4396 >Toxin: Apre_0211 9940 >Antitoxin: Apre_0210
    4397 >Toxin: Apar_0055 9941 >Antitoxin: Apar_0056
    4398 >Toxin: Dtox_2916 9942 >Antitoxin: Dtox_2917
    4399 >Toxin: Tfu_2052 9943 >Antitoxin: Tfu_2053
    4400 >Toxin: Cag_0777 9944 >Antitoxin: Cag_0776
    4401 >Toxin: Francci3_3169 9945 >Antitoxin: Francci3_3170
    4402 >Toxin: Acel_1257 9946 >Antitoxin: Acel_1258
    4403 >Toxin: Mflv_3524 9947 >Antitoxin: Mflv_3525
    4404 >Toxin: Strop_1894 9948 >Antitoxin: Strop_1893
    4405 >Toxin: TBFG_11676 9949 >Antitoxin: TBFG_11675
    4406 >Toxin: Franean1_1743 9950 >Antitoxin: Franean1_1742
    4407 >Toxin: Cphamn1_1332 9951 >Antitoxin: Cphamn1_1333
    4408 >Toxin: Bcav_2367 9952 >Antitoxin: Bcav_2368
    4409 >Toxin: Elen_2339 9953 >Antitoxin: Elen_2340
    4410 >Toxin: Svir_25570 9954 >Antitoxin: Svir_25580
    4411 >Toxin: Jden_1117 9955 >Antitoxin: Jden_1116
    4412 >Toxin: Mbar_A3694 9956 >Antitoxin: Mbar_A3695
    4413 >Toxin: Mhun_0050 9957 >Antitoxin: Mhun_0049
    4414 >Toxin: Mbur_1179 9958 >Antitoxin: Mbur_1180
    4415 >Toxin: Mthe_0027 9959 >Antitoxin: Mthe_0026
    4416 >Toxin: Tpen_0286 9960 >Antitoxin: Tpen_0287
    4417 >Toxin: Pisl_0703 9961 >Antitoxin: Pisl_0704
    4418 >Toxin: Mlab_1163 9962 >Antitoxin: Mlab_1164
    4419 >Toxin: Memar_0312 9963 >Antitoxin: Memar_0311
    4420 >Toxin: Pcal_2091 9964 >Antitoxin: Pcal_2090
    4421 >Toxin: MmarC5_0217 9965 >Antitoxin: MmarC5_0218
    4422 >Toxin: Pars_2321 9966 >Antitoxin: Pars_2320
    4423 >Toxin: Msed_0033 9967 >Antitoxin: Msed_0032
    4424 >Toxin: Mevan_0671 9968 >Antitoxin: Mevan_0670
    4425 >Toxin: Maeo_0791 9969 >Antitoxin: Maeo_0792
    4426 >Toxin: MmarC7_0606 9970 >Antitoxin: MmarC7_0605
    4427 >Toxin: Mboo_1934 9971 >Antitoxin: Mboo_1935
    4428 >Toxin: MmarC6_1312 9972 >Antitoxin: MmarC6_1313
    4429 >Toxin: Nmar_0347 9973 >Antitoxin: Nmar_0346
    4430 >Toxin: Tneu_1963 9974 >Antitoxin: Tneu_1962
    4431 >Toxin: Hlac_0107 9975 >Antitoxin: Hlac_0108
    4432 >Toxin: Huta_0784 9976 >Antitoxin: Huta_0785
    4433 >Toxin: Mpal_2159 9977 >Antitoxin: Mpal_2158
    4434 >Toxin: Hmuk_0515 9978 >Antitoxin: Hmuk_0516
    4435 >Toxin: Daro_2618 9979 >Antitoxin: Daro_2619
    4436 >Toxin: Ajs_1330 9980 >Antitoxin: Ajs_1331
    4437 >Toxin: Pnap_4568 9981 >Antitoxin: Pnap_4567
    4438 >Toxin: Mpe_A1650 9982 >Antitoxin: Mpe_A1651
    4439 >Toxin: Mpe_A2673 9983 >Antitoxin: Mpe_A2672
    4440 >Toxin: Daci_1741 9984 >Antitoxin: Daci_1740
    4441 >Toxin: Lcho_0143 9985 >Antitoxin: Lcho_0142
    4442 >Toxin: Tmz1t_2383 9986 >Antitoxin: Tmz1t_2384
    4443 >Toxin: Dtpsy_1143 9987 >Antitoxin: Dtpsy_1144
    4444 >Toxin: Avin_18560 9988 >Antitoxin: Avin_18550
    4445 >Toxin: Tfu_2917 9989 >Antitoxin: Tfu_2916
    4446 >Toxin: Francci3_0254 9990 >Antitoxin: Francci3_0255
    4447 >Toxin: Mmcs_2811 9991 >Antitoxin: Mmcs_2810
    4448 >Toxin: Arth_1620 9992 >Antitoxin: Arth_1621
    4449 >Toxin: Mkms_2855 9993 >Antitoxin: Mkms_2854
    4450 >Toxin: Mvan_3088 9994 >Antitoxin: Mvan_3087
    4451 >Toxin: Mjls_2838 9995 >Antitoxin: Mjls_2837
    4452 >Toxin: Mflv_3358 9996 >Antitoxin: Mflv_3357
    4453 >Toxin: Strop_0824 9997 >Antitoxin: Strop_0825
    4454 >Toxin: TBFG_11873 9998 >Antitoxin: TBFG_11874
    4455 >Toxin: Franean1_6582 9999 >Antitoxin: Franean1_6581
    4456 >Toxin: Sare_0768 10000 >Antitoxin: Sare_0769
    4457 >Toxin: Achl_1614 10001 >Antitoxin: Achl_1615
    4458 >Toxin: Ksed_01850 10002 >Antitoxin: Ksed_01860
    4459 >Toxin: Caci_0306 10003 >Antitoxin: Caci_0305
    4460 >Toxin: Svir_37270 10004 >Antitoxin: Svir_37280
    4461 >Toxin: Namu_3765 10005 >Antitoxin: Namu_3766
    4462 >Toxin: Syncc9605_2124 10006 >Antitoxin: Syncc9605_2125
    4463 >Toxin: Rru_A3295 10007 >Antitoxin: Rru_A3294
    4464 >Toxin: P9515_15031 10008 >Antitoxin: P9515_15041
    4465 >Toxin: P9301_15281 10009 >Antitoxin: P9301_15291
    4466 >Toxin: RoseRS_3359 10010 >Antitoxin: RoseRS_3358
    4467 >Toxin: Mboo_1484 10011 >Antitoxin: Mboo_1485
    4468 >Toxin: Rcas_4148 10012 >Antitoxin: Rcas_4149
    4469 >Toxin: Shewana3_2170 10013 >Antitoxin: Shewana3_2169
    4470 >Toxin: Veis_0272 10014 >Antitoxin: Veis_0273
    4471 >Toxin: Shew185_2063 10015 >Antitoxin: Shew185_2064
    4472 >Toxin: VIBHAR_02966 10016 >Antitoxin: VIBHAR_02965
    4473 >Toxin: Sbal195_2110 10017 >Antitoxin: Sbal195_2111
    4474 >Toxin: SeHA_C1627 10018 >Antitoxin: SeHA_C1628
    4475 >Toxin: SeSA_A1555 10019 >Antitoxin: SeSA_A1556
    4476 >Toxin: SeD_A1885 10020 >Antitoxin: SeD_A1884
    4477 >Toxin: SO_0503 10021 >Antitoxin: SO_0502
    4478 >Toxin: CPS_2452 10022 >Antitoxin: CPS_2453
    4479 >Toxin: Jann_3654 10023 >Antitoxin: Jann_3653
    4480 >Toxin: TM1040_0158 10024 >Antitoxin: TM1040_0157
    4481 >Toxin: Sfri_2516 10025 >Antitoxin: Sfri_2515
    4482 >Toxin: Shewana3_2338 10026 >Antitoxin: Shewana3_2337
    4483 >Toxin: Sputcn32_3823 10027 >Antitoxin: Sputcn32_3822
    4484 >Toxin: VIBHAR_05267 10028 >Antitoxin: VIBHAR_05266
    4485 >Toxin: EcE24377A_3989 10029 >Antitoxin: EcE24377A_3987
    4486 >Toxin: Dshi_0586 10030 >Antitoxin: Dshi_0587
    4487 >Toxin: Swoo_1498 10031 >Antitoxin: Swoo_1499
    4488 >Toxin: EcSMS35_A0147 10032 >Antitoxin: EcSMS35_A0148
    4489 >Toxin: EcSMS35_3797 10033 >Antitoxin: EcSMS35_3796
    4490 >Toxin: BR0925 10034 >Antitoxin: BR0924
    4491 >Toxin: Nwi_1669 10035 >Antitoxin: Nwi_1670
    4492 >Toxin: RPB_3001 10036 >Antitoxin: RPB_3002
    4493 >Toxin: Jann_1716 10037 >Antitoxin: Jann_1715
    4494 >Toxin: RPC_2847 10038 >Antitoxin: RPC_2848
    4495 >Toxin: RPD_2449 10039 >Antitoxin: RPD_2448
    4496 >Toxin: Nham_2333 10040 >Antitoxin: Nham_2334
    4497 >Toxin: TM1040_1092 10041 >Antitoxin: TM1040_1091
    4498 >Toxin: Meso_1786 10042 >Antitoxin: Meso_1787
    4499 >Toxin: RSP_2972 10043 >Antitoxin: RSP_2973
    4500 >Toxin: Mmar10_1307 10044 >Antitoxin: Mmar10_1306
    4501 >Toxin: Rsph17029_1618 10045 >Antitoxin: Rsph17029_1619
    4502 >Toxin: Rsph17025_1851 10046 >Antitoxin: Rsph17025_1852
    4503 >Toxin: BBta_3957 10047 >Antitoxin: BBta_3956
    4504 >Toxin: BOV_0919 10048 >Antitoxin: BOV_0918
    4505 >Toxin: Smed_1474 10049 >Antitoxin: Smed_1475
    4506 >Toxin: Oant_2261 10050 >Antitoxin: Oant_2262
    4507 >Toxin: Plav_3269 10051 >Antitoxin: Plav_3270
    4508 >Toxin: Xaut_4196 10052 >Antitoxin: Xaut_4195
    4509 >Toxin: Dshi_1179 10053 >Antitoxin: Dshi_1178
    4510 >Toxin: Mext_2985 10054 >Antitoxin: Mext_2984
    4511 >Toxin: Mrad2831_1807 10055 >Antitoxin: Mrad2831_1808
    4512 >Toxin: Bind_0013 10056 >Antitoxin: Bind_0012
    4513 >Toxin: Mpop_3169 10057 >Antitoxin: Mpop_3168
    4514 >Toxin: Rpal_2735 10058 >Antitoxin: Rpal_2734
    4515 >Toxin: M446_5848 10059 >Antitoxin: M446_5847
    4516 >Toxin: Rleg2_1909 10060 >Antitoxin: Rleg2_1910
    4517 >Toxin: Msil_1618 10061 >Antitoxin: Msil_1619
    4518 >Toxin: Mchl_3211 10062 >Antitoxin: Mchl_3210
    4519 >Toxin: Mnod_6053 10063 >Antitoxin: Mnod_6052
    4520 >Toxin: Avi_1887 10064 >Antitoxin: Avi_1886
    4521 >Toxin: Rleg_2116 10065 >Antitoxin: Rleg_2117
    4522 >Toxin: Tfu_2015 10066 >Antitoxin: Tfu_2014
    4523 >Toxin: Francci3_1639 10067 >Antitoxin: Francci3_1640
    4524 >Toxin: Mmcs_2406 10068 >Antitoxin: Mmcs_2407
    4525 >Toxin: Rxyl_2003 10069 >Antitoxin: Rxyl_2002
    4526 >Toxin: Arth_2089 10070 >Antitoxin: Arth_2090
    4527 >Toxin: Noca_2532 10071 >Antitoxin: Noca_2533
    4528 >Toxin: Mkms_2452 10072 >Antitoxin: Mkms_2453
    4529 >Toxin: Mvan_2705 10073 >Antitoxin: Mvan_2706
    4530 >Toxin: Mjls_2446 10074 >Antitoxin: Mjls_2447
    4531 >Toxin: Mflv_3708 10075 >Antitoxin: Mflv_3707
    4532 >Toxin: Strop_3096 10076 >Antitoxin: Strop_3095
    4533 >Toxin: Franean1_2065 10077 >Antitoxin: Franean1_2066
    4534 >Toxin: Sare_3323 10078 >Antitoxin: Sare_3322
    4535 >Toxin: BLD_0433 10079 >Antitoxin: BLD_0434
    4536 >Toxin: Achl_1830 10080 >Antitoxin: Achl_1831
    4537 >Toxin: Afer_0851 10081 >Antitoxin: Afer_0852
    4538 >Toxin: Amir_5197 10082 >Antitoxin: Amir_5196
    4539 >Toxin: Bcav_2169 10083 >Antitoxin: Bcav_2168
    4540 >Toxin: Bfae_15220 10084 >Antitoxin: Bfae_15230
    4541 >Toxin: Mlut_11370 10085 >Antitoxin: Mlut_11380
    4542 >Toxin: Ksed_13130 10086 >Antitoxin: Ksed_13140
    4543 >Toxin: Caci_5606 10087 >Antitoxin: Caci_5605
    4544 >Toxin: Svir_15890 10088 >Antitoxin: Svir_15900
    4545 >Toxin: Jden_1258 10089 >Antitoxin: Jden_1259
    4546 >Toxin: Apar_0922 10090 >Antitoxin: Apar_0921
    4547 >Toxin: Namu_2802 10091 >Antitoxin: Namu_2803
    4548 >Toxin: BRA0605 10092 >Antitoxin: BRA0606
    4549 >Toxin: Nwi_1713 10093 >Antitoxin: Nwi_1714
    4550 >Toxin: RPB_2418 10094 >Antitoxin: RPB_2417
    4551 >Toxin: RPC_2238 10095 >Antitoxin: RPC_2237
    4552 >Toxin: RPD_3034 10096 >Antitoxin: RPD_3035
    4553 >Toxin: Nham_2438 10097 >Antitoxin: Nham_2439
    4554 >Toxin: Meso_1353 10098 >Antitoxin: Meso_1352
    4555 >Toxin: BBta_4734 10099 >Antitoxin: BBta_4735
    4556 >Toxin: BOV_A0570 10100 >Antitoxin: BOV_A0571
    4557 >Toxin: Smed_0931 10101 >Antitoxin: Smed_0930
    4558 >Toxin: Oant_3672 10102 >Antitoxin: Oant_3673
    4559 >Toxin: Plav_2830 10103 >Antitoxin: Plav_2829
    4560 >Toxin: Xaut_3864 10104 >Antitoxin: Xaut_3863
    4561 >Toxin: Mext_2380 10105 >Antitoxin: Mext_2381
    4562 >Toxin: Mrad2831_3572 10106 >Antitoxin: Mrad2831_3573
    4563 >Toxin: Mpop_2337 10107 >Antitoxin: Mpop_2338
    4564 >Toxin: Rpal_3536 10108 >Antitoxin: Rpal_3537
    4565 >Toxin: M446_6672 10109 >Antitoxin: M446_6673
    4566 >Toxin: Rleg2_1294 10110 >Antitoxin: Rleg2_1293
    4567 >Toxin: Mchl_2657 10111 >Antitoxin: Mchl_2658
    4568 >Toxin: Mnod_7411 10112 >Antitoxin: Mnod_7412
    4569 >Toxin: Avi_1755 10113 >Antitoxin: Avi_1754
    4570 >Toxin: Rleg_1386 10114 >Antitoxin: Rleg_1385
    4571 >Toxin: Tery_4376 10115 >Antitoxin: Tery_4375
    4572 >Toxin: Dtur_0871 10116 >Antitoxin: Dtur_0872
    4573 >Toxin: Cyan7425_2516 10117 >Antitoxin: Cyan7425_2517
    4574 >Toxin: Elen_2553 10118 >Antitoxin: Elen_2552
    4575 >Toxin: Ccur_05120 10119 >Antitoxin: Ccur_05130
    4576 >Toxin: Shel_07820 10120 >Antitoxin: Shel_07830
    4577 >Toxin: Apar_0459 10121 >Antitoxin: Apar_0460
    4578 >Toxin: Moth_1329 10122 >Antitoxin: Moth_1330
    4579 >Toxin: Swol_1344 10123 >Antitoxin: Swol_1345
    4580 >Toxin: Cthe_0714 10124 >Antitoxin: Cthe_0713
    4581 >Toxin: Dred_1154 10125 >Antitoxin: Dred_1153
    4582 >Toxin: Daud_1178 10126 >Antitoxin: Daud_1179
    4583 >Toxin: Nther_1638 10127 >Antitoxin: Nther_1639
    4584 >Toxin: Emin_0546 10128 >Antitoxin: Emin_0545
    4585 >Toxin: Dhaf_3385 10129 >Antitoxin: Dhaf_3386
    4586 >Toxin: GWCH70_2169 10130 >Antitoxin: GWCH70_2170
    4587 >Toxin: Apre_1019 10131 >Antitoxin: Apre_1020
    4588 >Toxin: Dtox_2102 10132 >Antitoxin: Dtox_2101
    4589 >Toxin: Aaci_1703 10133 >Antitoxin: Aaci_1704
    4590 >Toxin: Dde_1453 10134 >Antitoxin: Dde_1452
    4591 >Toxin: Sfum_2116 10135 >Antitoxin: Sfum_2117
    4592 >Toxin: Dvul_1964 10136 >Antitoxin: Dvul_1965
    4593 >Toxin: Anae109_2310 10137 >Antitoxin: Anae109_2309
    4594 >Toxin: Dole_1983 10138 >Antitoxin: Dole_1982
    4595 >Toxin: DvMF_0908 10139 >Antitoxin: DvMF_0909
    4596 >Toxin: Ddes_0761 10140 >Antitoxin: Ddes_0760
    4597 >Toxin: Dbac_0507 10141 >Antitoxin: Dbac_0508
    4598 >Toxin: Dret_2197 10142 >Antitoxin: Dret_2196
    4599 >Toxin: RSp1366 10143 >Antitoxin: RS02091
    4600 >Toxin: Rfer_3196 10144 >Antitoxin: Rfer_3197
    4601 >Toxin: Rmet_4919 10145 >Antitoxin: Rmet_4918
    4602 >Toxin: Ajs_1142 10146 >Antitoxin: Ajs_1141
    4603 >Toxin: Rpic_4057 10147 >Antitoxin: Rpic_4058
    4604 >Toxin: Dtpsy_1063 10148 >Antitoxin: Dtpsy_1062
    4605 >Toxin: Rpic12D_4169 10149 >Antitoxin: Rpic12D_4170
    4606 >Toxin: Nham_4673 10150 >Antitoxin: Nham_4674
    4607 >Toxin: Smed_5395 10151 >Antitoxin: Smed_5394
    4608 >Toxin: Mrad2831_6101 10152 >Antitoxin: Mrad2831_6102
    4609 >Toxin: Mchl_5573 10153 >Antitoxin: Mchl_5574
    4610 >Toxin: Avi_8129 10154 >Antitoxin: Avi_8130
    4611 >Toxin: Avi_9187 10155 >Antitoxin: Avi_9188
    4612 >Toxin: Rleg_5516 10156 >Antitoxin: Rleg_5515
    4613 >Toxin: Bpro_4159 10157 >Antitoxin: Bpro_4158
    4614 >Toxin: Aave_4353 10158 >Antitoxin: Aave_4352
    4615 >Toxin: Ajs_3761 10159 >Antitoxin: Ajs_3760
    4616 >Toxin: Pnap_0473 10160 >Antitoxin: Pnap_0474
    4617 >Toxin: Mpe_A3358 10161 >Antitoxin: Mpe_A3357
    4618 >Toxin: Daci_5972 10162 >Antitoxin: Daci_5971
    4619 >Toxin: Dtpsy_3042 10163 >Antitoxin: Dtpsy_3041
    4620 >Toxin: Vapar_4721 10164 >Antitoxin: Vapar_4720
    4621 >Toxin: Nwi_1134 10165 >Antitoxin: Nwi_1135
    4622 >Toxin: RPB_3775 10166 >Antitoxin: RPB_3774
    4623 >Toxin: Jann_4196 10167 >Antitoxin: Jann_4195
    4624 >Toxin: RPD_1695 10168 >Antitoxin: RPD_1696
    4625 >Toxin: Nham_1394 10169 >Antitoxin: Nham_1395
    4626 >Toxin: TM1040_2963 10170 >Antitoxin: TM1040_2962
    4627 >Toxin: Rru_A2824 10171 >Antitoxin: Rru_A2823
    4628 >Toxin: RSP_1329 10172 >Antitoxin: RSP_1328
    4629 >Toxin: Pden_2112 10173 >Antitoxin: Pden_2113
    4630 >Toxin: Rsph17029_2990 10174 >Antitoxin: Rsph17029_2989
    4631 >Toxin: Rsph17025_2602 10175 >Antitoxin: Rsph17025_2603
    4632 >Toxin: Acry_2132 10176 >Antitoxin: Acry_2131
    4633 >Toxin: BBta_5495 10177 >Antitoxin: BBta_5494
    4634 >Toxin: Plav_2572 10178 >Antitoxin: Plav_2573
    4635 >Toxin: Dshi_3249 10179 >Antitoxin: Dshi_3250
    4636 >Toxin: Rpal_4409 10180 >Antitoxin: Rpal_4408
    4637 >Toxin: Gdia_3440 10181 >Antitoxin: Gdia_3441
    4638 >Toxin: Mnod_5536 10182 >Antitoxin: Mnod_5535
    4639 >Toxin: Moth_0849 10183 >Antitoxin: Moth_0848
    4640 >Toxin: Swol_0832 10184 >Antitoxin: Swol_0831
    4641 >Toxin: Cthe_0444 10185 >Antitoxin: Cthe_0443
    4642 >Toxin: Dred_0683 10186 >Antitoxin: Dred_0682
    4643 >Toxin: Haur_3503 10187 >Antitoxin: Haur_3504
    4644 >Toxin: Teth514_2004 10188 >Antitoxin: Teth514_2005
    4645 >Toxin: Ccel_2065 10189 >Antitoxin: Ccel_2066
    4646 >Toxin: GWCH70_1027 10190 >Antitoxin: GWCH70_1026
    4647 >Toxin: Dtox_1063 10191 >Antitoxin: Dtox_1062
    4648 >Toxin: Aaci_1287 10192 >Antitoxin: Aaci_1286
    4649 >Toxin: Teth514_1606 10193 >Antitoxin: Teth514_1605
    4650 >Toxin: SYO3AOP1_1148 10194 >Antitoxin: SYO3AOP1_1147
    4651 >Toxin: Dtur_1158 10195 >Antitoxin: Dtur_1157
    4652 >Toxin: Athe_1327 10196 >Antitoxin: Athe_1326
    4653 >Toxin: Afer_1349 10197 >Antitoxin: Afer_1350
    4654 >Toxin: Elen_1335 10198 >Antitoxin: Elen_1334
    4655 >Toxin: Ccur_06870 10199 >Antitoxin: Ccur_06860
    4656 >Toxin: Shel_09680 10200 >Antitoxin: Shel_09670
    4657 >Toxin: Pcar_1442 10201 >Antitoxin: Pcar_1443
    4658 >Toxin: Dde_2432 10202 >Antitoxin: Dde_2433
    4659 >Toxin: Moth_2390 10203 >Antitoxin: Moth_2389
    4660 >Toxin: Swol_2392 10204 >Antitoxin: Swol_2391
    4661 >Toxin: Dvul_1854 10205 >Antitoxin: Dvul_1855
    4662 >Toxin: Dred_3162 10206 >Antitoxin: Dred_3161
    4663 >Toxin: Dole_2082 10207 >Antitoxin: Dole_2081
    4664 >Toxin: DvMF_3097 10208 >Antitoxin: DvMF_3096
    4665 >Toxin: Ddes_1617 10209 >Antitoxin: Ddes_1618
    4666 >Toxin: Dbac_0064 10210 >Antitoxin: Dbac_0063
    4667 >Toxin: Dtox_4177 10211 >Antitoxin: Dtox_4176
    4668 >Toxin: Dret_1418 10212 >Antitoxin: Dret_1419
    4669 >Toxin: RSc2583 10213 >Antitoxin: RSc2582
    4670 >Toxin: Saro_0317 10214 >Antitoxin: Saro_0316
    4671 >Toxin: RPC_3540 10215 >Antitoxin: RPC_3539
    4672 >Toxin: RPC_3900 10216 >Antitoxin: RPC_3899
    4673 >Toxin: Rmet_1342 10217 >Antitoxin: Rmet_1343
    4674 >Toxin: Rmet_1551 10218 >Antitoxin: Rmet_1552
    4675 >Toxin: Sala_2613 10219 >Antitoxin: Sala_2612
    4676 >Toxin: Rru_A0807 10220 >Antitoxin: Rru_A0806
    4677 >Toxin: Meso_0508 10221 >Antitoxin: Meso_0507
    4678 >Toxin: Meso_2323 10222 >Antitoxin: Meso_2322
    4679 >Toxin: Meso_3852 10223 >Antitoxin: Meso_3853
    4680 >Toxin: Bamb_2005 10224 >Antitoxin: Bamb_2006
    4681 >Toxin: Shewana3_1283 10225 >Antitoxin: Shewana3_1284
    4682 >Toxin: Pden_1483 10226 >Antitoxin: Pden_1482
    4683 >Toxin: Pden_3195 10227 >Antitoxin: Pden_3196
    4684 >Toxin: Aave_0727 10228 >Antitoxin: Aave_0728
    4685 >Toxin: Ajs_2910 10229 >Antitoxin: Ajs_2909
    4686 >Toxin: Pnap_4196 10230 >Antitoxin: Pnap_4197
    4687 >Toxin: BBta_1501 10231 >Antitoxin: BBta_1502
    4688 >Toxin: BBta_7441 10232 >Antitoxin: BBta_7442
    4689 >Toxin: BBta_7742 10233 >Antitoxin: BBta_7743
    4690 >Toxin: Swit_3692 10234 >Antitoxin: Swit_3693
    4691 >Toxin: PSPA7_3705 10235 >Antitoxin: PSPA7_3704
    4692 >Toxin: Oant_3213 10236 >Antitoxin: Oant_3214
    4693 >Toxin: Plav_3542 10237 >Antitoxin: Plav_3543
    4694 >Toxin: Xaut_0596 10238 >Antitoxin: Xaut_0597
    4695 >Toxin: Xaut_0748 10239 >Antitoxin: Xaut_0747
    4696 >Toxin: Xaut_2879 10240 >Antitoxin: Xaut_2880
    4697 >Toxin: Xaut_3003 10241 >Antitoxin: Xaut_3002
    4698 >Toxin: Xaut_3999 10242 >Antitoxin: Xaut_3998
    4699 >Toxin: Daci_0437 10243 >Antitoxin: Daci_0436
    4700 >Toxin: Daci_2698 10244 >Antitoxin: Daci_2697
    4701 >Toxin: Bmul_2351 10245 >Antitoxin: Bmul_2352
    4702 >Toxin: Caul_2028 10246 >Antitoxin: Caul_2029
    4703 >Toxin: Caul_3828 10247 >Antitoxin: Caul_3829
    4704 >Toxin: Mpop_3087 10248 >Antitoxin: Mpop_3088
    4705 >Toxin: Rpic_2646 10249 >Antitoxin: Rpic_2647
    4706 >Toxin: Paes_2347 10250 >Antitoxin: Paes_2348
    4707 >Toxin: Gdia_1012 10251 >Antitoxin: Gdia_1011
    4708 >Toxin: Gdia_2825 10252 >Antitoxin: Gdia_2826
    4709 >Toxin: Tgr7_1896 10253 >Antitoxin: Tgr7_1897
    4710 >Toxin: Avi_9659 10254 >Antitoxin: Avi_9658
    4711 >Toxin: Dtpsy_3505 10255 >Antitoxin: Dtpsy_3504
    4712 >Toxin: Avin_35710 10256 >Antitoxin: Avin_35700
    4713 >Toxin: GM21_2350 10257 >Antitoxin: GM21_2349
    4714 >Toxin: BCE_A0182 10258 >Antitoxin: BCE_A0183
    4715 >Toxin: GBAA_3860 10259 >Antitoxin: GBAA_3861
    4716 >Toxin: BAS3576 10260 >Antitoxin: BAS3577
    4717 >Toxin: BT9727_3476 10261 >Antitoxin: BT9727_3477
    4718 >Toxin: BCZK3488 10262 >Antitoxin: BCZK3489
    4719 >Toxin: Tfu_0951 10263 >Antitoxin: Tfu_0952
    4720 >Toxin: Nmul_A2277 10264 >Antitoxin: Nmul_42278
    4721 >Toxin: Adeh_3496 10265 >Antitoxin: Adeh_3495
    4722 >Toxin: Rxyl_0131 10266 >Antitoxin: Rxyl_0132
    4723 >Toxin: Mlg_0323 10267 >Antitoxin: Mlg_0324
    4724 >Toxin: Mlg_0325 10268 >Antitoxin: Mlg_0324
    4725 >Toxin: Arth_4098 10269 >Antitoxin: Arth_4097
    4726 >Toxin: Acel_1651 10270 >Antitoxin: Acel_1650
    4727 >Toxin: Mvan_3669 10271 >Antitoxin: Mvan_3668
    4728 >Toxin: Dred_2140 10272 >Antitoxin: Dred_2141
    4729 >Toxin: Strop_0446 10273 >Antitoxin: Strop_0445
    4730 >Toxin: Krad_4588 10274 >Antitoxin: Krad_4587
    4731 >Toxin: Bcer98_0518 10275 >Antitoxin: Bcer98_0517
    4732 >Toxin: Sare_0534 10276 >Antitoxin: Sare_0533
    4733 >Toxin: BcerKBAB4_3497 10277 >Antitoxin: BcerKBAB4_3498
    4734 >Toxin: AnaeK_3584 10278 >Antitoxin: AnaeK_3583
    4735 >Toxin: BCAH187_A3780 10279 >Antitoxin: BCAH187_A3781
    4736 >Toxin: BCB4264_A3830 10280 >Antitoxin: BCB4264_A3831
    4737 >Toxin: BCG9842_B1471 10281 >Antitoxin: BCG9842_B1470
    4738 >Toxin: BCAH820_3742 10282 >Antitoxin: BCAH820_3743
    4739 >Toxin: BCAH820_B0280 10283 >Antitoxin: BCAH820_B0279
    4740 >Toxin: Achl_3872 10284 >Antitoxin: Achl_3871
    4741 >Toxin: A2cp1_3653 10285 >Antitoxin: A2cp1_3652
    4742 >Toxin: Bcav_4020 10286 >Antitoxin: Bcav_4021
    4743 >Toxin: Bcav_4022 10287 >Antitoxin: Bcav_4021
    4744 >Toxin: GWCH70_1801 10288 >Antitoxin: GWCH70_1802
    4745 >Toxin: Mlut_21360 10289 >Antitoxin: Mlut_21350
    4746 >Toxin: Ksed_10590 10290 >Antitoxin: Ksed_10580
    4747 >Toxin: Elen_1553 10291 >Antitoxin: Elen_1552
    4748 >Toxin: Cagg_3412 10292 >Antitoxin: Cagg_3411
    4749 >Toxin: Ccur_11220 10293 >Antitoxin: Ccur_11230
    4750 >Toxin: Svir_36750 10294 >Antitoxin: Svir_36760
    4751 >Toxin: Apre_0987 10295 >Antitoxin: Apre_0988
    4752 >Toxin: Jden_2500 10296 >Antitoxin: Jden_2499
    4753 >Toxin: Shel_01260 10297 >Antitoxin: Shel_01250
    4754 >Toxin: Apar_0439 10298 >Antitoxin: Apar_0438
    4755 >Toxin: Dtox_2078 10299 >Antitoxin: Dtox_2077
    4756 >Toxin: Aaci_1113 10300 >Antitoxin: Aaci_1112
    4757 >Toxin: Aaci_2657 10301 >Antitoxin: Aaci_2658
    4758 >Toxin: Moth_0866 10302 >Antitoxin: Moth_0865
    4759 >Toxin: Swol_0964 10303 >Antitoxin: Swol_0963
    4760 >Toxin: Teth514_1858 10304 >Antitoxin: Teth514_1859
    4761 >Toxin: Daud_1351 10305 >Antitoxin: Daud_1352
    4762 >Toxin: Nther_1322 10306 >Antitoxin: Nther_1321
    4763 >Toxin: Dhaf_4024 10307 >Antitoxin: Dhaf_4025
    4764 >Toxin: Hore_09300 10308 >Antitoxin: Hore_09290
    4765 >Toxin: Ccel_1852 10309 >Antitoxin: Ccel_1853
    4766 >Toxin: Athe_1363 10310 >Antitoxin: Athe_1362
    4767 >Toxin: Dtox_1126 10311 >Antitoxin: Dtox_1125
    4768 >Toxin: Aaci_1300 10312 >Antitoxin: Aaci_1299
    4769 >Toxin: Francci3_0449 10313 >Antitoxin: Francci3_0448
    4770 >Toxin: Mmcs_4529 10314 >Antitoxin: Mmcs_4528
    4771 >Toxin: Noca_4230 10315 >Antitoxin: Noca_4231
    4772 >Toxin: Mkms_4616 10316 >Antitoxin: Mkms_4615
    4773 >Toxin: Mvan_5101 10317 >Antitoxin: Mvan_5100
    4774 >Toxin: Mjls_4912 10318 >Antitoxin: Mjls_4911
    4775 >Toxin: Mflv_1646 10319 >Antitoxin: Mflv_1647
    4776 >Toxin: Franean1_6169 10320 >Antitoxin: Franean1_6170
    4777 >Toxin: Amir_6828 10321 >Antitoxin: Amir_6827
    4778 >Toxin: Svir_37710 10322 >Antitoxin: Svir_37700
    4779 >Toxin: Cthe_0333 10323 >Antitoxin: Cthe_0334
    4780 >Toxin: Tpet_1428 10324 >Antitoxin: Tpet_1429
    4781 >Toxin: Cphy_3800 10325 >Antitoxin: Cphy_3799
    4782 >Toxin: Teth514_2147 10326 >Antitoxin: Teth514_2146
    4783 >Toxin: TRQ2_1474 10327 >Antitoxin: TRQ2_1475
    4784 >Toxin: Nther_0091 10328 >Antitoxin: Nther_0092
    4785 >Toxin: Dtur_0910 10329 >Antitoxin: Dtur_0911
    4786 >Toxin: Athe_1290 10330 >Antitoxin: Athe_1291
    4787 >Toxin: Dtox_0180 10331 >Antitoxin: Dtox_0179
    4788 >Toxin: BRA0158 10332 >Antitoxin: BRA0159
    4789 >Toxin: RPC_1101 10333 >Antitoxin: RPC_1102
    4790 >Toxin: Meso_0291 10334 >Antitoxin: Meso_0290
    4791 >Toxin: BARBAKC583_1137 10335 >Antitoxin: BARBAKC583_1136
    4792 >Toxin: BOV_A0142 10336 >Antitoxin: BOV_A0143
    4793 >Toxin: Smed_0262 10337 >Antitoxin: Smed_0263
    4794 >Toxin: Oant_4205 10338 >Antitoxin: Oant_4204
    4795 >Toxin: Mext_0625 10339 >Antitoxin: Mext_0626
    4796 >Toxin: Mrad2831_1685 10340 >Antitoxin: Mrad2831_1684
    4797 >Toxin: Bind_3649 10341 >Antitoxin: Bind_3648
    4798 >Toxin: Mpop_0603 10342 >Antitoxin: Mpop_0604
    4799 >Toxin: Rleg2_0324 10343 >Antitoxin: Rleg2_0325
    4800 >Toxin: Msil_0031 10344 >Antitoxin: Msil_0032
    4801 >Toxin: Mchl_0636 10345 >Antitoxin: Mchl_0637
    4802 >Toxin: Avi_0744 10346 >Antitoxin: Avi_0745
    4803 >Toxin: Rleg_0356 10347 >Antitoxin: Rleg_0357
    4804 >Toxin: Rfer_0565 10348 >Antitoxin: Rfer_0566
    4805 >Toxin: Aave_4380 10349 >Antitoxin: Aave_4379
    4806 >Toxin: Ajs_3792 10350 >Antitoxin: Ajs_3791
    4807 >Toxin: Veis_2178 10351 >Antitoxin: Veis_2177
    4808 >Toxin: Mpe_A0583 10352 >Antitoxin: Mpe_A0584
    4809 >Toxin: Daci_0653 10353 >Antitoxin: Daci_0654
    4810 >Toxin: Lcho_1597 10354 >Antitoxin: Lcho_1598
    4811 >Toxin: Dtpsy_3066 10355 >Antitoxin: Dtpsy_3065
    4812 >Toxin: BMAA0399 10356 >Antitoxin: BMAA0400
    4813 >Toxin: BURPS1710b_A1204 10357 >Antitoxin: BURPS1710b_A1203
    4814 >Toxin: Bcep18194_B1592 10358 >Antitoxin: Bcep18194_B1591
    4815 >Toxin: BTH_II0254 10359 >Antitoxin: BTH_II0255
    4816 >Toxin: Jann_3035 10360 >Antitoxin: Jann_3036
    4817 >Toxin: Sde_1522 10361 >Antitoxin: Sde_1521
    4818 >Toxin: RSP_3487 10362 >Antitoxin: RSP_3488
    4819 >Toxin: Sfri_2373 10363 >Antitoxin: Sfri_2372
    4820 >Toxin: PA14_00910 10364 >Antitoxin: PA14_00900
    4821 >Toxin: Pden_2453 10365 >Antitoxin: Pden_2454
    4822 >Toxin: Aave_1473 10366 >Antitoxin: Aave_1474
    4823 >Toxin: BMASAVP1_1593 10367 >Antitoxin: BMASAVP1_1594
    4824 >Toxin: Rsph17029_3131 10368 >Antitoxin: Rsph17029_3132
    4825 >Toxin: BMA10247_A0448 10369 >Antitoxin: BMA10247_A0450
    4826 >Toxin: BURPS668_A2965 10370 >Antitoxin: BURPS668_A2963
    4827 >Toxin: BURPS1106A_A2840 10371 >Antitoxin: BURPS1106A_A2838
    4828 >Toxin: Pmen_2316 10372 >Antitoxin: Pmen_2315
    4829 >Toxin: Acry_2088 10373 >Antitoxin: Acry_2087
    4830 >Toxin: PSPA7_0148 10374 >Antitoxin: PSPA7_0147
    4831 >Toxin: Spro_3000 10375 >Antitoxin: Spro_3001
    4832 >Toxin: Daci_3846 10376 >Antitoxin: Daci_3847
    4833 >Toxin: Lcho_4057 10377 >Antitoxin: Lcho_4058
    4834 >Toxin: PXO_00250 10378 >Antitoxin: PXO_00249
    4835 >Toxin: Msil_2352 10379 >Antitoxin: Msil_2351
    4836 >Toxin: Mnod_3048 10380 >Antitoxin: Mnod_3049
    4837 >Toxin: Avi_6041 10381 >Antitoxin: Avi_6040
    4838 >Toxin: Avin_26710 10382 >Antitoxin: Avin_26700
    4839 >Toxin: Vapar_0213 10383 >Antitoxin: Vapar_0214
    4840 >Toxin: Vapar_0516 10384 >Antitoxin: Vapar_0517
    4841 >Toxin: CPS_4227 10385 >Antitoxin: CPS_4226
    4842 >Toxin: Reut_B5017 10386 >Antitoxin: Reut_B5016
    4843 >Toxin: Tbd_0071 10387 >Antitoxin: Tbd_0072
    4844 >Toxin: PA14_06670 10388 >Antitoxin: PA14_06680
    4845 >Toxin: Pden_2494 10389 >Antitoxin: Pden_2493
    4846 >Toxin: Ajs_1906 10390 >Antitoxin: Ajs_1907
    4847 >Toxin: Maqu_3130 10391 >Antitoxin: Maqu_3129
    4848 >Toxin: Memar_0876 10392 >Antitoxin: Memar_0877
    4849 >Toxin: PSPA7_0612 10393 >Antitoxin: PSPA7_0613
    4850 >Toxin: Mboo_0955 10394 >Antitoxin: Mboo_0956
    4851 >Toxin: Dshi_3173 10395 >Antitoxin: Dshi_3174
    4852 >Toxin: Lcho_1448 10396 >Antitoxin: Lcho_1447
    4853 >Toxin: Tmz1t_2609 10397 >Antitoxin: Tmz1t_2610
    4854 >Toxin: Dtpsy_1711 10398 >Antitoxin: Dtpsy_1712
    4855 >Toxin: Mpal_2629 10399 >Antitoxin: Mpal_2628
    4856 >Toxin: Jann_1701 10400 >Antitoxin: Jann_1702
    4857 >Toxin: TM1040_1114 10401 >Antitoxin: TM1040_1115
    4858 >Toxin: RSP_2955 10402 >Antitoxin: RSP_2954
    4859 >Toxin: Pden_1882 10403 >Antitoxin: Pden_1881
    4860 >Toxin: Rsph17029_1600 10404 >Antitoxin: Rsph17029_1599
    4861 >Toxin: Rsph17025_0890 10405 >Antitoxin: Rsph17025_0891
    4862 >Toxin: Dshi_1737 10406 >Antitoxin: Dshi_1736
    4863 >Toxin: Tfu_0674 10407 >Antitoxin: Tfu_0675
    4864 >Toxin: Francci3_3583 10408 >Antitoxin: Francci3_3582
    4865 >Toxin: Noca_3245 10409 >Antitoxin: Noca_3244
    4866 >Toxin: Mvan_2207 10410 >Antitoxin: Mvan_2208
    4867 >Toxin: Mflv_4135 10411 >Antitoxin: Mflv_4134
    4868 >Toxin: Franean1_1159 10412 >Antitoxin: Franean1_1160
    4869 >Toxin: Bcav_2509 10413 >Antitoxin: Bcav_2508
    4870 >Toxin: Bfae_10100 10414 >Antitoxin: Bfae_10110
    4871 >Toxin: Ksed_11170 10415 >Antitoxin: Ksed_11180
    4872 >Toxin: Namu_3115 10416 >Antitoxin: Namu_3114
    4873 >Toxin: SAG1651 10417 >Antitoxin: SAG1652
    4874 >Toxin: LMOf2365_2675 10418 >Antitoxin: LMOf2365_2676
    4875 >Toxin: SACOL0708 10419 >Antitoxin: SACOL0709
    4876 >Toxin: SERP2344 10420 >Antitoxin: SERP2343
    4877 >Toxin: MCAP_0235 10421 >Antitoxin: MCAP_0236
    4878 >Toxin: Rxyl_1067 10422 >Antitoxin: Rxyl_1068
    4879 >Toxin: SaurJH9_0674 10423 >Antitoxin: SaurJH9_0675
    4880 >Toxin: SaurJH1_0689 10424 >Antitoxin: SaurJH1_0690
    4881 >Toxin: Teth514_1026 10425 >Antitoxin: Teth514_1027
    4882 >Toxin: Hlac_1459 10426 >Antitoxin: Hlac_1460
    4883 >Toxin: Afer_0878 10427 >Antitoxin: Afer_0879
    4884 >Toxin: Amir_6113 10428 >Antitoxin: Amir_6112
    4885 >Toxin: GWCH70_1343 10429 >Antitoxin: GWCH70_1344
    4886 >Toxin: Rleg_2433 10430 >Antitoxin: Rleg_2434
    4887 >Toxin: Cagg_2004 10431 >Antitoxin: Cagg_2005
    4888 >Toxin: Apre_1081 10432 >Antitoxin: Apre_1082
    4889 >Toxin: Namu_1660 10433 >Antitoxin: Namu_1661
    4890 >Toxin: RSc2444 10434 >Antitoxin: RSc2443
    4891 >Toxin: Ajs_3450 10435 >Antitoxin: Ajs_3449
    4892 >Toxin: Pnap_3339 10436 >Antitoxin: Pnap_3338
    4893 >Toxin: Veis_1654 10437 >Antitoxin: Veis_1653
    4894 >Toxin: Mpe_A2695 10438 >Antitoxin: Mpe_A2694
    4895 >Toxin: Pnuc_1733 10439 >Antitoxin: Pnuc_1732
    4896 >Toxin: Daci_1904 10440 >Antitoxin: Daci_1905
    4897 >Toxin: Lcho_0695 10441 >Antitoxin: Lcho_0694
    4898 >Toxin: Dtpsy_2777 10442 >Antitoxin: Dtpsy_2776
    4899 >Toxin: Vapar_4033 10443 >Antitoxin: Vapar_4032
    4900 >Toxin: Plut_1586 10444 >Antitoxin: Plut_1587
    4901 >Toxin: Cag_0551 10445 >Antitoxin: Cag_0550
    4902 >Toxin: CHU_2016 10446 >Antitoxin: CHU_2017
    4903 >Toxin: Cpha266_0605 10447 >Antitoxin: Cpha266_0604
    4904 >Toxin: Fjoh_0491 10448 >Antitoxin: Fjoh_0492
    4905 >Toxin: Clim_1961 10449 >Antitoxin: Clim_1962
    4906 >Toxin: Cphamn1_0635 10450 >Antitoxin: Cphamn1_0634
    4907 >Toxin: Ppha_0731 10451 >Antitoxin: Ppha_0730
    4908 >Toxin: Paes_0590 10452 >Antitoxin: Paes_0589
    4909 >Toxin: Phep_4088 10453 >Antitoxin: Phep_4089
    4910 >Toxin: Dfer_4057 10454 >Antitoxin: Dfer_4056
    4911 >Toxin: Cpin_1428 10455 >Antitoxin: Cpin_1427
    4912 >Toxin: TM1040_1251 10456 >Antitoxin: TM1040_1250
    4913 >Toxin: Meso_0178 10457 >Antitoxin: Meso_0179
    4914 >Toxin: RSP_1412 10458 >Antitoxin: RSP_3802
    4915 >Toxin: Rsph17029_3482 10459 >Antitoxin: Rsph17029_3481
    4916 >Toxin: Rsph17025_3687 10460 >Antitoxin: Rsph17025_3686
    4917 >Toxin: VC0395_A2847 10461 >Antitoxin: VC0395_A2846
    4918 >Toxin: VIBHAR_00792 10462 >Antitoxin: VIBHAR_00791
    4919 >Toxin: VSAL_I0355 10463 >Antitoxin: VSAL_I0354
    4920 >Toxin: Dbac_0032 10464 >Antitoxin: Dbac_0031
    4921 >Toxin: BR1365 10465 >Antitoxin: BR1366
    4922 >Toxin: Dde_1190 10466 >Antitoxin: Dde_1191
    4923 >Toxin: RPC_1951 10467 >Antitoxin: RPC_1950
    4924 >Toxin: RPC_1959 10468 >Antitoxin: RPC_1960
    4925 >Toxin: Rru_A3500 10469 >Antitoxin: Rru_A3499
    4926 >Toxin: RSP_3207 10470 >Antitoxin: RSP_3208
    4927 >Toxin: Rsph17029_3947 10471 >Antitoxin: Rsph17029_3948
    4928 >Toxin: Rsph17025_3436 10472 >Antitoxin: Rsph17025_3435
    4929 >Toxin: BOV_1321 10473 >Antitoxin: BOV_1322
    4930 >Toxin: Ssed_2093 10474 >Antitoxin: Ssed_2094
    4931 >Toxin: Dole_2022 10475 >Antitoxin: Dole_2021
    4932 >Toxin: Ddes_1471 10476 >Antitoxin: Ddes_1472
    4933 >Toxin: Dbac_3138 10477 >Antitoxin: Dbac_3137
    4934 >Toxin: Dret_0756 10478 >Antitoxin: Dret_0757
    4935 >Toxin: Mbar_A3104 10479 >Antitoxin: Mbar_A3103
    4936 >Toxin: Pcar_2991 10480 >Antitoxin: Pcar_2992
    4937 >Toxin: Plut_1065 10481 >Antitoxin: Plut_1066
    4938 >Toxin: Nmul_A1659 10482 >Antitoxin: Nmul_A1658
    4939 >Toxin: Rfer_1163 10483 >Antitoxin: Rfer_1164
    4940 >Toxin: Patl_2670 10484 >Antitoxin: Patl_2671
    4941 >Toxin: Sfri_3051 10485 >Antitoxin: Sfri_3052
    4942 >Toxin: Ping_0465 10486 >Antitoxin: Ping_0466
    4943 >Toxin: Pnap_2334 10487 >Antitoxin: Pnap_2333
    4944 >Toxin: Mmwyl1_1956 10488 >Antitoxin: Mmwyl1_1957
    4945 >Toxin: Paes_0890 10489 >Antitoxin: Paes_0891
    4946 >Toxin: Dbac_0950 10490 >Antitoxin: Dbac_0949
    4947 >Toxin: Reut_B4434 10491 >Antitoxin: Reut_B4433
    4948 >Toxin: Nham_4181 10492 >Antitoxin: Nham_4182
    4949 >Toxin: Acid345_2997 10493 >Antitoxin: Acid345_2998
    4950 >Toxin: M446_5818 10494 >Antitoxin: M446_5819
    4951 >Toxin: Gdia_0829 10495 >Antitoxin: Gdia_0830
    4952 >Toxin: Mnod_6887 10496 >Antitoxin: Mnod_6888
    4953 >Toxin: Cagg_1523 10497 >Antitoxin: Cagg_1524
    4954 >Toxin: Pcar_0845 10498 >Antitoxin: Pcar_0846
    4955 >Toxin: Dde_2156 10499 >Antitoxin: Dde_2155
    4956 >Toxin: Csal_1619 10500 >Antitoxin: Csal_1618
    4957 >Toxin: CFF8240_0849 10501 >Antitoxin: CFF8240_0848
    4958 >Toxin: Ping_3346 10502 >Antitoxin: Ping_3345
    4959 >Toxin: Anae109_0871 10503 >Antitoxin: Anae109_0872
    4960 >Toxin: Dole_1735 10504 >Antitoxin: Dole_1736
    4961 >Toxin: AnaeK_1509 10505 >Antitoxin: AnaeK_1510
    4962 >Toxin: A2cp1_1604 10506 >Antitoxin: A2cp1_1605
    4963 >Toxin: Dret_1657 10507 >Antitoxin: Dret_1656
    4964 >Toxin: Noc_2709 10508 >Antitoxin: Noc_2708
    4965 >Toxin: RPC_1184 10509 >Antitoxin: RPC_1183
    4966 >Toxin: Mvan_1183 10510 >Antitoxin: Mvan_1182
    4967 >Toxin: Mflv_5149 10511 >Antitoxin: Mflv_5150
    4968 >Toxin: Franean1_1484 10512 >Antitoxin: Franean1_1485
    4969 >Toxin: M446_5114 10513 >Antitoxin: M446_5113
    4970 >Toxin: Amir_5089 10514 >Antitoxin: Amir_5088
    4971 >Toxin: Nwi_1704 10515 >Antitoxin: Nwi_1703
    4972 >Toxin: RPB_2386 10516 >Antitoxin: RPB_2387
    4973 >Toxin: RPC_3334 10517 >Antitoxin: RPC_3333
    4974 >Toxin: RPD_3068 10518 >Antitoxin: RPD_3067
    4975 >Toxin: Nham_2426 10519 >Antitoxin: Nham_2425
    4976 >Toxin: BBta_4785 10520 >Antitoxin: BBta_4784
    4977 >Toxin: Xaut_4457 10521 >Antitoxin: Xaut_4456
    4978 >Toxin: Rpal_3566 10522 >Antitoxin: Rpal_3565
    4979 >Toxin: Nwi_2202 10523 >Antitoxin: Nwi_2201
    4980 >Toxin: RPB_3675 10524 >Antitoxin: RPB_3674
    4981 >Toxin: RPC_1634 10525 >Antitoxin: RPC_1635
    4982 >Toxin: RPD_1785 10526 >Antitoxin: RPD_1786
    4983 >Toxin: Nham_2604 10527 >Antitoxin: Nham_2603
    4984 >Toxin: Rxyl_0115 10528 >Antitoxin: Rxyl_0116
    4985 >Toxin: Meso_1291 10529 >Antitoxin: Meso_1292
    4986 >Toxin: Acry_0472 10530 >Antitoxin: Acry_0473
    4987 >Toxin: BBta_5315 10531 >Antitoxin: BBta_5314
    4988 >Toxin: Smed_1484 10532 >Antitoxin: Smed_1483
    4989 >Toxin: Plav_2958 10533 >Antitoxin: Plav_2957
    4990 >Toxin: Rpal_4323 10534 >Antitoxin: Rpal_4322
    4991 >Toxin: M446_5446 10535 >Antitoxin: M446_5447
    4992 >Toxin: Mnod_1753 10536 >Antitoxin: Mnod_1754
    4993 >Toxin: BR0896 10537 >Antitoxin: BR0897
    4994 >Toxin: BOV_0892 10538 >Antitoxin: BOV_0893
    4995 >Toxin: Oant_2331 10539 >Antitoxin: Oant_2330
    4996 >Toxin: CCV52592_1895 10540 >Antitoxin: CCV52592_1894
    4997 >Toxin: Plav_2660 10541 >Antitoxin: Plav_2659
    4998 >Toxin: CCC13826_1098 10542 >Antitoxin: CCC13826_1097
    4999 >Toxin: Ssed_2356 10543 >Antitoxin: Ssed_2355
    5000 >Toxin: Swoo_2229 10544 >Antitoxin: Swoo_2230
    5001 >Toxin: M446_2238 10545 >Antitoxin: M446_2239
    5002 >Toxin: Rleg2_1503 10546 >Antitoxin: Rleg2_1504
    5003 >Toxin: Mnod_0371 10547 >Antitoxin: Mnod_0370
    5004 >Toxin: Avi_2480 10548 >Antitoxin: Avi_2479
    5005 >Toxin: Rleg_1701 10549 >Antitoxin: Rleg_1702
    5006 >Toxin: DET0050 10550 >Antitoxin: DET0051
    5007 >Toxin: Tfu_2070 10551 >Antitoxin: Tfu_2069
    5008 >Toxin: Francci3_3214 10552 >Antitoxin: Francci3_3213
    5009 >Toxin: Mmcs_2346 10553 >Antitoxin: Mmcs_2347
    5010 >Toxin: Rxyl_1359 10554 >Antitoxin: Rxyl_1360
    5011 >Toxin: Arth_2278 10555 >Antitoxin: Arth_2277
    5012 >Toxin: Acel_1330 10556 >Antitoxin: Acel_1329
    5013 >Toxin: Noca_2407 10557 >Antitoxin: Noca_2408
    5014 >Toxin: Mkms_2393 10558 >Antitoxin: Mkms_2394
    5015 >Toxin: Mvan_2640 10559 >Antitoxin: Mvan_2641
    5016 >Toxin: Mjls_2387 10560 >Antitoxin: Mjls_2388
    5017 >Toxin: Mflv_3763 10561 >Antitoxin: Mflv_3762
    5018 >Toxin: Strop_1828 10562 >Antitoxin: Strop_1829
    5019 >Toxin: DehaBAV1_0047 10563 >Antitoxin: DehaBAV1_0048
    5020 >Toxin: TBFG_12576 10564 >Antitoxin: TBFG_12575
    5021 >Toxin: Franean1_1694 10565 >Antitoxin: Franean1_1695
    5022 >Toxin: Sare_1819 10566 >Antitoxin: Sare_1820
    5023 >Toxin: BLD_0616 10567 >Antitoxin: BLD_0615
    5024 >Toxin: MARTH_orf202 10568 >Antitoxin: MARTH_orf203
    5025 >Toxin: UUR10_0378 10569 >Antitoxin: UUR10_0379
    5026 >Toxin: Dtur_1023 10570 >Antitoxin: Dtur_1024
    5027 >Toxin: Achl_2015 10571 >Antitoxin: Achl_2014
    5028 >Toxin: Afer_0951 10572 >Antitoxin: Afer_0952
    5029 >Toxin: Amir_5265 10573 >Antitoxin: Amir_5264
    5030 >Toxin: Bcav_2014 10574 >Antitoxin: Bcav_2015
    5031 >Toxin: Bfae_16360 10575 >Antitoxin: Bfae_16350
    5032 >Toxin: Mlut_12720 10576 >Antitoxin: Mlut_12710
    5033 >Toxin: Ksed_12510 10577 >Antitoxin: Ksed_12520
    5034 >Toxin: Elen_2555 10578 >Antitoxin: Elen_2554
    5035 >Toxin: Caci_2378 10579 >Antitoxin: Caci_2379
    5036 >Toxin: Ccur_05100 10580 >Antitoxin: Ccur_05110
    5037 >Toxin: Svir_15270 10581 >Antitoxin: Svir_15280
    5038 >Toxin: Jden_1351 10582 >Antitoxin: Jden_1350
    5039 >Toxin: Shel_07800 10583 >Antitoxin: Shel_07810
    5040 >Toxin: Apar_0457 10584 >Antitoxin: Apar_0458
    5041 >Toxin: Namu_3297 10585 >Antitoxin: Namu_3296
    5042 >Toxin: BR0669 10586 >Antitoxin: BR0670
    5043 >Toxin: Meso_0960 10587 >Antitoxin: Meso_0961
    5044 >Toxin: Smed_0707 10588 >Antitoxin: Smed_0708
    5045 >Toxin: Oant_2617 10589 >Antitoxin: Oant_2616
    5046 >Toxin: Rleg2_1014 10590 >Antitoxin: Rleg2_1015
    5047 >Toxin: Avi_1463 10591 >Antitoxin: Avi_1464
    5048 >Toxin: Rleg_1166 10592 >Antitoxin: Rleg_1167
    5049 >Toxin: Reut_B5756 10593 >Antitoxin: Reut_B5755
    5050 >Toxin: Bcep18194_C6667 10594 >Antitoxin: Bcep18194_C6668
    5051 >Toxin: Bcep18194_C7628 10595 >Antitoxin: Bcep18194_C7629
    5052 >Toxin: Rmet_3558 10596 >Antitoxin: Rmet_3557
    5053 >Toxin: Daci_1984 10597 >Antitoxin: Daci_1983
    5054 >Toxin: Bmul_6053 10598 >Antitoxin: Bmul_6054
    5055 >Toxin: Bphyt_4360 10599 >Antitoxin: Bphyt_4361
    5056 >Toxin: Gdia_0758 10600 >Antitoxin: Gdia_0759
    5057 >Toxin: Rfer_1402 10601 >Antitoxin: Rfer_1401
    5058 >Toxin: Bpro_1690 10602 >Antitoxin: Bpro_1689
    5059 >Toxin: Aave_1884 10603 >Antitoxin: Aave_1883
    5060 >Toxin: Ajs_3355 10604 >Antitoxin: Ajs_3356
    5061 >Toxin: Pnap_1461 10605 >Antitoxin: Pnap_1460
    5062 >Toxin: Veis_3939 10606 >Antitoxin: Veis_3938
    5063 >Toxin: Mpe_A1104 10607 >Antitoxin: Mpe_A1103
    5064 >Toxin: Daci_5345 10608 >Antitoxin: Daci_5346
    5065 >Toxin: Lcho_0681 10609 >Antitoxin: Lcho_0680
    5066 >Toxin: Dtpsy_2703 10610 >Antitoxin: Dtpsy_2704
    5067 >Toxin: Vapar_1473 10611 >Antitoxin: Vapar_1472
    5068 >Toxin: DET0199 10612 >Antitoxin: DET0198
    5069 >Toxin: Mbar_A2432 10613 >Antitoxin: Mbar_A2431
    5070 >Toxin: Dde_2738 10614 >Antitoxin: Dde_2739
    5071 >Toxin: Mhun_0927 10615 >Antitoxin: Mhun_0928
    5072 >Toxin: Mthe_0907 10616 >Antitoxin: Mthe_0908
    5073 >Toxin: Sfum_0349 10617 >Antitoxin: Sfum_0348
    5074 >Toxin: Dvul_2100 10618 >Antitoxin: Dvul_2101
    5075 >Toxin: Memar_2454 10619 >Antitoxin: Memar_2455
    5076 >Toxin: DehaBAV1_0154 10620 >Antitoxin: DehaBAV1_0155
    5077 >Toxin: Mboo_0831 10621 >Antitoxin: Mboo_0832
    5078 >Toxin: Dole_2967 10622 >Antitoxin: Dole_2968
    5079 >Toxin: DvMF_2934 10623 >Antitoxin: DvMF_2933
    5080 >Toxin: Dbac_0077 10624 >Antitoxin: Dbac_0078
    5081 >Toxin: Mpal_0502 10625 >Antitoxin: Mpal_0501
    5082 >Toxin: Dret_1407 10626 >Antitoxin: Dret_1406
    5083 >Toxin: Mbar_A0388 10627 >Antitoxin: Mbar_A0387
    5084 >Toxin: Mhun_1181 10628 >Antitoxin: Mhun_1182
    5085 >Toxin: Mbur_1241 10629 >Antitoxin: Mbur_1242
    5086 >Toxin: Mthe_1611 10630 >Antitoxin: Mthe_1610
    5087 >Toxin: Mlab_1220 10631 >Antitoxin: Mlab_1221
    5088 >Toxin: Memar_0285 10632 >Antitoxin: Memar_0286
    5089 >Toxin: MmarC7_0296 10633 >Antitoxin: MmarC7_0297
    5090 >Toxin: Mboo_2347 10634 >Antitoxin: Mboo_2346
    5091 >Toxin: Hlac_0279 10635 >Antitoxin: Hlac_0280
    5092 >Toxin: Huta_1435 10636 >Antitoxin: Huta_1436
    5093 >Toxin: Mpal_2679 10637 >Antitoxin: Mpal_2678
    5094 >Toxin: Hmuk_1279 10638 >Antitoxin: Hmuk_1280
    5095 >Toxin: Jann_1095 10639 >Antitoxin: Jann_1094
    5096 >Toxin: TM1040_1726 10640 >Antitoxin: TM1040_1727
    5097 >Toxin: RSP_0387 10641 >Antitoxin: RSP_0386
    5098 >Toxin: Rsph17029_2041 10642 >Antitoxin: Rsph17029_2040
    5099 >Toxin: Rsph17025_0849 10643 >Antitoxin: Rsph17025_0850
    5100 >Toxin: Dshi_0770 10644 >Antitoxin: Dshi_0769
    5101 >Toxin: Dd1591_2975 10645 >Antitoxin: Dd1591_2974
    5102 >Toxin: RPB_3184 10646 >Antitoxin: RPB_3183
    5103 >Toxin: Saro_0334 10647 >Antitoxin: Saro_0335
    5104 >Toxin: Jann_2923 10648 >Antitoxin: Jann_2924
    5105 >Toxin: RPC_1882 10649 >Antitoxin: RPC_1883
    5106 >Toxin: RPD_2314 10650 >Antitoxin: RPD_2315
    5107 >Toxin: TM1040_2218 10651 >Antitoxin: TM1040_2217
    5108 >Toxin: RSP_2821 10652 >Antitoxin: RSP_2822
    5109 >Toxin: Pden_2540 10653 >Antitoxin: Pden_2539
    5110 >Toxin: Rsph17029_1475 10654 >Antitoxin: Rsph17029_1474
    5111 >Toxin: Rsph17025_1525 10655 >Antitoxin: Rsph17025_1524
    5112 >Toxin: Daci_5844 10656 >Antitoxin: Daci_5845
    5113 >Toxin: Rpal_2374 10657 >Antitoxin: Rpal_2375
    5114 >Toxin: Mchl_5685 10658 >Antitoxin: Mchl_5686
    5115 >Toxin: Rru_A0999 10659 >Antitoxin: Rru_A0998
    5116 >Toxin: RSP_0542 10660 >Antitoxin: RSP_0543
    5117 >Toxin: Rsph17029_2193 10661 >Antitoxin: Rsph17029_2194
    5118 >Toxin: Rsph17025_1245 10662 >Antitoxin: Rsph17025_1244
    5119 >Toxin: BBta_5905 10663 >Antitoxin: BBta_5906
    5120 >Toxin: Xaut_0110 10664 >Antitoxin: Xaut_0109
    5121 >Toxin: M446_3597 10665 >Antitoxin: M446_3598
    5122 >Toxin: Gdia_1571 10666 >Antitoxin: Gdia_1572
    5123 >Toxin: Mnod_4011 10667 >Antitoxin: Mnod_4010
    5124 >Toxin: MCA0079 10668 >Antitoxin: MCA0080
    5125 >Toxin: BTH_II1184 10669 >Antitoxin: BTH_II1185
    5126 >Toxin: BBta_1297 10670 >Antitoxin: BBta_1298
    5127 >Toxin: Mext_4520 10671 >Antitoxin: Mext_4521
    5128 >Toxin: Mrad2831_3340 10672 >Antitoxin: Mrad2831_3341
    5129 >Toxin: Mpop_5032 10673 >Antitoxin: Mpop_5033
    5130 >Toxin: Mchl_4980 10674 >Antitoxin: Mchl_4981
    5131 >Toxin: BRA0156 10675 >Antitoxin: BRA0155
    5132 >Toxin: RPC_1099 10676 >Antitoxin: RPC_1098
    5133 >Toxin: Meso_0293 10677 >Antitoxin: Meso_0294
    5134 >Toxin: BARBAKC583_1139 10678 >Antitoxin: BARBAKC583_1140
    5135 >Toxin: Smed_0260 10679 >Antitoxin: Smed_0259
    5136 >Toxin: Oant_4207 10680 >Antitoxin: Oant_4208
    5137 >Toxin: Mext_0623 10681 >Antitoxin: Mext_0622
    5138 >Toxin: Mrad2831_1687 10682 >Antitoxin: Mrad2831_1688
    5139 >Toxin: Bind_3651 10683 >Antitoxin: Bind_3652
    5140 >Toxin: Mpop_0601 10684 >Antitoxin: Mpop_0600
    5141 >Toxin: M446_3678 10685 >Antitoxin: M446_3677
    5142 >Toxin: Rleg2_0322 10686 >Antitoxin: Rleg2_0321
    5143 >Toxin: Msil_0029 10687 >Antitoxin: Msil_0028
    5144 >Toxin: Mchl_0634 10688 >Antitoxin: Mchl_0633
    5145 >Toxin: Avi_0742 10689 >Antitoxin: Avi_0741
    5146 >Toxin: Rleg_0354 10690 >Antitoxin: Rleg_0353
    5147 >Toxin: Nwi_0763 10691 >Antitoxin: Nwi_0764
    5148 >Toxin: RPB_4586 10692 >Antitoxin: RPB_4585
    5149 >Toxin: RPC_4789 10693 >Antitoxin: RPC_4790
    5150 >Toxin: RPD_0816 10694 >Antitoxin: RPD_0817
    5151 >Toxin: Nham_3461 10695 >Antitoxin: Nham_3460
    5152 >Toxin: Mmar10_2232 10696 >Antitoxin: Mmar10_2231
    5153 >Toxin: BBta_0830 10697 >Antitoxin: BBta_0831
    5154 >Toxin: Smed_0520 10698 >Antitoxin: Smed_0521
    5155 >Toxin: Plav_0671 10699 >Antitoxin: Plav_0672
    5156 >Toxin: Xaut_4646 10700 >Antitoxin: Xaut_4645
    5157 >Toxin: Mext_3261 10701 >Antitoxin: Mext_3260
    5158 >Toxin: Mrad2831_0031 10702 >Antitoxin: Mrad2831_0030
    5159 >Toxin: Bind_0728 10703 >Antitoxin: Bind_0727
    5160 >Toxin: Mpop_3457 10704 >Antitoxin: Mpop_3456
    5161 >Toxin: Rpal_0901 10705 >Antitoxin: Rpal_0902
    5162 >Toxin: M446_2347 10706 >Antitoxin: M446_2346
    5163 >Toxin: Rleg2_0604 10707 >Antitoxin: Rleg2_0605
    5164 >Toxin: Msil_1239 10708 >Antitoxin: Msil_1240
    5165 >Toxin: Mchl_3585 10709 >Antitoxin: Mchl_3584
    5166 >Toxin: Mnod_0262 10710 >Antitoxin: Mnod_0263
    5167 >Toxin: Avi_1042 10711 >Antitoxin: Avi_1043
    5168 >Toxin: Rleg_0644 10712 >Antitoxin: Rleg_0645
    5169 >Toxin: WD1300 10713 >Antitoxin: WD1299
    5170 >Toxin: Tfu_0350 10714 >Antitoxin: Tfu_0349
    5171 >Toxin: ECH_0468 10715 >Antitoxin: ECH_0469
    5172 >Toxin: Ssed_3664 10716 >Antitoxin: Ssed_3665
    5173 >Toxin: Spea_3282 10717 >Antitoxin: Spea_3283
    5174 >Toxin: Swoo_3951 10718 >Antitoxin: Swoo_3952
    5175 >Toxin: Dret_0793 10719 >Antitoxin: Dret_0792
    5176 >Toxin: RSc2370 10720 >Antitoxin: RSc2369
    5177 >Toxin: Reut_A0685 10721 >Antitoxin: Reut_A0686
    5178 >Toxin: Bpro_0940 10722 >Antitoxin: Bpro_0941
    5179 >Toxin: Ajs_3461 10723 >Antitoxin: Ajs_3460
    5180 >Toxin: Pnap_0837 10724 >Antitoxin: Pnap_0838
    5181 >Toxin: Mpe_A1173 10725 >Antitoxin: Mpe_A1174
    5182 >Toxin: Pnuc_1395 10726 >Antitoxin: Pnuc_1394
    5183 >Toxin: Lcho_3725 10727 >Antitoxin: Lcho_3724
    5184 >Toxin: Rpic_2583 10728 >Antitoxin: Rpic_2582
    5185 >Toxin: Dtpsy_2788 10729 >Antitoxin: Dtpsy_2787
    5186 >Toxin: Vapar_4059 10730 >Antitoxin: Vapar_4058
    5187 >Toxin: Rpic12D_2179 10731 >Antitoxin: Rpic12D_2178
    5188 >Toxin: Mbar_A3386 10732 >Antitoxin: Mbar_A3387
    5189 >Toxin: Mhun_1603 10733 >Antitoxin: Mhun_1602
    5190 >Toxin: Mbur_0221 10734 >Antitoxin: Mbur_0220
    5191 >Toxin: Mthe_0333 10735 >Antitoxin: Mthe_0332
    5192 >Toxin: Pisl_0588 10736 >Antitoxin: Pisl_0587
    5193 >Toxin: Mlab_1499 10737 >Antitoxin: Mlab_1498
    5194 >Toxin: Memar_0719 10738 >Antitoxin: Memar_0720
    5195 >Toxin: MmarC5_0965 10739 >Antitoxin: MmarC5_0964
    5196 >Toxin: Pars_1758 10740 >Antitoxin: Pars_1757
    5197 >Toxin: Msed_0028 10741 >Antitoxin: Msed_0027
    5198 >Toxin: Mevan_1555 10742 >Antitoxin: Mevan_1554
    5199 >Toxin: Maeo_0079 10743 >Antitoxin: Maeo_0080
    5200 >Toxin: MmarC7_1663 10744 >Antitoxin: MmarC7_1664
    5201 >Toxin: Mboo_0688 10745 >Antitoxin: Mboo_0689
    5202 >Toxin: MmarC6_0250 10746 >Antitoxin: MmarC6_0249
    5203 >Toxin: Nmar_0227 10747 >Antitoxin: Nmar_0226
    5204 >Toxin: Tneu_1579 10748 >Antitoxin: Tneu_1578
    5205 >Toxin: Hlac_1844 10749 >Antitoxin: Hlac_1843
    5206 >Toxin: Huta_0752 10750 >Antitoxin: Huta_0751
    5207 >Toxin: Mpal_0525 10751 >Antitoxin: Mpal_0524
    5208 >Toxin: Hmuk_2623 10752 >Antitoxin: Hmuk_2622
    5209 >Toxin: BR1407 10753 >Antitoxin: BR1406
    5210 >Toxin: Nwi_2355 10754 >Antitoxin: Nwi_2354
    5211 >Toxin: RPB_2028 10755 >Antitoxin: RPB_2029
    5212 >Toxin: RPC_3273 10756 >Antitoxin: RPC_3272
    5213 >Toxin: RPD_3362 10757 >Antitoxin: RPD_3361
    5214 >Toxin: Nham_2734 10758 >Antitoxin: Nham_2733
    5215 >Toxin: RSP_2135 10759 >Antitoxin: RSP_2137
    5216 >Toxin: Pden_1906 10760 >Antitoxin: Pden_1907
    5217 >Toxin: Rsph17029_0810 10761 >Antitoxin: Rsph17029_0811
    5218 >Toxin: Rsph17025_0719 10762 >Antitoxin: Rsph17025_0720
    5219 >Toxin: BBta_6108 10763 >Antitoxin: BBta_6107
    5220 >Toxin: BOV_1364 10764 >Antitoxin: BOV_1363
    5221 >Toxin: Smed_2029 10765 >Antitoxin: Smed_2028
    5222 >Toxin: Oant_1770 10766 >Antitoxin: Oant_1771
    5223 >Toxin: Mext_2666 10767 >Antitoxin: Mext_2665
    5224 >Toxin: Mrad2831_4519 10768 >Antitoxin: Mrad2831_4520
    5225 >Toxin: Bind_0773 10769 >Antitoxin: Bind_0772
    5226 >Toxin: Mpop_2788 10770 >Antitoxin: Mpop_2787
    5227 >Toxin: Rpal_4016 10771 >Antitoxin: Rpal_4015
    5228 >Toxin: M446_1097 10772 >Antitoxin: M446_1096
    5229 >Toxin: Rleg2_2546 10773 >Antitoxin: Rleg2_2545
    5230 >Toxin: Msil_0597 10774 >Antitoxin: Msil_0598
    5231 >Toxin: Mchl_2893 10775 >Antitoxin: Mchl_2892
    5232 >Toxin: Mnod_0693 10776 >Antitoxin: Mnod_0694
    5233 >Toxin: Avi_2826 10777 >Antitoxin: Avi_2825
    5234 >Toxin: Rleg_2805 10778 >Antitoxin: Rleg_2804
    5235 >Toxin: RPC_4221 10779 >Antitoxin: RPC_4220
    5236 >Toxin: Ppro_3346 10780 >Antitoxin: Ppro_3347
    5237 >Toxin: P9515_14121 10781 >Antitoxin: P9515_14111
    5238 >Toxin: Mlab_1426 10782 >Antitoxin: Mlab_1425
    5239 >Toxin: Mext_0850 10783 >Antitoxin: Mext_0849
    5240 >Toxin: Mpop_0774 10784 >Antitoxin: Mpop_0773
    5241 >Toxin: PCC8801_3331 10785 >Antitoxin: PCC8801_3332
    5242 >Toxin: Mchl_0809 10786 >Antitoxin: Mchl_0808
    5243 >Toxin: Dbac_1869 10787 >Antitoxin: Dbac_1870
    5244 >Toxin: Cyan8802_2786 10788 >Antitoxin: Cyan8802_2785
    5245 >Toxin: Jann_2651 10789 >Antitoxin: Jann_2652
    5246 >Toxin: TM1040_1731 10790 >Antitoxin: TM1040_1732
    5247 >Toxin: RSP_0760 10791 >Antitoxin: RSP_0761
    5248 >Toxin: Pden_0936 10792 >Antitoxin: Pden_0937
    5249 >Toxin: Rsph17029_2419 10793 >Antitoxin: Rsph17029_2420
    5250 >Toxin: Rsph17025_0415 10794 >Antitoxin: Rsph17025_0414
    5251 >Toxin: Dshi_0765 10795 >Antitoxin: Dshi_0764
    5252 >Toxin: BR0096 10796 >Antitoxin: BR0097
    5253 >Toxin: Nwi_0320 10797 >Antitoxin: Nwi_0319
    5254 >Toxin: RPC_0207 10798 >Antitoxin: RPC_0208
    5255 >Toxin: RPD_0522 10799 >Antitoxin: RPD_0521
    5256 >Toxin: Nham_0411 10800 >Antitoxin: Nham_0412
    5257 >Toxin: Meso_3360 10801 >Antitoxin: Meso_3361
    5258 >Toxin: BBta_0379 10802 >Antitoxin: BBta_0378
    5259 >Toxin: BOV_0094 10803 >Antitoxin: BOV_0095
    5260 >Toxin: Smed_3086 10804 >Antitoxin: Smed_3087
    5261 >Toxin: Oant_0108 10805 >Antitoxin: Oant_0109
    5262 >Toxin: Rpal_0202 10806 >Antitoxin: Rpal_0203
    5263 >Toxin: Rleg2_3741 10807 >Antitoxin: Rleg2_3742
    5264 >Toxin: Avi_4191 10808 >Antitoxin: Avi_4192
    5265 >Toxin: Rleg_4064 10809 >Antitoxin: Rleg_4065
    5266 >Toxin: PP_4158 10810 >Antitoxin: PP_4157
    5267 >Toxin: PSPTO_2245 10811 >Antitoxin: PSPTO_2246
    5268 >Toxin: GSU2483 10812 >Antitoxin: GSU2484
    5269 >Toxin: Psyr_2050 10813 >Antitoxin: Psyr_2051
    5270 >Toxin: Reut_A2290 10814 >Antitoxin: Reut_A2289
    5271 >Toxin: RPB_4648 10815 >Antitoxin: RPB_4649
    5272 >Toxin: Nham_2991 10816 >Antitoxin: Nham_2990
    5273 >Toxin: Rmet_0041 10817 >Antitoxin: Rmet_0042
    5274 >Toxin: PA14_43350 10818 >Antitoxin: PA14_43340
    5275 >Toxin: Pnap_3601 10819 >Antitoxin: Pnap_3600
    5276 >Toxin: Spro_1245 10820 >Antitoxin: Spro_1244
    5277 >Toxin: PputGB1_3730 10821 >Antitoxin: PputGB1_3729
    5278 >Toxin: Avin_24520 10822 >Antitoxin: Avin_24530
    5279 >Toxin: Vapar_4991 10823 >Antitoxin: Vapar_4990
    5280 >Toxin: Jann_3967 10824 >Antitoxin: Jann_3968
    5281 >Toxin: RSP_3149 10825 >Antitoxin: RSP_3148
    5282 >Toxin: Veis_2514 10826 >Antitoxin: Veis_2513
    5283 >Toxin: Rsph17029_3886 10827 >Antitoxin: Rsph17029_3885
    5284 >Toxin: Mrad2831_5988 10828 >Antitoxin: Mrad2831_5987
    5285 >Toxin: Mnod_8672 10829 >Antitoxin: Mnod_8671
    5286 >Toxin: Vapar_5617 10830 >Antitoxin: Vapar_5616
    5287 >Toxin: MCA1644 10831 >Antitoxin: MCA1645
    5288 >Toxin: Daro_2905 10832 >Antitoxin: Daro_2904
    5289 >Toxin: Tbd_1531 10833 >Antitoxin: Tbd_1530
    5290 >Toxin: Noc_2387 10834 >Antitoxin: Noc_2386
    5291 >Toxin: Nmul_A2113 10835 >Antitoxin: Nmul_A2112
    5292 >Toxin: Mfla_1880 10836 >Antitoxin: Mfla_1879
    5293 >Toxin: Mlg_0717 10837 >Antitoxin: Mlg_0718
    5294 >Toxin: PXO_00875 10838 >Antitoxin: PXO_00874
    5295 >Toxin: Smal_2687 10839 >Antitoxin: Smal_2688
    5296 >Toxin: Lferr_0814 10840 >Antitoxin: Lferr_0815
    5297 >Toxin: Tmz1t_2263 10841 >Antitoxin: Tmz1t_2262
    5298 >Toxin: AFE_0665 10842 >Antitoxin: AFE_0666
    5299 >Toxin: PSPTO_5226 10843 >Antitoxin: PSPTO_5225
    5300 >Toxin: Psyr_0318 10844 >Antitoxin: Psyr_0319
    5301 >Toxin: Noc_2576 10845 >Antitoxin: Noc_2577
    5302 >Toxin: Sde_3516 10846 >Antitoxin: Sde_3515
    5303 >Toxin: Mlg_2539 10847 >Antitoxin: Mlg_2540
    5304 >Toxin: PA14_69030 10848 >Antitoxin: PA14_69020
    5305 >Toxin: Hhal_1182 10849 >Antitoxin: Hhal_1183
    5306 >Toxin: Maqu_3434 10850 >Antitoxin: Maqu_3433
    5307 >Toxin: Pmen_0322 10851 >Antitoxin: Pmen_0323
    5308 >Toxin: Pput_5110 10852 >Antitoxin: Pput_5109
    5309 >Toxin: PSPA7_5970 10853 >Antitoxin: PSPA7_5969
    5310 >Toxin: COXBURSA331_A0127 10854 >Antitoxin: COXBURSA331_A0126
    5311 >Toxin: PputGB1_5263 10855 >Antitoxin: PputGB1_5262
    5312 >Toxin: PputW619_5028 10856 >Antitoxin: PputW619_5027
    5313 >Toxin: Xfasm12_0963 10857 >Antitoxin: Xfasm12_0964
    5314 >Toxin: XfasM23_0845 10858 >Antitoxin: XfasM23_0846
    5315 >Toxin: PXO_02381 10859 >Antitoxin: PXO_02380
    5316 >Toxin: Smal_3280 10860 >Antitoxin: Smal_3279
    5317 >Toxin: Tgr7_2768 10861 >Antitoxin: Tgr7_2767
    5318 >Toxin: Avin_47360 10862 >Antitoxin: Avin_47350
    5319 >Toxin: SO_2677 10863 >Antitoxin: SO_2676
    5320 >Toxin: MCA2934 10864 >Antitoxin: MCA2935
    5321 >Toxin: Mfla_2702 10865 >Antitoxin: Mfla_2703
    5322 >Toxin: Rsph17025_2105 10866 >Antitoxin: Rsph17025_2106
    5323 >Toxin: VC0395_A0765 10867 >Antitoxin: VC0395_A0766
    5324 >Toxin: Shew185_2087 10868 >Antitoxin: Shew185_2086
    5325 >Toxin: Xaut_4504 10869 >Antitoxin: Xaut_4505
    5326 >Toxin: Sbal195_2134 10870 >Antitoxin: Sbal195_2133
    5327 >Toxin: Lcho_2251 10871 >Antitoxin: Lcho_2252
    5328 >Toxin: Rpal_0669 10872 >Antitoxin: Rpal_0668
    5329 >Toxin: Rpal_3387 10873 >Antitoxin: Rpal_3386
    5330 >Toxin: Tgr7_1643 10874 >Antitoxin: Tgr7_1644
    5331 >Toxin: Avi_3009 10875 >Antitoxin: Avi_3010
    5332 >Toxin: BR0179 10876 >Antitoxin: BR0180
    5333 >Toxin: RPB_1399 10877 >Antitoxin: RPB_1400
    5334 >Toxin: Saro_2556 10878 >Antitoxin: Saro_2557
    5335 >Toxin: Rru_A1933 10879 >Antitoxin: Rru_A1932
    5336 >Toxin: Meso_3989 10880 >Antitoxin: Meso_3990
    5337 >Toxin: BOV_0173 10881 >Antitoxin: BOV_0174
    5338 >Toxin: Smed_1099 10882 >Antitoxin: Smed_1100
    5339 >Toxin: Oant_0188 10883 >Antitoxin: Oant_0189
    5340 >Toxin: Caul_3269 10884 >Antitoxin: Caul_3268
    5341 >Toxin: Rpal_4696 10885 >Antitoxin: Rpal_4695
    5342 >Toxin: Rleg2_1636 10886 >Antitoxin: Rleg2_1637
    5343 >Toxin: Avi_2146 10887 >Antitoxin: Avi_2147
    5344 >Toxin: Rleg_1823 10888 >Antitoxin: Rleg_1824
    5345 >Toxin: Jann_0425 10889 >Antitoxin: Jann_0424
    5346 >Toxin: RPC_0267 10890 >Antitoxin: RPC_0266
    5347 >Toxin: RPD_0453 10891 >Antitoxin: RPD_0454
    5348 >Toxin: TM1040_0057 10892 >Antitoxin: TM1040_0056
    5349 >Toxin: RSP_1492 10893 >Antitoxin: RSP_1491
    5350 >Toxin: Rsph17029_0142 10894 >Antitoxin: Rsph17029_0141
    5351 >Toxin: Rsph17025_2897 10895 >Antitoxin: Rsph17025_2896
    5352 >Toxin: Swit_3664 10896 >Antitoxin: Swit_3665
    5353 >Toxin: Smed_3048 10897 >Antitoxin: Smed_3047
    5354 >Toxin: Xaut_2211 10898 >Antitoxin: Xaut_2212
    5355 >Toxin: Caul_4587 10899 >Antitoxin: Caul_4588
    5356 >Toxin: Rpal_0271 10900 >Antitoxin: Rpal_0270
    5357 >Toxin: Rleg2_3772 10901 >Antitoxin: Rleg2_3773
    5358 >Toxin: Avi_4242 10902 >Antitoxin: Avi_4243
    5359 >Toxin: Rleg_4096 10903 >Antitoxin: Rleg_4097
    5360 >Toxin: Pcar_2797 10904 >Antitoxin: Pcar_2798
    5361 >Toxin: Plut_0583 10905 >Antitoxin: Plut_0584
    5362 >Toxin: Francci3_3111 10906 >Antitoxin: Francci3_3112
    5363 >Toxin: Ppro_1965 10907 >Antitoxin: Ppro_1966
    5364 >Toxin: Dvul_0285 10908 >Antitoxin: Dvul_0284
    5365 >Toxin: Fnod_0467 10909 >Antitoxin: Fnod_0466
    5366 >Toxin: Franean1_1807 10910 >Antitoxin: Franean1_1806
    5367 >Toxin: Cmaq_0059 10911 >Antitoxin: Cmaq_0058
    5368 >Toxin: Emin_1036 10912 >Antitoxin: Emin_1037
    5369 >Toxin: DvMF_2367 10913 >Antitoxin: DvMF_2366
    5370 >Toxin: Tbd_0578 10914 >Antitoxin: Tbd_0579
    5371 >Toxin: Rfer_2118 10915 >Antitoxin: Rfer_2117
    5372 >Toxin: Bpro_2410 10916 >Antitoxin: Bpro_2409
    5373 >Toxin: Aave_3160 10917 >Antitoxin: Aave_3161
    5374 >Toxin: Ajs_1923 10918 >Antitoxin: Ajs_1922
    5375 >Toxin: Pnap_2167 10919 >Antitoxin: Pnap_2168
    5376 >Toxin: Veis_4920 10920 >Antitoxin: Veis_4921
    5377 >Toxin: Lcho_2593 10921 >Antitoxin: Lcho_2594
    5378 >Toxin: Dtpsy_1727 10922 >Antitoxin: Dtpsy_1726
    5379 >Toxin: Vapar_2732 10923 >Antitoxin: Vapar_2731
    5380 >Toxin: Francci3_3069 10924 >Antitoxin: Francci3_3068
    5381 >Toxin: Mmcs_5077 10925 >Antitoxin: Mmcs_5078
    5382 >Toxin: Mkms_5165 10926 >Antitoxin: Mkms_5166
    5383 >Toxin: Mjls_5456 10927 >Antitoxin: Mjls_5457
    5384 >Toxin: Franean1_1843 10928 >Antitoxin: Franean1_1844
    5385 >Toxin: Afer_0480 10929 >Antitoxin: Afer_0481
    5386 >Toxin: Amir_3816 10930 >Antitoxin: Amir_3815
    5387 >Toxin: Svir_26430 10931 >Antitoxin: Svir_26440
    5388 >Toxin: Namu_2498 10932 >Antitoxin: Namu_2499
    5389 >Toxin: Adeh_2402 10933 >Antitoxin: Adeh_2401
    5390 >Toxin: Anae109_0244 10934 >Antitoxin: Anae109_0245
    5391 >Toxin: CCC13826_1729 10935 >Antitoxin: CCC13826_1728
    5392 >Toxin: Oter_1803 10936 >Antitoxin: Oter_1804
    5393 >Toxin: SYO3AOP1_0447 10937 >Antitoxin: SYO3AOP1_0448
    5394 >Toxin: AnaeK_1461 10938 >Antitoxin: AnaeK_1462
    5395 >Toxin: Dhaf_0209 10939 >Antitoxin: Dhaf_0210
    5396 >Toxin: A2cp1_1556 10940 >Antitoxin: A2cp1_1557
    5397 >Toxin: Hlac_1165 10941 >Antitoxin: Hlac_1164
    5398 >Toxin: Daro_3986 10942 >Antitoxin: Daro_3985
    5399 >Toxin: Rmet_1293 10943 >Antitoxin: Rmet_1292
    5400 >Toxin: RSP_0499 10944 >Antitoxin: RSP_0500
    5401 >Toxin: Mlg_2026 10945 >Antitoxin: Mlg_2025
    5402 >Toxin: Pden_3101 10946 >Antitoxin: Pden_3102
    5403 >Toxin: Pnap_1971 10947 >Antitoxin: Pnap_1970
    5404 >Toxin: Mpe_A2822 10948 >Antitoxin: Mpe_A2821
    5405 >Toxin: Rsph17029_2150 10949 >Antitoxin: Rsph17029_2151
    5406 >Toxin: Rsph17025_3372 10950 >Antitoxin: Rsph17025_3371
    5407 >Toxin: Xaut_2176 10951 >Antitoxin: Xaut_2177
    5408 >Toxin: Bind_1153 10952 >Antitoxin: Bind_1154
    5409 >Toxin: Bphy_7262 10953 >Antitoxin: Bphy_7261
    5410 >Toxin: SNSL254_A1648 10954 >Antitoxin: SNSL254_A1647
    5411 >Toxin: SeHA_C1707 10955 >Antitoxin: SeHA_C1706
    5412 >Toxin: SeSA_A1640 10956 >Antitoxin: SeSA_A1639
    5413 >Toxin: SeAg_B1635 10957 >Antitoxin: SeAg_B1636
    5414 >Toxin: SeD_A1800 10958 >Antitoxin: SeD_A1801
    5415 >Toxin: Avin_50560 10959 >Antitoxin: Avin_50550
    5416 >Toxin: MCA0236 10960 >Antitoxin: MCA0237
    5417 >Toxin: Ava_3935 10961 >Antitoxin: Ava_3936
    5418 >Toxin: Ava_4253 10962 >Antitoxin: Ava_4254
    5419 >Toxin: Francci3_4482 10963 >Antitoxin: Francci3_4481
    5420 >Toxin: RPB_0975 10964 >Antitoxin: RPB_0976
    5421 >Toxin: RPC_4457 10965 >Antitoxin: RPC_4456
    5422 >Toxin: Bxe_B1473 10966 >Antitoxin: Bxe_B1474
    5423 >Toxin: RPD_1079 10967 >Antitoxin: RPD_1080
    5424 >Toxin: Pnap_2322 10968 >Antitoxin: Pnap_2321
    5425 >Toxin: BBta_5919 10969 >Antitoxin: BBta_5918
    5426 >Toxin: Xaut_0094 10970 >Antitoxin: Xaut_0095
    5427 >Toxin: Franean1_6875 10971 >Antitoxin: Franean1_6874
    5428 >Toxin: Lcho_1352 10972 >Antitoxin: Lcho_1353
    5429 >Toxin: Bind_0479 10973 >Antitoxin: Bind_0480
    5430 >Toxin: Rpal_5095 10974 >Antitoxin: Rpal_5094
    5431 >Toxin: Lferr_1232 10975 >Antitoxin: Lferr_1231
    5432 >Toxin: M446_3540 10976 >Antitoxin: M446_3541
    5433 >Toxin: Gdia_1564 10977 >Antitoxin: Gdia_1563
    5434 >Toxin: Msil_3626 10978 >Antitoxin: Msil_3625
    5435 >Toxin: PCC7424_2113 10979 >Antitoxin: PCC7424_2112
    5436 >Toxin: PCC8801_1791 10980 >Antitoxin: PCC8801_1792
    5437 >Toxin: AFE_1514 10981 >Antitoxin: AFE_1513
    5438 >Toxin: Cyan7425_4799 10982 >Antitoxin: Cyan7425_4800
    5439 >Toxin: Mnod_3990 10983 >Antitoxin: Mnod_3989
    5440 >Toxin: Cyan8802_1819 10984 >Antitoxin: Cyan8802_1820
    5441 >Toxin: BR0572 10985 >Antitoxin: BR0571
    5442 >Toxin: RPC_1776 10986 >Antitoxin: RPC_1775
    5443 >Toxin: Meso_0874 10987 >Antitoxin: Meso_0873
    5444 >Toxin: Mmar10_0895 10988 >Antitoxin: Mmar10_0894
    5445 >Toxin: BOV_0573 10989 >Antitoxin: BOV_0572
    5446 >Toxin: Oant_2687 10990 >Antitoxin: Oant_2688
    5447 >Toxin: Plav_0876 10991 >Antitoxin: Plav_0875
    5448 >Toxin: Xaut_0072 10992 >Antitoxin: Xaut_0073
    5449 >Toxin: Caul_1204 10993 >Antitoxin: Caul_1203
    5450 >Toxin: Avi_1289 10994 >Antitoxin: Avi_1287
    5451 >Toxin: Ent638_3494 10995 >Antitoxin: Ent638_3495
    5452 >Toxin: EcHS_A0931 10996 >Antitoxin: EcHS_A0930
    5453 >Toxin: Spro_0871 10997 >Antitoxin: Spro_0872
    5454 >Toxin: EcolC_2770 10998 >Antitoxin: EcolC_2771
    5455 >Toxin: SNSL254_A4372 10999 >Antitoxin: SNSL254_A4371
    5456 >Toxin: SeAg_B2861 11000 >Antitoxin: SeAg_B2862
    5457 >Toxin: SeD_A3043 11001 >Antitoxin: SeD_A3044
    5458 >Toxin: PC1_3178 11002 >Antitoxin: PC1_3177
    5459 >Toxin: Nwi_3022 11003 >Antitoxin: Nwi_3023
    5460 >Toxin: Nham_3394 11004 >Antitoxin: Nham_3395
    5461 >Toxin: Dgeo_2838 11005 >Antitoxin: Dgeo_2837
    5462 >Toxin: Pnap_2692 11006 >Antitoxin: Pnap_2691
    5463 >Toxin: Pnuc_1487 11007 >Antitoxin: Pnuc_1486
    5464 >Toxin: Acry_1978 11008 >Antitoxin: Acry_1979
    5465 >Toxin: Swit_0949 11009 >Antitoxin: Swit_0948
    5466 >Toxin: Xaut_1455 11010 >Antitoxin: Xaut_1454
    5467 >Toxin: Xaut_2672 11011 >Antitoxin: Xaut_2671
    5468 >Toxin: Mrad2831_4215 11012 >Antitoxin: Mrad2831_4216
    5469 >Toxin: Mrad2831_5267 11013 >Antitoxin: Mrad2831_5266
    5470 >Toxin: BamMC406_4627 11014 >Antitoxin: BamMC406_4628
    5471 >Toxin: SYO3AOP1_0084 11015 >Antitoxin: SYO3AOP1_0083
    5472 >Toxin: M446_6453 11016 >Antitoxin: M446_6452
    5473 >Toxin: Mchl_5388 11017 >Antitoxin: Mchl_5387
    5474 >Toxin: Mnod_8369 11018 >Antitoxin: Mnod_8370
    5475 >Toxin: Mnod_8664 11019 >Antitoxin: Mnod_8663
    5476 >Toxin: Mnod_2704 11020 >Antitoxin: Mnod_2703
    5477 >Toxin: BR1550 11021 >Antitoxin: BR1549
    5478 >Toxin: Meso_2169 11022 >Antitoxin: Meso_2170
    5479 >Toxin: BOV_1498 11023 >Antitoxin: BOV_1497
    5480 >Toxin: Smed_1549 11024 >Antitoxin: Smed_1548
    5481 >Toxin: Oant_1616 11025 >Antitoxin: Oant_1617
    5482 >Toxin: Rleg2_2786 11026 >Antitoxin: Rleg2_2785
    5483 >Toxin: Avi_3221 11027 >Antitoxin: Avi_3220
    5484 >Toxin: Rleg_3050 11028 >Antitoxin: Rleg_3049
    5485 >Toxin: Swol_0189 11029 >Antitoxin: Swol_0190
    5486 >Toxin: Cthe_2247 11030 >Antitoxin: Cthe_2246
    5487 >Toxin: Teth514_0448 11031 >Antitoxin: Teth514_0449
    5488 >Toxin: Nther_2239 11032 >Antitoxin: Nther_2238
    5489 >Toxin: Hore_17170 11033 >Antitoxin: Hore_17160
    5490 >Toxin: Ccel_0091 11034 >Antitoxin: Ccel_0092
    5491 >Toxin: Athe_1678 11035 >Antitoxin: Athe_1677
    5492 >Toxin: GWCH70_3034 11036 >Antitoxin: GWCH70_3033
    5493 >Toxin: Rru_A0101 11037 >Antitoxin: Rru_A0100
    5494 >Toxin: Smed_3679 11038 >Antitoxin: Smed_3678
    5495 >Toxin: Anae109_0022 11039 >Antitoxin: Anae109_0023
    5496 >Toxin: Rleg2_2305 11040 >Antitoxin: Rleg2_2306
    5497 >Toxin: PCC7424_1520 11041 >Antitoxin: PCC7424_1519
    5498 >Toxin: PCC8801_3755 11042 >Antitoxin: PCC8801_3756
    5499 >Toxin: Cyan7425_0809 11043 >Antitoxin: Cyan7425_0808
    5500 >Toxin: Tgr7_1598 11044 >Antitoxin: Tgr7_1597
    5501 >Toxin: Rleg_2644 11045 >Antitoxin: Rleg_2645
    5502 >Toxin: Cyan8802_3804 11046 >Antitoxin: Cyan8802_3805
    5503 >Toxin: Shewmr7_0736 11047 >Antitoxin: Shewmr7_0735
    5504 >Toxin: Mmwyl1_0574 11048 >Antitoxin: Mmwyl1_0575
    5505 >Toxin: YPK_0910 11049 >Antitoxin: YPK_0909
    5506 >Toxin: SNSL254_A2922 11050 >Antitoxin: SNSL254_A2923
    5507 >Toxin: SeHA_C3478 11051 >Antitoxin: SeHA_C347
    5508 >Toxin: SeAg_B2787 11052 >Antitoxin: SeAg_B2788
    5509 >Toxin: VSAL_I1041 11053 >Antitoxin: VSAL_I1040
    5510 >Toxin: RPB_3182 11054 >Antitoxin: RPB_3183
    5511 >Toxin: Saro_0336 11055 >Antitoxin: Saro_0335
    5512 >Toxin: Jann_2925 11056 >Antitoxin: Jann_2924
    5513 >Toxin: RPC_1884 11057 >Antitoxin: RPC_1883
    5514 >Toxin: RPD_2316 11058 >Antitoxin: RPD_2315
    5515 >Toxin: RSP_2823 11059 >Antitoxin: RSP_2822
    5516 >Toxin: Pden_2538 11060 >Antitoxin: Pden_2539
    5517 >Toxin: Rsph17029_1473 11061 >Antitoxin: Rsph17029_1474
    5518 >Toxin: Rpal_2376 11062 >Antitoxin: Rpal_2375
    5519 >Toxin: Mchl_5687 11063 >Antitoxin: Mchl_5686
    5520 >Toxin: Francci3_3214 11064 >Antitoxin: Francci3_3215
    5521 >Toxin: Arth_2278 11065 >Antitoxin: Arth_2279
    5522 >Toxin: Acel_1330 11066 >Antitoxin: Acel_1331
    5523 >Toxin: Noca_2407 11067 >Antitoxin: Noca_2406
    5524 >Toxin: Strop_1828 11068 >Antitoxin: Strop_1827
    5525 >Toxin: Franean1_1694 11069 >Antitoxin: Franean1_1693
    5526 >Toxin: Sare_1819 11070 >Antitoxin: Sare_1818
    5527 >Toxin: Achl_2015 11071 >Antitoxin: Achl_2016
    5528 >Toxin: Amir_5265 11072 >Antitoxin: Amir_5266
    5529 >Toxin: Bcav_2014 11073 >Antitoxin: Bcav_2013
    5530 >Toxin: Caci_2378 11074 >Antitoxin: Caci_2377
    5531 >Toxin: Svir_15270 11075 >Antitoxin: Svir_15260
    5532 >Toxin: Jden_1351 11076 >Antitoxin: Jden_1352
    5533 >Toxin: Namu_3297 11077 >Antitoxin: Namu_3298
    5534 >Toxin: Reut_C6182 11078 >Antitoxin: Reut_C6181
    5535 >Toxin: Reut_C6338 11079 >Antitoxin: Reut_C6339
    5536 >Toxin: Bxe_A1728 11080 >Antitoxin: Bxe_A1729
    5537 >Toxin: Bxe_A0064 11081 >Antitoxin: Bxe_A0063
    5538 >Toxin: Bpro_0341 11082 >Antitoxin: Bpro_0340
    5539 >Toxin: Nham_3807 11083 >Antitoxin: Nham_3806
    5540 >Toxin: Bcen2424_6839 11084 >Antitoxin: Bcen2424_6838
    5541 >Toxin: Smed_6339 11085 >Antitoxin: Smed_6338
    5542 >Toxin: Xaut_4609 11086 >Antitoxin: Xaut_4610
    5543 >Toxin: Bphy_7465 11087 >Antitoxin: Bphy_7466
    5544 >Toxin: Bphyt_6578 11088 >Antitoxin: Bphyt_6579
  • According to yet another aspect of the present invention there is provided an isolated bacterial population genetically modified to express a toxin and a cognate antitoxin thereof, the bacterial population being resistant to a lytic activity of a bacteriophage, wherein said toxin comprises an amino acid sequence at least 90% homologous to a sequence as set forth in SEQ ID NOs: 2773-3117, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 2773-2804, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8317-8348, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 2805-2871, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8349-8415, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 2872-2935, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8416-8479, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 2936-3030, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8480-8574, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 3031-3078, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO: 8575-8622, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 3079-3087, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8623-8631, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 3088-3094, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8632-8638, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 3095-3109, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8639-8653, wherein when said toxin comprises said amino acid sequence at least 90% homologous to SEQ ID NO: 3110-3117, said antitoxin comprises an amino acid sequence at least 90% homologous to the sequence as set forth in SEQ ID NO:8654-8661.
  • Thus for example, the present inventors have found that when the toxin comprises an amino acid sequence as set forth in SEQ ID NOs: 3031-3078, the bacterial population in which the toxin antitoxin system is expressed is protected from the lytic activity of the T7Δ4.3Δ4.5Δ4.7 bacteriophage.
  • Further, when the toxin comprises an amino acid sequence as set forth in SEQ ID NO: 2805-2871, the bacterial population in which the toxin antitoxin system is expressed is protected from the lytic activity of the WT T7 strain.
  • Methods of expressing polypeptides in microbial populations are known in the art and further described herein below.
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as those that direct inducible expression of the nucleotide sequence only under certain conditions. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence into mRNA. A promoter can have a transcription initiation region, which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter can also have a second domain called an operator, which can overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein can bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation can be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence.
  • An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression can therefore be either positive or negative, thereby either enhancing or reducing transcription. Other examples of positive and negative regulatory elements are well known in the art. Various promoters that can be included in the protein expression system include, but are not limited to, a T7/LacO hybrid promoter, a trp promoter, a T7 promoter, a lac promoter, and a bacteriophage lambda promoter. Any suitable promoter can be used to carry out the present invention, including the native promoter or a heterologous promoter. Heterologous promoters can be constitutively active or inducible. A non-limiting example of a heterologous promoter is given in U.S. Pat. No. 6,242,194 to Kullen and Klaenhammer.
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1987) Nature 198:1056), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; Yelverton et al. (1981) Nucleic Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publication Nos. 36,776 and 121,775). The beta-lactamase (bla) promoter system (Weissmann, (1981) “The Cloning of Interferon and Other Mistakes,” in Interferon 3 (ed. I. Gresser); bacteriophage lambda PL (Shimatake et al. (1981) Nature 292:128); the arabinose-inducible araB promoter (U.S. Pat. No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406) promoter systems also provide useful promoter sequences. See also Balbas (2001) Mol. Biotech. 19:251-267, where E. coli expression systems are discussed.
  • In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter can be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433). For example, the tac (Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21) and trc (Brosius et al. (1985) J. Biol. Chem. 260:3539-3541) promoters are hybrid trp-lac promoters comprised of both trp promoter and lac operon sequences that are regulated by the lac repressor. The tac promoter has the additional feature of being an inducible regulatory sequence. Thus, for example, expression of a coding sequence operably linked to the tac promoter can be induced in a cell culture by adding isopropyl-1-thio-.beta.-D-galactoside (IPTG). Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO Publication No. 267,851).
  • The vector can additionally contain a nucleotide sequence encoding the repressor (or inducer) for that promoter. For example, an inducible vector of the present invention can regulate transcription from the Lac operator (LacO) by expressing the nucleotide sequence encoding the Lad repressor protein. Other examples include the use of the lexA gene to regulate expression of pRecA, and the use of trpO to regulate ptrp. Alleles of such genes that increase the extent of repression (e.g., lacIq) or that modify the manner of induction (e.g., lambda CI857, rendering lambda pL thermo-inducible, or lambda CI+, rendering lambda pL chemo-inducible) can be employed.
  • In addition to a functioning promoter sequence, an efficient ribosome-binding site is also useful for the expression of the fusion construct. In prokaryotes, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine et al. (1975) Nature 254:34). The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ end of bacterial 16S rRNA (Steitz et al. (1979) “Genetic Signals and Nucleotide Sequences in Messenger RNA,” in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger, Plenum Press, NY).
  • The bacterial protecting peptides can also be secreted from the cell by creating chimeric DNA molecules that encode a protein comprising a signal peptide sequence fragment that provides for secretion of the two-component regulatory polypeptides in bacteria (U.S. Pat. No. 4,336,336). The signal sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids that direct the secretion of the protein from the cell. The protein is either secreted into the growth medium (Gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (Gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro, encoded between the signal peptide fragment and the protein of the invention.
  • DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb et al. (1984) EMBO J. 3:2437-2442) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212). Other prokaryotic signals include, for example, the signal sequence from penicillinase, Ipp, or heat stable enterotoxin II leaders.
  • Typically, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter, flank the coding sequence. These sequences direct the transcription of an mRNA that can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences (of about 50 nucleotides) that are capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.
  • Bacteria such as Lactobacillus acidophilus generally utilize the translation start codon ATG, which specifies the amino acid methionine (which is modified to N-formylmethionine in prokaryotic organisms). Bacteria also recognize alternative translation start codons, such as the codons GTG and TTG, which code for valine and leucine, respectively. However, when these alternative translation start codons are used as the initiation codon, these codons direct the incorporation of methionine rather than of the amino acid that they normally encode. Lactobacillus acidophilus NCFM recognizes these alternative translation start sites and incorporates methionine as the first amino acid.
  • It will be appreciated that for expressing toxin/antitoxin pairs in microbial populations the polynucleotide sequence encoding the toxin may be comprised in the same expression vector as that comprising the antitoxin or may be comprised on a separate expression vector from that of the antitoxin.
  • Exemplary microbial populations that may be protected from toxins and/or the lytic effect of bacteriophages according to this aspect of the present invention include those bacteria useful in dairy and fermentation processing. Thus, microorganisms in which the peptides described herein may be expressed are those that are useful in the manufacture of milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk. The microorganisms may be probiotic organisms.
  • According to one embodiment, the bacteria is a lactic acid bacteria.
  • As used herein the phrase “lactic acid bacteria” refers to a genus selected from the following: Aerococcus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Melissococcus, Alloiococcus, Dolosigranulum, Lactosphaera, Tetragenococcus, Vagococcus, and Weissella (Holzapfel et al. (2001) Am. J. Clin. Nutr. 73:365 S-373S; Bergey's Manual of Systematic Bacteriology, Vol. 2 (Williams and Wilkins, Baltimore (1986)) pp. 1075-1079).
  • The production of bacteria comprising the toxins and/or antitoxins of the present invention, the preparation of starter cultures of such bacteria, and methods of fermenting substrates, particularly food substrates such as milk, can be carried out in accordance with known techniques, including but not limited to those described in Mayra-Makinen and Bigret (1993) Lactic Acid Bacteria. Salminen and vonWright eds. Marcel Dekker, Inc. New York. 65-96; Sandine (1996) Dairy Starter Cultures Cogan and Accolas eds. VCH Publishers, New York. 191-206; Gilliland (1985) Bacterial Starter Cultures for Food. CRC Press, Boca Raton, Fla.
  • The term “fermenting” refers to the energy-yielding, metabolic breakdown of organic compounds by microorganisms that generally proceeds under anaerobic conditions and with the evolution of gas.
  • As used herein the term “about” refers to ±10%.
  • The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
  • The term “consisting of” means “including and limited to”.
  • The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
  • Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
    • EXAMPLES
  • Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
  • Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
    • Materials and Methods
  • Coverage Analysis of Pairs of Genes:
  • Mapping of sequencing clones on 360 bacteria and 28 archaea for which raw clone sequencing data was available was performed as described in (Kimelman et al., 2012). Gene positions and annotations were downloaded as described (Kimelman et al., 2012). For each consecutive pair of genes in each genome, three numbers were recorded:
  • x=<number of clones fully spanning the first and not the second gene>
  • y=<number of clones fully spanning the second gene but not the first gene>
  • z=<number of clones fully spanning both genes>
  • A pair of genes conforming with {x=0; y>0; z>0} or {x>0; y=0; z>0} was declared as “following the TA cloning pattern”. Pairs in which the putative toxin was identified as a ‘hitchhiker’ (Kimelman et al., 2012) were eliminated from further counts in order to avoid cases in which this clonability pattern was a byproduct of a nearby single, standalone toxic gene. Pairs residing in replicons lacking sufficient clone coverage were also ignored in further counts.
  • Aggregation of Pairs into Families:
  • Clustering of individual genes based on sequence homology was retrieved from IMG (worldwideweb(dot)img(dot)jgi(dot)doe(dot)gov/cgi-bin/w/main(dot)cgi) on November 2010 (based on the “IMG cluster” field). Cluster IDs were recorded for every consecutive pair of genes analyzed. Pairs containing a gene that lacked a cluster ID were discarded. All pairs having the same two cluster IDs (regardless of the order and the strand) were aggregated into a single “family of pairs”. Families containing less than 7 pairs were ignored, to ensure statistical power in next steps of the analysis. This resulted in 21,417 families, containing at least 7 pairs of consecutive genes sharing the same two cluster IDs, which were further analyzed. The following “TA cloning fraction” (F) parameters were calculated for each family:
  • F1=the fraction of family members that follow the TA cloning pattern {x=0; y>0; z>0}
  • F2=the fraction of family members that follow the TA cloning pattern {x>0; y=0; z>0}
  • Directionality of putative family (i.e., determining whether gene “x” is the putative toxin or the putative antitoxin) was determined using Fmax max{F1,F2}, such that:
  • Fmax=F1→x is the putative toxin
  • Fmax=F2→y is the putative toxin
  • For each family, the Genus names of all containing organisms were extracted. A “family diversity” (FD) measure was defined as the number of different genus names divided by the total number of family members (pairs). This measure was used to roughly assess the tendency of the family to undergo horizontal gene transfer (HGT) within a wide array of organisms, with higher FD corresponding to higher HGT tendency. For example, a 10-members family where all members are found in strains of Escherichia coli will receive a low FD of 0.1. Only families having FD>=0.5 were further analyzed, based on the empirical FD distribution among known families of TA systems (FIG. 9A=S3A).
  • A Statistical Framework to Detect TA Families:
  • Since a given pair of genes has the potential to follow the TA cloning pattern merely by chance (i.e., due to the random clone fragmentation) rather than reflecting a real toxin/antitoxin activity, clone distribution simulations were performed to asses statistical significance per family (FIG. 7=S1). For every pair of genes in each family, all clones covering its genome of origin were randomly distributed on the genome, shuffling clone positions but maintaining their number and sizes. Based on these random clone distributions, the x, y, and z values were measured for the gene pair, and a simulated “TA cloning fraction” (Fsim) was then calculated for the family. This procedure was repeated 1,000 times for each family, generating a distribution of Fsim (i) (i=1 . . . 1000). The real Fmax of the family was then compared to the distribution of Fsim values obtained from the simulations, generating an empirical p value for a family.
  • Since this procedure is computationally demanding to perform for >21000 families, only families where Fmax>=0.3 (i.e., at least 30% of family members followed the TA cloning pattern) were thus analyzed. Families presenting p-value <=0.05 were considered as following the TA cloning pattern in a statistically significantly manner.
  • To identify families significantly localized to Defense Islands (DIs) (Makarova et al., 2011), a value of ‘mean number of defense island genes in proximity’ (DIval) was calculated for each family based on a list of 132 COGs (DI genes) that were shown to be enriched in defense island regions (Makarova et al., 2011). For this, the average number of DI genes within a range of ±5 genes from each family member was calculated. Families having DIval>0.5 were defined as DI associated (FIG. 2), based on the empirical DIval distribution among known families of TA systems (FIG. 9B). Families were further reviewed manually to remove transposon-containing families possibly associated with defense islands due to their transposition-related properties rather than being genuine TA systems.
  • Analysis of Known TA Systems within the Identified Set:
  • The locus tags of all genes from the 24 final families (FIG. 2) were checked against a previously compiled list of known TA systems (Makarova et al., 2009). For a given family, if at least one pair was found in the list of known TA systems, the family was declared as ‘known’. Families that were not declared as ‘known’ were further similarly checked against a list of predicted TA genes downloaded from TADB (http://bioinfo-mml(dot)sjtu(dot)edu(dot)cn/TADB/) (Shao et al., 2011). Families in which at least one pair was found in this TADB list were declared as ‘predicted’. Families found in neither list were declared as ‘novel’.
  • Domain analysis of genes in the identified families was performed by searching their sequences against the Conserved Domain Database (CDD) (Marchler-Bauer et al., 2011) using rpsblast (ftp://ftp(dot)ncbi(dot)nih(dot)gov/blast/documents/rpsblast(dot)html) with e-value threshold of 0.05.
  • For the Phylogenetic distribution analysis of families (FIG. 4), the IMG cluster ids (http://img(dot)jgi(dot)doe(dot)gov/cgi-bin/w/main(dot)cgi) of the toxin and antitoxin for each family were used to retrieve all img adjacent pairs of genes that have the same two cluster ids. For each family, number and identity of organisms carrying members of the family were extracted (Tables 4 and 5, herein below; FIG. 4).
  • Experimental Evaluation of ‘TA Cloning Pattern’:
  • Toxin and antitoxin were amplified from their genomes of origin (in the case of sanaAT, psyrAT and sdenAT) or synthesized (GenScript) for the families pmenAT and rlegAT. The toxin was then directionally ligated into the pRSFDuet-1 vector (EMD Chemicals Inc.) and the antitoxin ligated into the pBAD/HisA plasmid (Invitrogen). Since transformation of the toxin gene alone resulted in mutations in the toxin due to toxicity, the two plasmids (carrying the toxin and antitoxin) were co-transformed into E. coli BL21(DE3)pLysS (Stratagene) in the presence of 0.3% arabinose to induce the antitoxin. The clones were verified by direct sequencing with primers on the pRSFduet-1 and pBAD/HisA vectors.
  • For the toxicity assay on plates, clones were cultured in LB medium with 100 μg/ml ampicillin, 50 μg/ml kanamycin, 34 μg/ml chloramphenicol and 0.3% arabinose overnight. The next day, a portion of each overnight culture was inoculated into fresh medium (10-fold dilution) and 100 were spotted on LB plates supplemented with 100 μg/ml ampicillin, 50 μg/ml kanamycin and 34 μg/ml chloramphenicol. Toxin, antitoxin or both were induced by 100 μM IPTG and 0.3% arabinose, respectively.
  • For the growth kinetics experiments 3 different colonies of each system were cultured in LB medium with 100 μg/ml ampicillin, 50 μg/ml kanamycin, 34 μg/ml chloramphenicol and 0.3% arabinose overnight. The next day cells were diluted 1:20 and measured for OD using 1 cm path cuvetts. Samples were equilibrated to the same OD and 5 μl of these samples were added to 175 μl LB medium supplemented with 100 μg/ml ampicillin, 50 μg/ml kanamycin, 34 μg/ml chloramphenicol in a 96-wells microplate. Cells were placed in Infinite M200 in a script employing a 60 cycles of ˜5 min interval. Measurements were done using 486ex/516em bandwidth (Gain 95, 105 and 110) and OD at 600 nm overnight. For each of the colonies the following treatments were applied: No induction, induction of 100 μM IPTG and/or 0.3% arabinose after ˜4 h, and induction of 100 μM IPTG after ˜4-h and of 0.3% arabinose after another ˜2.5 hours. The overnight growth replicate values were averaged and the measured OD values were plotted against time.
  • The viability assay (FIG. 3C) was performed as described by Pedersen et al (2002). Briefly, each strain was grown overnight at 37° C. in LB medium containing 100 μg/ml ampicillin, 50 μg/ml kanamycin, and 34 μg/ml chloramphenicol. In the next morning, cells were then diluted 1:1000 in the same medium as above and grown for 3 hours. At time zero cells were then washed once with LB, and then transcription of toxin was induced by 100 μM IPTG. At increasing time points after toxin induction (30, 60, 120, 180, 240 and 300 mins) cells were plated in several dilutions on LB-plates containing 100 μg/ml ampicillin, 50 μg/ml kanamycin, 34 μg/ml chloramphenicol and 0.3% arabinose. CFUs were determined in the next morning by colony counting.
  • Plaque Assays:
  • E. coli strains harboring the antitoxin only or the toxin-antitoxin of psyrAT, sanaAT, pmenAT, rlegAT, and sdenAT binary toxin-antitoxin systems were grown overnight in LB liquid medium supplemented with 35 μg/ml chloramphenicol, 100 μg/ml ampicillin and 0.3% L-arabinose with or without 50 μg/ml kanamycin, respectively. Overnight cultures were diluted 1:100 in fresh LB medium supplemented with inducers and antibiotics as above and aerated with shaking at 37° C. until reaching O.D600≈0.5. Cultures were then centrifuged for 10 min and re-suspended in LB to an O.D600 of exactly 0.5. Volumes of 200 μl of culture and 10 μl of the indicated T7 phages were mixed in 3 ml of warm 0.8% agar LB supplemented with 35 μg/ml chloramphenicol, 100 μg/ml ampicillin and 0.3% L-arabinose or 35 μg/ml chloramphenicol, 100 μg/ml ampicillin, 50 μg/ml kanamycin, 0.3% L-arabinose, and 100 μM IPTG for the antitoxin only- or toxin-antitoxin-harboring cultures, respectively. The mixtures were immediately overlaid on LB plates supplemented with the above indicated inducers and antibiotics. Overlaid plates were incubated at 37° C. for 3 h and plaques were then counted. For a given TA pair, efficiency of plating (EOP) was calculated by dividing the number of plaque forming units (PFU) obtained for bacterial lawn expressing the toxin+antitoxin by the number of PFU obtained on the bacterial lawn expressing the antitoxin alone. For the rlegAT system, additional experiments were done with resuspension of cultures in LB to a higher O.D600 of 2.5, to address the possibility that the observed smaller plaque sizes stemmed from lower lawn density resulting from partial toxicity of this system to E. coli when grown on plates.
  • Expression of Lon and 4.5 Proteins and Co-Immunoprecipitation Assay:
  • The 4.5 gene (3′ His-tagged) and full-length Lon protease gene (5′ Flag-tagged) were cloned into the 1st and 2nd expression cassettes, respectively, of the vector pRSFDuet-1 (EMD Chemicals, Inc.). DNA cloning was performed using the Restriction Free (RF) cloning procedure (Unger et al., 2010). Induction experiments were performed using E. coli BL21 (DE3) cells expressing only the Lon or the 4.5 proteins and cells expressing both Lon and 4.5 proteins. Induction was carried out at 37° C. for 3 hr by addition of 200 μM of IPTG. Cell pellets were lysed by sonication in a buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 μl/mL protease inhibitor cocktail (Set IV, EMD Chemicals, Inc.). Cell debris were removed by centrifugation at 4° C. for 15 minutes at 18,000 g. Clear supernatants were transferred to 1.5 ml tubes and incubated on a rotator shaker at 4° C. for 1 hr with 80 μl pre-washed anti-Flag M2-agarose beads (Sigma, # A2220). The beads were washed three times with the buffer described above and the Flag-tagged protein or protein complex were eluted using Flag-peptide (Sigma, #F3290) using the manufacturers' recommendations.
    • Example 1 Systematic Discovery of TA Families Based on Large-Scale Cloning Experiments
  • 360 bacterial and 28 archaeal genomes that were sequenced using the clone-based Sanger approach, and for which the raw sequencing data was accessible and mapped to the assembled genome (Kimelman et al., 2012) were analyzed. For the sequencing of each genome, an average of 22,313 different randomly fragmented clones (typically sized between 3-kb-8-kb, thus typically spanning ˜3 to ˜8 genes) were inserted into E. coli. Cumulatively, the analyzed genomes span over 1.5 million genes that were sequenced using over 8.5 million clones. The number of cloned DNA fragments that fully contain were recorded for each gene, and for each pair of consecutive genes.
  • To detect families of gene pairs in which one of the genes (putative toxin) is absent from clones unless the adjacent gene (putative antitoxin) is also present, the present inventors first searched for homologous gene pairs that repeatedly appear adjacent to each other in multiple genomes (at least 7 appearances) and aggregated them into families of pairs (Methods). To avoid the analysis of housekeeping genes that appear in conserved operons (e.g., ribosomal protein genes), the present inventors focused on families showing high tendency to undergo horizontal gene transfer (Methods). Each pair of genes (X and Y) in each family was considered as following the “TA cloning pattern” if the number of clones covering gene X (toxin) but not Y (antitoxin) was 0, the number of clones covering gene Y but not X was >0 and the number of clones covering both X and Y was >0 (FIGS. 1B-C).
  • Not all toxins are expected to manifest their toxicity when cloned in E. coli, because their expression depends on the ability of the E. coli host to recognize their native promoters and translate them using available tRNA pool (Sorek et al., 2007). Therefore, for a given toxin-antitoxin family of gene pairs, one may expect a significant fraction of pairs, but not necessarily all pairs, to follow the “TA cloning pattern” (FIG. 1C). To assign a statistical significance for a given family as possibly coding for a bona fide TA family, the present inventors performed, for each pair in each family, 1000 random simulations, where the clones used for sequencing of the relevant genome were randomly shuffled on the genome (Methods; FIG. 7). The results were then used to assign an empirical p-value per family, revealing families in which the fraction of pairs that follow the TA cloning pattern is significantly above the fraction expected by chance (p<=0.05). This yielded 188 candidate families (FIG. 2).
  • The identified families may include genes that follow the TA cloning pattern as a by-product of their functions, and not for reasons associated with classical TA systems. For example, a metabolic enzyme whose expression results in accumulation of toxic intermediates might be neutralized by an accompanying transcriptional repressor. This may be the case for the argininosuccinate synthase and the ArgR repressor of the arginine regulon that obey the TA cloning patterns in 4 out of 12 homologous pairs. To identify TA systems more likely to play phage defense-related roles, the present inventors focused on those families that had high tendency to appear within bacterial “defense islands”. It was recently shown that bacterial anti-phage immune systems such as restriction enzymes, CRISPR and Abi genes aggregate in such “defense island” loci in bacterial genomes (Makarova et al., 2011). Therefore, the present inventors selected those families in which the genomic neighborhood was enriched for defense genes (Methods). This analysis resulted in a set of 24 putative families of TA gene pairs, overall containing 400 pairs from 176 genomes (FIG. 2; Tables 4-5).
  • TABLE 4
    Families of previously known and bioinformatically
    predicted toxin-antitoxin systems retrieved by the
    TA discovery algorithm
    # COGs P value
    pairs Toxin associated COGs for TA
    in Antitoxin super- with associated cloning
    family superfamily family antitoxin with toxin pattern
    1 16 RelB RelE COG3905 COG3668 5.0 × 10−3
    2 25 RelB RelE COG3077 COG3041 3.0 × 10−3
    3 13 RelB RelE n/a n/a 1.1 × 10−2
    4 33 Xre RelE COG3620 COG4679 2.5 × 10−2
    5 28 Xre RelE n/a COG2944 2.5 × 10−2
    6 32 Xre RelE COG5499 COG4680 7.0 × 10−3
    7 27 Phd RelE COG2161 COG2026 3.4 × 10−2
    8 9 HicB HicA COG1598 COG1724 9.0 × 10−3
    9 11 HicB HicA n/a COG1724 3.4 × 10−2
    10 12 VapB VapC COG4456 COG1487 4.0 × 10−3
    11 10 HigA HigB COG3093 COG3549 1.0 × 10−3
    12 22 MosA MosT n/a COG2253 0
  • TABLE 5
    Novel families of toxin-antitoxin systems retrieved by the TA discovery algorithm
    # Domains P value
    pairs associated Domains for TA
    in Antitoxin Toxin with associated cloning
    fam. Antitoxin Toxin annotation annotation antitoxin with toxin pattern
    11 PsyrA PsyrT Nucleotide- RecQ- COG0758 COG0514 0
    SEQ SEQ ID binding family DNA
    ID NOs: protein helicase
    NOs: 2872-2935
    8416-8479
    10 SanaA SanaT S-adenosyl- Hypothetical n/a DUF1814 0
    SEQ SEQ ID homocysteine protein
    ID NO: NOs: hydrolase
    8575-8622 3031-3078
    10 PmenA PmenT ADP-ribose Hypothetical COG2110 n/a 0
    SEQ SEQ ID binding protein
    ID NO: NO: domain
    8480-8574 2936-3030 protein
    9 SEQ SEQ ID Hypothetical Adenine n/a COG2189 0
    ID NO: NOs: protein specific
    8623-8631 3079-3087 DNA
    methylase
    7 SEQ SEQ ID T/G Type II COG3727 PF09019 0
    ID NO: NOs: mismatch- restriction
    8632-8638 3088-3094 specific endonuclease
    endonuclease
    20 RlegA RlegT Predicted Hypothetical COG5340 DUF1814 0
    SEQ SEQ ID transcriptional protein
    ID NO: 2805-2871 regulator
    8349-8415
    8 SEQ SEQ ID Hypothetical RAMP n/a DUF324 2.9 × 10−2  
    ID NO: NOs: protein domain
    8654-8661 3110-3117 (associated protein
    with (Cmr6-like)
    cas/cmr
    genes)
    15 SEQ SEQ ID Uncharacterized Membrane n/a PF07916 2 × 10−3
    ID NO: NO: membrane protein,
    8639-8653 3095-3109 protein TraG-like
    N-terminal
    domain
    7 SdenA SdenT Hypothetical Hypothetical n/a DUF1814 3 × 10−3
    SEQ SEQ ID protein protein
    ID NO: NOs:
    8317-8348 2773-2804
    * TA system that is part of a larger operon putatively involved in bacterial defense (FIG. 8).
  • Of the 24 identified families, 12 families (50%) were already described as TA systems, either experimentally (12 “known” families) or by earlier bioinformatic predictions (3 “predicted” families (Shao et al., 2011)), providing strong validation to the present cloning-based approach for TA discovery (Table 4). Although a diverse set of known TA families is represented in the set retrieved by the present algorithm, due to limitations of the present approach not all known families were represented. For example, in the family consisting of HipAB gene pairs, 6 out of 19 pairs were found to obey the TA cloning pattern, but since the toxin is relatively large and the antitoxin is a short gene, such a pattern has high probability to occur by chance in the random simulations, and hence this family did not pass the present statistical threshold (p=0.43) (see Discussion).
    • Example 2 Experimental Validation of Novel TA Families
  • The present analysis retrieved 8 putative novel families of TA systems (Table 5). Six of these families, which appeared as “stand-alone” toxin-antitoxin pairs (see below), were selected for further experimental characterization (Table 6, herein below). A representative pair was selected from each family and co-transformed into E. coli BL21 (DE3) on a compatible two-vector system, so that the putative toxin was under the control of an IPTG-induced promoter, and the antitoxin was under the control of an arabinose-induced promoter (FIG. 3A). E. coli bacteria carrying these two plasmids were plated on agar plates containing IPTG, arabinose, or both IPTG and arabinose. In all six tested pairs, induction of toxin expression inhibited bacterial growth, while co-induction of the toxin and antitoxin resulted in bacterial survival (FIG. 3A). These results validate the genome-wide approach for discovery of novel TA systems.
  • The 6 novel validated families were named based on the species from which the validated system was taken: pmenAT (P. mendocina); sanaAT (S. sp. ana-3); rlegAT (R. leguminosarum); psyrAT (P. syringae); sdenAT (S. denitrificans); and hhalTA (H. halophila). The toxins in these new systems were further tested in order to analyze whether they have a bacteriocidic (cell-killing) or bacteriostatic (growth-inhibiting) effect. For this, IPTG was used to induce toxin expression for different time intervals (ranging from 30 to 300 mins). The cells were then plated on agar plates containing arabinose to activate antitoxin expression (FIG. 3C). For the rlegTA system, colony forming units dropped by 5 orders of magnitude following 120 minutes of toxin induction, implying that the rleg toxin has a bactericidal effect on the cells. These results were also supported by the kinetic assays, where induction of antitoxin expression 2.5 hours after toxin induction did not result in cell regrowth for rlegTA (FIG. 3B). A milder effect was observed for the remaining TA systems, with sdenTA and hhalTA showing almost no reduction in colony forming units following toxin induction, suggestive of a bacteriostatic effect for these systems (FIG. 3C).
  • TABLE 6
    Gene pairs selected for experimental verification in a dual-plasmid
    arabinose/IPTG expression induction system
    # clones # clones # clones
    Antitoxin A Toxin T that that that
    locus size Locus size cover cover cover Reversible
    Family tag (aa) tag (aa) Organism antitoxin toxin both toxicity?
    sdenAT Sden_0299 174 Sden_0300 295 Shewanella 8 0 31 Yes
    denitrificans
    OS217
    psyrAT Psyr_3805 455 Psyr_3804 698 Pseudomonas 13 0 1 Partial
    syringae
    pv.
    syringae
    B728a
    sanaAT Shewana3_4160 136 Shewana3_4161 313 Shewanella 5 0 29 Yes
    sp. ANA-3
    pmenAT Pmen_0566 360 Pmen_0565 217 Pseudomonas 3 0 10 Yes
    mendocinaymp
    rlegAT Rleg_6340 205 Rleg_6339 289 Rhizobium 11 0 31 No
    leguminosarum
    bv.
    trifolii
    WSM1325
    hhalTA Hhal_0686 96 Hhal_0685 119 Halorhodospira 2 0 23 Yes
    halophila
    SL1
    • Example 4 Characteristics of Novel TA Families
  • Most type II TA modules described to date share several typical characteristics: the antitoxin appears upstream of the toxin; both the toxin and antitoxin are small proteins (typically ˜100aa); and the antitoxin contains a DNA binding domain (Makarova et al., 2009). Since the present approach does not rely on such attributes for TA modules discovery it has the potential to expand the premises of TA modules properties. Indeed, some of the new families that were experimentally validated deviate significantly from the previously described characteristics. For example, the sizes of many new toxins and antitoxins are significantly larger than 100 aa, with a maximum of 698 aa in the toxin of the psyrAT system; in two families the toxin is located upstream of the antitoxin (FIG. 4A); and several antitoxins do not contain a known DNA binding domain (although the possibility cannot be ruled out that some antitoxins code for yet uncharacterized such domains) (FIG. 4A).
  • Although in most of the known TA systems the toxin is a ribonuclease, diverse domains within the new toxins detected, including DNA helicase, phosphoribosyl-transferase and nucleotidyl-transferase suggest novel mechanisms of toxicity (FIG. 4A). Similarly, the presence of an ADP-ribose-binding, S-adenosyl-homocysteine hydrolase and nucleotide-binding domains in some of the antitoxins suggest that these antitoxins perform a more complex function than simply masking the activity of the toxin by protein-protein interactions (FIG. 4A; Table 5). Finally, in four of the novel families, it seems that the TA system is part of a larger operon that is co-horizontally transferred between genomes in the context of defense islands, suggesting their involvement in more complex defense mechanisms (Table 5; FIG. 8).
  • Previous analyses have shown that toxin-antitoxin systems can be modular, such that members of one toxin family may be associated with several different types of antitoxin, and vice versa (Leplae et al., 2011; Makarova et al., 2009). Indeed, the toxins of three of the present new families carry the same domain, DUF1814, with a different antitoxin associated with this domain in each of the three families (Table 2; FIG. 4A). One of these families, rlegAT, where the DUF1814 toxin is accompanied by COG5340 as an antitoxin, was previously shown to be enriched in defense islands in bacterial genomes and was suggested as a new TA system based on its two-gene nature (Makarova et al., 2011). The DUF1814 domain was recently classified as a nucleotidyl-transferase domain based on structural information, but its specific substrates are yet unknown (Kuchta et al., 2009). Interestingly, the DUF1814 domain was also documented in AbiG, a two-gene system involved in abortive infection in Lactococcus lactis via an unknown mechanism (Makarova et al., 2011; O'Connor et al., 1996), although there is no direct homology between the AbiG system and any of the genes in the new TA systems that were presently detected. Therefore, the results point to DUF1814 domain-containing proteins as a widespread superfamily of toxins that might be involved in anti-phage defense (see below).
  • Overall, members of the novel systems that were detected appeared in 21% of the genomes analyzed, and in the vast majority of cases (93%) they appeared in the chromosomal DNA rather than on plasmids. These results suggest that such systems have important roles in bacterial physiology/defense rather than functioning as plasmid addiction molecules. The distribution of most families is spread across multiple bacterial phyla including several important human pathogens (Tables 4 and 5; FIGS. 4B-C). For example, the psyrAT, sanaAT, and pmenAT systems are abundant in enteropathogenic and uropathogenic E. coli strains; the pmenAT system also exists in many Mycobacterium tuberculosis isolates; psyrAT exists in Shigella and Pseudomonas aeruginosa strains; and the rlegAT and sanaAT systems exist in several pathogenic Legionella species. In addition, many resident bacteria of the human gut carry one or more of these TA modules, including bacteria belonging to the Bifidobacterium and Prevotella genera (Tables 7-11, herein below, FIG. 4C). This underscores the novel toxin-antitoxin families as potentially contributing to persistence, phage defense and stress responses of clinically important bacteria.
  • TABLE 7
    pmenAT Family
    Toxin Antitoxin
    IMG IMG
    Gene Gene
    Organism name Object ID Object ID
    Acidovorax avenae citrulli AAC00-1 639821371 639821372
    Actinosynnema mirum 101, DSM 43827 645948092 645948093
    Aliivibrio salmonicida LFI1238 643378769 643378768
    Alkalilimnicola ehrlichei MLHE-1 638125243 638125244
    Arthrospira maxima CS-328 643172840 643172839
    Arthrospira maxima CS-328 643172852 643172851
    Arthrospira platensis NIES-39 650387585 650387586
    Arthrospira platensis Paraca 646128269 646128268
    Arthrospira sp. PCC 8005 648389866 648389867
    Arthrospira sp. PCC 8005 648389879 648389880
    Aurantimonas manganoxydans SI85-9A1 639105350 639105351
    Bacillus thuringiensis IBL200 644713531 644713532
    Beggiatoa sp. PS 641099098 641099099
    Brachyspira hyodysenteriae WA1, ATCC 643738127 643738126
    49526
    Candidatus Accumulibacter phosphatis 645012050 645012051
    Type IIA UW-1
    Cyanothece sp. PCC 7822 648189618 648189617
    Dermacoccus sp. Ellin185 650227082 650227083
    Desulfonatronospira thiodismutans ASO3-1 644404121 644404120
    Desulfovibrio sp. 3_1_syn3 648927836 648927837
    Dickeya zeae Ech1591 644851325 644851326
    Edwardsiella ictaluri 93-146 644783480 644783479
    Edwardsiella tarda EIB202 646417146 646417147
    Edwardsiella tarda FL6-60 648267250 648267249
    Enhydrobacter aerosaccus SK60 646169120 646169121
    Escherichia coli O127:H6 E2348/69 (EPEC) 643442443 643442442
    Escherichia sp. 4_1_40B 645350401 645350402
    Eubacterium saburreum DSM 3986 650308116 650308115
    Geobacter lovleyi SZ 642677613 642677612
    Gloeobacter violaceus PCC 7421 637460780 637460781
    Gluconacetobacter diazotrophicus PAl 5, 641334933 641334934
    DSM 5601
    Klebsiella pneumoniae 342 643367815 643367814
    Leptotrichia goodfellowii F0264 647211994 647211995
    Lyngbya sp. CCY 8106 640014016 640014017
    Marinobacter sp. ELB17 640635553 640635552
    Methylobacter tundripaludum SV96 648842442 648842443
    Micromonospora aurantiaca ATCC 27029 648134607 648134606
    Mycobacterium bovis AF2122/97 637137397 637137398
    Mycobacterium bovis BCG Pasteur 1173P2 639828992 639828993
    Mycobacterium bovis BCG Tokyo 172 643732879 643732880
    Mycobacterium tuberculosis 02_1987 643025701 643025702
    Mycobacterium tuberculosis 210 647208652 647208653
    Mycobacterium tuberculosis 94_M4241A 643021508 643021509
    Mycobacterium tuberculosis 98-R604 645190033 645190034
    INH-RIF-EM
    Mycobacterium tuberculosis C 638728678 638728679
    Mycobacterium tuberculosis CDC1551 637094328 637094329
    Mycobacterium tuberculosis CPHL_A 646012704 646012705
    Mycobacterium tuberculosis EAS054 643034220 643034221
    Mycobacterium tuberculosis F11 (ExPEC) 640604798 640604799
    Mycobacterium tuberculosis GM 1503 643047494 643047495
    Mycobacterium tuberculosis H37Ra 640600718 640600719
    Mycobacterium tuberculosis H37Rv 637025228 637025229
    (lab strain)
    Mycobacterium tuberculosis Haarlem 641785646 641785647
    Mycobacterium tuberculosis K85 646016919 646016920
    Mycobacterium tuberculosis KZN 1435 644877737 644877738
    (MDR)
    Mycobacterium tuberculosis KZN 4207 647084598 647084599
    Mycobacterium tuberculosis KZN 4207 645118109 645118108
    (DS)
    Mycobacterium tuberculosis KZN R506 648334260 648334261
    Mycobacterium tuberculosis KZN V2475 647088790 647088791
    Mycobacterium tuberculosis SUMu001 648445593 648445594
    Mycobacterium tuberculosis SUMu002 648446131 648446132
    Mycobacterium tuberculosis SUMu003 648449945 648449946
    Mycobacterium tuberculosis SUMu004 648454358 648454359
    Mycobacterium tuberculosis SUMu005 648458950 648458951
    Mycobacterium tuberculosis SUMu006 648463059 648463060
    Mycobacterium tuberculosis SUMu007 648467445 648467446
    Mycobacterium tuberculosis SUMu008 648471678 648471679
    Mycobacterium tuberculosis SUMu009 648479444 648479445
    Mycobacterium tuberculosis SUMu010 648483724 648483725
    Mycobacterium tuberculosis SUMu011 648487988 648487989
    Mycobacterium tuberculosis SUMu012 648488272 648488273
    Mycobacterium tuberculosis T17 643047830 643047831
    Mycobacterium tuberculosis T85 643038464 643038465
    Mycobacterium tuberculosis T92 643026422 643026423
    Nitrosococcus halophilus Nc4 646693941 646693942
    Nitrosomonas europaea ATCC 19718 637427715 637427716
    Oscillatoria sp. PCC 6506 648860075 648860076
    Parvibaculum lavamentivorans DS-1 640878511 640878512
    Pectobacterium carotovorum carotovorum 644864144 644864143
    PC1
    Pedobacter sp. BAL39 641137361 641137360
    Prevotella timonensis CRIS 5C-B1 647314799 647314798
    Providencia rustigianii DSM 4541 643141873 643141874
    Pseudomonas entomophila L48 637999408 637999409
    Pseudomonas mendocina ymp 640501778 640501779
    Rhodopseudomonas palustris TIE-1 642711985 642711984
    Roseiflexus sp. RS-1 640593922 640593923
    Roseovarius nubinhibens ISM 638835723 638835722
    Ruminococcus flavefaciens FD-1 646114893 646114892
    Shewanella putrefaciens CN-32 640500731 640500732
    Stigmatella aurantiaca DW4/3-1 649685957 649685958
    Synechocystis sp. PCC 6803 637472042 637472041
    Syntrophothermus lipocalidus DSM 12680 646854986 646854985
    Thauera sp. MZ1T 643700535 643700534
    Thermus aquaticus Y51MC23 645188421 645188422
    Thiobacillus denitrificans ATCC 25259 637710175 637710174
    Zymomonas mobilis subsp. mobilis ZM4 648935037 648935036
    ZM4 plasmid pZZM405
  • TABLE 8
    psyrAT Family
    Toxin Antitoxin
    IMG IMG
    Gene Gene
    Organism name Object ID Object ID
    Acinetobacter johnsonii SH046 646301838 646301837
    Arthrospira platensis NIES-39 650383509 650383508
    Chlorobium phaeobacteroides DSM 266 639765722 639765723
    Cyanothece sp. PCC 8801 643474605 643474606
    Dehalogenimonas lykanthroporepellens 648069289 648069288
    BL-DC-9
    Desulfitobacterium hafniense DCB-2 643560590 643560591
    Desulfitobacterium hafniense Y51 637910440 637910441
    Desulfotalea psychrophila LSv54 637527656 637527657
    Desulfotomaculum acetoxidans 5575, 645033659 645033660
    DSM 771
    Escherichia coli 83972 644299713 644299712
    Escherichia coli ABU 83972 648235458 648235457
    Escherichia coli B088 647955723 647955722
    Escherichia coli B185 647960289 647960288
    Escherichia coli E22 (EPEC) 638663931 638663932
    Escherichia coli IAI39 643513743 643513742
    Escherichia coli M605 648348440 648348439
    Escherichia coli M718 648353998 648353997
    Escherichia coli MS 185-1 648599204 648599205
    Escherichia coli MS 200-1 648586960 648586959
    Escherichia coli MS 21-1 648564766 648564767
    Escherichia coli MS 45-1 648550266 648550267
    Escherichia coli MS 69-1 648553056 648553055
    Escherichia coli O103:H2 str. 12009 646343586 646343585
    Escherichia coli O111:NM B171 (EPEC2) 638674448 638674447
    Escherichia coli O139:H28 F11 (ETEC) 638660743 638660744
    Escherichia coli O150:H5 SE15 646872639 646872638
    Escherichia coli O157:H7 EC4042 642258307 642258308
    Escherichia coli O157:H7 EC4045 642254369 642254368
    Escherichia coli O157:H7 EC4076 642284244 642284243
    Escherichia coli O157:H7 EC4113 642276965 642276966
    Escherichia coli O157:H7 EC4196 642270878 642270879
    Escherichia coli O157:H7 EC4206 642245550 642245551
    Escherichia coli O157:H7 EC4401 642289554 642289553
    Escherichia coli O157:H7 EC4486 642295954 642295953
    Escherichia coli O157:H7 EC4501 642302598 642302597
    Escherichia coli O157:H7 EC508 642315404 642315403
    Escherichia coli O157:H7 EC869 642309592 642309591
    Escherichia coli O157:H7 EDL933 (EHEC) 637066109 637066108
    Escherichia coli O157:H7 TW14588 643013921 643013922
    Escherichia coli O6:K15:H31 536 (UPEC) 638064677 638064676
    Escherichia coli O6:K2:H1 CFT073 (UPEC) 637358068 637358067
    Escherichia coli SECEC SMS-3-5 641618544 641618543
    Kingella denitrificans ATCC 33394 650370405 650370406
    Lyngbya sp. CCY 8106 640016567 640016566
    Maribacter sp. HTCC2170 648162921 648162922
    Marinobacter aquaeolei VT8 639810511 639810512
    Microcoleus chthonoplastes PCC 7420 647568262 647568263
    Nitrococcus mobilis Nb-231 639000572 639000573
    Nitrosomonas europaea ATCC 19718 637428828 637428829
    Nostoc sp. PCC 7120 637233840 637233841
    Oscillochloris trichoides DG6 650114297 650114298
    Pelodictyon phaeoclathratiforme BU-1 642727669 642727668
    Photorhabdus luminescens laumondii TTO1 637466171 637466172
    Polaromonas naphthalenivorans CJ2 639835976 639835975
    Providencia stuartii ATCC 25827 642341526 642341525
    Pseudomonas aeruginosa 39016 650201418 650201417
    Pseudomonas syringae pv. syringae B728a 637654162 637654163
    Roseiflexus castenholzii HLO8, DSM 13941 640894598 640894597
    Shewanella baltica OS155 640121625 640121624
    Shigella dysenteriae 1617 650108289 650108288
    Shigella dysenteriae Sd197 640435823 640435822
    Sinorhizobium meliloti AK83, DSM 23913 648746639 648746638
    Teredinibacter turnerae T7901 644918750 644918749
    Trichodesmium erythraeum IMS101 638106714 638106713
    Vibrio furnissii CIP 102972 647177818 647177819
  • TABLE 9
    rlegAT Family
    Toxin Antitoxin
    IMG IMG
    Gene Gene
    Organism name Object ID Object ID
    Acidovorax delafieldii 2AN 645555203 645555202
    Acidovorax sp. JS42 639838035 639838036
    Agreia sp. PHSC20C1 638988138 638988137
    Agrobacterium sp. H13-3 649987843 649987844
    Asticcacaulis excentricus CB 48 649817025 649817026
    Bradyrhizobium japonicum USDA 110 637369489 637369490
    Brevibacterium mcbrellneri ATCC 49030 647495830 647495829
    Candidatus Accumulibacter phosphatis 645008063 645008062
    Type IIA UW-1
    Candidatus Protochlamydia amoebophila 637502933 637502934
    UWE25
    Chlorobium limicola DSM 245 642669528 642669529
    Chlorobium phaeobacteroides DSM 266 639765252 639765251
    Chlorobium phaeovibrioides DSM 265 640453070 640453069
    Conexibacter woesei DSM 14684 646506209 646506208
    Corynebacterium glutamicum R 640460410 640460411
    Delftia acidovorans SPH-1 641295145 641295146
    Desulfococcus oleovorans Hxd3 641265756 641265755
    Desulfomicrobium baculatum X, DSM 4028 645000532 645000533
    Eggerthella lenta VPI 0255, DSM 2243 645026066 645026065
    Eggerthella sp. 1_3_56FAA 650045752 650045751
    Ensifer medicae WSM419 640743222 640743221
    Ensifer medicae WSM419 640777216 640777215
    Ensifer medicae WSM419 640743222 640743221
    Gemmata obscuriglobus UQM 2246 642225335 642225336
    Gloeobacter violaceus PCC 7421 637460064 637460063
    Gordonia bronchialis DSM 43247 646396290 646396289
    Gordonibacter pamelaeae 7-10-1-bT, 650562241 650562242
    DSM 19378
    Intrasporangium calvum 7KIP, DSM 43043 649830949 649830948
    Legionella longbeachae NSW150 648036627 648036628
    Legionella longbeachae NSW150 648038896 648038897
    Lentisphaera araneosa HTCC2155 641129191 641129192
    Lentisphaera araneosa HTCC2155 641132412 641132413
    Mariprofundus ferrooxydans PV-1 639879700 639879701
    Methylobacterium extorquens AM1 644816407 644816408
    Mobiluncus mulieris ATCC 35239 648832456 648832455
    Mobiluncus mulieris ATCC 35243 644432065 644432064
    Mobiluncus mulieris FB024-16 648839977 648839978
    Mycobacterium leprae Br4923 643606676 643606675
    Mycobacterium leprae TN 637073074 637073073
    Mycobacterium marinum M, ATCC 641718837 641718836
    BAA-535
    Nakamurella multipartita Y-104, 645043556 645043555
    DSM 44233
    Nitrosococcus halophilus Nc4 646694275 646694276
    Olsenella uli VPI, DSM 7084 648106565 648106566
    Opitutaceae sp. TAV2 641180308 641180309
    Pelodictyon luteolum DSM 273 637768549 637768548
    Pelodictyon phaeoclathratiforme BU-1 642727042 642727041
    Phenylobacterium zucineum HLK1 642757721 642757722
    Polaromonas naphthalenivorans CJ2 639826420 639826421
    Polaromonas sp. JS666 639328971 639328972
    Polaromonas sp. JS666 639328971 639328972
    Propionibacterium freudenreichii shermanii 649639912 649639911
    CIRM-BIA1
    Raphidiopsis brookii D9 647110477 647110476
    Rhizobium leguminosarum bv. trifolii 644828134 644828135
    WSM1325
    Rhizobium leguminosarum bv. trifolii 644828341 644828342
    WSM1325
    Rhizobium leguminosarum bv. viciae 3841 639650816 639650815
    Rhizobium rhizogenes K84 643644497 643644496
    Rhizobium sp. NGR234 (ANU265) 643821930 643821929
    Rhodomicrobium vannielii ATCC 17100 649745264 649745263
    Rhodopseudomonas palustris DX-1 649838192 649838193
    Rhodopseudomonas palustris DX-1 649839250 649839249
    Slackia heliotrinireducens RHS 1, 644987249 644987248
    DSM 20476
    Synechococcus sp. RS9917 638961153 638961154
    Syntrophobacter fumaroxidans MPOB 639702820 639702821
    Variovorax paradoxus S110 644796233 644796232
    Verrucomicrobiales sp. DG1235 647600269 647600268
    Xanthomonas oryzae pv oryzae MAFF 637850518 637850519
    311018
    Xanthomonas oryzae pv oryzicola BLS256 641737990 641737989
    Xanthomonas oryzae pv. oryzae 637633303 637633304
    KACC10331
    Xanthomonas oryzae pv. oryzae PXO99A 642640643 642640644
    Xylanimonas cellulosilytica DSM 15894 646444306 646444305
  • TABLE 10
    sdenAT Family
    Toxin Antitoxin
    IMG IMG
    Gene Gene
    Organism name Object ID Object ID
    Acidithiobacillus ferrooxidans ATCC 53993 642789179 642789178
    Ammonifex degensii KC4 646360172 646360173
    Bifidobacterium longum DJO10A 642679488 642679489
    Bifidobacterium longum longum ATCC 644347132 644347131
    55813
    Bifidobacterium longum longum CCUG 643912156 643912157
    52486
    Bifidobacterium longum longum CCUG 643912885 643912886
    52486
    Bifidobacterium longum longum F8 650528372 650528373
    Bifidobacterium longum NCC2705 637327456 637327457
    Bifidobacterium sp. 12_1_47BFAA 650054808 650054809
    Burkholderia rhizoxinica HKI 454 649762359 649762358
    Cellvibrio japonicus Ueda 107 642704960 642704959
    delta proteobacterium sp. MLMS-1 639152971 639152970
    delta proteobacterium sp. MLMS-1 639153350 639153349
    delta proteobacterium sp. MLMS-1 639155398 639155397
    delta proteobacterium sp. MLMS-1 639158034 639158033
    Desulfurivibrio alkaliphilus AHT2 646845548 646845547
    Eggerthella lenta VPI 0255, DSM 2243 645023812 645023813
    Helicobacter canadensis MIT 98-5491, 643921049 643921048
    ATCC 700968
    Helicobacter canadensis MIT 98-5491, 647542139 647542138
    ATCC 700968
    Marinobacter sp. ELB17 640637051 640637050
    Marinomonas sp. MWYL1 640805575 640805574
    Pantoea sp. At-9b 649850365 649850364
    Parascardovia denticolens DSM 10105 650283875 650283876
    Pectobacterium wasabiae WPP163 646378766 646378765
    Photorhabdus luminescens laumondii TTO1 637462283 637462284
    Photorhabdus luminescens laumondii TTO1 637465673 637465672
    Proteus mirabilis ATCC 29906 644443817 644443818
    Proteus mirabilis HI4320 642578705 642578706
    Proteus mirabilis HI4320 642578886 642578885
    Providencia rustigianii DSM 4541 643143270 643143271
    Shewanella denitrificans OS217 637954281 637954280
    Verminephrobacter eiseniae EF01-2 639850605 639850606
    Xenorhabdus nematophila ATCC 19061 649646151 649646150
  • TABLE 11
    sanaAT Family
    Toxin Antitoxin
    IMG IMG
    Gene Gene
    Organism name Object ID Object ID
    Acidovorax delafieldii 2AN 645555430 645555431
    Acidovorax sp. JS42 639838656 639838657
    Burkholderia pseudomallei 1106a 640135680 640135679
    Burkholderia pseudomallei 14 641944057 641944056
    Burkholderia pseudomallei 9 641966075 641966074
    Burkholderia pseudomallei B7210 641976418 641976417
    Burkholderia pseudomallei Pakistan 9 644419997 644419996
    Chlorobium limicola DSM 245 642667845 642667846
    delta proteobacterium sp. MLMS-1 639154553 639154552
    Desulfatibacillum alkenivorans AK-01 643534322 643534323
    Escherichia coli 83972 644303823 644303822
    Escherichia coli ABU 83972 648234949 648234948
    Escherichia coli FVEC1302 648901279 648901278
    Escherichia coli FVEC1412 647965363 647965362
    Escherichia coli H591 648379532 648379531
    Escherichia coli MS 119-7 648651653 648651652
    Escherichia coli MS 198-1 648524147 648524146
    Escherichia coli MS 45-1 648547640 648547641
    Escherichia coli MS 69-1 648552519 648552520
    Escherichia coli MS 69-1 648554605 648554604
    Escherichia coli O17:K52:H18 UMN026 644760974 644760973
    Escherichia coli O6:K2:H1 CFT073 (UPEC) 637357531 637357530
    Escherichia coli W 648733316 648733317
    Escherichia coli W, ATCC 9739 650430781 650430780
    Escherichia sp. 1_1_43 646267384 646267383
    Legionella drancourtii LLAP12 645590921 645590922
    Legionella pneumophila Corby 640570188 640570189
    Legionella pneumophila Lens 637581974 637581973
    Legionella pneumophila Paris 637581109 637581110
    Methylovorus glucosetrophus SIP3-4 644899678 644899679
    Pelodictyon luteolum DSM 273 637768759 637768758
    Pelodictyon phaeoclathratiforme BU-1 642728170 642728169
    Photobacterium sp. SKA34 639052391 639052392
    Photorhabdus asymbiotica asymbiotica 644890382 644890383
    ATCC 43949
    Photorhabdus luminescens laumondii TTO1 637465992 637465993
    Pseudoalteromonas haloplanktis TAC125 637736049 637736048
    Rhodocista centenaria SW 643414024 643414025
    Shewanella baltica OS155 640110446 640110445
    Shewanella sp. ANA-3 639720519 639720518
    Synechocystis sp. PCC 6803 637471904 637471903
    Verminephrobacter eiseniae EF01-2 639851447 639851446
    Vibrio harveyi BB120, ATCC BAA-1116 640911279 640911280
    Vibrio tapetis CECT4600; CIP104856; 642914607 642914606
    B1090 plasmid pVT1
    Vibrio vulnificus CMCP6 637363648 637363647
    Vibrio vulnificus M06-24/O 649922394 649922393
    Vibrionales sp. SWAT-3 641059142 641059141
    Yersinia aldovae ATCC 35236 645336728 645336729
    Yersinia enterocolitica enterocolitica 8081 640077595 640077596
    • Example 5 Novel TA Systems Provide Phage Resistance
  • The present inventors next set out to explore whether any of the TA systems detected can provide defense against phage. For this, efficiency of plating assays of T7 phage on E. coli hosts were performed (FIG. 5A). Since these new TA systems are widespread in E. coli strains (but are not found in the lab strains E. coli K-12 and E. coli BL21), it was hypothesized that a successful coliphage, such as T7, might hold anti-defense mechanisms that mitigate the defense conferred by the TA systems. The present inventors therefore tested, in addition to the wild-type (WT) T7 phage, 12 additional T7 mutants lacking genes that are not-essential for infection of E. coli K-12 (Table 12). Each of these T7 mutants was used to infect E. coli BL21 or K-12 expressing the five verified new TA systems, as well as control clones expressing only the antitoxin of each system (FIG. 5A). One of the tested systems, sanaAT, was found to provide E. coli with resistance against T7Δ4.3Δ4.5Δ4.7, reducing sensitivity to this phage strain by almost 3 orders of magnitude (FIG. 5B). A second system, rlegAT, resulted in opaque plaques with plaque diameters reduced more than fourfold for the WT T7 strain (diameters of 0.47 mm±0.06 for E. coli expressing both the toxin and the antitoxin, as compared to 1.77 mm±0.03 for bacteria expressing the antitoxin only).
  • TABLE 12
    Genes deleted
    WT T7 none
    T7Δ0.3 0.3
    T7Δ0.7Δ1.7 0.7, 1.7
    T7 LG3 1.1, 1.2, 1.3
    T7Δ1.2 1.2
    T7Δ1.2Δ1.7 1.2, 1.7
    T7Δ1.7 1.7
    T7Δ2.8 2.8
    T7Δ3.8 3.8
    T7 HS33 4.3, 4.5, 4.7
    T7Δ4.5 4.5
    T7Δ5.3-5.9 5.3-5.9
    T7Δ5.9 5.9
  • Since the sanaAT system provides resistance to a T7 phage lacking 3 non-essential genes (genes 4.3, 4.5 and 4.7) but not to the WT T7 phage, it was hypothesized that one of these genes codes for an anti-defense mechanism that overcomes the abortive infection imposed by the TA system. Complementation assays in E. coli K-12 expressing gene 4.5 from a plasmid marked this gene as encoding the anti-Abi mechanism (FIG. 5C). The 4.5 gene codes for a peptide (85aa) with no functional annotation.
  • The present inventors asked whether the defense provided by the sanaTA system against the phage is Lon-dependent. The Lon protease is one of the major proteolytic machineries in the bacterial cell (Gottesman, 2003), and was implicated in degradation (“destabilization”) of many types of antitoxins in E. coli, thus enabling toxin activity (Christensen et al., 2001; Van Melderen et al., 1996; Wang et al., 2011). Indeed, T7 mutant growth on E. coli containing the sanaTA system was restored by two orders of magnitude when the E. coli also lacked lon (FIG. 5D), suggesting that the sanaTA protection from T7 phage depends on Lon activity.
  • It was hypothesized that the phage gene product (Gp) 4.5 interacts with Lon to prevent antitoxin degradation and thus hinders the sanaTA abortive infection activity. To test this hypothesis we co-expressed Lon (Flag-tagged) and Gp4.5 within E. coli. Indeed, we found that Lon and Gp4.5 co-immuno-precipitate, indicating that 4.5 tightly binds Lon (FIG. 5E). A reciprocal co-immuno-precipitation assay, where pull-down was performed on flag-tagged Gp4.5 protein, produced similar co-immuno-precipitation patterns (FIG. 5F). Overall, these results suggest that the T7 Gp4.5 neutralizes TA-system-mediated abortive infection by inhibiting the Lon protease activity, thus preventing antitoxin degradation and toxin activation.
    • Example 6 Phage Anti-Abi Gene Inhibits Persistence-Mediated Resistance to Antibiotics
  • Toxin-antitoxin systems were shown to mediate bacterial resistance to antibiotics through promotion of persister cells (Maisonneuve et al., 2011; Rotem et al., 2010). Persisters are non-growing bacterial cells found in a dormant state and are thus resistant to many antibiotics and other stress conditions (Smith and Romesberg, 2007). Such persisters are stochastically found in bacterial populations as a small fraction of the population. It was recently shown that persistence in E. coli depends on resident type II TA systems as well as on the Lon protease. Indeed, deletion of Lon in E. coli was shown to mitigate TA-system-mediated persistence and resulted in higher sensitivity to antibiotics (Maisonneuve et al., 2011).
  • The present discovery of a T7-encoded peptide that generally alleviates TA systems activity via inhibition of the Lon protease raised the hypothesis that this peptide can also inhibit persister formation. To test this hypothesis persistence was measured as the fraction of surviving E. coli cells following 5 hr exposure to antibiotics (ampicillin). Bacteria expressing the 4.5 gene were 5-fold more sensitive to ampicillin as compared to WT bacteria (manifested in 5-fold less persister cells for ampicillin) (FIG. 6). These results suggest possible utility of phage-derived peptides as a means to tackle bacterial persistence.
  • To search for additional phage genes that may function as Lon inhibitors, the present inventors looked for all phage genomes for genes showing homology to the N-terminus of Lon, roughly where the 4.5 aligns (FIG. 5D). 6 such genes were found (including gene 4.5) in a diverse set of phages.
  • It may be predicted that these phage proteins inhibit Lon protease and can thus function as inhibitors of persistence.
  • Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
  • All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
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Claims (7)

What is claimed is:
1. A method of killing a microbe, the method comprising contacting the microbe with an isolated peptide comprising an amino acid sequence of a toxin having a domain selected from the group consisting of COG0514, DUF1814, COG2189, PF09019, DUF1814, DUF324 and PF07916, thereby killing the microbe.
2. The method of claim 1, wherein when said peptide comprises said COG0514 domain said peptide is selected from the group consisting of SEQ ID NO: 2872-2935.
3. The method of claim 1, wherein when said peptide comprises said DUF1814 domain said peptide is selected from the group consisting of SEQ ID NO: 2773-2804, 3031-3078.
4. The method of claim 1, wherein when said peptide comprises said COG2189 domain said peptide is selected from the group consisting of SEQ ID NO: 3079-3087.
5. The method of claim 1, wherein when said peptide comprises said PF09019 domain said peptide is selected from the group consisting of SEQ ID NO: 3088-3094.
6. The method of claim 1, wherein when said peptide comprises said DUF324 domain said peptide is selected from the group consisting of SEQ ID NO: 3110-3117.
7. The method of claim 1, wherein when said peptide comprises said PF07916 domain said peptide is selected from the group consisting of SEQ ID NO: 3095-3109.
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