Patent Application
20160281053
The present invention, in some embodiments thereof, relates to polynucleotides encoding BREX system polypeptides and methods of using same.
The ongoing arms race between bacteria and bacteriophages (phages) has led to the rapid evolution of efficient resistance systems to protect bacteria from phage infection (Stern and Sorek, 2011). These systems include restriction-modification systems enzymes that recognize and cleave foreign DNA (King and Murray, 1994), abortive infection (Abi) mechanisms that lead to the suicide of the infected host, thus protecting the colony against phage spread (Chopin et al., 2005), and the CRISPR/Cas adaptive defense system, which uses small RNAs to target and destroy invading phage DNA (Deveau et al., 2010). On the counter arm, as part of this continuous bacteria and phages arms race, successful phages had also developed numerous counter-resistance mechanisms to overcome bacterial defense (Stern and Sorek, 2011). 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 genomes (Stern and Sorek, 2011).
A broad array of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various substrates. Enormous amounts of bacteria are being cultivated each day in large fermentation vats, thus phage 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.
Anti-microbial phage therapy dates back to the early 1900s, after their co-discovery by Frederick Twort and Felix d'Hérelle (Twort F W 1915; and D'Hérelle 1917). Over the last decade a marked increase in interest in the therapeutic use of phages has been observed, which has resulted due to a substantial rise in the prevalence of antibiotic resistance of bacteria, coupled with an inadequate number of new antibiotics (Miedzybrodzki R et al., 2012). Properly formulated and applied phages have sufficient potential to cure bacterial infections. The key advantage of phages as anti-microbial therapeutic agents is their potential to negatively impact only their specific bacterial targets. Other advantages include, for example, an increase in phage number over the course of treatment, tendency to only minimally disrupt normal flora, capability of disrupting bacterial biofilms, low inherent toxicities, and most importantly effectiveness against both antibiotic-sensitive and antibiotic-resistant bacteria.
In 1982, Chinenova and colleagues reported a unique phage defense phenotype in Streptomyces coelicolor A3(2), which was denoted Phage Growth Limitation (PGL) (Chinenova T. A. et al, 1982). In their work Chinenova et al. demonstrated that upon the first cycle of infection by the φ31 phage, Streptomyces coelicolor A3 was phage-sensitive and supported phage burst. However, phages emerging from this first cycle of infection could not successfully re-infect the Streptomyces coelicolor A3 host. Intriguingly, these phages were able to successfully infect strains of Streptomyces that do not carry the PGL system (Chinenova T. A. et al, 1982).
Further studies mapped the phenotype to a cluster of four genes, denoted pglW, pglX, pglY and pglZ, which were shown to reconstitute the above described PGL phenotype upon transfer to a PGL host (Sumby. P. & Smith, M. C. 2002). Of note, introduction of pglY and pglZ− was not sufficient to confer a PGL+ phenotype in all mutants tested (Laity et al., 1993; Sumby et al. 2002). The domains encoded within these four genes do not resemble any classical combination of genes currently known to be involved in phage defense: pglZ is a member of the alkaline phosphatase superfamily; pglW has a serine/threonine kinase domain; pglX is an adenine-specific DNA methyltransferase; and pglY contains a p-loop ATPase domain (Sumby, P. & Smith, M. C. 2002). The PGL system described to date was not active against any other phage except for φC31 and its homoimmune relatives (Sumby. P. & Smith, M. C. 2002; Laity, C. et al. 1993).
A major characteristic of the PGL system described to date is the initial release of phage from the first infectious cycle followed by the attenuation of phage growth in the second. Various combinations of genes belonging to the PGL system, and predominantly pglZ, were found to be enriched within ‘defense islands’ (typical clustering of genes encoding defense system components in microbial genomes), providing additional support to the general involvement of these genes in a complex anti-phage defense system in multiple species (Makarova, K. S. et al. 2011; Makarova, K. S. et al. 2013). The discovery of the PGL system as an additional line of defense in bacteria may shed more light on the complex bacteria and phage arms race. However, a molecular mechanism that explains the activity of the PGL system has not yet been solved. Profound understanding of the molecular mechanism of this system might prove to be a powerful and economically important tool in molecular engineering applications (as was previously demonstrated with other complex phage resistance systems such as CRISPR-Cas).
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a BREX system comprising:
According to some embodiments of the invention, the nucleic acid construct comprising the polynucleotide encoding the BREX system further comprises a cis-acting regulatory element for directing expression of the nucleic acid sequence.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.
According to an aspect of some embodiments of the present invention there is provided a phage defense composition, comprising as an active ingredient a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; and an acceptable carrier or diluent.
According to some embodiments of the invention, the nucleic acid construct or the composition comprises the BREX system formulated in a formulation suitable for cell penetration.
According to an aspect of some embodiments of the present invention there is provided an isolated cell genetically modified to express a BREX system selected from the group consisting of
(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW,
(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.
(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL,
(3ii) brxA, brxB, (brxC/pglY)x2, pglX, pglZ and brxHII,
(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI,
(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA,
(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI, or
(3vi) brxP, brxC/pglY, pglZ and brxL.
According to some embodiments of the invention, the genetically modified cell further being resistant to a first cycle phage infection.
According to some embodiments of the invention, the genetically modified cell being resistant to phage lysogeny.
According to some embodiments of the invention, the genetically modified cell being resistant to lytic phage.
According to some embodiments of the invention, the genetically modified cell being resistant to phage DNA replication.
According to an aspect of some embodiments of the present invention there is provided an isolated cell genetically modified to express a BREX system polypeptide selected from the group consisting of pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE.
According to some embodiments of the invention, the isolated cell does not express a BREX system endogenously.
According to some embodiments of the invention, there is provided a method of protecting bacteria from phage attack, the method comprising expressing in the bacteria the isolated polynucleotide or the nucleic acid construct, thereby protecting the bacteria from phage attack.
According to an aspect of some embodiments of the present invention there is provided a method of protecting first bacteria from phage attack, the method comprising contacting the first bacteria with second bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, wherein the first bacteria and the second bacteria are non identical; thereby protecting the bacteria from phage attack.
According to some embodiments of the invention, the first bacteria does not express a BREX system endogenously.
According to some embodiments of the invention, the bacteria does not express a BREX system endogenously.
According to some embodiments of the invention, the phage is selected from the group consisting of SPβ, SP16, Zeta, Φ3T and SPO2.
According to some embodiments of the invention, the phage is not Φ105, rho10 and rho14.
According to some embodiments of the invention, the phage is a lytic phage.
According to some embodiments of the invention, the lytic phage is SPO1 and/or SP82G.
According to an aspect of some embodiments of the present invention there is provided an isolated bacteria comprising a nucleic acid sequence encoding a BREX system and a transmissible genetic element expressing the BREX system, wherein the isolated bacteria do not endogenously express the BREX system and wherein the BREX system comprises brxC/pglY, pglZ and at least one of pglX, pglXL, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprises brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.
According to some embodiments of the invention, the BREX system is type 1 comprising brxA, brxB, brxC/pglY, pglX, pglZ and brxL.
According to some embodiments of the invention
According to an aspect of some embodiments of the present invention there is provided a method of inducing phage sensitivity in a bacterial cell, the method comprising contacting a bacterial cell which expresses a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby inducing sensitivity of the bacterial cell to phage infection.
According to some embodiments of the invention, the contacting is effected ex-vivo or in-vitro.
According to some embodiments of the invention, the contacting is effected in-vivo.
According to some embodiments of the invention, there is provided an isolated bacteria generated according to the method.
According to some embodiments of the invention, there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product the isolated polynucleotide, the construct, the composition, the isolated cell or the bacteria, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.
According to an aspect of some embodiments of the present invention there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product a bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.
According to some embodiments of the invention, the transmissible genetic element comprises a conjugative genetic element or mobilizable genetic element.
According to some embodiments of the invention, the food or feed is a dairy product.
According to some embodiments of the invention, the cell is a bacteria.
According to some embodiments of the invention, the bacteria is a species selected from the group consisting of Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, Brucella, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix, and Brevibacterium.
According to some embodiments of the invention, the bacteria is a lactic acid bacteria.
According to some embodiments of the invention, the bacteria is a species selected from the group consisting of Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus species, Pediococcus species, Bifidobacterium, and Propionibacterium species.
According to some embodiments of the invention, there is provided a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising the isolated polynucleotide, the construct, the composition, the isolated cell or the isolated bacteria.
According to some embodiments of the invention, the product further comprises a dairy product.
According to an aspect of some embodiments of the present invention there is provided a method of treating a microbial infection in a subject in need thereof, the method comprising contacting the bacteria with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby treating the infection.
According to some embodiments of the invention, the method further comprising administering to the subject a phage therapy.
According to some embodiments of the invention, the method further comprising administering to the subject an antibiotic.
According to some embodiments of the invention, the method further comprising administering to the subject a phage therapy and/or an antibiotic.
According to an aspect of some embodiments of the present invention there is provided an article of manufacture identified for killing a bacteria comprising a packaging material packaging an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and a phage.
According to an aspect of some embodiments of the present invention there is provided an anti-microbial composition comprising as active ingredient an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and an acceptable carrier or diluent.
According to some embodiments of the invention, the composition further comprising a phage.
According to some embodiments of the invention, the anti BREX system agent is administered in a formulation suitable for cell penetration.
According to some embodiments of the invention, the anti BREX system agent is selected from the group consisting of a nucleic acid suitable for silencing expression, aptamers, small molecules and inhibitory peptides.
According to some embodiments of the invention, the anti BREX system agent is directed against pglX.
According to some embodiments of the invention, the anti BREX system agent is directed against brxC/pglY or pglZ. According to an aspect of some embodiments of the present invention there is provided a method of screening for identifying phage useful for infecting a bacteria, the method comprising:
(a) contacting a phage with a bacteria expressing BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW or comprising pglY, brxC/pglZ, pglX, pglW and at least one of brxD and brxHI;
(b) monitoring phage sensitivity of the bacteria, wherein an increase in phage sensitivity of the bacteria in the presence of the phage compared to phage sensitivity in the absence of the phage is indicative of a phage useful for infecting the bacteria.
According to some embodiments of the invention, the carrier is a pharmaceutically acceptable carrier.
According to some embodiments of the invention, the BREX system is characterized by at least one of
(i) not being an abortive infection system;
(ii) not being a restriction modification system;
(iii) not preventing phage adsorption to a bacteria expressing same.
According to some embodiments of the invention, the pglX is a methylase.
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.
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:
The present invention, in some embodiments thereof, relates to polynucleotides encoding BREX system polypeptides and methods of using same.
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 continuous bacteria-phages arms race has led to rapid evolution of both anti-phage bacterial resistance systems and counter-resistance mechanisms developed by phages, many of which are yet uncharacterized. A broad array of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various substrates. Development of defense strategies and systems to curtail the propagation and evolution of phages in fermentation vats is therefore warranted. On the counter arm, properly formulated and applied phages have sufficient potential to cure bacterial infections addressing the therapeutic need for new antibiotics.
The PGL system has previously been reported as conferring phage resistance manifested by attenuation of phage growth in the second cycle.
Whilst reducing the present invention to practice, the present inventors have now uncovered a novel multi-gene phage resistance system broadly distributed in bacteria and archaea, which the present inventors denoted BREX (Bacteriophage Elusion) system. The newly discovered BREX system shares some structural and functional similarities with the previously described PGL system. The abundance of this system and the efficiency in which it protects against phages implies that it plays an important role as a major line of defense encoded by bacteria against phages.
Specifically, the present inventors have uncovered that BREX system confers complete or partial resistance against phages spanning a wide phylogeny of phage types, including lytic and temperate (also referred lysogenic) phages, even in the first cycle of infection. Alternatively, mutations (e.g.; frame shift in pglX) affecting the functionality of the BREX system abrogate phage resistance.
Taken together, the present teachings suggest that BREX system and functional portions thereof can be used for conferring phage resistance. Such naturally and engineered bacteria can be utilized for example 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. Alternatively, anti-BREX system agents can be used as antibiotics.
As is illustrated hereinunder and in the examples section which follows, the present inventors have uncovered that BREX system confers complete or partial resistance against phages spanning a wide phylogeny of phage types, including lytic and temperate phages, even in the first cycle of infection. Even more so, mutations (e.g.; frame shift in pglX) affecting the functionality of the BREX system abrogate phage resistance. Specifically, the present inventors have shown that the BREX system exists in almost 10% of sequenced microbial genomes, and can be divided into six coherent subtypes containing 4-8 genes each, two of which are core genes, pglZ and brxC/pglY, present in all subtypes (Examples 1-2, Tables 1-8,
The inventors have further demonstrated (Examples 3 and 4.
The present inventors have gained insight into BREX mechanism of action (Example 4,
Consequently, the present invention provides methods and compositions for use in the food, feed, medical and veterinary industries to confer phage resistance. On the other hand, the present invention provides methods suitable for use in the food, feed, medical and veterinary industries to generate phage with broader host range that can be used for more effective bio-control of bacteria.
Thus, according to a first aspect of the present invention, there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.
According to a second aspect of the present invention, there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.
According to another aspect of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a BREX system comprising:
(i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL;
(ii) brxA, brxB, (brxC/pglY)x2, pglX, pglZ and brxHII;
(iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI;
(iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA;
(v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHII; or
(vi) brxP, brxC/pglY, pglZ and brxL
As used herein the term “isolated” refers to at least partially separated from the natural environment, physiological environment e.g., a microorganism e.g., bacteria.
As used herein “BREX system” (previously denoted “PYZA system”) or a “functional BREX system”, refers to a multi-gene system which comprises BRX and/or PGL genes which expression confers phage resistance.
According to specific embodiments, the BREX system is characterized by at least one of
(i) not being an abortive infection system;
(ii) not being a restriction modification system;
(iii) not preventing phage adsorption to a bacteria expressing same.
The BREX system may be characterized by one, two or all i.e.: (i); (ii); (iii); (i)+(ii); (i)+(iii); (ii)+(iii) and (i)+(ii)+(iii).
According to a specific embodiment the BREX system is characterized by (i)+(ii)+(iii).
As used herein “abortive infection (Abi) system” refers to a controlled cell death of an infected bacterial cell which takes place prior to the production of phage progeny, thus protecting the culture from phage propagation. Methods of analyzing Abi include, but are not limited to cell survival assays using high multiplicity of infection, one step growth assays and determination of phage DNA replication by e.g. DNA sequencing and southern blot analysis as further described hereinbelow.
As used herein “restriction modification system” refers to the recognition and cleavage of foreign DNA. Typically, a restriction modification system comprises a restriction enzyme having an activity of cleaving DNA and a modification enzyme capable of protecting host DNA from the cleavage by the restriction enzyme e.g. by methylating the host DNA. Analyzing restriction modification mode of action include, but is not limited to, evaluation of host specific methylation, presence of degraded foreign DNA and host cell death in the absence of the modification enzyme by methods described infra.
As used herein “adsorption” refers to the attachment to the host (e.g. bacteria) cell surface via plasma membrane proteins and glycoproteins. Methods of analyzing phage adsorption include, but are not limited to enumerating free phages in bacterial cultures infected with the phages immediately after phage addition and at early time points (e.g. 30 minutes) following phage addition as further described hereinbelow.
As used herein “phage resistance” refers to a phage infection resistance which can be a first or a second cycle resistance. The phage can be a lytic phage or a temperate (lysogenic) phage. According to a specific embodiment the BREX system confers phage resistance to a first cycle phage infection. According to yet other specific embodiments, BREX system confers resistance to lytic phages.
According to a specific embodiment, BREX system confers resistance to phage lysogeny.
As used herein, the term “lysogeny” refers to the incorporation of the phage genetic material inside the genome of the host (e.g. bacteria). Methods of analyzing phage lysogent are well known in the art and include, but not limited to, DNA sequencing and PCR analysis.
According to another specific embodiment, BREX system confers resistance to phage DNA replication.
According to specific embodiments. BREX system does not confer resistance to phages Φ105, rho10 and rho14.
As used herein, “phage resistance” refers to an increase of at least 10% in bacterial resistance towards a phage in comparison to bacteria of the same species under the same developmental stage (culture state) which does not express a BREX system, as may be manifested in e.g. bacterial viability, phage lysogeny and phage DNA replication. According to a specific embodiment, the increase is in at least 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.
Assays for testing phage resistance are well known in the art and mentioned hereinbelow.
According to specific embodiments, BREX system confers resistance to a plasmid. The plasmid may undergo integration into the bacterial genome or may be episomal.
According to a specific embodiment, the plasmid is episomal.
As used herein, “plasmid resistance” refers to an increase of at least 5% in bacterial resistance towards a plasmid in comparison to bacteria of the same species under the same developmental stage (culture state) which does not express a BREX system, as may be manifested in e.g. viability. According to a specific embodiment, the increase is in at least 10%, 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.
Assays for testing plasmid resistance are well known in the art and include, but not limited to, a transformation assay such as described in Itaya and Tsuge [Methods Enzymol (2011) 498:427-47].
As used herein, “expressing” or “expression” refers to gene expression at the RNA and/or protein level.
As used herein the “Phage growth Limitation” abbreviated as PGL refers to a cluster of genes which were previously described in Streptomyces coelicolor A3(2) (Chinenova T. A. et al, 1982; Sumby, P. & Smith, M. C. 2002, herein incorporated by reference in its entirety).
As used herein the “Bacteriophage Exclusion” abbreviated as BREX refers to a cluster of genes some of which were previously described in Streptomyces coelicolor A3(2) (Chinenova T. A. et al, 1982; Sumby, P. & Smith, M. C. 2002,).
According to specific embodiments, the BREX genes which compose the BREX system comprise brxC/pglY, pglZ pglW, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, which can be divided into six coherent subtypes comprising 4-8 genes each, in which the gene order and composition is conserved.
Thus, the BREX subtypes according to some embodiments of the present invention are selected from the group consisting of:
(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the functional BREX system does not comprise pglW.
(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI,
(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL (also may be referred to as Type 1).
(3ii) brxA, brxB, (brxC/pglY)x2, pglX, pglZ and brxHII (also may be referred to as Type 5),
(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI (also may be referred to as Type 6),
(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA (also may be referred to as Type 3),
(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI (also may be referred to as Type 2), or
(3vi) brxP, brxC/pglY, pglZ and brxL (also may be referred to as Type 4).
Thus, specific examples of BREX systems which can be used according to the present teachings include but are not limited to BREX system type 1, BREX system type 2, BREX system type 3. BREX system type 4, BREX system type 5 and BREX system type 6 (see
According to specific embodiments, BREX system type 1 (previously denoted PYZA system type 1a) comprises brxA, brxB, brxC/pglY, pglX, pglZ and brxL; BREX system type 5 (previously denoted PYZA system type 1b) comprises brxA, brxB, (brxC/pglY)x2, pglX, pglZ and brxHII; BREX system type 6 (previously denoted PYZA system type 1c) comprises brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI; BREX system type 3 (previously denoted PYZA system type 2) comprises brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA; BREX system type 2 (previously denoted PYZA system type 3) comprises pglW, pglX, brxC/pglY, pglZ, brxD and brxHI; and BREX system type 4 (previously denoted PYZA system type 4) comprises brxP, brxC/pglY, pglZ and brxL.
According to specific embodiments the BREX system is type 1 comprising brxA, brxB, brxC/pglY, pglX, pglZ and brxL.
Two of the six genes found in type 1 BREX conserved cluster share homology with genes from the previously reported PGL system10,11: pglZ, coding for a protein with a predicted alkaline phosphatase domain, and pglX, coding for a protein with a putative methylase domain. The four additional genes include (i) a Ion-like protease-domain gene, denoted herein as brxL; (ii) brxA; (iii) brxB; and (iv) a ˜1200 amino acid protein with an ATP binding motif (GXXXXGK[T/S]), denoted herein as brxC. The preferential localization of this conserved gene cluster in the genomic vicinity of other defense genes suggests that it is a novel phage defense system.
The phage defense system originally described in Streptomyces coelicolor A3(2) as PGL is defined according to the present teachings as a type 2 BREX. While the PGL was described to be composed of four genes, pglW, pglX, pglY and pglZ, the present teaching suggest that 2 more genes, brxD and brxHI, are an integral part of the type 2 BREX system. In addition, pglW, an integral part of the previously described PGL, exists exclusively in type 2 BREX subtype.
The major phage resistance systems that were characterized to date, including the restriction-modification and CRISPR-Cas systems, encode mostly for proteins that interact with and manipulate DNA and RNA molecules. While the BREX system contains such proteins including methylases and helicases it also contains genes coding for proteins predicted to be involved in the manipulation of other proteins, such as the Ion-like protease, brxL, the predicted alkaline phosphatase, pglZ, and the serine/threonine kinase, brxW. Thus, according to specific embodiments, the defense mechanism employed by the BREX system takes place later in the infection where phage proteins are already produced and can be manipulated by pglZ and/or brxL.
According to other specific embodiments, BREX proteins target phage proteins co-injected with the phage DNA early in the infection cycle.
According to specific embodiments the BREX system acts before phage DNA replication.
According to specific embodiments, BREX proteins interact with other bacterial-encoded proteins to regulate BREX activity.
As used herein, the terms “pglY”, “brxC” and “brxC/pglY” refer to the polynucleotide and expression product e.g., polypeptide of the PGLY or BRXC gene. The polypeptide product of the PGLY and BRXC genes typically contains p-loop ATPase/ATP binding domain DUF2791 (pfam10923, SEQ ID NO: 6162) and a DUF499 domain. brxC/pglY together with pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, comprise a BREX system.
According to specific embodiments, brxC/pglY is selected from the group consisting of SEQ ID NO: 3155, 157-765 and 767-1175.
As used herein, the term “pglZ” refers to the polynucleotide and expression product e.g., polypeptide of the PGLZ gene. The polypeptide product of the PGLZ gene typically contains an alkaline phosphatase domain pfam08665. pglZ together with brxC/pglY and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, comprise a BREX system.
According to specific embodiments pglZ is selected from the group consisting of SEQ ID NO: 1176-1318, 1320-1856, 1858-2250, 6205 and 6204.
As used herein, the term “pglX” refers to the polynucleotide and expression product e.g., polypeptide of the PGLX gene. The polypeptide product of the PGLX gene typically contains an adenine-specific DNA methyltransferase domain pfam13659 (COG1002/COG0286). pglX together with at least brxC/pglY and pglZ and optionally at least one of pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system. pglX is a critical gene as the present inventors have shown that it presented high rates of irregularities in the BREX systems documented and a frame shift mutation in this gene in one of the type 1 BREX-containing Bacillus subtilis strains obtained was not active against any of the tested phages. In addition, Bacillus subtilis strains containing a type 1 BREX having a deletion of pglX were sensitive to all the phages tested.
According to specific embodiments pglX is selected from the group consisting of SEQ ID NO: 2251-3280 and 6186-6201.
According to specific embodiments, pglX is a methylase.
According to a specific embodiment, the pglX methylase and pglXI methylase are analogous in BREX systems types 1 and 3, respectively.
According to specific embodiments, the methylase of the BREX system methylates the bacterial DNA.
According to a specific embodiment, the methylase of the BREX system drives motif-specific (e.g. an adenine residue in TAGGAG motif) methylation on the genomic DNA of a bacteria expressing same. According to specific embodiments the methylation is non-polindromic. According to specific embodiments the BREX system methylase does not methylate a phage genome.
According to specific embodiments the methylation serves as part of the self/non-self recognition machinery of BREX.
According to a specific embodiment, type 4 BREX does not contain a methylase.
According to a specific embodiment the pglX methylase and brxP reductase are analogous in BREX systems types 1 and 4, respectively.
Methods of assessing DNA methylation and, more specifically, adenine-specific methylation are well known in the art and include e.g. the PacBio sequencing platform [Murray et al. Nucleic acids research (2012) 40: 11450-11462].
As used herein, the term “pglXI” refers to the polynucleotide and expression product e.g., polypeptide of the PGLXI gene. The polypeptide product of the PGLXI gene typically contains an adenine-specific DNA methylase COG0863/COG1743 (pfam 01555). pglXI together with at least brxC/pglY and pglZ and optionally at least one of pglX, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.
According to specific embodiments pglXI is selected from the group consisting of SEQ ID NO: 3281-3296, 3298-3356 and 3358-3403.
As used herein, the term “brxP” (previously denoted “pglPA”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXP gene. The polypeptide product of the BRXP gene typically contains a phosphoadenosine phosphosulfate reductase domain COG0175 (pfam01507), and a pfam13182 domain. brxP together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.
According to specific embodiments brxP is selected from the group consisting of SEQ ID NO: 3404-3440.
As used herein, the term “brxHI” (previously denoted “pglHI”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXHI gene. The polypeptide product of the BRXHI gene typically contains an Lhr-like a helicase domain COG1201. brxHI together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.
According to specific embodiments brxHI is selected from the group consisting of SEQ ID NO: 3543-3642.
As used herein, the term “brxHII” (previously denoted “pglHII”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXHII gene. The polypeptide product of the BRXHII gene typically contains a DNA/RNA helicase domain COG0553. brxHII together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.
According to specific embodiments brxHII is selected from the group consisting of SEQ ID NO: 3441-3460, 3462-3511, 3513-3542 and 6173-6185.
As used herein, the term “brxL” (previously denoted “pglL”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXL gene. The polypeptide product of the BRXL gene typically contains a ion-like protease domain COG4930. brxL together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.
According to specific embodiments brxL is selected from the group consisting of SEQ ID NO: 3643-4412, 6165, 6166, 6169, 6170, 6202 and 6203.
As used herein, the term “brxD” (previously denoted “pglD”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXD gene. The polypeptide product of the BRXD gene typically contains an ATP binding domain DUF2791 (pfam10923). brxD together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL brxA, brxB, brxF, brxE and pglW, comprise a BREX system.
According to specific embodiments brxD is selected from the group of consisting SEQ ID NO: 4413-4488.
As used herein, the term “brxA” (previously denoted “pglA”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXA gene. The polypeptide product of the BRXA gene typically contains a DUF1819 (pfam08849) domain. The brxA protein displays significant structural homology to NusB spanning the RNA-binding interface, as well as part of the protein:protein interaction interface of NusB with NusE. In light of this similarity, according to specific embodiments brxA is an RNA binding protein. According to specific embodiments, brxA has a role in interfering with the phage infection cycle by disrupting anti-termination events essential for the phage cycle. brxA together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxB, brxF, brxE and pglW comprise a BREX system.
According to specific embodiments brxA is selected from the group consisting of SEQ ID NO: 4489-4621, 4623-5086, 5088-5415, 6167, 6168, 6171 and 6172.
As used herein, the term “brxB” (previously denoted “pglB”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXB gene. The polypeptide product of the BRXB gene typically contains a DUF1788 (pfam08747) domain. brxB together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP. brxHI, brxHII, brxL, brxD, brxA, brxF, brxE and pglW, comprise a BREX system.
According to specific embodiments brxB is selected from the group consisting of SEQ ID NO: 5416-5947 and 6206-6209.
As used herein, the term “brxF” (previously denoted “pglC”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXF gene. The polypeptide product of the BRXF gene typically contains an ATPase domain, brxF together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxE and pglW, comprise a BREX system.
According to specific embodiments brxF is selected from the group consisting of SEQ ID NO: 5948-5957, 5959-5988 and 5990-6028.
As used herein, the term “brxE” (previously denoted “pglE”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXE gene. brxE together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and pglW, comprise a BREX system.
According to specific embodiments brxE is selected from the group consisting of SEQ ID NO: 6029-6040.
As used herein, the term “pglW” refers to the polynucleotide and expression product e.g., polypeptide of the PGLW gene. The polypeptide product of the PGLW gene typically contains a serine/threonine kinase domain COG0515. pglW together with brxC/pglY, pglZ and pglX, and at least one of brxD and brxHI, and optionally at least one of pglXI, brxP, brxHII, brxL, brxA, brxB, brxF, and brxE, comprise a BREX system.
According to specific embodiments pglW is selected from the group consisting of SEQ ID NO: 6041-6138.
The terms “brxC/pglY”, “pglZ”, “pglX”, “pglXI”, “brxP”, “brxHI”, “brxHII”, “brxL”, “brxD”, “brxA”, “brxB”, “brxF”, “brxE”, and “pglW” also refers to functional brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW homologues which exhibit the desired activity (i.e., conferring phage resistance). Such homologues can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the polypeptide SEQ ID NO: 614-765, 767-1175, 1714-1856, 1858-2250, 6204, 2766-3280, 6186, 6188, 6190, 6192, 6194, 6196, 6198, 6200, 3343-3356, 3358-3403, 3422-3440, 3492-3511, 3513-3542, 6173, 6175, 6178, 6180, 6182, 6184, 3593-3642, 4028-4412, 6165, 6169, 6202, 4438-4488, 4953-5086, 5088-5415, 6167, 6171, 5570-5947, 6206, 6208, 5979-5988, 5990-6028, 6035-6040 and 6090-6138, respectively, or 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding same (as further described hereinbelow). The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.
Alternatively or additionally, homology can be based on shared motifs [e.g., the p-loop motif GXXXXGK(T/S) (DUF2791, SEQ ID NO: 6162) and DUF499 motifs present in pglY] combined with the conserved size of the gene in the different subtypes and the location of the gene in the gene cluster.
Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, MUSCLE, and HHpred.
As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
According to specific embodiments the polynucleotides of the present invention are part of a nucleic acid construct comprising the polynucleotide encoding the BREX system and at least one cis-acting regulatory element for directing expression of the nucleic acid sequence.
Teachings of the invention further contemplate that the polynucleotides are part of a nucleic acid construct system where the BREX genes are expressed from a plurality of constructs.
Thus, the present invention further provides for a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW.
The present invention further provides for a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.
Thus according to specific embodiments, the nucleic acid construct system comprises an individual nucleic acid construct for each BREX system pgl and/or brx gene.
According to other specific embodiments a single construct comprises a number of BREX system pgl and/or brx genes.
Cis acting 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.
According to specific embodiments, the 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.
According to specific embodiments the promoter is a bacterial nucleic acid (e.g., expression) construct.
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. 1. 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 phage promoter can be joined with the operon sequences of another bacterial or phage 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 phage 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 phage promoter and an E. coli operator region (EPO Publication No. 267,851).
The nucleic acid construct 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 LacI 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., laclq) or that modify the manner of induction (e.g., lambda C1857, rendering lambda pL thermo-inducible, or lambda CI+, rendering lambda pL chemo-inducible) can be employed.
Various construct schemes can be utilized to express few genes from a single nucleic acid construct. For example, the genes can be co-transcribed as a polycistronic message from a single promoter sequence of the nucleic acid construct. To enable co-translation of all the genes from a single polycistronic message, the different polynucleotide segments can be transcriptionally fused via a linker sequence including an internal ribosome entry site (IRES) sequence which enables the translation of the polynucleotide segment downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule including the coding sequences of all the genes will be translated from both the capped 5′ end and the internal IRES sequence of the polycistronic RNA molecule to thereby produce the whole BREX system.
Alternatively, each two polynucleotide segments can be translationally fused via a protease recognition site cleavable by a protease expressed by the cell to be transformed with the nucleic acid construct. In this case, a chimeric polypeptide translated will be cleaved by the cell expressed protease to thereby generate the whole BREX system.
Still alternatively, the nucleic acid construct of some embodiments of the invention can include at least two promoter sequences each being for separately expressing a specific pgl or brx. These at least two promoters which can be identical or distinct can be constitutive, tissue specific or regulatable (e.g. inducible) promoters functional in one or more cell types.
The nucleic acid construct (also referred to herein as an “expression vector” or a “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, typical 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.
When secretion of the polypeptides is desired the polynucleotides of the invention can be expressed as fusion polypeptides comprising the nucleic acid sequence encoding the PGL or BRX gene ligated in frame to a nucleic acid sequence encoding a signal peptide that provides for secretion.
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.
According to a specific embodiment, the nucleic acid construct comprises a plurality of cloning sites for ligating a nucleic acid sequence of the invention such 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.
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.
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 polypeptide.
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.
Various methods known within the art 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, natural or induced transformation, 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, such as calcium chloride co-precipitation.
Introduction of nucleic acids by phage infection offers several advantages over other methods such as transformation, since higher transfection efficiency can be obtained due to the infectious nature of phages. These methods are especially useful for rendering bacteria more sensitive to phage attack for antibiotics purposes as further described hereinbelow.
It will be appreciated the BREX polypeptides can be introduced directly into the cell (e.g., bacterial cell) and not via recombinant expression to confer resistance. The term “polypeptide” 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.
The polypeptides of the present invention may be synthesized by any techniques known to those skilled in the art of peptide synthesis, for example but not limited to recombinant DNA techniques or solid phase peptide synthesis.
Thus, regardless of the method of introduction, the present teachings provide for an isolated cell (e.g., bacterial cell) which comprises a heterologous BREX system, as described herein.
According to specific embodiments, the isolated cell is transformed or transfected with the above-mentioned nucleic acid construct or nucleic acid construct system.
According to an aspect of the present invention, there is provided an isolated cell genetically modified to express a BREX system selected from the group consisting of
(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHI, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW,
(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI,
(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL.
(3ii) brxA, brxB, (brxC/pglY)x2, pglX, pglZ and brxHII.
(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI,
(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA,
(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI, or
(3vi) brxP, brxC/pglY, pglZ and brxL.
According to another aspect of the present invention there is provided an isolated cell genetically modified to express a BREX system polypeptide selected from the group consisting of pglX, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE.
According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of a COG0515 domain, said polypeptide conferring resistance to a first cycle phage infection.
According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of a pfam13659 domain, said polypeptide conferring resistance to a first cycle phage infection.
According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of DUF2791 and DUF499 domains, said polypeptide conferring resistance to a first cycle phage infection.
According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of a pfam08665 domain, said polypeptide conferring resistance to a first cycle phage infection.
According to specific embodiments there is provided an isolated cell genetically modified to express a pglW polypeptide with the proviso that said pglW polypeptide is not SEQ ID NO: 6110.
According to specific embodiments there is provided an isolated cell genetically modified to express a pglX polypeptide with the proviso that said pglX polypeptide is not SEQ ID NO: 2949.
According to specific embodiments there is provided an isolated cell genetically modified to express a brxC/pglY polypeptide with the proviso that said brxC/pglY polypeptide is not SEQ ID NO: 802.
According to specific embodiments there is provided an isolated cell genetically modified to express a pglZ polypeptide with the proviso that said pglZ polypeptide is not SEQ ID NO: 1890.
According to specific embodiment the isolated cell (e.g., bacterial cell) does not express a BREX system endogenously.
The term “endogenous” as used herein, refers to the expression of the native gene in its natural location and expression level in the genome of an organism.
The expression of the polynucleotide can be episomal or integrated into the chromosome of the cell.
According to specific embodiments the isolated cell is resistant to a first cycle phage infection.
According to specific embodiments the isolated cell is resistant to lytic phage.
According to specific embodiments the isolated cell is resistant to temperate (also referred as lysogenic) phage.
According to a specific embodiment the isolated cell is resistant to phage lysogeny.
According to another specific embodiment the isolated cell is resistant to phage DNA replication.
According to specific embodiments the isolated cell is a microbial cell such as a bacterial cell.
As used herein, the term “bacteria” refers to all prokaryotes and includes both bacteria and archaea.
Indeed, it is intended that any bacterial species (e.g., which does not express a PYZA system) will find use in the present invention. Thus, the bacteria may be for example gram-positive or gram-negative bacteria.
The phrase “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. Corvnebacteriwnum jeikeium, Enterococcus faecalis, Enterococcus faecium, Ervsipelothrix 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 colmii, 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 refers 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 xvlosoxidans, Bacteroides. Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamndia 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.
According to specific embodiments the bacteria is a species selected from the group consisting of Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, Brucella, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix, Bifidobacterium, Brevibacterium, Propionibacterium. Lactococcus, Lactobacillus, Pediococcus, Leuconostoc. Oenococcus, and Propionibacterium species.
Additionally, or alternatively the bacteria may be useful in the manufacture of dairy and fermentation processing such as, but not limited to, milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk.
According to specific embodiments the bacteria is a lactic bacteria. As used herein the term “lactic acid bacteria” refers to Gram positive, microaerophillic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid.
According to specific embodiments the bacteria is a species selected from the group of the industrially most useful lactic acid bacteria consisting of Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus species, Pediococcus species and Bifidobacterium species and Propionibacterium species.
As used herein, the term “phage” or “bacteriophage” refers to a virus that selectively infects one or more bacterial species. Many phages are specific to a particular genus or species or strain of bacteria.
According to specific embodiments, the phage is virulent to the bacteria.
According to some embodiments, the phage is a lytic phage.
According to other embodiments, the phage is temperate (also referred to as lysogenic).
A lytic phage is one that follows the lytic pathway through completion of the lytic cycle, rather than entering the lysogenic pathway. A lytic phage undergoes viral replication leading to lysis of the cell membrane, destruction of the cell, and release of progeny phage particles capable of infecting other cells.
A temperate phage is one capable of entering the lysogenic pathway, in which the phage becomes a dormant, passive part of the cell's genome through prior to completion of its lytic cycle.
Exemplary phages which fall under the scope of the invention include, but are not limited to, phages that belong to any of the following virus families: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae.
According to specific embodiments the phage is selected from the group consisting of SPβ, SP16, Zeta, Φ3T, and SPO2.
According to other specific embodiments the phage is not Φ105, rho10 and rho14.
According to specific embodiments, the lytic phage is SPO1 and/or SP82G.
According to specific embodiments, phage that infect bacteria that are pathogenic to plants and/or animals (including humans) find particular use.
According to specific embodiments, the resistance of a cell against a phage is improved as compared to a cell of the same species which was not treated according to the present teachings (i.e., with a BREX system).
The lysogenic activity of a phage can be assessed in multiple ways, including but not limited to PCR and DNA sequencing.
The DNA replication activity of a phage can be assessed in multiple ways, including but not limited to DNA sequencing and southern blot analysis.
The lytic activity of a phage can be assessed in multiple ways, including but not limited to optical density, plaque assay, and living dye indicators.
The lytic activity of a phage can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. This method involves introduction of phage into a fluid bacterial culture medium. After a period of incubation, the phage lyses the bacteria in the broth culture resulting in a clearing of the fluid medium resulting in decrease in optical density.
Another method, known as the plaque assay, introduces phage into a few milliliters of soft agar along with some bacterial host cells. This soft agar mixture is laid over a hard agar base (seeded-agar overlay). The phage adsorb onto the host bacterial cells, infect and lyse the cells, and then begin the process anew with other bacterial cells in the vicinity. After 6-24 hours, zones of clearing on the plate known as plaques, are observable within the lawn of bacterial growth on the plate. Each plaque represents a single phage particle in the original sample.
Yet another method is the one-step phage growth curve which allows determining the production of progeny virions by cells as a function of time after infection. The assay is based on the fact that cells in the culture are infected simultaneously with a low number of phages so that no cell can be infected with more than one phage. At various time intervals, samples are removed for a plaque assay allowing quantitative determination of the number of phages present in the medium.
Other methods use for example redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a dye (e.g., tetrazolium dye) and produces a color change that can be measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color.
Thus, the polynucleotides, polypeptides and nucleic acid constructs of the present invention can be used in conferring phage resistance.
As used herein, “confers phage resistance” refers to an increase of at least 10% in bacterial resistance towards a phage, as may be manifested in viability. According to a specific embodiment, the increase is in at least 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.
For the same culture conditions the bacterial susceptibility towards a phage of the present invention is generally expressed in comparison to the wild-type bacteria. As used herein, the phrase “increased resistance towards a phage” means that the level of phage infection and/or multiplication in the bacteria does not cause a deleterious effect to the bacteria e.g., growth arrest or death.
In some embodiments, the bacteria have about 100-100.000 times lower efficiency of plaquing ([EOP]=10-2), about 1000 times lower EOP (EOP=10=3), 10,000 times lower EOP (EOP=10-4), or 100,000 times lower EOP (EOP=10-5). In some embodiments, the level of phage multiplication in a culture is measured after about 6-14 hours incubation of the culture, e.g., after about 12 hours, after about 9 hours, after about 8 hours after about 7 hours, or after about 6 hours.
Thus, according to specific embodiments there is provided a method of protecting bacteria from phage attack, the method comprising introducing into or expressing in the bacteria a BREX system, thereby protecting the bacteria from phage attack.
According to specific embodiments the bacteria does not express a BREX system endogenously.
Various modalities may be used to introduce or express the BREX system in the bacteria.
Thus, according to specific embodiments, the method is effected by expressing in the bacteria, the isolated polynucleotides, nucleic acid construct or construct system or alternatively introducing the BREX polypeptides as described herein to confer protection.
According to another embodiment the BREX system is introduced into the bacteria via a transmissible genetic element in a process of bacterial conjugation.
As used herein, the phrase “bacterial conjugation” refers to a direct transfer of genetic material between bacterial cells by cell-to-cell contact or by bridge-like connection between the cells. During conjugation the donor bacteria provides a transmissible genetic element, typically a plasmid or a transposon. The transfer of the transmissible genetic element tale advantage of the complementary nature of double stranded DNA. Thus, one strand of the transmissible genetic element is transferred and the other remains in the original bacteria. Both strands have the complementary stranded added so that each bacteria ends up with a complete transmissible element.
According to a specific embodiment, there is provided a method of protecting first bacteria from phage attack, the method comprising contacting the first bacteria with second bacteria which expresses on a transmissible genetic element a BREX system, wherein the first bacteria and the second bacteria are non identical; thereby protecting the bacteria from phage attack.
As used herein, the term “contacting” refers to the step of incubation of the bacterial cell (e.g., first bacteria) with a substance or cell (e.g., second bacteria) such that the substance or a substance contained in the cell affects phage resistance of the bacterial cell.
According to specific embodiments the first bacteria does not express a BREX system endogenously.
As used herein the phrase “transmissible genetic element” refers to a nucleic acid sequence that can be transferred naturally from one bacteria to another.
According to specific embodiments the transmissible genetic element comprises a conjugative genetic element or a conjugative plasmid or mobilizable genetic element.
As used herein, a “conjugative plasmid” refers to a plasmid that is transferred from one bacterial cell to another during conjugation.
As used herein, the term “mobilizable element” refers to a transposon, which is a DNA sequence that can change its position within the genome.
According to a specific embodiment, the first bacteria is the industrially valuable bacteria such as those used for fermentation as described above.
Thus, following the above teachings there is provided an isolated bacteria comprising a nucleic acid sequence encoding a BREX system and a transmissible genetic element expressing the BREX system, wherein the isolated bacteria does not endogenously express the BREX system and wherein the BREX system comprises brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprises brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.
Cultures, and starter cultures, in particular are used extensively in the food industry in the manufacture of fermented products including milk products (e.g., yogurt, buttermilk, and cheese), meat products, bakery products, wine, and vegetable products. The preparation of cultures is labor intensive, occupying much space and equipment, and there is a considerable risk of contamination with spoilage bacteria and/or phages during the propagation steps. The failure of bacterial cultures due to phage infection and multiplication is a major problem with the industrial use of bacterial cultures. There are many different types of phages and new strains continue to emerge. Indeed, despite advances in culture development, there is a continuing need to improve cultures for use in industry.
Thus, according to an aspect of the present invention, there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product the isolated BREX system polynucleotide, the BREX system construct, the isolated cell or the isolated bacteria of the present invention, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.
Thus, following the above teachings there is provided a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising the isolated BREX system polynucleotide, the BREX system construct, the isolated cell or the isolated bacteria of the present invention.
According to another aspect of the present invention, there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product a bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.
According to specific embodiments the food or feed is a dairy product.
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.
Products produced by fermentation which have been known to experience phage infection, and the corresponding infected fermentation bacteria, include cheddar and cottage cheese (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris), yogurt (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus). Swiss cheese (S. thermophilus, Lactobacillus lactis, Lactobacillus helveticus), blue cheese (Leuconostoc cremoris), Italian cheese (L. bulgaricus, S. thermophilus), viili (Lactococcus lactis subsp. cremoris. Lactococcus lactis subsp. lactis biovar diacetylactis, Leuconostoc cremoris), yakult (Lactobacillus casei), casein (Lactococcus lactis subsp. cremoris), natto (Bacillus subtilis var. natto), wine (Leuconostoc oenos), sake (Leuconostoc mesenteroides), polymyxin (Bacillus polymyxa), colistin (Bacillus colistrium), bacitracin (Bacillus licheniformis), L-glutamic acid (Brevibacterium lactofermentum, Microbacterium ammoniaphilum), and acetone and butanol (Clostridium acetobutylicum. Clostridium saccharoperbutvlacetonicum).
The present inventors have uncovered that transformation of a Bacillus subtilis strain with a non-complete type 1 BREX (i.e. not expressing pglX) does not confer phage resistance. In addition it was also discovered that a frame shift mutation in a BREX gene (i.e., pglX) in one of the Bacillus subtilis strains transformed with type 1 BREX resulted in aberrant BREX system that was not active against any of the tested phages, indicating that down regulation of a BREX gene can render a bacteria resistant to phage infection. These results suggest the use of anti BREX agents as a method to induce phage sensitivity.
As used herein, “inducing phage sensitivity” refers to an increase of at least 10% in bacterial susceptibility towards a phage, as may be manifested in growth arrest or death. According to a specific embodiment, the increase is in at least 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.
For the same culture conditions, the bacterial susceptibility towards a phage of the present invention is generally expressed in comparison to the wild-type bacteria. As used herein, the phrase “increased susceptibility towards a phage” means that the level of phage infection and/or multiplication in the bacteria cause a deleterious effect to the bacteria e.g., growth arrest or death.
Thus, according to further aspect of the present invention, there is provided a method of inducing phage sensitivity in a bacterial cell, the method comprising contacting a bacterial cell which expresses a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglX, brxP, brxHI, brxHI, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby inducing sensitivity of the bacterial cell to phage infection.
As used herein the phrase “anti BREX system agent” is an agent capable of specifically inhibiting or “silencing” the expression of a target BREX gene or alternatively specifically impairs the functionality of the target BREX protein. According to specific embodiments the anti BREX system agent is directed against pglX. For example, the anti-BREX system may interfere with pglX expression (as described hereinbelow) or in its DNA methyltransferase function by the use of common inhibitors of such an enzyme e.g., 5-Azacytidine. Decitabine Zebularine, RG108, Hydralazine hydrochloride, and Psammaplin A.
According to other specific embodiments the anti BREX system agent is directed against brxC/pglY or pglZ.
Down regulation of BREX system can be effected on the genomic and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents), or on the protein level using e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide and the like.
According to specific embodiments the anti BREX system agent is selected from the group consisting of a nucleic acid suitable for silencing expression, aptamers, small molecules and inhibitory peptides.
As used herein the phrase “nucleic acid suitable for silencing expression” refers to regulatory mechanisms mediated by nucleic acid molecules which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene. Numerous methods are known in the art for gene silencing in prokaryotes. Examples include but are not limited to U.S. Patent Application 20040053289 which teaches the use of si hybrids to down-regulate prokaryotic genes, and
U.S. Patent Application PCT/US09/69258 which teaches the use of CRISPR to downregulate prokaryotic genes. Alternatively the inhibition can be carried out at the protein level which interferes with protein activity, such as by the use of aptamers. Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).
As used herein an “aptamer” refers to double stranded DNA or single stranded RNA molecule that binds to specific molecular target, such as a protein.
Alternatively or additionally, small molecule or peptides can be used which interfere with the BREX protein function (e.g., catalytic or interaction).
Specifically, contacting is effected such that the positioning of the anti BREX system agent is in direct or indirect contact with the bacterial cell. Thus, the present invention contemplates both applying the anti BREX system agents 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 bacteria 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 anti BREX agents per se or by transfecting the cells of the organism with the anti BREX agents of the present invention.
Thus, according to a specific embodiment contacting with an anti BREX system agent is effected in-vivo.
According to another specific embodiment contacting with an anti BREX system agent is effected ex-vivo.
According to another specific embodiment contacting with an anti BREX system agent is effected in-vitro.
According to specific embodiments, there is provided an isolated bacteria generated by contacting bacteria with anti BREX system agent in-vitro or ex-vivo.
According to some embodiments, a BREX system or an anti-BREX system agent is provided in a formulation suitable for cell penetration that enhances intracellular delivery of BREX system.
Any suitable penetrating agent for enhancing penetration of BREX system or anti BREX system agent to cell (e.g., bacteria) may be used, as known by those of skill in the art. Examples include but are not limited to:
Phages—Phages offer several advantages including lateral infection, higher efficiency of transformation, and targeting to, and propagation in, specific bacteria.
Cell-Penetrating Peptides (CPPs)—CPPs, for example TAT (transcription activator from HIV-1) are short peptides (≦40 amino acids), with the ability to gain access to the interior of almost any cell. They are highly cationic and usually rich in arginine and lysine amino acids. They have the exceptional property of carrying into the cells a wide variety of covalently and noncovalently conjugated cargoes such as proteins, oligonucleotides, and even 200 nm liposomes. Protocols for producing CPPs-cargos conjugates and for infecting cells with such conjugates can be found, for example L. Theodore et al. [The Journal of Neuroscience, (1995) 15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994) 91:664-668], and Jing Bian et al. [Circulation Research. (2007) 100: 1626-1633].
The expression level and/or activity level of the BREX system expressed in the cells of some embodiments of the invention can be determined using methods known in the arts, e.g. but not limited to selectable marker gene, Northern blot analysis, PCR analysis, DNA sequencing, RNA sequencing, Western blot analysis, and Immunohistochemistry.
According to another aspect of the present invention, there is provided a method of treating a microbial infection in a subject in need thereof, the method comprising contacting the bacteria with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby treating the infection.
As used herein, the term “treating” refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathogen infection.
As used herein, the phrase “subject in need thereof” includes mammals, preferably human beings at any age which suffer from pathogen infection.
The anti BREX system agent may be used alone or together with additional antimicrobial agents (e.g. phage therapy, antibiotic and/or additional anti microbial peptides).
According to specific embodiments the methods of the present invention further comprise administering to the subject a phage therapy.
According to other specific embodiments the methods of the present invention further comprise administering to the subject an antibiotic.
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 polypeptides 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 BREX system or the anti-BREX system agent of some embodiments of the invention can be administered to a starter culture, a fermentation vat or an organism per se, or in a composition where it is mixed with suitable carriers or excipients.
According to an aspect of the present invention there is provided a phage defense composition, comprising as an active ingredient a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHII, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; and an acceptable carrier or diluent.
According to another aspect of the present invention there is provided an anti-microbial composition comprising as active ingredient an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and an acceptable carrier or diluent.
As used herein, the phrase “anti-microbial activity”, refers to an ability to suppress, control, inhibit or kill a bacteria. Thus, for example the anti-microbial activity may comprise bactericidal or bacteriostatic activity, or both.
According to specific embodiments the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.
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.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used 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.
As used herein the term “active ingredient” refers to any one of BREX system polypeptide or polynucleotide, anti-BREX system agent capable of down regulating a BREX gene or cells generated according to the present teachings, accountable for the biological effect.
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 polypeptides, polynucleotides, or other agents 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 phage therapy, and antibiotics (e.g. rifampicin, chloramphenicol and spectinomycin).
According to specific embodiment the anti-microbial composition further comprises a phage.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
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, a gel and a patch.
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 (e.g., bacterial infection) 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.
Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
According to another aspect there is provided an article of manufacture or a kit identified for killing a bacteria comprising a packaging material packaging an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and a phage.
According to specific embodiments the anti BREX system agent and the phage are packaged in separate containers.
According to yet other specific embodiments the anti BREX system agent and the phage are in c-formulation.
According to further aspect of the present invention there is provided a method of screening for identifying phage useful for infecting a bacteria, the method comprising:
(a) contacting a phage with a bacteria expressing BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI;
(b) monitoring phage sensitivity of the bacteria, wherein an increase in phage sensitivity of the bacteria in the presence of the phage compared to phage sensitivity in the absence of the phage is indicative of a phage useful for infecting the bacteria.
The method comprising further isolating the phage characterizing it in terms of sequencing and compatibility with phages species and the ability to infect different bacterial species.
Tables 2-8 and 10-16 below demonstrate the six types of BREX system in a diverse array of bacteria and archaea genomes.
Acidiphilium
multivorum AIU301
Acidithiobacillus
ferrivorans SS3 uid67387
Acinetobacter baumannii
Anaeromyxobacter
dehalogenans 2CP C
Aromatoleum
aromaticum EbN1
Burkholderia CCGE1001
Burkholderia gladioli
Burkholderia
vietnamiensis G4
Calditerrivibrio
nitroreducens DSM
Carboxydothermus
hydrogenoformans Z
Chlorobium
phaeobacteroides BS1
Clostridium ljungdahlii
Clostridium
saccharolyticum WM1
Clostridium
saccharolyticum WM1
Clostridium sticklandii
Clostridium SY8519
Cupriavidus necator N 1
Cyanothece PCC 8802
Dehalococcoides VS
Dehalogenimonas
lykanthroporepellens BL
Desulfitobacterium
hafniense Y51 uid58605
Desulfomicrobium
baculatum DSM 4028
Desulfovibrio
magneticus RS 1
Desulfovibrio vulgaris
Hildenborough uid57645
Desulfurivibrio
alkaliphilus AHT2
Erwinia pyrifoliae Ep1
Erythrobacter litoralis
Escherichia coli HS
Escherichia coli O111 H
Escherichia fergusonii
Exiguobacterium
sibiricum 255 15
Gallionella
capsiferriformans ES 2
Gallionella
capsiferriformans ES 2
Geobacillus WCH70
Geobacter sulfurreducens
Haliscomenobacter
hydrossis DSM 1100
Lactobacillus casei
Lactobacillus johnsonii
Lactobacillus reuteri
Lactobacillus rhamnosus
Leuconostoc kimchii
Magnetospirillum
magneticum AMB 1
Marinobacter aquaeolei
Methanobrevibacter
smithii ATCC 35061
Methanosarcina
acetivorans C2A
Methanosarcina mazei
Methanospirillum
hungatei JF 1 uid58181
Methanospirillum
hungatei JF 1 uid58181
Microlunatus
phosphovorus NM 1
Moorella thermoacetica
Nostoc punctiforme PCC
Parvularcula
bermudensis HTCC2503
Pelobacter propionicus
Pelodictyon
phaeoclathratiforme BU
Photorhabdus
asymbiotica ATCC
Polaromonas JS666
Pseudomonas
brassicacearum NFM421
Psychrobacter
cryohalolentis K5
Rhodobacter sphaeroides
Rhodococcus
erythropolis PR4
Rhodopseudomonas
palustris TIE 1 uid58995
Runella slithyformis
Saccharophagus
degradans 2 40 uid57921
Salmonella enterica
Selenomonas sputigena
Shewanella ANA 3
Shewanella MR 4
Slackia
heliotrinireducens DSM
Spirosoma linguale DSM
Sulfuricurvum kujiense
Syntrophomonas wolfei
Syntrophomonas wolfei
Syntrophus
aciditrophicus SB
Tepidanaerobacter Re1
Thauera MZ1T uid58987
Thermoanaerobacterium
thermosaccharolyticum
Vibrio cholerae MJ 1236
Zymomonas mobilis
Thioalkalivibrio sp.
Haloarcula
hispanica
Halobacterium
salinarum R1
Halopiger
xanaduensis
Halorubrum
lacusprofundi
Halorhabdus
utahensis
Haloarcula
hispanica
Halobacterium
salinarum R1
Halopiger
xanaduensis
Halorubrum
lacusprofundi
Halorhabdus
utahensis
Anaeromyxobacter
dehalogenans 2CP
Haliangium
ochraceum DSM
Haliangium
ochraceum DSM
Planctomyces
limnophilus DSM
Anaeromyxobacter
dehalogenans 2CP
Haliangium
ochraceum DSM
Haliangium
ochraceum DSM
Planctomyces
limnophilus DSM
Acidothermus
cellulolyticus 11B
Parvibaculum
lavamentivorans DS 1
Parvibaculum
lavamentivorans DS 1
Chloroflexus aggregans
Desulfovibrio
aespoeensis Aspo 2
Methanosalsum zhilinae
Caldicellulosiruptor
kristjanssonii 177R1B
Pelotomaculum
thermopropionicum SI
Thermoanaerobacter
brockii finnii Ako 1
Thermoanaerobacter
pseudethanolicus ATCC
Thermoanaerobacterium
xylanolyticum LX 11
Thermoanaerobacter
italicus Ab9 uid46241
Syntrophothermus
lipocalidus DSM 12680
Acetohalobium
arabaticum DSM 5501
Dichelobacter nodosus
Nitrosococcus oceani
Nitrosococcus watsonii
Methylacidiphilum
infernorum V4
Thermanaerovibrio
acidaminovorans DSM
Planctomyces
brasiliensis DSM 5305
Tepidanaerobacter Re1
Candidatus
Accumulibacter
phosphatis clade IIA
Corynebacterium
variabile DSM 44702
Frankia CcI3
Frankia EuI1c
Hahella chejuensis
Haliangium
ochraceum DSM
Microlunatus
phosphovorus NM 1
Micromonospora
aurantiaca ATCC
Mycobacterium
gilvum PYR GCK
Polaromonas
naphthalenivorans
Saccharopolyspora
erythraea NRRL 2338
Sorangium
cellulosum So ce 56
Streptomyces
coelicolor A3 2
Streptomyces griseus
Thermobifida fusca
Burkholderia
thailandensis E264
Thermobispora
bispora DSM 43833
Saccharomonospora
viridis DSM 43017
Candidatus
Desulforudis
audaxviator
Coprothermobacter
proteolyticus DSM
Denitrovibrio
acetiphilus DSM
Geobacter M21
Prevotella
denticola F0289
Thermomicrobium
roseum DSM 5159
Thermotoga
petrophila RKU 1
Desulfovibrio vulgaris
Syntrophus aciditrophicus
Shewanella MR 4 uid58345
Acidiphilium multivorum
Nostoc punctiforme PCC
Aromatoleum aromaticum
Thauera MZ1T uid58987
Shewanella ANA 3
Salmonella enterica serovar
Typhimurium LT2
Desulfitobacterium
hafniense Y51 uid58605
Methanosarcina acetivorans
Methanosarcina mazei Go1
Saccharophagus degradans
Rhodococcus erythropolis
Geobacter sulfurreducens
Carboxydothermus
hydrogenoformans Z 2901
Exiguobacterium sibiricum
Moorella thermoacetica
Burkholderia vietnamiensis
Anaeromyxobacter
dehalogenans 2CP C
Polaromonas JS666
Dehalococcoides VS
Erythrobacter litoralis
Parvularcula bermudensis
Methanospirillum hungatei
Pelodictyon
phaeoclathratiforme BU 1
Escherichia coli HS
Chlorobium
phaeobacteroides BS1
Psychrobacter
cryohalolentis K5 uid58373
Syntrophomonas wolfei
Pelobacter propionicus
Magnetospirillum
magneticum AMB 1
Rhodobacter sphaeroides
Marinobacter aquaeolei
Gallionella
capsiferriformans ES 2
Rhodopseudomonas
palustris TIE 1 uid58995
Cyanothece PCC 8802
Methanobrevibacter smithii
Geobacillus WCH70
Slackia heliotrinireducens
Lactobacillus reuteri
Lactobacillus casei Zhang
Clostridium sticklandii
Spirosoma linguale DSM
Acinetobacter baumannii
Desulfomicrobium
baculatum DSM 4028
Selenomonas sputigena
Dehalogenimonas
lykanthroporepellens BL
Photorhabdus asymbiotica
Lactobacillus rhamnosus
Desulfovibrio magneticus
Thermoanaerobacterium
thermosaccharolyticum
Escherichia fergusonii
Escherichia coli O111 H
Desulfurivibrio alkaliphilus
Vibrio cholerae MJ 1236
Clostridium
saccharolyticum WM1
Zymomonas mobilis
Lactobacillus johnsonii
Erwinia pyrifoliae Ep1 96
Burkholderia CCGE1001
Sulfuricurvum kujiense
Acidithiobacillus
ferrivorans SS3 uid67387
Clostridium ljungdahlii
Haliscomenobacter
hydrossis DSM 1100
Runella slithyformis DSM
Leuconostoc kimchii
Calditerrivibrio
nitroreducens DSM 19672
Pseudomonas
brassicacearum NFM421
Burkholderia gladioli BSR3
Microlunatus phosphovorus
Clostridium SY8519
Cupriavidus necator N 1
Tepidanaerobacter Re1
Halorubrum lacusprofundi
Halobacterium salinarum
Halorhabdus utahensis
Haloarcula hispanica ATCC
Halopiger xanaduensis SH
Anaeromyxobacter
dehalogenaris 2CP 1
Haliangium ochraceum
Planctomyces limnophilus
Nitrosococcus watsonii C
Dichelobacter nodosus
Nitrosococcus oceani
Chloroflexus aggregans
Thermoanaerobacter
pseudethanolicus ATCC
Acidothermus cellulolyticus
Pelotomaculum
thermopropionicum SI
Parvibaculum
lavamentivorans DS 1
Methylacidiphilum
infernorum V4 uid59161
Thermoanaerobacter brockii
finnii Ako 1 uid55639
Thermanaerovibrio
acidaminovorans DSM
Acetohalobium arabaticum
Thermoanaerobacter
italicus Ab9 uid46241
Caldicellulosiruptor
kristjanssonii 177R1B
Desulfovibrio aespoeensis
Syntrophothermus
lipocalidus DSM 12680
Methanosalsum zhilinae
Planctomyces brasiliensis
Thermoanaerobacterium
xylanolyticum LX 11
Streptomyces coelicolor A3
Frankia CcI3 uid58397
Thermobifida fusca YX
Burkholderia thailandensis
Frankia EuI1c uid42615
Hahella chejuensis KCTC
Mycobacterium gilvum
Polaromonas
naphthalenivorans CJ2
Saccharopolyspora
erythraea NRRL 2338
Sorangium cellulosum So
Streptomyces griseus
Thermobispora bispora
Candidatus Accumulibacter
phosphatis clade IIA UW 1
Micromonospora aurantiaca
Corynebacterium variabile
Coprothermobacter
proteolyticus DSM 5265
Thermomicrobium roseum
Thermotoga petrophila
Geobacter M21 uid59037
Candidatus Desulforudis
audaxviator MP104C
Denitrovibrio acetiphilus
Prevotella denticola F0289
Thioalkalivibrio sp.
Saccharomonospora viridis
Acidiphilium
multivorum AIU301
Acidithiobacillus
ferrivorans SS3
Acinetobacter
baumannii AYE
Alteromonas macleodii
Anaeromyxobacter
dehalogenans 2CP C
Aromatoleum
aromaticum EbN1
Arthrobacter
nitroguajacolicus
Azospirillum lipoferum
Bifidobacterium
animalis ATCC 25527
Bordetella parapertussis
Burkholderia
Burkholderia gladioli
Burkholderia
vietnamiensis G4
Calditerrivibrio
nitroreducens DSM
Carboxydothermus
hydrogenoformans Z
Chlorobium
phaeobacteroides BS1
Clostridium clariflavum
Clostridium clariflavum
Clostridium ljungdahlii
Clostridium
saccharolyticum WM1
Clostridium
saccharolyticum WM1
Clostridium sticklandii
Clostridium SY8519
Cupriavidus necator N 1
Cyanothece PCC 8802
Dehalococcoides VS
Dehalogenimonas
lykanthroporepellens
Desulfitobacterium
hafniense Y51
Desulfobacula toluolica
Desulfomicrobium
baculatum DSM 4028
Desulfosporosinus
meridiei DSM 13257
Desulfovibrio
magneticus RS 1
Desulfovibrio vulgaris
Desulfovibrio vulgaris
Desulfurivibrio
alkaliphilus AHT2
Enterobacter cloacae
Erwinia Ejp617
Erwinia pyrifoliae DSM
Erwinia pyrifoliae Ep1
Erythrobacter litoralis
Escherichia coli clone
Escherichia coli clone
Escherichia coli HS
Escherichia coli O111
Escherichia fergusonii
Exiguobacterium
sibiricum 255 15
Flavobacterium
branchiophilum FL 15
Gallionella
capsiferriformans ES 2
Gallionella
capsifeniformans ES 2
Geobacillus WCH70
Geobacter
sulfurreducens PCA
Haliscomenobacter
hydrossis DSM 1100
Halobacillus halophilus
Halobacteroides
halobius DSM 5150
Klebsiella oxytoca
Lactobacillus
amylovorus GRL1118
Lactobacillus casei
Lactobacillus helveticus
Lactobacillus helveticus
Lactobacillus johnsonii
Lactobacillus reuteri
Lactobacillus
rhamnosus GG
Lactobacillus
rhamnosus GG
Leuconostoc kimchii
Methanobrevibacter
smithii ATCC 35061
Methanoculleus
bourgensis MS2
Methanolobus
psychrophilus R15
Methanomethylovorans
hollandica DSM 15978
Methanosarcina
acetivorans C2A
Methanosarcina mazei
Methanospirillum
hungatei JF 1 uid58181
Methanospirillum
hungatei JF 1 uid58181
Microlunatus
phosphovorus NM 1
Moorella thermoacetica
Parvularcula
bermudensis
Pectobacterium
carotovorum PCC21
Pelobacter propionicus
Pelodictyon
phaeoclathratiforme BU
Photorhabdus
asymbiotica uid59243
Polaromonas JS666
Pseudomonas
brassicacearum
Pseudomonas stutzeri
Psychrobacter
cryohalolentis K5
Rhodococcus
erythropolis PR4
Rhodopseudomonas
palustris TIE 1
Saccharophagus
degradans 2 40
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Selenomonas sputigena
Shewanella ANA 3
Shewanella MR 4
Slackia
heliotrinireducens DSM
Spirosoma linguale
Sulfuricurvum kujiense
Synechococcus PCC
Syntrophomonas wolfei
Syntrophomonas wolfei
Syntrophus
aciditrophicus SB
Tepidanaerobacter
acetatoxydans Re1
Tepidanaerobacter Re1
Thauera MZ1T
Thermacetogenium
phaeum DSM 12270
Thermoanaerobacterium
thermosaccharolyticum
Thiocystis violascens
Thioflavicoccus mobilis
Vibrio cholerae MJ
Vibrio cholerae O1
Zymomonas mobilis
Magnetospirillum
magneticum AMB 1
Marinobacter aquaeolei
Nostoc punctiforme
Rhodobacter
sphaeroides ATCC
Runella slithyformis
Acidovorax sp. NO-1
Acinetobacter
baumannii OIFC098
Acinetobacter
baumannii WC-136
Acinetobacter sp. P8-3-
Actinomyces neuii
Aurantimonas
manganoxydans SI85-
Bacillus cereus
Bacteroides ovatus SD
Bacteroides sp. 2_1_7
Bacteroides sp.
Bacteroides sp. D2
Bacteroides sp. 2_1_22
Clostridium perfringens
Collinsella aerofaciens
Coprobacillus sp.
Desulfonatronospira
thiodismutans ASO3-1
Enterobacter
hormaechei ATCC
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Escherichia coli 4.0522
Escherichia coli B41
Escherichia coli B799
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Faecalibacterium
prausnitzii M21/2
Fusobacterium
necrophorum subsp
Fusobacterium sp.
Fusobacterium sp. 7_1
Glaciecola lipolytica E3
Lactobacillus casei
Lactobacillus helveticus
Lactobacillus zeae
Magnetospirillum sp.
Magnetospirillum sp.
Marinobacter sp.
Methanoplanu
Methanoplanu
Methylophaga
aminisulfidivorans MP
Oribacterium sp. ACB7
Oribacterium sp. ACB8
Photobacterium sp.
Proteus mirabilis
Pseudomonas fragi A22
Pseudomonas
fuscovaginae UPB0736
Roseburia inulinivorans
Salmonella enterica
Enteritidi
Salmonella enterica
Salmonella enterica
Typhimuriu
Salmonella enterica
Uganda str
Sinorhizobium meliloti
Sphingobium
yanoikuyae XLDN2-5
Vibrio cholera CIRS
Vibrio cholerae
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae
Vibrio cholerae MO10
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Xanthomonas
vesicatoria ATCC
Acetivibrio
cellulolyticus CD2
Acinetobacter
baumannii AB5256
Acinetobacter
baumannii Naval-18
Acinetobacter johnsonii
Acinetobacter junii
Acinetobacter lwoffii
Acinetobacter
radioresistens DSM
Acinetobacter sp. SH024
Actinomyces sp. ICM47
Actinomyces sp. oral
Alcanivorax pacificus
Alcanivorax sp. DG881
Bacillus cereus
Bacillus cereus HuB2-9
Bacteroides coprophilus
Bacteroides ovatus
Bacteroides ovatus SD
Bacteroides sp. 1_1_14
Bacteroides sp. D1
Bacteroides
xylanisolvens SD CC
Bifidobacterium
angulatum DSM 20098
Bifidobacterium
bifidum IPL
Bifidobacterium
bifidum NCIMB 41171
Brachybacterium
paraconglomeratum
Brevibacterium
mcbrellneri ATCC
Cellvibrio sp. BR
Clostridium butyricum
Clostridium perfringens
Collinsella intestinalis
Collinsella stercoris
Coprobacillus sp.
Cylindrospermopsis
raciborskii CS-505
Desulfotomaculum
gibsoniae DS
Enterobacter sp. SST3
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Enterococcus faecium
Escherichia coli 2534-
Escherichia coli 3.3884
Escherichia coli 96.154
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli DEC9E
Escherichia coli JB1-95
Escherichia coli KD2
Escherichia coli KTE12
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli MS 69-
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli S17
Escherichia coli
Escherichia coli
Eubacterium
cellulosolvens 6
Faecalibacterium
prausnitzii A2-165
Fulvimarina pelagi
Fusobacterium
necrophorum subsp
Fusobacterium
nucleatum subsp.
nucleatum ATCC
Fusobacterium sp.
Fusobacterium sp.
Fusobacterium sp.
Fusobacterium ulcerans
Geobacillus sp.
Geobacillus
thermoglucosidan
Glaciecola polaris LMG
Glaciecola punicea
Haloarcula japonica
Halomonas sp. HAL1
Holdemania filiformis
Holophaga foetida
Johnsonella ignava
Lachnoanaerobaculum
saburreum DSM 3986
Lachnoanaerobaculum
saburreum F0468
Lactobacillus casei Lpc-37
Lactobacillus
rhamnosus LRHMDP2
Lactobacillus
rhamnosus LRHMDP3
Lactobacillus johnsonii
Lactobacillus reuteri
Microcystis aeruginosa
Nitratireductor indicus
Opitutacea
Oribacterium sp. ACB1
Paenibacillus elgii B69
Pantoea sp. GM01
Parabacteroides sp. D25
Pectobacterium
wasabiae CFBP 3304
Pseudanabaena biceps
Pseudoalteromona
Pseudoalteromonas
luteoviolacea B =
Pseudomonas mandelii
Pseudomonas
psychrotolerans L19
Pseudomonas syringae
Roseobacter sp.
Salmonella enterica
Infanti
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Selenomonas sputigena
Shewanella baltica
Sphingobium indicum
Sporolactobacillus
vineae DSM 21990 =
Sporosarcina
newyorkensis 2681
Stomatobaculum
longum
Stomatobaculum
longum
Streptomyces sp.
Thiorhodovibrio sp. 970
Thiothrix nivea DSM
Vibrio cholerae 4260B
Vibrio cholerae B33
Vibrio cholerae B33
Vibrio cholerae
Vibrio cholerae
Vibrio cholerae H1
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HC-
Vibrio cholerae HFU-
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae Ol str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae O1 str.
Vibrio cholerae
Vibrio harveyi CAIM
Vibrio shilonii AK1
Vibrio tubiashii ATCC
Vibrio cholerae O1 str.
Yersinia ruckeri ATCC
Thioalkalivibrio sp.
Halopiger xanaduensis SH6
Halorhabdus utahensis DSM
Halobacterium salinarum R1
Halorubrum lacusprofundi
Haloarcula hispanica ATCC
Natrinema pellirubrum DSM
Natronorubrum tibetense
Haloarcula argentinensis
Halosimplex carlsbadense 2-
Planctomyces limnophilus
Anaeromyxobacter
dehalogenans 2CP 1
Haliangium ochraceum
Haliangium ochraceum
Rhodopirellula
Pseudanabaena biceps
Planctomyces limnophilus
Anaeromyxobacter
dehalogenans 2CP 1
Haliangium ochraceum
Haliangium ochraceum
Rhodopirellula
Pseudanabaena biceps
Acetohalobium arabaticum
Acidothermus
cellulolyticus 11B
Anaerobaculum mobile
Bacteroides vulgatus
Caldicellulosiruptor
kristjanssonii 177R1B
Chloroflexus aggregans
Desulfovibrio aespoeensis
Dichelobacter nodosus
Methanocaldococcus
Methanosalsum zhilinae
Methylacidiphilum
infernorum V4 uid59161
Nitrosococcus oceani
Nitrosococcus watsonii C
Parvibaculum
lavamentivorans DS 1
Parvibaculum
lavamentivorans DS 1
Planctomyces brasiliensis
Syntrophothermus
lipocalidus DSM 12680
Tepidanaerobacter
acetatoxydans Re1
Tepidanaerobacter Re1
Thermanaerovibrio
acidaminovorans DSM
Thermoanaerobacter
brockii finnii Ako 1
Thermoanaerobacter
italicus Ab9 uid46241
Thermoanaerobacter
pseudethanolicus ATCC
Thermoanaerobacterium
saccharolyticum JW SL
Thermoanaerobacterium
thermosaccharolyticum
Thermoanaerobacterium
xylanolyticum LX 11
Pelotomaculum
thermopropionicum SI
Bacillus cereus W
Clostridium thermocellum
Enterococcus faecium
Caloramator australicus
Pseudoalteromonas marina
Desulfotomaculum
nigrificans DSM 574
Dethiosulfovibrio
peptidovorans DSM 11002
Kingella denitrificans
Alcanivorax
hongdengensis A-11-3
Bacillus cereus BAG2X1-
Nitrosococcus oceani
Methyloversatilis
universalis FAM5
Thermoplasmatales
Pseudomonas sp. GM55
Bacillus cereus 03BB108
Treponema primitia ZAS-
Bacillus methanolicus
Vibrio scophthalmi LMG
Thiorhodovibrio sp. 970
Ectothiorhodospir
Clostridium papyrosolvens
Thermoanaerobacter
ethanolicus JW 200
Thermoanaerobacter
ethanolicus CCSD1
Nitrococcus mobilis Nb-
Burkholderia thailandensis
Candidatus Accumulibacter
phosphalis clade IIA UW 1
Corallococcus coralloides
Corynebacterium variabile
Frankia CcI3 uid58397
Frankia EuI1c uid42615
Haliangium ochraceum DSM
Microlunatus phosphovorus
Micromonospora aurantiaca
Mycobacterium gilvum PYR
Nocardia cyriacigeorgica
Polaromonas
naphthalenivorans CJ2
Saccharomonospora viridis
Saccharopolyspora erythraea
Saccharothrix espanaensis
Singulisphaera acidiphila
Sorangium cellulosum So ce
Streptomyces coelicolor A3 2
Streptomyces griseus NBRC
Thermobifida fusca YX
Thermobispora bispora DSM
Hahella chejuensis KCTC
Saccharomonospora glauca
Rhodococcus triatomae BKS
Saccharomonospora cyanea
Gordonia polyisoprenivorans
Amycolatopsis azurea DSM
Marinobacter sp. ELB17
Saccharopolyspora erythraea
Streptomyces turgidiscabies
Gemma taobscuriglobus
Rhodococcus ruber BKS 20-
Mycobacterium
xenopi RIVM700367
Micromonospora sp. ATCC
Saccharomonospora
xinjiangensis XJ-54
Bradyrhizobium sp. ORS 375
Burkholderia thailandensis
Planctomyces maris DSM
Streptomyces gancidicus BKS
Gordonia amicalis NBRC
Mycobacterium intracellulare
Phaeospirillum molischianum
Nitrococcus mobilis Nb-231
Frankia sp. EUN1f
Pseudomonas stutzeri NF13
Dietzia cinnamea P4
Aciduliprofundum MAR08
Anaerobaculum mobile DSM
Candidatus Desulforudis
audaxviator MP104C
Coprothermobacter
proteolyticus DSM 5265
Cyanobacterium stanieri PCC
Denitrovibrio acetiphilus
Desulfitobacterium
dichloroeliminans LMG P
Geobacter M21 uid59037
Prevotella denticola F0289
Thermotoga petrophila RKU
Thermomicrobium roseum
Pseudanabaena biceps PCC 7429
Moorella thermoacetica ATCC 39073
Tepidanaerobacter acetatoxydans Re1
Tepidanaerobacter Re1 uid66873
Thiorhodovibrio sp. 970
Marinobacter sp. ELB17
Microlunatus phosphovorus NM 1 uid68055
Acetivibrio cellulolyticus CD2
Acidiphilium multivorum AIU301 uid63345
Acidithiobacillus ferrivorans SS3 uid67387
Acidovorax sp. NO-1
Acinetobacter baumannii AB5256
Acinetobacter baumannii AYE uid61637
Acinetobacter baumannii Naval-18
Acinetobacter baumannii OIFC098
Acinetobacter baumannii WC-136
Acinetobacter johnsonii SH046
Acinetobacter junii SH205
Acinetobacter lwoffii SH145
Acinetobacter radioresistens DSM 6976 =
Acinetobacter sp. P8-3-8
Acinetobacter sp. SH024
Actinomyces neuii BVS029A5
Actinomyces sp. ICM47
Actinomyces sp. oral taxon 178 str
Alcanivorax pacificus W11-5
Alcanivorax sp. DG881
Alteromonas macleodii Black Sea 11
Anaeromyxobacter dehalogenans 2CP C
Aromatoleum aromaticum EbN1 uid58231
Arthrobacter nitroguajacolicus Rue61a
Aurantimonas manganoxydans SI85-9A1
Azospirillum lipoferum 4B uid82343
Bacillus cereus BAG6X1-2
Bacillus cereus H3081.97
Bacillus cereus HuB2-9
Bacteroides coprophilus DSM 18228
Bacteroides ovatus CL02T12C04
Bacteroides ovatus SD CC 2a
Bacteroides ovatus SD CMC 3f
Bacteroides sp. 1_1_14
Bacteroides sp. 2_1_7
Bacteroides sp. 3_1_33FAA
Bacteroides sp. D1
Bacteroides sp. D2
Bacteroides sp. 2_1_22
Bacteroides xylanisolvens SD CC 1b
Bifidobacterium angulatum DSM 20098
Bifidobacterium animalis ATCC 25527
Bifidobacterium bifidum IPL
Bifidobacterium bifidum NCIMB 41171
Bordetella parapertussis Bpp5 uid177516
Brachybacterium paraconglomeratum LC44
Brevibacterium mcbrellneri ATCC 49030
Burkholderia CCGE1001 uid42975
Burkholderia gladioli BSR3 uid66301
Burkholderia vietnamiensis G4 uid58075
Calditerrivibrio nitroreducens DSM 19672
Carboxydothermus hydrogenoformans Z
Cellvibrio sp. BR
Chlorobium phaeobacteroides BS1
Clostridium butyricum E4 str. BoNT E
Clostridium ljungdahlii DSM 13528
Clostridium perfringens C str. JGS1495
Clostridium perfringens D str. JGS1721
Clostridium sticklandii DSM 519 uid59585
Clostridium SY8519 uid68705
Collinsella aerofaciens ATCC 25986
Collinsella intestinalis DSM 13280
Collinsella stercoris DSM 13279
Coprobacillus sp. 3_3_56FAA
Coprobacillus sp. 8_2_54BFAA
Cupriavidus necator N 1 uid68689
Cyanothece PCC 8802 uid59143
Cylindrospermopsis raciborskii CS-505
Dehalococcoides VS uid42393
Dehalogenimonas lykanthroporepellens BL
delta proteobacterium NaphS2
Desulfitobacterium hafniense Y51 uid58605
Desulfobacula toluolica Tol2 uid175777
Desulfomicrobium baculatum DSM 4028
Desulfonatronospira thiodismutans ASO3-1
Desulfosporosinus meridiei DSM 13257
Desulfotomaculum gibsoniae DS
Desulfovibrio magneticus RS 1 uid59309
Desulfovibrio vulgaris Hildenborough
Desulfovibrio vulgaris RCH1 uid161961
Desulfurivibrio alkaliphilus AHT2 uid49487
Enterobacter cloacae ENHKU01 uid172463
Enterobacter hormaechei ATCC 49162
Enterobacter sp. SST3
Enterococcus faecalis TX0109
Enterococcus faecalis TX1302
Enterococcus faecium 509
Enterococcus faecium 511
Enterococcus faecium 514
Enterococcus faecium C1904
Enterococcus faecium C497
Enterococcus faecium E0679
Enterococcus faecium E1731
Enterococcus faecium E1904
Enterococcus faecium E2883
Enterococcus faecium ERV99
Enterococcus faecium P1123
Enterococcus faecium P1137
Enterococcus faecium P1139
Enterococcus faecium TX0133A
Enterococcus faecium TX0133a01
Enterococcus faecium TX0133a04
Enterococcus faecium TX0133B
Enterococcus faecium TX0133C
Erwinia Ejp617 uid159955
Erwinia pyrifoliae DSM 12163 uid159693
Erwinia pyrifoliae Ep1 96 uid40659
Erythrobacter litoralis HTCC2594 uid58299
Escherichia coli clone D i14 uid162049
Escherichia coli clone D i2 uid162047
Escherichia coli 2534-86
Escherichia coli 3.3884
Escherichia coli 4.0522
Escherichia coli 96.154
Escherichia coli B41
Escherichia coli B799
Escherichia coli DEC10E
Escherichia coli DEC10F
Escherichia coli DEC13A
Escherichia coli DEC13B
Escherichia coli DEC13C
Escherichia coli DEC13D
Escherichia coli DEC13E
Escherichia coli DEC14B
Escherichia coli DEC14C
Escherichia coli DEC14D
Escherichia coli DEC7B
Escherichia coli DEC8A
Escherichia coli DEC8B
Escherichia coli DEC9A
Escherichia coli DEC9B
Escherichia coli DEC9C
Escherichia coli DEC9D
Escherichia coli DEC9E
Escherichia coli HS uid58393
Escherichia coli JB1-95
Escherichia coli KD2
Escherichia coli KTE12
Escherichia coli KTE139
Escherichia coli KTE153
Escherichia coli KTE211
Escherichia coli KTE218
Escherichia coli KTE234
Escherichia coli KTE47
Escherichia coli KTE53
Escherichia coli KTE6
Escherichia coli MS 69-1
Escherichia coli O10:K5(L):H4 str. ATCC
Escherichia coli O111 H 11128 uid41023
Escherichia coli O111:H8 str. CVM9570
Escherichia coli O111:H8 str. CVM9574
Escherichia coli O111:H8 str. CVM9602
Escherichia coli O111:H8 str. CVM9634
Escherichia coli O113:H21 str. CL-3
Escherichia coli O25b:ST131 str. JIE186
Escherichia coli OK1180
Escherichia coli S17
Escherichia coli STEC_94C
Escherichia coli TW10828
Escherichia fergusonii ATCC 35469
Eubacterium cellulosolvens 6
Exiguobacterium sibiricum 255 15 uid58053
Faecalibacterium prausnitzii A2-165
Faecalibacterium prausnitzii M21/2
Flavobacterium branchiophilum FL 15
Fulvimarina pelagi HTCC2506
Fusobacterium nucleatum subsp. nucleatum
Fusobacterium sp. 11_3_2
Fusobacterium sp. 2_1_31
Fusobacterium sp. 3_1_27
Fusobacterium sp. 3_1_5R
Fusobacterium sp. 7_1
Fusobacterium ulcerans ATCC 49185
Geobacillus sp. G11MC16
Geobacillus thermoglucosidan
Geobacillus WCH70 uid59045
Geobacter sulfurreducens PCA uid57743
Glaciecola lipolytica E3
Glaciecola polaris LMG 21857
Glaciecola punicea DSM 14233 = ACAM
Haliscomenobacter hydrossis DSM 1100
Haloarcula japonica DSM 6131
Halobacillus halophilus DSM 2266
Halobacteroides halobius DSM 5150
Halomonas sp. HAL1
Holdemania filiformis DSM 12042
Holophaga foetida DSM 6591
Johnsonella ignava ATCC 51276
Klebsiella oxytoca E718 uid170256
Lachnoanaerobaculum (Eubacterium)
saburreum DSM 3986
Lachnoanaerobaculum (Eubacterium)
saburreum F0468
Lactobacillus amylovorus GRL1118
Lactobacillus casei Lpc-37
Lactobacillus casei UW4
Lactobacillus casei Zhang uid50673
Lactobacillus helveticus DSM 20075
Lactobacillus helveticus H10 uid162017
Lactobacillus helveticus R0052 uid174439
Lactobacillus johnsonii ATCC 33200
Lactobacillus johnsonii FI9785 uid41735
Lactobacillus reuteri CF48-3A
Lactobacillus reuteri SD2112 uid55357
Lactobacillus rhamnosus GG uid161983
Lactobacillus rhamnosus GG uid59313
Lactobacillus rhamnosus LRHMDP2
Lactobacillus rhamnosus LRHMDP3
Lactobacillus zeae KCTC 3804
Leuconostoc kimchii IMSNU 11154
Magnetospirillum magneticum AMB 1
Marinobacter aquaeolei VT8 uid59419
Methanobrevibacter smithii ATCC 35061
Methanoculleus bourgensis MS2 uid171377
Methanolobus psychrophilus R15
Methanomethylovorans hollandica DSM
Methanosarcina acetivorans C2A uid57879
Methanosarcina mazei Go1 uid57893
Methylophaga aminisulfidivorans MP
Microcystis aeruginosa PCC 9443
Nitratireductor indicus C115
Nostoc punctiforme PCC 73102 uid57767
Oribacterium sp. ACB1
Oribacterium sp. ACB7
Oribacterium sp. ACB8
Paenibacillus elgii B69
Pantoea sp. GM01
Parabacteroides sp. D25
Parvularcula bermudensis HTCC2503
Pectobacterium carotovorum PCC21
Pectobacterium wasabiae CFBP 3304
Pelobacter propionicus DSM 2379 uid58255
Pelodictyon phaeoclathratiforme BU 1
Photobacterium sp. SKA34
Photorhabdus asymbiotica uid59243
Polaromonas JS666 uid58207
Proteus mirabilis WGLW6
Pseudoalteromona
Pseudoalteromonas luteoviolacea B =
Pseudomonas brassicacearum NFM421
Pseudomonas fragi A22
Pseudomonas fuscovaginae UPB0736
Pseudomonas mandelii JR-1
Pseudomonas psychrotolerans L19
Pseudomonas stutzeri CCUG 29243
Pseudomonas syringae Lz4W
Psychrobacter cryohalolentis K5 uid58373
Rhodobacter sphaeroides ATCC 17025
Rhodococcus erythropolis PR4 uid59019
Rhodopseudomonas palustris TIE 1
Roseburia inulinivorans DSM 16841
Roseobacter sp. MED193
Runella slithyformis DSM 19594 uid68317
Saccharophagus degradans 2 40 uid57921
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica serovar Typhimurium
Salmonella enterica subsp. enterica serovar
Salmonella enterica subsp. enterica serovar
Enteritidi
Salmonella enterica subsp. enterica serovar
Infanti
Salmonella enterica subsp. enterica serovar
Saintpau
Salmonella enterica subsp. enterica serovar
Typhimuriu
Salmonella enterica subsp. enterica serovar
Uganda str
Selenomonas sputigena ATCC 35185
Selenomonas sputigena ATCC 35185
Shewanella ANA 3 uid58347
Shewanella baltica OS625
Shewanella MR 4 uid58345
Sinorhizobium meliloti CCNWSX0020
Slackia heliotrinireducens DSM 20476
Sphingobium indicum B90A
Sphingobium yanoikuyae XLDN2-5
Spirosoma linguale DSM 74 uid43413
Sporolactobacillus vineae DSM 21990 =
Sporosarcina newyorkensis 2681
Streptomyces sp. SPB78
Sulfuricurvum kujiense DSM 16994
Synechococcus PCC 6312 uid182934
Syntrophus aciditrophicus SB uid58539
Thauera MZ1T uid58987
Thermacetogenium phaeum DSM 12270
Thermoanaerobacterium
thermosaccharolyticum DSM 571 uid51639
Thiocystis violascens DSM 198 uid74025
Thioflavicoccus mobilis 8321 uid184343
Thiothrix nivea DSM 5205
Vibrio cholera CIRS 101
Vibrio cholerae 4260B
Vibrio cholerae CP1035(8)
Vibrio cholerae CP1048(21)
Vibrio cholerae CP1050(23)
Vibrio cholerae H1
Vibrio cholerae HC-17A1
Vibrio cholerae HC-17A2
Vibrio cholerae HC-19A1
Vibrio cholerae HC-22A1
Vibrio cholerae HC-23A1
Vibrio cholerae HC-28A1
Vibrio cholerae HC-32A1
Vibrio cholerae HC-37A1
Vibrio cholerae HC-38A1
Vibrio cholerae HC-39A1
Vibrio cholerae HC-40A1
Vibrio cholerae HC-41A1
Vibrio cholerae HC-43A1
Vibrio cholerae HC-46A1
Vibrio cholerae HC-47A1
Vibrio cholerae HC-48A1
Vibrio cholerae HC-48B2
Vibrio cholerae HC-49A2
Vibrio cholerae HC-55B2
Vibrio cholerae HC-56A2
Vibrio cholerae HC-57A2
Vibrio cholerae HC-60A1
Vibrio cholerae HC-61A1
Vibrio cholerae HC-61A2
Vibrio cholerae HC-62A1
Vibrio cholerae HC-62B1
Vibrio cholerae HC-64A1
Vibrio cholerae HC-65A1
Vibrio cholerae HC-67A1
Vibrio cholerae HC-68A1
Vibrio cholerae HC-69A1
Vibrio cholerae HC-70A1
Vibrio cholerae HC-71A1
Vibrio cholerae HC-72A2
Vibrio cholerae HC-77A1
Vibrio cholerae HC-7A1
Vibrio cholerae HC-80A1
Vibrio cholerae HC-81A1
Vibrio cholerae HC-81A2
Vibrio cholerae HCUF01
Vibrio cholerae HFU-02
Vibrio cholerae MJ 1236 uid59387
Vibrio cholerae MO10
Vibrio cholerae O1 2010EL 1786 uid78933
Vibrio cholerae O1 str. 2010EL-1792
Vibrio cholerae O1 str. 2010EL-1798
Vibrio cholerae O1 str. EC-0009
Vibrio cholerae O1 str. EC-0012
Vibrio cholerae O1 str. EC-0027
Vibrio cholerae O1 str. EDC-020
Vibrio cholerae O1 str. EM-1546
Vibrio cholerae O1 str. Inaba G4222
Vibrio cholerae O1 str. Nep-21106
Vibrio cholerae O1 str. Nep-21113
Vibrio cholerae O1 str. NHCC-004A
Vibrio cholerae O1 str. NHCC-006C
Vibrio cholerae O1 str. NHCC-010F
Vibrio cholerae O1 str. PCS-023
Vibrio cholerae VC4370
Vibrio harveyi CAIM 1792
Vibrio shilonii AK1
Vibrio tubiashii ATCC 19109
Vibrio cholerae HC-20A2
Vibrio cholerae HC-21A1
Vibrio cholerae HC-42A1
Vibrio cholerae HC-51A1
Vibrio cholerae O1 str. 3582-05
Xanthomonas vesicatoria ATCC 35937
Yersinia ruckeri ATCC 29473
Zymomonas mobilis NCIMB 11163
Clostridium clariflavum DSM 19732
Clostridium saccharolyticum WM1
Fusobacterium necrophorum subsp
Gallionella capsiferriformans ES 2 uid51505
Magnetospirillum sp. SO-1
Methanoplanu
Methanospirillum hungatei JF 1 uid58181
Salmonella enterica serovar Typhimurium
Stomatobaculum longum (Lachnospiraceae
bacterium ACC2)
Syntrophomonas wolfei Goettingen
Vibrio cholerae B33
Haloarcula argentinensis DSM 12282
Haloarcula hispanica ATCC 33960
Halobacterium salinarum R1 uid61571
halophilic archaeon DL31 uid72619
Halopiger xanaduensis SH6 uid68105
Halorhabdus utahensis DSM 12940
Halorubrumlacus profundi ATCC 49239
Halosimplex carlsbadense 2-9-1
Natrinema pellirubrum DSM 15624
Natronorubrum tibetense GA33
Anaeromyxobacter dehalogenans 2CP 1
Planctomyces limnophilus DSM 3776
Rhodopirellula sp. SWK7
Haliangium ochraceum DSM 14365
Nitrococcus mobilis Nb-231
Anaerobaculum mobile DSM 13181
Acetohalobium arabaticum DSM 5501
Acidothermus cellulolyticus 11B uid58501
Alcanivorax hongdengensis A-11-3
Bacillus cereus 03BB108
Bacillus cereus BAG2X1-1
Bacillus methanolicus MGA3
Bacillus cereus W
Bacteroides vulgatus ATCC 8482 uid58253
Caldicellulosiruptor kristjanssonii 177R1B
Caloramator australicus RC3]Length = 15
Chloroflexus aggregans DSM 9485
Clostridium papyrosolvens DSM 2782
Clostridium thermocellum YS
Desulfotomaculum nigrificans DSM 574
Desulfovibrio aespoeensis Aspo 2 uid42613
Dethiosulfovibrio peptidovorans DSM
Dichelobacter nodosus VCS1703A
Ectothiorhodospir
Enterococcus faecium 1,231,501
Kingella denitrificans ATCC 33394
Methanocaldococcus FS406 22 uid42499
Methanosalsum zhilinae DSM 4017
Methylacidiphilum infernorum V4 uid59161
Methyloversatilis universalis FAM5
Nitrosococcus oceani AFC27
Nitrosococcus oceani ATCC 19707
Nitrosococcus watsonii C 113 uid50331
Pelotomaculum thermopropionicum SI
Planctomyces brasiliensis DSM 5305
Pseudoalteromonas marina mano4
Pseudomonas sp. GM55
Syntrophothermus lipocalidus DSM 12680
Thermanaerovibrio acidaminovorans DSM
Thermoanaerobacter brockii finnii Ako 1
Thermoanaerobacter ethanolicus CCSD1
Thermoanaerobacter ethanolicus JW 200
Thermoanaerobacter italicus Ab9 uid46241
Thermoanaerobacter pseudethanolicus
Thermoanaerobacterium saccharolyticum
Thermoanaerobacterium
thermosaccharolyticum M0795 uid184821
Thermoanaerobacterium xylanolyticum LX
Thermoplasmatales archaeon SCGC AB-
Treponema primitia ZAS-1
Vibrio scophthalmi LMG 19158
Parvibaculum lavamentivorans DS 1
Amycolatopsis azurea DSM 43854
Bradyrhizobium sp. ORS 375
Burkholderia thailandensis E264
Burkholderia thailandensis E264 uid58081
Corallococcus coralloides DSM 2259
Corynebacterium variabile DSM 44702
Dietzia cinnamea P4
Frankia CcI3 uid58397
Frankia EuI1c uid42615
Frankia sp. EUN1f
Gemmata obscuriglobus UQM 2246
Gordonia amicalis NBRC 100051 = JCM
Gordonia polyisoprenivorans NBRC 16320
Hahella chejuensis KCTC 2396 uid58483
Micromonospora aurantiaca ATCC 27029
Micromonospora sp. ATCC 39149
Mycobacterium gilvum PYR GCK
Mycobacterium xenopi RIVM700367
Mycobacterium intracellulare ATCC 13950
Nocardia cyriacigeorgica GUH 2 uid89395
Phaeospirillum molischianum DSM 120
Planctomyces maris DSM 8797
Polaromonas naphthalenivorans CJ2
Pseudomonas stutzeri NF13
Rhodococcus triatomae BKS 15-14
Rhodococcus ruber BKS 20-38
Saccharomonospora cyanea NA-134
Saccharomonospora glauca K62
Saccharomonospora viridis DSM 43017
Saccharomonospora xinjiangensis XJ-54
Saccharopolyspora erythraea NRRL 2338
Saccharopolyspora erythraea NRRL 2338
Saccharothrix espanaensis DSM 44229
Singulisphaera acidiphila DSM 18658
Sorangium cellulosum So ce 56 uid61629
Streptomyces coelicolor A3 2 uid57801
Streptomyces griseus NBRC 13350
Streptomyces turgidiscabies Car8
Streptomyces gancidicus BKS 13-15
Thermobifida fusca YX uid57703
Thermobispora bispora DSM 43833
Aciduliprofundum MAR08 339 uid184407
Candidatus Desulforudis audaxviator
Coprothermobacter proteolyticus DSM 5265
Cyanobacterium stanieri PCC 7202
Denitrovibrio acetiphilus DSM 12809
Desulfitobacterium dichloroeliminans LMG
Geobacter M21 uid59037
Prevotella denticola F0289 uid65091
Thermomicrobium roseum DSM 5159
Thermotoga petrophila RKU 1 uid58655
Thioalkalivibrio sp. K90mix
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.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
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.
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. No. 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.
Genomic Data and Molecular Phylogeny of the pglZ Protein—
A set of 1447 completely sequenced prokaryotic genomes (1336 bacterial and 111 archaeal genomes) were downloaded from the NCBI FTP site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) and used for subsequent analyses. Several pglZ protein sequences were used as a query in a PSI-BLAST search against the 1447 prokaryotic genomes with an inclusion threshold e-value of 0.001. Proteins that did not contain the pglZ domain or that were <600 amino acids length were filtered out. The remaining protein sequences were used to build a pglZ tree as follows: Amino acid sequences were aligned using the MAFFT algorithm [Katoh et al. Nucleic acids research (2002) 30: 3059-3066]. The Fourier transform approximation was disabled, and substitution rates were modeled with JTT [Jones et al. Computer applications in the biosciences: CABIOS (1992) 8: 275-282] and BLOSUM45 matrix, which is suitable for diverged sequences. The gene tree was reconstructed using the probabilistic RA×ML algorithm, with 100 bootstrap replicates, substitutions modeled with JTT (Jones et al. 1992), while allowing for rate variability among sites. For simplicity, the tree presented in
Identification of Bacteriophage Exclusion (BREX) Types 1-6—
System types were characterized based on manual observation of phyletic clusters in the pglZ tree. The specific genes associated with each pglZ phyletic type were defined using the IMG genome browser (www://img.jgi.doe.gov/cgi-bin/w/main.cgi). A representative protein sequence of each of the individual genes (Table 1 below) was then used as query in a PSI-BLAST search with an inclusion threshold e-value of 0.05. Only gene clusters containing the two core genes (pglz and brxC/pglY) and at least two additional genes were considered, under the added constraint that the genomic distance between the first and last genes in the system be under 30 kb. In the case of pglY, homology was based on the shared motifs (the p-loop motif GXXXXGK(T/S) (DUF2791, SEQ ID NO: 6162) and DUF499 combined with the conserved size of the gene in the different subtypes (˜1200 amino acids). The filtered clusters were manually assigned to systems according to gene content. Only clusters containing the complete set of genes or missing one non-core gene were included in the final set (Tables 2-7 below). In the case of BREX type 2, systems missing both brxD and brxHI were also included in the final set. The blastx program was used to scan intergenic regions in the clusters for unnanotated genes. Protein domains were annotated using the conserved domain database (CDD)34 and HHpred35. In the latter case, queries were carried out using representative sequences against the PDB, SCOP, interpro, pfam, smart, tigrfam and COG databases using default search parameters. The blastx program was used to scan intergenic regions in the clusters for un-annotated genes.
The consensus organisms tree (represented in
Extensive Identification of BREX Systems in Prokaryotic Genomes
A set of 2263 completely sequenced prokaryotic genomes and 5493 draft genomes was downloaded from the NCBI FTP site (ftp://ftp.ncbi.nih.gov/genomes/Bacterial and ftp://ftp.ncbi.nih.gov/genomes/Bacteria_DRAFT/, respectively) and used for subsequent analyses. A representative protein sequence of each of the 13 genes (Table 1 below) was then used as query in a PSI-BLAST search against the 7756 completely sequences and draft genomes with an inclusion threshold e-value of 0.05. Only gene clusters containing the two core genes (pglZ and brxC/pglY) and at least two additional genes were considered and listed in tables 10-15 below.
Strain Construction—
The type 1 BREX system was amplified in fragments from the Bacillus cereus H3081.97 genome from position 89,288-103,514 (GenBank ABDL02000007.1, SEQ ID NO: 6164). The PCR-amplified fragments were assembled to a circular plasmid in S. cerevisiae using the pYES1L vector (Invitrogen), transformed into Eccherichia coli BL21 AI and amplified, and then integrated into the proB gene in Bacillus subtilis BEST7003, along with a chloramphenicol resistance cassette. The DNA sequence of the plasmid used for the integration is depicted in SEQ ID NO: 6139. The primers used for construction are depicted in SEQ ID NO: 6140-6151. The presence of the intact BREX system within Basillus subtilis BEST7003 was confirmed by PCR and Illumina-based whole genome sequencing. Primers sequences are depicted in SEQ ID NO: 6152-6161. Control strains contain only the chloramphenicol resistance cassette integrated at the proB locus. The pglX deletion strain was constructed in a similar manner with PCR fragments that created a deletion from position 94,655-98,163 (GenBank ABDLO2000007.1, SEQ ID NO: 6164), leaving only 31 nucleotides of the pglX gene. The DNA sequence of the plasmid used for the integration is depicted in SEQ ID NO: 6210.
Growth Dynamics of Phage Infected Cultures—
Overnight cultures were diluted 1:100 in LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 and then grown to an OD600 of 0.06 in a 96-well plate format. Phages were added at a multiplicity of infection (MOI) ranging from 10−3 to 10−4. High concentration phage infections were performed at MOI ranging from 0.05 to 5. Optical density measurements at a wavelength of 600 nm were taken every 13 minutes using a TECAN infinite 2000 plate reader.
Plaque Assays—
Small drop plaque assays were initially performed using 0.75% agar plates containing bacterial cultures that were diluted 1:13 in LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl2. Serial dilutions of the phage between 2×100 and 2×105 plaque-forming units (pfu) were spotted on these plates and plaques were counted after overnight growth at room temperature. Further confirmation of plaque numbers was performed by an agar overlay assay. The bottom agar was composed of LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 and 1.5% agar. The top agar was prepared by diluting overnight bacterial cultures 1:30 in LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 and 0.5% agar with the addition of serial dilutions of the phage. Plaques were counted after overnight growth at room temperature.
One-Step Phage Growth Curve Assays—
One-step phage growth curve experiments were performed as described by Carlson [E. Kutter and A. Sulakvelidze (ed.), Bacteriophages: biology and applications, CRC Press, Boca Raton. Fla. Appendix p. 437-494]. Logarithmic phase cultures were infected with either phage SPO1 or φ3T at an MOI of 0.05. Following 18 minutes of growth at 37° C. the infected culture was diluted 1:10,000, to reduce the likelihood of phage infection following cell lysis. To evaluate the number of infective centers and extracellular phage present in the infected culture, samples were taken at specific time points throughout the incubation period, mixed with a phage-sensitive Basillus subtilis strain and plated using the agar overlay method described hereinabove. Phage adsorption was inferred by evaluating the number of extracellular phage present in the mixture 15 minutes following infection. This was assayed by mixing the infection mixture with chloroform, incubating it at 37° C. for 4 minutes, followed by incubation for 4 minutes on ice, and 30 minutes at room temperature. The aqueous phase was then mixed with a phage-sensitive Basillus subtilis strain and plated using the agar overlay method described hereinabove. The addition of chloroform leads to bacteria killing, including phage adsorbed bacteria. At early time points, phage have not yet assembled inside the cell, and are therefore unable to form plaques. Thus, the derived results allow evaluation of the extracellular phage levels. A drop in extracellular phage levels indicates that adsorption has occurred.
DNA Extraction—
DNA extraction was performed by suspending cells in 50 mM EDTA pH 8.0 with a lytic enzyme (lysozyme. Sigma) for 90 minutes at 37° C. followed by centrifugation for 2 minutes at 13,000 g and removal of supernantat. The cells were then lysed by adding a Nuclei Lysis Solution (Promega, cat no. A7941) for 5 minutes at 80° C. followed by addition of Rnase A (10 mg/ml) for 30-60 minutes at 37° C. The protein fraction was precipitated by adding 200 μl Protein Precipitation Solution (Promega, cat no. A795A), incubating the suspension for 5 minutes on ice followed by centrifugation at 13,000-16,000 g for 3 minutes. The supernatant was then transferred to a clean tube containing 600 μl isopropanol, mixed and centrifuged at 13,000-16,000 g for 3 minutes. The supernatant was removed and 600 μl 70% Ethanol was added to the pellet mixed and centrifuged at 13,000-16,000 g for 3 minutes. The ethanol was then aspirated and the pellet was air dried for a couple of minutes followed by resuspension in Qiagen Elution buffer.
Phage Infection Time Courses, Genomic DNA Sequencing and Methylation Analysis—
Phage infection time course cultures for both methylome analysis, detection of lysogeny and relative phage abundance were performed at an MOI of 4. Phage infection time course cultures are practically cultures infected by phage and analyzed at specific time points (e.g. 0, 5, 10, 15, 20, 30 and 40 minutes following infection). Uninfected cultures analyzed at the same time points served as control. Cell pellets were washed three times in 10 mM Tris pH 7.4 to remove unadsorbed phage, followed by DNA extraction as described hereinabove. DNA library preparations and sequencing for methylome analysis were performed at the Yale Center for Genome Analysis (see Murray I A et al. (2012) Nucleic acids research 40: 11450-11462). To determine the relative abundance of bacterial and phage φ3T DNA levels, DNA was first fragmented using NEBNext® dsDNA Fragmentase (New England Biolabs Ibc.) according to manufacturer's instructions, followed by Illumina sequencing of the DNA libraries of φ3T phage-infected time course cultures. The sequences were mapped to the phage and host genomes as previously described [Wurtzel et al. PloS one (2010) 5: e15628]. Sequences shared by both Basillus subtilis BEST7003 and phage φ3T DNA were discarded from the dataset. The remaining mapped sequences were enumerated at each time point to compare the number of sequences mapped to the Basillus subtilis BEST7003 DNA relative to phage φ3T DNA and normalized to the genome size.
Detection of Phage Lysogeny—
Genomic DNA sequencing of a lysogen containing phage φ3T was performed using Illumina sequencing to determine the DNA sequence of the φ3T phage and the site of phage integration in the genome. The integration of the φ3T phage was determined at a GTAGG site on the Basillus subtilis BEST7003 bacterial genome at position 2106060-2106064. Multiplex PCR assays were used to detect phage φ3T DNA, Basillus subtilis BEST7003 DNA, and the novel junction created in the lysogenized strain. Primers used to detect phage φ3T were GAGGTTCGCTACGGGCGAAAT (SEQ ID NO: 6211) and TCTCTGCTTGATITCGTCCATGA (SEQ ID NO: 6212). Primers for detection of Basillus subtilis BEST7003 and the unique junction found in the lysogen were TGCCTGCATGAGCTGATITG (SEQ ID NO: 6213) and GCAGGAATGAATGGTGGATATTG (SEQ ID NO: 6214); and TCATGCTCCGGATTTGCGAT (SEQ ID NO: 6215) and TGCCTCCITTCGATITTGTTACC (SEQ ID NO: 6216), respectively.
Structural Homology Between brxA and NusB—
Alignment between brxA from Magnetospirillum magneticum (PDB entry 3BHW) and NusB from Aquifex aelicus (PDB entry 3R2C) was performed using the MultiProt web server and presented using PyMol (Schrödinger, Inc, Portland, Oreg., USA).
Agarose Gel and Southern Blot Analysis—
200 ng of undigested genomic DNA was run on a TAE agarose gel. The agarose gel was depurinated in 0.25 N HCl for 20 minutes, rinsed in ddH2O, and soaked in denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 10 minutes. The DNA was then transferred onto HybondXL membrane (Amersham) by capillary transfer in denaturation buffer and the membranes were baked for 2 hours at 80° C. DNA for probes was labeled with ∝32P-dCTP using the High Prime Kit (Roche Cat no. 11 585 584 001) according to manufacturer's instructions. Phage φ3T specific primers were PTG111: TGGATTTCAGCTGGGGAAGA (SEQ ID NO: 6217) and PTG112: AACTTGTCTCTATCTTATCACCTGT (SEQ ID NO: 6218). The membranes were incubated overnight with the probe at 65° C. in hybridization buffer (7% SDS, 0.5 M NaPhosphate pH 7.2, 10 mM EDTA), washed twice with 2×SSC, 0.1% w/v SDS, washed twice with 1×SSC, 0.1% w/v SDS, then four times with 0.2×SSC, 0.1% w/v SDS and exposed to phosphorimager screen and visualized.
RNA sequencing and 5′ and 3′ RACE—were performed as described in Wurtzel O. et al. (2012) Molecular Systems Biology, 8:583.
Previous reports demonstrated that various combinations of genes belonging to the Phage Growth Limitation (PGL) system, and predominantly pglZ, were enriched within ‘defense islands’ of bacteria and archaea9,13. The present inventors have initially performed homology searches in 1447 bacterial and archaeal genomes in order to understand whether there is higher order organization amongst pglZ and its associated genes. These homology searches found 144 occurrences of pglZ amongst the 1447 bacterial and archaeal genomes analyzed. Phylogenetic tree reconstruction of these pglZ proteins showed clear clustering of pglZ into several defined phyletic groups (
The present inventors termed this overall system as ‘BREX’ (Bacteriophage Exclusion, previously termed PYZA), and defined six major BREX types according to the phylogeny and operon organization (
Taken together pan genomic analysis revealed a novel broadly distributed multi-gene system which the present inventors denoted BREX system. This family of systems exists in almost 10% of sequenced microbial genomes, and can be divided into six coherent subtypes in which the gene composition and order is conserved (for further details see Example 2 below). Each BREX subtype contains 4-8 genes. By definition, all BREX subtypes contain a pglZ-domain gene. In addition, all of them harbor a large protein with a P-loop motif. The P-loop motif (GXXXXGK[T/S]) is a conserved ATP/GTP binding motif that is ubiquitously found in many ATP-utilizing proteins such as kinases, helicases, motor proteins and proteins with multiple other functions [Thomsen and Berger Molecular microbiology (2008) 69: 1071-1090]. In general, the P-loop containing genes in the various BREX subtypes share little homology: for example, the brxC gene of BREX type 1 and pglY gene of BREX type 2 share homology only across 4% of their protein sequence, and this homology is concentrated around the P-loop motif (
Six types of BREX system were characterized based on manual observation of phyletic clusters in the pglZ tree (
Type 1 BREX—
The most common BREX system identified comprises a 6-gene cluster arranged in a highly conserved order in a diverse array of bacteria and archaea (
The brxA family of proteins are, on average, 232 amino acids long and do not share sequence similarity with any domain of known function. However, as part of the protein structure initiative the structure of the type 1 brxA protein from Magnetospirillum sp. SO-1 was solved (PDB entry 3BHW). A significant structural similarity, spanning 44 amino acids of the brxA protein, was found between the Magnetospirillum brxA and the 148 amino acids RNA binding protein NusB (PDB entry 3R2C)[Stagno et al. Nucleic acids research (2011) 39, 7803-7815]. NusB is part of an anti-termination complex that enables proper ribosomal RNA transcription in E. coli. The anti-termination complex is initiated by binding of NusB and NusE to a BOXA site, a specific sequence on the nascent rRNA. The complex, which assembles additional proteins such as NusE, NusG and NusA, modifies RNA polymerase to enable readthrough past Rho-dependent transcriptional terminators that are present in the rRNA sequence [Luttgen et al. Journal of molecular biology (2002) 316, 875-885]. NusB was also shown to be essential for the life cycle of bacteriophage λ, and specifically for the transition from early transcription into late transcription. In the middle stages of infection, the phage N protein couples with NusB. NusE, NusA and NusG to direct the host RNA polymerase to read through the terminators of the phage immediate early genes and proceed to transcription of middle genes [Stagno et al. Nucleic acids research (2011) 39, 7803-7815]. As demonstrated in
Type 2 BREX—
Type 2 BREX system encloses the phage defense system originally described as PGL10 (
Type 3 BREX—
The type 3 BREX system was observed in 20 of the genomes analyzed (
Type 4 BREX—
The type 4 BREX is composed of four genes (
Type 5 BREX and Type 6 BREX—
The two least common BREX subtypes, type 5 and type 6, are similar to the type 1 BREX system but contain some additional variations (
Taken together, 135 of the 144 (94%) pglZ genes detected in microbial genomes were found to be embedded as part of one of the six BREX systems described (Table 8 above), and 7 of the remaining pglZ genes were clearly part of degraded (probably pseudogenized) systems. In most cases a single BREX system per organism was found, with only 8 (6.5%) of genomes harboring more than one subtype (Table 8 above). In addition, in 14% (19/135) of the identified systems, one of the genes was either missing or has become a pseudogene (tables 2-7 above), possibly representing inactivated systems. A similar tendency for gene loss was observed for the CRISPR-Cas system, and it was suggested that CRISPR-Cas inactivation is caused by fitness cost imposed by this defense system2,19,20. Phage defense systems often encode toxic genes21, and it is possible that such toxic genes encoded by BREX systems impose fitness cost and lead to gene loss in the absence of phage pressure.
To determine whether the BREX system provides protection against phage infection, the complete type 1 BREX system from Bacillus cereus H3081.97 (
Ten Bacillus subtilis phages were selected for phage infection experiments, spanning a wide range of phage phylogeny, from T4-like Myoviridae (SPO1 and SP82G), lambda-like Siphoviridae (φ105, rho10, rho14 and SPO2) and SPβ-like Siphoviridae (Φ3T, SPβ, SP16 and Zeta). Two of the phages are obligatory lytic (SPO1 and SP82G), while the remaining are temperate (See Table 9 below). The sensitivity of Bacillus subtilis strains either lacking or containing the BREX type 1 system to infection by the different phages was evaluated using both optical density measurements in a 96-well plate format, and double agar overlay and plaque assays (Table 9 below).
Upon phage infection, the Bacillus subtilis strain containing the BREX system showed complete resistance to five of the eight temperate phages tested (
To further evaluate the level of protection provided by the type 1 BREX system against the tested temperate phages, plaque assays using increasing dilutions of phage were performed. For five of the temperate phages, no plaques were observed when the type 1 BREX-containing strain was challenged even with the highest phage concentrations, indicating that the type 1BREX system provides at least a 105 fold protection against cell lysis upon phage infection (Table 9 below). The plaque assays also confirmed that phage Φ105 and its relatives evade type 1 BREX defense, with similar efficiencies of plating and plaque morphology observed in both type 1 BREX-containing and wild-type control strains (Table 9 below).
aProtection efficiency was calculated as the ratio between the number of plaques formed on the BREX-lacking strain divided by the number of plaques formed on the BREX-containing strain with the same phage titer, using increasing titers. Standard deviation was calculated from a biological triplicate of the plaque experiment.
The type 1 BREX-containing Bacillus subtilis strain also displayed some protection from the lytic SPO1 and SP82G phages in liquid culture experiments. Growth curves of the strain containing the BREX type 1 system infected with either SPO1 or SP28G phages were similar to the uninfected strains when evaluated for up to 12 hours following infection, while complete lysis was observed in infected control strains lacking the BREX system (
To gain further insight into the nature of the incomplete type 1 BREX defense against these lytic phages, a one-step phage growth curve assay [Carlson Bacteriophages, Biology and Applications (eds. E Kutter, A Sulakvelidze) (2005) pp. 437-494. CRC Press, Florida.] was performed with SPO1. Briefly, this experiment involves mixing SPO1-infected cells with a SPO1-sensitive B. subtilis cells and plating them together using an agar overlay method. Phage bursts from successful infections are visualized as a single plaque on a lawn from the SPO1-sensitive B. subtilis strain, enabling an evaluation of the number of phages that have adsorbed and completed a successful infection cycle. As demonstrated in
Taken together, these results suggest that the type 1 BREX system provides significant protection from infection by the lytic phages SPO1 and SP82G.
Due to the homology of a subset of the genes in the BREX system to genes in the previously described Pgl system10, it was necessary to examine whether BREX also functions through the described Pgl mechanism. The Pgl phenotype observed in S. coelicolor A3 predicts that the Pgl system does not confer resistance to phage first cycle of infection. One-step phage growth curve assays were used to examine the first infection cycle of phage Φ3T in type 1 BREX-containing cells. As demonstrated in
Previous experiments with the S. coelicolor Pgl system also demonstrated that although the Pgl defense system prevents continued propagation of the temperate phage ΦC31, it does not block lysogeny of the phage10. To determine whether BREX also permits lysogeny, phage Φ3T integration into the Bacillus subtilis genome during infection was examined using a PCR assay. In control Bacillus subtilis strains lacking BREX, lysogeny was first detected 10 minutes following phage infection (
One of the common forms of phage defense is abortive infection (Abi), where infected cells commit “suicide” before phage progeny are produced, thus protecting the culture from phage propagation4. To test whether the type 1 BREX system acts via an Abi mechanism, the type 1 BREX-containing Bacillus subtilis strains were infected with increasing concentrations of Φ3T phage. Using high multiplicity of infection (MOI) where nearly all bacteria are infected in the first cycle, massive cell lysis should be observed in the culture in the case of Abi. The results demonstrated that even at an MOI>1, no significant growth arrest or culture decline was found in the liquid culture (
In the next step, BREX ability to prevent phage adsorption and phage DNA replication were evaluated. As illustrated in
To further test whether BREX leads to cleavage or degradation of phage DNA, the integrity of phage DNA was examined using Southern blot analysis on total cellular DNA extracted from phage-infected cells at increasing time points following infection. This analysis showed extensive replication of phage DNA in control Bacillus subtilis strains lacking type 1 BREX and affirmed no phage DNA replication in Bacillus subtilis strains containing type 1 BREX (
These results indicate that phage DNA replication does not occur in type 1 BREX-containing cells, that type 1 BREX does not lead to the degradation of phage DNA and that this system exerts its function at the early stages of the infection cycle.
As type 1 BREX contain an m6A DNA adenine methylase (pglX), the present inventors have evaluated whether either bacterial or phage DNA are methylated in a BREX-dependent manner. To this end, the PacBio sequencing platform that directly detects m6A modifications in sequenced DNA [Murray et al. Nucleic acids research (2012) 40: 11450-11462] was used. As demonstrated in
To examine whether BREX also methylates the invading phage DNA, total cellular DNA (including chromosomal DNA and intracellular phage DNA) was extracted at 10 and 15 minutes following a high-MOI infection by Φ3T and analysed PacBio sequencing. The results affirmed that the TAGGAG motifs in the bacterial genome were methylated throughout the infection. However, there was no methylation on these motifs in the phage genome at the time points tested during infection (data not shown).
The presence of bacterial-specific methylation could suggest that the type 1 BREX system encodes some kind of restriction/modification activity, and that the methylation of TAGGAG motifs in the bacterial genome may serve to differentiate between self and non-self DNA. This suggests that deletion of the methylase gene, pglX, would be detrimental to the cell, as the genomic TAGGAG motifs will no longer be protected from the putative restriction activity of BREX. However, as can be seen in
Taken together, these results suggest that phage adsorption occurs in type 1 BREX-containing strains. This system does not display the Pgl phenotype, and hence probably functions through a novel mechanism different than that of the Pgl system. In addition, the system methylates the host chromosomal DNA at a specific motif, and that this methylation is likely to be essential for the system's activity.
An examination of the distribution of BREX systems across microbial species showed that these systems undergo extensive horizontal transfer (
The individual clades demonstrated in
Within the 1447 genomes, the relative frequency of BREX in archaea (10%) was similar to that observed in bacteria (8.5%). Only subtypes 1, 3 and 5 were represented in the 111 archaeal genomes analyzed by the present inventors. However, the absence of subtypes 2, 4 and 6 from archaeal genomes could be the result of their rarity and the relative paucity of sequenced archaeal genomes, comprising only 111 out of the 1447 genomes analyzed.
Taken together, the BREX systems undergo extensive horizontal transfer, with subtype 1 possibly the ancestral form of BREX.
One of the type 1 BREX-containing Bacillus subtilis strains obtained was not active against any of the tested phages although PCR analysis showed that it contained the complete BREX system. Upon Illumina whole-genome re-sequencing of the engineered strain, a frameshift mutation in the adenine-specific methylase gene pglX was observed, resulting from a single nucleotide deletion occurring in a stretch of seven guanine (G) residues at position 2128 (out of 3539 bp) of this gene. These results further support that the pglX gene is essential for the function of the type 1 BREX system. Therefore more broadly additional evidence for genetic variability of pglX in nature was examined.
In 11% (15/135) of the BREX systems that were documented, the pglX gene presented irregularities with respect to the common BREX organization (
DNA shuffling via recombination events was previously shown to control phase variation in bacterial defense-related genes to alter the specificity or to mitigate toxic effects of specific genes in the absence of phage pressure4-26. Taken together, since no other gene except for pglX presented such high rates of irregularities, these results marked pglX as possibly undergoing frequent phase-variation, suggesting that this gene might confer specificity in the BREX system, or, alternatively, is particularly toxic.
Following the initial homology searches in 1447 genomes described in details hereinabove, the present inventors performed an extensive homology search on a bigger set of genomes, 2263 complete and 5493 draft genomes, using the 14 genes associated with BREX systems (Table 1 above). Only gene clusters containing the two core genes (pglX and brxC/pglY) and at least two additional genes were considered (Tables 10-16 above).
The homology searches of the BREX genes in the 5493 genomes found 536 BREX systems in 9.3% (513/5493) of all genomes analyzed.
BREX type 1, the most common form of BREX, appeared 409 times in 398 genomes (Tables 10 and 16 above).
In most cases a single BREX system per organism was found, with only 21 (4%) of genomes harboring more than one subtype (Table 16 above).
In addition, in 25% (134/536) of the identified systems, one of the genes was either missing or has become a pseudogene (Tables 10-15 above), possibly representing inactivated systems.
Furthermore, in 11.5% (62/536) of the BREX systems that were documented, the pglX gene presented irregularities with respect to the common BREX organization.
Taken together, the broader analysis of the 7756 genomes reinforced all findings obtained with the 1447 set of genomes described hereinabove.
Taken together, the above results described a phage resistance system widespread in bacteria and archaea, which the present inventors denoted BREX system. The BREX family of systems can be divided into six coherent subtypes containing 4-8 genes each, two of which are core genes, pglZ and brxC/pglY, present in all systems. The results also suggested pglX might confer specificity in the BREX system, or, alternatively, is particularly toxic. Moreover, the BREX systems undergo extensive horizontal transfer, with subtype 1, the most frequent subtype of this system, possibly the ancestral form of BREX.
In addition, the results demonstrated that the BREX type 1 system confers complete or partial resistance against phages spanning a wide phylogeny of phage types, including lytic and temperate phages, even in the first cycle of infection. The abundance of this system and the efficiency in which it protects against phages implies that it plays an important role as a major line of defense encoded by bacteria against phages.
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.
This application claims the benefit of priority under 35 USC §119(e) of U.S. Provisional Patent Application No. 61/894,993 filed Oct. 24, 2013, the contents of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2014/050902 | 10/14/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61894993 | Oct 2013 | US |
