PROPAGATOR CELLS AND METHODS FOR PROPAGATING PHAGE, IN PARTICULAR FOR DELIVERING CRISPR-CAS COMPONENTS VIA PROBIOTIC ORGANISMS

BACKGROUND

Bacteriophages (phages) are a phylum of viruses that infect bacteria, and are distinct from the animal and plant viruses. Phages can have either a “lytic” life cycle, a “lysogenic” life cycle that can potentially become lytic, or a “non-lytic” life cycle. Phages replicating through the lytic cycle cause lysis of the host target bacterial cell as a normal part of their life cycles. Phages replicating through the lysogenic cycles are called temperate phages, and can either replicate by means of the lytic life cycle and cause lysis of the host bacterium, or they can incorporate their DNA into the host bacterial DNA and become noninfectious prophages. Bacteriophages are bacterial viruses that only infect and multiply within their specific bacterial hosts. Host specificity is generally found at strain level, species level, or, more rarely, at genus level. This specificity allows for directed targeting of dangerous bacteria using phages. The adsorption of bacteriophages onto host cells is, in all but a few rare cases, a sine qua non condition for the onset of the infection process.

The natural capability of phages to infect and kill bacteria, together with the specificity of the phage-bacterial interactions, is the basic phenomena on which the concept of phage therapy is built. Therefore, phages that possess lytic life cycle are suitable candidates for phage therapy. The use of phage in food production has recently become an option for the food industry as a novel method for biocontrol of unwanted pathogens, enhancing the safety of especially fresh and ready-to-eat food products.

International Patent Application No. WO 00/69269 discloses the use of certain phage strain for treating infections caused by Vancomycin-sensitive as well as resistant strains of Enterococcus faecium, and International Patent Application No. WO 01/93904 discloses the use of bacteriophage, alone or in combination with other anti-microbial means, for preventing or treating gastrointestinal diseases associated with the species of the genus Clostridium.

US Patent Application No. 2001/0026795 describes methods for producing bacteriophage modified to delay inactivation by the host defense system, and thus increasing the time period in which the phage is active in killing the bacteria.

US Patent Application No. 2002/0001590 discloses the use of phage therapy against multi-drug resistant bacteria, specifically methicillin-resistant Staphylococcus aureus, and International Patent Application No. WO 02/07742 discloses the development of bacteriophage having multiple host range.

The use of phage therapy for the treatment of specific bacterial-infectious disease is disclosed, for example, in US Patent Application Nos. 2002/0044922; 2002/0058027 and International Patent Application No. WO 01/93904.

However, commercial scale production of bacteriophage compositions for therapeutic use is still limited. In current techniques, the titer of the phage composition is low, usually in the range of 10⁹-10¹¹pfu/ml on a laboratory scale, and 10⁷-10⁹on a commercial scale, whereas the titer typically required for therapeutic use is still limited. In current techniques, the titer of the phage composition is low, usually in the range of 10⁹-10¹¹pfu/ml on a laboratory scale, and 10⁷-10⁹on a commercial scale, whereas the titer typically required for phage therapy is 10¹²pfu/ml. Additionally, to reach the desirable titer, very large volumes of liquid are required.

US20160333348 describes the use of CRISPR/Cas systems delivered to host target bacterial cells using phage as vectors. In principle, phage can be grown at volume in the cognate host cell using standard bacterial culture techniques and equipment. Growth of such phage or lytic phage in the target host cells may, however, be hampered by host cell killing by the resident phage by lysis and/or by CRISPR/Cas targeting of host DNA or by any other anti-host mechanism or agent encoded by the phage nucleic acid and which is active in host cells.

As bacteriophage use in industrial application grows there is a need for commercial quantities of identified bacteriophage. Therefore, there is a need for a method for production of phage that provides good yield titer and/or reduces manufacturing volume.

SUMMARY OF THE INVENTION

The invention provides a solution by providing propagator cells for propagating phage. To this end, the invention provides:

In a First Configuration

A method of producing a population of phage, wherein the phage are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the method comprising

- (a) Providing a population of second cells comprising the receptor on the surface thereof, wherein the second cells are of a second species or strain, wherein the second species or strain is different from the first species or strain;
- (b) Infecting the second cells with phage of said first type;
- (c) Propagating the phage in the second cells, thereby producing the population of phage; and
- (d) Optionally isolating phage of said population.

In a Second Configuration

A cell (propagator cell) for propagating phage, wherein the phage are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the propagator cell comprising the receptor on the surface thereof, wherein the propagator cell is of a second species or strain, wherein the second species or strain is different from the first species or strain, whereby the propagator cell is capable of being infected by phage of said first type for propagation of phage therein.

In a Third Configuration

A population of propagator cells according to the invention, optionally comprised in a fermentation vessel for culturing the propagator cells and propagating phage of said first type.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A bacterial production strain (1) was engineered to express a receptor (2) recognized by a helper phage (line X) while non-receptor containing cells (line Y) served as control. Both lines were then transformed with a CGV and infected with a helper phage to produce CGC-PLP (3). Only in line X harbouring the helper phage receptor, CGV-PLP was produced (4) that could be used to deliver DNA to a target cell population expressing the phage receptor

FIG. 2. Delivery of CGV to target cells ATCC43888 (obtained from ATCC) or EMG-2 (obtained from Coli Genetic Stock Center, CGSC) both expressing the receptor recognized by the CGV-PLP. Lysates used for the infection was produced on production strains harboring the receptor for the helper phage (filled bars) or the control strain with no receptor (open bars). Only the production strain with the receptor where the helper phage was able to infect and produce CGV-PLP was able to produce a CGV-PLP lysate capable of infecting the target cell population.

DETAILED DESCRIPTION

The invention recognises the advantage of artificially altering receptors expressed by bacterial cells (or selecting cells according to the profile of receptors naturally expressed), for example in the use of cells that can be cultured at scale and are useful for propagating and growing up useful phage populations at scale (eg, commercial scale). Such phage, for example, may encode a HM-crRNA or gRNA as described in US20160333348, which phage are useful for killing host bacterial cells comprised by humans, animals, plants, foodstuffs, beverages, cosmetics, environments (eg, soil, waterway, water reservoir or oil recovery environments), such as those applications described in US20160333348, the disclosure of which is incorporated herein by reference.

Proteinaceous receptors are mainly outer membrane proteins; sugar moieties include those that compose the cell wall, pellicles, teichoic and LTA. The receptor of the invention is, for, example selected from any of these.

Bacteriophage adsorption initiates the infection process. Through a series of interactions between binding proteins of the bacteriophage (phage) and receptors on the bacterial cell surface, the virus recognizes a potentially sensitive host and then positions itself for DNA ejection. Phage adsorption is thus not only a crucial step in the infection process, but also represents the initial point of contact between virus and host and dictates host range specificity.

Bacteriophage adsorption generally consists of three steps: initial contact, reversible binding and irreversible attachment (Duckworth 1987). The first step involves random collisions between phage and host caused by Brownian motion, dispersion, diffusion or flow (Kokjohn and Miller 1992). In the reversible step, binding to bacterial surface components is not definitive and the phage can desorb from the host. This process, firstly identified by Garen and Puck (1951) through experimental observations of phage detachment after elution, may serve to keep the phage close to the cell surface as it searches for a specific receptor (Kokjohn and Miller 1992). The specific connection between bacterial receptor and phage-binding domains is sometimes mediated by an enzymatic cleavage. This step triggers conformational rearrangements in other phage molecules that allow the insertion of the genetic material into the host (for further details on the mechanism of phage genome ejection, see the review by Molineux and Panja (2013)).

Numerous review studies have highlighted the extensive range of host-associated receptors (proteins, sugars and cell surface structures) that bacteriophages target during adsorption (Lindberg 1977; Schwartz 1980; Wright, McConnell and Kanegasaki 1980; Heller 1992; Frost 1993; Henning and Hashemolhosseini 1994; Vinga et al. 2006; Rakhuba et al. 2010; Chaturongakul and Ounjai 2014). The nature and location of the host cell receptors recognised by bacteriophages varies greatly depending on the phage and host. They range from peptide sequences to polysaccharide moieties. In fact, bacteriophages have been shown to bind to receptors located in the walls of both Gram-positive (Xia et al. 2011) and Gram-negative bacteria (Marti et al. 2013), in bacterial capsules or slime layers (Fehmel et al. 1975), and in appendages [e.g. pili (Guerrero-Ferreira et al. 2011) and flagella (Shin et al. 2012)]. This diversity in receptors and structures involved is a testament to the multiplicity of mechanisms developed by phages and hosts to overcome the evolutionary strategies adopted by their counterparts. It is not unexpected to encounter so many possibilities considering the diversity and staggering amount of phages estimated to populate the different environments of the planet (Clokie et al. 2011). Nevertheless, in all cases, adsorption has so far been shown to involve either constituents of the bacterial cell wall or protruding structures. In an embodiment, therefore, a receptor in the present invention can be any such receptor mentioned in this paragraph or elsewhere in this disclosure.

Optionally, the receptor comprises lipopolysaccharide (LPS), a heptose moiety, the host's glucosylated cell wall teichoic acid (WTA), YueB, or a receptor recognized by a tail fiber protein of the phage or gp21 of the phage.

Receptors in the Cell Wall of Gram-Positive Bacteria

Peptidoglycan, or murein, is an important component of the bacterial cell wall and is often involved in bacteriophage adsorption. It is a polymer composed of multiple units of amino acids and sugar derivatives—N-acetylglucosamine and N-acetylmuramic acid. These sugar constituents are connected through glycosidic bonds, forming glycan tetrapeptide sheets that are joined together through the cross-linking of amino acids. The cross-linking occurs through peptide bonds between diaminopimelic acid (an amino acid analog) and D-alanine, or through short peptide interbridges. These interbridges are more numerous in Gram-positive bacteria, leading to their characteristically thicker cell walls.

Another main component of the cell wall of Gram-positive bacteria that can be involved in phage adsorption is teichoic acid—polysaccharides composed of glycerol phosphate or ribitol phosphate and amino acids. They are bonded to the muramic acid of peptidoglycans. When teichoic acids are bonded to the lipids of the plasma membrane, they are called lipoteichoic acids (LTA). Further details of the composition of cell walls of bacteria can be found in Tortora, Funke and Case (2007), Willey, Sherwood and Woolverton (2008), Pommerville (2010) and Madigan et al. (2012).

The majority of the receptors so far identified are associated either with peptidoglycan or teichoic acid structures (Table 1). Out of 30 phages targeting Gram-positive bacteria reported in Table 1, only 10 utilize other structures for adsorption. Among these 10 phages, 9 display interactions with residues of either teichoic acid (phage SPP1) or peptidoglycan (phages 5, 13, c2, h, ml 3, kh, L and p2) for reversible binding. This highlights the important role these structures may play in the adsorption of phage to Gram-positive bacteria.

Optionally, the receptor of the invention is peptidoglycan, murein, teichoic acid or lipoteichoic acid (LTA). Optionally, the phage is a phage of a family listed in Table 1 (and optionally the host is the host for the phage as listed in Table 1 and/or the receptor is the receptor for the phage as listed in Table 1). Optionally, the phage is a phage listed in Table 1 (and optionally the host is the host for the phage as listed in Table 1 and/or the receptor is the receptor for the phage as listed in Table 1). In an embodiment, the host and second cells are gram-positive cells. Optionally the host and/or second cells are of a species or strain listed in Table 1 (where the host and second cell species or strains are different). Preferably when the host is a gram-positive bacteria, the receptor is a peptidoglycan. Alternatively, preferably when the host is a gram-positive bacteria, the receptor is a teichoic acid.

TABLE 1

Phages
Family
Main host
Recetpor(s)
References

γ
Siphoviridae

Bacillus anthracis

Membrane surface-anchored protein
Davison et al. (2005)

gamma phage receptor (GamR)

SPP1
Siphoviridae

Bacillus subtilis

Glucosyl residues of poly(glycerophosphate)
São-José, Baptista

on WTA for reversible binding and
and Santos (2004),

membrane protein YueB for
Baptista, Santos

irreversible binding
and São-José (2008)

ϕ29
Podoviridae

Bacillus subtilis

Cell WTA (primary receptor)
Xiang et al. (2009)

Bam35
Tectiviridae

Bacillus thuringiensis

N-acetyl-muramic acid (MurNAc) of
Gaidelyte et al. (2006)

peptidoglycan in the cell wall

LL-H
Siphoviridae

Lactobacillus delbrueckii

Glucose moiety of LTA for reversible
Munsch-Alatossava

adsorption and negatively charged
and Alatossava (2013)

glycerol phosphate group of the LTA for

irreversible binding

B1
Siphoviridae

Lactobacillus plantarum

Galactose component of the
Douglas and Wolin (1971)

wall polysaccharide

B2
Siphoviridae

Lactobacillus plantarum

Glucose substituents in teichoic acid
Douglas and Wolin (1971)

513c2hml3khL
Siphoviridae

Lactococcus lactis

Rhamnose^amoieties in
Monteville, Ardestani

the cell wall peptidoglycan for reversible
and Geller (1994)

binding and membrane phage infection protein

(PIP) for irreversible binding

ϕLC3TP901ermTP901-1
Siphoviridae

Lactococcus lactis

Cell wall polysaccharides
Ainsworth, Sadovskaya

and Vinogradov (2014)

p2
Siphoviridae

Lactococcus lactis

Cell wall saccharides for reversible
Bebeacua et al. (2013)

attachment and

pellicle^bphosphohexasaccharide

motifs for irreversible

adsorption

A511
Myoviridae

Listeria monocytogenes

Peptidoglycan (murein)
Wendlinger, Loessner

and Scherer (1996)

A118
Siphoviridae

Listeria monocytogenes

Glucosaminyl and rhamnosyl components of
Wendlinger, Loessner

ribitol teichoic acid
and Scherer (1996)

A500
Siphoviridae

Listeria monocytogenes

Glucosaminyl residues in teichoic acid
Wendlinger, Loessner

and Scherer (1996)

ϕ812ϕK
Myoviridae

Staphylococcus aureus

Anionic backbone of WTA
Xia et al. (2011)

52A
Siphoviridae

Staphylococcus aureus

O-acetyl group from the 6-position of muramic
Shaw and Chatterjee (1971)

acid residues in murein

Wϕ13ϕ47ϕ77ϕSa2m
Siphoviridae

Staphylococcus aureus

N-acetylglucosamine (GlcNAc)
Xia et al. (2011)

glycoepitope on WTA

ϕSLT
Siphoviridae

Staphylococcus aureus

Poly(glycerophosphate) moiety of LTA
Kaneko et al. (2009)

^aMonteville, Ardestani and Geller (1994) noted that since phages can also bind to glucose and galactose moieties in the cell wall, these might, to a lesser extent, be involved in the adsorption mechanism;

^bPellicle is a protective polysaccharide layer that covers the cell surface of Lactococcus lactis(Chapot-Chartier et al. 2010).

RECEPTORS IN THE CELL WALL OF GRAM-NEGATIVE BACTERIA

In Gram-negative bacteria, the peptidoglycan layer is relatively thin and is located inward of the outer membrane, the major component of the cell wall. These two layers are connected by Braun's lipoproteins. The outer membrane is a sophisticated structure composed of a lipid bilayer ornamented with proteins, polysaccharides and lipids; the latter two molecules form the LPS layer. LPSs are complexes that consist of three parts: lipid A, the core polysaccharide and the O-polysaccharide. Lipid A is, in general, composed of fatty acids attached to glucosamine phosphate disaccharides. The core polysaccharide is connected to the lipid A through a ketodeoxyoctonate linker. The core polysaccharide and the O-polysaccharide (O-chain or O-antigen) contain several units of sugar residues extending outward to the outer membrane. Cells that contain all three components of the LPS are denominated as smooth (S) type and those that lack the O-polysaccharide portion are distinguished as rough (R) type. In general, the saccharides composing the O-antigen are highly variable and those of the core polysaccharide are more conserved among species. Because of this, phages specific to only S-type strains tend to target the O-polysaccharide and, thus, have generally a narrower host range when compared to those able to adsorb to R-type cells (Rakhuba et al. 2010).

Table 2(a) compiles Gram-negative bacterial receptors located in the cell wall that interact with phage receptor-binding proteins (RBPs). Interestingly, in coliphages there is no preference for proteinaceous or polysaccharide receptors: some phages adsorb on cell wall proteins, some on sugar moieties and others require both structures for adsorption. In the case of Salmonella phages, the picture is not so different: some use proteins, some sugar moieties and some both types of receptors. On the other hand, Pseudomonas phages commonly adsorb onto polysaccharide receptors. Although definitive conclusions cannot be drawn from such a small sample size, it should be noted that Pseudomonas can have two LPS moieties, a short chain LPS named A band and a longer B-band LPS (Beveridge and Graham 1991).

Optionally, the receptor is a host cell wall protein. Optionally, the receptor is a saccharide. Optionally, the receptor comprises O-antigen, LPS lipid A or LPS core polysaccharide. In an example, the receptor is smooth LPS or rough LPS. Optionally, the host cells are S-type bacteria and the receptor comprises O-antigen of the host. Optionally, the host cells are R-type bacteria and the receptor comprises LPS lipid A of the host.

In an example, the host is E coli and the phage are coliphage, wherein the receptor is a polysaccharide receptor and/or a host cell wall protein receptor. In an example, the second cells are engineered to express E coli polysaccharide receptor and/or an E coli cell wall protein receptor, wherein the E coli is optionally of the same strain as the host cells.

In an example, the host is Salmonella, wherein the receptor is a polysaccharide receptor and/or a host cell wall protein receptor. In an example, the second cells are engineered to express Salmonella polysaccharide receptor and/or a Salmonella cell wall protein receptor, wherein the Salmonella is optionally of the same strain as the host cells.

In an example, the host is Pseudomonas, wherein the receptor is a polysaccharide receptor. In an example, the second cells are engineered to express Pseudomonas polysaccharide receptor, wherein the Psendomonas is optionally of the same strain as the host cells.

Optionally, the phage is a phage of a family listed in Table 2 (and optionally the host is the host for the phage as listed in Table 2 and/or the receptor is the receptor for the phage as listed in Table 2). Optionally, the phage is a phage listed in Table 2 (and optionally the host is the host for the phage as listed in Table 2 and/or the receptor is the receptor for the phage as listed in Table 2).

In an embodiment, the host and second cells are gram-negative cells. Preferably, the second cells are E coli cells. Optionally the host and/or second cells are of a species or strain listed in Table 2 (where the host and second cell species or strains are different).

In an example, the host is Pseudomonas, wherein the receptor is a polysaccharide receptor. In an example, the second cells are engineered to express Pseudomonas polysaccharide receptor, wherein the Pseudomonas is optionally of the same strain as the host cells.

Table 2(b) reports cases where phages not only adsorb onto bacterial surfaces but also enzymatically degrade the sugar moieties in the O-chain structure. It should be noted that all these phages belong to the Podoviridae family.

TABLE 2

Receptors in the cell wall of Gram-negative bacteria. Host names are ordered alphabetically.

Phages
Family
Main host
Receptor(s)
References

(a) Receptors that bind to RBP of phages

ϕCr30
Myoviridae

Caulobacter crescentus

Paracrystalline surface
Edwards and Smit (1991)

(S) layer protein

434
Siphoviridae

Escherichia coli

Protein Ib (OmpC)
Hantke (1978)

BF23
Siphoviridae

Escherichia coli

Protein BtuB (vitamin
Bradbeer, Woodrow and

B₁₂receptor)
Khalifah (1976)

K3
Myoviridae

Escherichia coli

Protein d or 3A (OmpA) with LPS
Skurray, Hancock and

Reeves (1974);

Manning and Reeves

(1976); Van Alphen,

Havekes and

Lugtenberg (1977)

K10
Siphoviridae

Escherichia coli

Outer membrane protein LamB
Roa (1979)

(maltodextran selective channel)

Me1
Myoviridae

Escherichia coli

Protein c (OmpC)
Verhoef, de Graaff

and Lugtenberg (1977)

Mu G(+)
Myoviridae

Escherichia coli

Terminal Glcα-2Glcα1- or
Sandulache,

GlcNAcα1-2Glcα1- of the LPS
Prehm and Kamp (1984)

Mu G(−)
Myoviridae

Escherichia coli

Terminal glucose with
Sandulache et al. (1985)

a βl,3 glycosidic linkage

Erwinia

Terminal glucose linked

in βl,6 configuration

M1
Myoviridae

Escherichia coli

Protein OmpA
Hashemolhosseini et al. (1994)

Ox2
Myoviridae

Escherichia coli

Protein OmpA^a
Morona and Henning (1984)

ST-1
Microviridae

Escherichia coli

Terminal Glcα-2Glcα1-
Sandulache,

or GlcNAcα1-
Prehm and Kamp (1984)

2Glcα1- of the LPS

TLS
Siphoviridae

Escherichia coli

Antibiotic efflux protein TolC
German and

and the inner core of LPS
Misra (2001)

TuIa
Myoviridae

Escherichia coli

Protein Ia (OmpF)
Datta, Arden and

with LPS
Henning (1977)

TuIb
Myoviridae

Escherichia coli

Protein Ib (OmpC)

with LPS

TuII*
Myoviridae

Escherichia coli

Protein II* (OmpA)

with LPS

T1
Siphoviridae

Escherichia coli

Proteins TonA (FhuA, involved in
Hantke and Braun (1975, 1978);

ferrichrome uptake) and TonB^b
Hancock and Braun (1976)

T2
Myoviridae

Escherichia coli

Protein Ia (OmpF) with
Hantke (1978); Morona and

LPS and the outer
Henning (1986); Black (1988)

membrane protein

FadL (involved in

the uptake of

long-chain fatty acids)

T3
Podoviridae

Escherichia coli

Glucosyl-α-1,3-glucose
Prehm et al. (1976)

terminus of rough LPS

T4
Myoviridae

Escherichia coli
K-12

Protein O-8 (OmpC) with LPS
Prehm et al. (1976);

Mutoh, Furukawa and

Mizushima (1978); Goldberg,

Grinius and Letellier (1994)

Escherichia coli B

Glucosyl-α-1,3-glucose

terminus of rough LPS

T5
Siphoviridae

Escherichia coli

Polymannose sequence in the
Braun and Wolff (1973);

O-antigen and protein FhuA
Braun, Schaller and

Wolff (1973); Heller

and Braun (1982)

T6
Myoviridae

Escherichia coli

Outer membrane protein Tsx
Manning and Reeves (1976, 1978)

(involved in nucleoside uptake)

T7
Podoviridae

Escherichia coli

LPS^c
Lindberg (1973)

U3
Microviridae

Escherichia coli

Terminal galactose residue in LPS
Picken and Beacham (1977)

λ
Siphoviridae

Escherichia coli

Protein LamB
Randall-Hazelbauer

and Schwartz (1973)

ϕX174
Microviridae

Escherichia coli

Terminal galactose in the core
Feige and Stirm (1976)

oligosaccharide of rough LPS

ϕ80
Siphoviridae

Escherichia coli

Proteins FhuA and TonB^b
Hantke and Braun

(1975, 1978); Wayne and

Neilands (1975);

Hancock and Braun (1976)

PM2
Corticoviridae

Pseudoalteromonas

Sugar moieties on the
Kivela et al. (2008)

cell surface^d

E79
Myoviridae

Pseudomonas aeruginosa

Core polysaccharide of LPS
Meadow and Wells (1978)

JG004
Myoviridae

Pseudomonas aeruginosa

LPS
Garbe et al. (2011)

ϕCTX
Myoviridae

Pseudomonas aeruginosa

Core polysaccharide of
Yokota, Hayashi

LPS, with emphasis
and Matsumoto (1994)

on L-rhamnose and D-glucose

residues in the outer core

ϕPLS27
Podoviridae

Pseudomonas aeruginosa

Galactosamine-alanine region
Jarrell and Kropinski (1981)

of the LPS core

ϕ13
Cystoviridae

Pseudomonas syringae

Truncated O-chain of LPS
Mindich et al. (1999);

Daugelavicius et al. (2005)

ES18
Siphoviridae

Salmonella

Protein FhuA
Killmann et al. (2001)

Gifsy-1Gifsy-2
Siphoviridae

Salmonella

Protein OmpC
Ho and Slauch (2001)

SPC35
Siphoviridae

Salmonella

BtuB as the main receptor
Kim and Ryu (2012)

and O12-antigen as

adsorption-assisting apparatus

SPN1SSPN2TCWSPN4B
Podoviridae

Salmonella

O-antigen of LPS
Shin et al. (2012)

SPN6TCW SPN8TCW

SPN9TCW SPN13U

SPN7CSPN9C SPN10H
Siphoviridae

Salmonella

Protein BtuB

SPN12C SPN14

SPN17T SPN18

vB_SenM-S16 (S16)
Myoviridae

Salmonella

Protein OmpC
Marti et al. (2013)

L-413CP2 vir1
Myoviridae

Yersinia pestis

Terminal GlcNAc residue
Filippov et al. (2011)

of the LPS outer

core. HepII/HepIII and

HepI/Glc residues

are also involved in

receptor activity^e

ϕJA1
Myoviridae

Yersinia pestis

Kdo/Ko pairs of inner

core residues.

LPS outer and inner

core sugars are also

involved in receptor

activity^e

T7_YpY (YpP-Y)
Podoviridae

Yersinia pestis

HepI/Glc pairs of inner

core residues.

HepII/HepIII and

Kdo/Ko pairs are also

involved in receptor

activity^e

Pokrovskaya
Podoviridae

Yersinia pestis

HepII/HepIII pairs of

YepE2YpP-G

inner core residues.

HepI/Glc residues

are also involved in

receptor activity^e

ϕA1122
Podoviridae

Yersinia pestis

Kdo/Ko pairs of inner

core residues.

HepI/Glc residues are

also involved in

receptor activity^e

PST
Myoviridae

Yersinia pseudotuberculosis

HepII/HepIII pairs of inner

core residues^e

(b) Receptors in the O-chain structure that are enzymatically cleaved by phages

Ω8
Podoviridae

Escherichia coli

The α-1,3-mannosyl linkages between
Reske, Wallenfels

the trisaccharide repeating unit
and Jann (1973)

α-mannosyl-1,2-α-mannosyl-

1,2-mannose

c341
Podoviridae

Salmonella

The O-acetyl group in the mannosyl-
Iwashita and

rhamnosyl-O-acetylgalactose
Kanegasaki (1976)

repeating sequence

P22
Podoviridae

Salmonella

α-Rhmanosyl 1-3 galactose
Iwashita and Kanegasaki (1973)

linkage of the O-chain

ε³⁴
Podoviridae

Salmonella

[-β-Gal-Man-Rha-]
Takeda and Uetake (1973)

polysaccharide units of the O-antigen

Sf6
Podoviridae

Shigella

Rha II 1-α-3 Rha III linkage
Lindberg et al. (1978)

of the O-polysaccharide.

^aSukupolvi (1984) suggested that LPS is also required for adsorption of phage Ox2 on E. coli and S. typhimurium, although the study verified that isolated OmpA is enough to inactivate the phage and that the binding is not increased with the addition of LPS to the protein.

^bAccording to Rakhuba et al. (2010), TonB is not a receptor itself, but acts as a mediator of electrochemical potential transmission; Vinga et al. (2006) stated that TonB is a membrane protein required for genome entry; Letellier et al. (2004) explained that TonB is part of a protein complex involved in the energy transduction from the electron transfer chain in the cytoplasmic membrane to the outer membrane receptors and speculated that it possibly might be critical for the genome injection through its interaction with FhuA.

^cRhakuba et al. (2010) mentioned proteins FhuA and TonB as the receptors for T7; Molineux (2001) reported that ‘Bayer patches’, described as adhesion sites between the cytoplasmic membrane and the outer envelope of Gram-negative bacteria, are the proposed receptors for T7.

^dIn 2010 the same group suggested that the adsorption of the phage on the sugar moieties of the host is an initial interaction, and that the true receptor is a protein molecule or protein complex (Cvirkaite-Krupovic 2010).

^eKdo, 2-keto-3-deoxy-octulosonic acid; Ko, D-glycero-D-talo-oct-2-ulosonic acid; Hep, heptulose (ketoheptose); Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine (from Filippov et al. 2011).

RECEPTORS IN OTHER STRUCTURES OF GRAM-NEGATIVE BACTERIA

In this section, bacterial structures, other than cell wall moieties, that also serve as receptors for phages are discussed. These include structures such as flagella, pili and capsules. They can be found in species from both Gram stains. See Table 3 for examples.

Optionally, the receptor of the invention is a flagellum, pilus or capsule component (eg, a component listed in Table 3 in the listed species or as found in a host that is of a different species to that listed). Optionally, the phage is a phage of a family listed in Table 3 (and optionally the host is the host for the phage as listed in Table 3 and/or the receptor is the receptor for the phage as listed in Table 3). Optionally, the phage is a phage listed in Table 3 (and optionally the host is the host for the phage as listed in Table 3 and/or the receptor is the receptor for the phage as listed in Table 3).

Flagella are long thin helical structures that confer motility to cells. They are composed of a basal body, a flagellar hook and a flagellar filament composed of subunits of flagellin proteins (Willey, Sherwood and Woolverton 2008). Table 3(a) reports phages attaching to flagellal proteins. The adhesion of phages to the filament structure is generally reversible and the flagellum's helical movement causes the phage to move along its surface until they reach the bacterial wall. Irreversible adsorption occurs, then, on receptors located on the surface of the bacterium, near the base of the flagellum (Schade, Adler and Ris 1967; Lindberg 1973; Guerrero-Ferreira et al. 2011). Interestingly, some phages (ϕCbK and ϕCb13) were observed to contain filaments protruding from their capsids that are responsible for reversible binding onto the host's flagellum; irreversible adsorption occurs only when the phage's tails interact with pili portals on the cell pole (Guerrero-Ferreira et al. 2011). Because for these phages irreversible adsorption occurs on the pilus, even if they interact with the flagellum, they were reported in Table 3(b), which focuses on phages interacting with receptors in pili and mating pair formation structures.

Pili are rod-shaped filamentous appendages used for bacterial conjugation (Lindberg 1973). They extend from the donor cell and attach to receptors on the wall of the recipient cell. A depolymerization of the pilus causes its retraction, bringing both cells closer to each other. Further adhesion of the cells is achieved through binding proteins on their surfaces; genetic material is transferred through this conjugating junction (Madigan et al. 2012). Adsorption to the pilus structure has been so far associated with phages that belong to orders different from Caudovirales (Table 3b). In fact, according to Frost (1993), the families Cystoviridae and Inoviridae compose the majority of phages that adsorb onto pili structures. Interestingly, phages can be selective towards certain parts of the pili. That is the case for F-type phages, whose adsorption occur only on the tip of the pilus (Click and Webster 1998). In other phages, such as ϕ6, the attachment happens at the sides (shaft) of the structure (Daugelavicius et al. 2005).

TABLE 3

Receptors in bacterial complexes other than cell wall structures. Host names are ordered alphabetically.

Phages
Family
Main host
Receptor(s)
References

(a) Receptors in flagella

SPN2T SPN3C
Siphoviridae

Salmonella

Flagellin protein FliC
Shin et al. (2012) -

SPN8T SPN9T

SPN11T SPN13B

SPN16C

SPN4SSPN5T
Siphoviridae

Salmonella

Flagellin proteins FliC or FliB

SPN6T SPN19

iEPS5
Siphoviridae

Salmonella

Flagellal molecular ruler protein FliK
Choi et al. (2013);

Chaturongakul and Ounjai

(2014)

(b) Receptors in pili and mating pair formation structures

ϕCbK ϕCb13
Siphoviridae

Caulobacter

Initial contact between phage head
Guerrero-Ferreira et al. (2011)

crescentus

filament and host's flagellum followed

by pili portals on the cell pole

FdFff1M13
Inoviridae

Escherichia

Tip of the F pilus followed by TolQRA
Loeb (1960); Caro and Schnos

coli

complex in membrane after pilus
(1966); Russel et al. (1988);

retraction
Click and Webster (1998)

PRD1
Tectiviridae

Escherichia

Mating pair formation (Mpf) complex in
Daugelavicius et al. (1997)

coli

the membrane

ϕ6
Cystoviridae

Pseudomonas

Sides of the type IV pilus
Vidaver, Koski and Van Etten

(1973); Daugelavicius et al.

(2005)

MPK7
Podoviridae

Pseudomonas

Type IV pili (TFP)
Bae and Cho (2013)

aeruginosa

MP22
Siphoviridae

Pseudomonas

Type IV pili (TFP)
Heo et al. (2007)

aeruginosa

DMS3
Siphoviridae

Pseudomonas

Type IV pili (TFP)
Budzik et al. (2004)

aeruginosa

(c) Receptors in bacterial capsules

29
Podoviridae

Escherichia

Endoglycosidase hydrolysis in β-D-
Stirm et al. (1971); Fehmel et

coli

glucosido-(1-3)-D-glucoronic acid bonds
al. (1975)

in the capsule composed of

hexasaccharides repeating units

K11
Podoviridae

Klebsiella

Hydrolysis of β-D-glucosyl-(1-3)-β-D-
Thurow, Niemann and Stirm

glucuronic acid linkages. The phage is
(1975)

also able to cleave α-D-galactosyl-(1-3)-

β-D-glucose bonds

Vi I
Myoviridae

Salmonella

Acetyl groups of the Vi
Pickard et al. (2010) -

exopolysaccharide capsule (a polymer of

α-1,4-linked N-acetyl

galactosaminuronate)

Vi II
Siphoviridae

Salmonella

Acetyl groups of the Vi

exopolysaccharide capsule (a polymer of

α-1,4-linked N-acetyl

galactosaminuronate)

Vi IIIVi IVVi VVi
Podoviridae

Salmonella

Acetyl groups of the Vi
—

VIVi VII

exopolysaccharide capsule (a polymer of

α-1,4-linked N-acetyl

galactosaminuronate)

Capsules are flexible cementing substances that extend radially from the cell wall. They act as binding agents between bacteria and/or between cells and substrates (Beveridge and Graham 1991). Slime layers are similar to capsules, but are more easily deformed. Both are made of sticky substances released by bacteria, and their common components are polysaccharides or proteins (Madigan et al. 2012). Adsorption of phages to capsules or slime layers is mediated by enzymatic cleavage of the exopolysaccharides that compose the layers. The hydrolysis of the layer is a reversible step, whereas irreversible binding is achieved through bonding of the phage with receptors on the cell wall (Rakhuba et al. 2010). As can be seen in Table 3(c), the few phages identified to have RBP recognizing exopolysaccharides are mostly of Podoviridae morphology.

In an example, the host is Salmonella (eg, S enterica Serovar Typhimurium) and the receptor is selected from flagella, vitamin B₁₂uptake outer membrane protein, BtuB and lipopolysaccharide-related O-antigen. In an example the receptor is a flagellum or BtuB and the phage are Siphoviridae phage. In an example the receptor is O-antigen of LPS and the phage are Podoviridae phage. Optionally, the receptor is FliC host receptor or FljB receptor.

Optionally, the host is S enterica or P aeruginosa. Optionally, the receptor is the receptor of the host as listed in Table 4.

TABLE 4

Specific host receptors for Salmonella and P. aeruginosa phages.

Specific host

receptors
Reference

Flagellar proteins

S. enterica

FliC and
Shin et al. (2012)

FljB

FliK
Choi et al. (2013)

Outermembrane proteins

OmpC
Ho and Slauch (2001), Marti et al. (2013)

BtuB
Kim and Ryu (2011)

TolC
Ricci and Piddock (2010)

FhuA
Casjens et al. (2005)

Surface antigens

O-antigen
Shin et al. (2012)

Vi-antigen
Pickard et al. (2010)

Surface antigens

P. aeruginosa

O-antigen
Le et al. (2013)

Vi-antigen
Temple et al. (1986), Hanlon et al. (2001)

Type IV pili

PilA
Bae and Cho (2013), Heo et al. (2007)

The O-antigen structure of Salmonella O66 has been established, which reportedly differs from the known O-antigen structure of Escherichia coli O166 only in one linkage (most likely the linkage between the O-units) and O-acetylation. The O-antigen gene clusters of Salmonella O66 and E. coli O166 were found to have similar organizations, the only exception being that in Salmonella O66, the wzy gene is replaced by a non-coding region. The function of the wzy gene in E. coli O166 was confirmed by the construction and analysis of deletion and trans-complementation mutants. It is proposed that a functional wzy gene located outside the O-antigen gene cluster is involved in Salmonella O66 0-antigen biosynthesis, as has been reported previously in Salmonella serogroups A, B and D1. The sequence identity for the corresponding genes between the O-antigen gene clusters of Salmonella O66 and E. coli O166 ranges from 64 to 70%, indicating that they may originate from a common ancestor. It is likely that after the species divergence, Salmonella O66 got its specific O-antigen form by inactivation of the wzy gene located in the O-antigen gene cluster and acquisition of two new genes (a wzy gene and a prophage gene for O-acetyl modification) both residing outside the O-antigen gene cluster.

In an example, the second cells are engineered to comprise an expressible E coli (eg, Escherichia coli O166) wzy gene. In an example, the second cells do not comprise an expressible E coli (eg, Escherichia coli O166) wzy gene. Optionally, the host cells are E coli or Salmonella (eg, Salmonella 066) cells.

In an example, the phage or particle comprises a phage genome or a phagemid, eg, wherein the genome or phagemid comprises DNA encoding one or more proteins or nucleic acids of interest, such as crRNAs for targeting host cell genomes or antibiotics for killing host cells.

In an alternative, instead of bacteria, the host and second cells (propagator cells) are archaeal cells and the disclosure herein relating to bacteria instead can be read as applying mutatis mutandis to archaea.

Target host strains or species of bacteria may comprise restriction-modification system (R-M system), such as R-M comprising restriction endonucleases, that can recognize and cut or otherwise destroy or degrade invading nucleic acid. Host DNA is protected by the action of methyltransferases that methylate host DNA and protect it from the R-M system. It may be desirable, therefore, to provide second bacterial cells (propagator cells) that do not comprise an R-M system or whose genome is devoid of nucleic acid encoding one or more restriction endonucleases which are encoded by host cells. Additionally or alternatively, the second cells comprise nucleic acid encoding one or more methyltransferases which are encoded by host cells, optionally all or substantially all (eg, at least 50, 60, 70 80 or 90%) of all of the methyltransferases encoded by host cells. Optionally, the second cells comprise nucleic acid encoding 1, 2,3 4, 5, 6, 7, 8, 9 or 10 or (or at least 1, 2,3 4, 5, 6, 7, 8, 9 or 10) methyltransferases encoded by host cells.

Advantageously, to produce phage or transduction particles targeting a specific bacterial host population, it may be beneficial to produce the phage or particles in a strain of bacteria related to the target host strain, for example to produce phage or particle nucleic acid (eg, DNA) that can evade host cell defence mechanisms, such as R-M systems or restriction endonuclease action. Optionally, therefore, the host cells and second cells (propagator cells) are cells of the same species (or the same strain of species except that the second cells comprise one or more genetic modifications that are not found in the genomes of host cells; such modification can be deletion of one or more protospacer sequences, for example wherein the host cells comprise such sequence(s) and the phage or particles express crRNA that recognize the sequences in the host cells to guide Cas and to modify the protospacer sequence(s)). For example, modification of the DNA of the phage or particles by methyltransferases in the second bacteria can be useful to shield the DNA against restriction modification once the phage or particles subsequently infect the target host cells where the latter also comprise methyltranferases in common with the second cells. By adapting (or choosing) the second cells as per the invention to display a surface receptor that is also displayed on the host cells, the invention enables phage or particle production in a strain that may display beneficial DNA modification against restriction modification subsequently by the target host bacteria. Usefully, the protospacer sequence(s) to which (in one embodiment) crRNAs encoded by the phage or particles are targeted in the target host bacteria may be deleted or naturally absent in the genome of the second bacteria, such that Cas-mediated cutting of the second cell genomes does not take place during the production of the phage or particles.

A heterologous methyltransferase (MTase) can be used to confer on a production bacterium (propagator bacterium or second cells herein) a similar methylation pattern as that of a target host bacterium. See, for example, WO2016205276, which incorporated herein by reference, for example to provide illustration of how to provide production strain genomes comprising desirable MTases for use in the present invention). In bacteria and archaea, some DNA methyltransferases can be separated into three distinct classes depending on the location of the modification and type of reaction they catalyze. N6-methyladenine (m6A) and N4-methylcytosine (m4C) result from methylation of the amino moiety of adenine and cytosine, respectively, while 5-methylcytosine (m5C) is the result of methylation at the C5 position of cytosine.

A non-limiting example of a DNA MTase useful with the invention includes LlaPI from phage Φ50, which can be introduced to protect against type II R-M systems in lactococci (Hill et al. J Bacteriol. 173(14):4363-70 (1991)). Optionally, the production bacterium encodes and expresses one or more DNA modification enzymes that catalyse methylation of adenines, eg, to produce N6-methyladenine (m6A). Optionally, the production bacterium encodes and expresses one or more DNA modification enzymes that catalyse methylation of cytosines, eg, to produce N4-methylcytosine (m4C) or 5-methylcytosine (m5C). Optionally, the production bacterium encodes and expresses one or more DNA modification enzymes that catalyse acetimidation of adenine residues. Some R-M systems are sensitive to adenine methylation. Polypeptides that acetimidate the adenine residues in the phage or particle DNA will protect the DNA against such systems. Non-limiting examples of polypeptides that can acetimidate adenine residues in the production host bacteria include the mom gene from phage Mu and the Mu-like prophage sequences (see, Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnmel) and H. influenzae biotype aegyptius ATCC 111 16), which converts adenine residues to N(6)-methyladenine, thereby protecting against adenine sensitive restriction enzymes. The methylation patterns conferred by individual methyltransferases can be assessed using established DNA sequencing technologies such as Pacbio SMRT sequencing (O'Loughlin et al. PLoS One. 2015:e0118533). Once generated, the production strain can be used to produce bacteriophage particles for DNA delivery into the target strain.

Bacterial “restriction-modification systems” (R-M systems) comprise (1) methyltransferases that methylate DNA at specific sequences and/or (2) restriction enzymes that cleave DNA that are unmethylated (Types I, II, and III) or methylated (Type IV). The R-M systems constitute a bacterial defence system wherein DNA with foreign methylation patterns is cleaved in multiple locations by the restriction enzymes of the R-M systems. Most: bacteria comprise more than one R-M system. Roberts, R. J. et al. Nucleic Acids Res . 31 , 1805-1812 (2003). Type I methyltransferases require the presence of a compatible specificity protein for functionality. Type II and type III methyltransferases do not require any additional proteins to function. Thus, methyltransferases and restriction enzymes useful with this invention (either as targets for modification or inhibition, or as heterologous polypeptides to be expressed in a production bacterium, thereby modifying the R-M system of the production bacterium) can include any methyltransferase or restriction enzyme comprised in a bacterial restriction-modification system (e.g. Type I, II, III, or IV). Thus, in an example, the genome of the production bacterium (second or propagator cell) encodes a Type I methyltransferase that is also encoded by the host bacterium. Additionally or alternatively, in an example, the genome of the production bacterium (second or propagator cell) encodes a Type II methyltransferase that is also encoded by the host bacterium. Additionally or alternatively, in an example, the genome of the production bacterium (second or propagator cell) encodes a Type III methyltransferase that is also encoded by the host bacterium. Additionally or alternatively, in an example, the genome of the production bacterium (second or propagator cell) encodes a Type IV methyltransferase that is also encoded by the host bacterium.

In an example, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) nucleic acid sequences encoding enzymes of the endogenous restriction modification system of a production bacterium are disrupted or altered in activity (eg, reduced or eliminated in activity).

A production bacterium (ie, second cell or propagator cell) can be a gram positive or gram negative bacterium. Thus, for example, production bacterium is an Escherichia coli, Bacillus subtilis, Lactobacillus rhamnosus, Salmonella enteria, Streptococcus thermophilus, Listeria, Campylobacter or Staphylococcus aureus bacterium. In an example, the production bacterium is an E. coli strain MG1655, Nissle, BW25113, BL21, TOP10, or MG1655 Δdam Δdcm ΔhsdRMS.

The activity of an enzyme of an endogenous R-M system may be disrupted using methods well known in the art or later developed for disrupting the function and activity of a polypeptide. Such methods can include, but are not limited to, generating point mutations (e.g., missense, or nonsense, or insertions or deletions of single base pairs that result in frame shifts), insertions, deletions, and/or truncations. In some embodiments, a polypeptide inhibitor may be used to disrupt or suppress the activity of an enzyme of a bacterial restriction modification system (R-M system). Such polypeptide inhibitors are known in the art. Polypeptide inhibitors may be encoded, for example, within the phage or particle DNA and/or packaged as proteins in the phage or particle. For example, P1 phage encodes two polypeptide inhibitors that inhibit Type I restriction enzymes found in E. coli (Lobocka et al. J. Bacteriol. 186, 7032-7068 (2004)). In some embodiments, an endogenous R-M system may be inhibited or disrupted by the introduction of polypeptide inhibitors, polypeptides that stimulate the activity of the host methylation enzymes to accelerate the methylation and protection of the delivered DNA.

Inhibitors of R-M system enzymes include but are not limited to proteins that degrade a REase (restriction endonuclease), thereby preventing the host R-M enzyme system from cleaving the phage or particle DNA. Non-limiting examples of an R-M enzyme inhibitor that may be used with this invention to disrupt or modify the activity of an endogenous bacterial R-M system enzyme include (a) orf18 from Enterococcus faecalis, which produces the protein ArdA that inhibits all major classes of type I R-M systems; and (b) gp0.3 from bacteriophage T7 produces the protein Ocr that sequesters the type I R-M enzyme EcoKI. Additional non-limiting examples of proteins that may be used to block the activity of an enzyme of an R-M system include masking proteins. Masking proteins are packaged into the phage head and upon DNA injection bind the phage DNA, thereby masking R-M recognition sites. Non-limiting examples of masking proteins useful with this invention include DarA and DarB proteins (Iida et al. Virology. 1 57(1): 156-66 (1987)). These proteins are expressed by the P1 bacteriophage during the lytic cycle and are packaged into the head. Upon DNA injection to a host bacterium, they bind and mask the Type I R-M recognition sites.

In addition to or in the alternative, an endogenous R-M system of a production bacterium can be altered/modified through the expression of at least one heterologous methyltransferase. Any methyltransferase that alters the endogenous methylation pattern of a production host bacterium so that the methylation pattern of the production host bacterium is substantially similar to the methylation pattern of the target host bacterium can be used with this invention. The heterologous methyltransferase may be from the same or a different organism as long as it confers a methylation pattern substantially similar to the production host bacterium as the target bacterial strain. A non-limiting example of a DNA MTase useful with the invention includes LlaPI from phage Φ50, which can be introduced to protect against type II R-M systems in lactococci (McGrath et al. Applied Environmental Microbiology. 65:1891 -1899 (1999)). The methylation patterns conferred by individual methyltransferases are then assessed using established DNA sequencing technologies such as Pacbio SMRT sequencing (O'Loughlin et al. Pl.oS One. 2015:e0118533). Once generated, the production strain is used to produce phage or particles for DNA delivery into the target host strain.

Further heterologous DNA modification enzymes can be expressed in a production bacterium so that the R-M system of the production bacterium is made substantially similar to the R-M system of the target host bacterium. Examples of such DNA modification enzymes useful for this purpose include those that encode polypeptides that convert the adenine residues in the DNA to acetamidoadenine. Polypeptides that convert the adenine residues in the phage or particle DNA to acetamidoadenine will protect the DNA against restriction enzymes that are sensitive to adenine methylation. Non-limiting examples of polypeptides that can convert the adenine residues in the DNA to acetamidoadenine in the production bacteria include the mom gene from phage Mu and the Mu-like prophage sequences (see, Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme 1) and H. influenzae biotype aegyptius ATCC 1 1116; (Drozdz et al. Nucleic Acids Res. 40(5):2119-30 (2012)), which converts adenine residues to N(6)-methyladenine, thereby protecting against adenine-sensitive restriction enzymes.

In some embodiments, the polynucleotides encoding polypeptide inhibitors and other DNA modification enzymes as described herein can be introduced into the phage or particle genome directly for use in protecting the delivered DNA from the R-M system of the target host bacterium.

Accordingly, in some embodiments, the invention provides a method of increasing the efficiency of introducing a heterologous nucleic acid of interest into a target host bacterium via bacteriophage or transduction particles, comprising introducing at least one heterologous nucleic acid of interest into a phage or particle DNA prior to introduction of a production bacterium, wherein the production host bacterium has been modified to disrupt at least one enzyme of an endogenous R-M system and/or to comprise a polynucleotide encoding at least one heterologous methyltransferase, thereby methylating said phage or particle DNA and producing phage or particle DNA comprising the at least one heterologous nucleic acid of interest having a modified methylation pattern (as compared to phage or particle DNA produced in a production bacterium without said altered methylating activity); producing a phage or particle comprising said recombinant DNA comprising the at least one heterologous nucleic acid of interest; and infecting a target host bacterium with said bacteriophage or particle, wherein the target host bacterium has a methylation pattern (or R-M system(s)) that is identical, similar to or substantially similar to that of the production bacterium, thereby increasing the efficiency of introducing said heterologous nucleic acid of interest into said target host bacterium as compared to introducing said heterologous nucleic acid of interest using a bacteriophage grown in a control production bacterium (wherein the control production host bacterium has not had its methylation activity altered to be identical, similar or substantially similar with that of the target host bacterium). In some aspects, the production bacterium can be modified to alter its R-M system (e.g., disrupt at least one enzyme of an endogenous R-M system and/or to comprise a polynucleotide encoding at least one heterologous methyl transferase) after infection by the phage or particle.

In some embodiments a method of increasing the efficiency of introducing a heterologous nucleic acid of interest into a target host bacterium via a phage or transduction particle is provided, comprising: infecting a production bacterium with a bacteriophage or particle comprising DNA comprising at least one heterologous nucleic acid of interest, wherein the production bacterium has altered methylating activity via disruption of at least one enzyme of an endogenous R-M system and/or expression of at least one heterologous methyltransferase, thereby methylating said DNA; producing a bacteriophage particle comprising bacteriophage or particle DNA having a modified methylation pattern and comprising/encoding the at least one heterologous nucleic acid of interest; and infecting a target host bacterium with said bacteriophage or particle, wherein the target host bacterium has a methylation pattern (or R-M system(s)) that is identical, similar or substantially similar with that of the production host bacterium, thereby increasing the efficiency of introducing said heterologous nucleic acid of interest into said target host bacterium as compared to introducing said heterologous nucleic acid of interest using a bacteriophage or particle produced in a control production bacterium (wherein the control production bacterium has not had its methylation activity altered to be identical, similar substantially similar to that of the target host bacterium as described herein). In some aspects, the production bacterium can be modified to alter its R-M system (e.g., disrupt at least one enzyme of an endogenous R-M system and/or to comprise a polynucleotide encoding at least one heterologous methyltransferase) after infection by the bacteriophage or particle.

In an example, the target host bacterium is chosen on the basis of having a DNA methylation pattern substantially similar to a production host bacterium's restriction-modification system(s) (R-M system).

A methylation pattern is determined by the type of methylation (e.g. m4C) present in the bacterium as well as the particular sequence that is methylated (e.g. GmATC). Thus, the level of similarity (whether it is natural or the result of modifications) between methylation patterns refers to the frequency by which target sites having the appropriate type of methylation. Thus, a substantially similar methylation pattern means having at least about 20% or greater similarity (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more, or any range or value therein) between the target sites having the appropriate type of methylation as described herein. Thus, in some embodiments, a methylation pattern can be between about 20% to 99% or more similar, about 30% to 99% or more % similar, about 40% to 99% or more similar, about 50% to 99% or more similar, about 60% to 99% or more similar, about 70% to 99% or more similar, about 80% to 99% or more similar, about 85% to 99% or more similar, about 90% to 99% or more similar, or about 95% to 99% or more similar, between host and target bacteria. Substantial similarity between methylation patterns of a target host bacterium and the introduced DNA (bacteriophage or particle DNA that has been modified) means that the introduced DNA is less degraded than that of an introduced DNA that does not share a substantially similar methylation pattern with the target host bacterium. In some embodiments, the methylation pattern of a production bacterium and a target bacterium can be identical.

In some embodiments, the invention provides a bacteriophage or particle comprising DNA that comprise a modified DNA methylation pattern that is identical, similar or substantially similar to a target host bacterium's R-M system(s) and wherein at least one heterologous nucleic acid of interest is integrated into the bacteriophage or particle DNA (genome). Thus, for example, a bacteriophage or transduction particle DNA having a modified methylation pattern (that is substantially similar to a target host bacterium's R-M system(s)) can comprise (1) a polynucleotide encoding a CRISPR array or (2) a Type II CRISPR-Cas system comprising: (a) a polynucleotide encoding a Cas9 polypeptide; (b) a polynucleotide encoding a CRISPR array; and/or c) a tracr nucleic acid. In some embodiments, the polynucleotide encoding a CRISPR array (a) and the tracr nucleic acid (c) can be fused to one another. In additional embodiments, a bacteriophage or particle DNA having a modified methylation pattern (that is identical, similar or substantially similar to a target host bacterium's R-M system(s)) can comprise (1) a polynucleotide encoding a CRISPR array or (2) a recombinant Type I CRISPR-Cas system comprising: (a) a polynucleotide encoding a CRISPR array; and/or (b) at least one polynucleotide encoding one or more Type I CRISPR polypeptides. In some embodiments, the at least one heterologous nucleic acid of interest can be integrated into the bacteriophage or particle DNA (e.g., genome) at a dispensable site of integration or at a complemented site of integration.

As used herein, “dispensable site” means a site in the DNA or genome that is not necessary for maintenance of the bacteriophage or particle genome, the generation of phage or particles, and the delivery of packaged DNA. Thus, any site in a bacteriophage or particle genome that is not required for carrying out such functions can be used as a “landing” site for integrating a nucleic acid of interest. Some exemplary dispensable sites include, but are not limited to, (a) a phage-encoded restriction-modification system (e.g., res/mod in P1 phage), (b) a gene that blocks superinfection (e.g., simABC), (c) an inhibitor of a restriction-modification system (e.g., darA in P1 phage), (d) an insertion sequence element (e.g., IS1 in P1 phage), (e) an addiction system (e.g., phd/doc in P1 phage) or (f) any combination thereof.

A “complemented site” or a “complementable site” as used herein means an

indispensible site in the bacteriophage or particle DNA or genome that is necessary for maintenance of the bacteriophage or particle genome, the generation of phage or particles, and the delivery of packaged DNA but which can be complemented by a complementing polynucleotide encoding the nucleic acid that is disrupted by the integration (complemented site of integration) of the nucleic acid of interest. The complementing polynucleotide can be integrated into the genome of the production bacterium or it can be comprised on a plasmid in the production bacterium. Accordingly, when the nucleic acid of interest is integrated into a complemented site of a bacteriophage or particle DNA, the production bacterium can comprise on a plasmid or in its genome a polynucleotide encoding a complement to the complemented site in the bacteriophage or particle DNA. Exemplary complemented sites can include, but are not limited to, (a) an activator of the lytic cycle (e.g., coi in P1 phage), (b) a lytic gene (e.g., kilA in P1 phage), (c) a tRNA (e.g., tRNA1 ,2 in P1 phage), (d) a particle component (e.g., cixL and cixR tail fiber genes in P1 phage), or (e) any combination thereof.

In an embodiment, the methylation pattern of a production strain, such as Escherichia coli MG1655 or Bacillus subtilis 168, is altered by deleting its endogenous restriction-modification systems and introducing heterologous methyltransferase genes as follows. The restriction-modification genes are identified through means that are known in the art, such as through the online REBASE database (Roberts et al. Nucleic Acids Res 43:D298-D299. doi.org/10.1093/nar/gku1046). These restriction-modification systems can be deleted using standard recombineering strategies known in the art. Once deleted, foreign methyltransferase genes are inserted into replicative plasmids or recombineered into the host genome under the control of a constitutive or inducible promoter. These genes are obtained directly from the target strain using the natural sequence or a sequence codon-optimized for the production host. Alternatively, heterologous methyltransferase genes can be used to confer a similar methylation patterns as the target strain. The methylation patterns conferred by individual methyltransferases are then assessed using established DNA sequencing technologies such as PacBio SMRT sequencing (O'Loughlin et al. PLoS One. 2015:e0118533.). Once generated, the production strain is used to produce bacteriophage or transduction particles for DNA delivery into the target host strain.

Promoters:

Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated promoters for use in the preparation of recombinant nucleic acid constructs, polynucleotides, expression cassettes and vectors comprising the polynucleotides and recombinant nucleic acid constructs of the invention. These various types of promoters are known in the art.

Thus, in some embodiments, expression herein according to the invention can be made constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated promoters using the recombinant nucleic acid constructs of the invention operatively linked to the appropriate promoter functional in an organism of interest. In representative embodiments, repression can be made reversible using the recombinant nucleic acid constructs of the invention operatively linked to, for example, an inducible promoter functional in an organism of interest.

The choice of promoter will vary depending on the quantitative, temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.

Exemplary promoters include useful with this invention include promoters functional in bacteria. A promoter useful with bacteria can include, but is not limited to, L-arabinose inducible (araBAD, P_BAD) promoter, any lac promoter, L-rhamnose inducible (rhaP_BAD) promoter, T7 RNA polymerase promoter, trc promoter, tac promoter, lambda phage promoter (P_L,P_L-9G-50), anhydrotetracycline-inducible (tetA) promoter, tip, lpp, phoA, recA, proU, cst-1, cadA, nar, lpp-lac, cspA, T7-lac operator, T3-lac operator, T4 gene 32, T5-lac operator, nprM-lac operator, Vhb, Protein A, corynebacterial-E. coli like promoters, thr, horn, diphtheria toxin promoter, sig A, sig B, nusG, SoxS, katb, alpha-amylase (Pamy), Ptms, P43 (comprised of two overlapping RNA polymerase σfactor recognition sites, σA, σB), Ptms, P43, rplK-rplA, ferredoxin promoter, and/or xylose promoter. (See, K. Terpe Appl. Microbiol, Biotechnol. 72:211-222 (2006); Hannig et al. Trends in Biotechnology 16:54-60 (1998); and Srivastava Protein Expr Purif 40:221-229 (2005)).

In some embodiments of the invention, inducible promoters can be used. Thus, for example, chemical-regulated promoters can be used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences of the invention via promoters that are chemically regulated enables the RNAs and/or the polypeptides of the invention to be synthesized only when, for example, an organism is treated with the inducing chemicals. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. In some aspects, a promoter can also include a light-inducible promoter, where application of specific wavelengths of light induce gene expression (Levskaya et al. 2005. Nature 438:441-442).

Statements

By way of illustration, the invention provides the following Statements.

- 1. A method of producing a population of phage, wherein the phage are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the method comprising
  - (a) Providing a population of second cells comprising the receptor on the surface thereof, wherein the second cells are of a second species or strain, wherein the second species or strain is different from the first species or strain;
  - (b) Infecting the second cells with phage of said first type;
  - (c) Propagating the phage in the second cells, thereby producing the population of phage; and
  - (d) Optionally isolating phage of said population.

Preferably, the second cells are bacterial cells. Alternatively, the second cells are archaeal cells; eukaryotic cells, yeast cells, CHO cells or HEK293 cells.

In an embodiment, the receptor comprises a protein that is encoded by an expressible exogenous nucleotide sequence (ie a non wild-type sequence of the second bacteria), wherein the exogenous sequence is comprises by the genome of the second bacteria. For example, the nucleotide sequence is identical to or at least 85, 90, 95 or 98% identical to a nucleotide sequence comprised by host cells.

In another embodiment, the receptor comprises a sugar moiety that is produced by the action of one or more enzymes in the second bacteria, wherein the genome of the second bacteria comprise one or more expressible exogenous nucleotide sequences (ie a non wild-type sequence of the second bacteria) encoding one or more of the enzyme(s). For example, each nucleotide sequence is identical to or at least 85, 90, 95 or 98% identical to a nucleotide sequence comprised by host cells.

Optionally, the second species or strain do not naturally express the receptor. The host and/or second cells may be engineered cells. The host and/or second cells may be non-naturally-occurring bacterial cells. The host and/or second cells may be non-wild-type cells.

Optionally the host cells comprise an expressible exogenous nucleotide sequence (eg, chromosomally integrated) encoding the receptor.

In an alternative, instead of infecting the second cells with the phage in step (b), phage-encoding DNA is introduced by other means into the second cells, eg, by electroporation. In an example, step (c) comprises culturing the second cells, eg, in a culture vessel, such as a steel fermentation tank.

The second cells comprise cellular machinery operable to replicate DNA encoding the phage.

In an example, the host cells are pathogenic to humans (eg, the host cells are C difficile cells) and/or the second cells are non-pathogenic to humans or are cells of a gut commensal species (eg, the second cells are Lactobacillus cells, such as L lactis or reuteri cells). For example, the second cells are carrier cells, eg, as described in US20160333348 (this specific dislosure being incorporated herein by reference). In an example, the invention provides a method of treating or preventing a host cell infection in a human or animal subject (eg, an infecton of the gut of the subject), the method comprising administering a population of said second cells to the subject (eg, to populate the gut of the subject) wherein the cells are carrier cells comprising said phage (eg, prophage) of the first type, wherein the phage encode cRNAs or gRNAs that target a protospacer sequence in host cells comprised by the subject (eg, host cells comprised by the gut of the subject), wherein the second cells are carriers for phage that infect host cells in the subject, wherein phage nucleic acid encoding said crRNAs or gRNAs are produced in host cells thereby forming an active CRISPR/Cas system in the host cells, whereby Cas is guided by the crRNAs or gRNAs to a protospacer sequence comprised by the host cells genome to modify (eg, cut) host cell DNA thereby killing host cells or inhibiting host cell growth or proliferation, whereby the infection is treated or preveneted. In an embodiment, such a method is for treating or preventing a disease or condition of the subject, wherein the disease or condition is associated or caused by the host cell infection, whereby the disease or condition is treated or prevented. The host cells and/or the second cells can be any such cells disclosed herein.

- 2. The method of Statement 1, wherein the phage comprise a nucleotide sequence encoding crRNAs (or single guide RNAs) that are operable with Cas (and tracrRNA where necessary) in bacteria of said host cell strain or species to form an active CRISPR/Cas system that is capable of targeting one or more protospacer nucleotide sequences, wherein each target sequence is comprised by the genome of said host cells, whereby the crRNAs (or gRNAs) guide Cas in host cells to modify (eg, cut) the target sequence(s), thereby killing host cells or reducing host cell population growth.

In an example, the phage comprise a HM-array or gRNA-encoding nucleotide sequence as disclosed in US20160333348, the specific disclosure of which is incorporated herein by reference.

- 3. The method of Statement 2, wherein when infected by the phage, the second cells do not comprise said active CRISPR/Cas system.

For example, one or more Cas is repressed, inactivated or knocked-out in the second cells, wherein the second cells comprise a defective CRISPR/Cas system that is not operable with the crRNAs or gRNAs.

In an example, the active CRISPR/Cas system is as disclosed in US20160333348, the specific disclosure of which is incorporated herein by reference.

- 4. The method of Statement 2 or 3, wherein the genome of each second bacterial cell does not comprise a said target sequence.

In an example, the target sequence is as disclosed in US20160333348, the specific disclosure of which is incorporated herein by reference.

- 5. The method of any one of Statements 2 to 4, wherein
  - (a) Cas (eg, Cas3, 9, cpfl and/or CASCADE Cas) of said second cells is not operable with said crRNAs (or gRNAs);
  - (b) tracrRNA of said second cells is not operable with said crRNAs; and/or
  - (c) said second cells are not operable to produce said crRNAs from said crRNA-encoding nucleotide sequence (or are not operable to produce said gRNAs from said gRNA-encoding nucleotide sequence).
- 6. The method of any one of Statements 2 to 5, wherein the crRNAs (or gRNAs) comprise repeat sequences that are not operable with Cas of the second cells (eg, Cas3, 9, cpfl and/or CASCADE Cas of the second cells).

In an example, the repeat(s) is (are) as disclosed in US20160333348, the specific disclosure of which is incorporated herein by reference.

- 7. The method of any one of Statements 2 to 6, wherein said phage nucleotide sequence is operably connected with a promoter for transcription of crRNAs (or gRNAs) in bacteria of said host species or strain, but not in said second species or strain.

In an example, the promoter is constitutively active in the second cells.

- 8. The method of any preceding Statement, wherein bacteria of said host species or strain comprise an anti-phage toxin or mechanism for killing or reducing the propagation of phage of said first type that infect host bacteria, wherein the second bacteria do not comprise said toxin or mechanism.
- 9. The method of any preceding Statement, wherein bacteria of said host species or strain comprise a CRISPR/Cas system that is active for killing or reducing the propagation of phage of said first type that infect host bacteria, wherein the second bacteria do not comprise said system.
- 10. The method of any preceding Statement, wherein the second bacterial cells are engineered to produce the receptor, wherein wild-type bacteria of said second species or strain do not produce said receptor.
- 11. The method of any preceding Statement, wherein the phage comprise an origin of replication that is operable in said second cells and in cells of said first species or strain.
- 12. The method of any preceding Statement, wherein the second cells are E coli cells. For example, the second cells are not pathogenic to humans. For example, the second cells are Hazard Group 1 or 2 cells (eg, such of a species noted as Group 2 in the table herein).

13. The method of any preceding Statement, wherein the first and second cells are of the same species (eg, E coli strains).

For example, the second cells are engineered versions of the host cells, eg, wherein the second cells comprise a defective CRISPR/Cas system as mentioned herein and/or do not comprise a said protospacer sequence and/or do not express a toxin that is expressed by host cells.

- 14. The method of Statement 13, wherein the strain of host cells is a human pathogenic strain (eg, C difficile) and the second cell strain is not human pathogenic strain (eg, a Lactobacillus, such as L reuteri or lactis).
- 15. The method of any preceding Statement, wherein the second cells are cells of a lower hazard category (eg, Hazard Group 1 or 2) compared to cells of the host species or strain (eg, Hazard Group 3 or 4). See Tables 5 and 6.
- 16. The method of any preceding Statement, wherein the receptor is selected from lipopolysaccharides, teichoic acids (eg, a ManNAc(β→4)GlcNAc disaccharide with one to three glycerol phosphates attached to the C4 hydroxyl of the ManNAc residue followed by a long chain of glycerol- or ribitol phosphate repeats), proteins and flagella.
- 17. The method of any preceding Statement, wherein the receptor comprises an O-antigen of the host cells.
- 18. The method of any preceding Statement, wherein the phage are operable to express an endolysin or holin in second cells, eg, when phage replicate in second cells.
- 19. A cell (propagator cell) for propagating phage, wherein the phage are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the propagator cell comprising the receptor on the surface thereof, wherein the propagator cell is of a second species or strain, wherein the second species or strain is different from the first species or strain, whereby the propagator cell is capable of being infected by phage of said first type for propagation of phage therein.

In an example, the genome of the propagator cell (second cell in the method of the invention) comprises an exogenous nucleotide sequence that encodes the receptor, wherein wild-type cells of the species or strain of the cell do not comprise said nucleotide sequence.

- 20. The propagator cell of Statement 19, wherein the receptor comprises a protein that is encoded by an expressible nucleotide sequence comprised by the genome of the propagator cell, wherein wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequence.
- 21. The propagator cell of Statement 19, wherein the receptor comprises a sugar moiety that is the product of the action of one or more enzymes in the propagator cell, wherein the genome of the propagator cell comprises one or expressible nucleotide sequences encoding said one or more enzymes, wherein wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequence(s).
- 22. The propagator cell of Statement 19, wherein the receptor comprises a teichoic acid moiety that is the product of the action of one or more enzymes in the propagator cell, wherein the genome of the propagator cell comprises one or expressible nucleotide sequences encoding said one or more enzymes, wherein wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequence(s).
- 23. The propagator cell of Statement 22, wherein the enzyme(s) are selected from TarO, TarA, TarB, TarF, TarK, and TarL (or a homologue thereof expressed by cells of the host and/or second cells).
- 24. The propagator cell of any one of Statements 19 to 23 in combination with phage of said first type.
- 25. The propagator cell of any one of Statements 19 to 24, wherein the cell comprises one or more prophage of said first type (eg, chromosomally integrated in the propagator cell).
- 26. The propagator cell of any one of Statements 19 to 25, wherein the propagator cell is a gram-negative bacterial cell and optionally the host cells are gram-negative bacterial cells.
- 27. The propagator cell of any one of Statements 19 to 25, wherein the propagator cell is a gram-positive bacterial cell and optionally the host cells are gram-positive bacterial cells.
- 28. A population of propagator cells according to any one of Statements 19 to 27, optionally comprised in a fermentation vessel for culturing the propagator cells and propagating phage of said first type.
- 29. The propagator cell or population of any one of Statements 19 to 28, wherein each propagator cell is a second cell as defined in any one of Statements 1 to 18.
- 30. The propagator cell or population of any one of Statements 19 to 28, wherein each host cells is a host cell as defined in any one of Statements 1 to 18.
- 31. The propagator cell or population of any one of Statements 19 to 28, wherein the phage are phage as defined in any one of Statements 1 to 18.
- 32. A method of treating or preventing a disease or condition in a human or animal subject, the disease or condition being mediated by host cells comprised by the subject (eg, comprised by the gut of the subject), the method comprising administering propagator cells to the subject (eg, to populate the gut of the subject), wherein the propagator cells are according to any one of Statements 19 to 31, wherein the propagator cells produce phage and phage infect host cells in the patient (eg, in the gut thereof), thereby killing host cells or inhibiting growth or proliferation of host cells in the subject, whereby the disease or condition is treated or prevented.
- 33. The method of Statement 32, wherein the propagator cells are Lactobacillus (eg, L reuteri) cells.
- 34. The method of Statement 32 or 33, wherein the phage encode anti-host cell crRNAs or gRNAs that guide Cas in the host cells to modify (eg, cut) host cell DNA, thereby carrying out said killing or inhibiting.

Concepts

The invention also provides the following Concepts:

- 1. A method of producing a population of phage, wherein the phage are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the method comprising
  - (a) Providing a population of second bacterial cells comprising the receptor on the surface thereof, wherein the second cells are of a second species or strain, wherein the second species or strain is different from the first species or strain;
  - (b) Infecting the second cells with phage of said first type;
  - (c) Propagating the phage in the second cells, thereby producing the population of phage; and
  - (d) Optionally isolating phage of said population.
- 2. A method of producing a population of transduction particles comprising nucleic acid packaged by phage coat proteins, wherein the particles are capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, whereby host cells are transduced with the nucleic acid, the method comprising
  - (a) Providing a population of second bacterial cells comprising the receptor on the surface thereof, wherein the second cells are of a second species or strain, wherein the second species or strain is different from the first species or strain, and wherein the second cells comprise DNA that is capable of producing copies of said nucleic acid;
  - (b) Infecting the second cells with phage by binding the phage to the receptor comprised by the second bacterial cells;
  - (c) Propagating the phage in the second cells, wherein phage coat proteins are produced that package copies of said nucleic acid, thereby producing the population of particles; and
  - (d) Optionally isolating particles of said population.

In an example, the nucleic acid comprised by the particles is DNA. In an example, the nucleic acid is RNA. In an example, the phage used to infect the second cells in step (b) are helper phage, optionally that are different from the transduction particles (when the transduction particles are phage). Optionally, the helper phage are defective for self-replication in the second cells.

For example, the DNA comprised by the second cells is comprised by chromosomal DNA of each second cell. In another example, the DNA is comprised by one or more episomes (eg, plasmids) comprised by each second cell.

“Transduction particles” may be phage or smaller than phage and are particles that are capable of transducing nucleic acid (eg, encoding an antibiotic or component thereof, such as a CRISPR array) into host bacterial cells.

The particles comprise phage coat proteins and optionally other phage structural proteins encoded by the phage used in step (b). Examples of structural proteins are phage proteins selected from one, more or all of the major head and tail proteins, the portal protein, tail fibre proteins, and minor tail proteins.

The particles comprise nucleic acid (eg, DNA, such as DNA encoding the array or antibiotic), wherein the nucleic acid comprises a packaging signal sequence operable with proteins encoded by the phage of step (b) to package the nucleic acid or copies thereof into transduction particles that are capable of infecting host cells.

In an example, each transduction particle is a non-self replicative transduction particle. A “non-self replicative transduction particle” refers to a particle, (eg, a phage or phage-like particle; or a particle produced from a genomic island (eg, a S aureus pathogenicity island (SaPI)) or a modified version thereof) capable of delivering a nucleic acid molecule of the particle (eg, encoding an antibacterial agent or component) into a bacterial cell, but does not package its own replicated genome into the transduction particle.

Optionally, the nucleic acid of each particle comprises a modified genomic island. Optionally, the genomic island is an island that is naturally found in bacterial cells of the host species or strain. In an example, the genomic island is selected from the group consisting of a SaPI, a SaPI1, a SaPI2, a SaPIbov1 and a SaPibov2 genomic island. Optionally, the nucleic acid of each particle comprises a modified pathogenicity island. Optionally, the pathogenicity island is an island that is naturally found in bacterial cells of the first species or strain, eg, a Staphylococcus SaPI or a Vibro PLE or a P. aeruginosa pathogenicity island (eg, a PAPI or a PAGI, eg, PAPI-1, PAGI-5, PAGI-6, PAGI-7, PAGI-8, PAGI-9 or PAGI-10). Optionally, the pathogenicity island is a SaPI (S aureus pathogenicity island).

Optionally, the transcription of transduction particle nucleic acid is under the control of an inducible promoter, for transcription of copies of the antibacterial agent or component or array in a host cell. This may be useful, for example, to control switching on of the antibacterial activity or production of anti-host cell crRNAs for use against target bacterial cells, such as in an environment (eg, soil or water) or in an industrial culture or fermentation container containing the target cells. For example, the host cells may be useful in an industrial process (eg, for fermentation, eg, in the brewing or dairy industry) and the induction enables the process to be controlled (eg, stopped or reduced) by using the antibacterial agent or crRNAs against the host bacteria.

- 3. The method of Concept 2, wherein the particles are non-replicative transduction particles or phage.
- 4. The method of any preceding Concept, wherein the phage or particles comprise a nucleotide sequence encoding crRNAs (or single guide RNAs) that are operable with Cas in bacteria of said host cell strain or species to form an active CRISPR/Cas system that is capable of targeting one or more protospacer nucleotide sequences, wherein each target sequence is comprised by the genome of said host cells, whereby the crRNAs (or gRNAs) guide Cas in host cells to modify (optionally cut) the target sequence(s), thereby killing host cells or reducing host cell population growth.
- 5. The method of Concept 4, wherein when infected by the phage, the second cells do not comprise said active CRISPR/Cas system.
- 6. The method of Concept 4 or 5, wherein the genome of each second bacterial cell does not comprise a said target sequence.
- 7. The method of any one of Concepts 4 to 6, wherein
  - (a) Cas (optionally Cas3, 9, cpfl and/or CASCADE Cas) of said second cells is not operable with said crRNAs (or gRNAs);
  - (b) tracrRNA of said second cells is not operable with said crRNAs; and/or
  - (c) said second cells are not operable to produce said crRNAs from said crRNA-encoding nucleotide sequence (or are not operable to produce said gRNAs from said gRNA-encoding nucleotide sequence).
- 8. The method of any one of Concepts 4 to 7, wherein the crRNAs (or gRNAs) comprise repeat sequences that are not operable with Cas of the second cells (optionally Cas3, 9, cpfl and/or CASCADE Cas of the second cells).
- 9. The method of any one of Concepts 4 to 8, wherein said nucleotide sequence is operably connected with a promoter for transcription of crRNAs (or gRNAs) in bacteria of said host species or strain, but not in said second species or strain.
- 10. The method of any preceding Concept, wherein
  - (a) the phage or particles comprise a nucleotide sequence encoding crRNAs (or single guide RNAs) that are operable with Cas in bacteria of said host cell strain or species to form an active CRISPR/Cas system that is capable of targeting one or more protospacer nucleotide sequences, wherein each target sequence is comprised by the genome of said host cells, whereby the crRNAs (or gRNAs) guide Cas in host cells to modify (optionally cut) the target sequence(s), thereby killing host cells or reducing host cell population growth;
  - (b) the host and second cells are of the same species (optionally E coli strains); and
  - (c) the genome of each second bacterial cell does not comprise a said target sequence, wherein the first and second cells are different strains of the same species.
- 11. The method of any preceding Concept, wherein bacteria of said host species or strain comprise an anti-phage toxin or mechanism for killing or reducing the propagation of phage of said first type or particles that infect host bacteria, wherein the second bacteria do not comprise said toxin or mechanism.
- 12. The method of any preceding Concept, wherein bacteria of said host species or strain comprise a CRISPR/Cas system that is active for killing or reducing the propagation of phage of said first type or particles that infect host bacteria, wherein the second bacteria do not comprise said system.
- 13. The method of any preceding Concept, wherein the second bacterial cells are engineered to produce the receptor, wherein wild-type bacteria of said second species or strain do not produce said receptor.
- 14. The method of any preceding Concept, wherein the phage or particles comprise an origin of replication that is operable in said second cells and in cells of said first species or strain.
- 15. The method of any preceding Concept, wherein the second cells are E coli cells.
- 16. The method of any preceding Concept, wherein the first and second cells are of the same species (optionally E coli strains).
- 17. The method of Concept 16, wherein the strain of host cells is a human pathogenic strain and the second cell strain is not human pathogenic strain.
- 18. The method of any preceding Concept, wherein the second cells are cells of a lower hazard category (optionally Hazard Group 1 or 2) compared to cells of the host species or strain (optionally Hazard Group 3 or 4).
- 19. The method of any preceding Concept, wherein the receptor is selected from lipopolysaccharides, teichoic acids (optionally a ManNAc(β1 →4)GlcNAc disaccharide with one to three glycerol phosphates attached to the C4 hydroxyl of the ManNAc residue followed by a long chain of glycerol- or ribitol phosphate repeats), proteins and flagella.
- 20. The method of any preceding Concept, wherein the receptor comprises an O-antigen of the host cells.
- 21. The method of any preceding Concept, wherein the phage or particles are operable to express an endolysin or holin in second cells, optionally when phage or particles replicate in second cells.
- 22. A cell (propagator cell) for propagating phage or transduction particles comprising nucleic acid packaged by phage coat proteins, wherein the phage or particles are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the propagator cell comprising the receptor on the surface thereof, wherein the propagator cell is of a second species or strain, wherein the second species or strain is different from the first species or strain, whereby the propagator cell is capable of being infected by phage of said first type or said particles for propagation of phage or particles respectively therein.
- 23. The propagator cell of Concept 22, wherein the receptor comprises a protein that is encoded by an expressible nucleotide sequence comprised by the genome of the propagator cell, wherein wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequence.
- 24. The propagator cell of Concept 22, wherein the receptor comprises a sugar moiety that is the product of the action of one or more enzymes in the propagator cell, wherein the genome of the propagator cell comprises one or expressible nucleotide sequences encoding said one or more enzymes, wherein wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequence(s).
- 25. The propagator cell of Concept 22, wherein the receptor comprises a teichoic acid moiety that is the product of the action of one or more enzymes in the propagator cell, wherein the genome of the propagator cell comprises one or expressible nucleotide sequences encoding said one or more enzymes, wherein wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequence(s).
- 26. The propagator cell of Concept 25, wherein the enzyme(s) are selected from TarO, TarA, TarB, TarF, TarK, and TarL (or a homologue thereof expressed by cells of the host and/or second cells).
- 27. The propagator cell of any one of Concepts 22 to 26 in combination with phage of said first type or a said transduction particle.
- 28. The propagator cell of any one of Concepts 22 to 27, wherein the cell comprises one or more prophage of said first type (optionally chromosomally integrated in the propagator cell) or DNA that is capable of producing copies of said nucleic acid of the transducing particles (optionally chromosomally integrated in the propagator cell).
- 29. The propagator cell of any one of Concepts 22 to 28, wherein the propagator cell is a gram-negative bacterial cell and optionally the host cells are gram-negative bacterial cells.
- 30. The propagator cell of any one of Concepts 22 to 28, wherein the propagator cell is a gram-positive bacterial cell and optionally the host cells are gram-positive bacterial cells.
- 31. A population of propagator cells according to any one of Concepts 22 to 30, optionally comprised in a fermentation vessel for culturing the propagator cells and propagating phage of said first type or said transduction particles.
- 32. The propagator cell or population of any one of Concepts 22 to 31, wherein each propagator cell is a second cell as defined in any one of Concepts 1 to 21.
- 33. The propagator cell or population of any one of Concepts 22 to 31, wherein each host cells is a host cell as defined in any one of Concepts 1 to 21.
- 34. The propagator cell or population of any one of Concepts 22 to 31, wherein the phage or particles are phage or particles as defined in any one of Concepts 1 to 21.
- 35. A method of treating or preventing a disease or condition in a human or animal subject, the disease or condition being mediated by host cells comprised by the subject (optionally comprised by the gut of the subject), the method comprising administering propagator cells to the subject (optionally to populate the gut of the subject), wherein the propagator cells are according to any one of Concepts 22 to 34, wherein the propagator cells produce phage or transduction partiles and phage or particles respectively infect host cells in the patient (optionally in the gut thereof), thereby killing host cells or inhibiting growth or proliferation of host cells in the subject, whereby the disease or condition is treated or prevented.
- 36. The method of Concept 35, wherein the propagator cells are Lactobacillus (optionally L reuteri) cells.
- 37. The method of Concept 35 or 36, wherein the phage encode anti-host cell crRNAs or gRNAs that guide Cas in the host cells to modify (optionally cut) host cell DNA, thereby carrying out said killing or inhibiting.

EXAMPLE 1
Engineering of Production Strain to Become Susceptible to Helper Phage
Summary:

We engineered a production strain of bacteria (in this case an E coli production strain) to express a phage receptor rendering the strain susceptible to infection by a helper phage. The production bacteria harboured a vector containing a CRISPR array and a phage packaging site so that the vector could be packaged in cells that had been infected by the helper (but not in cells that are not so infected), thereby enabling use of the bacteria as a production strain for phage-like particles encoding crRNAs. We further showed that a lysate produced by such production strain contains phage-like particles that could be used to deliver the CRISPR array to other related E coli target populations. Here we call the vectors CRISPR Guided Vectors (CGVs™)

Advantageously, to produce CGV-charged phage-like particles (CGV-PLP) targeting a specific bacterial population, it may be beneficial to produce the CGV-PLPs in a strain related to the target strain, for example to produce CGV-PLPs that avoid host defence mechanisms in the target strain. For example, modification of the DNA of CGV-PLPs by methyltransferases in the production bacteria can be useful to shield the DNA against restriction modification once the PLP subsequently infects the target cells where the species or strains of the production and target bacteria are the same or closely related (or at any rate comprise common methyltranferases). By adapting the production strain as per the invention to display a surface receptor, the invention enables PLP production in a strain that may display beneficial DNA modification against restriction modification subsequently by the target bacteria. Usefully, the protospacer sequence(s) to which crRNAs of the PLP are targeted in the target bacteria may be deleted or naturally absent in the genome of the production bacteria, such that Cas-mediated cutting of the production bacteria genomes does not take place during the production of the PLPs.

Methods and Results:

As a production strain, we used the Escherichia coli strain MG1655 that was transformed with a plasmid expressing the receptor for helper phage M13KO7 (FIG. 1_X) while a strain line not receiving the receptor (FIG. 1_Y) served as control. The receptor was a F-pilus expressed from the plasmid pCJ105 obtained from New England Biolabs. Both strains were transformed with a CGV (FIG. 1_3) and infected with helper phage M13KO7 for the production of CGV-PLP.

In line X, CGV-PLP lysate was produced due to presence of receptor while in line Y no lysate was produced (FIG. 1_4). The resulting lysate was shown to be able to deliver the CGV to different target populations related to the production strain and harbouring the phage receptor (FIG. 1_5 and FIG. 2). The control strain line did not produce CGV-PLP that was able to deliver the CGV to the target population (FIG. 2).

TABLE 5

HAZARD GROUPS

Group 1
Unlikely to cause human disease.

Group 2
Can cause human disease and may be

a hazard to employees; it is unlikely to

spread to the community and there is

usually effective prophylaxis or

treatment available.

Group 3
Can cause severe human disease and

may be a serious hazard to employees;

it may spread to the community, but

there is usually effective prophylaxis

or treatment available.

Group 4
Causes severe human disease and is a

serious hazard to employees; it is

likely to spread to the community and

there is usually no effective

prophylaxis or treatment available.

TABLE 6

THE APPROVED LIST OF BIOLOGICAL AGENTS (HSE CLASSIFICATION)

BACTERIA
HAZARD GROUP

Arcobacter butzleri (formerly
2

Campylobacter butzleri)

Actinobacillus

2

actinomycetemcomitans

Actinomadura madurae

2

Actinomadura pelletieri

2

Actinomyces gerencseriae

2

Actinomyces israelii

2

Actinomyces pyogenes

2

Actinomyces spp
2

Alcaligenes spp
2

Arcanobacterium haemolyticum

2

(Corynebacterium haemolyticum)

Arcanobacterium pyogenes (formerly
2

Actinomyces pyogenes)

Bacillus anthracis

3

Bacillus cereus

2

Bacteroides fragilis

2

Bacteroides spp
2

Bartonella bacilliformis

2

Bartonella quintana (Rochalimaea
2

quintana)

Bartonella spp (Rochalimaea spp)
2

Bordetella bronchiseptica

2

Bordetella parapertussis

2

Bordetella pertussis

2

Bordetella spp
2

Borrelia burgdorferi

2

Borrelia duttonii

2

Borrelia recurrentis

2

Borrelia spp
2

Brachispira spp (formerly Serpulina
2

spp)

Brucella abortus

3

Brucella canis

3

Brucella melitensis

3

Brucella suis

3

Burkholderia cepacia

2

Burkholderia mallei (formerly
3

Pseudomonas mallei)

Burkholderia pseudomallei (formerly
3

Pseudomonas pseudomallei)

Campylobacter fetus

2

Campylobacter jejuni

2

Campylobacter spp
2

Cardiobacterium hominis

2

Chlamydophila pneumoniae

2

Chlamydophila psittaci (avian strains)
3

Chlamydophila psittaci (non-avian
2

strains)

Chlamydophila trachomatis

2

Clostridium botulinum

2

Clostridium perfringens

2

Clostridium spp
2

Clostridium tetani

2

Corynebacterium diphtheriae

2

Corynebacterium haemolyticum

2

Corynebacterium minutissimum

2

Corynebacterium pseudotuberculosis

2

Corynebacterium pyogenes

2

Corynebacterium spp
2

Corynebacterium ulcerans

2

Coxiella burnetti

3

Edwardsiella tarda

2

Ehrlichia sennetsu (Rickettsia
3

sennetsu)

Ehrlichia spp
2

Eikenella corrodens

2

Elizabethkingia meningoseptica

2

(formerly Flavobacterium

meningosepticum)

Enterobacter aerogenes/cloacae
2

Enterobacter spp
2

Enterococcus spp
2

Erysipelothrix rhusiopathiae

2

Escherichia coli (with the exception of
2

non-pathogenic strains)

Escherichia coli, verocytotoxigenic
3

strains (eg O157:H7 or O103)

Flavobacterium meningosepticum

2

Fluoribacter bozemanae (formerly
2

Legionella)

Francisella tularensis (Type A)
3

Francisella tularensis (Type B)
2

Fusobacterium necrophorum

2

Fusobacterium spp
2

Gardnerella vaginalis

2

Haemophilus ducreyi

2

Haemophilus influenzae

2

Haemophilus spp
2

Helicobacter pylori

2

Klebsiella oxytoca

2

Klebsiella pneumoniae

2

Klebsiella spp
2

Legionella pneumophila

2

Legionella spp
2

Leptospira interrogans (all serovars)
2

Listeria ivanovii

2

Listeria monocytogenes

2

Moraxella catarrhalis

2

Morganella morganii

2

Mycobacterium africanum

3

Mycobacterium avium/intracellulare
2

Mycobacterium bovis

3

Mycobacterium bovis (BCG strain)
2

Mycobacterium chelonae

2

Mycobacterium fortuitum

2

Mycobacterium kansasii

2

Mycobacterium leprae

3

Mycobacterium malmoense

3

Mycobacterium marinum

2

Mycobacterium microti

3

Mycobacterium paratuberculosis

2

Mycobacterium scrofulaceum

2

Mycobacterium simiae

2

Mycobacterium szulgai

3

Mycobacterium tuberculosis

3

Mycobacterium ulcerans

3

Mycobacterium xenopi

2

Mycoplasma caviae

2

Mycoplasma hominis

2

Mycoplasma pneumoniae

2

Neisseria gonorrhoeae

2

Neisseria meningitidis

2

Nocardia asteroids

2

Nocardia braziliensis

2

Nocardia farcinica

2

Nocardia nova

2

Nocardia otitidiscaviarum

2

Pasteurella multocida

2

Pasteurella spp
2

Peptostreptococcus anaerobius

2

Peptostreptococcus spp
2

Plesiomonas shigelloides

2

Porphyromonas spp
2

Prevotella spp
2

Proteus mirabilis

2

Proteus penneri

2

Proteus vulgaris

2

Providencia alcalifaciens

2

Providencia rettgeri

2

Providencia spp
2

Pseudomonas aeruginosa

2

Pseudomonas mallei

3

Pseudomonas pseudomallei

3

Rhodococcus equi

2

Rickettsia akari

3

Rickettsia canada

3

Rickettsia conorii

3

Rickettsia montana

3

Rickettsia mooseri

3

Rickettsia prowazekii

3

Rickettsia rickettsii

3

Rickettsia sennetsu

3

Rickettsia spp
3

Rickettsia tsutsugamushi

3

Rickettsia typhi (Rickettsia mooseri)
3

Rochalimaea quintana

2

Rochalimaea spp
2

Salmonella arizonae

2

Salmonella enterica serovar
2

enteritidis

Salmonella enterica serovar
2

typhimurium 2

Salmonella paratyphi A
3

Salmonella paratyphi B/java
3

Salmonella paratyphi C/Choleraesuis
3
Serovars other than arizonae,

enterica serovar enteritidis, enterica

serovar typhimurium 2, paratyphi A,

Salmonella spp
2
B, C, typhi

Serovars arizonae, enterica serovar

enteritidis, enterica serovar

typhimurium 2, paratyphi A, B, C,

Salmonella typhi

3

typhi

Serpulina spp
2

Shigella boydii

2

Shigella dysenteriae (other than Type 1)
2

Shigella dysenteriae (Type 1)
3

Shigella flexneri

2

Shigella sonnei

2

Staphylococcus aureus

2

Streptobacillus moniliformis

2

Streptococcus agalactiae

2

Streptococcus dysgalactiaeequisimilis

2

Streptococcus pneumoniae

2

Streptococcus pyogenes

2

Streptococcus spp
2

Streptococcus suis

2

Treponema carateum

2

Treponema pallidum

2

Treponema pertenue

2

Treponema spp
2

Ureaplasma parvum

2

Ureaplasma urealyticum

2

Vibrio cholerae (including El Tor)
2

Vibrio parahaemolyticus

2

Vibrio spp
2

Yersinia enterocolitica

2

Yersinia pestis

3

Yersinia pseudotuberculosis

2

Yersinia spp
2

TABLE 7

Example Bacteria

Optionally, the host cells are selected from this Table and/or the second cells are selected from this Table (wherein the host and second cells are of a

different species; or of the same species but are a different strain or the host cells are engineered but the second cells are wild-type or vice versa).

Abiotrophia

Acidocella

Actinomyces

Alkalilimnicola

Aquaspirillum

Abiotrophia defectiva

Acidocella aminolytica

Actinomyces bovis

Alkalilimnicola ehrlichii

Aquaspirillum polymorphum

Acaricomes

Acidocella facilis

Actinomyces denticolens

Alkaliphilus

Aquaspirillum

Acaricomes phytoseiuli

Acidomonas

Actinomyces europaeus

Alkaliphilus oremlandii

putridiconchylium

Acetitomaculum

Acidomonas methanolica

Actinomyces georgiae

Alkaliphilus transvaalensis

Aquaspirillum serpens

Acetitomaculum ruminis

Acidothermus

Actinomyces gerencseriae

Allochromatium

Aquimarina

Acetivibrio

Acidothermus cellulolyticus

Actinomyces

Allochromatium vinosum

Aquimarina latercula

Acetivibrio cellulolyticus

Acidovorax

hordeovulneris

Alloiococcus

Arcanobacterium

Acetivibrio ethanolgignens

Acidovorax anthurii

Actinomyces howellii

Alloiococcus otitis

Arcanobacterium

Acetivibrio multivorans

Acidovorax caeni

Actinomyces hyovaginalis

Allokutzneria

haemolyticum

Acetoanaerobium

Acidovorax cattleyae

Actinomyces israelii

Allokutzneria albata

Arcanobacterium pyogenes

Acetoanaerobium noterae

Acidovorax citrulli

Actinomyces johnsonii

Altererythrobacter

Archangium

Acetobacter

Acidovorax defluvii

Actinomyces meyeri

Altererythrobacter ishigakiensis

Archangium gephyra

Acetobacter aceti

Acidovorax delafieldii

Actinomyces naeslundii

Altermonas

Arcobacter

Acetobacter cerevisiae

Acidovorax facilis

Actinomyces neuii

Altermonas haloplanktis

Arcobacter butzleri

Acetobacter cibinongensis

Acidovorax konjaci

Actinomyces odontolyticus

Altermonas macleodii

Arcobacter cryaerophilus

Acetobacter estunensis

Acidovorax temperans

Actinomyces oris

Alysiella

Arcobacter halophilus

Acetobacter fabarum

Acidovorax valerianellae

Actinomyces radingae

Alysiella crassa

Arcobacter nitrofigilis

Acetobacter ghanensis

Acinetobacter

Actinomyces slackii

Alysiella filiformis

Arcobacter skirrowii

Acetobacter indonesiensis

Acinetobacter baumannii

Actinomyces turicensis

Aminobacter

Arhodomonas

Acetobacter lovaniensis

Acinetobacter baylyi

Actinomyces viscosus

Aminobacter aganoensis

Arhodomonas aquaeolei

Acetobacter malorum

Acinetobacter bouvetii

Actinoplanes

Aminobacter aminovorans

Arsenophonus

Acetobacter nitrogenifigens

Acinetobacter calcoaceticus

Actinoplanes auranticolor

Aminobacter niigataensis

Arsenophonus

Acetobacter oeni

Acinetobacter gerneri

Actinoplanes brasiliensis

Aminobacterium

nasoniae

Acetobacter orientalis

Acinetobacter haemolyticus

Actinoplanes consettensis

Aminobacterium mobile

Arthrobacter

Acetobacter orleanensis

Acinetobacter johnsonii

Actinoplanes deccanensis

Aminomonas

Arthrobacter agilis

Acetobacter pasteurianus

Acinetobacter junii

Actinoplanes derwentensis

Aminomonas paucivorans

Arthrobacter albus

Acetobacter pornorurn

Acinetobacter lwoffi

Actinoplanes digitatis

Ammoniphilus

Arthrobacter aurescens

Acetobacter senegalensis

Acinetobacter parvus

Actinoplanes durhamensis

Ammoniphilus oxalaticus

Arthrobacter chlorophenolicus

Acetobacter xylinus

Acinetobacter radioresistens

Actinoplanes ferrugineus

Ammoniphilus oxalivorans

Arthrobacter citreus

Acetobacterium

Acinetobacter schindleri

Actinoplanes globisporus

Amphibacillus

Arthrobacter crystallopoietes

Acetobacterium bakii

Acinetobacter soli

Actinoplanes humidus

Amphibacillus xylanus

Arthrobacter cumminsii

Acetobacterium carbinolicum

Acinetobacter tandoii

Actinoplanes italicus

Amphritea

Arthrobacter globiformis

Acetobacterium dehalogenans

Acinetobacter tjernbergiae

Actinoplanes liguriensis

Amphritea balenae

Arthrobacter

Acetobacterium fimetarium

Acinetobacter towneri

Actinoplanes lobatus

Amphritea japonica

histidinolovorans

Acetobacterium malicum

Acinetobacter ursingii

Actinoplanes missouriensis

Amycolatopsis

Arthrobacter ilicis

Acetobacterium paludosum

Acinetobacter venetianus

Actinoplanes palleronii

Amycolatopsis alba

Arthrobacter luteus

Acetobacterium tundrae

Acrocarpospora

Actinoplanes philippinensis

Amycolatopsis albidoflavus

Arthrobacter methylotrophus

Acetobacterium wieringae

Acrocarpospora corrugata

Actinoplanes rectilineatus

Amycolatopsis azurea

Arthrobacter mysorens

Acetobacterium woodii

Acrocarpospora

Actinoplanes regularis

Amycolatopsis coloradensis

Arthrobacter nicotianae

Acetofilamentum

macrocephala

Actinoplanes

Amycolatopsis lurida

Arthrobacter nicotinovorans

Acetofilamentum rigidum

Acrocarpospora pleiomorpha

teichomyceticus

Amycolatopsis mediterranei

Arthrobacter oxydans

Acetohalobium

Actibacter

Actinoplanes utahensis

Amycolatopsis rifamycinica

Arthrobacter pascens

Acetohalobium arabaticum

Actibacter sediminis

Actinopolyspora

Amycolatopsis rubida

Arthrobacter

Acetomicrobium

Actinoalloteichus

Actinopolyspora halophila

Amycolatopsis sulphurea

phenanthrenivorans

Acetomicrobium faecale

Actinoalloteichus

Actinopolyspora mortivallis

Amycolatopsis tolypomycina

Arthrobacter

Acetomicrobium flavidum

cyanogriseus

Actinosynnema

Anabaena

polychromogenes

Acetonema

Actinoalloteichus

Actinosynnema mirum

Anabaena cylindrica

Atrhrobacter protophormiae

Acetonema longum

hymeniacidonis

Actinotalea

Anabaena flos-aquae

Arthrobacter

Acetothermus

Actinoalloteichus spitiensis

Actinotalea fermentans

Anabaena variabilis

psychrolactophilus

Acetothermus paucivorans

Actinobaccillus

Aerococcus

Anaeroarcus

Arthrobacter ramosus

Acholeplasma

Actinobacillus capsulatus

Aerococcus sanguinicola

Anaeroarcus burkinensis

Arthrobacter sulfonivorans

Acholeplasma axanthum

Actinobacillus delphinicola

Aerococcus urinae

Anaerobaculum

Arthrobacter sulfureus

Acholeplasma brassicae

Actinobacillus hominis

Aerococcus urinaeequi

Anaerobaculum mobile

Arthrobacter uratoxydans

Acholeplasma cavigenitalium

Actinobacillus indolicus

Aerococcus urinaehominis

Anaerobiospirillum

Arthrobacter ureafaciens

Acholeplasma equifetale

Actinobacillus lignieresii

Aerococcus viridans

Anaerobiospirillum

Arthrobacter viscosus

Acholeplasma granularum

Actinobacillus minor

Aeromicrobium

succiniciproducens

Arthrobacter woluwensis

Acholeplasma hippikon

Actinobacillus muris

Aeromicrobium erythreum

Anaerobiospirillum thomasii

Asaia

Acholeplasma laidlawii

Actinobacillus

Aeromonas

Anaerococcus

Asaia bogorensis

Acholeplasma modicum

pleuropneumoniae

Aeromonas

Anaerococcus hydrogenalis

Asanoa

Acholeplasma morum

Actinobacillus porcinus

allosaccharophila

Anaerococcus lactolyticus

Asanoa ferruginea

Acholeplasma multilocale

Actinobacillus rossii

Aeromonas bestiarum

Anaerococcus prevotii

Asticcacaulis

Acholeplasma oculi

Actinobacillus scotiae

Aeromonas caviae

Anaerococcus tetradius

Asticcacaulis biprosthecium

Acholeplasma palmae

Actinobacillus seminis

Aeromonas encheleia

Anaerococcus vaginalis

Asticcacaulis excentricus

Acholeplasma parvum

Actinobacillus succinogenes

Aeromonas

Anaerofustis

Atopobacter

Acholeplasma pleciae

Actinobaccillus suis

enteropelogenes

Anaerofustis stercorihominis

Atopobacter phocae

Acholeplasma vituli

Actinobacillus ureae

Aeromonas eucrenophila

Anaeromusa

Atopobium

Achromobacter

Actinobaculum

Aeromonas ichthiosmia

Anaeromusa acidaminophila

Atopobium fossor

Achromobacter denitrificans

Actinobaculum massiliense

Aeromonas jandaei

Anaeromyxobacter

Atopobium minutum

Achromobacter insolitus

Actinobaculum schaalii

Aeromonas media

Anaeromyxobacter

Atopobium parvulum

Achromobacter piechaudii

Actinobaculum suis

Aeromonas popoffii

dehalogenans

Atopobium rimae

Achromobacter ruhlandii

Actinomyces urinale

Aeromonas sobria

Anaerorhabdus

Atopobium vaginae

Achromobacter spanius

Actinocatenispora

Aeromonas veronii

Anaerorhabdus furcosa

Aureobacterium

Acidaminobacter

Actinocatenispora rupis

Agrobacterium

Anaerosinus

Aureobacterium barkeri

Acidaminobacter

Actinocatenispora

Agrobacterium

Anaerosinus glycerini

Aurobacterium

hydrogenoformans

thailandica

gelatinovorum

Anaerovirgula

Aurobacterium liquefaciens

Acidaminococcus

Actinocatenispora sera

Agrococcus

Anaerovirgula multivorans

Avibacterium

Acidaminococcus fermentans

Actinocorallia

Agrococcus citreus

Ancalomicrobium

Avibacterium avium

Acidaminococcus intestini

Actinocorallia aurantiaca

Agrococcus jenensis

Ancalomicrobium adetum

Avibacterium gallinarum

Acidicaldus

Actinocorallia aurea

Agromonas

Ancylobacter

Avibacterium paragallinarum

Acidicaldus organivorans

Actinocorallia cavernae

Agromonas oligotrophica

Ancylobacter aquaticus

Avibacterium volantium

Acidimicrobium

Actinocorallia glomerata

Agromyces

Aneurinibacillus

Azoarcus

Acidimicrobium ferrooxidans

Actinocorallia herbida

Agromyces fucosus

Aneurinibacillus aneurinilyticus

Azoarcus indigens

Acidiphilium

Actinocorallia libanotica

Agromyces hippuratus

Aneurinibacillus migulanus

Azoarcus tolulyticus

Acidiphilium acidophilum

Actinocorallia longicatena

Agromyces luteolus

Aneurinibacillus

Azoarcus toluvorans

Acidiphilium angustum

Actinomadura

Agromyces mediolanus

thermoaerophilus

Azohydromonas

Acidiphilium cryptum

Actinomadura alba

Agromyces ramosus

Angiococcus

Azohydromonas australica

Acidiphilium multivorum

Actinomadura atramentaria

Agromyces rhizospherae

Angiococcus disciformis

Azohydromonas lata

Acidiphilium organovorum

Actinomadura

Akkermansia

Angulomicrobium

Azomonas

Acidiphilium rubrum

bangladeshensis

Akkermansia muciniphila

Angulomicrobium tetraedrale

Azomonas agilis

Acidisoma

Actinomadura catellatispora

Albidiferax

Anoxybacillus

Azomonas insignis

Acidisoma sibiricum

Actinomadura chibensis

Albidiferax ferrireducens

Anoxybacillus pushchinoensis

Azomonas macrocytogenes

Acidisoma tundrae

Actinomadura chokoriensis

Albidovulum

Aquabacterium

Azorhizobium

Acidisphaera

Actinomadura citrea

Albidovulum inexpectatum

Aquabacterium commune

Azorhizobium caulinodans

Acidisphaera rubrifaciens

Actinomadura coerulea

Alcaligenes

Aquabacterium parvum

Azorhizophilus

Acidithiobacillus

Actinomadura echinospora

Alcaligenes denitrificans

Azorhizophilus paspali

Acidithiobacillus albertensis

Actinomadura fibrosa

Alcaligenes faecalis

Azospirillum

Acidithiobacillus caldus

Actinomadura formosensis

Alcanivorax

Azospirillum brasilense

Acidithiobacillus ferrooxidans

Actinomadura hibisca

Alcanivorax borkumensis

Azospirillum halopraeferens

Acidithiobacillus thiooxidans

Actinomadura kijaniata

Alcanivorax jadensis

Azospirillum irakense

Acidobacterium

Actinomadura latina

Algicola

Azotobacter

Acidobacterium capsulatum

Actinomadura livida

Algicola bacteriolytica

Azotobacter beijerinckii

Actinomadura

Alicyclobacillus

Azotobacter chroococcum

luteofluorescens

Alicyclobacillus

Azotobacter nigricans

Actinomadura macra

disulfidooxidans

Azotobacter salinestris

Actinomadura madurae

Alicyclobacillus

Azotobacter vinelandii

Actinomadura oligospora

sendaiensis

Actinomadura pelletieri

Alicyclobacillus vulcanalis

Actinomadura rubrobrunea

Alishewanella

Actinomadura rugatobispora

Alishewanella fetalis

Actinomadura umbrina

Alkalibacillus

Actinomadura

Alkalibacillus

verrucosospora

haloalkaliphilus

Actinomadura vinacea

Actinomadura viridilutea

Actinomadura viridis

Actinomadura yumaensis

Bacillus

Bacteroides

Bibersteinia

Borrelia

Brevinema

[see below]

Bacteroides caccae

Bibersteinia trehalosi

Borrelia afzelii

Brevinema andersonii

Bacteriovorax

Bacteroides coagulans

Bifidobacterium

Borrelia americana

Brevundimonas

Bacteriovorax stolpii

Bacteroides eggerthii

Bifidobacterium adolescentis

Borrelia burgdorferi

Brevundimonas alba

Bacteroides fragilis

Bifidobacterium angulatum

Borrelia carolinensis

Brevundimonas aurantiaca

Bacteroides galacturonicus

Bifidobacterium animalis

Borrelia coriaceae

Brevundimonas diminuta

Bacteroides helcogenes

Bifidobacterium asteroides

Borrelia garinii

Brevundimonas intermedia

Bacteroides ovatus

Bifidobacterium bifidum

Borrelia japonica

Brevundimonas subvibrioides

Bacteroides pectinophilus

Bifidobacterium boum

Bosea

Brevundimonas vancanneytii

Bacteroides pyogenes

Bifidobacterium breve

Bosea minatitlanensis

Brevundimonas variabilis

Bacteroides salyersiae

Bifidobacterium catenulatum

Bosea thiooxidans

Brevundimonas vesicularis

Bacteroides stercoris

Bifidobacterium choerinum

Brachybacterium

Brochothrix

Bacteroides suis

Bifidobacterium coryneforme

Brachybacterium

Brochothrix campestris

Bacteroides tectus

Bifidobacterium cuniculi

alimentarium

Brochothrix thermosphacta

Bacteroides thetaiotaomicron

Bifidobacterium dentium

Brachybacterium faecium

Brucella

Bacteroides uniformis

Bifidobacterium gallicum

Brachybacterium

Brucella canis

Bacteroides ureolyticus

Bifidobacterium gallinarum

paraconglomeratum

Brucella neotomae

Bacteroides vulgatus

Bifidobacterium indicum

Brachybacterium rhamnosum

Bryobacter

Balnearium

Bifidobacterium longum

Brachybacterium

Bryobacter aggregatus

Balnearium lithotrophicum

Bifidobacterium

tyrofermentans

Burkholderia

Balneatrix

magnumBifidobacterium

Brachyspira

Burkholderia ambifaria

Balneatrix alpica

merycicum

Brachyspira alvinipulli

Burkholderia andropogonis

Balneola

Bifidobacterium minimum

Brachyspira hyodysenteriae

Burkholderia anthina

Balneola vulgaris

Bifidobacterium

Brachyspira innocens

Burkholderia caledonica

Barnesiella

pseudocatenulatum

Brachyspira murdochii

Burkholderia caryophylli

Barnesiella viscericola

Bifidobacterium

Brachyspira

Burkholderia cenocepacia

Bartonella

pseudolongum

pilosicoli

Burkholderia cepacia

Bartonella alsatica

Bifidobacterium pullorum

Bradyrhizobium

Burkholderia cocovenenans

Bartonella bacilliformis

Bifidobacterium ruminantium

Bradyrhizobium canariense

Burkholderia dolosa

Bartonella clarridgeiae

Bifidobacterium saeculare

Bradyrhizobium elkanii

Burkholderia fungorum

Bartonella doshiae

Bifidobacterium subtile

Bradyrhizobium japonicum

Burkholderia glathei

Bartonella elizabethae

Bifidobacterium

Bradyrhizobium liaoningense

Burkholderia glumae

Bartonella grahamii

thermophilum

Brenneria

Burkholderia graminis

Bartonella henselae

Bilophila

Brenneria alni

Burkholderia kururiensis

Bartonella rochalimae

Bilophila wadsworthia

Brenneria nigrifluens

Burkholderia multivorans

Bartonella vinsonii

Biostraticola

Brenneria quercina

Burkholderia phenazinium

Bavariicoccus

Biostraticola tofi

Brenneria quercina

Burkholderia plantarii

Bavariicoccus seileri

Bizionia

Brenneria salicis

Burkholderia pyrrocinia

Bdellovibrio

Bizionia argentinensis

Brevibacillus

Burkholderia silvatlantica

Bdellovibrio bacteriovorus

Blastobacter

Brevibacillus agri

Burkholderia stabilis

Bdellovibrio exovorus

Blastobacter capsulatus

Brevibacillus borstelensis

Burkholderia thailandensis

Beggiatoa

Blastobacter denitrificans

Brevibacillus brevis

Burkholderia tropica

Beggiatoa alba

Blastococcus

Brevibacillus centrosporus

Burkholderia unamae

Beijerinckia

Blastococcus aggregatus

Brevibacillus choshinensis

Burkholderia vietnamiensis

Beijerinckia derxii

Blastococcus saxobsidens

Brevibacillus invocatus

Buttiauxella

Beijerinckia fluminensis

Blastochloris

Brevibacillus laterosporus

Buttiauxella agrestis

Beijerinckia indica

Blastochloris viridis

Brevibacillus parabrevis

Buttiauxella brennerae

Beijerinckia mobilis

Blastomonas

Brevibacillus reuszeri

Buttiauxella ferragutiae

Belliella

Blastomonas natatoria

Brevibacterium

Buttiauxella gaviniae

Belliella baltica

Blastopirellula

Brevibacterium abidum

Buttiauxella izardii

Bellilinea

Blastopirellula marina

Brevibacterium album

Buttiauxella noackiae

Bellilinea caldifistulae

Blautia

Brevibacterium aurantiacum

Buttiauxella warmboldiae

Belnapia

Blautia coccoides

Brevibacterium celere

Butyrivibrio

Belnapia moabensis

Blautia hansenii

Brevibacterium epidermidis

Butyrivibrio fibrisolvens

Bergeriella

Blautia producta

Brevibacterium

Butyrivibrio hungatei

Bergeriella denitrificans

Blautia wexlerae

frigoritolerans

Butyrivibrio proteoclasticus

Beutenbergia

Bogoriella

Brevibacterium halotolerans

Beutenbergia cavernae

Bogoriella caseilytica

Brevibacterium iodinum

Bordetella

Brevibacterium linens

Bordetella avium

Brevibacterium lyticum

Bordetella bronchiseptica

Brevibacterium mcbrellneri

Bordetella hinzii

Brevibacterium otitidis

Bordetella holmesii

Brevibacterium oxydans

Bordetella parapertussis

Brevibacterium paucivorans

Bordetella pertussis

Brevibacterium stationis

Bordetella petrii

Bordetella trematum

Bacillus

B. acidiceler

B. aminovorans

B. glucanolyticus

B. taeanensis

B. lautus

B. acidicola

B. amylolyticus

B. gordonae

B. tequilensis

B. lehensis

B. acidiproducens

B. andreesenii

B. gottheilii

B. thermantarcticus

B. lentimorbus

B. acidocaldarius

B. aneurinilyticus

B. graminis

B. thermoaerophilus

B. lentus

B. acidoterrestris

B. anthracis

B. halmapalus

B. thermoamylovorans

B. licheniformis

B. aeolius

B. aquimaris

B. haloalkaliphilus

B. thermocatenulatus

B. ligniniphilus

B. aerius

B. arenosi

B. halochares

B. thermocloacae

B. litoralis

B. aerophilus

B. arseniciselenatis

B. halodenitrificans

B. thermocopriae

B. locisalis

B. agaradhaerens

B. arsenicus

B. halodurans

B. thermodenitrificans

B. luciferensis

B. agri

B. aurantiacus

B. halophilus

B. thermoglucosidasius

B. luteolus

B. aidingensis

B. arvi

B. halosaccharovorans

B. thermolactis

B. luteus

B. akibai

B. aryabhattai

B. hemicellulosilyticus

B. thermoleovorans

B. macauensis

B. alcalophilus

B. asahii

B. hemicentroti

B. thermophilus

B. macerans

B. algicola

B. atrophaeus

B. herbersteinensis

B. thermoruber

B. macquariensis

B. alginolyticus

B. axarquiensis

B. horikoshii

B. thermosphaericus

B. macyae

B. alkalidiazotrophicus

B. azotofixans

B. horneckiae

B. thiaminolyticus

B. malacitensis

B. alkalinitrilicus

B. azotoformans

B. horti

B. thioparans

B. mannanilyticus

B. alkalisediminis

B. badius

B. huizhouensis

B. thuringiensis

B. marisflavi

B. alkalitelluris

B. barbaricus

B. humi

B. tianshenii

B. marismortui

B. altitudinis

B. bataviensis

B. hwajinpoensis

B. trypoxylicola

B. marmarensis

B. alveayuensis

B. beijingensis

B. idriensis

B. tusciae

B. massiliensis

B. alvei

B. benzoevorans

B. indicus

B. validus

B. megaterium

B. amyloliquefaciens

B. beringensis

B. infantis

B. vallismortis

B. mesonae

B.

B. berkeleyi

B. infernus

B. vedderi

B. methanolicus

a. subsp. amyloliquefaciens

B. beveridgei

B. insolitus

B. velezensis

B. methylotrophicus

B. a. subsp. plantarum

B. bogoriensis

B. invictae

B. vietnamensis

B. migulanus

B. boroniphilus

B. iranensis

B. vireti

B. mojavensis

B. dipsosauri

B. borstelensis

B. isabeliae

B. vulcani

B. mucilaginosus

B. drentensis

B. brevis Migula

B. isronensis

B. wakoensis

B. muralis

B. edaphicus

B. butanolivorans

B. jeotgali

B. weihenstephanensis

B. murimartini

B. ehimensis

B. canaveralius

B. kaustophilus

B. xiamenensis

B. mycoides

B. eiseniae

B. carboniphilus

B. kobensis

B. xiaoxiensis

B. naganoensis

B. enclensis

B. cecembensis

B. kochii

B. zhanjiangensis

B. nanhaiensis

B. endophyticus

B. cellulosilyticus

B. kokeshiiformis

B. peoriae

B. nanhaiisediminis

B. endoradicis

B. centrosporus

B. koreensis

B. persepolensis

B. nealsonii

B. farraginis

B. cereus

B. korlensis

B. persicus

B. neidei

B. fastidiosus

B. chagannorensis

B. kribbensis

B. pervagus

B. neizhouensis

B. fengqiuensis

B. chitinolyticus

B. krulwichiae

B. plakortidis

B. niabensis

B. firmus

B. chondroitinus

B. laevolacticus

B. pocheonensis

B. niacini

B. flexus

B. choshinensis

B. larvae

B. polygoni

B. novalis

B. foraminis

B. chungangensis

B. laterosporus

B. polymyxa

B. oceanisediminis

B. fordii

B. cibi

B. salexigens

B. popilliae

B. odysseyi

B. formosus

B. circulans

B. saliphilus

B. pseudalcalophilus

B. okhensis

B. fortis

B. clarkii

B. schlegelii

B. pseudofirmus

B. okuhidensis

B. fumarioli

B. clausii

B. sediminis

B. pseudomycoides

B. oleronius

B. funiculus

B. coagulans

B. selenatarsenatis

B. psychrodurans

B. oryzaecorticis

B. fusiformis

B. coahuilensis

B. selenitireducens

B. psychrophilus

B. oshimensis

B. galactophilus

B. cohnii

B. seohaeanensis

B. psychrosaccharolyticus

B. pabuli

B. galactosidilyticus

B. composti

B. shacheensis

B. psychrotolerans

B. pakistanensis

B. galliciensis

B. curdlanolyticus

B. shackletonii

B. pulvifaciens

B. pallidus

B. gelatini

B. cycloheptanicus

B. siamensis

B. pumilus

B. pallidus

B. gibsonii

B. cytotoxicus

B. silvestris

B. purgationiresistens

B. panacisoli

B. ginsengi

B. daliensis

B. simplex

B. pycnus

B. panaciterrae

B. ginsengihumi

B. decisifrondis

B. siralis

B. qingdaonensis

B. pantothenticus

B. ginsengisoli

B. decolorationis

B. smithii

B. qingshengii

B. parabrevis

B. globisporus (eg, B.

B. deserti

B. soli

B. reuszeri

B. paraflexus

g. subsp. Globisporus; or B.

B. solimangrovi

B. rhizosphaerae

B. pasteurii

g. subsp. Marinus)

B. solisalsi

B. rigui

B. patagoniensis

B. songklensis

B. ruris

B. sonorensis

B. safensis

B. sphaericus

B. salarius

B. sporothermodurans

B. stearothermophilus

B. stratosphericus

B. subterraneus

B. subtilis (eg, B.

s. subsp. Inaquosorum, or B.

s. subsp. Spizizenr, or B.

s. subsp. Subtilis)

Caenimonas

Campylobacter

Cardiobacterium

Catenuloplanes

Curtobacterium

Caenimonas koreensis

Campylobacter coli

Cardiobacterium hominis

Catenuloplanes atrovinosus

Curtobacterium albidum

Caldalkalibacillus

Campylobacter concisus

Carnimonas

Catenuloplanes castaneus

Curtobacterium citreus

Caldalkalibacillus uzonensis

Campylobacter curvus

Carnimonas nigrificans

Catenuloplanes crispus

Caldanaerobacter

Campylobacter fetus

Carnobacterium

Catenuloplanes indicus

Caldanaerobacter subterraneus

Campylobacter gracilis

Carnobacterium alterfunditum

Catenuloplanes japonicus

Caldanaerobius

Campylobacter helveticus

Carnobacterium divergens

Catenuloplanes nepalensis

Caldanaerobius fijiensis

Campylobacter hominis

Carnobacterium funditum

Catenuloplanes niger

Caldanaerobius

Campylobacter hyointestinalis

Carnobacterium gallinarum

Chryseobacterium

polysaccharolyticus

Campylobacter jejuni

Carnobacterium

Chryseobacterium

Caldanaerobius zeae

Campylobacter lari

maltaromaticum

balustinum

Caldanaerovirga

Campylobacter mucosalis

Carnobacterium mobile

Citrobacter

Caldanaerovirga acetigignens

Campylobacter rectus

Carnobacterium viridans

C. amalonaticus

Caldicellulosiruptor

Campylobacter showae

Caryophanon

C. braakii

Caldicellulosiruptor bescii

Campylobacter sputorum

Caryophanon latum

C. diversus

Caldicellulosiruptor kristjanssonii

Campylobacter upsaliensis

Caryophanon tenue

C. farmeri

Caldicellulosiruptor owensensis

Capnocytophaga

Catellatospora

C. freundii

Capnocytophaga canimorsus

Catellatospora citrea

C. gillenii

Capnocytophaga cynodegmi

Catellatospora

C. koseri

Capnocytophaga gingivalis

methionotrophica

C. murliniae

Capnocytophaga granulosa

Catenococcus

C. pasteurii
^[1]

Capnocytophaga haemolytica

Catenococcus thiocycli

C. rodentium

Capnocytophaga ochracea

C. sedlakii

Capnocytophaga sputigena

C. werkmanii

C. youngae

Clostridium

(see below)

Coccochloris

Coccochloris elabens

Corynebacterium

Corynebacterium flavescens

Corynebacterium variabile

Clostridium

Clostridium absonum, Clostridium aceticum, Clostridium acetireducens, Clostridium acetobutylicum, Clostridium acidisoli, Clostridium aciditolerans, Clostridium acidurici, Clostridium aerotolerans, Clostridium

aestuarii, Clostridium akagii, Clostridium aldenense, Clostridium aldrichii, Clostridium algidicarni, Clostridium algidixylanolyticum, Clostridium algifaecis, Clostridium algoriphilum, Clostridium alkalicellulosi,

Clostridium aminophilum, Clostridium aminovalericum, Clostridium amygdalinum, Clostridium amylolyticum, Clostridium arbusti, Clostridium arcticum, Clostridium argentinense, Clostridium asparagiforme,

Clostridium aurantibutyricum, Clostridium autoethanogenum, Clostridium baratii, Clostridium barkeri, Clostridium bartlettii, Clostridium beijerinckii, Clostridium bifermentans, Clostridium bolteae, Clostridium

bornimense, Clostridium botulinum, Clostridium bowmanii, Clostridium bryantii, Clostridium butyricum, Clostridium cadaveris, Clostridium caenicola, Clostridium caminithermale, Clostridium carboxidivorans,

Clostridium carnis, Clostridium cavendishii, Clostridium celatum, Clostridium celerecrescens, Clostridium cellobioparum, Clostridium cellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi,

Clostridium cellulovorans, Clostridium chartatabidum, Clostridium chauvoei, Clostridium chromiireducens, Clostridium citroniae, Clostridium clariflavum, Clostridium clostridioforme, Clostridium coccoides,

Clostridium cochlearium, Clostridium colletant, Clostridium colicanis, Clostridium colinum, Clostridium collagenovorans, Clostridium cylindrosporum, Clostridium difficile, Clostridium diolis, Clostridium

disporicum, Clostridium drakei, Clostridium durum, Clostridium estertheticum, Clostridium estertheticum estertheticum, Clostridium estertheticum laramiense, Clostridium fallax, Clostridium felsineum, Clostridium

fervidum, Clostridium fimetarium, Clostridium formicaceticum, Clostridium frigidicarnis, Clostridium frigoris, Clostridium ganghwense, Clostridium gasigenes, Clostridium ghonii, Clostridium glycolicum,

Clostridium glycyrrhizinilyticum, Clostridium grantii, Clostridium haemolyticum, Clostridium halophilum, Clostridium hastiforme, Clostridium hathewayi, Clostridium herbivorans, Clostridium hiranonis,

Clostridium histolyticum, Clostridium homopropionicum, Clostridium huakuii, Clostridium hungatei, Clostridium hydrogeniformans, Clostridium hydroxybenzoicum, Clostridium hylemonae, Clostridium jejuense,

Clostridium indolis, Clostridium innocuum, Clostridium intestinale, Clostridium irregulare, Clostridium isatidis, Clostridium josui, Clostridium kluyveri, Clostridium lactatifermentans, Clostridium lacusfryxellense,

Clostridium laramiense, Clostridium lavalense, Clostridium lentocellum, Clostridium lentoputrescens, Clostridium leptum, Clostridium limosum, Clostridium litorale, Clostridium lituseburense, Clostridium ljungdahlii,

Clostridium lortetii, Clostridium lundense, Clostridium magnum, Clostridium malenominatum, Clostridium mangenotii, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium methylpentosum,

Clostridium neopropionicum, Clostridium nexile, Clostridium nitrophenolicum, Clostridium novyi, Clostridium oceanicum, Clostridium orbiscindens, Clostridium oroticum, Clostridium oxalicum, Clostridium

papyrosolvens, Clostridium paradoxum, Clostridium paraperfringens (Alias: C. welchii), Clostridium paraputrificum, Clostridium pascui, Clostridium pasteurianum, Clostridium peptidivorans, Clostridium perenne,

Clostridium perfringens, Clostridium pfennigii, Clostridium phytofermentans, Clostridium piliforme, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium propionicum, Clostridium proteoclasticum,

Clostridium proteolyticum, Clostridium psychrophilum, Clostridium puniceum, Clostridium purinilyticum, Clostridium putrefaciens, Clostridium putrificum, Clostridium quercicolum, Clostridium quinii,

Clostridium ramosum, Clostridium rectum, Clostridium roseum, Clostridium saccharobutylicum, Clostridium saccharogumia, Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum, Clostridium

sardiniense, Clostridium sartagoforme, Clostridium scatologenes, Clostridium schirmacherense, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,

Clostridium sporogenes, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium stercorarium leptospartum, Clostridium stercorarium stercorarium, Clostridium stercorarium thermolacticum,

Clostridium sticklandii, Clostridium straminisolvens, Clostridium subterminale, Clostridium sufflavum, Clostridium sulfidigenes, Clostridium symbiosum, Clostridium tagluense, Clostridium

tepidiprofundi, Clostridium termitidis, Clostridium tertium, Clostridium tetani, Clostridium tetanomorphum, Clostridium thermaceticum, Clostridium thermautotrophicum, Clostridium thermoalcaliphilum,

Clostridium thermobutyricum, Clostridium thermocellum, Clostridium thermocopriae, Clostridium thermohydrosulfuricum, Clostridium thermolacticum, Clostridium thermopalmarium,

Clostridium thermopapyrolyticum, Clostridium thermosaccharolyticum, Clostridium thermosuccinogenes, Clostridium thermosulfurigenes, Clostridium thiosulfatireducens, Clostridium tyrobutyricum,

Clostridium uliginosum, Clostridium ultunense, Clostridium villosum, Clostridium vincentii, Clostridium viride, Clostridium xylanolyticum, Clostridium xylanovorans

Dactylosporangium

Deinococcus

Delftia

Echinicola

Dactylosporangium aurantiacum

Deinococcus aerius

Delftia acidovorans

Echinicola pacifica

Dactylosporangium fulvum

Deinococcus apachensis

Desulfovibrio

Echinicola vietnamensis

Dactylosporangium matsuzakiense

Deinococcus aquaticus

Desulfovibrio desulfuricans

Dactylosporangium roseum

Deinococcus aquatilis

Diplococcus

Dactylosporangium thailandense

Deinococcus caeni

Diplococcus pneumoniae

Dactylosporangium vinaceum

Deinococcus radiodurans

Deinococcus radiophilus

Enterobacter

Enterobacter kobei

Faecalibacterium

Flavobacterium

E. aerogenes

E. ludwigii

Faecalibacterium prausnitzii

Flavobacterium antarcticum

E. amnigemis

E. mori

Fangia

Flavobacterium aquatile

E. agglomerans

E. nimipressuralis

Fangia hongkongensis

Flavobacterium aquidurense

E. arachidis

E. oryzae

Fastidiosipila

Flavobacterium balustinum

E. asburiae

E. pulveris

Fastidiosipila sanguinis

Flavobacterium croceum

E. cancerogenous

E. pyrinus

Fusobacterium

Flavobacterium cucumis

E. cloacae

E. radicincitans

Fusobacterium nucleatum

Flavobacterium daejeonense

E. cowanii

E. taylorae

Flavobacterium defluvii

E. dissolvens

E. turicensis

Flavobacterium degerlachei

E. gergoviae

E. sakazakii Enterobacter soli

Flavobacterium

E. helveticus

Enterococcus

denitrificans

E. hormaechei

Enterococcus durans

Flavobacterium filum

E. intermedins

Enterococcus faecalis

Flavobacterium flevense

Enterococcus faecium

Flavobacterium frigidarium

Erwinia

Flavobacterium mizutaii

Erwinia hapontici

Flavobacterium

Escherichia

okeanokoites

Escherichia coli

Gaetbulibacter

Haemophilus

Ideonella

Janibacter

Gaetbulibacter saemankumensis

Haemophilus aegyptius

Ideonella azotifigens

Janibacter anophelis

Gallibacterium

Haemophilus aphrophilus

Idiomarina

Janibacter corallicola

Gallibacterium anatis

Haemophilus felis

Idiomarina abyssalis

Janibacter limosus

Gallicola

Haemophilus gallinarum

Idiomarina baltica

Janibacter melonis

Gallicola barnesae

Haemophilus haemolyticus

Idiomarina fontislapidosi

Janibacter terrae

Garciella

Haemophilus influenzae

Idiomarina loihiensis

Jannaschia

Garciella nitratireducens

Haemophilus paracuniculus

Idiomarina ramblicola

Jannaschia cystaugens

Geobacillus

Haemophilus parahaemolyticus

Idiomarina seosinensis

Jannaschia helgolandensis

Geobacillus thermoglucosidasius

Haemophilus parainfluenzae

Idiomarina zobellii

Jannaschia

Geobacillus stearothermophilus

Haemophilus

Ignatzschineria

pohangensis

Geobacter

paraphrohaemolyticus

Ignatzschineria

Jannaschia rubra

Geobacter bemidjiensis

Haemophilus parasuis

larvae

Janthinobacterium

Geobacter bremensis

Haemophilus pittmaniae

Ignavigranum

Janthinobacterium

Geobacter chapellei

Hafnia

Ignavigranum ruoffiae

agaricidamnosum

Geobacter grbiciae

Hafnia alvei

Ilumatobacter

Janthinobacterium lividum

Geobacter hydrogenophilus

Hahella

Ilumatobacter fluminis

Jejuia

Geobacter lovleyi

Hahella ganghwensis

Ilyobacter

Jejuia pallidilutea

Geobacter metallireducens

Halalkalibacillus

Ilyobacter delafieldii

Jeotgalibacillus

Geobacter pelophilus

Halalkalibacillus halophilus

Ilyobacter insuetus

Jeotgalibacillus

Geobacter pickeringii

Helicobacter

Ilyobacter polytropus

alimentarius

Geobacter sulfurreducens

Helicobacter pylori

Ilyobacter tartaricus

Jeotgalicoccus

Geodermatophilus

Jeotgalicoccus halotolerans

Geodermatophilus obscurus

Gluconacetobacter

Gluconacetobacter xylinus

Gordonia

Gordonia rubripertincta

Kaistia

Labedella

Listeria ivanovii

Micrococcus

Nesterenkonia

Kaistia adipata

Labedella gwakjiensis

L. marthii

Micrococcus luteus

Nesterenkonia holobia

Kaistia soli

Labrenzia

L. monocytogenes

Micrococcus lylae

Nocardia

Kangiella

Labrenzia aggregata

L. newyorkensis

Moraxella

Nocardia argentinensis

Kangiella aquimarina

Labrenzia alba

L. riparia

Moraxella bovis

Nocardia corallina

Kangiella

Labrenzia alexandrii

L. rocourtiae

Moraxella nonliquefaciens

Nocardia

koreensis

Labrenzia marina

L. seeligeri

Moraxella osloensis

otitidiscaviarum

Kerstersia

Labrys

L. weihenstephanensis

Nakamurella

Kerstersia gyiorum

Labrys methylaminiphilus

L. welshimeri

Nakamurella multipartita

Kiloniella

Labrys miyagiensis

Listonella

Nannocystis

Kiloniella laminariae

Labrys monachus

Listonella anguillarum

Nannocystis pusilia

Klebsiella

Labrys okinawensis

Macrococcus

Natranaerobius

K. gramilomatis

Labrys

Macrococcus bovicus

Natranaerobius

K. oxytoca

portucalensis

Marinobacter

thermophilus

K. pneumoniae

Lactobacillus

Marinobacter algicola

Natranaerobius trueperi

K. terrigena

[see below]

Marinobacter bryozoorum

Naxibacter

K. variicola

Laceyella

Marinobacter flavimaris

Naxibacter alkalitolerans

Kluyvera

Laceyella putida

Meiothermus

Neisseria

Kluyvera ascorbata

Lechevalieria

Meiothermus ruber

Neisseria cinerea

Kocuria

Lechevalieria aerocolonigenes

Methylophilus

Neisseria denitrificans

Kocuria roasea

Legionella

Methylophilus methylotrophus

Neisseria gonorrhoeae

Kocuria varians

[see below]

Microbacterium

Neisseria lactamica

Kurthia

Listeria

Microbacterium

Neisseria mucosa

Kurthia zopfii

L. aquatica

ammoniaphilum

Neisseria sicca

L. booriae

Microbacterium arborescens

Neisseria subflava

L. cornellensis

Microbacterium liquefaciens

Neptunomonas

L. fleischmannii

Microbacterium oxydans

Neptunomonas japonica

L. floridensis

L. grandensis

L. grayi

L. innocua

Lactobacillus

L. acetotolerans

L. catenaformis

L. mali

L. parakefiri

L. sakei

L. acidifarinae

L. ceti

L. manihotivorans

L. paralimentarius

L. salivarius

L. acidipiscis

L. coleohominis

L. mindensis

L. paraplantarum

L. sanfranciscensis

L. acidophilus

L. collinoides

L. mucosae

L. pentosus

L. satsumensis

Lactobacillus agilis

L. composti

L. murinus

L. perolens

L. secaliphilus

L. algidus

L. concavus

L. nagelii

L. plantarum

L. sharpeae

L. alimentarius

L. coryniformis

L. namurensis

L. pontis

L. siliginis

L. amylolyticus

L. crispatus

L. nantensis

L. protectus

L. spicheri

L. amylophilus

L. crustorum

L. oligofermentans

L. psittaci

L. suebicus

L. amylotrophicus

L. curvatus

L. oris

L. rennini

L. thailandensis

L. amylovorus

L. delbrueckii subsp. bulgaricus

L. panis

L. reuteri

L. ultunensis

L. animalis

L. delbrueckii subsp.

L. pantheris

L. rhamnosus

L. vaccinostercus

L. antri

delbrueckii

L. parabrevis

L. rimae

L. vaginalis

L. apodemi

L. delbrueckii subsp. lactis

L. parabuchneri

L. rogosae

L. versmoldensis

L. aviarius

L. dextrinicus

L. paracasei

L. rossiae

L. vini

L. bifermentans

L. diolivorans

L. paracollinoides

L. ruminis

L. vitulinus

L. brevis

L. equi

L. parafarraginis

L. saerimneri

L. zeae

L. buchneri

L. equigenerosi

L. homohiochii

L. jensenii

L. zymae

L. camelliae

L. farraginis

L. iners

L. johnsonii

L. gastricus

L. casei

L. farciminis

L. ingluviei

L. kalixensis

L. ghanensis

L. kitasatonis

L. fermentum

L. intestinalis

L. kefiranofaciens

L. graminis

L. kunkeei

L. fornicalis

L. fuchuensis

L. kefiri

L. hammesii

L. leichmannii

L. fructivorans

L. gallinarum

L. kimchii

L. hamsteri

L. lindneri

L. frumenti

L. gasseri

L. helveticus

L. harbinensis

L. malefermentans

L. hilgardii

L. hayakitensis

Legionella

Legionella adelaidensis

Legionella drancourtii

Candidatus Legionella jeonii

Legionella quinlivanii

Legionella anisa

Legionella dresdenensis

Legionella jordanis

Legionella rowbothamii

Legionella beliardensis

Legionella drozanskii

Legionella lansingensis

Legionella rubrilucens

Legionella birminghamensis

Legionella dumoffii

Legionella londiniensis

Legionella sainthelensi

Legionella bozemanae

Legionella erythra

Legionella longbeachae

Legionella santicrucis

Legionella brunensis

Legionella fairfieldensis

Legionella lytica

Legionella shakespearei

Legionella busanensis

Legionella fallonii

Legionella maceachernii

Legionella spiritensis

Legionella cardiaca

Legionella feeleii

Legionella massiliensis

Legionella steelei

Legionella cherrii

Legionella geestiana

Legionella micdadei

Legionella steigerwaltii

Legionella cincinnatiensis

Legionella genomospecies

Legionella monrovica

Legionella taurinensis

Legionella clemsonensis

Legionella gormanii

Legionella moravica

Legionella tucsonensis

Legionella donaldsonii

Legionella gratiana

Legionella nagasakiensis

Legionella tunisiensis

Legionella gresilensis

Legionella nautarum

Legionella wadsworthii

Legionella hackeliae

Legionella norrlandica

Legionella waltersii

Legionella impletisoli

Legionella oakridgensis

Legionella worsleiensis

Legionella israelensis

Legionella parisiensis

Legionella yabuuchiae

Legionella jamestowniensis

Legionella pittsburghensis

Legionella pneumophila

Legionella quateirensis

Oceanibulbus

Paenibacillus

Prevotella

Quadrisphaera

Oceanibulbus indolifex

Paenibacillus thiaminolyticus

Prevotella albensis

Quadrisphaera

Oceanicaulis

Pantoea

Prevotella amnii

granulorum

Oceanicaulis alexandrii

Pantoea

Prevotella bergensis

Quatrionicoccus

Oceanicola

agglomerans

Prevotella bivia

Quatrionicoccus

Oceanicola batsensis

Paracoccus

Prevotella brevis

australiensis

Oceanicola granulosus

Paracoccus alcaliphilus

Prevotella bryantii

Quinella

Oceanicola nanhaiensis

Paucimonas

Prevotella buccae

Quinella

Oceanimonas

Paucimonas lemoignei

Prevotella buccalis

ovalis

Oceanimonas baumannii

Pectobacterium

Prevotella copri

Ralstonia

Oceaniserpentilla

Pectobacterium aroidearum

Prevotella dentalis

Ralstonia eutropha

Oceaniserpentilla haliotis

Pectobacterium atrosepticum

Prevotella denticola

Ralstonia insidiosa

Oceanisphaera

Pectobacterium betavasculorum

Prevotella disiens

Ralstonia mannitolilytica

Oceanisphaera donghaensis

Pectobacterium cacticida

Prevotella histicola

Ralstonia pickettii

Oceanisphaera litoralis

Pectobacterium carnegieana

Prevotella intermedia

Ralstonia

Oceanithermus

Pectobacterium carotovorum

Prevotella maculosa

pseudosolanacearum

Oceanithermus desulfurans

Pectobacterium chrysanthemi

Prevotella marshii

Ralstonia syzygii

Oceanithermus profundus

Pectobacterium cypripedii

Prevotella melaninogenica

Ralstonia solanacearum

Oceanobacillus

Pectobacterium rhapontici

Prevotella micans

Ramlibacter

Oceanobacillus caeni

Pectobacterium wasabiae

Prevotella multiformis

Ramlibacter henchirensis

Oceanospirillum

Planococcus

Prevotella nigrescens

Ramlibacter

Oceanospirillum linum

Planococcus citreus

Prevotella oralis

tataouinensis

Planomicrobium

Prevotella oris

Raoultella

Planomicrobium okeanokoites

Prevotella oulorum

Raoultella ornithinolytica

Plesiomonas

Prevotella pallens

Raoultella planticola

Plesiomonas shigelloides

Prevotella salivae

Raoultella terrigena

Proteus

Prevotella stercorea

Rathayibacter

Proteus vulgaris

Prevotella tannerae

Rathayibacter caricis

Prevotella timonensis

Rathayibacter festucae

Prevotella veroralis

Rathayibacter iranicus

Providencia

Rathayibacter rathayi

Providencia stuartii

Rathayibacter toxicus

Pseudomonas

Rathayibacter tritici

Pseudomonas aeruginosa

Rhodobacter

Pseudomonas alcaligenes

Rhodobacter sphaeroides

Pseudomonas anguillispetica

Ruegeria

Pseudomonas fluorescens

Ruegeria gelatinovorans

Pseudoalteromonas

haloplanktis

Pseudomonas mendocina

Pseudomonas

pseudoalcaligenes

Pseudomonas putida

Pseudomonas tutzeri

Pseudomonas syringae

Psychrobacter

Psychrobacter faecalis

Psychrobacter

phenylpyruvicus

Saccharococcus

Sagittula

Sanguibacter

Stenotrophomonas

Tatlockia

Saccharococcus thermophilus

Sagittula stellata

Sanguibacter keddieii

Stenotrophomonas

Tatlockia maceachernii

Saccharomonospora

Salegentibacter

Sanguibacter suarezii

maltophilia

Tatlockia micdadei

Saccharomonospora azurea

Salegentibacter salegens

Saprospira

Streptococcus

Tenacibaculum

Saccharomonospora cyanea

Salimicrobium

Saprospira grandis

[also see below]

Tenacibaculum

Saccharomonospora viridis

Salimicrobium album

Sarcina

Streptomyces

amylolyticum

Saccharophagus

Salinibacter

Sarcina maxima

Streptomyces

Tenacibaculum discolor

Saccharophagus degradans

Salinibacter ruber

Sarcina ventriculi

achromogenes

Tenacibaculum

Saccharopolyspora

Salinicoccus

Sebaldella

Streptomyces

gallaicum

Saccharopolyspora erythraea

Salinicoccus alkaliphilus

Sebaldella

cesalbus

Tenacibaculum

Saccharopolyspora gregorii

Salinicoccus hispanicus

termitidis

Streptomyces cescaepitosus

lutimaris

Saccharopolyspora hirsuta

Salinicoccus roseus

Serratia

Streptomyces cesdiastaticus

Tenacibaculum

Saccharopolyspora hordei

Salinispora

Serratia fonticola

Streptomyces cesexfoliatus

mesophilum

Saccharopolyspora rectivirgula

Salinispora arenicola

Serratia marcescens

Streptomyces fimbriatus

Tenacibaculum

Saccharopolyspora spinosa

Salinispora tropica

Sphaerotilus

Streptomyces fradiae

skagerrakense

Saccharopolyspora taberi

Salinivibrio

Sphaerotilus natans

Streptomyces fulvissimus

Tepidanaerobacter

Saccharothrix

Salinivibrio costicola

Sphingobacterium

Streptomyces griseoruber

Tepidanaerobacter

Saccharothrix australiensis

Salmonella

Sphingobacterium multivorum

Streptomyces griseus

syntrophicus

Saccharothrix coeruleofusca

Salmonella bongori

Staphylococcus

Streptomyces lavendulae

Tepidibacter

Saccharothrix espanaensis

Salmonella enterica

[see below]

Streptomyces

Tepidibacter

Saccharothrix longispora

Salmonella subterranea

phaeochromogenes

formicigenes

Saccharothrix mutabilis

Salmonella typhi

Streptomyces

Tepidibacter thalassicus

Saccharothrix syringae

thermodiastaticus

Thermus

Saccharothrix tangerinus

Streptomyces tubercidicus

Thermus aquaticus

Saccharothrix texasensis

Thermus filiformis

Thermus thermophilus

Staphylococcus

S. arlettae

S. equorum

S. microti

S. schleiferi

S. agnetis

S. felis

S. muscae

S. sciuri

S. aureus

S. fleurettii

S. nepalensis

S. simiae

S. auricularis

S. gallinarum

S. pasteuri

S. simulans

S. capitis

S. haemolyticus

S. petrasii

S. stepanovicii

S. caprae

S. hominis

S. pettenkoferi

S. succinus

S. carnosus

S. hyicus

S. piscifermentans

S. vitulinus

S. caseolyticus

S. intermedius

S. pseudintermedius

S. warneri

S. chromogenes

S. kloosii

S. pseudolugdunensis

S. xylosus

S. cohnii

S. leei

S. pulvereri

S. condimenti

S. lentus

S. rostri

S. delphini

S. lugdunensis

S. saccharolyticus

S. devriesei

S. lutrae

S. saprophyticus

S. epidermidis

S. lyticans

S. massiliensis

Streptococcus

Streptococcus agalactiae

Streptococcus infantarius

Streptococcus orisratti

Streptococcus thermophilus

Streptococcus anginosus

Streptococcus iniae

Streptococcus parasanguinis

Streptococcus sanguinis

Streptococcus bovis

Streptococcus intermedius

Streptococcus peroris

Streptococcus sobrinus

Streptococcus canis

Streptococcus lactarius

Streptococcus pneumoniae

Streptococcus suis

Streptococcus constellatus

Streptococcus milleri

Streptococcus

Streptococcus uberis

Streptococcus downei

Streptococcus mitis

pseudopneumoniae

Streptococcus vestibularis

Streptococcus dysgalactiae

Streptococcus mutans

Streptococcus pyogenes

Streptococcus viridans

Streptococcus equines

Streptococcus oralis

Streptococcus ratti

Streptococcus

Streptococcus faecalis

Streptococcus tigurinus

Streptococcus salivariu

zooepidemicus

Streptococcus ferus

Uliginosibacterium

Vagococcus

Vibrio

Virgibacillus

Xanthobacter

Uliginosibacterium

Vagococcus carniphilus

Vibrio aerogenes

Virgibacillus

Xanthobacter agilis

gangwonense

Vagococcus elongatus

Vibrio aestuarianus

halodenitrificans

Xanthobacter

Ulvibacter

Vagococcus fessus

Vibrio albensis

Virgibacillus

aminoxidans

Ulvibacter litoralis

Vagococcus fluvialis

Vibrio alginolyticus

pantothenticus

Xanthobacter

Umezawaea

Vagococcus lutrae

Vibrio campbellii

Weissella

autotrophicus

Umezawaea tangerina

Vagococcus salmoninarum

Vibrio cholerae

Weissella cibaria

Xanthobacter flavus

Undibacterium

Variovorax

Vibrio cincinnatiensis

Weissella confusa

Xanthobacter tagetidis

Undibacterium pigrum

Variovorax boronicumulans

Vibrio coralliilyticus

Weissella halotolerans

Xanthobacter viscosus

Ureaplasma

Variovorax dokdonensis

Vibrio cyclitrophicus

Weissella hellenica

Xanthomonas

Ureaplasma

Variovorax paradoxus

Vibrio diazotrophicus

Weissella kandleri

Xanthomonas

urealyticum

Variovorax soli

Vibrio fluvialis

Weissella koreensis

albilineans

Ureibacillus

Veillonella

Vibrio furnissii

Weissella minor

Xanthomonas alfalfae

Ureibacillus composti

Veillonella atypica

Vibrio gazogenes

Weissella

Xanthomonas

Ureibacillus suwonensis

Veillonella caviae

Vibrio halioticoli

paramesenteroides

arboricola

Ureibacillus terrenus

Veillonella criceti

Vibrio harveyi

Weissella soli

Xanthomonas

Ureibacillus thermophilus

Veillonella dispar

Vibrio ichthyoenteri

Weissella thailandensis

axonopodis

Ureibacillus thermosphaericus

Veillonella montpellierensis

Vibrio mediterranei

Weissella viridescens

Xanthomonas

Veillonella parvula

Vibrio metschnikovii

Williamsia

campestris

Veillonella ratti

Vibrio mytili

Williamsia marianensis

Xanthomonas citri

Veillonella rodentium

Vibrio natriegens

Williamsia maris

Xanthomonas codiaei

Venenivibrio

Vibrio navarrensis

Williamsia serinedens

Xanthomonas

Venenivibrio stagnispumantis

Vibrio nereis

Winogradskyella

cucurbitae

Verminephrobacter

Vibrio nigripulchritudo

Winogradskyella

Xanthomonas

Verminephrobacter eiseniae

Vibrio ordalii

thalassocola

euvesicatoria

Verrucomicrobium

Vibrio orientalis

Wolbachia

Xanthomonas fragariae

Verrucomicrobium spinosum

Vibrio parahaemolyticus

Wolbachia persica

Xanthomonas fuscans

Vibrio pectenicida

Wolinella

Xanthomonas gardneri

Vibrio penaeicida

Wolinella succinogenes

Xanthomonas hortorum

Vibrio proteolyticus

Zobellia

Xanthomonas hyacinthi

Vibrio shilonii

Zobellia galactanivorans

Xanthomonas perforans

Vibrio splendidus

Zobellia uliginosa

Xanthomonas phaseoli

Vibrio tubiashii

Zoogloea

Xanthomonas pisi

Vibrio vulnificus

Zoogloea ramigera

Xanthomonas populi

Zoogloea resiniphila

Xanthomonas theicola

Xanthomonas

translucens

Xanthomonas

vesicatoria

Xylella

Xylella fastidiosa

Xylophilus

Xylophilus ampelinus

Xenophilus

Yangia

Yersinia mollaretii

Zooshikella

Zobellella

Xenophilus azovorans

Yangia pacifica

Yersinia philomiragia

Zooshikella ganghwensis

Zobellella denitrificans

Xenorhabdus

Yaniella

Yersinia pestis

Zunongwangia

Zobellella taiwanensis

Xenorhabdus beddingii

Yaniella flava

Yersinia pseudotuberculosis

Zunongwangia profunda

Zeaxanthinibacter

Xenorhabdus bovienii

Yaniella halotolerans

Yersinia rohdei

Zymobacter

Zeaxanthinibacter

Xenorhabdus cabanillasii

Yeosuana

Yersinia ruckeri

Zymobacter palmae

enoshimensis

Xenorhabdus doucetiae

Yeosuana aromativorans

Yokenella

Zymomonas

Zhihengliuella

Xenorhabdus griffiniae

Yersinia

Yokenella regensburgei

Zymomonas mobilis

Zhihengliuella

Xenorhabdus hominickii

Yersinia aldovae

Yonghaparkia

Zymophilus

halotolerans

Xenorhabdus koppenhoeferi

Yersinia bercovieri

Yonghaparkia alkaliphila

Zymophilus paucivorans

Xylanibacterium

Xenorhabdus nematophila

Yersinia enterocolitica

Zavarzinia

Zymophilus raffinosivorans

Xylanibacterium ulmi

Xenorhabdus poinarii

Yersinia entomophaga

Zavarzinia compransoris

Xylanibacter

Yersinia frederiksenii

Xylanibacter oryzae

Yersinia intermedia

Yersinia kristensenii

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PROPAGATOR CELLS AND METHODS FOR PROPAGATING PHAGE, IN PARTICULAR FOR DELIVERING CRISPR-CAS COMPONENTS VIA PROBIOTIC ORGANISMS

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