Patent Application
20240384279
The contents of the electronic sequence listing (786212001600SEQLIST.xml; Size: 18,622 bytes; and Date of Creation: Jan. 24, 2024) is herein incorporated by reference in its entirety.
The invention relates to methods and means for temporally regulating the production of products of interest (eg, proteins or RNAs) in microbiota of subjects. such as gut microbiota of humans or animals. For example, in this way microbiota can usefully be modified for expression of products in a controlled and optionally reversible manner.
Mounting evidence undoubtedly links homeostasis in human microbiotas to human health and conversely, microbial dysbiosis is linked to a variety of disease conditions. For example, dysbiosis such as overgrowth of the bacterial pathogen C. difficile in the gut microbiota is linked to severe diarrhoea caused by production of toxins secreted from the C. difficile. Another example of disease related to microbial dysbiosis is inflammatory bowel disease (TBD), in which an inflammatory state of the intestines is at least partly caused by the metabolome associated with overgrowth of Enterobacteriaceae and under representation of bacterial species capable of producing short-chain fatty acids.
Reverting a disease condition by restoring a healthy microbiota by faecal microbial transplants (FMT), in which the faecal microbiota from a healthy donor is transplanted into the gut of the patient suffering from microbial dysbiosis has been proven effective to treat for example recurrent C. difficile infections. However, FMT is a relatively uncontrolled process and also associated with risks as demonstrated by fatalities caused by bacterial infections that could be traced back to a FMT. A more controlled approach in which single strains or consortia of defined strains are added to the dysbiotic gut environment is currently being heavily investigated. Such approaches require the displacement of strains by colonizing new strains added in the consortia
In some cases, target compounds of interest are identified, such as the presence of bacterial toxins (against which antibodies have been generated) or the absence of IL-22 associated with disease. The delivery, however, of relevant proteinaceous compounds locally in the intestinal tract is inefficient and cumbersome due to instability of the proteins in the gut environment.
Hence, local in situ production of relevant molecules (e.g. enzymes, antibodies, nanobodies, small molecules) intracellularly or secreted by live bacteria which are established and already present in the microbiota would allow targeted intervention with minimal impact on the native microbiota. The invention addresses this need and how to control such production in situ in microbiota.
The invention thus provides the following configurations:—
At least one nucleic acid vector for transfer into a host cell of a microbiota. the vector(s) comprising nucleic acid that comprises
In an embodiment there is provided:—
At least one nucleic acid vector for transfer into a host cell of a microbiota, the vector(s) comprising nucleic acid that comprises
At least one nucleic acid vector for transfer into a host cell of a microbiota, the vector(s) comprising nucleic acid that comprises
Preferably, NS1 and NS2 are comprised by the same nucleic acid vector. In another configuration, exposure of a regulator agent (R) to the vector nucleic acid regulates the second promoter, thereby regulating the expression of P2 and P1.
Preferably, P2 is operable in the host cell to bind to vector nucleic acid to regulate expression of P1.
In an embodiment, the invention provides a host cell comprising said at least one vector.
In an embodiment, P1 is a protein (eg, an enzyme) of a metabolic pathway in the host cell, wherein said activity is an activity (eg, enzymatic activity) of P1 in the pathway. In an example, P1 is a protein inhibitor and the activity is inhibitor activity (eg, inhibitor of a component of a metabolic pathway). In an example, Pt is a binding agent, eg, an antibody or antibody fragment, such as a single domain antibody (eg, a nanobody) or an scFv.
In an embodiment, P2 is an inhibitor of P1 expression. In an embodiment, P2 activity is inhibition of P1 expression. In an embodiment, P2 is an enhancer of P1 expression. In an embodiment, P2 activity is enhancement of P1 expression.
A nucleic acid vector for transfer into a host cell of a microbiota, the vector comprising a nucleic acid that comprises
P2 is expressible in the host cell for the formation of a nuclease that is operable in the host cell to cut the vector nucleic acid, wherein the nucleic acid is degraded, thereby downregulating the expression of P1. This is useful to control the expression of P1 temporally (ie, for a predetermined window of time) in a subject, such as in a human or animal microbiota. For example, in this way microbiota can usefully be modified for expression of P1 in a controlled and optionally reversible manner.
Each vector (such as according to the First Aspect) is a conjugative plasmid. This is useful to enable spreading of the vector nucleic acid—and thus expression of P1—within a targeted microbiota. When coupled with the First Aspect of the First Configuration, there is provided a powerful way to controllably modify the extent and timing of P1 expression in the microbiota.
The host cell is a cell of commensal or probiotic bacterial cell species of a human or animal microbiota, preferably a Bacteroides species. Such species are present and maintained in native microbiota of humans and animals and thus, the invention provides means for relatively stable and controllable microbiota modification for temporally regulating the P1 expression.
A method of temporally regulating the production of an expression product in a human or animal subject, the method comprising
A method of temporally regulating the production of an expression product in a human or animal subject, the method comprising
A method of temporally regulating the production of an expression product in a human or animal subject, the method comprising
A method of
A nucleic acid vector for transfer into a host cell of a microbiota, wherein the vector is comprised by a carrier cell (eg, a bacterial cell) and encodes
A method of engineering a microbiome, the method comprising contacting the microbiome with a plurality of vectors as described herein and optionally allowing transfer of said vector nucleic acid into target cells of the microbiota.
A modified microbiota obtained or obtainable by the method herein, optionally wherein the microbiota is comprised by a pharmaceutical composition for use as a medicament to treat a disease or condition in a human or animal subject.
A host cell comprising nucleic acid that comprises
A cell (optionally according to any other Configuration), comprising a nucleic acid, wherein the nucleic acid comprises a gene encoding a product of interest (P1), the gene comprising a nucleotide sequence (NS1) encoding P1 and a regulatory region 5′ of NS1 that comprises a promoter (Px) for controlling the expression of NS1, wherein the combination of Px and NS1 is heterologous to the cell and Px is regulatable by xylitol.
A cell (optionally according to any other Configuration), comprising a nucleic acid, wherein the nucleic acid comprises a gene encoding a product of interest (P1), the gene comprising a nucleotide sequence (NS1) encoding P1 and a regulatory region 5′ of NS1 that comprises a promoter (Px) for controlling the expression of NS1, wherein the combination of Px and NS1 is heterologous to the cell and Px is regulatable by xylose.
In a third Aspect
A nucleic acid vector comprising a gene as recited in the first or second Aspect.
The invention relates to methods and means for temporally regulating the production of products of interest (herein called P1, eg, proteins or RNAs) in microbiota of subjects. such as gut microbiota of humans or animals. For example, in this way microbiota can usefully be modified for expression of products in a controlled and possibly reversible manner. This can be useful to enable controlled engineering of microbiota of subjects in a way that enables, for example, production of desirable levels of P1 followed by controlled reduction in expression, eg, for reversion to a pre-engineered level of P1. To this end, the invention provides the following illustrative embodiments.
In a First Configuration, there is provided:
At least one nucleic acid vector for transfer into a host cell of a microbiota, the vector(s) comprising nucleic acid that comprises
Said “at least one vector” may relate to one vector or a plurality of vectors, eg, first and second vectors, eg, 2 vectors. Herein, where features are described in the context of one vector or “the vector”, the skilled person will realise that the features may apply mutatis mutandis to said “at least one vector” such as a first and a second vector, or such as a plurality of vectors. The one vector may be further according to the vector of the Fourth Configuration. Each of said first and second, 2 or plurality of vectors may be further according to the vector of the Fourth Configuration. This is advantageous to limit the presence or spread of NS1 and/or NS2 (thus limiting P1 and/or P2 respectively) in the microbiota or subject comprising the microbiota.
For example, said at least one vector comprises a first vector and a second vector, wherein the first vectors comprises NS1 (and optionally not NS2) and the second vector comprises NS2 (and optionally not NS1) and the vectors are capable of co-existing in the host cell for expression of Pt and P2. Thus, the first vector may comprise NS1 and not NS2; and the second vector may comprise NS2 and not NS1. The first and second vectors are capable of being transferred into the same host cell and co-existing in the cell, whereby P1 and P2 can be expressed in the cell. For example, said at least one vector is one vector that comprises both NS1 and NS2.
A microbiota may be in any environment, eg, in soil or a waterway, comprised by a plant, or comprised by a human or animal subject. As the skilled person will know, a microbiota may comprise bacteria, archaea, fungi and viruses.
A First Aspect of the First Configuration provides:
A nucleic acid vector for transfer into a host cell of a microbiota, the vector comprising a nucleic acid that comprises
As more fully described herein, some advantages of specific aspects of the invention may be
Each host cell may be a bacterial, fungal (eg, yeast) or archaeal cell. Preferably, each host cell is a microbial cell. Preferably, each host cell is a bacterial cell. Preferably, each host cell is an archaeal cell.
Preferably, each host cell is a cell of a commensal or probiotic bacterial cell species found in human or animal microbiota. Preferably, each host cell is a cell of a commensal or probiotic bacterial cell species of a human or animal microbiota. Preferably, each host cell is a microbial (eg, bacterial) cell of a human or animal gut microbiota species.
The nucleic acid may be DNA or RNA. For example, the nucleic acid is DNA.
A host cell with reference to the First to Third Configurations is interchangeably referred to herein as a target cell. A host cell with reference to the Fourth Configurations is interchangeably referred to herein as a carrier or donor cell.
In an example, P1 is secreted from the host cell(s). In an example, P1 is expressed from NS1 in the host cell as a amino acid sequence comprising a signal peptide for secretion of P1 from the cell. In an example, P1 is not secreted from the host cell(s). P1 may be a protein or RNA (eg, a mRNA).
In an embodiment, P1 is not expressed in the microbiota prior to carrying out the method of the invention.
P2 may upregulate P1 expression. In an embodiment, P1 is expressed in the subject prior to carrying out the method of the invention, wherein carrying out the method causes at least a 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000% increase in the expression of Pt in the subject. The increase in expression may be determined by determining the relative levels of P1 in a tissue or fluid sample (eg, blood sample) that has been obtained from the patient. The increase in expression may be determined by determining the relative levels of P1 in a faecal sample that has been obtained from the patient.
P2 may upregulate P1 expression. In an embodiment, P1 is expressed in the microbiota (eg, gut microbiota of a human or animal subject) prior to carrying out the method of the invention, wherein carrying out the method causes at least a 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000% increase in the expression of P1 in the microbiota. The increase in expression may be determined by determining the relative levels of P1 in a sample of the microbiota that has been previously obtained from the patient. The increase in expression may be determined by determining the relative levels of P1 in a tissue or fluid sample (eg, blood sample) that has been obtained from the patient. The increase in expression may be determined by determining the relative levels of P1 in a faecal sample that has been obtained from the patient (eg, wherein the microbiota is a gut microbiota).
P2 may downregulate P1 expression. In an embodiment, the method comprises administering R to the subject in step (c) of the method of the invention, whereby the expression of P1 in the subject is decreased by at least a 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000% compared to the expression of P1 immediately before carrying out step (c). The decrease in expression may be determined by determining the relative level of P1 in a tissue or fluid sample (eg, blood sample) that has been obtained from the patient immediately before carrying out step (c) and comparing the level with the level of P1 in a similar sample (ie, tissue or fluid sample respectively) obtained after step (c) has been performed (eg, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 21, 22, 23 or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, or 3, 4, 5, 6, 7, or 8 weeks after the commencement of step (c)). The decrease in expression may alternatively be determined by determining the relative levels of P1 in faecal samples that have been obtained from the patient.
P2 may downregulate P1 expression. In an embodiment, the method comprises administering R to the subject in step (c) of the method of the invention, whereby the expression of P1 is decreased in the microbiota by at least a 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000% compared to the expression of P1 immediately before carrying out step (c). The decrease in expression may be determined by determining the relative level of P1 in a tissue or fluid sample (eg, blood sample) that has been obtained from the patient immediately before carrying out 5 step (c) and comparing the level with the level of P1 in a similar sample (ie, tissue or fluid sample respectively) obtained after step (c) has been performed (eg, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 21, 22, 23 or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, or 3, 4, 5, 6, 7, or 8 weeks after the commencement of step (c)). The decrease in expression may alternatively be determined by determining the relative levels of P1 in faecal samples that have been obtained from the patient.
The second promoter may be an inducible promoter (wherein induction of promoter causes an increase in P2 expression) or a repressible promoter (wherein repression of promoter causes a reduction in P2 expression). Thus, binding of R to the nucleic acid may induce or repress the second promoter.
For example, a promoter herein is selected from Plac and Ptac (comprising lacO operator, lacI repressor), Ptet (comprising tetO operator, tetR repressor) and ParaBad (comprising araO operator, araC repressor). As will be familiar to the skilled person, it is possible to synthetically produce repressible promotors from native constitutive promoters by adding the tetO operator (or an analogue thereof) to the sequence and express tetR or analogues to repress them.
P2 may comprise a nucleic acid (eg, an RNA) or a protein (eg, a peptide). For example, P2 is a silencing RNA or protein that is capable of binding to the nucleic acid to inhibit expression of P1, eg, by binding to the first promoter a sequence that overlaps with the first promoter, or by binding to NS1.
P2 may be operable in the host cell to bind to the vector nucleic acid to downregulate expression of P1; and/or R may upregulate expression of P2. P2 may be operable in the host cell to bind to the vector nucleic acid to upregulate expression of P1.
P2 may be expressible in the host cell for the formation of a nuclease that is operable in the host cell to cut the nucleic acid. Preferably, the cut nucleic acid is degraded (optionally the vector comprising the nucleic acid is degraded), thereby downregulating the expression of P1 in the host cell. Preferably, the cut nucleic acid is degraded, thereby downregulating the expression of P1 in the microbiota. P2 itself can be the nuclease or a component thereof (wherein the component combines with one or more other components in the host cell to form the nuclease, such as an RNA-guided nuclease). Examples of suitable nucleases are an RNA-guided endonuclease or restriction endonuclease. For example the nuclease is a restriction nuclease selected from AatII, AbaSI, Acc65I, AccI, AciI, AciI, AcuI, AfeI, AfIII, AfIIII, AgeI, AhdI, AeI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BglI, BglII, BipI, BmgBI, BmrI, BmtI, BpmI, BpuEI, Bpul0I, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspEI, BspHI, Bsp1286I, BspMI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ171, Bsu36I, BtgI, BtgZI, BtsCI, BtsIMutI, BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DraI, DralII, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoOI09I, EcoP15I, EcoRI, EcoRV, Esp3I, FatI, FauI, Fnu4HI, FokI, FseI, FspEI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HphI, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, Hpy99I, Hpy188I, Hpy 166II, Hpy188III, I-CeuI, I-SceI, KasI, KpnI, LpnPI, MboI, MboH, MfeI, MluCI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspAII, MspI, MspJI, MwoI, NaeI, NarI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaqCI, PciI, PflMI, PI-PspI, PI-SceI, PleI, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SrfI, SspI, StuI, StyD4I, StyI, SwaI, TaqI, TfiI, TseI, Tsp45I, TspRI, Tth111I PflFI, XbaI, XcmI, XhoI, XhoI, PaeR71, XmaI, XmaI TspMI, XmnI and ZraI.
In an example, P2 comprises a guided nuclease that is programmable in the host cell to guide the nuclease to a target nucleotide sequence comprised by the nucleic acid, wherein the nuclease is capable of cutting the target sequence, whereby the nucleic acid sequence is degraded and P1 expression is inhibited from the nucleic acid.
In an example, the nuclease is operable to cut a target site comprised by the vector nucleic acid, wherein the target site is
A CRISPR/cas system comprises at least one Cas (eg, Cas3 (optionally also Cascade Cas, eg, CasA-E), Cas9, Cas12 or Cas13) and a cognate guide RNA that is capable of forming a Cas/guide RNA complex for recognising and binding to a protospacer sequence. In the above examples, the protospacer is comprised by the target site. The guide RNA may be a single guide RNA.
P2 may comprise
Optionally, the precursor is an RNA, pre-cRNA or tracrRNA.
In an example, P2 is capable of binding to an operator (O) that is comprised by the nucleic acid and operatively connected to the first promoter (eg O is 5′ of the first promoter, eg, withing 200 or 100 kb 5′ of the promoter), wherein when P2 is bound to O the expression of P1 is reduced (eg, eliminated). In an example, P2 comprises a dead Cas nuclease (eg, a dCas9 or dCas3 or dCas12 or dCas13) wherein the dCas is capable of forming a dCas/guide RNA complex in the host cell that recognises and binds to a protospacer comprised by the nucleic acid to interrupt expression of P1 (eg, by interrupting promoter function of the first promoter).
Optionally, the guided nuclease is a Cas nuclease, TALEN, meganuclease or zinc finger nuclease, preferably a Cas nuclease. The nuclease may cut DNA or RNA, preferably DNA.
The nuclease may be operable to cut the nucleic acid at a predetermined sequence motif (a target site), optionally a protospacer sequence or restriction site. The protospacer may be a CRISPR/Cas protospacer. The restriction site may be cut by a restriction endonuclease or any other restriction nuclease disclosed herein. The nucleic acid may comprise a plurality (eg, at least 2 or 3, eg, 2, 3, 4, 5, 6, 7, 8, or 9) of said motifs. This may be useful for efficiency of cutting and destruction of the nucleic acid (or vector comprising the nucleic acid).
P1 may be an amino acid, protein (eg, peptide or a polypeptide) or RNA (eg, mRNA or silencing RNA) for human or animal therapy. For example P1 is a cytokine, growth factor, enzyme, hormone or antibody (or antibody chain or antibody fragment). For example, P1 is an antibody chain or antibody fragment, eg, a single domain antibody (AKA a dAb) or a nanobody. For example, the chain or fragment is a human antibody chain or fragment. For example, P1 is an antibody heavy chain that forms an antibody with an antibody light chain that is also expressed in the host cell. Preferably. the antibody, chain or fragment is capable of being secreted from the host cell. Preferably, the antibody, chain or fragment is secreted from the host cell. For example, P1 is an incretin, eg, an incretin peptide or a multimer thereof. Optionally, the incretin in selected from GLP-1, GIP, exendin-4 and insulin. For example, P1 comprises an antigen binding site of an antibody or a variable domain (eg, VH and/or VL domain) of an antibody, eg, wherein the antibody is selected from the group consisting of ReoPro™; Abciximab; Rituxan™; Rituximab; Zenapax™; Daclizumab; Simulect™; Basiliximab; Synagis™; Palivizumab; Remicade™; Infliximab; Herceptin®; Mylotarg™; Gemtuzumab; Campath®: Alemtuzumab; Zevalin™; Ibritumomab; Humira™; Adalimumab; Xolair™; Omalizumab; Bexxar™; Tositumomab; Raptiva™; Efalizumab; Erbitux™; Cetuximab; Avastin™; Bevacizumab; Tysabri™; Natalizumab; Actemra™; Tocilizumab; Vectibix™; Panitumumab; Lucentis™; Ranibizumab; Soliris™; Eculizumab; Cimzia™; Certolizumab; Simponi™; Golimumab, Ilaris™; Canakinumab; Stelara™; Ustekinumab; Arzerra™; Ofatumumab; Prolia™; Denosumab; Numax™; Motavizumab; ABThrax™; Raxibacumab; Benlysta™; Belimumab; Yervoy™; Ipilimumab; Adcetris™; Brentuximab; Vedotin™; Perjeta™; Pertuzumab; pembrolizumab, nivolumab, atezolizumab, Kadcyla™; Ado-trastuzumab; Keytruda™, Opdivo™, Gazyva™ and Obinutuzumab. For example, P is selected from an insulin peptide, incretin peptide or peptide hormone. For example, the antibody is adalimumab. For example, the antibody is pembrolizumab. For example, the antibody is nivolumab. For example, the antibody is atezolizumab. For example, the antibody is dupilumab. For example, the antibody is tocilizumab. For example, the antibody is sarilumab. For example, the antibody is alirocumab. For example, the antibody is evolocumab. In an alternative the antibody is an anti-CD38 antibody, an anti-TNFa antibody, an anti-TNFR antibody, an anti-IL-4Ra antibody, an anti-IL-6R antibody, an anti-IL-6 antibody, an anti-VEGF antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PCSK9 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD138 antibody, an anti-IL-1 antibody. In an alternative the antibody is selected from the antibodies disclosed in WO2007024715 at page 40, line 23 to page 43, line 23, the disclosure of which is incorporated herein by reference.
An antigen herein may be an antigen selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AWI; AIGI; AKAP1; AKAP2; AIYIH; AMHR2; ANGPT1; ANGPT2; ANGFTL3; ANGPTL4; ANPEP: APC; APOC1; AR; AZGPI (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR 15); BlyS; BM P1; BMP2; BMP3B (GDFIO); BMP4; BMP6; BM P8; BMPRIA; BMPRIB: BM PR2; BPAG1 (plectin); BRCA1; CI9orflO (IL27w); C3; C4A; C5; C5R1; CANT1; CASPi; CASP4; CAVi; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (M IP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (M PIF-1); CCL24 (MPIF-2 I eotaxin-2); CCL25 (TECK): CCL26 (eotaxin-3): CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-la); CCL4 (M IP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA 1; CCNA2; CCND1; CCNEl; CCNE2; CCR I (CKR I/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CM KBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBIl); CCR8 (CM KBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN I A (p2fWapl/Cipl); CDKN I B (p27Kipl); CDKNIC; CDKN2A (pl61NK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR 1; CMKOR1 (RDCI); CNR1; COL18AI; COL1AI; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNBI (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDi); CX3CR1 (V28); CXCL1 (GROI); CXCLIO (IP-10); CXCL1I (1-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78 I LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR ISTRL33 I Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451JO118; DNCL1; DPP4; E2F1; ECGF1; EDGi; EFNAI; EFNA3: EFNB2; EGF; EGFR: ELAC2; ENG; EN01; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGFI (aFGF); FGF10; FGF11; FGF12; FGFI2B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGP23; FGF3 (int-2): FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FILI (EPSILON); FILl (ZETA); FUl2584; FU25530: FLRTI (fibronectin): FLTI: FOS; FOSLI (FRA-1); FY (DARC); GABRP (GABAa); GAGEBI; GAGECI; GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCRIO); GPR31; GPR44; GPR81 (FKSG80); GRCCIO (CIO); GRP; GSN (Gelsolin); GSTPI; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9; HGF; HIFlA; HIPi; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; TFNW1; IGBP1; TGF1; IGF1R; TGF2; IGFBP2; IGFBP3; TCFBP6; IL-1; IL10; IL10RA; IL10RB; rL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; 116; IL17; TL17B; TL17C; IL 17R; IL18; TL18BP; IL18R1; IL18RAP; IL19; ILIA; TL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; I L26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; TL7R; IL8; IL8RA; IL8RB; IL8RB; L9; IL9R; ILK; INHA; INHBA; TNSL3; INSL4; TRAKI; IRAK2; ITGA; ITGA2; 1TGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAGi; JAKi; JAK3; JUN; K6HF; KAIl; KDR; MTLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; M IB1; midkine; M rF; M IP-2; MK167 (Ki-67); MMP2; M MP9; MS4A1; MSMB; MT3 (metallothionectin-ifi); MTSS 1; M UC 1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB 1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NM El (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR 1D2; NR 1H2; NR1H3; NR1H4; NR 112; NR 1I3; NR2C1; NR2C2; NR2EI; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZI; OPRDI; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; P1K3CG; PLAU (uPA); PLG; PLXDCI; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p2TRac2); RARB; RGSI; RGS13; RGS3; RNF110 (ZNF144); ROB02; S100A2; SCGBID2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINAI; SERPTNIA3; SERPTNB5 (maspin); SERPINEI (PAT-i); SERPTNFI; SHBG; SLA2; SLC2A2; SLC33AI; SLC43A1; SLIT2; SPPI; SPRRIB (Spri); ST6GAL1; STABI; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCPIO; TDGF1; TEK; TGFA; TGFB1; TGFBII; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; THIL; THBSI(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-i); T]MP3; tissue factor; TLRIO; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-α; TNFAIP2 (B94); TNFAIP3; TNFRSFI IA; TNFRSFIA; TNFRSFIB; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL); TNFSFI I (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSFI 5 (VEGI); TNFSFI 8; TNFSF4 (0X40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); ThFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53; TPM 1; TPM2; TRADD; TRAFi; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM 1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB: VEGFC; versican; VHL C5; VLA-4; XCLI (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YYI; and ZFPM2.
For example, P1 comprises a VEGF binding site of aflibercept, eg, anti-VEGF FLT1 and/or KDR domain(s).
For example, P1 comprises (or is) an incretin, an insulin peptide, a GLP-1 (glucagon-like peptide-1 (GLP-1) peptide, a GIP (glucose-dependent insulinotropic polypeptide) peptide, an exendin (eg, exendin-4) peptide, a peptide hormone, a prolactin or prolactin peptide, a ACTH or ACTH peptide, a growth hormone or growth hormone peptide, a vasopressin or vasopressin peptide, an oxytocin or oxytocin peptide, a glucagon or glucagon peptide, a insulin or insulin peptide, a somatostatin or somatostatin peptide, a cholecystokinin or cholecystokinin peptide, a gastrin or gastrin peptide, a leptin or leptin peptide, an antibody binding site (eg, a scFv or Fab) or variable domain thereof, a TCR binding site (eg, a scTCR) or domain thereof, a TCR Vα/Vβ binding site or variable domain thereof, a TCR Vγ/Vδ binding site or variable domain thereof, an antibody single variable domain binding site, or an FcAb binding site. In an example, P1 comprises at least one copy of a GLP-1 and at least one copy of another incretin (eg, an Exendin-4). For example, there is one copy of GLP-1 and one copy of the other increting. Preferably, in the examples in this paragraph, P1 is a secreted or host cell surface-exposed protein. Optionally. any GLP-1 herein is GLP-l(7-37)-Pro9. Optionally, any incretin herein is an Exendn-4 or Peptide Y. Optionally, any P1 or incretin herein is DURAGLUTIDE™.
P1 may comprise an antigen binding site. A binding site herein may, for example, be an antigen (eg, cytokine or growth factor, eg, VEGF or EGFR) binding site of a receptor (eg, KDR or FIt). A binding site herein may, for example, be a binding site of Eyelea™, Avastin™ or Lucentis™, eg, for ocular or oncological medical use in a human or animal. When the antigen is VEGF, the vector or method may be for treatment or prevention of a cancer or ocular condition (eg, wet or dry AMD or diabetic retinopathy) or as an inhibitor of neovascularisation in a human or animal subject.
For example, P1 is a metabolism pathway component, eg, an enzyme or reagent in the pathway. For example, P1 is an intracellular enzyme in the target cell (ie, host cell). For example, P1 is a secreted enzyme (eg, secreted from the target cell). The pathway herein may be in the target cell or it may be outside the target cell. For example, the pathway is a pathway inside a different cell comprised by the microbiota of which the target cell is a component. The different cell may be a carrier cell or it may be an endogenous cell of the microbiota (ie, any cell of the microbiota except the target cell or a carrier cell). The pathway may comprise one or more as a product or intermediate:—
Optionally, R upregulates P2 expression, P2 downregulates P1 expression and optionally P1 is a component in a metabolic pathway (eg, in the microbiota or target cell or in a subject or environment comprising the microbiota or target cell) wherein a production product (X) of the pathway downstream from P1 causes regulation of P1 or P2 expression. See, for example, Scenarios 1, 3 and 4 (
Optionally, R upregulates P2 expression, P2 upregulates P1 expression and optionally P1 is a component in a metabolic pathway (eg, in the microbiota or target cell or in a subject or environment comprising the microbiota or target cell) wherein a production product (X) of the pathway downstream from P1 causes regulation of P1 or P2 expression. See, for example, Scenario 2 (
In an example, P1 is a secreted or cell-surface exposed protein antigen. This is useful, for example, to vaccinate the subject, eg, where the antigen is an antigen of a pathogen (such as a bacterium or virus, eg, a coat protein, such as spike protein, eg, a SARS-Cov or SARS-Cov-2 or influenza antigen).
In an example, P1 is a secreted antagonist of a target ligand in the subject. For example, binding of P1 to the ligand may inhibit or neutralise the ligand or mark it for destruction in the subject (eg, by immune cells of the subject). For example, the antagonist may comprise an antibody fragment (such as a nanobody or any other antibody single variable domain) comprising a binding site for the target ligand. The ligand may be, for example,
In an embodiment, P1 is toxic to cells of the same species as the host cell. This may be useful for killing cells of such species in the microbiota or for reducing the growth or proliferation of such cells, such as when the cells are detrimental to the health of the subject.
In an embodiment, P1 is a transcription or translation regulator in cells of the same species as the host cell. Thus, up- or down-regulation of P1 expression may advantageously act on one or a plurality of genes in the genome of the host cell.
Optionally, R is an amino acid, protein, carbohydrate (eg, a sugar), lipid, metal ion or nucleic acid (eg, RNA). R may be a sugar alcohol. eg, xylitol, glycerol, arabitol, erythritol, isomalt, HSHs, lactitol, maltitol, mannitol or sorbitol, preferably xylitol. R may be an antibiotic. R may be a metabolite of a metabolic pathway that operates in the subject or in the microbiota. R is optionally a metabolite of P1 or a metabolite produced in a pathway comprising P1. For example, P1 can be a secreted enzyme (ie, secreted from the host cell) that is capable of acting in a pathway in the subject that produces R. For example, P1 can be metabolised in a pathway that produces R (eg, P1 is metabolised to directly produce R, or to indirectly produce R). The pathway may be inside the target host cell or may be outside the target host cell (eg, in a neighbouring cell in the microbiota of which the target cell is a component). The effect of R on P1 expression may be dose dependent (or it may not). The skilled person will readily be able to determine (eg, by way of titrating doses in assays) the appropriate amount of R to use to provide a desired effect on the microbiota.
As exemplified herein, R may be xylitol. Operons of genes related to the conversion/utilization of sugars can be induced by the sugars themselves by virtue of their binding to the relevant transcriptional repressor proteins. For the present study it was decided to investigate a negatively repressible promoter predicted to be induced by xylitol. This 5-carbon sugar alcohol has several advantages. First of all, xylitol is generally regarded as safe for human consumption by the FDA (Xiang et al., 2021). Furthermore, over half of ingested xylitol is not adsorbed by human cells instead reaching the gastrointestinal tract where it is taken up by the microbiome (Livesey, 2003). Finally, xylitol serves as a metabolite for some bacteria: It is either taken up directly through an ABC-type transporter complex (Madigan et al., 2015) and/or generated through reduction of the corresponding sugar D-xylose. Followingly, xylitol is typically dehydrogenated and phosphorylated to xylulose-5-phosphate which is further catabolized in the pentose phosphate pathway.
For example, R is an amino acid. For example, R is selected from Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and Valine. In an embodiment, R is Tryptophan. For example, R is a protein (eg, a peptide), eg, R is selected from a nisin, sakacin A and sakacin P, For example, R is a bacteriocin. For example, R is a carbohydrate, eg, R is a sugar, such as selected from 1-arabinose, 1-rhamnose, xylose and sucrose. For example R is a metal ion, eg, R is selected from Fe2+, Mn2+, Co2+, Hg2+ and Cu2+. For example, R is a lipid, eg, proprionate. For example, R is a fatty acid. For example, R is a nucleic acid, eg, R is an RNA. For example, R is a benzene compound, eg, a substituted benzene compound or benzoic acid.
Optionally, the vector is an ICE (integrative and conjugative element), plasmid (eg, a conjugative plasmid), transduction particle (eg, a phage or non-self-replicative transduction particle) or nanoparticle. In an embodiment, the plasmid comprises an oriT and oriV. The plasmid may be self-conjugating. The plasmid may be a shuttle plasmid, ie, a plasmid that can propagate in at least two different host species. The plasmid may comprise a transposon or an ICE (or mobilizable part thereof) that comprises the nucleic acid comprising NS1 and NS2. For example, the transposon or ICE may be a Barteroides transposon or ICE. The plasmid may comprise an oriT and be mobilisable in the presence of a conjugative system, eg, a system found on other plasmids or integrative and conjugative element (ICE) in the host cell or microbiota. In an example, a such conjugative system is comprised by the genome of the donor cell or carrier cell. In an example, additionally or alternatively, a conjugative system is comprised by the genome of a recipient cell. The conjugative system may be carried on a chromosome or episome of a carrier cell as described herein (ie, system in trans to the plasmid). The conjugative system may be comprised by the plasmid itself (ie, system in cis). The system may be comprised by cells in the microbiota that neighbour the host cell into which the plasmid has been transferred; in this way the plasmid can be spread between neigbouring cells, thereby propagating the plasmid vector in the microbiota. This can be useful to amplify the P1 expression in the microbiota. Similarly, with a self-conjugating plasmid (ie, the conjugative system is provided along with oriT on the plasmid), the plasmid can spread in the microbiota.
The vector may be a conjugative plasmid comprised by a carrier cell (eg, a bacterial carrier cell). For example, the carrier cell and vector is for administration to a microbiota of a human or animal subject. For example, the carrier cell is a cell of commensal or probiotic bacterial cell species of a human or animal microbiota. Additionally or alternatively, the carrier cell is a cell of a human or animal gut microbiota species.
The conjugation genes of conjugative plasmids such as those of the Inc groups: P, N, W, or X show similarity at the protein level to the VirB system of Agrobacterium, itself constituting a prototypic Type IV secretion system (T4SS). These are often smaller and probably the most minimal systems around. The genes necessary for conjugation of the prototypical VirB plasmid pTI and the protein homologs in the incN (somewhat related) and incF (distantly related) plasmid families are shown in Table 4.
Optionally, the conjugation system is a VirB, IncN or IncF conjugation system, or a homologue or orthologue thereof.
Each vector may be a self-conjugative plasmid comprising an oriT and a conjugation system for transferring the plasmid between cells in the microbiota, wherein the conjugation system is a VirB, IncN or IncF conjugation system, or a homologue or orthologue thereof. The carrier (or donor) cell and said plasmid vector(s) may between them comprise a conjugation system for transferring the plasmid between cells in the microbiota, wherein the conjugation system is a VirB, IncN or IncF conjugation system, or a homologue or orthologue thereof, wherein each vector comprises an oriT. The host (or recipient) cell and said plasmid vector(s) may between them comprise a conjugation system for transferring the plasmid between cells in the microbiota, wherein the conjugation system is a VirB, IncN or IncF conjugation system, or a homologue or orthologue thereof, wherein each vector comprises an oriT.
For example, the system is the system of VirB pasmid pTIm, an IncN plasmid or an IncF plasmid, or the system is a homologue or orthologue thereof.
For example, the system comprises (i) VirB genes virB1-11 and virD4, or homologues or orthologues of said genes; (ii) IncN genes traA-G, traJ, traL, traM, traN and traO, or homologues or orthologues 30 of said genes; (iii) Inc F genes traA-E, traG, traH, traK, traL and ORF196, or homologues or orthologues of said genes; or (iv) IncF genes traA-T, traK, traL, traM, traW and traU (and optionally at least one or all of traX, traN, finO, trbI and trbB), or homologues or orthologues of said genes. For option (iv), reference is made to Front Mol Biosci., 2016 Nov. 10; 3:71. doi: 10.3389/fmolb.2016.00071. eCollection 2016, “Comparative Genomics of the Conjugation Region of F-like Plasmids: Five Shades of F”, Raul Fernandez-Lopez et al.
The invention also provides, according to the Fourth Configuration:
A nucleic acid vector for transfer into a host cell of a microbiota, wherein the vector is comprised by a carrier cell (eg, a bacterial cell) and encodes
The vector may have any of the vector features disclosed herein. The episome may be a plasmid.
In an embodiment, the vector comprises an inducible or repressible promoter that regulates expression of the nuclease of (a) and/or the vector comprises an inducible or repressible promoter that regulates expression of the RNA or component of (b), preferably wherein the promoter(s) are inducible promoters. In an alternative, the promoter(s) are repressible promoters. In an example, components (a) and (b) are under the control of different promoters. In an example, components (a) and (b) are under the control a common promoter
In an embodiment, the vector comprises an inducible promoter that regulates expression of the nuclease of (a). In an embodiment, the vector comprises an inducible promoter that regulates expression of the RNA or component of (b). Alternatively, the vector comprises an repressible promoter that regulates expression of the RNA or component of (b).
In an embodiment, the vector comprises an repressible promoter that regulates expression of the nuclease of (a). In an embodiment, the vector comprises an inducible promoter that regulates expression of the RNA or component of (b). Alternatively, the vector comprises an repressible promoter that regulates expression of the RNA or component of (b).
Optionally, the guided nuclease is a Cas nuclease, TALEN, meganuclease or zinc finger nuclease, preferably a Cas nuclease. For example, the guided nuclease is a Cas3. For example, the guided nuclease is a Cas9. For example, the guided nuclease is a Cas12 (eg, Cas12a). For example, the guided nuclease is a Cas13 (eg, Cas13a).
The nuclease in the Fourth Configuration is the same guided nuclease as in the First Configuration.
The nuclease in the Fourth Configuration is operable to cut the chromosome or episome in the carrier cell at a predetermined sequence motif, optionally a protospacer sequence or restriction site. Preferably, cutting of the carrier cell chromosome or episome kills the carrier cell or reduces growth or proliferation of the carrier cell, most preferably wherein the cell is killed. This is useful to reduce the transmission of the vector nucleic acid, such as wherein a microbiota or environment has been exposed to the vector. This provides a useful way of regulating expression of products from the vector, such as expression in recipient cells into which the vector nucleic acid has been transferred.
In an example, the vector comprises an oriT for transfer into the host cell, optionally wherein the vector is a conjugative plasmid.
In a preferred embodiment
According to the Fourth Configuration, there is provided:
A nucleic acid vector for transfer into a host cell of a microbiota, wherein the vector is comprised by a carrier cell (eg, a bacterial cell) and encodes
There is also provides a plurality of carrier cells comprising the vector. The cutting may reduce the number of carrier cells of said plurality at least 105, 106 or 107-fold, eg, between 105 and 107-fold, or between 105 and 108-fold or between 105 and 109-fold. The skilled person will be familiar with determining fold-killing or reduction in cells, eg, using a cell sample that is representative of a microbiota or cell population. For example, the extent of killing or reduction is determined using a cell sample, eg, a sample obtained from a subject to which the carrier cells of the invention have been administered, or an environmental sample (eg, aqueous, water or soil sample) obtained from an environment (eg, a water source, waterway or field) that has been contacted with the carrier cells of the invention. For example, the cutting reduces the number of carrier cells of said plurality at least 105, 106 or 107-fold and optionally the plurality comprises at least 100,000; 1,000,000; or 10,000.000 carrier cells respectively. Optionally, the plurality of carrier cells is comprised by a cell population of the microbiota, wherein at least 5, 6 or 7 log 10 of cells of the population are killed by the cutting, and optionally the plurality comprises at least 100,000; 1,000,000; or 10,000,000 carrier cells respectively. Optionally, the cutting kills at least 99%, 99.9%, 99.99%, 99.999%, 99.9999% or 99.99999% cells of said plurality of carrier cells.
There is provided a method of killing a plurality of carrier cells comprised by a microbiota, the carrier cells comprising vectors of the Fourth Configurations, wherein the method comprises upregulating expression of the nuclease of (a) and/or the RNA or component of (b) in the carrier cells (eg, by inducing the regulatable promoter(s)) whereby the genomes of the carrier cells are cut and the cells are killed. Preferably, chromosomes of the cells are cut. The method optionally kills at least 99%, 99.9%, 99.99%, 99.999%, 99.9999% or 99.99999% cells of said plurality of carrier cells. In an example, the method kills all (or essentially all) of the cells of said plurality of carrier cells. In an example, the method is kills 100% (or about 100%) of the plurality of carrier cells. Preferably all carrier cells comprised by the microbiota are cells of said plurality.
Preferably, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the carrier cells are killed.
A conjugative plasmid herein may be a self-conjugative plasmid (ie, wherein the plasmid comprises an oriT and encodes all proteins required to mobilise the plasmid for conjugative transfer between cells).
For any Configuration herein, the host cell (eg, donor, carrier or recipient cell) is a cell of a species found in a microbiota (eg, gut microbiota) of humans or animals. In a preferred example, the species is a Bacteroides species. In a preferred example, the species is E col. The host cell may be a cell of commensal or probiotic bacterial cell species of a human or animal microbiota. For example, the species is selected from any species in Table 1, preferably a Bacteroides species (eg, Bacteroides thetaiomaomicron, Bacteroides vulgatus, Bacteroides uniformis or Bacteroides ovatus) or Clostridales species, (eg, Clostridioides difficile or Clostridium disporicum).
In an embodiment,
Human or animal therapy herein may be treatment or prophylaxis of a disease or condition in the human or animal.
For any Configuration herein, a crRNA may be comprised by a guide RNA, such as a single guide RNA. For example, the single guide RNA comprises a crRNA and a tracrRNA that are operable in the recipient and/or carrier (donor) cell with a Cas9 to cut a cognate target nucleic acid sequence.
The invention provides:
A vector according to any Configuration herein for use as a medicament.
The medicament may be for treating or preventing a disease in a human or animal subject, eg, when comprised by a formulation for oral-administration to the subject.
A pharmaceutical composition comprising a vector according to any Configuration herein and a pharmaceutically-acceptable carrier, diluent or excipient, optionally an antacid.
A tablet, suppository, pill. capsule, or liquid formulation for administration to the gastrointestinal tract of a human or animal subject, wherein the table, suppository, pill, capsule or liquid formulation comprises a vector according to any Configuration herein.
Optionally, the tablet pill, or capsule comprises an enteric coating. Optionally, the tablet, pill, capsule or liquid formulation is for use as an orally-administered medicament.
In an example, the composition. tablet, suppository, pill, capsule or formulation herein comprises a medicament selected from the medicaments listed in Table 3.
The invention provides:
A method of temporally regulating the production of an expression product in a human or animal subject, the method comprising
The invention provides:
A method of temporally regulating the production of an expression product in a human or animal subject, the method comprising
Step (b) may be for a desired time between time points T1 and T2. For example for any of the methods, step (a) of the method is commenced at a first time (T1) and step (c) at a second time (T2). Optionally, T2 is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 21, 22, 23 or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, or 3, 4, 5, 6, 7, or 8 weeks after T1. Preferably, T2 is at least 0.5 hours after T1. Preferably, T2 is at least 1 day after T1. Preferably, T2 is at least 1 week after T1. Preferably, T2 is at least 1 month after T1. Preferably, T2 is at least 2 months after T1. Preferably, T2 is at least 3 months after T1. Preferably, T2 is at least 3 months after T1. Preferably, T2 is at least 4 months after Ti. Preferably, T2 is at least 5 months after TI. Preferably, T2 is at least 6 months after T1. Preferably, T2 is at least 12 months after T1. Preferably, T2 is at least 18 months after T1. For example, T2 is 1-6 (eg, 1, 2, 3, 4, 5 or 6) months after T1. For example, T2 is 1-8 weeks after TI. Thus, in this way the method is a method of temporally regulating the expression.
There is provided:
A method of
For example, there is provided a method of modifying a metabolome of a human or animal subject by carrying out the method of the invention. For example, expression of P1 causes the secreting or sequestering of one or more metabolites in the subject (eg, in the target or cell comprising the vector nucleic acid). For example, expression of P1 causes altering of a pathway intracellularly in the microbiota of the subject (eg, in the target cell) to cause compound metabolism (e.g. for producing a tryptophan sink, such as wherein the tryptophan is used in the microbiota (eg, in the target cell) for the production of AhR (Aryl Hydrocarbon Receptor) ligands). For example, expression of P1 causes the metabolizing or modifying of a chemical, such as a therapeutic drugs, in the microbiota. P1 may do such “causing” since it is a component of a metabolic pathway in the microbiota (eg, in the target cell), such as wherein P1 is a protein, like an enzyme.
The disease or condition may be any disease or condition described herein. The microbiota of a human or animal may be any microbiota described herein.
The administration of step (a) may be oral, topical (eg, by application on skin), buccal, rectal, vaginal, parenteral, intravenously, intramuscularly, inhaled, subcutaneously, ocular or intranasal administration. Preferably oral administration is used. Preferably topical administration is used.
In an example, the vector is comprised by a Faecal Microbial Transplat (FMT). The administration of step (a) may be by rectal administration of an enema or FMT comprising the vector.
In step (b) the vector (eg, a plasmid) comprising the nucleic acid can be transferred.
P1 may be a therapeutically or prophylactically useful expression product in the subject.
In an embodiment, n step (c) P1 expressed from the nucleic acid is the regulator agent (R) or is a component of a pathway that produces R, whereby a P1 expression feedback loop negatively regulates further expression of P1. For example, Pt is a metabolism pathway enzyme. For example, P1 is an intracellular enzyme in the target cell. For example, P1 is a secreted enzyme (eg, secreted from the target cell). In an example, expression of P1 causes the production (eg, in the microbiota, eg, in the target cell) of R.
A pathway as mentioned herein may be in the target cell or it may be outside the target cell. For example, the pathway is a pathway inside a different cell comprised by the microbiota of which the target cell is a component. The different cell may be a carrier cell (ie, donor cell) or it may be an endogenous cell of the microbiota (ie, any cell of the microbiome except the target cell or the carrier cell).
In an example, R upregulates the second promoter in step (c) and P2 downregulates the expression of P1; and optionally wherein the upregulation of the second promoter causes the production of a guided nuclease or restriction endonuclease that cuts the nucleic acid in the host cell, wherein the nucleic acid is degraded, thereby downregulating the expression of P1.
In an example, P2 is capable of upregulating the expression of P1 in the host cell, wherein R downregulates the second promoter in step (c) whereby the expression of P1 is downregulated.
Downregulation of P1 in step (c) may be by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% compared to P1 expression in step (b). For example, downregulation of P1 in step (c) may be expression of P1 for a period (PD1) in the presence of R that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% less compared to P1 expression in step (b) for a period (PD2), wherein PD1 and PD2 are the same length of time. For example, PD1 and PD2 each is 1 minute, 1 hour, 1 day, 1 week, 1 month, 6 months or 12 months. Expression of P1 may be determined in steps (b) and (c) by assessing P1 expression in a sample of the microbiota taken during steps (b) and (c) respectively. For example, when the microbiota is a gut microbiota of a human or animal subject, each sample may be a faecal sample of the subject.
Any upregulation of P1 expression may be an increase in P1 by at least 10, 20, 30, 40, 50, 60, 70, 80, 90. or 95%, such as compared to P1 expression immediately prior step (b). Any downregulation of P1 expression may be an decrease in P1 by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, such as compared to P1 expression immediately prior step (b).
Any upregulation of P2 expression may be an increase in P2 by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, such as compared to P2 expression immediately prior step (c). Any downregulation of P2 expression may be an decrease in P2 by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, such as compared to P2 expression immediately prior step (c). The vector, composition, tablet, suppository, pill, capsule, or liquid formulation described herein may be for use in the method described herein, such as wherein the vector, composition, suppository, pill, capsule or formulation is administered to the subject by oral or topical administration.
The host, carrier and/or target cells may be bacterial cells. In an alternative, instead of bacterial cells, the host, carrier and/or target cells may be archaea.
In an example, the method modifies the genome of the cell, eg, modifies a chromosome or episome (eg, a plasmid) of the host cell. For example, following transfer of the vector nucleic acid into the host cell, a copy of NS1 is inserted into a chromosome or episome of the host cell. In an example, the expression of a nuclease (eg, P2 or a nuclease that is operable with P2, such as wherein P2 comprises guide RNA) leads to cutting of the NS1 in the chromosome or episome and optionally host cell death.
The carrier cell and target cell may be cells of the same order, family or genus, such as shown in the Examples.
Preferably, the agent comprises a CRISPR/Cas system or component thereof. The agent may be a crRNA or guide RNA that guides a Cas nuclease in the target cell to a target protospacer sequence, wherein the Cas cuts the target sequence and the target cell is killed. For example, the plasmid may encode a plurality of different crRNAs or guide RNAs, such as a first cRNA or gRNA that comprises a spacer sequence that is capable of guiding a Cas in the target cell to a first protospacer sequence and a second cRNA or gRNA that comprises a spacer sequence that is capable of guiding a Cas in the target cell to a second protospacer sequence wherein the protospacer sequences are different (eg, different chromosomal sequences of the target cell). Each protospacer may be comprised by an essential gene, virulence gene or antibiotic resistance gene of the target cell genome. Each protospacer sequence may be from 10 to 60 nucleotides in length, eg, 15 to 50, 15 to 40, 15 to 30 or 15 to 20 nucleotides in length. The target sequence may be a chromosomal sequence of the target cell. The target sequence may be an episomal sequence of the target cell. The plasmid may encode a or said Cas nuclease, optionally a Cas9, Cas3 or Cpf1.
For example, the target cell is comprised by a plant microbiota. The carrier cell may be a Pseudomonas cell, optionally a P fluorescens cell. Optionally, the carrier and target cells are cells of the same genus or species, optionally both are Pseudomonas cells. For example, the target cell is a P syringae or aeruginosa cell and the carrier is a Pseudomonas (eg, P fluorescens) cell.
Preferably, the carrier cells are of a strain or species that is not pathogenic to an organism (eg, a plant, animal or human) that comprises the target cells. The carrier cells may be of a strain or species that is symbiotic or probiotic to an organism (eg, a plant, animal or human) that comprises the target cells, eg, probiotic or symbiotic in the gut of the organism.
For example, the target cell is comprised by a plant microbiota. In an example, the carrier cell comprises a Chitinase class I exoenzyme and/or the carrier cell genome encodes a Chitinase class I exoenzyme. Optionally, the carrier cell in this example is a Pseudomonas, eg, P fluorescens, cell. In an example, the carrier cell comprises a pep1 gene. Optionally, the carrier cell in this example is a Pseudomonas, eg, P fluorescens, cell.
In an example, the carrier cell is a motile bacterial cell. Optionally, the target cell is comprised by a plant microbiota and the carrier cell in this example is a Pseudomonas, eg, P fluorescens, cell.
For example, each target cell is a lag phase cell, exponential phase cell or a stationary phase cell. For example, each carrier cell is a lag phase cell, exponential phase cell or a stationary phase cell.
For example, the target cell is comprised by a plant microbiota. Optionally, the target cell is a Pseudomonas (optionally a P fluorescens or P aeruginosa) cell, Erwinia (optionally E carotovora), Xanthomonas, Agrobacterium, Burkholdi, Clavibacterium, Enterobacteria, Pantoae, Pectobacterium (eg. P atrosepticum). Rhizobium, Streptomyces (eg, S scabies), Xylella (eg, X fastidiosa), Candidatus (eg, C liberibacter), Phytoplasma, Ralstonia (eg, R solanacearum). or Dickeya (eg, D dadantii) cell.
Each target cell (eg, the plurality of target cells) may be a cell of a genus or species disclosed in Table 1 or 2. Each target cell (eg, the plurality of target cells) may be comprised by a plant or a plant environment (such as soil) and selected from a genus or species disclosed in Table 1. Each carrier cell (eg, the plurality of carrier cells) may be a cell of a genus or species disclosed in Table 1 or 2.
The method may be carried out in vitro or ex vivo.
The target cell may be comprised by
For example, the target cell is comprised by a plant leaf, stem, root, seed, bulb, flower or fruit microbiota.
Optionally, a microbiota herein is a gut, lung, kidney, urethral, bladder. blood, vaginal, eye, ear, nose, penile, bowel, liver, heart, tongue, hair or skin microbiota.
For example, the target cell is a cell of a species found in soil.
The method may be carried out using a first cell population comprising a plurality of carrier cells that are contacted with a second cell population comprising a plurality of target cells, wherein copies of said plasmid are conjugatively transferred from carrier cells into target cells, whereby some or all of the cells of the second population are killed or growth or proliferation of cells of the second population is inhibited (eg, by at least 50, 60, 70, 80, 90 or 95%, such as after 5 or 10 hours or 1 day after commencing the method). Preferably cells of the second population are killed.
Optionally, the method of the Second and Third Configurations is carried out on a plurality of target cells comprised by the microbiota by exposing the plurality of target cells to a plurality of the vectors (eg, a plurality of copies of a vector comprising NS1 and NS2; or a plurality of copies of a first vector comprising NS1 with a plurality of copies of a second vector comprising NS2). The method optionally modifies at least 99%, 99.9%, 99.99%, 99.999%, 99.9999% or 99.99999% cells of said plurality of target cells so that they are capable of expressing P1. In an example, the method is carried out on a population (or said plurality) of said target cells and the method modifies all (or essentially all) of the cells of said population (or said plurality). In an example, the method is carried out on a population (or said plurality) of said target cells and the method modifies 100% (or about 100%) of the cells of said population (or plurality).
Preferably, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the target cells are modified.
Optionally, the target cells are Pseudomonas (eg, P syringae) cells, eg, wherein the cells are comprised by a crop plant, such as a tomato plant. For example, leaf, fruit, ear, seed, grain, head, pod, stem, trunk, tuber and/or root biomass is increased by the method. For example, leaf or fruit dry biomass, leaf or fruit wet biomass or number of flowers is increased by the method, eg, wherein expression of P1 is beneficial to the health or growth of the plant. For example, average biomass or number is increased over a plurality of plants on which the method of the invention has been practised.
An increase in biomass (eg, average biomass or number) may be an increase by at least 5, 10, 15, 20, 25, 30, 40, or 50% compared to the biomass of plant(s) that have not been exposed to the carrier bacteria, but which comprise the target bacteria. Increases in plant biomass may be determined by measuring the weight of harvested material (eg, fruit, grain, cane, leaves, tubers, nuts or seeds) per area harvested and comparing the measurement of harvested material from plants that have been treated per the invention versus the same area of harvested material from plants of the same species and strain grown that have not been treated per the invention, where all plants are grown under the same conditions, eg, in the same field. In some systems units of volume, such as bushels, are used instead of units of weight.
In an Aspect the method is a method of promoting growth of a plant or germination of a plant seed, wherein the method is carried out using a first cell population comprising a plurality of carrier cells that are contacted with a second cell population comprising a plurality of target cells, wherein copies of said nucleic acid are transferred from carrier cells into target cells, whereby some or all of the cells of the second population are modified to express P1, wherein the seed comprises said target cells and said growth or germination is promoted.
Promoting germination may be decreasing the time to onset of germination and/or decreasing the duration of germination. Promoting germination may be increasing the percentage (eg, by at least 5, 10, 15 or 20%) of germination of seeds comprised by a plurality of seeds that are exposed to the carrier cells in the method.
Each seed may comprise target cells on the seed surface.
An increase in germination (eg, average germination) in a plurality of seeds exposed to the carrier cells in the method may be obtained, which is an increase by at least 5, 10, 15, 20, 25, 30, 40, or 50% compared to the germination of seeds that have not been exposed to the carrier cells, but which seeds comprise the target bacteria.
The method may be useful for treating pre-emergent seedlings have pathogens present which stop successful germination. Each seedling may comprise target cells on leaves and/or stems of the seedling.
An increase in growth (eg, average growth) in a plurality of seedlings exposed to the carrier cells in the method may be obtained, which is an increase by at least 5, 10, 15, 20, 25, 30, 40, or 50% compared to the growth of seedlings that have not been exposed to the carrier cells, but which seedlings comprise the target bacteria.
In an Aspect the method is a method of increasing leaf chlorophyll (eg, chlorophyll a and/or b) production in a plant, wherein the method is carried out using a first cell population comprising a plurality of carrier cells that are contacted with a second cell population comprising a plurality of target cells, wherein copies of said nucleic acid are transferred from carrier cells into target cells, whereby some or all of the cells of the second population are modified to express P1, wherein the plant comprises said target cells (optionally on leaves and/or stems thereof, or comprised by the apoplast of the plant), whereby target cells are modified and chlorophyll is increased in the plant. Chlorophyll measurement may be measured, for example, by spectrophotometry, high performance liquid chromatography (HPLC) or fluorometry.
In an Aspect the method is a method of modifying target cells comprised by a biofilm, wherein the biofilm is comprised by a subject or comprised on a surface, wherein the biofilm comprises target cells, wherein the method is carried out using a first cell population comprising a plurality of carrier cells that are contacted with a second cell population comprising a plurality of target cells, wherein copies of said nucleic acid are transferred from carrier cells into target cells, thereby modifying the target cells in the biofilm to express P1, optionally wherein the method is carried out ex vivo or in vitro.
The subject may be a human or animal, optionally wherein the surface is a lung surface.
The subject may be a plant, optionally wherein the biofilm is comprised by a leaf, trunk, root or stem of the plant.
The surface may be comprised by a domestic or industrial apparatus or container, eg, a fermentation vessel.
There is further provided:—
A carrier bacterial cell (eg, for use in a method according to the invention) for administration to a microbiota comprising a target cell, wherein the carrier cell comprises a conjugative plasmid, the plasmid being a vector of the invention, wherein the carrier cell is capable of conjugating to the target cell wherein the plasmid is transferred into the target cell to modify the target cell to express P1.
The carrier cell may be any carrier cell or carrier cell disclosed herein. The target cell may be any carrier cell or target cell disclosed herein.
There is provided:—
A carrier cell (eg, a bacterial cell) comprising a vector of the invention. The invention also provides a plurality of such carrier cells (eg, wherein the cells are genetically identical or wherein all of the cells encode the same P1). In an alternative, cells of the plurality encode different P1 proteins.
A pharmaceutical composition comprising a plurality of carrier cells of the invention for administration to a human or animal subject for modifying a plurality of bacterial target cells comprised by the subject to express P1, wherein vectors of the invention (eg, conjugative plasmids) encoding P1 are capable of being introduced from carrier cells into target cells (eg, by conjugation) and P1 is produced in target cells.
Preferably, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the target cells are so modified.
The plurality of target cells may comprise at least 107, 108, 109, 1010, 1011 or 1012 target cells. For example, the plurality of target cells is comprised by a gut, blood, lung, oral cavity, liver, kidney, bladder, urethra or skin microbiota of the subject.
There is provided:—
A method of treating or preventing a disease or condition in a subject, the method comprising contacting the subject (eg, a gut microbiota where the subject is a human or animal) with a composition comprising a plurality of carrier cells of the invention, wherein vectors of the invention (eg, conjugative plasmids) encoding P1 are introduced from carrier cells into target cells (eg, by conjugation) and P1 is produced in target cells, whereby the disease or condition is treated or prevented.
Use of a plurality of carrier cells of the invention in the manufacture of a composition for administration to a subject or environment (eg, soil), for modifying bacterial target cells comprised by the subject or environment to express P1, wherein the target cells are contacted with the carrier cells and vectors of the invention (eg, conjugative plasmids) encoding P1 are introduced from carrier cells into target cells (eg, by conjugation) and P1 is produced in target cells.
For example, the subject is a human or animal. For example, the subject is a mammal. For example, the subject is a bird, fish, protozoan or insect. For example, the animal is a livestock anima. For example, the animal is a dog, cat, horse, cow, sheep or pig.
For example, the subject is a plant and optionally the method comprises contacting the plant (eg, one or more stems and/or one or more leaves of the plant, or the plant apoplast) with the composition comprising a plurality of carrier cells.
There is provided:
Use of a carrier cell of the invention in the manufacture of a composition, for modifying a bacterial target cell ex vivo or wherein the target cell is not comprised by a human or animal (eg, the target cell is comprised by a plant or soil or a human microbiota sample ex vivo), wherein the target cell is contacted with the carrier cell and the carrier cell conjugates to the target cell, whereby the vector nucleic acid is introduced into the target cell, wherein P1 is expressed in the target cell.
For example, each vector is a conjugative plasmid, wherein the carrier cell conjugates (or is capable of conjugating) with the target cell and transfers the plasmid into the target cell by conjugation. Optionally, the plasmid comprises an origin or transer (oriT) and genes for self-conjugation, whereby the plasmid is capable of conjugative transfer from the target cell to a further cell (eg, wherein the target and further cells are comprised by a microbiota).
Optionally, the use comprises using a plurality of said carrier cells to modify a plurality of said target cells, wherein the target cells are comprised by a plant or plant environment (eg, soil) and the modifying
Optionally, the target cell or plurality of target cells is in an environment, eg, soil, or in an environment for growing plants. For example, P1 is a plant growth promoter (eg, a fertilizer).
For example, each target cell is a Bacteroides cell, eg, comprised by a human or animal subject. For example, each target cell is a Clostridiales cell, eg, comprised by a human or animal subject.
For example, each target cell is a gram-positive bacterial cell (eg, a Staphylococcus (such as S aureus, eg, methicillin-resistant Staphylococcus aureus (MRSA)), Streptococcus pneumonia, Clostridium difficile. Enterococcus spp. or Listeria monocytogenes cell). For example, each target cell is a gram-negative bacterial cell (eg, a Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Campylobacter jejuni, Pseudonmoas aeruginosa, Neisseria gonorrhoeae. Vibrio cholerae or Salmonella spp. Cell) For example, each target cell is a cell of a genus or species disclosed in Table 1 herein or Table 2 herein.
Reference is made to Journal of Plant Pathology (2010), 92 (3), 551-592 Edizioni ETS Pisa, 2010 551, LETTER TO THE EDITOR. “COMPREHENSIVE LIST OF NAMES OF PLANT PATHOGENIC BACTERIA, 1980-2007”, C. T. Bull et al, the disclosure of which is incorporated herein by reference to provide examples of bacterial genera, species and strains of importance to plants and which may be genera, species and strains of target cells of the invention. Examples are disclosed in Table 1 herein.
For example, each target cell is resistant to a fluoroquinolone. β-lactam (eg, methicillin), tetracycline or linezolid antibiotic. For example, each target cell is resistant to vancomycin, eg, wherein the cell is a vancomycin-resistant Enterococcus cell.
For example, each target cell is an Azotobacter, Burkwideria, Cupriavidus, Enterococcus, Lysobacter, Paucimonas, Paraburkholderia, Ralstonia, Stenotrophomonas, Variovorax, Xanthomonas or Pseudomonas cell, eg, wherein the target cell is comprised by a plant.
For example, each target cell is an E coli cell.
For example, each target cell is Klebsiella cell, eg, wherein the target cell is comprised by a plant.
For example, each target cell is an Azotobacter, Burkholderia, Cupriavidus. Lysobacter, Paraburkholderia, Ralstonia, Variovorax, Xanthomonas or Pseudomonas cell, eg, wherein the target cell is comprised by a plant.
For example, each target cell is a cell of a Pseudomonas species, optionally wherein the species is selected from Pseudomonas aeruginosa Pseudomonas anmygdali, Pseudomonas asturiensis, Pseudomonas avellanae, Pseudomonas cerasi, Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas otitidis, Pseudomonas putida, Pseudomonas salegens Pseudomonas savastanoi, Pseudomonas syringae and Pseudomonas viridiflava, eg, wherein the target cell is comprised by a plant.
For example, each target cell is a cell of a species selected from Azotobacter chroococcum, Azotobacter salinestris, Burkholderia ambifaria, Burkholderia cenocepacia, Burkholderia lata, Burkholderia pyrrocinia, Cupriavidus basilensis, Cupriavidus necator, Cupriavidus taiwanensis, Lysobacter gunmosus, Paraburkholderia sprentiae, Paraburkholderia terricola, Ralstonia pseudosolanacearum, Ralstonia solanacearum, Variovorax paradoxus, Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas campestris Xanthomonas citri, Xanthomonas euvesicatoria and Xanthomonas perforans, eg, wherein the target cell is comprised by a plant.
For example, each target cell is a Stenotrophomonas, Enterococcus, Paucimonas or Pseudomonas cell, eg, wherein the target cell is comprised by a plant.
For example, each target cell is a cell of a Pseudomonas species, optionally wherein the species is selected from Pseudomonas amygdali. Pseudomonas asturiensis, Pseudomonas avellanae. Pseudomonas cerasi, Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas putida, Pseudomonas savastanoi, Pseudomonas syringae and Pseudomonas viridiflava, eg, wherein the target cell is comprised by a plant.
For example, each target cell is a cell of a species selected from Stenotrophomonas rhizophila. Enterococcus faecalis, Paucimonas lemoignei, Pseudomonas amygdali, Pseudomonas asturiensis, Pseudomonas avellanae, Pseudomonas cerasi, Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas putida, Pseudomonas savastanoi, Pseudomonas syringae and Pseudomonas viridiflava, eg, wherein the target cell is comprised by a plant.
For example, the carrier is an E coli cell (eg, E coli, K12, Nissle or S17 cell). eg, wherein the cell is for administration to a human or animal subject, such as to treat or prevent a disease or condition. For example, each carrier cell is an Bacteroides cell, eg, wherein the cell is for administration to a human or animal subject, such as to treat or prevent a disease or condition. For example, each carrier cell is an Clostridiales cell, eg, wherein the cell is for administration to a human or animal subject, such as to treat or prevent a disease or condition.
For example, each carrier cell is a gram-positive bacterial cell. For example, each carrier cell is a gram-negative bacterial cell. For example, the carrier cell is a cell of a genus or species disclosed in Table 1 of WO2017211753 (the disclosure of this table and each genus and species individually being incorporated herein for disclosure of cell genus or species that may be used in the present invention).
For example, the carrier cell is a cell of phylum Proteobacteria, class Gammaproteobacteria, order Pseudomonadales or family Pseudomonadaceae. In a preferred example, the carrier is a Pseudomonas (eg, P fluorscens) cell, eg, wherein the target cell is comprised by a plant.
For example, each carrier cell is Klebsiella cell, eg, wherein the target cell is comprised by a plant.
For example, the carrier is a gram positive cell. eg, a Bacillus (such as Bacillus subtilis) or Cloistridiales (such as Clostridium butyricum) cell.
In an example, the subject is a shellfish. The shellfish may be selected from shrimp, crayfish, crab, lobster, clam, scallop, oyster, prawn and mussel.
The subject may be any subject disclosed herein. The subject may be an animal, such as a livestock animal, eg, a bird (such as a poultry bird; or a chicken or a turkey) or swine.
In an alternative, the subject is a plant, eg, and the target bacteria are plant pathogen bacteria. In an example, the target bacteria are Pseudomonas, eg, P syringae or P aeruginosa.
In an alternative, the carrier and target cells are archaeal cells. For example the target cells are methanobacterium cells. For example the target cells are methanogen cells. For example, the target cells comprise one or more species of cell selected from:
Optionally, the target cells are not pathogenic to the subject, for example when the method is a non-medical method. In an example, the method is a cosmetic method.
In the example, optionally the subject or animal is a livestock animal, such as a cow, sheep. goat or chicken (preferably a cow). Optionally, eg, wherein the subject is an animal (eg, a livestock animal or a wild animal), the target cells are zoonotic bacterial cells, such as cells of a species selected from Bacillus anthracis, Mycobacterium bovis (eg, wherein the animal is a cow), Campylobacter spp (eg. wherein the animal is a poultry animal), Mycobacterium marinum (eg. wherein the animal is a fish), Shiga toxin-producing E. coli (eg. wherein the animal is a ruminant), Listeria spp (eg, wherein the animal is a cow or sheep), Chlamydia abortus (eg, wherein the animal is a sheep), Coxiella burnetii (eg, wherein the animal is a cow, sheep or goat), Salmonella spp (eg, wherein the animal is a poultry animal), Streptococcus suis (eg, wherein the animal is a pig) and Corynebacterium (eg, C ulcerans) (eg, wherein the animal is a cow).
In an example, a plurality of carrier cells as described herein (eg, carrier cells of any configuration, aspect, example or embodiment described herein) is administered to the subject, wherein the carrier cells comprise the nucleic acid encoding P1.
In an example, each animal is a chicken. In an example, each animal is a cow (eg, a beef or dairy cow).
Optionally the method modifies target cells in the gastrointestinal tract of the subject human or animal; optionally the method modifies target cells in the jejunum, ileum, colon, liver, spleen or caecum of the subject: optionally wherein the animal is a bird and the method modifies target cells in the caecum of the bird. In an example the method is carried out on a group (optionally a flock or herd) of animals, wherein some or all of the animals comprise target cells.
Optionally, the plasmid comprises a RP4 origin of transfer (oriT). The plasmid may be any type of plasmid disclosed herein.
P2 may be any antibacterial agent disclosed herein or a component thereof, preferably a guided nuclease that is programmed to cut one or more target sequences in target cells. A suitable nuclease may be a TALEN, meganuclease, zinc finger nuclease or Cas nuclease. For example, the agent comprises one or more components (eg, a Cas nuclease and/or a guide RNA or a crRNA) of a CRTSPR/Cas system that is operable in a target cell to cut a protospacer sequence comprised by the target cell (eg, comprised by the vector, such that cut vector is degraded in the target cell). For example, the system is operable to cut at least 2 or 3 different protospacer sequences comprised by the vector of the invention. Optionally, P2 is operable to cut a plurality of different protospacer sequences comprised by the vector (and optionally further operable to cut the carrier cell genome, eg, a carrier cell chromosomal or episomal protospacer sequence, wherein cutting thereof is lethal to the carrier cell—as explained elsewhere, this is useful to reduce or remove carrier cells from the subject or microbiota when desired, such as after a desired level of P1 expression in target cells has been obtained). Optionally, the agent comprises one or more components of a CRISPR/Cas system that is operable to cut at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different protospacer sequences comprised by the vector and/or carrier cell genome (eg, comprised by the carrier cell chromosome).
In an embodiment, P2
This is useful as an “off switch” to remove the vector from the target cell, eg, for downregulating the capability of P1 expression in a plurality of target cells that have been previously contacted with carrier cells and into which target cells vectors of the invention have been transferred for P1 expression. For example, this usefully can be used to clear the microbiota of a subject so that it can produce less or no P1 after cutting of vector nucleic acid has taken place. In this way, for example, it is possible in a first step to modify a microbiota of a subject (eg, a human gut microbiota) so that it produces P1 (eg, where P1 expression is useful for treatment or prevention of a disease or condition or alternatively for non-medical utility) and thereafter P1 expression can be reduced or removed and the microbiota can be restored to a partially modified or unmodified condition.
Any administration of cells to a subject herein may be by oral administration. Any administration of cells to a subject herein may preferably be by administration to the GI tract. Any administration of cells to a subject herein may be by systemic, intranasal or inhaled administration.
There is provided the following definitions:—
Homologue: A gene, nucleotide or protein sequence related to a second gene, nucleotide or protein sequence by descent from a common ancestral DNA or protein sequence. The term, homologue, may apply to the relationship between genes separated by the event of or to the relationship between genes separated by the event of genetic duplication.
Orthologue: Orthologues are genes, nucleotide or protein sequences in different species that evolved from a common ancestral gene, nucleotide or protein sequence by speciation. Normally, orthologues retain the same function in the course of evolution.
Optionally, each P2 is a guide RNA. Optionally, each vector (eg, plasmid) encodes a plurality of guide RNAs or crRNAs of a CRISPR/Cas system wherein the guide RNAs or crRNAs are operable with Cas nuclease in the target cell to recognise a plurality of protospacer sequences comprised by the vector and/or carrier cell genome, optionally wherein the protospacer sequences comprise one or more nucleotide sequences of genes selected. In an example, the vector additionally or alternatively encodes a Cas, eg, a Cas9, Cas3, Cpf1, Cas12, Cas13, CasX or CasY.
In an embodiment, a Cas herein is a Type I, H, HI, IV, V or VI Cas, preferably a Type I or II Cas.
In an example, the vector also encodes a Cas3 and cognate Cascade proteins (eg, CasA, B, C, D and E). Optionally, the Cas (and Cascade of present) are E coli Cas (and Cascade).
The plasmid may comprise one or more CRISPR spacers, wherein each spacer consists of 20-40, 25-35, or 30-35 consecutive nucleotides, eg, consecutive nucleotides selected from
Optionally, the plasmid comprises a RP4 origin of transfer (oriT) and/or a p15A origin of replication.
In an example, the plasmid is a conjugative phagemid.
In an example, the vector encodes a Cas3 and optionally one or more Cascade proteins (eg, one or more of CasA, B, C, D and E). In an embodiment, the vector encodes a Cas3 and CasA, B, C, D and E. In an embodiment, the vector encodes an E coli Cas3 and CasA, B, C, D and E. Optionally, the guided nuclease (eg, Cas3) is a Type I-A, -B, -C, -D, -E, -F or -U Cas.
In an example, P2 in any configuration, aspect, example, option or embodiment herein comprises one or more components of a CRISPR/Cas system that is operable in the target cell to cut a protospacer sequence comprised by the vector. Additionally, in an example, P2 in any configuration, aspect, example, option or embodiment herein comprises one or more components of a CRISPR/Cas system (eg, the same system as in the first sentence in this paragraph) that is operable in the carrier cell to cut a protospacer sequence comprised by the carrier cell genome (eg, a chromosomal or episomal sequence whose cutting is lethal to the carrier cell). This is useful to clear the carrier cell from the subject (eg, from a gut microbiota) when expression of P1 is no longer required.
In an example, the system is operable to cut at least 3 different protospacer sequences comprised by the vector or carrier cell genome.
In an example, the vector
The expression of (a) and/or (b) may be inducible by exposure of the carrier cell to a regulator agent, eg, R. For example, exposure of the carrier and target cells to R may induce production of P2, wherein P2 comprises component (a) and/or (b) whereby vector nucleic acid is cut in the target cell and the genome of the carrier cell is also cut. This kills the carrier cell and leads to degradation of the vector in the target cell. This usefully can then clear the subject of the vector and the carrier cell, eg after a desirable amount of P1 has been expressed in the subject.
Optionally, the Cas, Cascade proteins, gRNAs and crRNAs are E. coli K12 (MG1655) Cas, Cascade proteins, gRNAs and crRNAs respectively. Optionally, the vector is devoid of nucleotide sequences encoding Cas1 and Cas2 proteins.
In embodiments, by action of P2 (eg, components (a) and/or (b)) in the carrier cells, growth or proliferation of carrier cells is reduced (eg, by at least 40, 50, 60, 70, 80, or 90% compared to growth in the absence of P2 therein).
For example, the carrier cells may be comprised by a medicament for treating or preventing a disease or condition in a human or animal; a growth promoting agent for administration to animals for promoting growth thereof; killing zoonositic bacteria in the animals; for administration to livestock as a pesticide; a pesticide to be applied to plants; or a plant fertilizer.
An advantage may be that the carrier cells may be used as producer cells in which vectors of the invention can be replicated (eg, before (eg, in vitro) and/or following administration to the subject).
A method of delivery of a vector can be by bacterial conjugation, a natural process whereby a donor bacterium (carrier bacterium) transfers plasmid DNA from itself to a recipient bacterium (target bacterium). Donor bacteria elaborate a surface structure, the pilus which can be considered to be like a syringe or drinking straw through which the DNA is delivered. The donor pilus binds to the surface of a receptive recipient and this event triggers the process of DNA transfer. Plasmids are suitable for this conjugative process, where the plasmid comprises DNA encoding the agent of the invention.
DNA transfer by conjugation may only take place with a ‘susceptible recipient’ but does not generally occur with a recipient carrying a similar type of plasmid. Because conjugation is via pilus bridge, it is possible for that bridge to attach itself not to a recipient but to the donor bacterium. This could result in a futile cycle of transfer of the plasmid DNA to itself. Plasmids thus naturally encode incompatibility factors. One is a surface arrayed protein that prevents the pilus binding to bacterium displaying that surface protein such as itself or any other bacterium carrying the same plasmid. Additionally, plasmids naturally encode another incompatibility system that closely regulates the copy number of the plasmid inside a bacterium. Thus, should a conjugation event manage to evade surface exclusion and start to transfer DNA by conjugation, the recipient will prevent that plasmid establishing as it already maintains the current copy number and will not accept and maintain a further unwanted additional copy.
In an example of the invention, the plasmid is a member of a plasmid incompatibility group, wherein the target cell does not comprise a plasmid of said group. Optionally. the plasmid of the invention is a member of the incompatibility group P (ie, the plasmid is an incP plasmid). For example within the Enterobacteriaceae the following is a non-exclusive list of potential plasmids that could use for delivery: IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Incl, IncA, IncB, IncC, IncH, IncIa, InclIc, Incl2, Incly, IncJ, InwL, IncN, Inc2e, IncO, IncP, IncS, IncT and/or IncW. Thus, optionally, the target cell is an Enterobacteriaceae cell and the vector of the invention is a plasmid, wherein the plasmid is selected from an IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Incl, IncA, IncB, IncC, IncH, Incla, InclIc, IncII, Incl2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT and IncW plasmid.
Preferably, the subject is a human or animal and the plasmid is an IncI plasmid, eg, an IncIH or IncI2 plasmid.
In an example, the carrier cell of the invention comprises two or more plasmids, each plasmid comprising a DNA that encodes a respective P1 and P2 (P1/P2 may be the same in the cells or the cells may comprise different P1 and/or P2). Optionally, a first of said plasmids is a member of a first incompatibility group, wherein the target cell does not comprise a plasmid of said first group, and wherein a second of said plasmids is a member of a second incompatibility group, wherein the target cell does not comprise a plasmid of said second group. For example, a carrier cell may comprise an incP plasmid encoding P1 and P2 (eg, a CRISPR-Cas system or a component thereof (eg, encoding a first crRNA or guide RNA that targets a first protospacer sequence of the vector)) and wherein the carrier cell further comprises an incF1 plasmid (eg, encoding P1 and P2 (eg, an anti-carrier cell CRISPR-Cas system or a component thereof (eg, encoding a second crRNA or guide RNA that targets a protospacer sequence of the carrier cell genome)). The protospacers may comprise different nucleotide sequences. Optionally, the carrier cell comprises a group of plasmids comprising 2, 3, 4, 5, 6 or more different types of plasmid, wherein each plasmid is capable of being conjugatively transferred into a target cell, wherein the plasmids encode different P1 and/or P2 products. For example, the plasmids encode different cRNAs or gRNAs that target different protospacers comprised by the vector, carrier cell and/or target cell genome. For example, the group of plasmids comprises up to n different types of plasmid, wherein the plasmids are members of up to n different incompatibility groups, eg, groups selected from IncFI, ItncFlI, IncFIll, IncFIV, hncFV, lncM, lnc9, InclO, Incl, IncA, IncB, IncC, IncH, IncIa, IclIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, lncT and IncW. For example, n=2, 3, 4, 5, 6, 7, 8, 9 or 10.
For example, the carrier cell comprises (i) a first vector (eg, plasmid) that encodes a P2 which is a first type of CRISPR/Cas system that targets a first protospacer comprised by the vector, or encodes a component of said system; and (ii) a second vector (eg, plasmid) that encodes a P2 which is a second type of CRISPR/Cas system that targets a second protospacer comprised by the vector or carrier cell genome (eg, a carrier cell chromosomal sequence), or encodes a component of said system, wherein the first and second types are different. For example, the first type is a Type I system, and the second type is a Type H system (eg, the first vector encodes a Cas3, Cascade and a crRNA or guide RNA that is operable with the Cas3 and Cascade in the target cell to modify the first protospacer; and the second vector encodes a Cas9 and a crRNA or guide RNA that is operable with the Cas9 in the target cell to modify the second protospacer). In an alternative, the Cas3 and Cascade are encoded by endogenous target cell genes, wherein the first vector encodes the crRNA or guide RNA that is operable with the endogenous Cas3 and Cascade in the target cell to modify the first protospacer. In an alternative, the Cas9 is encoded by an endogenous target cell gene, wherein the second vector encodes the crRNA or guide RNA that is operable with the endogenous Cas9 in the target cell to modify the second protospacer. Optionally, the Cas3 and Cascade are encoded by endogenous genes of the target cell and the Cas9 is encoded by the second vector.
Instead of a Type I and Type II system, the invention alternatively provides in an embodiment a first vector (eg, plasmid) encoding a Type I CRISPR/Cas system (or component thereof, eg, a Cas3 or a crRNA or a gRNA) and a second vector (eg, plasmid) encoding a Type III CRISPR/Cas system (or a component thereof). Instead of a Type I and Type II system, the invention alternatively provides in an embodiment a first vector encoding a Type I CRISPR/Cas system (or component thereof) and a second vector encoding a Type IV CRISPR/Cas system (or a component thereof). Instead of a Type I and Type II system, the invention alternatively provides in an embodiment a first vector encoding a Type I CRISPR/Cas system (or component thereof) and a second vector encoding a Type V CRISPR/Cas system (or a component thereof). Instead of a Type I and Type II system, the invention alternatively provides in an embodiment a first vector encoding a Type I CRISPR/Cas system (or component thereof) and a second vector encoding a Type VI CRISPR/Cas system (or a component thereof).
Instead of a Type I and Type 11 system, the invention alternatively provides in an embodiment a first vector encoding a Type II CRISPR/Cas system (or component thereof, eg, a Cas9 or a crRNA or a gRNA) and a second vector encoding a Type III CRISPR/Cas system (or a component thereof). Instead of a Type I and Type 11 system, the invention alternatively provides in an embodiment a first vector encoding a Type II CRISPR/Cas system (or component thereof) and a second vector encoding a Type IV CRISPR/Cas system (or a component thereof). Instead of a Type I and Type H system, the invention alternatively provides in an embodiment a first vector encoding a Type H CRISPR/Cas system (or component thereof) and a second vector encoding a Type V CRISPR/Cas system (or a component thereof). Instead of a Type I and Type II system, the invention alternatively provides in an embodiment a first vector encoding a Type II CRISPR/Cas system (or component thereof) and a second vector encoding a Type VI CRISPR/Cas system (or a component thereof).
Instead of a Type I and Type 11 system, the invention alternatively provides in an embodiment a first vector encoding a Type V CRISPR/Cas system (or component thereof, eg, a Cas12a or a crRNA) and a second vector encoding a Type III CRISPR/Cas system (or a component thereof). Instead of a Type I and Type H system, the invention alternatively provides in an embodiment a first vector encoding a Type V CRISPR/Cas system (or component thereof) and a second vector encoding a Type IV CRISPR/Cas system (or a component thereof). Instead of a Type I and Type II system, the invention alternatively provides in an embodiment a first vector encoding a Type V CRISPR/Cas system (or component thereof) and a second vector encoding a Type V CRISPR/Cas system (or a component thereof). Instead of a Type I and Type H system, the invention alternatively provides in an embodiment a first vector encoding a Type V CRISPR/Cas system (or component thereof) and a second vector encoding a Type VI CRISPR/Cas system (or a component thereof).
Instead of a Type I and Type II system, the invention alternatively provides in an embodiment first and second vectors (eg, plasmids), each encoding a Type I CRISPR/Cas system (or a component thereof). Instead of a Type I and Type H system, the invention alternatively provides in an embodiment first and second vectors, each encoding a Type H CRISPR/Cas system (or a component thereof). Instead of a Type I and Type 11 system, the invention alternatively provides in an embodiment first and second vectors, each encoding a Type III CRISPR/Cas system (or a component thereof). Instead of a Type I and Type II system, the invention alternatively provides in an embodiment first and second vectors, each encoding a Type IV CRISPR/Cas system (or a component thereof). Instead of a Type I and Type II system, the invention alternatively provides in an embodiment first and second vectors, each encoding a Type V CRISPR/Cas system (or a component thereof). Instead of a Type I and Type H system, the invention alternatively provides in an embodiment first and second vectors, each encoding a Type VI CRISPR/Cas system (or a component thereof).
Optionally, the plasmids are members of different incompatibility groups, eg, groups selected from IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Incl, IncA, IncB, IncC, IncH, IncIa, InclIc, Incll, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT and IncW. In an example here, the target cell is an Enterobacteriaceae cell.
Advantageously, the carrier cells are for treating or preventing a target cell infection in a human or an animal subject (eg, a dog, cat, horse, chicken, cow, sheep, goat, pig, fish or shellfish).
Advantageously, the carrier cells are of a species that is probiotic to said subject or is probiotic to humans or animals (eg, chickens). For example, the carrier cells are probiotic Bacteriodetes (eg, Bacteroides) cell, eg, wherein the subject is a human. For example, the carrier cells are probiotic Clostridiales cell, eg, wherein the subject is a human. For example, the carrier cells are probiotic E coli cell. For example, the carrier cells are probiotic Bacillus cell, eg, wherein the subject is a plant. Advantageously, each vector (eg, virus or plasmid) encodes one or more guide RNAs or one or more crRNAs that are capable of hybridizing in the target cell to respective vector target nucleic acid sequence(s). For example, each vector encodes 2, 3, 4, 5, 6, 7, 7, 9, or 10 (or more than 10) different gRNAs or different crRNAs that hybridise to a respective target sequence, wherein the target sequences are different from each other. For example, 3 different gRNAs or crRNAs are encoded by each vector. For example, 2 different gRNAs or crRNAs are encoded by each vector. For example, 3 different gRNAs or crRNAs are encoded by each vector. For example, 4 different gRNAs or crRNAs are encoded by each vector. For example, 3 different gRNAs or crRNAs are encoded by each vector. For example, 5 different gRNAs or crRNAs are encoded by each vector. For example, 6 different gRNAs or crRNAs are encoded by each vector. For example, 7 different gRNAs or crRNAs are encoded by each vector. For example, 8 different gRNAs or crRNAs are encoded by each vector. For example, 9 different gRNAs or crRNAs are encoded by each vector. For example, 10 different gRNAs or crRNAs are encoded by each vector. For example, 11 different gRNAs or crRNAs are encoded by each vector. For example, 12 different gRNAs or crRNAs are encoded by each vector. For example, 13 different gRNAs or crRNAs are encoded by each vector.
In an example, the target cells are Salmonella cells (eg, wherein the subject is a chicken). In an example, the target cells are Campylobacter cells (eg, wherein the subject is a chicken). In an example, the target cells are Edwardsiella cells (eg, wherein the subject is a fish or shellfish. eg, a catfish or a shrimp or prawn). In an example, the target cells are Bacteriodetes (eg, Bacteroides) cells. In an example the target cells are Clostridiales cells. In an example, the target cells are E coli cells.
Optionally, each plasmid comprises an expressible tra1 and/or tra2 module or a homologue thereof for conjugative transfer of the plasmid between cells. Any episome herein may be a plasmid.
Optionally, each plasmid comprises an expressible operon of a tra1 and/or tra2 module or a homologue thereof for conjugative transfer of the plasmid between cells.
Optionally, each plasmid is a modified RK2 or R6K plasmid. The modification comprises an insertion of nucleotide sequences encoding P1 and P2.
Optionally, each plasmid comprises an oriV, eg, oriV of an IncI (eg, IncI1 or IncI2), RK2 or R6K plasmid, or a homologue thereof. Optionally, each plasmid comprises an oriV of an IncI (eg, IncI1 or IncI2), RK2 or R6K plasmid, or a homologue thereof
Optionally, each plasmid comprises an oriT, eg, oriT of an IncI1 or IncI2 plasmid. Optionally, each plasmid is a modified an IncI (eg, IncI1 or IncI2) plasmid. The modification (for the First to Third Configurations, and optionally for the Fourth Configuration) comprises an insertion of nucleotide sequences encoding P1 and P2.
Optionally, P2 comprises one or more components of a CRISPR/Cas system that is operable in the target cell to cut a protospacer sequence comprised by the vector or carrier cell genome, eg, wherein the protospacer sequence is comprised by the carrier cell chromosome.
In an embodiment, the cutting herein kills the carrier cell or causes degradation of the vector in the target cell. In an alternative, the cutting inhibits the growth or proliferation of the target cell.
Optionally, P2 encodes a guide RNA or crRNA of a CRISPR/Cas system that is operable with a Cas nuclease in the target cell to cut a protospacer sequence comprised by the vector.
In an example of the First to Third Configurations, the protospacer is comprised by a gene required for vector viability or maintenance in the cell. In an example of the Fourth Configuration, the protospacer is comprised by a gene required for carrier cell viability.
Optionally, each vector (eg, virus (eg, phage) or plasmid) comprises a gene that encodes a product, wherein the product is essential for survival or proliferation of the carrier cell when in an environment that is devoid of the product, wherein the carrier cell chromosome does not comprise an expressible gene encoding the product and optionally the vector nucleic acid is the only episomal nucleic acid comprised by the carrier cell that encodes the product. For example, the gene is selected from an aroA, argiH, hisD, leuB, lysA, retB, proC, thrC, pheA, tyrA, trpC and pflA gene; or wherein the gene is an anti-toxin gene and optionally the vector encodes a cognate toxin.
For example, the carrier cell is an E coli (eg, Nissle, F18 or S17 E coli strain) cell. For example, the carrier cell is a Bacillus (eg, B subtilis), Enterococcus or Lactobacillus cell, eg, wherein the subject is a plant.
Optionally, each carrier cell is for administration to a microbiota of a human or animal subject for medical use.
For example, the medical use is for treating or preventing a disease disclosed herein. For example, the medical use is for treating or preventing a condition disclosed herein.
Optionally, the medical use is for the treatment or prevention of a disease or condition mediated by said target cells. Optionally, the medical use is for the treatment or prevention of a disease or condition mediated by cells of a microbiota that also comprises said target cells. For example, P1 is secreted from target cells and acts to kill or modify the growth or metabolism of neighbouring cells in the microbiota.
Optionally, the carrier cell(s) is(are) for administration to a human or animal for enhancing growth or weight of the human or animal. Optionally, the carrier cell(s) is(are) for administration to a human or animal for reducing growth or weight of the human or animal. Optionally, the carrier cell(s) is(are) for administration to a human or animal for reducing obesity in the human or animal.
In an embodiment, the administration is to a human for enhancing the growth or weight of the human. Optionally, the enhancing is not a medical therapy. Optionally, the enhancing is a medical therapy.
Optionally, the use comprises the administration of a plurality of carrier cells to a microbiota (eg, a gut microbiota) of a human or animal subject, wherein the microbiota comprises target cells and the vector nucleic acid is transferred into target cells for expression therein of P1, thereby killing microbiota cells in the subject or reducing the growth or proliferation of microbiota cells.
Optionally, the use comprises the administration of a plurality of carrier cells to a microbiota (eg, a gut microbiota) of a human or animal subject, wherein the microbiota comprises target cells and the vector nucleic acid is transferred into target cells for expression therein of P1, thereby promoting growth or metabolism of microbiota cells in the subject.
Optionally, the use comprises the administration of a plurality of carrier cells to a microbiota (eg, a gut microbiota) of a human or animal subject, wherein the microbiota comprises target cells and the vector nucleic acid is transferred into target cells for expression therein of P1, wherein P1 is a protein (eg, an enzyme) in a metabolic pathway in cells of the microbiota. For example, P1 is secreted by target cells and is taken up by further cells in the microbiota for use of P1 in a metabolic pathway in the further cells.
Optionally, the use comprises the administration of a plurality of carrier cells to a microbiota (eg, a gut microbiota) of a human or animal subject, wherein the microbiota comprises target cells and the vector nucleic acid is transferred into target cells for expression therein of P1, wherein P1 is a protein that is capable of sequestering a substance (eg, a protein, peptide, nucleic acid (eg, RNA), carbohydrate (eg, a sugar or precursor thereof), amino acid, lipid, fatty acid, ion or chemical compound) in the subject. For example, P1 is secreted by target cells and is taken up by further cells in the microbiota for use of P1 as a sequestering agent in the further cells. For example, P1 is secreted by target cells for use of P1 as a sequestering agent in the subject, eg, in the microbiota, an organ, tissue, cell or bloodstream of the subject.
For example, a plant herein in any configuration or embodiment of the invention is selected from a tomato plant, a potato plant, a wheat plant, a corn plant, a maize plant, an apple tree, a bean-producing plant, a pea plant, a beetroot plant, a stone fruit plant, a barley plant, a hop plant and a grass. For example, the plant is a tree, eg, palm, a horse chestnut tree, a pine tree, an oak tree or a hardwood tree.
For example the plant is a plant that produces fruit selected from strawberries, raspberries, blackberries, redcurrants, kiwi fruit, bananas, apples, apricots, avocados, cherries, oranges, clementines, satsumas, grapefruits, plus, dates, figs, limes, lemons, melons, mangos, pears, olives or grapes. Optionally, the plant is a dicotyledon. Optionally, the plant is a flowering plant. Optionally, the plant is a monocotyledon.
In an example, the weight (ie, biomass) of a plant is dry weight. For example, the method is for increasing plant dry weight (eg, within 1 or 2 weeks of said administration). Optionally, the increase is an increase of at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% compared to a control plant of the same species or strain to which the administration if carrier cells has not taken place, wherein all plants are kept under the same environmental conditions. For example, such an increase is within 1, 2, 3, 4, 5, 6, or 8 weeks following the first administration of the carrier cells. In an example, the method is for increasing the dry weight of a leaf and/or fruit of the plant, such as a tomato plant.
In an example, the weight is wet weight. For example, the method is for increasing plant wet weight (eg, within 1 or 2 weeks of said administration). Optionally, the increase is an increase of at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% compared to a control plant of the same species or strain to which the administration if carrier cells has not taken place, wherein all plants are kept under the same environmental conditions. For example, such an increase is within 1, 2, 3, 4, 5, 6, or 8 weeks following the first administration of the carrier cells. In an example, the method is for increasing the dry weight of a leaf and/or fruit of the plant, such as a tomato plant.
For example, the microbiota is comprised by a leaf, trunk, root or stem of the plant.
The target bacteria (or target cell) may be comprised by a microbiota of a plant. In an example, the microbiota is comprised by a leaf. In an example, the microbiota is comprised by a xylem. In an example, the microbiota is comprised by a phloem. In an example, the microbiota is comprised by a root. In an example, the microbiota is comprised by a tuber. In an example, the microbiota is comprised by a bulb. In an example, the microbiota is comprised by a seed. In an example, the microbiota is comprised by an exocarp, epicarp, mesocarp or endocarp. In an example, the microbiota is comprised by a fruit, eg, a simple fruits; aggregate fruits; or multiple fruits. In an example, the microbiota is comprised by a seed or embryo, eg, by a seed coat; a seed leaf; cotyledons; or a radicle. In an example, the microbiota is comprised by a flower, eg, comprised by a peduncle; sepal: petals; stamen; filament; anther or pistil. In an example, the microbiota is comprised by a root; eg, a tap root system, or a fibrous root system. In an example, the microbiota is comprised by a leaf or leaves, eg, comprised by a leaf blade, petiole or stipule. In an example, the microbiota is comprised by a stem, eg, comprised by bark, epidermis, phloem, cambium. xylem or pith.
For example, the biofilm is comprised by a lung of the subject, eg, wherein the target cells are Pseudomonas (eg, P aeruginosa) cells. This may be useful wherein the subject is a human suffering from a lung disease or condition, such as pneumonia or cystic fibrosis, wherein P1 is a therapeutic protein that is expressed by modified target cells of the biofilm. For example, the biofilm is comprised by an animal or human organ disclosed herein. For example, the biofilm is comprised by a microbiota of a human or animal disclosed herein.
Optionally, said surface is a surface ex vivo, such as a surface comprised by a domestic or industrial apparatus or container.
Optionally, the target cells are comprised by a biofilm, eg, a biofilm as disclosed herein.
There is provided:—
A pharmaceutical composition, livestock growth promoting composition, soil improver, herbicide, plant fertilizer, food or food ingredient sterilizing composition, dental composition, personal hygiene composition or disinfectant composition (eg, for domestic or industrial use) comprising a plurality of the carrier cells.
Herein, a carrier cell is, eg, a commensal or probiotic cell for administration to a human or animal subject. For example, the carrier cell is commensal in a microbiota (eg, gut or blood microbiota) of a human or animal subject, wherein the carrier is for administration to the subject. In an example, a carrier cell is a prokaryotic cell. In an example, a carrier cell is a bacterial cell (and optionally the target cell is a bacterial cell). In an example, a carrier cell is an archaeal cell (and optionally the target cell is an archaeal cell)
Optionally, the carrier cell is a gram-positive bacterial cell and the target cell is a gram-positive bacterial cell.
Optionally, the carrier cell is a gram-positive bacterial cell and the target cell is a gram-negative bacterial cell.
Optionally, the carrier cell is a gram-negative bacterial cell and the target cell is a gram-positive bacterial cell.
Optionally, the carrier cell is a gram-negative bacterial cell and the target cell is a gram-negative bacterial cell.
Optionally, the carrier cell is a Bacteroides bacterial cell and the target cell is a gram-positive bacterial cell. Optionally, the carrier cell is a Bacteroides bacterial cell and the target cell is a gram-negative bacterial cell. Optionally, the carrier cell is a Bacteroides bacterial cell and the target cell is a Bacteroides bacterial cell. Optionally, the carrier cell is a Bacteroides bacterial cell and the target cell is an E coli bacterial cell. Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Bacteroides bacterial cell. For example, in these options the subject is a human or animal, preferably a human.
Optionally, the carrier cell is a Clostridiales bacterial cell and the target cell is a gram-positive bacterial cell. Optionally, the carrier cell is a Clostridiales bacterial cell and the target cell is a gram-negative bacterial cell. Optionally, the carrier cell is a Bacteroides bacterial cell and the target cell is a Clostridiales bacterial cell. Optionally, the carrier cell is a Clostridiales bacterial cell and the target cell is an E coli bacterial cell. Optionally. the carrier cell is an E coli bacterial cell and the target cell is a Clostridiales bacterial cell. For example, in these options the subject is a human or animal, preferably a human.
Optionally, the carrier cell is an Escherichia (eg, E coli) bacterial cell and the target cell is a gram-positive bacterial cell. Optionally, the carrier cell is an Escherichia (eg, E coli) bacterial cell and the target cell is a gram-negative bacterial cell. Optionally, the carrier cell is a Bacteroides bacterial cell and the target cell is an Escherichia (eg, E coli) bacterial cell. Optionally. the carrier cell is an Escherichia (eg, E coli) bacterial cell and the target cell is an E coli bacterial cell. Optionally, the carrier cell is an E coli bacterial cell and the target cell is an Escherichia (eg, E coli) bacterial cell. For example, in these options the subject is a human or animal, preferably a human.
Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a gram-positive bacterial cell. Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a gram-negative bacterial cell. Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a Salmonella bacterial cell. Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is an E coli bacterial cell. Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Pseudomonas bacterial cell. For example, in these options the subject is a plant.
Optionally, the carrier cell is an E coli bacterial cell and the target cell is a gram-positive bacterial cell.
Optionally, the carrier cell is an E coli bacterial cell and the target cell is a gram-negative bacterial cell.
Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Salmonella bacterial cell.
Optionally, the carrier cell is an E coli bacterial cell and the target cell is an E coli bacterial cell.
Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Pseudomonas bacterial cell.
A Bacillus cell herein is optionally a B subtilis cell.
Optionally, the carrier cell is a probiotic or commensal Bacteroides bacterial cell for administration to a human or animal subject. Optionally. the carrier cell is a probiotic or commensal Clostridiales bacterial cell for administration to a human or animal subject. Optionally, the carrier cell is a probiotic or commensal E coli bacterial cell for administration to a human or animal subject. Optionally, the carrier cell is a probiotic or commensal Bacillus bacterial cell for administration to a human or animal subject.
Herein, optionally the plasmid is a closed circular DNA.
In an embodiment, the vector (eg, plasmid) nucleic acid is DNA. Optionally, the DNA is dsDNA. In an embodiment, the vector DNA is ssDNA. In an embodiment, the vector (eg, plasmid) nucleic acid is RNA.
In an example, the target cell is a cell of a species that does not cause nosocomial infection in humans.
Optionally, the target cell is comprised by an animal (eg, poultry animal (such as chicken), swine, cow, fish (eg, catfish or salmon) or shellfish (eg, prawn or lobster)) microbiota. Optionally, the microbiota is a gut microbiota. For example, the target cell is a cell comprised by a human or animal (eg, chicken) gut biofilm. For example, the target cell is a cell comprised by a gut biofilm sample ex vivo. For example, the target cell is a cell comprised by a human or animal (eg, chicken) lung biofilm. For example, the target cell is a cell comprised by a lung biofilm sample ex vivo. For example, the target cell is a cell comprised by a human or animal (eg, chicken) skin biofilm. For example, the target cell is a cell comprised by a skin biofilm sample ex vivo.
In an embodiment, each plasmid comprises an oriV and/or an oriT. In an embodiment, each plasmid comprises a bacterial oriV and/or an oriT.
In an embodiment, the plasmid comprises an oriV and does not encode any replication protein (eg, pir or trfA) that is operable with the oriV to initiate replication of the plasmid.
In an example, the invention relates to a composition comprising a plurality of carrier cells of the invention. Optionally, all of the carrier cells comprise identical said vectors (eg, plasmids). Optionally, the plurality comprises a first sub-population of carrier cells (first cells) and a second sub-population of carrier cells (second cells) wherein the first cells comprise identical first said vectors and the second cells comprise identical second said vectors (which are different from the first vectors of the first cells). For example, the first vectors encode a first guide RNA or crRNA and the second vectors encode a second guide RNA or crRNA, wherein the first guide RNA/crRNA is capable of hybridizing to a first protospacer sequence comprised by the vector in first target cells; and the second guide RNA/crRNA is capable of hybridizing to a second protospacer sequence in carrier or target cells, wherein the protospacers are different.
Optionally, the composition is comprised by a liquid (eg, an aqueous liquid or in water), the composition comprising the carrier cells at an amount of from 1×103 to 1×1010 (eg, from 1×104 to 1×1010; from 1×104 to 1×109; from 1×104 to 1×108; from 1×104 to 1×107; from 1×104 to 1×1010; from 1×103 to 1×109; from 1×103 to 1×108; from 1×103 to 1×107; from 1×105 to 1×1010; from 1×105 to 1×109; from 1×105 to 1×108; from 1×105 to 1×107; from 1×106 to 1×1010; from 1×106 to 1×109; from 1×106 to 1×108; or from 1×106 to 1×107) cfu/ml. For example, the liquid is a beverage, such for human or animal consumption. For example, the beverage is a livestock 30 beverage, eg, a poultry beverage (ie, a beverage for consumption by poultry, such as chicken).
In an example, the composition is a dietary (eg, dietary supplement) composition for consumption by humans or animals. In an example, the composition is a slimming composition for consumption by humans or animals. In an example, the composition is a growth promotion composition for consumption by humans or animals. In an example, the composition is a body building composition for consumption by humans. In an example, the composition is a probiotic composition for consumption by humans or animals. In an example, the composition is a biocidal composition for consumption by humans or animals. In an example, the composition is a pesticidal composition for consumption by humans or animals. In an example, the composition is a zoonosis control composition for consumption by animals.
In an example, the composition comprises vitamins in addition to the carrier cells. In an example, the composition comprises vitamin A, B (eg, B12), C, D, E and/or K in addition to the carrier cells. In an example, the composition comprises lipids in addition to the carrier cells. In an example, the composition comprises carbohydrates in addition to the carrier cells. In an example, the composition comprises proteins and/or amino acids in addition to the carrier cells. In an example, the composition comprises minerals in addition to the carrier cells. In an example, the composition comprises metal ions (eg, Mg2+, Cu2+ and/or Zn2+) in addition to the carrier cells. In an example, the composition comprises sodium ions, potassium ions, magnesium ions, calcium ions, manganese ions, iron ions, cobalt ions, copper ions, zinc ions and/or molybdenum ions.
In an example, the composition is a plant fertilizer composition. In an example, the composition is a herbicide. In an example, the composition is a pesticide composition for application to plants.
In any embodiment or example, where appropriate: The plants are, for example, crop plants. The plants are, for example, wheat. The plants are, for example, corn. The plants are, for example, maize. The plants are, for example, fruiting plants. The plants are, for example, vegetable plants. The plants are, for example, tomato plants. The plants are, for example, potato plants. The plants are, for example, grass plants. The plants are, for example, flowering plants. The plants are, for example, trees. The plants are, for example, shrubs.
In an example, the composition is for environmental application, wherein the environment is an outdoors environment (eg, application to a field or waterway or reservoir). In an example, the composition is comprised by a food or food ingredient (eg, for human or animal consumption). In an example, the composition is comprised by a beverage or beverage ingredient (eg, for human or animal consumption).
In an example the target cell(s) are biofilm cells found in a human, eg, wherein the biofilm is a gut, skin, lung, eye, nose, ear, gastrointestinal tract (GI tract), stomach, hair, kidney, urethra, bronchiole, oral cavity, mouth, liver, heart, anus, rectum, bladder, bowel, intestine, penis, vagina or scrotum biofilm. In an example the target cell(s) are animal biofilm cells, eg, wherein the biofilm is a gut, skin, lung, eye, nose, ear, gastrointestinal tract (GI tract), caecum, jejunum, ileum, colon, stomach, hair, feather, scales, kidney, urethra, bronchiole, oral cavity, mouth, liver, spleen, heart, anus, rectum, bladder, bowel, intestine, penis, vagina or scrotum biofilm. For example, the biofilm is a bird (eg, chicken) caecum biofilm. For example, the biofilm is a bird (eg, chicken) gastrointestinal tract (GI tract), caecum, jejunum, ileum. colon or stomach biofilm.
In an example, any method herein is ex vivo. In an example, a method herein is in vivo. In an example, a method herein is in vitro. In an example, a method herein is carried out in an environment, eg, in a domestic (such as in a house), industrial (such as in a factory) or agricultural environment (such as in a field). In an example, a method herein is carried out in or on a container; or on a surface.
In an example each vector (eg, plasmid) encodes one or more components of a CRTSPR/Cas system operable to perform vector protospacer cutting in the target cell (eg, wherein the protospacer comprises 10-20, 10-30, 10-40, 10-100, 12-15 or 12-20 consecutive nucleotides that are capable of hybridizing in the target cell with a crRNA or gRNA encoded by the vector). For example, the system is a Type I, II, III, IV or V CRISPR/Cas system.
In an example, each vector encodes a Cas9 (and optionally a second, different, Cas, such as a Cas3, Cas9, Cpf1, Cas13a, Cas13b or Cas10); and/or a Cas3 (and optionally a second, different. Cas, such as a Cas3, Cas9, Cpf1, Cas13a, Cas13b or Cas10). In an example, each vector encodes a Cas selected from a Cas3, Cas9, Cpf1, Cas13a, Cas13b and Cas10. Additionally or alternatively, the vector encodes a guide RNA or crRNA or tracrRNA. For example, the guide RNA or crRNA or tracrRNA is cognate to (ie, operable with in the target cell) the first Cas.
In an example, a Cas herein is a Cas9. In an example, a Cas herein is a Cas3. The Cas may be identical to a Cas encoded by the target bacteria.
In an embodiment, each plasmid is a shuttle vector.
Optionally, the target cell is devoid of a functional endogenous CRISPR/Cas system before transfer therein of the vector, eg, wherein the vector encodes a component of an exogenous CRISPR/Cas system that is functional in the target cell. An embodiment provides a medicament comprising a plurality of carrier cells of the invention, wherein each target cell is optionally according to this paragraph, for administration to a human or animal subject for medical use.
In an example, the composition of the invention is a herbicide, pesticide, insecticide, plant fertilizer or cleaning agent.
Optionally, target bacteria herein are comprised by a microbiota of the subject, eg, a gut microbiota. Alternatively, the microbiota is a skin, scalp, hair, eye, ear, oral, throat, lung, blood, rectal, anal, vaginal, scrotal, penile. nasal or tongue microbiota.
In an example the subject (eg, human or animal) is further administered a medicament simultaneously or sequentially with the carrier cell administration. In an example, the medicament is an antibiotic, antibody or antibody fragment (eg, an scFv, nanobody or Fab), immune checkpoint inhibitor (eg, an anti-PD-1, anti-PD-L1 or anti-CTLA4 antibody), adoptive cell therapy (eg. CAR-T therapy) or a vaccine.
In an embodiment, the vector encodes a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease. Thus, P2 may comprise a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease. Optionally, the vector encodes a restriction nuclease that is capable of cutting the vector and/or genome of the carrier cell (eg, chromosome of the carrier cell).
Optionally, the composition is a pharmaceutical composition for use in medicine practised on a human or animal subject. Optionally, the composition is not a medicament.
In an example, the animal is a livestock or companion pet animal (eg, a cow, pig, goat, sheep, horse. dog, cat or rabbit). In an example, the animal is an insect (an insect at any stage of its lifecycle, eg, egg, larva or pupa). In an example, the animal is a protozoan. In an example, the animal is a cephalopod.
Optionally, the composition is a herbicide, pesticide, food or beverage processing agent, food or beverage additive, petrochemical or fuel processing agent, water purifying agent, cosmetic additive, detergent additive or environmental (eg, soil) additive or cleaning agent.
For example, the carrier bacteria are Lactobacillus (eg, L reuteri or L lactis), E coli, Bacillus or Streptococcus (eg, S thermophilus) bacteria. Usefully, the carrier can provide protection for the plasmid from the surrounding environment. The use of a carrier may be useful for oral administration or other routes where the carrier can provide protection for the vector from the acid stomach or other harsh environments in the subject. Furthermore, the carrier can be formulated into a beverage, for example, a probiotic drink, eg, an adapted Yakult (trademark), Actimel (trademark), Kevita (trademark), Activia (trademark), Jarrow (trademark) or similar drink for human consumption.
Optionally, the carrier cell(s) or composition are for administration to a human or animal subject for medical use, comprising killing target bacteria using P1 or a metabolite thereof that is produced in the target cell, wherein the target bacteria mediate as disease or condition in the subject. In an example, when the subject is a human, the subject is not an embryo. In an example, the carrier cells are probiotic in the subject.
Optionally, the environment is a microbiota of soil; a plant, part of a part (e.g., a leaf, fruit, vegetable or flower) or plant product (e.g., pulp); water: a waterway; a fluid; a foodstuff or ingredient thereof; a beverage or ingredient thereof; a medical device; a cosmetic; a detergent; blood; a bodily fluid; a medical apparatus; an industrial apparatus; an oil rig; a petrochemical processing, storage or transport apparatus; a vehicle or a container.
Optionally, the environment is an ex vivo bodily fluid (e.g., urine, blood, blood product, sweat, tears, sputum or spit), bodily solid (e.g., faeces) or tissue of a human or animal subject that has been administered the composition.
Optionally, the environment is an in vivo bodily fluid (e.g., urine, blood, blood product, sweat, tears, sputum or spit), bodily solid (e.g., faeces) or tissue of a human or animal subject that has been administered the composition.
In an embodiment, the plasmid is a phagemid or cloning vector (eg, a shuttle vector, eg, a pUC vector). In an embodiment, the plasmid is a conjugative plasmid.
Optionally, P2 comprises one or more components of a CRISPR/Cas system, eg, a DNA sequence encoding one or more components of Type I Cascade (eg, CasA).
Optionally, P2 comprises a DNA sequence encoding guided nuclease, such as a Cas nuclease. TALEN, zinc finger nuclease or meganuclease.
In an example, the carrier cell(s) or composition are comprised by a medical container, eg, a syringe, vial, TV bag, inhaler, eye dropper or nebulizer. In an example, the carrier cell(s) or composition are comprised by a sterile container. In an example, the carrier cell(s) or composition are comprised by a medically-compatible container. In an example, the carrier cell(s) or composition are comprised by a fermentation vessel, eg, a metal, glass or plastic vessel. In an example, the carrier cell(s) or composition are comprised by an agricultural apparatus. In an example, the carrier cell(s) or composition are comprised by food production or processing apparatus. In an example, the carrier cell(s) or composition are comprised by a horticultural apparatus. In an example, the carrier cell(s) or composition are comprised by a farming apparatus. In an example, the carrier cell(s) or composition are comprised by petrochemicals recovery or processing apparatus. In an example, the carrier cell(s) or composition are comprised by a distillation apparatus. In an example, the carrier cell(s) or composition are comprised by cell culture vessel (eg, having a capacity of at least 50, 100, 1000, 10000 or 100000 litres). Additionally or alternatively, the target cell(s) are comprised by any of these apparatus etc.
In an example, the carrier cell(s) or composition are comprised by a medicament, e,g in combination with instructions or a packaging label with directions to administer the medicament by oral, IV, subcutaneous, intranasal, intraocular, vaginal, topical, rectal or inhaled administration to a human or animal subject. In an example, the carrier cell(s) or composition are comprised by an oral medicament formulation. In an example, the carrier cell(s) or composition are comprised by an intranasal or ocular medicament formulation. In an example, the carrier cell(s) or composition are comprised by a personal hygiene composition (eg, shampoo, soap or deodorant) or cosmetic formulation. In an example, the the carrier cell(s) or composition are comprised by a detergent formulation. In an example, the carrier cell(s) or composition are comprised by a cleaning formulation, eg, for cleaning a medical or industrial device or apparatus. In an example, the carrier cell(s) or composition are comprised by foodstuff, foodstuff ingredient or foodstuff processing agent. In an example, the carrier cell(s) or composition are comprised by beverage, beverage ingredient or beverage processing agent. In an example, the carrier cell(s) or composition are comprised by a medical bandage, fabric, plaster or swab. In an example, the carrier cell(s) or composition are comprised by a herbicide or pesticide. In an example, the carrier cell(s) or composition are comprised by an insecticide.
In an example, the CRISPR/Cas component(s) are component(s) of a Type I CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type II CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type III CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type IV CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type V CRISPR/Cas system. In an example, the CRISPR/Cas component(s) comprise a Cas9-encoding nucleotide sequence (eg, S pyogenes Cas9, S aureus Cas9 or S thermophilus Cas9). In an example, the CRISPR/Cas component(s) comprise a Cas3-encoding nucleotide sequence (eg, E coli Cas3, C dificile Cas3 or Salmonella Cas3). In an example, the CRISPR/Cas component(s) comprise a Cpf-encoding nucleotide sequence. In an example, the CRISPR/Cas component(s) comprise a CasX-encoding nucleotide sequence. In an example, the CRISPR/Cas component(s) comprise a CasY-encoding nucleotide sequence.
Optionally, target bacteria are gram negative bacteria (eg, a spirilla or vibrio). Optionally, target bacteria are gram positive bacteria. Optionally, target bacteria are mycoplasma, chlamydiae, spirochete or mycobacterium bacteria. Optionally, target bacteria are Streptococcus (eg, pyogenes or thermophilus). Optionally, target bacteria are Staphylococcus (eg, aureus, eg, MRSA). Optionally, target bacteria are E. coli (eg, O157: H7), eg, wherein the Cas is encoded by the vecor or an endogenous target cell Cas nuclease (eg, Cas3) activity is de-repressed. Optionally, target bacteria are Pseudomonas (eg, syringae or aeruginosa). Optionally, target bacteria are Vibro (eg, cholerae (eg, O139) or vulnificus). Optionally, target bacteria are Neisseria (eg, gonnorrhoeae or meningitidis). Optionally, target bacteria are Borderella (eg, pertussis). Optionally, target bacteria are Haemophilus 10 (eg, influenzae). Optionally, target bacteria are Shigella (eg, dysenteriae). Optionally, target bacteria are Brucella (eg, abortus). Optionally, target bacteria are Francisella host. Optionally, target bacteria are Xanthomonas. Optionally, target bacteria are Agrobacterium. Optionally, target bacteria are Erwinia. Optionally, target bacteria are Legionella (eg, pneumophila). Optionally, target bacteria are Listeria (eg, monocytogenes). Optionally, target bacteria are Campylobacter (eg, jejuni). Optionally, target bacteria are Yersinia (eg, pestis). Optionally, target bacteria are Borelia (eg, burgdorferi). Optionally, target bacteria are Helicobacter (eg, pylori). Optionally, target bacteria are Clostridium (eg, dificile or botulinum). Optionally, target bacteria are Erlichia (eg, chaffeensis). Optionally, target bacteria are Salmonella (eg, typhi or enterica, eg, serotype typhimurium, eg, DT 104). Optionally, target bacteria are Chlamydia (eg, pneumoniae). Optionally, target bacteria are Parachlanmydia host. Optionally, target bacteria are Corynebacterium (eg, amycolatum). Optionally, target bacteria are Klebsiella (eg, pneumoniae). Optionally, target bacteria are Enterococcus (eg, faecalis or faecim, eg, linezolid-resistant). Optionally, target bacteria are Acinetobacter (eg, baumannii, eg, multiple drug resistant).
Further examples of target cells are as follows:—
In an example, the target cell(s) is a cell of a species selected from Shigella, E coli. Salmonella. Serratia, Klebsiella. Yersinia, Pseudomonas and Enterobacter, eg, wherein the subject is a plant. Optionally, the composition comprises carrier cells that are each or in combination capable of conjugative transfer of the vector nucleic acid into target cells of species selected from two or more of Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter, eg, wherein the subject is a plant.
In an example, the reduction in growth or proliferation of target cells is at least 50, 60, 70, 80, 90 or 95%.
In embodiments, the plasmid contains a screenable or selectable marker gene. For example, the selectable marker gene is an antibiotic resistance gene.
The carrier bacteria can be bacteria of a species or genus as follows. For example, the species is found in warm-blooded animals (eg, livestock vertebrates). For example, the species is found in humans. For example, the species is found in plants. Preferably. non-pathogenic bacteria that colonize the non-sterile parts of the human or animal body (e.g., skin, digestive tract, urogenital region, mouth. nasal passages, throat and upper airway, ears and eyes) are utilized as carrier cells, and in an example the methodology of the invention is used to combat a target cell bacterial infection of such a part of the body of a human or animal. In another embodiment, the infection is systemic infection. Examples of carrier bacterial species include, but are not limited to: non-pathogenic strains of Escherichia coli (E. coli F18, S17 and E. coli. strain Nissle), various species of Lactobacillus (such as L casei, L plantarum, L paracasei, L. acidophilus. L fermentum, L. zeae and L. gasseri), or other nonpathogenic or probiotic skin- or GI colonizing bacteria such as Lactococcus, Bifidobacteria, Eubacteria, and bacterial mini-cells, which are a nucleoid cells destined to die but still capable of transferring plasmids (see: e.g., Adler et al., Proc. Natl. Acad. Sci. USA 57; 321-326, 1970: Frazer and Curtiss III, Current Topics in Microbiology and Immunology 69: 1-84, 1975; U.S. Pat. No. 4,968,619 to Curtiss III). In some embodiments, the target recipient cells are pathogenic bacteria comprised by a human, animal or plant, eg, on the skin or in the digestive tract, urogenital region, mouth, nasal passage, throat and upper airway, eye(s) and ear(s). Of particular interest for targeting and eradication are pathogenic strains of Pseudomonas aeruginosa. Escherichia coli, Staphylococcus pneumoniae and other species, Enterobacter spp., Enterococcus spp. and Mycobacterium tuberculosis.
The present invention finds use with a wide array of settings or environments, eg, in therapeutic, agricultural, or other settings, including, but not limited to, those described in U.S. Pat. Nos. 6,271,359, 6,261,842, 6,221,582, 6,153,381, 6,106,854, and 5,627,275. Others are also discussed herein, and still others will be readily apparent to those of skill in the art.
A single carrier bacterial strain might harbor more than one type of such vector (eg, differing in the P1 that they encode). Further, in another example two or more different carrier bacterial strains, each containing one or more such vectors. may be combined for producing a plurality of different P1 products in the subject.
The present invention finds utility for treatment of humans and in a variety of veterinary, agronomic, horticultural and food processing applications. For human and veterinary use, and depending on the cell population or tissue targeted for protection, the following modes of administration of the carrier bacteria of the invention are contemplated: topical, oral. nasal, ocular, aural, pulmonary (e.g., via an inhaler), ophthalmic, rectal, urogenital, subcutaneous, intraperitoneal and intravenous. The bacteria may be supplied as a pharmaceutical composition, in a delivery vehicle suitable for the mode of administration selected for the patient being treated. The term “patient” or “subject” as used here may refer to humans or animals (animals being particularly useful as models for clinical efficacy of a particular donor strain, for example, or being farmed or livestock animals). Commercially-relevant animals are chicken, turkey, duck, catfish, salmon, cod, herring, lobster, shrimp, prawns, cows, sheep, goats. pigs, goats, geese or rabbits.
For example, to deliver the carrier bacteria to the gastrointestinal tract or to the nasal passages, the preferred mode of administration may be by oral ingestion or nasal aerosol, or by feeding (alone or incorporated into the subject's feed or food and/or beverage, such as drinking water). In this regard, the carrier cells may be comprised by a food of livestock (or farmed or companion animal), eg, the carrier bacteria are comprised by a feed additive for livestock. Alternatively, the additive is a beverage (eg, water) additive for livestock. It should be noted that probiotic bacteria, such as Lactobacillus acidophilus, are sold as gel capsules containing a lyophilized mixture of bacterial cells and a solid support such as mannitol. When the gel capsule is ingested with liquid, the lyophilized cells are re-hydrated and become viable, colonogenic bacteria. Thus, in a similar fashion, carrier bacterial cells of the present invention can be supplied as a powdered. lyophilized preparation in a gel capsule, or in bulk, eg, for sprinkling onto food or beverages. The re-hydrated, viable bacterial cells will then populate and/or colonise sites throughout the upper and/or lower gastrointestinal system, and thereafter come into contact with the target bacteria.
For topical applications, the carrier bacteria may be formulated as an ointment or cream to be spread on the affected skin surface. Ointment or cream formulations are also suitable for rectal or vaginal delivery, along with other standard formulations, such as suppositories. The appropriate formulations for topical, vaginal or rectal administration are well known to medicinal chemists.
The present invention may be of utility for topical or mucosal administrations to treat a variety of bacterial infections or bacterially related undesirable conditions. Some representative examples of these uses include treatment of (1) conjunctivitis, caused by Haemophilus sp., and corneal ulcers, caused by Pseudomonas aeruginosa; (2) otitis externa, caused by Pseudomonas aeruginosa; (3) chronic sinusitis, caused by many Gram-positive cocci and Gram-negative rods, or for general decontamination of bronchii; (4) cystic fibrosis, associated with Pseudomonas aeruginosa; (5) 35 enteritis, caused by Helicobacter pylori (eg, to treat or prevent gastric ulcers), Escherichia coli, Salmonella typhimurium, Campylobacter or Shigella sp.; (6) open wounds, such as surgical or non-surgical, eg, as a prophylactic measure; (7) burns to eliminate Pseudomonas aeruginosa or other Gram-negative pathogens; (8) acne, eg, caused by Propionobacter acnes; (9) nose or skin infection, eg, caused by methicillin resistant Staphylococcus aureus (MSRA); (10) body odor, eg, caused by Gram-positive anaerobic bacteria (i.e., use of carrier cells in deodorants); (11) bacterial vaginosis, eg, associated with Gardnerella vaginalis or other anaerobes; and (12) gingivitis and/or tooth decay caused by various organisms.
In one example, the target cells are E coli cells and the disease or condition to be treated or prevented in a human is a uterine tract infection or a ventilator associated infection, eg, pneumonia. sepsis, septicaemia or HUS.
In other embodiments, the carrier cells of the present invention find application in the treatment of surfaces for the removal or attenuation of unwanted target bacteria or for modification of bacteria on the surfaces, for example use in a method of treating such a surface or an environment comprising target bacteria, wherein the method comprises contacting the surface or environment with carrier bacteria of the invention, allowing transfer of the vector nucleic acid of the invention from the carrier to the target bacteria, and expressing P1 in target cells. For example, surfaces that may be used in invasive treatments such as surgery, catheterization and the like may be treated to prevent infection of a subject by bacterial contaminants on the surface. It is contemplated that the methods and compositions of the present invention may be used to treat numerous surfaces, objects, materials and the like (e.g., medical or first aid equipment, nursery and kitchen equipment and surfaces) to control bacterial contamination thereon.
Pharmaceutical preparations or other compositions comprising the carrier bacteria may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient or plant or environment or surface undergoing treatment. Each dosage should contain a quantity of the carrier bacteria calculated to produce the desired antibacterial effect in association with the selected carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of a patient, plant. surface or environment. Appropriate concentrations for achieving eradication of pathogenic target cells (eg, comprised by a tissue of the patient) may be determined by dosage concentration curve calculations, as known in the art.
Other uses for the carrier bacteria of the invention are also contemplated. These include a variety agricultural, horticultural, environmental and food processing applications. For example, in agriculture and horticulture, various plant pathogenic bacteria may be targeted in order to minimize plant disease. One example of a plant pathogen suitable for targeting is Erwinia (eg, E amylovora, the causal agent of fire blight). Similar strategies may be utilized to reduce or prevent wilting of cut flowers. For veterinary or animal farming, the carrier cells of the invention may be incorporated into animal feed (chicken, swine, poultry, goat, sheep, fish, shellfish or cattle feed) to reduce bio-burden or to eliminate certain pathogenic organisms (e.g., Salmonella, such as in chicken, turkey or other poultry). In other embodiments, the invention may be applied on meat or other foods to eliminate unwanted or pathogenic bacteria (e.g., E. coli O157:H7 on meat, or Proteus spp., one cause of “fishy odour” on seafood).
Environmental utilities comprise, for example, engineering carrier bacteria, eg, Bacillus thuringiensis and one of its conjugative plasmids, to deliver and conditionally express an insecticidal agent in addition to or instead of an antibacterial agent (e.g., for the control of mosquitos that disseminate malaria or West Nile virus). In such applications, as well as in the agricultural and horticultural or other applications described above, formulation of the carrier bacteria as solutions, aerosols, or gel capsules are contemplated.
In an example the plasmid (eg, where the microbiota is a human, anima or plant microbiota) is an engineered RK2 plasmid (ie, a RK2 plasmid that has been modified by recombinant DNA technology or a progeny of such a modified plasmid). Plasmid RK2 is a promiscuous plasmid that can replicate in 29 (and probably many more) gram-negative species (Guiney and Lanka, 1989, p 27-54. In C. M. Thomas (ed) Promiscous plasmids in gram-negative bacteria. London, Ltd London United Kingdom.). Plasmid RK2 is a 60-kb self-transmissible plasmid with a complete nucleotide sequence known (Pansegrau et al., 1994. J. Mol. Biol. 239, 623-663). A minimal replicon derived from this large plasmid has been obtained that is devoid of all its genes except for a trfA gene, that encodes plasmid's Rep protein called TrfA, and an origin of vegetative replication oriV For a review of RK2 replication and its control by TrfA protein, see Helinski et al., 1996 (In Escherichia coli and Salmonella Cellular and Molecular Biology, Vol. 2 (ed. F. Neidhardt, et al., 2295-2324, ASM Press, Washington D.C.).
In an example the plasmid (eg, where the microbiota is a human, anima or plant microbiota) is an engineered R6K plasmid (ie, a R6K plasmid that has been modified by recombinant DNA technology or a progeny of such a modified plasmid).
The present invention is optionally for an industrial or domestic use, or is used in a method for such use. For example, it is for or used in agriculture, oil or petroleum industry, food or drink industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, medical industry, healthcare industry, dentistry industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal hide industry, tobacco industry or steel industry.
The present invention is optionally for use in an industry or the environment is an industrial environment, wherein the industry is an industry of a field selected from the group consisting of the medical and healthcare; pharmaceutical; human food; animal food; plant fertilizers; beverage; dairy; meat processing; agriculture; livestock farming; poultry farming; fish and shellfish farming; veterinary; oil; gas; petrochemical; water treatment; sewage treatment; packaging; electronics and computer; personal healthcare and toiletries; cosmetics; dental; non-medical dental; ophthalmic; non-medical ophthalmic; mineral mining and processing; metals mining and processing; quarrying; aviation; automotive; rail; shipping; space; environmental; soil treatment; pulp and paper; clothing manufacture; dyes; printing; adhesives; air treatment; solvents; biodefence; vitamin supplements; cold storage; fibre retting and production; biotechnology; chemical; industrial cleaning products; domestic cleaning products; soaps and detergents; consumer products: forestry; fishing; leisure; recycling; plastics; hide, leather and suede; waste management; funeral and undertaking; fuel; building; energy; steel; and tobacco industry fields.
In an example, the plasmid comprises a CRISPR array, wherein the array comprises one, or two or 20 more different spacers (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more spacers) for targeting the vector and/or the genome of a carrier bacterium.
In an example, the target bacteria are comprised by an environment as follows. In an example, the environment is a microbiota of a human, eg, the oral cavity microbiota or gut microbiota or the bloodstream. In an example, the environment is not an environment in or on a human. In an example, the environment is not an environment in or on a non-human animal. In an embodiment, the environment is an air environment. In an embodiment, the environment is an agricultural environment. In an embodiment, the environment is an oil or petroleum recovery environment, eg, an oil or petroleum field or well. In an example, the environment is an environment in or on a foodstuff or beverage for human or non-human animal consumption. In an example, the environment is a maritime environment, eg, in seawater or on a boat (eg, in ship or boat ballast water).
In an example, the environment is a human or animal microbiota (eg, gut, vaginal, scalp, armpit, skin or oral cavity microbiota). In an example, the target bacteria are comprised by a human or animal microbiota (eg, gut, vaginal, scalp, armpit, skin or oral cavity microbiota).
In an example, the carrier bacteria or composition of the invention are administered intranasally, topically or orally to a human or non-human animal, or is for such administration. The skilled person aiming to treat a microbiota of the human or animal will be able to determine the best route of administration, depending upon the microbiota of interest. For example, when the microbiota is a gut microbiota, administration can be intranasally or orally. When the microbiota is a scalp or armpit microbiota, administration can be topically. When the microbiota is in the mouth or throat, the administration can be orally.
In an example, the environment is harboured by a beverage or water (eg, a waterway or drinking water for human consumption) or soil. The water is optionally in a heating. cooling or industrial system, or in a drinking water storage container.
In an example, the carrier and/or target bacteria are Firmicutes selected from Anaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus, Leuconostoc, Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter, Papillibacter, Proprionispira, Pseudobutvrivibrio. Pseudoramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttleworthia, Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum, Syntrophococcus, Thernobacillus, Turibacter and Weisella.
In an example, the carrier bacteria, composition, use or method is for reducing pathogenic infections or for re-balancing gut or oral biofilm eg, for treating or preventing obesity or disease in a human or animal; or for treating or preventing a GI condition (such as Crohn's disease, IBD or colitis). For example, the vector, carrier bacteria, composition, use or method is for knocking-down Salmonella, Campylobacter, Erwinia, Xanthomonas, Edwardsiella, Pseudomonas, Klebsiella, Pectobacterium, Clostridium dificile or E coli bacteria in a gut biofilm of a human or animal or a plant, preferably in a human or animal.
In an example, the animal is a chicken, eg, and the target bacteria are Salmonella or Campylobacter. In an example, the animal is a fish (eg, catfish or salmon) or shellfish (eg, prawn or lobster), eg, and the target bacteria are Edwardsiella. In an example, the plant is a potato plant and, eg, the target bacteria are Pectobacterium. In an example, the plant is a cabbage plant and, eg, the target bacteria are Xanthomonous (eg, X campestris). In an example, the plant is a marijuana plant and, eg, the target bacteria are Pseudomonas (eg, P cannabina or P amygdali), Agrobacterium (eg, A tumefaciens) or Xanthomonas (eg, X campestris). In an example, the plant is a hemp plant and, eg, the target bacteria are Pseudomonas (eg, P cannabina or P amygdali), Agrobacterium (eg, A tumefaciens) or Xanthomonas (eg, X campestris).
Optionally, the environment is comprised by, or the target bacteria are comprised by, a gut biofilm, skin biofilm, oral cavity biofilm, throat biofilm, hair biofilm, armpit biofilm, vaginal biofilm, rectal biofilm, anal biofilm, ocular biofilm, nasal biofilm, tongue biofilm, lung biofilm, liver biofilm, kidney biofilm, genital biofilm, penile biofilm, scrotal biofilm, mammary gland biofilm, ear biofilm, urethra biofilm, labial biofilm, organ biofilm or dental biofilm. Optionally, the environment is comprised by, or the target bacteria are comprised by, a plant (eg, a tobacco, crop plant, fruit plant, vegetable plant or tobacco, eg on the surface of a plant or contained in a plant) or by an environment (eg, soil or water or a waterway or aqueous liquid).
In an example, the carrier cell(s) or composition is for treating a disease or condition in an animal or human. In an example, the disease or condition is caused by or mediated by the presence of a protein or metabolite in the human or animal subject and expression of P1 by target cells comprising the vector nucleic acid causes a reduction in the protein or metabolite in the subject. In an example, the disease or condition is caused by or mediated by the absence of a protein or metabolite in the human or animal subject and expression of P1 by target cells comprising the vector nucleic acid causes an increase in the protein or metabolite in the subject. In an example, the disease or condition is caused by an undesirably high level of a protein or metabolite in the human or animal subject and expression of P1 by target cells comprising the vector nucleic acid causes an decrease in the protein or metabolite in the subject. In an example, the disease or condition is caused by an undesirably low level of a protein or metabolite in the human or animal subject and expression of P1 by target cells comprising the vector nucleic acid causes an increase in the protein or metabolite in the subject. The metabolite may be a protein, peptide, amino acid, carbohydrate, sugar, lipid, fatty acid or ion (eg, a metal ion). The metabolite may be toxic to human cells of the human subject. The metabolite may be a hormone, growth factor or antibiotic. The metabolite may be a mineral. The metabolite may be a salt. The metabolite may be a nucleic acid, eg, a RNA (eg, a mRNA) or DNA.
In an example, the disease or condition is a cancer, inflammatory or autoimmune disease or condition, eg, obesity, diabetes IBD, a GI tract condition or an oral cavity condition.
Optionally, the disease or condition of a human or animal subject is selected from
In an example, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer disease, geriopsychosis, Down syndrome, Parkinson's disease, Creutzfeldt-jakob disease, diabetic neuropathy, Parkinson syndrome, Huntington's disease. Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt Creutzfeldt-Jakob disease. For example, the disease is Alzheimer disease. For example, the disease is Parkinson syndrome.
In an example, wherein the method of the invention is practised on a human or animal subject for treating a CNS or neurodegenerative disease or condition, the method causes downregulation of Treg cells in the subject, thereby promoting entry of systemic monocyte-derived macrophages and/or Treg cells across the choroid plexus into the brain of the subject, whereby the disease or condition (eg, Alzheimer's disease) is treated, prevented or progression thereof is reduced. In an embodiment the method causes an increase of IFN-gamma in the CNS system (eg, in the brain and/or CSF) of the subject. In an example, the method restores nerve fibre and/or reduces the progression of nerve fibre damage. In an example, the method restores nerve myelin and/or reduces the progression of nerve myelin damage. In an example, the method of the invention treats or prevents a disease or condition disclosed in WO2015136541 and/or the method can be used with any method disclosed in WO2015136541 (the disclosure of this document is incorporated by reference herein in its entirety, eg, for providing disclosure of such methods, diseases, conditions and potential therapeutic agents that can be administered to the subject for effecting treatment and/or prevention of CNS and neurodegenerative diseases and conditions, eg, agents such as immune checkpoint inhibitors, eg, anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed therein).
Cancers that may be treated include tumours that are not vascularized, or not substantially vascularized, as well as vascularized tumours. The cancers may comprise non-solid tumours (such as haematological tumours, for example, leukaemias and lymphomas) or may comprise solid tumours. Types of cancers to be treated with the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukaemia or lymphoid malignancies, benign and malignant tumours, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers and paediatric tumours/cancers are also included.
Haematologic cancers are cancers of the blood or bone marrow. Examples of haematological (or hematogenous) cancers include leukaemias, including acute leukaemias (such as acute lymphocytic leukaemia, acute myelocytic leukaemia, acute myelogenous leukaemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronic leukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronic myelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukaemia and myelodysplasia.
Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumours, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer, testicular tumour, seminoma, bladder carcinoma, melanoma, and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medu!loblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).
For example, the composition comprising carrier cells is a human or animal food and/or beverage (eg, mixed in drinking water for livestock consumption). When supplied in a beverage, the vector may be comprised by carrier bacteria, wherein the carrier bacteria are comprised in the beverage at an amount of from 1×103 to 1×1010 (eg, from 1×104 to 1×1010; from 1×104 to 1×109; from 1×104 to 1×108; from 1×104 to 1×107; from 1×105 to 1×1010; from 1×105 to 1×109; from 1×103 to 1×108; from 1×105 to 1×107; from 1×106 to 1×1010; from 1×105 to 1×109; from 1×105 to 1×108; from 1×105 to 1×107; from 1×106 to 1×1010; from 1×106 to 1×109; from 1×106 to 1×108; or from 1×106 to 1×107) cfu/ml. When supplied in a beverage, the vector may be comprised by carrier bacteria, wherein the carrier bacteria are comprised in the beverage at an amount of at least 1×101 cfu/ml, eg, wherein the subject is a human or animal (eg, a poultry bird, such as a chicken).
Optionally, the guided nuclease is any guided nuclease disclosed herein, eg, a Cas, TALEN, meganuclease or a zinc finger nuclease. In an example, the component is a crRNA or guide RNA that is operable in target cells with a cognate Cas nuclease. The Cas nuclease can be any Cas nuclease disclosed herein. The Cas nuclease may be an endogenous Cas of the target cells or may be encoded by an exogenous nucleic acid that is administered to the animal.
There is provided according to the Fifth Configuration:
A method of engineering a microbiome (eg, any microbiome disclosed herein), the method comprising contacting the microbiome with a plurality of vectors as described herein (eg, by combining the microbiome with carrier cells as described) and optionally allowing transfer of said vector nucleic acid into target cells of the microbiota.
A modified microbiota obtained or obtainable by the method herein, optionally wherein the microbiota is comprised by a pharmaceutical composition for use as a medicament to treat a disease or condition in a human or animal subject.
Any of the features disclosed in the context of the First to Fourth Configurations may apply mutatis mutandis to the Fifth Configuration.
The invention also provides the following Concepts.
1. A host cell comprising nucleic acid that comprises
Optionally, the nucleic acid is comprised by at least one nucleic acid vector for transfer from the host cell into a cell of a microbiota. For example the host cell and microbiota cell are bacterial cells.
For example, the fungal cell is a yeast cell. The bacterial cell may be a cell of any bacterial species or genus disclosed herein.
2. The cell of Concept 1, wherein
3. The cell of any one of Concept 2(a)-(c) wherein the vector or each vector is a conjugative plasmid for transfer to a cell of a microbiota comprised by a human or animal subject.
4. The cell of any preceding Concept, wherein the cell is a cell of commensal or probiotic bacterial species of a human or animal microbiota, optionally an E coli cell or a Bacteroides cell.
5. The cell of any preceding Concept, wherein NS2 is under the control of a second promoter that is regulatable for expression of P2, wherein binding of a regulator agent (R) to the vector nucleic acid regulates the second promoter, thereby regulating the expression of P2 and P1.
6. The cell of any preceding Concept, wherein P2 comprises an RNA-guided nuclease (optionally a Cas nuclease), wherein the nuclease is operable to cut the nucleic acid at a predetermined sequence motif.
7. The cell of Concept 6, wherein
This is useful for promoting degradation of the vector, thereby reducing or inhibiting the production of P1. For example, the cut vector is degraded in the cell.
8. The cell of any preceding Concept for treating or preventing a disease or condition in a human or animal subject, wherein the cell is administered to a microbiota (optionally a gut microbiota) of the subject to produce P1 in the subject thereby treating or preventing the disease or condition in the subject.
Over half of ingested xylitol in humans is not adsorbed by human cells instead reaching the gastrointestinal tract where it is taken up by the microbiome (Livesey, 2003). making this useful to control our switches in the GI tract. As exemplified herein, we advantageously found that xylitol induces expression by lifting the repression that the transcriptional regulator exerts on the promoter. Furthermore, E. coli strains generally lack the xylose reductase and xylitol dehydrogenase typically necessary for xylitol metabolism (Ge et al., 2018). We realised that this may be useful for more precise or lasting control by xylitol when the strain cannot metabolise xylitol. Use of lower doses of xylitol may be possible when the strain cannot metabolise xylitol. Thus, in an example the cell of the invention is an E coli cell and the promoter is regulatable by xylitol.
1. A cell (optionally according to any preceding Concept), comprising a nucleic acid, wherein the nucleic acid comprises a gene encoding a product of interest (P1), the gene comprising a nucleotide sequence (NS1) encoding P1 and a regulatory region 5′ of NS1 that comprises a promoter (Px) for controlling the expression of NS1, wherein the combination of Px and NS1 is heterologous to the cell and Px is regulatable by xylitol or xylose.
For example, NS1 is not found in a wild-type cell of the same species as the cell of the invention. For example, NS1 is a non-bacterial (eg, an animal, human, mammal or plant) sequence. Preferably, NS1 is a human sequence.
For example, NS1 is an endogenous sequence of the cell and Px is heterologous to the cell.
2. The cell of Concept 1, wherein the promoter is a xylitol or xylose regulatable promoter of a Morganella species, optionally M morganii.
For example, the M morganii is Morganella morganii strain ZJG812.
3. The cell of Concept I or 2, wherein the promoter comprises SEQ ID NO: 3 or a nucleotide sequence that is at least 70% identical to SEQ ID NO: 3.
For example, any percent identity herein is at least 70, 80, 90, 95, 96, 97, 98 or 99%.
Optionally, the cell comprises SEQ ID NO: 4 or a nucleotide sequence encoding a xylitol regulatable promoter. For example, the sequence further encodes a repressor that is cognate to the promoter. For example, the sequence further encodes a xylitol isomerase.
4. The cell of any one of Concepts 1-3, wherein Px is homologous to a xylitol or xylose regulatable promoter of Morganella morganii.
For example, the promoter is a xylitol regulatable promoter. For example, the promoter is repressible by a repressor and xylitol is capable of de-repressing the repressor.
5. The cell of any one of Concepts 1-4, wherein the cell genome encodes a repressor that is capable of repressing Px, wherein xylitol and/or xylose is capable of de-repressing the repressor.
6. The cell of Concept 5, wherein the repressor is encoded by SEQ ID NO: I or a nucleotide sequence that is at least 70% identical to SEQ ID NO: 1.
For example, the identity is at least 70, 80, 90, 95, 96, 97, 98 or 99%.
For example, the repressor comprises SEQ ID NO: 2 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 2.
7. The cell of any one of Concepts 1-6, wherein the cell comprises a xylitol transporter, optionally a xylitol ABC transporter.
Our data surprisingly suggest that the presence of a xylitol transporter increases the sensitivity of the donor strains towards xylitol in the growth medium.
8. The cell of any one of Concepts 1-7, wherein the cell is devoid of a xylitol isomerase gene.
For example, the cell genome comprises a xylitol inducible promoter of a Morganii species and excludes a sugar isomerase gene. For example, the cell comprises a xylitol inducible promoter of a Morganii species and excludes a nucleotide sequence encoding a xylitol isomerase.
In an example, the cell genome comprises a xylABC operon from Morganella morganii. Optionally the operon is devoid of a nucleotide sequence encoding a xylitol isomerase.
9. A nucleic acid vector comprising a gene as recited in any one of Concepts 1-8.
10. The vector of Concept 9, wherein the vector is a plasmid (optionally a conjugative plasmid), transposon, phagemid or a phage.
In an example, the cell of the invention comprises any vector of the invention as described herein.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications and all US equivalent patent applications and patents are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps The term “or combinations thereof” or similar as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB. AC. BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The order of the components:
Kanamycin resistance gene, pBBR1-msc2, oriT from RP4 plasmid, araC. pBad promoter, E. coli Cas3 Type I-E sequence, Cascade sequence, S. pyogenes terminator, rhamnose promoter, array comprising spacer that can hybridise to the sfGFP sequence, rmtB (16S rRNA methylase gene) which gives resistance to amikacin and gentamicin, sfGFP gene
p1075 containing the pBBR1 replicon and CRISPR cas system, was used as a template for cloning sfGFP gene, S. pyogenes terminator after the E. coli Type I-E Cas3 and Cascade, CRISPR array with sfGFP target and rhamnose promoter to control CRISPR array with sfGFP target (see plasmid map
Transformation, selection and sequence verification
Transformation technique: The final construct (p1364) is electroporated to the strain of interest (NEB 10-beta cells for cloning or JKE201 strain for further conjugation). Correct colonies are selected on 3 antibiotic markers (kanamycin, gentamicin, amikacin), colonies will have a distinctive green color (due to expression of GFP) and with confirmation using colony PCR with the construct specific primers.
Sequence verification is performed by subjecting the purified plasmid to sequencing with construct specific primers to cover the whole construct.
See
The JKE201 strain was transformed with p1364 and a positive clone was selected on kanamycin LB plates (supplemented with DAP) and further verified by the positive GFP signal. One colony of the transformed JKE201/p1364 donor strain as well as the b52 (strain C-la-CGSC, obtained from the Coli Genetic Stock Center) recipient strain was grown to exponential phase in liquid LB (JKE201 supplemented with DAP). The two cultures were then mixed 5:1 (JKE201/p1364: b52) and spotted solid LB agar to incubate overnight.
After overnight conjugation, the mix was scraped off and dissolved in liquid LB and plated on LB plates containing kanamycin but without DAP to select only b52 transconjugants. These were enumerated compared to total recipient counts on LB plates.
Results are depicted in
The p1364 transfers at high frequency into the b52 model strain.
Transconjugant recipient cells (b52 cells containing plasmid p1364) were produced as described in Example 2. The transconjugants, containing the sfGFP expressing construct with a self-targeting CRISPR array, conjugative plasmid p1364, was grown in LB medium to and OD600 of 1. A plasmid-free b52 strain was grown to the same level for use as a negative/autofluorescence control. Each culture was transferred to a microtiter plate and measured in a Synergy™ H1 plate reader. sfGFP production was quantified using and excitation wavelength of 485 nm and an emission wavelength of 510 nm.
Results are depicted in
Transconjugants b52 cells (from Example 2) carrying the p1364 plasmid were grown with selection for p1364 (kanamycin) for 2 h and subsequently washed in LB to remove residual antibiotics.
The resuspended culture was then split in two 1 ml portions and inducers (I % arabinose, 10 mM of rhamnose) were added to one of the tubes to induce the self-targeting Cas mechanism. Just after induction, cells from both cultures were plated on LB plates (without inducers added, timepoint 0). Subsequently, samples from each tube (induced and non-induced) were plated on LB plates at timepoints: 1 h and 24 hrs after induction. All plates were incubated overnight after which the proportion of sfGFP expressing cells was determined by colony counting in a blue light transilluminator (365 nm, UV benchtop transilluminator, VWR®).
Results are depicted in
To validate that the loss of sfGFP was indeed due to plasmid loss, PCR specific for the plasmid backbone was performed on 14 randomly selected colonies from the plate of the 24 h timepoint. See the resulting gel image in
A conjugative plasmid construct was made that could be introduced into host cells to produce donor cells. The plasmid will be a vector for a gene of interest (eg, GFP in the examples above) and a self-targeting CRISPR/array to target the plasmid in recipient cells after it has been transferred by conjugation from donor to recipient cells.
The plasmid was engineered so that, on induction of the CRISPR/cas system with arabinose, the plasmid was able to target the genome of the host cells. The plasmid was made using the RP4 plasmid as a starting point. The arabinose inducible E. coli Type I-E Cas3 Cascade followed by a CRISPR array for producing a guide RNA targeting the E. coli chromosomal gene lptA was integrated into the RP4 plasmid using recombineering.
The Cas system and array with the arabinose promoter was amplified along with a spectinomycin resistance gene and araC from a cloning vector containing these.
The PCR product were flanked with homology arms that allowed insertion of the CRISPR/cas-araC-Spectinomycin cassette into the tetracycline gene of RP4.
Recombineering was performed. Briefly, JKE201 cells with an lptA mutant allele, not targeted by the array, that contained apSIM5 recombineering plasmid were grown to an OD of 0.5 in LB supplemented with DAP and chloramphenicol. Then, the lambda RED system was induced at 42 C degrees for 15 min and the culture was placed on ice to prepare the cells for transformation by washing in cold MiliQ water. Cells were transformed with the PCR product at 18 kv and recovered for 12 h prior to plating on selective plates containing DAP and spectinomycin. The resulting recombinant clones were confirmed by PCR.
A positive clone as well as JKE201 with the wildtype RP4 plasmid (negative control without CRISPR/Cas) was grown in liquid LB with spectinomycin for 4 hrs. Wildtype MG1655 cells were grown to the same OD and mixed with the JKE201/RP4::Cas and JKE201/RP4 to allow plasmid transfer and amplification in this strain. The mixes were resuspended in LB without DAP to remove the initial JKE201 donor strain. The resulting population of MG1655 (newly formed plasmid donor cells) containing the plasmid were now plated on LB plates with and without arabinose (arabinose for CRISPR/cas induction) to active self-targeting and killing of donor cells by cutting the lptA target comprised by the donor chromosome. The population containing the wildtype RP4 plasmid was plated on the same media as a control. After overnight incubation, the surviving cells were enumerated and presented in
The results depicted in
Various metabolic pathway schemes can be envisaged where the invention functions. For example, the pathway may be in any environment comprising a microbiota, such as in a human or animal subject. Examples of schemes are provided in
R may upregulate P2 expression, P2 downregulates P1 expression and optionally P1 is a component in a metabolic pathway (eg, in the microbiota or target cell or in a subject or environment comprising the microbiota or target cell) wherein a production product (X) of the pathway downstream from P1 causes regulation of P1 or P2 expression. See, for example, Scenarios 1, 3 and 4 (
R may upregulate P2 expression, P2 upregulates Pt expression and optionally P1 is a component in a metabolic pathway (eg, in the microbiota or target cell or in a subject or environment comprising the microbiota or target cell) wherein a production product (X) of the pathway downstream from P1 causes regulation of P1 or P2 expression. See, for example, Scenario 2 (
In this study we developed and tested a plasmid comprising a CRISPR/Cas system (which we call a CRISPR-guided vector (CGV)) that could be removed from a population of Bacteroides thetaiotaomicron using vector-borne spacers targeting the CGV itself (self-targeting CGV). The CGV harbored an inducible type I-B CRISPR/Cas operon originating from Clostridioides difficile alongside five CGV-targeting spacers. We demonstrated that upon induction, the CRISPR/Cas system caused the CGV to be removed from more than 99.9% of the Bacteroides population without killing the host cells in the process.
Deliver a self-targeting CGV to Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) via conjugation from an Escherichia coli donor to show that the CGV can be efficiently removed upon CRISPR/Cas induction.
Demonstrate that self-targeting CGVs are removed from a population of Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) without off-target effects.
2.1 Bacterial strains and growth conditions
Escherichia coli strains were grown at 37° C. in lysogeny broth (LB) at 250 RPM, or on solid LB-agar plates (made with 1.5% (w/v) agar). For plasmid selection, the medium was supplemented with ampicillin (Amp, 100 μg ml−1) or spectinomycin (Spc, 400 μg ml−1). For growth of auxotrophic strains, the medium was further supplemented with diaminopimelic acid (DAP, 40 μg ml−1). Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) only grows anaerobically but does not die under aerobic conditions. For this reason, the “M45 variable atmosphere workstation” (Don Whitley, Yorkshire, UK) was used and set to contain a mixture of gasses N2, H2, and CO2 in the ratio 92:6:2. All solutions and media intended for Bacteroides were allowed to reduce under these conditions for ˜24 hours before being used.
All Bacteroides strains were routinely grown at 37° C. in Brain Heart Infusion (BHI) broth supplemented with cysteine (1 g 1−1), hemin (5 mg−1), and NaHCO3 (0.2% (w/v)). This medium, denoted as BHIpp, is based on a recent Bacteroides protocol (Bacic & Smith, 2008). When growth on solid media was required, the BHIpp broth was further supplemented with agar (1.5% (w/v)). For plasmid selection, the medium was supplemented with erythromycin (Erm, 10 μg 1−1). For counterselection of the E. coli donor strain, the BHIpp medium was supplemented with gentamicin (Gm, 30 μg−1).
2.2 CGV design and assembly
CGV pSNP1599 is based on a shuttle plasmid, which can be delivered into the Bacteroides thetaiotaomicron VPI-5482 recipient strain (bSNP2978) via conjugation from an E. coli (bSNP3235)-based donor strain.
To construct pSNP1599, five spacers complementary to protospacers found within the plasmid itself (details found in Appendix C) were first assembled into a CRISPR array and then cloned into the shuttle plasmid. In the same cloning reaction, the type 1-B CRISPR/Cas operon from the Clostridioides dificile strain 630Δerm (denoted as CdCas) was also cloned into the plasmid. Expression of the vector-borne CRISPR/Cas system was regulated by a rhamnose-inducible promoter (Prha), which allowed the plasmid removal to be controlled. Detailed information on the construction of pSNP1599 is found in Appendix B.
2.3 Conjugative transfer of CGV from E. coli to B. thetaiotaomicron
First, the bSNP3235-based donor strain carrying the plasmid of interest was grown aerobically in 30 ml LB medium supplemented with DAP while the bSNP2978 recipient was grown anaerobically in 5 ml BHIpp broth. Once the donor and recipient cultures reached optical densities (OD600) of 0.4±0.2 and 0.15±0.05, respectively, they were mixed, using 25 ml of the donor and 2.5 ml of the recipient. The mixture was spun down (9000×g for 10 minutes), supernatant removed, and the pellet resuspended in 100 μl PBS. The resuspended pellet was spotted onto a BHIpp agar plate and allowed to incubate aerobically at 37° C. for 18 hours to facilitate conjugation.
The mating spot was collected and resuspended in 500 μl pre-reduced BHIpp broth (from this point, the rest of the experiment was performed in the anaerobic work station). A 10-fold serial dilution was prepared in PBS (spanning dilutions 101 to 101) and then spotted onto selective media (BHIpp+Gm+Erm agar) with and without inducer. Plates were packed in plastics bags and allowed to incubate anaerobically at 37° C. for 2-3 days. Any transconjugant colony forming units (CFUs) emerging from the plates were enumerated. Two examples of plates after 3 days of incubation are given in
2.4 Plasmid removal assay
A colony of bSNP2978 containing the intact self-targeting CGV pSNP1599 (verified by sequencing) was inoculated into 5 ml BHIpp broth supplemented with gentamicin and erythromycin and allowed to grow anaerobically overnight. The culture was then washed three times (each wash consisting of a spin-down at 4500×g for 2 min, removal of supernatant, and resuspension in 5-ml PBS) to remove all traces of erythromycin.
Two 5-μl aliquots of the washed culture were transferred to separate tubes containing 5 ml fresh pre-reduced BHIpp broth. One tube was supplemented with the inducer rhamnose (10 mM) while the other tube was given an equal volume of milliQ H2O. The induced and non-induced cultures were allowed to grow anaerobically over the course of a day, with 200-p1 aliquots being taken from each culture at 0-, 1-, 3-, 6- and 24 hours after induction.
10-fold serial dilutions were prepared in PBS for each collected aliquot (spanning dilutions 100 to 10−9) and spotted (2.5 μl per dilution) onto BHIpp agar plates either supplemented with gentamicin (non-selective plates) or gentamicin and erythromycin (selective plates). Plates were packed in plastic bags and allowed to incubate anaerobically for 2-3 days. CFUs were counted. Experiment was performed with biological triplicates. Two examples of plates after 3 days of incubation are given in
3.1 Estimating the efficacy of CRISPR-mediated CGV removal
A self-targeting CGV, denoted as pSNP1599, was generated, comprising a type I CRISPR/Cas system from C. difficile (CdCas) and five spacers which target the CGV itself. Upon induction with 30 rhamnose, the CdCas system is activated which lead to CRISPR targeting and, in turn, removal of the CGV.
The removal efficiency was tested by conjugating the CGV into the model strain Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) via a mating with an E. coli (bSNP3235)-based donor strain and plating the mating mixture onto solid medium that selecting for the CGV with and without rhamnose. As a control, the empty plasmid pSNP1380 was conjugated via a separate mating.
Conjugation experiments showed that the CGV was efficiently removed, as 1000-fold less transconjugants emerged on the selective media with induction compared to the plates without induction (˜104 CFU/ml and ˜107 CFU/ml, respectively) (
3.2 Testing for off-target effects of self-targeting CGVs
To test if the self-targeting CGV is removed without killing the Bacteroides host, we grew bSNP2978 (pSNP1599) with and without rhamnose (
One hour after induction, around 99% of the cells in the population lost their CGVs (
A new CGV carrying a self-targeting CRISPR/Cas system (CdCas) can efficiently was removed from more than 99.9% of cells in a population of B. thetaiotaomicron upon induction with rhamnose.
It was also demonstrated that survival and growth of the B. thetaiotaomicron cells was unaffected by the induced self-targeting CGV, suggesting that the CRISPR/Cas-mediated CGV removal had no off-target effects.
E. coli NEB ® 10-beta.
Bacteroides thetaiotaomicron VPI-5482.
Design and assembly of CRISPR array
A CRISPR array was designed by identifying five 37-bp protospacers in the backbone of the shuttle plasmid (details in Appendix C), each positioned adjacent to the consensus type I-B PAM sequence (5′-CCW-3′). A preliminary sequence similarity search (BLASTn, not shown) did not predict any off-targets for any of the five chosen spacers to the chromosome of bSNP2978. The repeat sequence (5′-GTTTTATATTAACTAAGTGGTATGTAAAT-3′ (SEQ ID NO: 5)) compatible with type I-B CRISPR/Cas system was chosen.
difficile strain 630Δerm (CdCas) and a
A predicted xylitol-inducible promoter originating from the bacterium Morganella morganii was cloned and investigated within various Escherichia coli strain backgrounds.
It was demonstrated that the promoter is repressed by a transcriptional regulator protein IZ184_04885 and that repression could be lifted either gradually by adding xylitol to the growth medium or completely by mutating the transcriptional regulator. Sensitivity of the promoter towards xylitol increased when the xylitol ABC transporter system was included as well.
Demonstrate that the repression of the xylitol-inducible promoter can be lifted by adding increasing amounts of xylitol to the growth medium.
Demonstrate that the repression of the xylitol-inducible promoter is avoided by mutating the transcriptional regulator protein IZ184_04885.
Investigate whether expression of the xylitol ABC transporter increases the sensitivity of the xylitol-inducible promoter towards xylitol in the medium.
All strains of Escherichia coli used in this study are listed in Table 5, Appendix A. For cloning, E. coli strains were grown at 37° C. in lysogeny broth (LB) at 250 RPM or on solid LB-agar plates (made with 1.5% (w/v) agar). For plasmid selection, the medium was supplemented with tetracycline (10 μg ml−1) and/or spectinomycin (Spc, 100 μg ml−1).
GFP reporter plasmids
Based on an annotated genome sequence of Morganella morganii strain ZJG812 (Genome ID: CP064831.1), a 1213-bp DNA sequence encoding the xylitol-inducible regulatory system (excluding the sugar isomerase gene IZ184_04880) was defined and ordered as a gBlock fragment from Integrated DNA Technologies™ (Coralville, IA, USA). The gBlock DNA fragment was further amplified by PCR using the CloneAmp HiFi PCR Premix from Takara Bio (Mountain View, CA, USA) with primers SEM1770 and SEM1771. In parallel, the backbone of the plasmid pSNP1248 was PCR amplified using the CloneAMP HiFi PCR premix with primers SEM1282 and SEM1769, yielding a 32.50-bp DNA fragment that contain the fluorescent reporter gene (gfp), a low copy-number origin of replication (cloDF13) and a spectinomycin resistance marker (Spc®). The full list of primers used in this study is found in Table 6, Appendix B.
The amplified DNA fragments were routinely treated with DpnI (1 hour at 37° C. followed by 20 minutes at 80° C.) and purified by gel extraction using the MinElute kit from Qiagen (Hilden, Germany) according to the manufacturer's instructions. The purified regulatory system DNA was inserted into the linearized pSNP1248 backbone with the In-Fusion® HD Cloning kit from Takara Bio according to the manufacturer's instruction. Chemically competent bSNP2480 cells were transformed with the DNA from the In-Fusion® reaction mixture. Transformed cells were spread onto LB+Spc100 agar plates and allowed to incubate overnight. Plasmids were purified from individual colonies using the QIAprep Spin Miniprep Kit from Qiagen according to the manufacturer's instructions and followingly verified by Sanger sequencing. The positive clone was registered as plasmid pSNP1902. In addition, a serendipitous clone containing a 595-bp deletion located within the transcriptional regulator open reading frame (ORF) (IZ184_04885) was identified and registered as plasmid pSNP1903.
xylitol ABC transporter plasmid
The 3728-bp.xylABC operon from Morganella morganii strain ZJG812 (Genome ID: CP064831.1) was ordered as two gBlock DNA fragments from Integrated DNA Technologies™. The plasmid backbone was prepared by PCR amplification of plasmid pSNP639 using the CloneAmp HiFi PCR Premix with primers SEM1814 and SEM1815. The amplified fragment was DpnI treated and purified by gel extraction, following the same method as described in section 3.2.1.
The two gBlock DNA fragments and the linearized backbone DNA were fused together using the In-Fusion® HD Cloning kit from Takara Bio according to the manufacturer's instruction. Chemically competent bSNP2522 cells were transformed with the DNA from the In-Fusion@ reaction mixture. Transformed cells were spread onto LB+Tet10 agar plates and allowed to incubate overnight. Plasmids were purified from individual colonies using the QIAprep Spin Miniprep Kit from Qiagen according to the manufacturer's instructions and followingly verified by Sanger sequencing. The positive clone was registered as plasmid pSNP1939. The full list of plasmids used in this study is found in Table 7, Appendix C.
The constructed GFP reporter plasmids (pSNP1902 and pSNP1903), the xylitol ABC transporter-containing plasmid (pSNP1939) as well as the control plasmids (see Table 7, Appendix C) were transformed individually or in combination into the model strains E. coli MG1655 (bSNP230) and a probiotic E. coli isolate (bSNP463) via electroporation. Transformed cells were spread onto LB+Spc100-, LB+Tet10- or LB+Spc100+Tet100 agar plates depending on whether the cells were transformed with the GFP reporter plasmid, xylitol ABC transporter plasmid, or both, respectively. The plates were allowed to incubate overnight. Three colonies of each strain were picked and used as biological triplicates in the ensuing 24-hour gfp expression assay.
24-hour gfp expression assay Triplicate colonies were inoculated into 5-ml LB medium or 5-ml M9 minimal medium broth (recipe found in Appendix D) supplemented with Tet (10 μg ml−1) and/or Spc (100 μg ml−1) and allowed to incubate overnight (˜16 hours) at 37° C. and 250 RPM. Cultures would have reached optical densities (OD600) of 0.8±0.3 in M9 minimal medium and 1.4±0.2 in LB medium.
Each overnight culture was then diluted 100× in fresh medium supplemented with appropriate antibiotics (as above) and distributed into 3-6 new cultures which were given xylitol at different concentrations within the range of 0% to 5% (the exact concentrations of xylitol are listed in the related figures in section 4). The cultures were then transferred as 200-μl aliquots into a black 96-well microtiter plate with transparent bottom. To normalize the fluorescence emissions, a row of wells in the plate were dedicated to cells that harbor the control plasmids pSNP958 and pSNP1617 which do not encode gfp (to estimate autofluorescence) and another row dedicated to media without any cells (to estimate background fluorescence). The microtiter plate was sealed with a Breath-Easy® sealing membrane from Merck (Whitehouse station, NJ, USA) and placed in the Synergy H1 microplate reader from Agilent Biotek (Winooski, VT, USA) programmed to incubate for 24 hours at 37° C. with a constant agitation. Both OD600 and GFP fluorescence (excitation at 485 nm, emission at 516 nm, gain of 90) were measured every 10 minutes throughout the incubation. The recorded fluorescence emissions were normalized according to the corresponding cell densities and corrected for the background and autofluorescence levels.
Criteria and choice of the inducible promoter We have developed a biocontainment strategy using CRISPR/Cas systems as off-switches of our production circuits or strains. This requires that the CRISPR/Cas system is under tight transcriptional control from a promoter which is activated upon addition of an external inducer molecule.
Within the field of molecular microbiology, operons of genes related to the conversion/utilization of sugars may be induced by the sugars themselves as they bind and influence the transcriptional repressor proteins. For the present study, it was decided to investigate a negatively inducible promoter predicted to be induced by xylitol. This 5-carbon sugar alcohol has several advantages. First of all, xylitol is generally regarded as safe for human consumption by the FDA (Xiang et al., 2021). Furthermore, over half of ingested xylitol is not adsorbed by human cells instead reaching the gastrointestinal tract where it is taken up by the microbiome (Livesey, 2003), making this useful to control our switches in the GI tract. Finally, xylitol is a known metabolite for some bacteria: It is either taken up directly through an ABC-type transporter complex (Madigan et al., 2015) and/or generated through reduction of the corresponding sugar D-xylose. Subsequently, xylitol can be dehydrogenated and phosphorylated to xylulose-5-phosphate which is further catabolized in the pentose phosphate pathway.
From a dated PhD thesis (Gallo, 1991), we found an operon in the genome of the bacterium Morganella morganii strain ZJG812 which contain genes related to xylitol uptake and metabolism (details summarized in
Estimating promoter activity in response to xylitol in growth medium
For this study. two reporter plasmids were constructed consisting of a green fluorescent protein (GFP) reporter gene located downstream from the xylitol-inducible regulatory system either without (pSNP1902,
The reporter plasmids consist of a green fluorescent protein (GFP) reporter gene located downstream from the predicted xylitol-inducible promoter which in turn is controlled by the LacI family DNA-binding transcriptional regulator (IZ184_04885 gene). Two reporter plasmids are made, either (A) with a full-length transcriptional regulator (plasmid pSNP1902) or (B) with a 595-bp loss-of-function (LOF) deletion within the ORF of the transcriptional regulator (plasmid pSNP1903).
The GFP reporter plasmids pSNP1902 and pSNP1903 were transformed into the probiotic isolate strain bSNP463 and tested by measuring the optical density (OD600) and green fluorescence emitted from cultures after a 24-hour incubation in the presence of increasing amounts of xylitol. The results from these tests are summarized in
The test of the GFP reporter plasmid with the full-length transcriptional regulator (pSNP1902) was repeated in a minimal medium broth using the E. coli strain MG1655 (bSNP230) which gave similar results (as shown in
Testing whether the presence of the xylitol ABC transporter increases the sensitivity of the xylitol-inducible promoter
The activity of a negatively inducible promoter will depend on the import of the inducer molecule into the cell. In this context, it was hypothesized that expression of the predicted xylitol ABC transporter operon (mentioned in section 4.1) would allow more xylitol to enter the cell and hence increase the sensitivity of the promoter. To test this, plasmid pSNP1939 was transformed into the bSNP230-construct already containing the GFP reporter plasmid pSNP1902 and tested by the same 24-hour assay as used previously. For these experiments, the well-defined M9 minimal media were used either with glycerol or glucose as the carbon source for growth. Glucose was chosen as it is the preferred sugar for bacterial growth, but it might also have an inhibitory effect on promoters which regulate alternative metabolic pathways such as those related to xylitol. Glycerol was chosen as it had previously been shown by us (data not shown) not to influence the expression from the xylitol-inducible promoter. The results are summarized in
We used bSNP230 strains containing the GFP reporter plasmid (pSNP1902) either with or without a co-resident plasmid containing the xylitol ABC transporter (pSNP1939) were grown for 24-hours in minimal medium supplemented with 0.4% (v/v) glycerol (A) or 0.4% (w/v) glucose (B) in the presence or absence of xylitol. The cell-density adjusted fluorescence emission after 24 hours of incubation are shown above. Error bars indicate standard deviations based on two (A) and three (B) biological replicates, respectively. The corresponding growth curves are shown in
At these low concentrations of xylitol, the strain bSNP230/pSNP1902 did not exhibit any change in fluorescence intensity (but higher concentrations of xylitol in a different experiment may lead to increased fluorescence). For the strain bSNP230/pSNP1902p+pSNP1939, however, it was observed that the fluorescence intensity increased as xylitol was added—an effect which was more pronounced when the minimal medium was supplemented with glycerol (
The xylitol-inducible promoter originating from Morganella morganii is repressed by the transcriptional regulator protein IZ184_04885 when tested in various E. coli strain backgrounds. This repression was lifted gradually by adding xylitol to the growth medium or fully by mutating IZ184_04885. The sensitivity of the promoter towards xylitol in the growth medium was further enhanced by including the xylitol ABC transporter system.
E. coli MG1655
E. coli probiotic isolate
E. coli Takara Stellar
E. coli NEB 10-beta
Morganella morganii but without the sugar
morganii. Encodes a tetracycline resistance marker
The M9 minimal medium used in this study report consists of a medium salts solution supplemented with a carbon source (glucose or glycerol). B1 vitamin, magnesium, calcium and a source of iron.
The medium salts solution is a composition of four crucial salts. We recommend preparing it as a 5× stock solution according to Table 8 below:
All remaining stock solutions required to make the M9 medium salts solution are prepared routinely by weighing the appropriate amount of the component, dissolving it in distilled H2O under constant agitation and filter-sterilizing it (pore size=0.2 μm). Once prepared, mix the stock solutions together according to the instructions from Table 9 below:
This sequence can be used in the invention and provides the repressor and promoter.
Acidovorax avenae subsp. cattleyae
Acidovorax avenae subsp.
Acidovorax konjaci
Acidovorax valerianellae
Agrobacterium
Agrobacterium larrymoorei
Agrobacterium radiobacter
Agrobacterium rhizogenes
Agrobacterium rubi
Agrobacterium tumefaciens
Agrobacterium vitis
Arthrobacter
Arthrobacter ilicis
Bacillus
Bacillus megaterium
Bacillus megaterium pv. cerealis
Bacillus pumilus
Brenneria
Brenneria alni
Brenneria nigrifluens
Brenneria quercina
Brenneria rubrifaciens
Brenneria salicis
Burkholderia
Burkholderia andropogonis
Burkholderia caryophylli
Burkholderia cepacia
Burkholderia gladioli
Burkholderia gladioli pv. agaricicola
Burkholderia gladioli pv. alliicola
Burkholderia gladioli pv. gladioli
Burkholderia glumae
Burkholderia
Clavibacter
Clavibacter michiganensis
Clavibacter michiganensis subsp.
Clavibacter michiganensis subsp. michiganensis
Clavibacter michiganensis subsp. nebraskensis
Clavibacter michiganensis subsp. sepedonicus
Clavibacter michiganensis subsp. tessellarius
Clavibacter rathayi
Clavibacter toxicus
Clavibacter tritici
Clavibacter xyli
Clavibacter xyli subsp. cynodontis
Clavibacter xyli subsp. xyli
Clostridium
Clostridium puniceum
Corynebacterium
Corynebacterium betae
Corynebacterium beticola
Corynebacterium fascians
Corynebacterium flaccumfaciens
Corynebacterium flaccumfaciens pv. betae
Corynebacterium flaccumfaciens pv. flaccumfaciens
Corynebacterium flaccumfaciens pv. oortii
Corynebacterium flaccumfaciens pv. poinsettiae
Corynebacterium flaccumfaciens subsp.
Corynebacterium flaccumfaciens subsp. flaccumfaciens
Corynebacterium flaccumfaciens subsp. oortii
Corynebacterium flaccumfaciens subsp. poinsettiae
Corynebacterium ilicis
Corynebacterium insidiosum
Corynebacterium iranicum
Corynebacterium michiganense
Corynebacterium michiganensis pv. insidiosus
Corynebacterium michiganensis pv. iranicum
Corynebacterium michiganense pv. nebraskense
Corynebacterium michiganense pv. rathayi
Corynebacterium michiganense pv. sepedonicum
Corynebacterium michiganense pv. tritici
Corynebacterium michiganense subsp. insidiosum
Corynebacterium michiganense subsp.
Corynebacterium michiganense subsp. nebraskense
Corynebacterium michiganense subsp. sepedonicum
Corynebacterium michiganense subsp. tessellarius
Corynebacterium oortii
Corynebacterium
Corynebacterium rathayi
Corynebacterium sepedonicum
Corynebacterium tritici
Curtobacterium
Curtobacterium flaccumfaciens
Curtobacterium flaccumfaciens pv.
Curtobacterium flaccumfaciens pv. flaccumfaciens
Curtobacterium flaccumfaciens pv. ilicis
Curtobacterium flaccumfaciens pv. oortii
Curtobacterium flaccumfaciens pv. poinsettiae
Dickeya
Dickeya chrysanthemi
Dickeya chrysanthemi pv. chrysanthemi
Dickeya chrysanthemi pv. parthenii
Dickeya dadantii
Dickeya dianthicola
Dickeya dieffenbachiae
Dickeya paradisiaca
Dickeya zeae
Enterobacter
Enterobacter agglomerans
Enterobacter cancerogenus
Enterobacter cloacae
Enterobacter cloacae subsp. dissolvens
Enterobacter nimipressuralis
Enterobacter pyrinus
Erwinia
Erwinia alni
Erwinia amylovora.
Erwinia amylovora pv. pyri
Erwinia ananatis corrig.
Erwinia ananatis pv. ananatis
Erwinia ananas pv. uredovora
Erwinia cacticida
Erwinia cancerogena
Erwinia carnegieana
Erwinia carotovora
Erwinia carotovora pv. atroseptica
Erwinia carotovora pv. carotovora
Erwinia carotovora subsp. atroseptica
Erwinia carotovora subsp. carotovora
Erwinia carotovora subsp. betavasculorum
Erwinia carotovora subsp. odorifera
Erwinia carotovora subsp. wasabiae
Erwinia chrysanthemi
Erwinia chrysanthemi pv. chrysanthemi
Erwinia chrysanthemi pv.
Erwinia chrysanthemi pv. dieffenbachiae
Erwinia chrysanthemi pv. paradisiaca
Erwinia chrysanthemi pv. parthenii
Erwinia chrysanthemi pv. zeae
Erwinia cypripedii
Erwinia dissolvens
Erwinia herbicola
Erwinia herbicola f. sp.
Erwinia herbicola pv. millettiae
Erwinia mallotivora
Erwinia nigrifluens
Erwinia nimipressuralis
Erwinia papayae
Erwinia proteamaculans
Erwinia persicina
Enterobacter pyrinus
Erwinia psidii
Erwinia pyrifoliae
Erwinia rhapontici
Erwinia rubrifaciens
Erwinia salicis
Erwinia stewartii
Erwinia tracheiphila
Erwinia uredovora
Ewingella
Ewingella americana
Gluconobacter Asai
Gluconobacter oxydans
Herbaspirillum
Herbaspirillum rubrisubalbicans
Janthinobacterium
Janthinobacterium agaricidamnosum
Leifsonia
Leifsonia cynodontis
Leifsonia xyli
Leifsonia xyli subsp. cynodontis
Leifsonia xyli subsp. xyli
Nocardia
Nocardia vaccinii
Pantoea
Pantoea agglomerans
Pantoea agglomerans pv. gypsophilae
Pantoea agglomerans pv. millettiae
Pantoea ananatis
Pantoea ananatis pv. ananatis
Pantoea ananatis pv. uredovora
Pantoea stewartii
Pantoea stewartii subsp. indologenes
Pantoea stewartii subsp. stewartii
Pectobacterium
Pectobacterium
Pectobacterium
Pectobacterium cacticida corrig
Pectobacterium
Pectobacterium carotovorum
Pectobacterium carotovorum subsp. atrosepticum
Pectobacterium carotovorum subsp. betavasculorum
Pectobacterium carotovorum subsp. brasiliensis
Pectobacterium carotovorum subsp. carotovorum
Pectobacterium carotovorum subsp. odoriferum
Pectobacterium carotovorum subsp. wasabiae
Pectobacterium chrysanthemi
Pectobacterium chrysanthemi pv. chrysanthemi
Pectobacterium chrysanthemi pv. dianthicola
Pectobacterium chrysanthemi pv. dieffenbachiae
Pectobacterium chrysanthemi pv. parthenii
Pectobacterium chrysanthemi pv. zeae
Pectobacterium cypripedii
Pectobacterium rhapontici
Pectobacterium wasabiae
Pseudomonas
Pseudomonas agarici
Pseudomonas amygdali
Pseudomonas andropogonis pv. andropogonis
Pseudomonas andropogonis pv. sojae
Pseudomonas andropogonis pv. stizolobii
Pseudomonas asplenii
Pseudomonas avellanae
Pseudomonas avenae
Pseudomonas avenae subsp. avenae
Pseudomonas avenae subsp. citrulli
Pseudomonas avenae subsp. konjaci
Pseudomonas beteli corrig.
Pseudomonas cannabina
Pseudomonas caricapapayae
Pseudomonas caryophylli
Pseudomonas cattleyae
Pseudomonas cepacia
Pseudomonas cichorii
Pseudomonas cissicola
Pseudomonas coronafaciens
Pseudomonas corrugata
Pseudomonas costantinii
Pseudomonas dodoneae
Pseudomonas ficuserectae
Pseudomonas flectens
Pseudomonas fuscovaginae
Pseudomonas gingeri
Pseudomonas gladioli
Pseudomonas gladioli pv. agaricicola
Pseudomonas gladioli pv. alliicola
Pseudomonas gladioli pv. gladioli
Pseudomonas glumae
Pseudomonas hibiscicola
Pseudomonas marginalis
Pseudomonas marginalis pv. alfalfae
Pseudomonas marginalis pv. marginalis
Pseudomonas marginalis pv. pastinacae
Pseudomonas mediterranea
Pseudomonas meliae
Pseudomonas palleroniana
Pseudomonas plantarii
Pseudomonas pomi
Pseudomonas pseudoalcaligenes subsp. citrulli
Pseudomonas pseudoalcaligenes subsp. konjaci
Pseudomonas rubrilineans
Pseudomonas rubrisubalbicans
Pseudomonas salomonii
Pseudomonas savastanoi
Pseudomonas savastanoi pv. fraxini
Pseudomonas savastanoi pv. glycinea
Pseudomonas savastanoi pv. nerii
Pseudomonas savastanoi pv. phaseolicola
Pseudomonas savastanoi pv. retacarpa
Pseudomonas savastanoi pv. savastanoi
Pseudomonas syringae
Pseudomonas syringae pv. aceris
Pseudomonas syringae pv. actinidiae
Pseudomonas syringae pv. aesculi
Pseudomonas syringae pv. alisalensis
Pseudomonas syringae pv. antirrhini
Pseudomonas syringae pv. apii
Pseudomonas syringae pv. aptata
Pseudomonas syringae pv.
Pseudomonas syringae pv. atropurpurea
Pseudomonas syringae pv. avellanae
Pseudomonas syringae pv. avii
Pseudomonas syringae pv. berberidis
Pseudomonas syringae pv. broussonetiae
Pseudomonas syringae pv. castaneae
Pseudomonas syringae pv. cerasicola
Pseudomonas syringae pv. ciccaronei
Pseudomonas syringae pv. coriandricola
Pseudomonas syringae pv. coronafaciens
Pseudomonas syringae pv. coryli
Pseudomonas syringae pv. cunninghamiae
Pseudomonas syringae pv. daphniphylli
Pseudomonas syringae pv. delphinii
Pseudomonas syringae pv. dendropanacis
Pseudomonas syringae pv. dysoxyli
Pseudomonas syringae pv. eriobotryae
Pseudomonas syringae pv. garcae
Pseudomonas syringae pv. glycinea
Pseudomonas syringae pv. helianthi
Pseudomonas syringae pv.
Pseudomonas syringae pv.
Pseudomonas syringae pv.
Pseudomonas syringae pv. lapsa
Pseudomonas syringae pv. maculicola
Pseudomonas syringae pv.
Pseudomonas syringae pv. mori
Pseudomonas syringae pv. morsprunorum.
Pseudomonas syringae pv. myricae
Pseudomonas syringae pv.
Pseudomonas syringae pv. papulans
Pseudomonas syringae pv. passiflorae
Pseudomonas syringae pv.
Pseudomonas syringae pv. philadelphi
Pseudomonas syringae pv. photiniae
Pseudomonas syringae pv. pisi
Pseudomonas syringae pv. porri
Pseudomonas syringae pv. primulae
Pseudomonas syringae pv. rhaphiolepidis
Pseudomonas syringae pv. ribicola
Pseudomonas syringae pv. sesami
Pseudomonas syringae pv. solidagae
Pseudomonas syringae pv. spinaceae
Pseudomonas syringae pv. syringae
Pseudomonas syringae pv. tagetis
Pseudomonas syringae pv. theae
Pseudomonas syringae pv. tomato
Pseudomonas syringae pv. ulmi
Pseudomonas syringae pv. viburni
Pseudomonas syringae pv.
Pseudomonas syzygii
Pseudomonas tolaasii
Pseudomonas tremae
Pseudomonas viridiflava
Ralstonia
Ralstonia solanacearum
Ralstonia syzygii
Rathayibacter
Rathayibacter iranicus
Rathayibacter rathayi
Rathayibacter
Rathayibacter tritici
Rhizobacter
Rhizobacter dauci corrig.
Rhizobium
Rhizobium larrymoorei
Rhizobium radiobacter
Rhizobium rhizogenes
Rhizobium rubi
Rhizobium vitis
Rhodococcus
Rhodococcus fascians
Samsonia
Samsonia erythrinae
Serratia
Serratia marcescens
Serratia proteamaculans
Sphingomonas
Sphingomonas melonis Buonaurio
Sphingomonas suberifaciens
Spiroplasma
Spiroplasma citri
Spiroplasma kunkelii
Spiroplasma phoeniceum
Streptomyces
Streptomyces acidiscabies
Streptomyces albidoflavus
Streptomyces candidus
Streptomyces caviscabies
Streptomyces collinus
Streptomyces europaeiscabiei
Streptomyces intermedius
Streptomyces ipomoeae
Streptomyces luridiscabiei
Streptomyces niveiscabiei
Streptomyces puniciscabiei
Streptomyces reticuliscabei
Streptomyces scabiei corrig.
Streptomyces setonii
Streptomyces steliiscabiei
Streptomyces turgidiscabies
Streptomyces wedmorensis
Xanthomonas
Xanthomonas albilineans
Xanthomonas alfalfae
Xanthomonas alfalfae subsp. alfalfae
Xanthomonas alfalfae subsp. citrumelonis
Xanthomonas arboricola
Xanthomonas axonopodis
Xanthomonas bromi
Xanthomonas campestris
Xanthomonas cassavae
Xanthomonas citri
Xanthomonas cucurbitae
Xanthomonas euvesicatoria
Xanthomonas fragariae
Xanthomonas fuscans
Xanthomonas fuscans
Xanthomonas gardneri
Xanthomonas hortorum
Xanthomonas hortorum
Xanthomonas hyacinthi
Xanthomonas oryzae
Xanthomonas populi
Xanthomonas sacchari
Xanthomonas theicola
Xanthomonas translucens
Xanthomonas vasicola
Xylella
Xylella fastidiosa
Xylophilus
Xylophilus ampelinus
Abiotrophia
Acidocella
Actinomyces
Alkalilimnicola
Aquaspirillum
Abiotrophia defectiva
Acidocella aminolytica
Actinomyces bovis
Alkalilimnicola ehrlichii
Aquaspirillum polymorphum
Acaricomes
Acidocella facilis
Actinomyces denticolens
Alkaliphilus
Aquaspirillum putridiconchylium
Acaricomes phytoseiuli
Acidomonas
Actinomyces europaeus
Alkaliphilus oremlandii
Aquaspirillum serpens
Acetitomaculum
Acidomonas methanolica
Actinomyces georgiae
Alkaliphilus transvaalensis
Aquimarina
Acetitomaculum ruminis
Acidothermus
Actinomyces gerencseriae
Allochromatium
Aquimarina latercula
Acetivibrio
Acidothermus cellulolyticus
Actinomyces hordeovulneris
Allochromatium vinosum
Arcanobacterium
Acetivibrio cellulolyticus
Acidovorax
Actinomyces howellii
Alloiococcus
Arcanobacterium haemolyticum
Acetivibrio ethanolgignens
Acidovorax anthurii
Actinomyces hyovaginalis
Alloiococcus otitis
Arcanobacterium pyogenes
Acetivibrio multivorans
Acidovorax caeni
Actinomyces israelii
Allokutzneria
Archangium
Acetoanaerobium
Acidovorax cattleyae
Actinomyces johnsonii
Allokutzneria albata
Archangium gephyra
Acetoanaerobium noterae
Acidovorax citrulli
Actinomyces meyeri
Altererythrobacter
Arcobacter
Acetobacter
Acidovorax defluvii
Actinomyces naeslundii
Altererythrobacter ishigakiensis
Arcobacter butzleri
Acetobacter aceti
Acidovorax delafieldii
Actinomyces neuii
Altermonas
Arcobacter cryaerophilus
Acetobacter cerevisiae
Acidovorax facilis
Actinomyces odontolyticus
Altermonas haloplanktis
Arcobacter halophilus
Acetobacter cibinongensis
Acidovorax konjaci
Actinomyces oris
Altermonas macleodii
Arcobacter nitrofigilis
Acetobacter estunensis
Acidovorax temperans
Actinomyces radingae
Alysiella
Arcobacter skirrowii
Acetobacter fabarum
Acidovorax valerianellae
Actinomyces slackii
Alysiella crassa
Arhodomonas
Acetobacter ghanensis
Acinetobacter
Actinomyces turicensis
Alysiella filiformis
Arhodomonas aquaeolei
Acetobacter indonesiensis
Acinetobacter baumannii
Actinomyces viscosus
Aminobacter
Arsenophonus
Acetobacter lovaniensis
Acinetobacter baylyi
Actinoplanes
Aminobacter aganoensis
Arsenophonus nasoniae
Acetobacter malorum
Acinetobacter bouvetii
Actinoplanes auranticolor
Aminobacter aminovorans
Arthrobacter
Acetobacter nitrogenifigens
Acinetobacter calcoaceticus
Actinoplanes brasiliensis
Aminobacter niigataensis
Arthrobacter agilis
Acetobacter oeni
Acinetobacter gerneri
Actinoplanes consettensis
Aminobacterium
Arthrobacter albus
Acetobacter orientalis
Acinetobacter haemolyticus
Actinoplanes deccanensis
Aminobacterium mobile
Arthrobacter aurescens
Acetobacter orleanensis
Acinetobacter johnsonii
Actinoplanes derwentensis
Aminomonas
Arthrobacter chlorophenolicus
Acetobacter pasteurianus
Acinetobacter junii
Actinoplanes digitatis
Aminomonas paucivorans
Arthrobacter citreus
Acetobacter pornorurn
Acinetobacter lwoffi
Actinoplanes durhamensis
Ammoniphilus
Arthrobacter crystallopoietes
Acetobacter senegalensis
Acinetobacter parvus
Actinoplanes ferrugineus
Ammoniphilus oxalaticus
Arthrobacter cumminsii
Acetobacter xylinus
Acinetobacter radioresistens
Actinoplanes globisporus
Ammoniphilus oxalivorans
Arthrobacter globiformis
Acetobacterium
Acinetobacter schindleri
Actinoplanes humidus
Amphibacillus
Arthrobacter histidinolovorans
Acetobacterium bakii
Acinetobacter soli
Actinoplanes italicus
Amphibacillus xylanus
Arthrobacter ilicis
Acetobacterium carbinolicum
Acinetobacter tandoii
Actinoplanes liguriensis
Amphritea
Arthrobacter luteus
Acetobacterium dehalogenans
Acinetobacter tjernbergiae
Actinoplanes lobatus
Amphritea balenae
Arthrobacter methylotrophus
Acetobacterium fimetarium
Acinetobacter towneri
Actinoplanes missouriensis
Amphritea japonica
Arthrobacter mysorens
Acetobacterium malicum
Acinetobacter ursingii
Actinoplanes palleronii
Amycolatopsis
Arthrobacter nicotianae
Acetobacterium paludosum
Acinetobacter venetianus
Actinoplanes philippinensis
Amycolatopsis alba
Arthrobacter nicotinovorans
Acetobacterium tundrae
Acrocarpospora
Actinoplanes rectilineatus
Amycolatopsis albidoflavus
Arthrobacter oxydans
Acetobacterium wieringae
Acrocarpospora corrugata
Actinoplanes regularis
Amycolatopsis azurea
Arthrobacter pascens
Acetobacterium woodii
Acrocarpospora macrocephala
Actinoplanes teichomyceticus
Amycolatopsis coloradensis
Arthrobacter phenanthrenivorans
Acetofilamentum
Acrocarpospora pleiomorpha
Actinoplanes utahensis
Amycolatopsis lurida
Arthrobacter polychromogenes
Acetofilamentum rigidum
Actibacter
Actinopolyspora
Amycolatopsis mediterranei
Atrhrobacter protophormiae
Acetohalobium
Actibacter sediminis
Actinopolyspora halophila
Amycolatopsis rifamycinica
Arthrobacter psychrolactophilus
Acetohalobium arabaticum
Actinoalloteichus
Actinopolyspora mortivallis
Amycolatopsis rubida
Arthrobacter ramosus
Acetomicrobium
Actinoalloteichus cyanogriseus
Actinosynnema
Amycolatopsis sulphurea
Arthrobacter sulfonivorans
Acetomicrobium faecale
Actinoalloteichus hymeniacidonis
Actinosynnema mirum
Amycolatopsis tolypomycina
Arthrobacter sulfureus
Acetomicrobium flavidum
Actinoalloteichus spitiensis
Actinotalea
Anabaena
Arthrobacter uratoxydans
Acetonema
Actinobaccillus
Actinotalea fermentans
Anabaena cylindrica
Arthrobacter ureafaciens
Acetonema longum
Actinobacillus capsulatus
Aerococcus
Anabaena flos-aquae
Arthrobacter viscosus
Acetothermus
Actinobacillus delphinicola
Aerococcus sanguinicola
Anabaena variabilis
Arthrobacter woluwensis
Acetothermus paucivorans
Actinobacillus hominis
Aerococcus urinae
Anaeroarcus
Asaia
Acholeplasma
Actinobacillus indolicus
Aerococcus urinaeequi
Anaeroarcus burkinensis
Asaia bogorensis
Acholeplasma axanthum
Actinobacillus lignieresii
Aerococcus urinaehominis
Anaerobaculum
Asanoa
Acholeplasma brassicae
Actinobacillus minor
Aerococcus viridans
Anaerobaculum mobile
Asanoa ferruginea
Acholeplasma cavigenitalium
Actinobacillus muris
Aeromicrobium
Anaerobiospirillum
Asticcacaulis
Acholeplasma equifetale
Actinobacillus pleuropneumoniae
Aeromicrobium erythreum
Anaerobiospirillum succiniciproducens
Asticcacaulis biprosthecium
Acholeplasma granularum
Actinobacillus porcinus
Aeromonas
Anaerobiospirillum thomasii
Asticcacaulis excentricus
Acholeplasma hippikon
Actinobacillus rossii
Aeromonas allosaccharophila
Anaerococcus
Atopobacter
Acholeplasma laidlawii
Actinobacillus scotiae
Aeromonas bestiarum
Anaerococcus hydrogenalis
Atopobacter phocae
Acholeplasma modicum
Actinobacillus seminis
Aeromonas caviae
Anaerococcus lactolyticus
Atopobium
Acholeplasma morum
Actinobacillus succinogenes
Aeromonas encheleia
Anaerococcus prevotii
Atopobium fossor
Acholeplasma multilocale
Actinobaccillus suis
Aeromonas enteropelogenes
Anaerococcus tetradius
Atopobium minutum
Acholeplasma oculi
Actinobacillus ureae
Aeromonas eucrenophila
Anaerococcus vaginalis
Atopobium parvulum
Acholeplasma palmae
Actinobaculum
Aeromonas ichthiosmia
Anaerofustis
Atopobium rimae
Acholeplasma parvum
Actinobaculum massiliense
Aeromonas jandaei
Anaerofustis stercorihominis
Atopobium vaginae
Acholeplasma pleciae
Actinobaculum schaalii
Aeromonas media
Anaeromusa
Aureobacterium
Acholeplasma vituli
Actinobaculum suis
Aeromonas popoffii
Anaeromusa acidaminophila
Aureobacterium barkeri
Achromobacter
Actinomyces urinale
Aeromonas sobria
Anaeromyxobacter
Aurobacterium
Achromobacter denitrificans
Actinocatenispora
Aeromonas veronii
Anaeromyxobacter dehalogenans
Aurobacterium liquefaciens
Achromobacter insolitus
Actinocatenispora rupis
Agrobacterium
Anaerorhabdus
Avibacterium
Achromobacter piechaudii
Actinocatenispora thailandica
Agrobacterium gelatinovorum
Anaerorhabdus furcosa
Avibacterium avium
Achromobacter ruhlandii
Actinocatenispora sera
Agrococcus
Anaerosinus
Avibacterium gallinarum
Achromobacter spanius
Actinocorallia
Agrococcus citreus
Anaerosinus glycerini
Avibacterium paragallinarum
Acidaminobacter
Actinocorallia aurantiaca
Agrococcus jenensis
Anaerovirgula
Avibacterium volantium
Acidaminobacter hydrogenoformans
Actinocorallia aurea
Agromonas
Anaerovirgula multivorans
Azoarcus
Acidaminococcus
Actinocorallia cavernae
Agromonas oligotrophica
Ancalomicrobium
Azoarcus indigens
Acidaminococcus fermentans
Actinocorallia glomerata
Agromyces
Ancalomicrobium adetum
Azoarcus tolulyticus
Acidaminococcus intestini
Actinocorallia herbida
Agromyces fucosus
Ancylobacter
Azoarcus toluvorans
Acidicaldus
Actinocorallia libanotica
Agromyces hippuratus
Ancylobacter aquaticus
Azohydromonas
Acidicaldus organivorans
Actinocorallia longicatena
Agromyces luteolus
Aneurinibacillus
Azohydromonas australica
Acidimicrobium
Actinomadura
Agromyces mediolanus
Aneurinibacillus aneurinilyticus
Azohydromonas lata
Acidimicrobium ferrooxidans
Actinomadura alba
Agromyces ramosus
Aneurinibacillus migulanus
Azomonas
Acidiphilium
Actinomadura atramentaria
Agromyces rhizospherae
Aneurinibacillus thermoaerophilus
Azomonas agilis
Acidiphilium acidophilum
Actinomadura bangladeshensis
Akkermansia
Angiococcus
Azomonas insignis
Acidiphilium angustum
Actinomadura catellatispora
Akkermansia muciniphila
Angiococcus disciformis
Azomonas macrocytogenes
Acidiphilium cryptum
Actinomadura chibensis
Albidiferax
Angulomicrobium
Azorhizobium
Acidiphilium multivorum
Actinomadura chokoriensis
Albidiferax ferrireducens
Angulomicrobium tetraedrale
Azorhizobium caulinodans
Acidiphilium organovorum
Actinomadura citrea
Albidovulum
Anoxybacillus
Azorhizophilus
Acidiphilium rubrum
Actinomadura coerulea
Albidovulum inexpectatum
Anoxybacillus pushchinoensis
Azorhizophilus paspali
Acidisoma
Actinomadura echinospora
Alcaligenes
Aquabacterium
Azospirillum
Acidisoma sibiricum
Actinomadura fibrosa
Alcaligenes denitrificans
Aquabacterium commune
Azospirillum brasilense
Acidisoma tundrae
Actinomadura formosensis
Alcaligenes faecalis
Aquabacterium parvum
Azospirillum halopraeferens
Acidisphaera
Actinomadura hibisca
Alcanivorax
Borrelia
Azospirillum irakense
Acidisphaera rubrifaciens
Actinomadura kijaniata
Alcanivorax borkumensis
Borrelia afzelii
Azotobacter
Acidithiobacillus
Actinomadura latina
Alcanivorax jadensis
Borrelia americana
Azotobacter beijerinckii
Acidithiobacillus albertensis
Actinomadura livida
Algicola
Borrelia burgdorferi
Azotobacter chroococcum
Acidithiobacillus caldus
Actinomadura luteofluorescens
Algicola bacteriolytica
Borrelia carolinensis
Azotobacter nigricans
Acidithiobacillus ferrooxidans
Actinomadura macra
Alicyclobacillus
Borrelia coriaceae
Azotobacter salinestris
Acidithiobacillus thiooxidans
Actinomadura madurae
Alicyclobacillus disulfidooxidans
Borrelia garinii
Azotobacter vinelandii
Acidobacterium
Actinomadura oligospora
Alicyclobacillus sendaiensis
Borrelia japonica
Brevinema
Acidobacterium capsulatum
Actinomadura pelletieri
Alicyclobacillus vulcanalis
Bosea
Brevinema andersonii
Bacillus
Actinomadura rubrobrunea
Alishewanella
Bosea minatitlanensis
Brevundimonas
[see below]
Actinomadura rugatobispora
Alishewanella fetalis
Bosea thiooxidans
Brevundimonas alba
Bacteriovorax
Actinomadura umbrina
Alkalibacillus
Brachybacterium
Brevundimonas aurantiaca
Bacteriovorax stolpii
Actinomadura verrucosospora
Alkalibacillus haloalkaliphilus
Brachybacterium alimentarium
Brevundimonas diminuta
Bacillus
Actinomadura vinacea
Bibersteinia
Brachybacterium faecium
Brevundimonas intermedia
B. acidiceler
Actinomadura viridilutea
Bibersteinia trehalosi
Brachybacterium paraconglomeratum
Brevundimonas subvibrioides
B. acidicola
Actinomadura viridis
Bifidobacterium
Brachybacterium rhamnosum
Brevundimonas vancanneytii
B. acidiproducens
Actinomadura yumaensis
Bifidobacterium adolescentis
Brachybacterium tyrofermentans
Brevundimonas variabilis
B. acidocaldarius
Bacteroides
Bifidobacterium angulatum
Brachyspira
Brevundimonas vesicularis
B. acidoterrestris
Bacteroides caccae
Bifidobacterium animalis
Brachyspira alvinipulli
Brochothrix
B. aeolius
Bacteroides coagulans
Bifidobacterium asteroides
Brachyspira hyodysenteriae
Brochothrix campestris
B. aerius
Bacteroides eggerthii
Bifidobacterium bifidum
Brachyspira innocens
Brochothrix thermosphacta
B. aerophilus
Bacteroides fragilis
Bifidobacterium boum
Brachyspira murdochii
Brucella
B. agaradhaerens
Bacteroides galacturonicus
Bifidobacterium breve
Brachyspira pilosicoli
Brucella canis
B. agri
Bacteroides helcogenes
Bifidobacterium catenulatum
Bradyrhizobium
Brucella neotomae
B. aidingensis
Bacteroides ovatus
Bifidobacterium choerinum
Bradyrhizobium canariense
Bryobacter
B. akibai
Bacteroides pectinophilus
Bifidobacterium coryneforme
Bradyrhizobium elkanii
Bryobacter aggregatus
B. alcalophilus
Bacteroides pyogenes
Bifidobacterium cuniculi
Bradyrhizobium japonicum
Burkholderia
B. algicola
Bacteroides salyersiae
Bifidobacterium dentium
Bradyrhizobium liaoningense
Burkholderia ambifaria
B. alginolyticus
Bacteroides stercoris
Bifidobacterium gallicum
Brenneria
Burkholderia andropogonis
B. alkalidiazotrophicus
Bacteroides suis
Bifidobacterium gallinarum
Brenneria alni
Burkholderia anthina
B. alkalinitrilicus
Bacteroides tectus
Bifidobacterium indicum
Brenneria nigrifluens
Burkholderia caledonica
B. alkalisediminis
Bacteroides thetaiotaomicron
Bifidobacterium longum
Brenneria quercina
Burkholderia caryophylli
B. alkalitelluris
Bacteroides uniformis
Bifidobacterium
Brenneria quercina
Burkholderia cenocepacia
B. altitudinis
Bacteroides ureolyticus
magnumBifidobacterium
Brenneria salicis
Burkholderia cepacia
B. alveayuensis
Bacteroides vulgatus
merycicum
Brevibacillus
Burkholderia cocovenenans
B. alvei
Balnearium
Bifidobacterium minimum
Brevibacillus agri
Burkholderia dolosa
B. amyloliquefaciens
Balnearium lithotrophicum
Bifidobacterium pseudocatenulatum
Brevibacillus borstelensis
Burkholderia fungorum
B. a. subsp. amyloliquefaciens
Balneatrix
Bifidobacterium pseudolongum
Brevibacillus brevis
Burkholderia glathei
B. a. subsp. plantarum
Balneatrix alpica
Bifidobacterium pullorum
Brevibacillus centrosporus
Burkholderia glumae
B. dipsosauri
Balneola
Bifidobacterium ruminantium
Brevibacillus choshinensis
Burkholderia graminis
B. drentensis
Balneola vulgaris
Bifidobacterium saeculare
Brevibacillus invocatus
Burkholderia kururiensis
B. edaphicus
Barnesiella
Bifidobacterium subtile
Brevibacillus laterosporus
Burkholderia multivorans
B. ehimensis
Barnesiella viscericola
Bifidobacterium thermophilum
Brevibacillus parabrevis
Burkholderia phenazinium
B. eiseniae
Bartonella
Bilophila
Brevibacillus reuszeri
Burkholderia plantarii
B. enclensis
Bartonella alsatica
Bilophila wadsworthia
Brevibacterium
Burkholderia pyrrocinia
B. endophyticus
Bartonella bacilliformis
Biostraticola
Brevibacterium abidum
Burkholderia silvatlantica
B. endoradicis
Bartonella clarridgeiae
Biostraticola tofi
Brevibacterium album
Burkholderia stabilis
B. farraginis
Bartonella doshiae
Bizionia
Brevibacterium aurantiacum
Burkholderia thailandensis
B. fastidiosus
Bartonella elizabethae
Bizionia argentinensis
Brevibacterium celere
Burkholderia tropica
B. fengqiuensis
Bartonella grahamii
Blastobacter
Brevibacterium epidermidis
Burkholderia unamae
B. firmus
Bartonella henselae
Blastobacter capsulatus
Brevibacterium frigoriTolerans
Burkholderia vietnamiensis
B. flexus
Bartonella rochalimae
Blastobacter denitrificans
Brevibacterium halotolerans
Buttiauxella
B. foraminis
Bartonella vinsonii
Blastococcus
Brevibacterium iodinum
Buttiauxella agrestis
B. fordii
Bavariicoccus
Blastococcus aggregatus
Brevibacterium linens
Buttiauxella brennerae
B. formosus
Bavariicoccus seileri
Blastococcus saxobsidens
Brevibacterium lyticum
Buttiauxella ferragutiae
B. fortis
Bdellovibrio
Blastochloris
Brevibacterium mcbrellneri
Buttiauxella gaviniae
B. fumarioli
Bdellovibrio bacteriovorus
Blastochloris viridis
Brevibacterium otitidis
Buttiauxella izardii
B. funiculus
Bdellovibrio exovorus
Blastomonas
Brevibacterium oxydans
Buttiauxella noackiae
B. fusiformis
Beggiatoa
Blastomonas natatoria
Brevibacterium paucivorans
Buttiauxella warmboldiae
B. galactophilus
Beggiatoa alba
Blastopirellula
Brevibacterium stationis
Butyrivibrio
B. galactosidilyticus
Beijerinckia
Blastopirellula marina
B. taeanensis
Butyrivibrio fibrisolvens
B. galliciensis
Beijerinckia derxii
Blautia
B. tequilensis
Butyrivibrio hungatei
B. gelatini
Beijerinckia fluminensis
Blautia coccoides
B. thermantarcticus
Butyrivibrio proteoclasticus
B. gibsonii
Beijerinckia indica
Blautia hansenii
B. thermoaerophilus
B. lautus
B. ginsengi
Beijerinckia mobilis
Blautia producta
B. thermoamylovorans
B. lehensis
B. ginsengihumi
Belliella
Blautia wexlerae
B. thermocatenulatus
B. lentimorbus
B. ginsengisoli
Belliella baltica
Bogoriella
B. thermocloacae
B. lentus
B. globisporus
Bellilinea
Bogoriella caseilytica
B. thermocopriae
B. licheniformis
Bellilinea caldifistulae
Bordetella
B. thermodenitrificans
B. ligniniphilus
Belnapia
Bordetella avium
B. thermoglucosidasius
B. litoralis
Caenimonas
Belnapia moabensis
Bordetella bronchiseptica
B. thermolactis
B. locisalis
Caenimonas koreensis
Bergeriella
Bordetella hinzii
B. thermoleovorans
B. luciferensis
Caldalkalibacillus
Bergeriella denitrificans
Bordetella holmesii
B. thermophilus
B. luteolus
Caldalkalibacillus uzonensis
Beutenbergia
Bordetella parapertussis
B. thermoruber
B. luteus
Caldanaerobacter
Beutenbergia cavernae
Bordetella pertussis
B. thermosphaericus
B. macauensis
Caldanaerobacter subterraneus
B. aminovorans
Bordetella petrii
B. thiaminolyticus
B. macerans
Caldanaerobius
B. amylolyticus
Bordetella trematum
B. thioparans
B. macquariensis
Caldanaerobius fijiensis
B. andreesenii
B. glucanolyticus
B. thuringiensis
B. macyae
Caldanaerobius polysaccharolyticus
B. aneurinilyticus
B. gordonae
B. tianshenii
B. malacitensis
Caldanaerobius zeae
B. anthracis
B. gottheilii
B. trypoxylicola
B. mannanilyticus
Caldanaerovirga
B. aquimaris
B. graminis
B. tusciae
B. marisflavi
Caldanaerovirga acetigignens
B. arenosi
B. halmapalus
B. validus
B. marismortui
Caldicellulosiruptor
B. arseniciselenatis
B. haloalkaliphilus
B. vallismortis
B. marmarensis
Caldicellulosiruptor bescii
B. arsenicus
B. halochares
B. vedderi
B. massiliensis
Caldicellulosiruptor kristjanssonii
B. aurantiacus
B. halodenitrificans
B. velezensis
B. megaterium
Caldicellulosiruptor owensensis
B. arvi
B. halodurans
B. vietnamensis
B. mesonae
B. aryabhattai
B. halophilus
B. vireti
B. methanolicus
B. asahii
B. halosaccharovorans
B. vulcani
B. methylotrophicus
B. atrophaeus
B. hemicellulosilyticus
B. wakoensis
B. migulanus
B. axarquiensis
B. hemicentroti
B. weihenstephanensis
B. mojavensis
B. azotofixans
B. herbersteinensis
B. xiamenensis
B. mucilaginosus
B. azotoformans
B. horikoshii
B. xiaoxiensis
B. muralis
B. badius
B. horneckiae
B. zhanjiangensis
B. murimartini
B. barbaricus
B. horti
B. peoriae
B. mycoides
B. bataviensis
B. huizhouensis
B. persepolensis
B. naganoensis
B. beijingensis
B. humi
B. persicus
B. nanhaiensis
B. benzoevorans
B. hwajinpoensis
B. pervagus
B. nanhaiisediminis
B. beringensis
B. idriensis
B. plakortidis
B. nealsonii
B. berkeleyi
B. indicus
B. pocheonensis
B. neidei
B. beveridgei
B. infantis
B. polygoni
B. neizhouensis
B. bogoriensis
B. infernus
B. polymyxa
B. niabensis
B. boroniphilus
B. insolitus
B. popilliae
B. niacini
B. borstelensis
B. invictae
B. pseudalcalophilus
B. novalis
B. brevis Migula
B. iranensis
B. pseudofirmus
B. oceanisediminis
B. butanolivorans
B. isabeliae
B. pseudomycoides
B. odysseyi
B. canaveralius
B. isronensis
B. psychrodurans
B. okhensis
B. carboniphilus
B. jeotgali
B. psychrophilus
B. okuhidensis
B. cecembensis
B. kaustophilus
B. psychrosaccharolyticus
B. oleronius
B. cellulosilyticus
B. kobensis
B. psychrotolerans
B. oryzaecorticis
B. centrosporus
B. kochii
B. pulvifaciens
B. oshimensis
B. cereus
B. kokeshiiformis
B. pumilus
B. pabuli
B. chagannorensis
B. koreensis
B. purgationiresistens
B. pakistanensis
B. chitinolyticus
B. korlensis
B. pycnus
B. pallidus
B. chondroitinus
B. kribbensis
B. qingdaonensis
B. pallidus
B. choshinensis
B. krulwichiae
B. qingshengii
B. panacisoli
B. chungangensis
B. laevolacticus
B. reuszeri
B. panaciterrae
B. cibi
B. larvae
B. rhizosphaerae
B. pantothenticus
B. circulans
B. laterosporus
B. rigui
B. parabrevis
B. clarkii
B. salexigens
B. ruris
B. paraflexus
B. clausii
B. saliphilus
B. safensis
B. pasteurii
B. coagulans
B. schlegelii
B. salarius
B. patagoniensis
B. coahuilensis
B. sediminis
Catenuloplanes
Curtobacterium
B. cohnii
B. selenatarsenatis
Catenuloplanes atrovinosus
Curtobacterium albidum
B. composti
B. selenitireducens
Catenuloplanes castaneus
Curtobacterium citreus
B. curdlanolyticus
B. seohaeanensis
Catenuloplanes crispus
B. cycloheptanicus
B. shacheensis
Catenuloplanes indicus
B. cytotoxicus
B. shackletonii
Catenuloplanes japonicus
B. daliensis
B. siamensis
Catenuloplanes nepalensis
B. decisifrondis
B. silvestris
Catenuloplanes niger
B. decolorationis
B. simplex
Chryseobacterium
B. deserti
B. siralis
Chryseobacterium balustinum
Campylobacter
B. smithii
Citrobacter
Campylobacter coli
B. soli
C. amalonaticus
Campylobacter concisus
B. solimangrovi
C. braakii
Campylobacter curvus
B. solisalsi
C. diversus
Campylobacter fetus
B. songklensis
C. farmeri
Campylobacter gracilis
B. sonorensis
C. freundii
Campylobacter helveticus
B. sphaericus
C. gillenii
Campylobacter hominis
B. sporothermodurans
C. koseri
Campylobacter hyointestinalis
B. stearothermophilus
C. murliniae
Campylobacter jejuni
B. stratosphericus
C. pasteurii
Campylobacter lari
B. subterraneus
C. rodentium
Campylobacter mucosalis
B. subtilis
C. sedlakii
Campylobacter rectus
C. werkmanii
Campylobacter showae
C. youngae
Campylobacter sputorum
Clostridium
Campylobacter upsaliensis
Cardiobacterium
Capnocytophaga
Cardiobacterium hominis
Coccochloris
Capnocytophaga canimorsus
Carnimonas
Coccochloris elabens
Capnocytophaga cynodegmi
Carnimonas nigrificans
Corynebacterium
Capnocytophaga gingivalis
Carnobacterium
Corynebacterium flavescens
Capnocytophaga granulosa
Carnobacterium alterfunditum
Corynebacterium variabile
Capnocytophaga haemolytica
Carnobacterium divergens
Capnocytophaga ochracea
Carnobacterium funditum
Capnocytophaga sputigena
Carnobacterium gallinarum
Carnobacterium maltaromaticum
Carnobacterium mobile
Carnobacterium viridans
Caryophanon
Caryophanon latum
Caryophanon tenue
Catellatospora
Catellatospora citrea
Catellatospora methionotrophica
Catenococcus
Catenococcus thiocycli
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
Nesterenkonia
Dactylosporangium aurantiacum
Deinococcus aerius
Delftia acidovorans
Echinicola pacifica
Nesterenkonia holobia
Dactylosporangium fulvum
Deinococcus apachensis
Desulfovibrio
Echinicola vietnamensis
Nocardia
Dactylosporangium matsuzakiense
Deinococcus aquaticus
Desulfovibrio desulfuricans
Flavobacterium
Nocardia argentinensis
Dactylosporangium roseum
Deinococcus aquatilis
Diplococcus
Flavobacterium antarcticum
Nocardia corallina
Dactylosporangium thailandense
Deinococcus caeni
Diplococcus pneumoniae
Flavobacterium aquatile
Nocardia otitidiscaviarum
Dactylosporangium vinaceum
Deinococcus radiodurans
Faecalibacterium
Flavobacterium aquidurense
L. sakei
Enterobacter
Deinococcus radiophilus
Faecalibacterium prausnitzii
Flavobacterium balustinum
L. salivarius
E. aerogenes
Enterobacter kobei
Fangia
Flavobacterium croceum
L. sanfranciscensis
E. amnigenus
E. ludwigii
Fangia hongkongensis
Flavobacterium cucumis
L. satsumensis
E. agglomerans
E. mori
Fastidiosipila
Flavobacterium daejeonense
L. secaliphilus
E. arachidis
E. nimipressuralis
Fastidiosipila sanguinis
Flavobacterium defluvii
L. sharpeae
E. asburiae
E. oryzae
Fusobacterium
Flavobacterium degerlachei
L. siliginis
E. cancerogenous
E. pulveris
Fusobacterium nucleatum
Flavobacterium denitrificans
L. spicheri
E. cloacae
E. pyrinus
Ideonella
Flavobacterium filum
L. suebicus
E. cowanii
E. radicincitans
Ideonella azotifigens
Flavobacterium flevense
L. thailandensis
E. dissolvens
E. taylorae
Idiomarina
Flavobacterium frigidarium
L. ultunensis
E. gergoviae
E. turicensis
Idiomarina abyssalis
Flavobacterium mizutaii
L. vaccinostercus
E. helveticus
E. sakazakii
Idiomarina baltica
Flavobacterium okeanokoites
L. vaginalis
E. hormaechei
Enterobacter soli
Idiomarina fontislapidosi
Janibacter
L. versmoldensis
E. intermedius
Enterococcus
Idiomarina loihiensis
Janibacter anophelis
L. vini
Gaetbulibacter
Enterococcus durans
Idiomarina ramblicola
Janibacter corallicola
L. vitulinus
Gaetbulibacter saemankumensis
Enterococcus faecalis
Idiomarina seosinensis
Janibacter limosus
L. zeae
Gallibacterium
Enterococcus faecium
Idiomarina zobellii
Janibacter melonis
L. zymae
Gallibacterium anatis
Erwinia
Ignatzschineria
Janibacter terrae
L. gastricus
Gallicola
Erwinia hapontici
Ignatzschineria larvae
Jannaschia
L. ghanensis
Gallicola barnesae
Escherichia
Ignavigranum
Jannaschia cystaugens
L. graminis
Garciella
Escherichia coli
Ignavigranum ruoffiae
Jannaschia helgolandensis
L. hammesii
Garciella nitratireducens
Haemophilus
Ilumatobacter
Jannaschia pohangensis
L. hamsteri
Geobacillus
Haemophilus aegyptius
Ilumatobacter fluminis
Jannaschia rubra
L. harbinensis
Geobacillus thermoglucosidasius
Haemophilus aphrophilus
Ilyobacter
Janthinobacterium
L. hayakitensis
Geobacillus stearothermophilus
Haemophilus felis
Ilyobacter delafieldii
Janthinobacterium agaricidamnosum
Tatlockia
Geobacter
Haemophilus gallinarum
Ilyobacter insuetus
Janthinobacterium lividum
Tatlockia maceachernii
Geobacter bemidjiensis
Haemophilus haemolyticus
Ilyobacter polytropus
Jejuia
Tatlockia micdadei
Geobacter bremensis
Haemophilus influenzae
Ilyobacter tartaricus
Jejuia pallidilutea
Tenacibaculum
Geobacter chapellei
Haemophilus paracuniculus
Listeria ivanovii
Jeotgalibacillus
Tenacibaculum amylolyticum
Geobacter grbiciae
Haemophilus parahaemolyticus
L. marthii
Jeotgalibacillus alimentarius
Tenacibaculum discolor
Geobacter hydrogenophilus
Haemophilus parainfluenzae
L. monocytogenes
Jeotgalicoccus
Tenacibaculum gallaicum
Geobacter lovleyi
Haemophilus paraphrohaemolyticus
L. newyorkensis
Jeotgalicoccus halotolerans
Tenacibaculum lutimaris
Geobacter metallireducens
Haemophilus parasuis
L. riparia
Micrococcus
Tenacibaculum mesophilum
Geobacter pelophilus
Haemophilus pittmaniae
L. rocourtiae
Micrococcus luteus
Tenacibaculum skagerrakense
Geobacter pickeringii
Hafnia
L. seeligeri
Micrococcus lylae
Tepidanaerobacter
Geobacter sulfurreducens
Hafnia alvei
L. weihenstephanensis
Moraxella
Tepidanaerobacter syntrophicus
Geodermatophilus
Hahella
L. welshimeri
Moraxella bovis
Tepidibacter
Geodermatophilus obscurus
Hahella ganghwensis
Listonella
Moraxella nonliquefaciens
Tepidibacter formicigenes
Gluconacetobacter
Halalkalibacillus
Listonella anguillarum
Moraxella osloensis
Tepidibacter thalassicus
Gluconacetobacter xylinus
Halalkalibacillus halophilus
Macrococcus
Nakamurella
Thermus
Gordonia
Helicobacter
Macrococcus bovicus
Nakamurella multipartita
Thermus aquaticus
Gordonia rubripertincta
Helicobacter pylori
Marinobacter
Nannocystis
Thermus filiformis
Kaistia
Labedella
Marinobacter algicola
Nannocystis pusilla
Thermus thermophilus
Kaistia adipata
Labedella gwakjiensis
Marinobacter bryozoorum
Natranaerobius
Xanthobacter
Kaistia soli
Labrenzia
Marinobacter flavimaris
Natranaerobius
Xanthobacter agilis
Kangiella
Labrenzia aggregata
Meiothermus
thermophilus
Xanthobacter aminoxidans
Kangiella aquimarina
Labrenzia alba
Meiothermus ruber
Natranaerobius trueperi
Xanthobacter autotrophicus
Kangiella koreensis
Labrenzia alexandrii
Methylophilus
Naxibacter
Xanthobacter flavus
Kerstersia
Labrenzia marina
Methylophilus methylotrophus
Naxibacter alkalitolerans
Xanthobacter tagetidis
Kerstersia gyiorum
Labrys
Microbacterium
Neisseria
Xanthobacter viscosus
Kiloniella
Labrys methylaminiphilus
Microbacterium ammoniaphilum
Neisseria cinerea
Xanthomonas
Kiloniella laminariae
Labrys miyagiensis
Microbacterium arborescens
Neisseria denitrificans
Xanthomonas albilineans
Klebsiella
Labrys monachus
Microbacterium liquefaciens
Neisseria gonorrhoeae
Xanthomonas alfalfae
K. granulomatis
Labrys okinawensis
Microbacterium oxydans
Neisseria lactamica
Xanthomonas arboricola
K. oxytoca
Labrys portucalensis
L. mali
Neisseria mucosa
Xanthomonas axonopodis
K. pneumoniae
Lactobacillus
L. manihotivorans
Neisseria sicca
Xanthomonas campestris
K. terrigena
L. mindensis
Neisseria subflava
Xanthomonas citri
K. variicola
Laceyella
L. mucosae
Neptunomonas
Xanthomonas codiaei
Kluyvera
Laceyella putida
L. murinus
Neptunomonas japonica
Xanthomonas cucurbitae
Kluyvera ascorbata
Lechevalieria
L. nagelii
L. parakefiri
Xanthomonas euvesicatoria
Kocuria
Lechevalieria aerocolonigenes
L. namurensis
L. paralimentarius
Xanthomonas fragariae
Kocuria roasea
Legionella
L. nantensis
L. paraplantarum
Xanthomonas fuscans
Kocuria varians
L. oligofermentans
L. pentosus
Xanthomonas gardneri
Kurthia
Listeria
L. oris
L. perolens
Xanthomonas hortorum
Kurthia zopfii
L. aquatica
L. panis
L. plantarum
Xanthomonas hyacinthi
Lactobacillus
L. booriae
L. pantheris
L. pontis
Xanthomonas perforans
L. acetotolerans
L. cornellensis
L. parabrevis
L. protectus
Xanthomonas phaseoli
L. acidifarinae
L. fleischmannii
L. parabuchneri
L. psittaci
Xanthomonas pisi
L. acidipiscis
L. floridensis
L. paracasei
L. rennini
Xanthomonas populi
L. acidophilus
L. grandensis
L. paracollinoides
L. reuteri
Xanthomonas theicola
Lactobacillus agilis
L. grayi
L. parafarraginis
L. rhamnosus
Xanthomonas translucens
L. algidus
L. innocua
L. homohiochii
L. rimae
Xanthomonas vesicatoria
L. alimentarius
L. catenaformis
L. iners
L. rogosae
Xylella
L. amylolyticus
L. ceti
L. ingluviei
L. rossiae
Xylella fastidiosa
L. amylophilus
L. coleohominis
L. intestinalis
L. ruminis
Xylophilus
L. amylotrophicus
L. collinoides
L. fuchuensis
L. saerimneri
Xylophilus ampelinus
L. amylovorus
L. composti
L. gallinarum
L. jensenii
Zobellella
L. animalis
L. concavus
L. gasseri
L. johnsonii
Zobellella denitrificans
L. antri
L. coryniformis
Candidatus Legionella jeonii
L. kalixensis
Zobellella taiwanensis
L. apodemi
L. crispatus
Legionella jordanis
L. kefiranofaciens
Zeaxanthinibacter
L. aviarius
L. crustorum
Legionella lansingensis
L. kefiri
Zeaxanthinibacter enoshimensis
L. bifermentans
L. curvatus
Legionella londiniensis
L. kimchii
Zhihengliuella
L. brevis
L. delbrueckii subsp. bulgaricus
Legionella longbeachae
L. helveticus
Zhihengliuella halotolerans
L. buchneri
L. delbrueckii subsp. delbrueckii
Legionella lytica
L. hilgardii
Xylanibacterium
L. camelliae
L. delbrueckii subsp. lactis
Legionella maceachernii
Legionella quinlivanii
Xylanibacterium ulmi
L. casei
L. dextrinicus
Legionella massiliensis
Legionella rowbothamii
L. kitasatonis
L. diolivorans
Legionella micdadei
Legionella rubrilucens
L. kunkeei
L. equi
Legionella monrovica
Legionella sainthelensi
L. leichmannii
L. equigenerosi
Legionella moravica
Legionella santicrucis
L. lindneri
L. farraginis
Legionella nagasakiensis
Legionella shakespearei
L. malefermentans
L. farciminis
Legionella nautarum
Legionella spiritensis
Legionella
L. fermentum
Legionella norrlandica
Legionella steelei
Legionella adelaidensis
L. fornicalis
Legionella oakridgensis
Legionella steigerwaltii
Legionella anisa
L. fructivorans
Legionella parisiensis
Legionella taurinensis
Legionella beliardensis
L. frumenti
Legionella pittsburghensis
Legionella tucsonensis
Legionella birminghamensis
Legionella drancourtii
Legionella pneumophila
Legionella tunisiensis
Legionella bozemanae
Legionella dresdenensis
Legionella quateirensis
Legionella wadsworthii
Legionella brunensis
Legionella drozanskii
Prevotella
Legionella waltersii
Legionella busanensis
Legionella dumoffii
Prevotella albensis
Legionella worsleiensis
Legionella cardiaca
Legionella erythra
Prevotella amnii
Legionella yabuuchiae
Legionella cherrii
Legionella fairfieldensis
Prevotella bergensis
Quadrisphaera
Legionella cincinnatiensis
Legionella fallonii
Prevotella bivia
Quadrisphaera granulorum
Legionella clemsonensis
Legionella feeleii
Prevotella brevis
Quatrionicoccus
Legionella donaldsonii
Legionella geestiana
Prevotella bryantii
Quatrionicoccus australiensis
Oceanibulbus
Legionella genomospecies
Prevotella buccae
Quinella
Oceanibulbus indolifex
Legionella gormanii
Prevotella buccalis
Quinella ovalis
Oceanicaulis
Legionella gratiana
Prevotella copri
Ralstonia
Oceanicaulis alexandrii
Legionella gresilensis
Prevotella dentalis
Ralstonia eutropha
Oceanicola
Legionella hackeliae
Prevotella denticola
Ralstonia insidiosa
Oceanicola batsensis
Legionella impletisoli
Prevotella disiens
Ralstonia mannitolilytica
Oceanicola granulosus
Legionella israelensis
Prevotella histicola
Ralstonia pickettii
Oceanicola nanhaiensis
Legionella jamestowniensis
Prevotella intermedia
Ralstonia pseudosolanacearum
Oceanimonas
Paenibacillus
Prevotella maculosa
Ralstonia syzygii
Oceanimonas baumannii
Paenibacillus thiaminolyticus
Prevotella marshii
Ralstonia solanacearum
Oceaniserpentilla
Pantoea
Prevotella melaninogenica
Ramlibacter
Oceaniserpentilla haliotis
Pantoea agglomerans
Prevotella micans
Ramlibacter henchirensis
Oceanisphaera
Paracoccus
Prevotella multiformis
Ramlibacter tataouinensis
Oceanisphaera donghaensis
Paracoccus alcaliphilus
Prevotella nigrescens
Raoultella
Oceanisphaera litoralis
Paucimonas
Prevotella oralis
Raoultella ornithinolytica
Oceanithermus
Paucimonas lemoignei
Prevotella oris
Raoultella planticola
Oceanithermus desulfurans
Pectobacterium
Prevotella oulorum
Raoultella terrigena
Oceanithermus profundus
Pectobacterium aroidearum
Prevotella pallens
Rathayibacter
Oceanobacillus
Pectobacterium atrosepticum
Prevotella salivae
Rathayibacter caricis
Oceanobacillus caeni
Pectobacterium betavasculorum
Prevotella stercorea
Rathayibacter festucae
Oceanospirillum
Pectobacterium cacticida
Prevotella tannerae
Rathayibacter iranicus
Oceanospirillum linum
Pectobacterium carnegieana
Prevotella timonensis
Rathayibacter rathayi
Saccharococcus
Pectobacterium carotovorum
Prevotella veroralis
Rathayibacter toxicus
Saccharococcus thermophilus
Pectobacterium chrysanthemi
Providencia
Rathayibacter tritici
Saccharomonospora
Pectobacterium cypripedii
Providencia stuartii
Rhodobacter
Saccharomonospora azurea
Pectobacterium rhapontici
Pseudomonas
Rhodobacter sphaeroides
Saccharomonospora cyanea
Pectobacterium wasabiae
Pseudomonas aeruginosa
Ruegeria
Saccharomonospora viridis
Planococcus
Pseudomonas alcaligenes
Ruegeria gelatinovorans
Saccharophagus
Planococcus citreus
Pseudomonas anguillispetica
Stenotrophomonas
Saccharophagus degradans
Planomicrobium
Pseudomonas fluorescens
Stenotrophomonas maltophilia
Saccharopolyspora
Planomicrobium okeanokoites
Pseudoalteromonas haloplanktis
Streptococcus
Saccharopolyspora erythraea
Plesiomonas
Pseudomonas mendocina
Saccharopolyspora gregorii
Plesiomonas shigelloides
Pseudomonas pseudoalcaligenes
Streptomyces
Saccharopolyspora hirsuta
Proteus
Pseudomonas putida
Streptomyces achromogenes
Saccharopolyspora hordei
Proteus vulgaris
Pseudomonas tutzeri
Streptomyces cesalbus
Saccharopolyspora rectivirgula
Sagittula
Pseudomonas syringae
Streptomyces cescaepitosus
Saccharopolyspora spinosa
Sagittula stellata
Psychrobacter
Streptomyces cesdiastaticus
Saccharopolyspora taberi
Salegentibacter
Psychrobacter faecalis
Streptomyces cesexfoliatus
Saccharothrix
Salegentibacter salegens
Psychrobacter phenylpyruvicus
Streptomyces fimbriatus
Saccharothrix australiensis
Salimicrobium
Sanguibacter
Streptomyces fradiae
Saccharothrix coeruleofusca
Salimicrobium album
Sanguibacter keddieii
Streptomyces fulvissimus
Saccharothrix espanaensis
Salinibacter
Sanguibacter suarezii
Streptomyces griseoruber
Saccharothrix longispora
Salinibacter ruber
Saprospira
Streptomyces griseus
Saccharothrix mutabilis
Salinicoccus
Saprospira grandis
Streptomyces lavendulae
Saccharothrix syringae
Salinicoccus alkaliphilus
Sarcina
Streptomyces phaeochromogenes
Saccharothrix tangerinus
Salinicoccus hispanicus
Sarcina maxima
Streptomyces thermodiastaticus
Saccharothrix texasensis
Salinicoccus roseus
Sarcina ventriculi
Streptomyces tubercidicus
Staphylococcus
Salinispora
Sebaldella
S. schleiferi
S. arlettae
Salinispora arenicola
Sebaldella termitidis
S. sciuri
S. agnetis
Salinispora tropica
Serratia
S. simiae
S. aureus
Salinivibrio
Serratia fonticola
S. simulans
S. auricularis
Salinivibrio costicola
Serratia marcescens
S. stepanovicii
S. capitis
Salmonella
Sphaerotilus
S. succinus
S. caprae
Salmonella bongori
Sphaerotilus natans
S. vitulinus
S. carnosus
Salmonella enterica
Sphingobacterium
S. warneri
S. caseolyticus
Salmonella subterranea
Sphingobacterium multivorum
S. xylosus
S. chromogenes
Salmonella typhi
Staphylococcus
Streptococcus thermophilus
S. cohnii
S. equorum
Streptococcus sanguinis
S. condimenti
S. felis
S. microti
Streptococcus sobrinus
S. delphini
S. fleurettii
S. muscae
Streptococcus suis
S. devriesei
S. gallinarum
S. nepalensis
Streptococcus uberis
S. epidermidis
S. haemolyticus
S. pasteuri
Streptococcus vestibularis
Streptococcus
S. hominis
S. petrasii
Streptococcus viridans
Streptococcus agalactiae
S. hyicus
S. pettenkoferi
Streptococcus zooepidemicus
Streptococcus anginosus
S. intermedius
S. piscifermentans
Virgibacillus
Streptococcus bovis
S. kloosii
S. pseudintermedius
Virgibacillus halodenitrificans
Streptococcus canis
S. leei
S. pseudolugdunensis
Virgibacillus pantothenticus
Streptococcus constellatus
S. lentus
S. pulvereri
Weissella
Streptococcus downei
S. lugdunensis
S. rostri
Weissella cibaria
Streptococcus dysgalactiae
S. lutrae
S. saccharolyticus
Weissella confusa
Streptococcus equines
S. lyticans
S. saprophyticus
Weissella halotolerans
Streptococcus faecalis
S. massiliensis
Streptococcus orisratti
Weissella hellenica
Streptococcus ferus
Streptococcus infantarius
Streptococcus parasanguinis
Weissella kandleri
Uliginosibacterium
Streptococcus iniae
Streptococcus peroris
Weissella koreensis
Uliginosibacterium gangwonense
Streptococcus intermedius
Streptococcus pneumoniae
Weissella minor
Ulvibacter
Streptococcus lactarius
Streptococcus pseudopneumoniae
Weissella paramesenteroides
Ulvibacter litoralis
Streptococcus milleri
Streptococcus pyogenes
Weissella soli
Umezawaea
Streptococcus mitis
Streptococcus ratti
Weissella thailandensis
Umezawaea tangerina
Streptococcus mutans
Streptococcus salivariu
Weissella viridescens
Undibacterium
Streptococcus oralis
Vibrio
Williamsia
Undibacterium pigrum
Streptococcus tigurinus
Vibrio aerogenes
Williamsia marianensis
Ureaplasma
Vagococcus
Vibrio aestuarianus
Williamsia maris
Ureaplasma urealyticum
Vagococcus carniphilus
Vibrio albensis
Williamsia serinedens
Ureibacillus
Vagococcus elongatus
Vibrio alginolyticus
Winogradskyella
Ureibacillus composti
Vagococcus fessus
Vibrio campbellii
Winogradskyella thalassocola
Ureibacillus suwonensis
Vagococcus fluvialis
Vibrio cholerae
Wolbachia
Ureibacillus terrenus
Vagococcus lutrae
Vibrio cincinnatiensis
Wolbachia persica
Ureibacillus thermophilus
Vagococcus salmoninarum
Vibrio coralliilyticus
Wolinella
Ureibacillus thermosphaericus
Variovorax
Vibrio cyclitrophicus
Wolinella succinogenes
Xenophilus
Variovorax boronicumulans
Vibrio diazotrophicus
Zobellia
Xenophilus azovorans
Variovorax dokdonensis
Vibrio fluvialis
Zobellia galactanivorans
Xenorhabdus
Variovorax paradoxus
Vibrio furnissii
Zobellia uliginosa
Xenorhabdus beddingii
Variovorax soli
Vibrio gazogenes
Zoogloea
Xenorhabdus bovienii
Veillonella
Vibrio halioticoli
Zoogloea ramigera
Xenorhabdus cabanillasii
Veillonella atypica
Vibrio harveyi
Zoogloea resiniphila
Xenorhabdus doucetiae
Veillonella caviae
Vibrio ichthyoenteri
Zooshikella
Xenorhabdus griffiniae
Veillonella criceti
Vibrio mediterranei
Zooshikella ganghwensis
Xenorhabdus hominickii
Veillonella dispar
Vibrio metschnikovii
Zunongwangia
Xenorhabdus koppenhoeferi
Veillonella montpellierensis
Vibrio mytili
Zunongwangia profunda
Xenorhabdus nematophila
Veillonella parvula
Vibrio natriegens
Zymobacter
Xenorhabdus poinarii
Veillonella ratti
Vibrio navarrensis
Zymobacter palmae
Xylanibacter
Veillonella rodentium
Vibrio nereis
Zymomonas
Xylanibacter oryzae
Venenivibrio
Vibrio nigripulchritudo
Zymomonas mobilis
Venenivibrio stagnispumantis
Vibrio ordalii
Zymophilus
Verminephrobacter
Vibrio orientalis
Zymophilus paucivorans
Verminephrobacter eiseniae
Vibrio parahaemolyticus
Zymophilus raffinosivorans
Verrucomicrobium
Vibrio pectenicida
Verrucomicrobium spinosum
Vibrio penaeicida
Yangia
Vibrio proteolyticus
Yangia pacifica
Vibrio shilonii
Yaniella
Vibrio splendidus
Yaniella flava
Vibrio tubiashii
Yaniella halotolerans
Vibrio vulnificus
Yeosuana
Yersinia mollaretii
Yeosuana aromativorans
Yersinia philomiragia
Yersinia
Yersinia pestis
Yersinia aldovae
Yersinia pseudotuberculosis
Yersinia bercovieri
Yersinia rohdei
Yersinia enterocolitica
Yersinia ruckeri
Yersinia entomophaga
Yokenella
Yersinia frederiksenii
Yokenella regensburgei
Yersinia intermedia
Yonghaparkia
Yersinia kristensenii
Yonghaparkia alkaliphila
Zavarzinia
Zavarzinia compransoris
| Number | Date | Country | Kind |
|---|---|---|---|
| 2111068.9 | Aug 2021 | GB | national |
| 2116398.5 | Nov 2021 | GB | national |
This application is a U.S. national stage application under 35 U.S.C. § 371 of International Application No. PCT/EP2022/071562, filed internationally on Aug. 1, 2022, which claims priority to GB Application No. 2111068.9, filed Aug. 1, 2021, and GB Application No. 2116398.5. filed Nov. 14, 2021, the disclosures of which are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/071562 | 8/1/2022 | WO |
