Bacillus plasmid expressing a fluorescent reporter gene

Abstract

The present invention relates to a fluorescent reporter plasmid system for making Bacillus strains fluoresce, and to a method for visualizing the state of Bacillus strains.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a fluorescent reporter gene plasmid system for causing a Bacillus strain to emit fluorescence, and a method for visualizing the status of the Bacillus strain. Background Art

[0004] The paper "Molecular Kinetics of Reviving Bacterial Spores" (Segev E. et al., 2013. J Bacteriol 195:1875-82) utilized two different strains containing translational fusions of green fluorescent protein (GFP) with either RplA or SspA in Bacillus subtilis. The original construction of these strains was detailed in previous work by the same research group (Rosenberg A, Sinai L, Smith Y, Ben-Yehuda S. 2012. Dynamic expression of the translational machinery during Bacillus subtilis life cycle at a single cell level. PLoS One 7: e41921), and similar fusions have been used to track Bacillus colony morphology (Mamou G, Malli Mohan GB, Rouvinski A, Rosenberg A, Ben-Yehuda S. 2016. Early Developmental Program Shapes Colony Morphology in Bacteria. Cell Rep 14: 1850-7). In these previous studies, the strains of interest contained a single genetic circuit to allow only one type of fluorescence; multiple strains were developed to elicit fluorescence from more than one gene. Summary of the Invention

[0005] The present invention provides a fluorescent reporter plasmid system for causing a Bacillus strain to fluoresce, wherein the Bacillus strain fluoresces in its dormant spore state and / or in its metabolically active vegetative state, and wherein the plasmid system is designed to function extrachromosomally. In one aspect, upon transformation of the plasmid into a Bacillus cell, the plasmid system causes the Bacillus strain to fluoresce, wherein the Bacillus strain fluoresces in its dormant spore state and / or in its metabolically active vegetative state. In another or additional aspect, the Bacillus strain fluoresces a first fluorescent color in its dormant spore state and a second fluorescent color in its metabolically active vegetative state, and wherein the first fluorescent color is different from the second fluorescent color.

[0006] The present invention further provides a method for visualizing the state of a Bacillus strain in an animal, a plant or a plant seed, the method comprising

[0007] a. combining a Bacillus strain and a cell comprising a single or dual reporter gene plasmid system to cause the Bacillus strain to emit fluorescence of one fluorescent color when in a dormant spore state and / or emit fluorescence of another fluorescent color when in a metabolically active vegetative state,

[0008] b. treating an animal or plant seed with the Bacillus strain of step (a),

[0009] c. collecting a sample from the treated animal or plant seed of step (b),

[0010] d. optionally staining the sample of step (c) with a DNA dye, and

[0011] e. Imaging the sample of step (c) or (d) to visualize the status of the Bacillus strain.

[0012] Also included herein are methods for visualizing the state of Bacillus strains in vitro or in vivo, wherein a plasmid system is used to cause the Bacillus strains to fluoresce in a dormant spore state and / or a metabolically active vegetative state, wherein the fluorescence of the Bacillus strain is indicative of the state of the strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The plasmid map of the dual reporter gene plasmid pJDH20 (SEQ ID No: 5) is shown.

[0014] Figure 2Shown is after hatching in the LB culture medium containing 5ug / ml erythromycin and 25ug / ml lincomycin, the fluorescence microscopy of the double reporter gene fluorescent bacterial strain of the bacillus subtilis strain (described in WO2016 / 118864) of DSM 29870 is preserved.Shown is the representative image of sample after 0h (top row) and 24h (bottom row).Use phase contrast (left column), GFP filter (middle column) and DsRed filter (right column), capture each sample image.

[0015] Figure 3 Shown is a fluorescence microscopy of a wild-type strain of the bacillus subtilis strain deposited as DSM 29870 after incubation in LB medium containing 5 ug / ml erythromycin and 25 ug / ml lincomycin. Shown are representative images of samples taken after 0 hour (top row) and 24 hours (bottom row). Each sample image was captured using phase contrast (left column), a GFP filter (middle column), and a DsRed filter (right column).

[0016] Figure 4 Flow cytometry of the percentage of green fluorescent events during germination of wild-type (◯) and fluorescent (+) spores of Bacillus subtilis strain deposited as DSM 29870 after incubation in LB medium containing 5 μg / ml erythromycin and 25 μg / ml lincomycin is shown. A blank sample (◯) containing no spores is also shown as a negative control. The data shown are scatter plots of three replicates. Also shown are shaded linear regressions for the blank (solid line), wild-type (dashed line), and fluorescent (dash-dot line) strains, which indicate 95% confidence intervals.

[0017] Figure 5 Flow cytometry of the percentage of red fluorescent events during germination of wild-type (◯) and fluorescent (+) spores of Bacillus subtilis strain deposited as DSM 29870 after incubation in LB medium containing 5 μg / ml erythromycin and 25 μg / ml lincomycin is shown. A blank sample (◯) containing no spores is also shown as a negative control. The data shown are scatter plots of three replicates. Also shown are shaded linear regressions for the blank (solid line), wild-type (dashed line), and fluorescent (dash-dot line) strains, which indicate 95% confidence intervals.

[0018] Figure 6Shown are examples of images of gastrointestinal (GI) content samples from Example 3. The top four images show samples from the duodenum, ileum, cecum, and a pure Bacillus subtilis strain culture stained with SytoxGreen DNA, respectively, and the bottom four images show samples from the duodenum, ileum, cecum, and a reference strain stained with Cy3-dsRED, respectively. Pure strain cultures demonstrated heterogeneous expression of RFP populations compared to total cellular DNA staining.

[0019] Figure 7 After detecting a single object from all collected images, the inset shows the entire dataset with DNA as the object identifier on the x-axis and the measured RFP intensity on the y-axis. All data used for this comparison are from sample dilutions. A cluster of RFP bright objects was observed.

[0020] Figure 8 A subset of individual objects selected based on high RFP intensity levels is shown. Numerous Cy3-bright objects were detected in all wells containing pure samples of the RFP-expressing strain. Furthermore, RFP-bright objects were detected in ileal samples from birds fed an RFP-expressing Bacillus subtilis strain, but not in ileal samples from control birds fed a different diet.

[0021] Figure 9 . Image of exemplary ileal content samples from birds fed an RFP-expressing Bacillus subtilis strain and select antibiotics compared to ileal content samples from control birds.

[0022] Figure 10. Fluorescent reporter gene strains show that Bacillus is strain-dependent to the root colonization of germinated corn seeds. Shown are Bacillus megaterium O83AN1 (A), Bacillus megaterium O8337C (B), Bacillus amyloliquefaciens O44EAY (C) and Bacillus thuringiensis O84YVJ (D), after 4-5 days of seed germination, fluorescence microscopy of radicle top, middle and bottom (see illustration mark) sections. Shown are representative images of at least 10 replicates of every bacterial strain, and each image shows the fluorescence superposition of sections with green and red filters. Viable bacterial cells emit red fluorescence (light-colored rods in the figure), and plant cells naturally emit green fluorescence (light-colored rectangular cells in the figure). All images are 400x magnification.

[0023] definition

[0024] The following includes definitions of selected terms that may be used throughout the disclosure and claims. These definitions include various examples and / or component forms that fall within the scope of the terms and can be used for implementation. These examples are not intended to be limiting. Both the singular and plural forms of the terms fall within the definitions.

[0025] Capable of germination: With respect to bacterial spores, the term "capable of germination" means that at least some of the spores in a population will germinate when sufficient germinants are provided.

[0026] About: This term means ±10% with respect to a stated value or property.

[0027] Plasmids: Small double-stranded DNA molecules that remain within the cell but are physically separate from the larger chromosomal DNA molecules. They replicate independently and are circular.

[0028] Plasmid Backbone: A "plasmid backbone" or "plasmid backbone" refers to a genetically engineered plasmid designed as a template for easy additional genetic manipulation. The backbone contains, at a minimum, one or more selectable markers for selection of the plasmid in a bacterial host, one or more multiple cloning site regions for integration of foreign DNA into the plasmid, and any origins of replication required for replication in the plasmid's potential host. It may also contain an origin of transfer (oriT) for transfer of the plasmid from one bacterial host to another.

[0029] Transformation: The process of introducing foreign DNA (such as a plasmid) into a cell.

[0030] Bacteria: refers to prokaryotic organisms that have peptidoglycan in their cell walls and lipids containing fatty acids in their cell membranes.

[0031] Bacterial spores are structures formed by certain bacteria during a process called sporulation. Typically, bacterial spores are resistant to environmental conditions, metabolically inactive, and unable to reproduce. Bacterial spores are often capable of germinating into vegetative cells.

[0032] Custom-made: The term "custom-made" plasmid is used to describe a plasmid designed for a specific recipient bacterial species. Thus, a non-customized plasmid system is one that is designed for a recipient bacterial genus rather than species.

[0033] Dormant spore state: A state in the life cycle of spore-forming bacteria in which they are in a stable, resting, non-reproductive and enzymatically inert (called "dormant") form.

[0034] Spore: refers to a type of spore that develops inside a bacterium and is a dormant, non-reproductive and enzymatically inert form of the bacterial vegetative cell.

[0035] Fluorescent reporter plasmid system: A plasmid-based system that contains one or more reporter genes that fluoresce different colors depending on the state of the organism (host of the plasmid).

[0036] Germination: refers to the process by which bacterial spores become vegetative cells.

[0037] Gram-positive: Refers to bacteria that stain purple during Gram staining. Gram-positive bacteria typically have a different structure and / or arrangement of their cell membrane and cell wall than Gram-negative bacteria. Specifically, Gram-positive organisms in their cellular state contain a thick layer of peptidoglycan surrounding a cell membrane.

[0038] Metabolically active vegetative state: The active state in the life cycle of spore-forming bacteria in which they grow and divide by binary fission.

[0039] Recipient strain: Same as "target strain" herein, and refers to the bacterial species or subspecies that will accept and maintain the genetic elements on the plasmids of the present invention.

[0040] Reporter gene: This refers to a reporter gene encoding, for example, a fluorescent protein. One or more reporter genes can be introduced into a plasmid system; thus, the term "single reporter plasmid system" refers to a plasmid system containing one reporter gene, and "dual reporter plasmid system" refers to a plasmid system containing two reporter genes, etc.

[0041] Vegetative cell: refers to a bacterial cell that is metabolically active and / or actively growing / dividing. Vegetative bacterial cells are not spore cells.

[0042] Method for visualizing a strain state: This term refers to a method for displaying a state in the life cycle of a spore-forming bacterial strain, wherein the state can be, for example, a dormant spore state, a metabolically active vegetative state, and / or a germinating state. In one aspect, the method is used to visualize germination of a strain. DETAILED DESCRIPTION

[0043] According to the present invention, the inventors have developed an advantageous plasmid system suitable for visualizing the state of a Bacillus strain, such as the germination of a Bacillus strain. The plasmid system of the present invention causes Bacillus strains to emit fluorescence, depending on the cell state of these strains. In one aspect of the invention, the plasmid system is used to visualize a Bacillus strain in its dormant spore state. In one aspect of the invention, the plasmid system is used to visualize a Bacillus strain in its metabolically active vegetative state. In one aspect of the invention, the plasmid system is used to visualize a Bacillus strain in its dormant spore state and its metabolically active vegetative state. In one aspect of the invention, the plasmid system is used to visualize the transition of a Bacillus strain from the dormant spore state to the metabolically active vegetative state.

[0044] Plasmid systems can be used intrachromosomally or extrachromosomally. It has been found that when used extrachromosomally, plasmid systems are surprisingly stable. The inventors have determined several additional advantages over traditional intrachromosomal methods of genetic modification. For example, plasmid systems do not necessarily have to be customized for recipient strains, and if the recipient organism can maintain the Bacillus replication origin and use the σ factor present in Bacillus, then the genomic sequence does not necessarily have to be known. Compared with intrachromosomal genetic modification, the phenotype in the strain can be changed faster because no integration step is required after conversion, and therefore no additional selection and screening steps are required. The plasmid copy number of the present invention is higher than the plasmid copy number that causes the expression of fluorescent genes, and these fluorescent genes are likely to be larger than a single copy of the gene on the chromosome. If it is necessary to remove the plasmid, the plasmid can be removed from the strain. For example, removing antibiotic selection and repeating subculture for many generations (a simple task in the laboratory) can restore wild-type genotype and phenotype.

[0045] In one aspect of the present invention, the plasmid system is designed to function extrachromosomally. In another aspect, the plasmid system is not customized for the recipient strain. In another aspect, the genomic sequence is unknown, provided that the recipient organism can maintain the Bacillus origin of replication and use the σ factor present in Bacillus. The plasmid system of the present invention can be maintained in most Bacillus strains after transformation. The plasmid system can be used for analysis, such as gastrointestinal or plant seed samples. In one aspect, the plasmid system is used for gastrointestinal samples, such as gastrointestinal samples from animals. In another aspect or another aspect, the plasmid system is used for plant seed samples.

[0046] Plasmid system

[0047] In conjunction with the present invention, a fluorescent reporter plasmid system is provided for causing a Bacillus strain to fluoresce, wherein the Bacillus strain fluoresces in its dormant spore state and / or in its metabolically active vegetative state.

[0048] The level of control provided by the plasmid system of the present invention depends on the promoter structure upstream of the fluorescent marker gene. For example, the plasmid system of the present invention can include a fluorescent protein gene fused to a spore-specific gene, resulting in fluorescence in the dormant spore state, and / or another fluorescent protein gene expressed from a promoter region that is strongly expressed during vegetative cell growth, resulting in fluorescence in the metabolically active vegetative state.

[0049] In one aspect of the present invention, upon plasmid transformation into Bacillus cells, a fluorescent reporter plasmid system causes the Bacillus strain to fluoresce, wherein the Bacillus strain fluoresces in its dormant spore state and / or in its metabolically active vegetative state. In one aspect, the Bacillus strain fluoresces at one fluorescent color when in the dormant spore state and at another fluorescent color when in the metabolically active vegetative state. In one aspect of the present invention, the Bacillus strain fluoresces at two different fluorescent colors, the two colors being selected from the group consisting of green, red, yellow, cyan, blue, far-red, and orange.

[0050] In one aspect of the invention, the plasmid system is designed to function extrachromosomally.

[0051] The backbone of the plasmid system can be derived from any suitable backbone known to those skilled in the art.

[0052] The backbone of the plasmid system of the present invention can comprise any suitable components known to those skilled in the art, including but not limited to one or more vectors (e.g., such as plasmids), one or more origins of replication (ori), one or more origins of transfer (oriT), one or more antibiotic resistance markers, one or more promoter regions, one or more reporter genes, and one or more multiple cloning sites. In one aspect of the invention, the plasmid system comprises an origin of replication, a selectable marker for erythromycin resistance, an origin of transfer, and two marker genes (GFP and dsRed) under the control of specific promoters that are dependent on transcriptional events within the host cell.

[0053] In one aspect of the invention, the backbone of the plasmid system comprises an origin of replication that functions in Bacillus strains. In one aspect of the invention, the backbone of the plasmid system comprises an origin of transfer. In one aspect of the invention, the backbone of the plasmid system comprises an antibiotic resistance cassette. In another or additional aspect of the invention, the plasmid system is derived from a backbone comprising an origin of replication and, optionally, an antibiotic resistance marker. In another aspect of the invention, the plasmid system is derived from a backbone comprising an origin of replication and an antibiotic resistance marker. Multiple origins of replication and / or antibiotic resistance markers known to those skilled in the art can be used to allow the plasmid to be hosted in a variety of organisms during the cloning process. In another aspect of the invention, the backbone of the plasmid is derived from pBM317 (SEQ ID No: 1) or pBG2 (SEQ ID No: 2).

[0054] In one aspect of the invention, the plasmid system comprises one or more plasmids selected from the group consisting of: pE194, pUC, pBAD, and any combination thereof. In another aspect of the invention, the plasmid system comprises an origin of transfer (oriT). In yet another aspect, oriT is derived from pUB110.

[0055] The plasmid system can be transformed by any suitable means, such as but not limited to conjugation, natural competence, electroporation or chemical competence. In one aspect, the plasmid system is transformed by conjugation or natural competence.

[0056] The plasmid system of the present invention comprises one or more reporter genes encoding fluorescent proteins. On the one hand, the plasmid system comprises two or more reporter genes, wherein each of these reporter genes encodes a fluorescent protein. In other aspects, the one or more fluorescent proteins encoded by one or more reporter genes are selected from the group consisting of: green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), DsRedexpress2, mCherry, mOrange, mPlum yellow fluorescent protein (EYFP), cyan fluorescent protein (ECFP) and Sapphire. In other aspects, the one or more fluorescent proteins encoded by one or more reporter genes are green fluorescent protein (GFP) or red fluorescent protein (RFP), such as DsRedexpress2 protein or DsRed protein. In preferred aspects, the plasmid system comprises a first reporter gene and a second reporter gene. In other preferred aspects, the plasmid system comprises a first reporter gene encoding a fluorescent protein and a second reporter gene encoding a fluorescent protein different from the fluorescent protein encoded by the first reporter gene. In other aspects, the first reporter gene is the gene encoding green fluorescent protein (GFP), or the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen. In alternative or in other aspects, the second reporter gene is the gene encoding green fluorescent protein (GFP), or the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen. In preferred aspects, the first reporter gene is the gene encoding green fluorescent protein (GFP), and the second reporter gene is the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen. In another preferred aspect, the first reporter gene is the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen, and the second reporter gene is the gene encoding green fluorescent protein (GFP). In other aspects, reporter gene (such as the first reporter gene or the second reporter gene) is expressed in dormant spores.

[0057] In a preferred aspect of the invention, the reporter gene (e.g., the first reporter gene) is translationally fused to a gene encoding a small acid-soluble protein (SASP), such as sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as sspA, sspB, or sspE. In another aspect, the reporter gene (e.g., the first reporter gene) comprises a promoter region and a region encoding a small acid-soluble protein (SASP). The promoter region of the plasmid system can comprise any suitable promoter known to those skilled in the art. In one aspect of the invention, the promoter region comprises one or more promoters selected from the group consisting of: amyLp, amyQp, and cryp. In one aspect, the promoter region is sspB. The reporter gene can be any suitable gene. In one aspect, the reporter gene (e.g., the first reporter gene) is selected from the group consisting of sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, and sspP. In another aspect, the reporter gene (e.g., the first reporter gene) is selected from the group consisting of sspA, sspB, and sspE. In yet another aspect, the reporter gene (e.g., the first reporter gene) is sspB. In still other aspects, a linker encoding 1 to 24 amino acids, such as 6 to 20 amino acids, 8 to 20 amino acids, 8 to 16 amino acids, 9 to 15 amino acids, 10 to 14 amino acids, or 11 to 13 amino acids, is located between the reporter gene (such as the first reporter gene) and the gene encoding the SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB or sspE). In yet other aspects, a linker encoding about 12 amino acids is located between the reporter gene (e.g., the first reporter gene) and the gene encoding a SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB or sspE).In alternative aspects, a linker of 3 to 72 base pairs, such as 18 to 60 base pairs, 24 to 60 base pairs, 24 to 48 base pairs, 27 to 45 base pairs, 30 to 42 base pairs, or 33 to 39 base pairs is located between the reporter gene (such as the first reporter gene) and the gene encoding the SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB or sspE). In further alternative aspects, a linker of about 36 base pairs is located between the reporter gene (e.g., the first reporter gene) and the gene encoding the SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB, or sspE). In one aspect of the invention, the plasmid system can be transformed by conjugation or naturally competent, electroporation, or chemically competent. In further aspects of the invention, the plasmid system can be transformed by conjugation or naturally competent. In one aspect of the invention, the promoter region of the plasmid system comprises one or more promoters that are expressed during vegetative growth.

[0058] In one aspect of the invention, a plasmid system is used to translate a SASP protein and a fluorescent protein fusion.

[0059] In one aspect of the invention, the plasmid system is selected from the group consisting of: pJDH11 (SEQ ID No: 3), pJDH14 (SEQ ID No: 4), pJDH20 (SEQ ID No: 5), pJDH21 (SEQ ID No: 6), pJDH22 (SEQ ID No: 7), pJDH23 (SEQ ID No: 8), pJDH24 (SEQ ID No: 9), pJDH25 (SEQ ID No: 10), pJDH26 (SEQ ID No: 11), pJDH29 (SEQ ID No: 12), and pBG3 (SEQ ID No: 13).

[0060] Plasmid systems can be used to make Bacillus strains fluoresce. In one aspect of the invention, the Bacillus strain is any Bacillus species strain. In another aspect of the invention, the Bacillus strain is selected from the group consisting of: Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velez, Bacillus pumilus, Bacillus licheniformis, Bacillus simplex, Bacillus thuringiensis, and Bacillus thuringiensis. In yet another aspect of the invention, the Bacillus strain is selected from the group consisting of: Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velez, Bacillus pumilus, and Bacillus licheniformis.

[0061] Plasmid systems can be added to plasmids capable of chromosomal integration, such as pMUTIN, by using regions of homology tailored to the desired Bacillus host.

[0062] Method for visualizing the status of Bacillus strains

[0063] The present invention also encompasses methods for visualizing the state of a Bacillus strain in vitro or in vivo. In one aspect, the method can be used to visualize the germination of a Bacillus strain. In one aspect, a method for visualizing the state of a Bacillus strain in an animal, plant, or plant seed is provided. In another aspect, a method for visualizing the germination of a Bacillus strain in an animal, plant, or plant seed is provided. In yet another aspect, a method for visualizing the state of a Bacillus strain in an animal is provided. In still another aspect, a method for visualizing the germination of a Bacillus strain in an animal is provided. In another aspect, a method for visualizing the state of a Bacillus strain in a plant or plant seed is provided. In another aspect, a method for visualizing the germination of a Bacillus strain in a plant or plant seed is provided.

[0064] In one aspect of the present invention, a method for visualizing the status (e.g., germination) of a Bacillus strain in an animal, a plant, or a plant seed comprises the following steps:

[0065] a) combining a Bacillus strain and a cell comprising a single or dual reporter gene plasmid system for causing the Bacillus strain to fluoresce in one fluorescent color when in a dormant spore state and / or to fluoresce in another fluorescent color when in a metabolically active vegetative state,

[0066] b) treating animal or plant seeds with the Bacillus strain of step (a),

[0067] c) collecting a sample from the treated animal or plant seed of step (b),

[0068] d) optionally staining the sample of step (c) with a DNA dye, and

[0069] e) imaging the stained sample of step (c) or (d) to visualize germination.

[0070] In another aspect of the present invention, a method for visualizing the status (e.g., germination) of a Bacillus strain in an animal comprises the following steps:

[0071] a) combining a Bacillus strain and a cell comprising a single or dual reporter gene plasmid system for causing the Bacillus strain to fluoresce in one fluorescent color when in a dormant spore state and / or to fluoresce in another fluorescent color when in a metabolically active vegetative state,

[0072] b) feeding the Bacillus strain of step (a) to an animal,

[0073] c) collecting samples from the duodenum, ileum and / or cecum tissue of the animal and preparing these samples for analysis,

[0074] d) optionally staining the sample of step (c) with a DNA dye, and

[0075] e) imaging the stained sample of step (c) or (d) to visualize germination.

[0076] In yet another aspect of the present invention, a method for visualizing the status (e.g., germination) of a Bacillus strain in a plant or plant seed comprises the following steps:

[0077] a) combining a Bacillus strain and cells containing a single or dual reporter gene plasmid system for causing the Bacillus strain to fluoresce in one fluorescent color when in a dormant spore state and another fluorescent color when in a metabolically active vegetative state,

[0078] b) treating the plant seeds with the Bacillus strain of step (a),

[0079] c) optionally planting the treated plant seeds of step (b) to allow plants to grow,

[0080] d) collecting samples for analysis by washing the treated plant seeds of step (b) which have been allowed to germinate, or the plants of step (c) or fractions thereof,

[0081] e) preparing the samples of step (d) for analysis,

[0082] f) optionally staining the sample of step (e) with a DNA dye, and

[0083] g) imaging the stained sample of step (e) or (f).

[0084] In one aspect of the method, the cells of step (a) comprise a single reporter plasmid system for causing the Bacillus strain to fluoresce a single fluorescent color when in a dormant spore state or when in a metabolically active vegetative state. In another aspect of the method, the cells of step (a) comprise a dual reporter plasmid system for causing the Bacillus strain to fluoresce a single fluorescent color when in a dormant spore state and a second fluorescent color when in a metabolically active vegetative state.

[0085] In one aspect of the present invention, methods for visualizing the state of a Bacillus strain (e.g., the germination of Bacillus spores) are contemplated, wherein the methods comprise using a plasmid system for causing the Bacillus strain to fluoresce. In another aspect, the plasmid system is a fluorescent reporter plasmid system. In yet another aspect, the Bacillus strain fluoresces in its dormant spore state and / or in its metabolically active vegetative state.

[0086] In one aspect of the present invention, after plasmid transformation into Bacillus cells, the plasmid system used in the method causes the Bacillus strain to fluoresce, wherein the Bacillus strain fluoresces in its dormant spore state and / or in its metabolically active vegetative state. In one aspect, the Bacillus strain fluoresces at one fluorescent color when in the dormant spore state and at another fluorescent color when in the metabolically active vegetative state. In one aspect of the present invention, the Bacillus strain fluoresces at two different fluorescent colors, the two colors being selected from the group consisting of green, red, yellow, cyan, blue, far-red, and orange.

[0087] In one aspect of the invention, the plasmid system used in this method is designed to function extrachromosomally.

[0088] The backbone of the plasmid system used in this method may be derived from any suitable backbone known to those skilled in the art.

[0089] The backbone of the plasmid system used in this method can comprise any suitable components known to those skilled in the art, including but not limited to one or more vectors (such as, for example, plasmids), one or more origins of replication (ori), one or more origins of transfer (oriT), one or more antibiotic resistance markers, one or more promoter regions, one or more reporter genes, and one or more multiple cloning sites. In one aspect of the invention, the plasmid system used in this method comprises an origin of replication, a selectable marker for erythromycin resistance, an origin of transfer, and two marker genes (GFP and dsRed) under the control of specific promoters that depend on transcriptional events in the host cell.

[0090] In one aspect of the invention, the backbone of the plasmid system used in the method comprises an origin of replication that functions in Bacillus strains. In one aspect of the invention, the backbone of the plasmid system used in the method comprises an origin of transfer. In one aspect of the invention, the backbone of the plasmid system used in the method comprises an antibiotic resistance cassette. In another or additional aspect of the invention, the plasmid system is derived from a backbone comprising an origin of replication and optionally an antibiotic resistance marker. In another aspect of the invention, the plasmid system is derived from a backbone comprising an origin of replication and an antibiotic resistance marker. Multiple origins of replication and / or antibiotic resistance markers known to those skilled in the art can be used to allow the plasmid to be hosted in a variety of organisms during the cloning process. In another aspect of the invention, the backbone of the plasmid is derived from pBM317 (SEQ ID No: 1) or pBG2 (SEQ ID No: 2).

[0091] In one aspect of the invention, the plasmid system used in the method comprises one or more plasmids selected from the group consisting of: pE194, pUC, pBAD, and any combination thereof. In another aspect of the invention, the plasmid system used in the method comprises an origin of transfer (oriT). In yet another aspect, oriT is derived from pUB110.

[0092] The plasmid system used in the method can be transformed by any suitable means, such as but not limited to conjugation, natural competence, electroporation or chemical competence. In one aspect, the plasmid system used in the method is transformed by conjugation or natural competence.

[0093] The plasmid system used in the method comprises one or more reporter genes encoding fluorescent proteins. On the one hand, the plasmid system used in the method comprises two or more reporter genes, wherein each of these reporter genes encodes a fluorescent protein. In other aspects, one or more fluorescent proteins encoded by one or more reporter genes are selected from the group consisting of: green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), DsRedexpress2, mCherry, mOrange, mPlum yellow fluorescent protein (EYFP), cyan fluorescent protein (ECFP) and Sapphire. In other aspects, one or more fluorescent proteins encoded by one or more reporter genes are green fluorescent protein (GFP) or red fluorescent protein (RFP), such as DsRedexpress2 albumen or DsRed albumen. In preferred aspects, the plasmid system used in the method comprises the first reporter gene and the second reporter gene. In other preferred aspects, the plasmid system used in the method comprises the first reporter gene encoding a fluorescent protein and the second reporter gene encoding a fluorescent protein different from the fluorescent protein encoded by the first reporter gene. In other aspects, the first reporter gene is the gene encoding green fluorescent protein (GFP), or the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen. In alternative or in other aspects, the second reporter gene is the gene encoding green fluorescent protein (GFP), or the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen. In preferred aspects, the first reporter gene is the gene encoding green fluorescent protein (GFP), and the second reporter gene is the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen. In another preferred aspect, the first reporter gene is the gene encoding red fluorescent protein (RFP), such as the gene encoding DsRedexpress2 albumen or DsRed albumen, and the second reporter gene is the gene encoding green fluorescent protein (GFP). In other aspects, reporter gene (such as the first reporter gene or the second reporter gene) is expressed in dormant spores.

[0094] In a preferred aspect of the invention, the reporter gene used in the method (e.g., the first reporter gene) is translationally fused to a gene encoding a small acid-soluble protein (SASP), such as sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB, or sspE. In another aspect, the reporter gene (e.g., the first reporter gene) comprises a promoter region and a region encoding a small acid-soluble protein (SASP). The promoter region of the plasmid system used in the method can comprise any suitable promoter known to those skilled in the art. In one aspect of the invention, the promoter region comprises one or more promoters selected from the group consisting of: amyLp, amyQp, and cryp. In one aspect, the promoter region is sspB. The reporter gene can be any suitable gene. In one aspect, the reporter gene (e.g., the first reporter gene) is selected from the group consisting of sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, and sspP. In another aspect, the reporter gene (e.g., the first reporter gene) is selected from the group consisting of sspA, sspB, and sspE. In yet another aspect, the reporter gene (e.g., the first reporter gene) is sspB. In still other aspects, a linker encoding 1 to 24 amino acids, such as 6 to 20 amino acids, 8 to 20 amino acids, 8 to 16 amino acids, 9 to 15 amino acids, 10 to 14 amino acids, or 11 to 13 amino acids, is located between the reporter gene (such as the first reporter gene) and the gene encoding the SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB or sspE). In yet other aspects, a linker encoding about 12 amino acids is located between the reporter gene (e.g., the first reporter gene) and the gene encoding a SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB or sspE).In alternative aspects, a linker of 3 to 72 base pairs, such as 18 to 60 base pairs, 24 to 60 base pairs, 24 to 48 base pairs, 27 to 45 base pairs, 30 to 42 base pairs, or 33 to 39 base pairs is located between the reporter gene (such as the first reporter gene) and the gene encoding the SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB or sspE). In another alternative aspect, a linker of about 36 base pairs is located between the reporter gene (such as the first reporter gene) and the gene encoding the SASP (e.g., sspA, sspB, sspC, sspD, sspE, sspF, sspG, sspH, sspI, sspJ, sspK, sspL, sspM, sspN, sspO, or sspP, such as, for example, sspA, sspB or sspE).

[0095] In one aspect of the invention, the plasmid system used in this method can be transformed by conjugation or natural competence, electroporation or chemical competence. In another aspect of the invention, the plasmid system can be transformed by conjugation or natural competence. In one aspect of the invention, the promoter region of the plasmid system used in this method is included in one or more promoters expressed during vegetative growth.

[0096] In one aspect of the invention, the plasmid system used in this method translates to a fusion of a SASP protein and a fluorescent protein.

[0097] In one aspect of the invention, the plasmid system used in the method is selected from the group consisting of: pJDH11 (SEQ ID No: 3), pJDH14 (SEQ ID No: 4), pJDH20 (SEQ ID No: 5), pJDH21 (SEQ ID No: 6), pJDH22 (SEQ ID No: 7), pJDH23 (SEQ ID No: 8), pJDH24 (SEQ ID No: 9), pJDH25 (SEQ ID No: 10), pJDH26 (SEQ ID No: 11), pJDH29 (SEQ ID No: 12), and pBG3 (SEQ ID No: 13).

[0098] Methods for visualizing the state of Bacillus strains in vitro or in vivo (e.g., germination) can be used to visualize the germination of any Bacillus species strain. In one aspect of the invention, the Bacillus strain is selected from the group consisting of: Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velez, Bacillus pumilus, Bacillus licheniformis, Bacillus simplex, Bacillus cyrosaccharolyticus, and Bacillus thuringiensis. In another aspect of the invention, the Bacillus strain is selected from the group consisting of: Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velez, Bacillus pumilus, and Bacillus thuringiensis.

[0099] The plasmid system used in this method can be augmented with a plasmid capable of chromosomal integration, such as pMUTIN, by using regions of homology tailored to the desired Bacillus host.

[0100] Preferred embodiment

[0101] The following is a list of preferred embodiments of the present invention:

[0102] 1. A fluorescent reporter gene plasmid system for causing a Bacillus strain to fluoresce, wherein the Bacillus strain fluoresces in its dormant spore state and / or in its metabolically active vegetative state, and wherein the plasmid system is designed to function extrachromosomally.

[0103] 2. The plasmid system of Example 1, wherein after the plasmid is transformed into a Bacillus cell, the plasmid system causes the Bacillus strain to emit fluorescence, wherein the Bacillus strain emits fluorescence in its dormant spore state and / or in its metabolically active vegetative state.

[0104] 3. The plasmid system of any one of embodiments 1-2, wherein the Bacillus strain fluoresces a first fluorescent color in its dormant spore state and fluoresces a second fluorescent color in its metabolically active vegetative state, and wherein the first fluorescent color is different from the second fluorescent color.

[0105] 4. The plasmid system of any one of embodiments 1 to 3, wherein the one or more fluorescent colors are selected from the group consisting of green, red, yellow, cyan, blue, far red, and orange.

[0106] 5. The plasmid system of any one of embodiments 1 to 4, wherein the backbone comprises an origin of replication functional in a Bacillus strain.

[0107] 6. The plasmid system as described in embodiment 5, wherein the backbone further comprises an antibiotic resistance marker.

[0108] 7. The plasmid system of any one of embodiments 1 to 6, wherein the backbone comprises an origin of transfer.

[0109] 8. The plasmid system of any one of embodiments 1 to 7, wherein the backbone comprises an antibiotic resistance cassette.

[0110] 9. The plasmid system of any one of embodiments 1 to 8, comprising one or more reporter genes encoding fluorescent proteins.

[0111] 10. The plasmid system of embodiment 9, wherein the fluorescent protein encoded by the reporter gene is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), DsRedexpress2, mCherry, mOrange, mPlum yellow fluorescent protein (EYFP), cyan fluorescent protein (ECFP), and Sapphire.

[0112] 11. The plasmid system of any one of embodiments 1 to 10, wherein the reporter gene is a gene encoding green fluorescent protein (GFP), or a gene encoding red fluorescent protein (RFP), such as a gene encoding DsRedexpress2 protein or DsRed protein.

[0113] 12. The plasmid system of any one of embodiments 1 to 11, wherein the reporter gene is expressed in dormant spores.

[0114] 13. The plasmid system of any one of embodiments 1 to 12, wherein the reporter gene is translationally fused to a gene encoding a small acid-soluble protein (SASP).

[0115] 14. The plasmid system of any one of embodiments 1 to 13, wherein the reporter gene comprises a promoter region and a region encoding a small acid-soluble protein (SASP).

[0116] 15. The plasmid system of embodiment 14, wherein the promoter region is sspB.

[0117] 16. The plasmid system of any one of embodiments 1 to 15, wherein the reporter gene is selected from the group consisting of sspA, sspB, and sspE.

[0118] 17. The plasmid system of any one of embodiments 1 to 16, wherein the reporter gene is sspB having SEQ ID No: 14.

[0119] 18. The plasmid system of any one of embodiments 1 to 17, wherein a linker encoding 1 to 24 amino acids, such as 6 to 20 amino acids, 8 to 20 amino acids, 8 to 16 amino acids, 9 to 15 amino acids, 10 to 14 amino acids, or 11 to 13 amino acids, is located between the first reporter gene and the gene encoding SASP.

[0120] 19. The plasmid system of any one of embodiments 1 to 18, wherein a linker encoding about 12 amino acids is located between the first reporter gene and the gene encoding SASP.

[0121] 20. The plasmid system of any one of embodiments 1 to 19, wherein a linker of 3 to 72 base pairs, such as 18 to 60 base pairs, 24 to 60 base pairs, 24 to 48 base pairs, 27 to 45 base pairs, 30 to 42 base pairs, or 33 to 39 base pairs is located between the first reporter gene and the gene encoding SASP.

[0122] 21. The plasmid system of any one of embodiments 1 to 20, wherein a linker of about 36 base pairs is located between the first reporter gene and the gene encoding SASP.

[0123] 22. The plasmid system of any one of embodiments 18 to 21, which is translated into a fusion of a SASP protein and a fluorescent protein.

[0124] 23. The plasmid system of any one of embodiments 1 to 22, wherein the reporter gene is a first reporter gene and the plasmid system further comprises a second reporter gene, wherein the second reporter gene encodes a fluorescent protein that is different from the fluorescent protein encoded by the first reporter gene.

[0125] 24. The plasmid system of embodiment 23, wherein the first reporter gene encodes a fluorescent protein that is designed to fluoresce in its dormant spore state, and the second reporter gene encodes a fluorescent protein that is designed to fluoresce in its metabolically active vegetative state.

[0126] 25. The plasmid system of embodiment 23 or 24, wherein the fluorescent protein encoded by the second reporter gene is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), DsRedexpress2, mCherry, mOrange, mPlum yellow fluorescent protein (EYFP), cyan fluorescent protein (ECFP), and Sapphire.

[0127] 26. The plasmid system of any one of embodiments 1 to 25, wherein the second reporter gene is a gene encoding green fluorescent protein (GFP), or a gene encoding red fluorescent protein (RFP), such as a gene encoding DsRedexpress2 protein or DsRed protein.

[0128] 27. The plasmid system of embodiment 26, wherein the first reporter gene is a gene encoding green fluorescent protein (GFP), and the second reporter gene is a gene encoding red fluorescent protein (RFP), such as a gene encoding DsRedexpress2 protein or DsRed protein.

[0129] 28. The plasmid system of embodiment 26, wherein the first reporter gene is a gene encoding red fluorescent protein (RFP), such as a gene encoding DsRedexpress2 protein or DsRed protein, and the second reporter gene is a gene encoding green fluorescent protein (GFP).

[0130] 29. The plasmid system of any one of embodiments 1 to 28, wherein the backbone of the plasmid is derived from pBM317 (SEQ ID No: 1) or pBG2 (SEQ ID No: 2).

[0131] 30. The plasmid system of any one of embodiments 1 to 29, comprising vector pE194, pUC or pBAD, and an origin of transfer (oriT) from pUB110.

[0132] 31. The plasmid system of any one of embodiments 1 to 30, which is transformable by conjugation, natural competence, electroporation, or chemical competence.

[0133] 32. The plasmid system of any one of embodiments 1 to 31, which is capable of transformation by conjugation or natural competence.

[0134] 33. The plasmid system of any one of embodiments 1 to 32, wherein the promoter region comprises one or more promoters expressed during vegetative growth.

[0135] 34. The plasmid system of any one of embodiments 1 to 33, wherein the promoter region comprises one or more promoters selected from the group consisting of amyLp, amyQp, and cryp.

[0136] 35. The plasmid system of any one of embodiments 1 to 34, wherein the Bacillus strain is selected from the group consisting of Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus pumilus, Bacillus licheniformis, Bacillus simplex, Bacillus refrigerated saccharolyticus, and Bacillus thuringiensis.

[0137] 36. The plasmid system of any one of embodiments 1 to 35, wherein the plasmid system is not customized for a recipient strain.

[0138] 37. The plasmid system of any one of embodiments 1 to 36, wherein the genomic sequence is unknown, provided that the recipient organism can maintain a Bacillus origin of replication and utilizes a sigma factor present in Bacillus.

[0139] 38. The plasmid system of any one of embodiments 1 to 37, which can be maintained in most Bacillus strains after transformation.

[0140] 39. Use of the plasmid system of any one of embodiments 1 to 38 for analyzing a gastrointestinal sample or a plant seed sample.

[0141] 40. Use of the plasmid system of any one of embodiments 1 to 38 for analyzing a gastrointestinal sample, such as a gastrointestinal sample of an animal.

[0142] 41. Use of the plasmid system of any one of embodiments 1 to 38 for analyzing a plant sample or a plant seed sample.

[0143] 42. A cell comprising the plasmid system of any one of embodiments 1 to 38.

[0144] 43. The cell of embodiment 37, wherein the plasmid system is stably integrated into the genome of the cell.

[0145] 44. A method for visualizing the status of a Bacillus strain in an animal, a plant, or a plant seed, the method comprising:

[0146] a) combining a Bacillus strain and a cell comprising a single or dual reporter gene plasmid system for causing the Bacillus strain to fluoresce in one fluorescent color when in a dormant spore state and / or to fluoresce in another fluorescent color when in a metabolically active vegetative state,

[0147] b) treating animal or plant seeds with the Bacillus strain of step (a),

[0148] c) collecting a sample from the treated animal or plant seed of step (b),

[0149] d) optionally staining the sample of step (c) with a DNA dye, and

[0150] e) imaging the sample of step (c) or (d) to visualize the status of the Bacillus strain.

[0151] 45. A method for visualizing the status of a Bacillus strain in an animal, the method comprising the steps of:

[0152] a) combining a Bacillus strain and cells containing a single or dual reporter gene plasmid system for causing the Bacillus strain to fluoresce in one fluorescent color when in a dormant spore state and / or another fluorescent color when in a metabolically active vegetative state.

[0153] b) feeding the Bacillus strain of step (a) to an animal,

[0154] c) collecting samples from the duodenum, ileum and / or cecum tissue of the animal and preparing these samples for analysis,

[0155] d) optionally staining the sample of step (c) with a DNA dye, and

[0156] e) imaging the sample of step (c) or (d) to visualize the status of the Bacillus strain.

[0157] 46. ​​A method for visualizing the status of a Bacillus strain in a plant or plant seed, the method comprising the steps of:

[0158] a) combining a Bacillus strain and cells containing a single or dual reporter gene plasmid system for causing the Bacillus strain to fluoresce in one fluorescent color when in a dormant spore state and another fluorescent color when in a metabolically active vegetative state,

[0159] b) treating the plant seeds with the Bacillus strain of step (a),

[0160] c) optionally planting the treated plant seeds of step (b) to allow plants to grow,

[0161] d) collecting samples for analysis by washing the treated plant seeds of step (b) which have been allowed to germinate, or the plants of step (c) or fractions thereof,

[0162] e) preparing the samples of step (d) for analysis,

[0163] f) optionally staining the sample of step (e) with a DNA dye, and

[0164] g) imaging the sample of step (e) or (f) to visualize the status of the Bacillus strain.

[0165] 47. The method of any one of embodiments 44 to 46, wherein the cells of step (a) comprise a single reporter plasmid system for causing the Bacillus strain to fluoresce a fluorescent color when in a dormant spore state or in a metabolically active vegetative state.

[0166] 48. The method of any one of embodiments 44 to 47, wherein the cells of step (a) comprise a dual reporter plasmid system for causing the Bacillus strain to fluoresce one fluorescent color when in a dormant spore state and another fluorescent color when in a metabolically active vegetative state.

[0167] 49. A method of visualizing the state of a Bacillus strain in vitro or in vivo, wherein the Bacillus strain is made to fluoresce in a dormant spore state and / or a metabolically active vegetative state using the plasmid system of any one of embodiments 1 to 38, wherein the fluorescence of the Bacillus strain is indicative of the state of the strain.

[0168] 50. The method of embodiment 49, wherein the Bacillus strain fluoresces one fluorescent color when in a dormant spore state and another fluorescent color when in a metabolically active vegetative state.

[0169] 51. The method of any one of embodiments 38 to 50, used to visualize germination of a Bacillus strain.

[0170] Examples

[0171] The plasmid system of the present invention has been used herein to transform six strains of Bacillus subtilis, four strains of Bacillus amyloliquefaciens, two strains of Bacillus megaterium, two strains of Bacillus pumilus, one strain of Bacillus thuringiensis, and even one Bacillus strain whose species designation we do not know as exemplified below.

[0172] Example 1 - Plasmid Design and Function

[0173] The backbone of one or more plasmids is derived from pBM317 (SEQ ID No: 1). This backbone is composed of pE194 and pUC plus oriT from pUB110. Therefore, this vector can be maintained in E. coli and most Bacillus species without the need for chromosomal integration. Furthermore, it can be transformed using a variety of methods, including conjugation, native competence, electroporation, and chemical competence. The first two methods have proven most effective in our work. For this plasmid including a green and / or red fluorescence system, our versions are pJDH11 (SEQ ID No: 3), pJDH14 (SEQ ID No: 4), pJDH20 (SEQ ID No: 5), pJDH21 (SEQ ID No: 6), pJDH22 (SEQ ID No: 7), pJDH23 (SEQ ID No: 8), pJDH24 (SEQ ID No: 9), pJDH25 (SEQ ID No: 10), pJDH26 (SEQ ID No: 11), pJDH29 (SEQ ID No: 12), and pBG3 (SEQ ID No: 13).

[0174] In most of these constructs, the GFP gene serves as a reporter gene for the presence of spores. The gene is translationally fused to a gene encoding a small acid-soluble protein (SASP). Specifically, this gene is sspB (SEQ ID No: 14) from Bacillus amyloliquefaciens. A small region lies between the SASP and GFP genes, encoding a short amino acid linker that should form a small loop between these proteins. The linker ensures that the proteins remain connected, but their folding remains largely independent, unaffected by the fusion. This last item is considered critical, as we need both domains of this fusion to remain functional. SASP binds to DNA with high affinity and is unique to dormant spores. Therefore, our SASP-GFP fusion encapsulates sporulated DNA in a green fluorescent protein, and our work demonstrates that fluorescence persists as long as the spores are stable. Shortly after germination is triggered, a protease called germination protease (Gpr) rapidly degrades the SASP. Gpr also degrades the GFP fused to SspB. This allows for a smooth transition from green fluorescent spores to red fluorescent cells during or shortly after spore germination.

[0175] In most of these plasmid constructs, there is a second reporter gene sequence that encodes the DsRedexpress2 (DsRed) protein. In most cases, this protein is controlled by a multipartite promoter. The entire promoter results in high expression of any immediately downstream genes during vegetative state. The complete promoter region consists of one or more of the following: amyLp, amyQp, and cryp. In addition, some constructs use rplKp from Bacillus amyloliquefaciens (O7SKR). Various combinations of these promoters are used to fine-tune the expression of DsRed up or down, depending on the needs of the final recipient strain of the plasmid.

[0176] Ultimately, we used the aforementioned plasmid backbone and two genetic circuits to generate a variety of plasmids, which we term "reporter plasmids" because they tend to confer fluorescence on their hosts; this property allows transformed cells to report their cellular and metabolic status. The spore-specific reporter system is activated during sporulation and results in both dormant and stable spores uniformly emitting green fluorescence. Due to its translational fusion nature, green fluorescence is rapidly degraded during spore germination and decreases significantly as the organism transitions to a vegetative state. During the vegetative state, the promoters driving red fluorescence become highly active. Furthermore, we can determine whether an organism is in a spore or vegetative state and, when the latter is true, gain a relative understanding of its growth level and protein synthesis. A major advantage of these plasmids is that they function extrachromosomally, meaning they do not need to integrate into the host chromosome to function. Furthermore, their origin of replication allows them to be stably maintained in most Bacillus species. Therefore, a single plasmid can be rapidly transformed and evaluated in new strains within a short timeframe.

[0177] Example 2 - In vitro examination of dual reporter strain manipulation

[0178] To ensure that the plasmid is functioning as designed, use in vitro assays to observe the Bacillus subtilis strain carrying pJDH20, e.g. Figure 1 This new reporter strain was generated from a Bacillus subtilis strain deposited as DSM 29870 and described in WO 2016 / 118864 (a commercial probiotic for poultry). We used fluorescence microscopy and flow cytometry to track the spore population as it transitioned from dormant spores with green fluorescence (GFP) to vegetative cells with red fluorescence (DsRed). The present invention can be measured by any instrument capable of detecting fluorescence in both green and red wavelengths.

[0179] Purified spores of the wild-type Bacillus subtilis strain deposited as DSM 29870 (WT) and the fluorescent Bacillus subtilis strain deposited as DSM 29870 (Fluor) (the latter having a dual reporter plasmid pJDH20) were used for this test. The spores were heat-induced to germinate and then aliquoted into the wells of a transparent 96-well plate (Corning (Costar) 3595) containing Miller LB medium (Fisher (Fisher) BP1426) with 5 ug / ml erythromycin and 25 ug / ml lincomycin. Each strain was aliquoted into a total of 12 wells to allow for destructive testing at defined time points, and three replicates were performed. These time points were 0, 5, 18, and 24 hours after the medium and spores were combined. Immediately after the spores and medium were combined, the plates were incubated at 35°C and stirred continuously during the experiment. A control without spores was also included in the test. Samples were removed from the 96-well plates after 0, 5, 18, and 24 hours of incubation and assayed using fluorescence microscopy and flow cytometry (BD Accuri C6).

[0180] To observe whether both GFP and DsRed were expressed in the Fluor strain, we used fluorescence microscopy. Microscopy clearly showed that both the WT and Fluor strains contained phase-bright dormant spores at the beginning of the examination, which germinated into vegetative cells during the incubation period. Figure 2 and Figure 3 In addition, the spores and cells of the Fluor strain are fluorescent ( Figure 2 These spores are distinctly green ( Figure 2 middle column), while these cells are red ( Figure 2 Right column). Other filter sets are typically used to detect fluorescence of one color, and this was also the case here, but after 24 hours of incubation, the most intense green was observed from the spores and the most intense red was observed from the cells. At intermediate time points, grown mature spores and young cells exhibited varying levels of both green and red fluorescence (data not shown). Although WT spores and cells were clearly present, germinated, and growing, they never exhibited any fluorescence ( Figure 3 The non-spore control contained no spores or cells and produced no fluorescence (data not shown).

[0181] Although aesthetically pleasing, it is difficult to accurately determine the fluorescence intensity of a spore or cell population using microscopy alone. Therefore, we used flow cytometry to quantify the number of spore-associated and cell-associated events in each sample that fluoresced green or red in the same 96-well plate assay described above. A blank sample containing no spores is essential to determine the level of background fluorescence due to the culture medium or other particles in the assay that are not part of the Bacillus subtilis strain deposited as DSM 29870. As the incubation progressed, WT spores and later developing cells did not produce significantly more fluorescent green ( Figure 4 ) or red ( Figure 5 ) events. These Fluor spores did clearly produce green and red fluorescence events ( Figure 4 and Figure 5 ). In addition, the percentage of fluorescent events varied with the incubation of spores and the resulting vegetative cells. At 0 h of incubation while the spores were still dormant, green fluorescence was highest in the Fluor strain and steadily decreased as incubation continued and the spores germinated into vegetative cells ( Figure 4 The green fluorescence never decreased to background levels, indicating that dormant spores were still present after 24 h; this was consistent with the microscopic findings, which clearly showed dormant spores in the 24 h sample ( Figure 2 The red fluorescence at 0 h was not significantly higher than the background, but it increased steadily and exceeded the background at 5 h of incubation, and continued to increase after 18 and 24 h of incubation ( Figure 5 The red fluorescence detected during flow cytometry was consistent with microscopic examination, which showed no red fluorescent cells at 0 h and the appearance of many red blood cells at later time points, with a maximum after 24 h ( Figure 3 ).

[0182] In this in vitro experiment, the novel fluorescent properties conferred by pJDH20 to the Bacillus subtilis strain deposited as DSM 29870 were clearly demonstrated. Due to the translational fusion of the SASP gene with the GFP gene, the spores emitted green fluorescence. As expected, as the spore population germinated, the green fluorescence diminished, and the SASP protein was degraded through the normal spore growth process. Also as expected, over time, the resulting vegetative cells produced red fluorescence from the DsRed gene driven by a constitutive vegetative promoter. This plasmid never required integration into the host strain's chromosome. Ultimately, the fluorescent strain of the Bacillus subtilis strain deposited as DSM 29870 served as an in vitro indicator of spore dormancy, germination, and vegetative growth using a dual reporter gene system.

[0183] Example 3 - Examination of gastrointestinal samples harvested from chickens fed a dual reporter strain

[0184] The plasmid of Example 1 was transformed into Bacillus subtilis, engineered to express green fluorescent protein (GFP) during the spore life stage, and red fluorescent protein (RFP) during the vegetative life stage to visualize the conversion of spores fed to chickens into nutrients in gastrointestinal samples.

[0185] Chickens were fed Bacillus subtilis-treated feed samples, then sacrificed and the duodenum, ileum, and cecum tissues harvested. The contents of each tissue were collected in separate tubes and frozen. The tissues were fixed in 10% buffered formalin. Upon receipt, GI content samples were processed for visualization by thawing and resuspending them in phosphate-buffered saline containing 0.01% Tween 20 and 0.25% Triton-X 100. The main solids were then separated by low-speed centrifugation, and the supernatant samples were fixed in buffered formalin for 1 hour. After fixation, the samples were stained with the DNA dye SytoxGreen and DAPI, followed by a final 25μm filtration and imaging using the INCell 2200 High Content Imaging System. Three color DAPI / FITC / Cy3 images were collected for each sample at different final dilutions. SytoxGreen DNA staining data were collected from FITC images, DAPI DNA staining data were collected from DAPI channels, and RFP data were collected from Cy3 images. As a control, a pure RFP-expressing Bacillus subtilis strain ( Figure 6 ).

[0186] After detecting individual objects from all collected images, the entire dataset was plotted using DNA as the object identifier (x-axis) and the measured RFP intensity on the y-axis ( Figure 7 All data used for this comparison were from sample dilutions. A cluster of RFP bright objects was observed and this group of object data was separated for further analysis ( Figure 8 In all wells containing pure samples of the RFP-expressing strain, a large number of Cy3-bright objects were detected. In addition, we were able to detect RFP-bright objects only in ileum samples from birds fed the RFP-expressing Bacillus species strain, but not from control birds fed a different diet ( Figure 9 ).

[0187] in conclusion

[0188] Analysis of gastrointestinal (GI) content samples revealed the presence of RFP-expressing Bacillus subtilis cells in the ileum of birds.

[0189] Example 4 - Examination of Germinated Corn Seeds Treated with a Reporter Strains

[0190] The following four strains were transformed with the reporter gene plasmid described in Example 1: Bacillus amyloliquefaciens (O44EAY), Bacillus megaterium (O83AN1), Bacillus megaterium (O8337C), and Bacillus thuringiensis (O84YVJ). The fluorescent properties of these organisms revealed that each strain had different colonization behaviors on the roots of germinated corn seeds. These results indicate that the reporter gene plasmid can be used to detect the presence of the recipient organism when using fluorescence microscopy. In addition, the reporter gene system reveals the relative amount of the organism in the environment, the state of the organism as a cell or spore, and the position of the organism in the tested environment.

[0191] Corn seeds (Viking) were treated with a liquid suspension of dormant spores of O44EAY, O83AN1, O8337C or O84YVJ. Non-fluorescent versions of these strains were also used for treatment and used as controls. Individual treatment consisted of 50 seeds, which were mixed with 100 μL of spores at a concentration of 1e8 cfu / mL, with an estimated rate of application of 5e5 cfu / mL. After treatment, these seeds were placed on seedling paper (Anchor Paper Co., Ltd.) and germinated, soaked in water, and incubated for 4-5 days in the dark at 22°C. During incubation, the seeds germinated and produced the initials of radicles and root systems. After incubation, the radicles were divided into three parts: the top portion was the radicle closest to the crown, the middle portion was the center of the radicle, and the bottom portion was the radicle including the tip. Using a scalpel, the top, middle, and bottom of each radicle were made into thin slices suitable for fluorescence microscopy. For each treatment, at least 10 seeds were observed.

[0192] After seed germination, each strain was observed in some areas of the corn root (Figure 10). The fluorescent properties of the organisms are essential for locating these organisms on the slices of the root material. Using a green filter, the cell walls of the plant tissue are clearly visible, and a red filter shows the presence of vegetative cells with strong red fluorescence properties. Control seeds that were not treated with the organisms did not produce any root sections with detectable red fluorescence (data not shown).

[0193] These results suggest that during seed germination, these spores can germinate on seeds and / or roots, in some cases colonizing areas significantly distant from their application site, and that some strains are better suited for germination and potentially root growth than others. Germination on seeds is supported by the appearance of red cells after treatment with dormant spores (Figure 10). Colonization along the entire root is also supported by the fact that even when O83AN1 and O8337C were applied directly to seeds as dormant spores, they were clearly observed near the root tip (Figure 10 left and Figure 10 center). Strain-specific properties are supported by the fact that we consistently observed more fluorescent vegetative cells for O8337C than for O83AN1 on root sections, rarely for O44EAY, and many for O84YVJ, but only in the very apical region of the root (Figure 10). Similarly, these differences occurred even when the same spore dose was applied to all strains during seed treatment. Notably, for O44EAY, we never observed cells in the middle or basal regions of the root. These results suggest that O44EAY is not a candidate for seed inoculant like O83AN1, O8337C, or O84YVJ because it struggles to germinate or maintain viability in this environment.

[0194] The fluorescent reporter gene system can be inserted into four different Bacillus strains without the need for custom modifications to the plasmids, which then produces spores that can be applied to seeds before germination, which clearly indicates whether the spores germinated, and subsequently whether these spores survived, the location of these spores, and the relative abundance of these spores on the newly formed maize roots.

Claims

1. A fluorescent reporter gene plasmid system for causing a Bacillus strain to emit fluorescence after the plasmid is transformed into a Bacillus cell, wherein the Bacillus strain emits fluorescence in its dormant spore state and / or in its metabolically active vegetative state, and wherein the plasmid system is designed to function extrachromosomally; and wherein the plasmid system comprises a first reporter gene and a second reporter gene, wherein the first reporter gene encodes a fluorescent protein, the fluorescent protein being designed to fluoresce in its dormant spore state, and the second reporter gene encodes a fluorescent protein being designed to fluoresce in its metabolically active vegetative state, the fluorescent protein being different from the fluorescent protein encoded by the first reporter gene, wherein the first reporter gene is translationally fused to a gene encoding a small acid-soluble protein (SASP) and wherein a linker encoding 1 to 24 amino acids is located between the reporter gene and the gene encoding the SASP. 2 . The plasmid system of claim 1 , wherein the fluorescent protein encoded by the reporter gene is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (ECFP), and Sapphire. 3 . The plasmid system of claim 1 , wherein the fluorescent protein encoded by the reporter gene is selected from the group consisting of: DsRedexpress2, mCherry, mOrange, and mPlum yellow fluorescent protein (EYFP).

4. The plasmid system of claim 1, wherein the linker encodes 6 to 20 amino acids.

5. The plasmid system of claim 1, wherein the linker encodes 8 to 20 amino acids.

6. The plasmid system of claim 4, wherein the linker encodes 8 to 16 amino acids.

7. The plasmid system of claim 4, wherein the linker encodes 9 to 15 amino acids.

8. The plasmid system of claim 4, wherein the linker encodes 10 to 14 amino acids.

9. The plasmid system of claim 4, wherein the linker encodes 11 to 13 amino acids.

10. The plasmid system according to any one of claims 1 to 9, which is translated into a fusion of a SASP protein and a fluorescent protein.

11. The plasmid system of any one of claims 1 to 9, wherein the second reporter gene comprises a promoter region, wherein the promoter region comprises one or more promoters expressed during vegetative growth.

12. The plasmid system of any one of claims 1 to 9, wherein the Bacillus strain is selected from the group consisting of Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus pumilus, Bacillus licheniformis, Bacillus simplex, Bacillus refrigerated saccharolyticus, and Bacillus thuringiensis.

13. A method for visualizing the status of a Bacillus strain in an animal, a plant, or a plant seed, the method comprising: a) combining by transformation a Bacillus strain and cells comprising a dual reporter plasmid system for causing the Bacillus strain to fluoresce one fluorescent color when in a dormant spore state and / or another fluorescent color when in a metabolically active vegetative state, and wherein the plasmid system comprises a first reporter gene and a second reporter gene, wherein the first reporter gene encodes a fluorescent protein that is designed to fluoresce in its dormant spore state, and the second reporter gene encodes a fluorescent protein that is designed to fluoresce in its metabolically active vegetative state, which fluorescent protein is different from the fluorescent protein encoded by the first reporter gene, wherein the first reporter gene is translationally fused to a gene encoding a small acid-soluble protein (SASP) and wherein a linker encoding 1 to 24 amino acids is located between the reporter gene and the gene encoding the SASP; b) treating animal or plant seeds with the Bacillus strain of step (a), c) collecting a sample from the treated animal or plant seed of step (b), d) optionally staining the sample of step (c) with a DNA dye, and e) imaging the sample of step (c) or (d) to visualize the status of the Bacillus strain.

14. A method for visualizing the state of a Bacillus strain in vitro or in vivo, wherein the Bacillus strain is made to fluoresce in a dormant spore state and / or a metabolically active vegetative state using the plasmid system of any one of claims 1 to 12, wherein the fluorescence of the Bacillus strain indicates the state of the strain.

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