A method for regulating the viability of microorganisms in soil, a method for producing microorganisms with increased viability in soil, and a soil composition containing microorganisms with reduced gene expression.

Abstract

A method for regulating the viability of microorganisms in soil, a method for producing microorganisms with increased viability in soil, and a soil composition containing microorganisms with reduced gene expression. [Solution] A method for regulating the viability of a microorganism in soil, comprising regulating the expression of a gene in the microorganism, wherein the gene is one or more genes selected from cbl, nhaR, crp, cadC, nemR, citR, and csuR.

Description

Technical Field

[0001] The present disclosure relates to a method for regulating the viability of microorganisms in soil, a method for producing microorganisms with increased viability in soil, and a soil composition containing microorganisms with reduced gene expression.

Background Art

[0002] The emission of greenhouse gases from soil is mainly due to the activities of microorganisms growing in the soil. If these microorganisms can be killed or inactivated, it is possible to reduce greenhouse gas emissions. However, since microorganisms in the soil are responsible for reactions that supply essential nitrogen sources for the growth of terrestrial plants that absorb and fix carbon dioxide in the atmosphere, such as grains and trees, simply reducing the activities of all microorganisms in the soil will have the opposite effect on reducing greenhouse gas emissions. In addition, when fertilizers are applied to the soil to promote plant growth, the activities of all microorganisms in the soil will change simultaneously along with the promotion of plant growth.

[0003] Therefore, there is a need for technologies that improve the survival of microorganisms with useful functions (e.g., nitrogen cycling) among the microorganisms in the soil, or that only reduce the survival of microorganisms that emit greenhouse gases.

[0004] For such purposes, it is conceivable to use methods that change the nutrients added to the soil and modify the microbial species growing in the soil (Non-Patent Document 1). However, with such methods, it is not possible to regulate only specific microorganisms.

[0005] Also, it is also conceivable to use a method of adding an inhibitor to the soil to inhibit the activities of microorganisms (Non-Patent Document 2). However, with such methods, it is not possible to regulate the activities of microorganisms for which appropriate inhibitors for inhibiting the activities cannot be obtained.

[0006] Also, for example, when a specific microorganism used for industrial purposes leaks into the soil, it may be desirable to operate so that it cannot reproduce.

[0007] Therefore, there is a need to identify proteins (or genes encoding them) involved in the survival of microorganisms in soil and modify their function to individually regulate the survival of target microorganisms. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Noah Fierer, Christian L Lauber, Kelly S Ramirez, Jesse Zaneveld, Mark A Bradford and Rob Knight. Comparative metagenomic, phylogenetic and physiological analyzes of soil microbial communities across nitrogen gradients. The ISME Journal (2012) 6, 1007-1017, https: / / www.nature.com / articles / ismej2011159%EF%BC%89 [Non-Patent Document 2] Yuki Yamamoto, Western Plant Protection, Volume 65, No. 6, 2011, http: / / jppa.or.jp / archive / pdf / 65_06_20.pdf [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] This disclosure is made to solve the above-mentioned problems and aims to provide a method for regulating the viability of microorganisms in soil, a method for producing microorganisms with increased viability in soil, and a soil composition containing microorganisms with reduced gene expression. [Means for solving the problem]

[0010] One aspect of the present disclosure is a method for regulating the viability of a microbial organism in soil, comprising regulating the expression of a gene in the organism, wherein the gene is one or more genes selected from nemR, citR, csuR, cbl, nhaR, crp, and cadC. One aspect of the present disclosure is a method for producing a microorganism with increased viability in soil, comprising reducing the expression of a gene in the microorganism, wherein the gene is one or more genes selected from nemR, citR, csuR, cbl, nhaR, crp, and cadC. One aspect of the present disclosure is a soil composition comprising a microorganism in which gene expression is reduced, wherein the gene is one or more genes selected from nemR, citR, csuR, cbl, nhaR, crp, and cadC. [Effects of the Invention]

[0011] This disclosure provides a method for regulating the viability of microorganisms in soil, a method for producing microorganisms with increased viability in soil, and a soil composition containing microorganisms with reduced gene expression. [Brief explanation of the drawing]

[0012] [Figure 1] The survival rate of gene-deficient K-12 strains in soil was compared to the survival rate of wild-type E. coli K-12 strains at day 42, which was set to 100%. Three independent measurements were performed, and the survival rates from each measurement are shown. [Modes for carrying out the invention]

[0013] The following describes non-limiting embodiments of this disclosure. This disclosure is not limited to the embodiments described below.

[0014] <Methods for regulating the viability of microorganisms in soil> In one embodiment, a method is provided for regulating the viability of a microbial organism in soil, comprising regulating the expression of a gene in the microbial organism, wherein the gene is one or more genes selected from nemR, citR, csuR, cbl, nhaR, crp, and cadC.

[0015] The microorganisms covered in this disclosure are not particularly limited and may include, for example, bacteria, archaea, fungi, protists, or algae (especially unicellular algae). The microorganisms may be soil microorganisms originally collected from soil, or soil bacteria. The microorganisms in the embodiments are preferably bacteria, and may be Escherichia coli. In embodiments, the microorganisms may be non-E. coli microorganisms, particularly non-E. coli bacteria.

[0016] In the method of the embodiment, regulating the survivability of microorganisms in soil may mean increasing or decreasing the survivability of microorganisms in soil compared to control microorganisms. Therefore, the method of regulating the survivability of microorganisms in soil in the embodiment may be a method that increases the survivability of microorganisms in soil, or a method that decreases the survivability of microorganisms in soil. The survivability of microorganisms in soil can be measured, for example, by adding the target microorganism to the soil, quantifying the number of living microorganisms present in the soil after a certain period of time, and comparing this with the value for control microorganisms. As a control microorganism, for example, wild-type microorganisms in which the expression of the above gene is not regulated can be used.

[0017] The soils described herein may, but are not limited to, clayey soils, sandy soils, silt soils, loamy soils, volcanic ash soils, calcareous soils, or moist soils. The moist soils may include black soil or black volcanic soil. In the embodiments, soils containing black soil are preferred. The soils described herein may be parent materials of these exemplified soils or soils that are mixtures of several of these parent materials.

[0018] The method of the embodiment includes regulating gene expression in a microorganism, that is, artificially modifying gene expression. In an embodiment, regulating gene expression may include reducing gene expression or may include increasing gene expression.

[0019] In an embodiment, reducing gene expression may include deleting the gene. Deleting the gene in an embodiment may be introducing a deletion or point mutation that completely or partially loses the function of the gene in the gene possessed by the microorganism. These mutations may be introduced into the coding region or the non-coding region as long as the function of the gene is lost. These mutations can be introduced into the genes of the microorganism by methods known to those skilled in the art including chemical mutation, radiation irradiation, introduction of transposons, and the CRISPR / CAS9 method. The gene to be deleted in an embodiment is preferably an endogenous gene of the microorganism.

[0020] In an embodiment, reducing gene expression may include introducing an antisense nucleic acid complementary to the gene transcript. The antisense nucleic acid is a nucleic acid containing a sequence complementary to all or part of the gene transcript, and may be RNA or a nucleic acid containing a modified nucleic acid. The antisense nucleic acid may be introduced by expressing the antisense nucleic acid in the microorganism cell by transformation of the microorganism. Alternatively, the antisense nucleic acid may be directly introduced into the target microorganism as a complex with a suitable carrier.

[0021] In an embodiment, increasing the expression of a gene may include transforming a microorganism or transducing a microorganism. Transformation in the present disclosure includes increasing the expression of the gene compared to the corresponding non-transformed individual by introducing a gene from the outside. Transformation can be performed by various methods known to those skilled in the art, including chemical transformation, electroporation, homologous recombination, and various genome editing techniques such as the CRISPR / CAS9 method. Transduction in the present disclosure includes introducing a gene into a microorganism using a bacteriophage containing the gene to be introduced into the microorganism. Transduction can be performed by infecting the microorganism with a bacteriophage by a method known to those skilled in the art.

[0022] The method of an embodiment, wherein the method of regulating the viability of a microorganism in soil is a method of increasing the viability of the microorganism in soil, and regulating the expression of a gene may be a method including decreasing the expression of the gene. In this case, the microorganism can be a soil bacterium and can be a beneficial bacterium, for example, a beneficial microorganism that provides an environment suitable for the growth of crops.

[0023] Alternatively, the method of an embodiment, wherein the method of regulating the viability of a microorganism in soil is a method of decreasing the viability of the microorganism in soil, and regulating the expression of a gene may be a method including increasing the expression of the gene. Also in this case, the microorganism can be a bacterium that is not preferably leaked into the soil environment.

[0024] The gene whose expression is regulated in an embodiment can be a gene that directly inhibits the survival, growth or reproduction of a microorganism in soil, or a gene that suppresses the expression and / or function of a gene responsible for a function necessary for the survival, growth or reproduction of a microorganism in soil.

[0025] For example, increasing or decreasing the viability of a microorganism in soil is 1x10 with respect to 1 g of the corresponding soil 7The CFU can be measured as the ability to increase or decrease the number of microbial cells (or CFU) compared to a control microorganism whose gene expression is not regulated, after mixing the target microorganism with the target microorganism and allowing static culture at 25°C and 60% humidity for 6 weeks. This increase or decrease in the number of microbial cells may be, for example, 10% or more, 20% or more, 50% or more, or 90% or more compared to the control microorganism. Here, for example, an increase of 10% or more means that the number of cells is 110% or more compared to the control microorganism, and a decrease of 10% or more means that the number of cells is 90% or less compared to the control microorganism.

[0026] In this embodiment, the genes whose expression is regulated are one or more genes selected from nemR, citR, csuR, cbl, nhaR, crp, and cadC. In this embodiment, the genes whose expression is regulated may be one or more of these genes. By reducing the expression of these genes, the viability of microorganisms in soil may be increased. Conversely, by increasing the expression of these genes, the viability of microorganisms in soil may be decreased.

[0027] In this specification, the genes represented by nemR, citR, csuR, cbl, nhaR, crp, and cadC include these genes in Escherichia coli, and also include genes from other bacterial species that are orthologues of these genes. Thus, the microorganisms of this disclosure may be microorganisms having orthologues of at least one of these genes. An orthologue is understood by those skilled in the art to be two genes that have separated and been conserved through speciation and belong to the same cluster in the molecular phylogenetic tree.

[0028] In this specification, the genes nemR, citR, csuR, cbl, nhaR, crp, and cadC include the genes of the E. coli K12 strain corresponding to NCBI gene IDs 946166, 945216, 948107, 946502, 944757, 947867, and 948653, respectively. In addition, the genes nemR, citR, csuR, cbl, nhaR, crp, and cadC each include genes from other bacterial species that are orthologues of the genes of the E. coli K12 strain corresponding to the above NCBI gene IDs.

[0029] By regulating the survival of only the target microorganism from among the countless microbial species that can potentially grow in the soil, the method disclosed herein can specifically regulate the particular functions and roles that the microorganism plays in the soil.

[0030] <Method for creating microorganisms with increased viability in soil>

[0031] In one embodiment, a method is provided for producing microorganisms with increased viability in soil, the method comprising reducing gene expression in the microorganisms, wherein the gene is one or more genes selected from nemR, citR, csuR, cbl, nhaR, crp, and cadC. The explanations provided in the section on <Methods for Regulating the Viability of Microorganisms in Soil> regarding the elements included in this embodiment (soil, viability, microorganisms, reducing gene expression, and genes, etc.) can also be applied to this embodiment unless otherwise stated.

[0032] The microorganisms in the method for producing microorganisms with increased viability in soil according to the embodiment may be non-E. coli microorganisms.

[0033] <Soil composition containing microorganisms with reduced gene expression> In one embodiment, a soil composition is provided that contains microorganisms in which gene expression is reduced, wherein the gene is one or more genes selected from nemR, citR, csuR, cbl, nhaR, crp, and cadC. The explanations provided in the sections on "Methods for Regulating the Viability of Microorganisms in Soil" and "Methods for Producing Microorganisms with Increased Viability in Soil" regarding the elements included in this embodiment (soil, soil, microorganisms, reducing gene expression, and genes, etc.) can also be applied to this embodiment unless otherwise stated.

[0034] The soil composition in this embodiment may include at least soil containing the above-mentioned microorganisms, and may also include a mixture of soil and fertilizer, or may include a field or a section thereof containing them.

[0035] In the soil composition of the embodiment, the microorganisms in which gene expression is reduced may be non-E. coli microorganisms. [Examples]

[0036] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0037] <Introduction> To identify genes involved in the survival of microorganisms in soil, we used Escherichia coli, a model microorganism. The approximately 4,700 genes of E. coli are regulated by approximately 300 transcription factors. These transcription factors sense environmental changes and regulate the expression of genes (or groups of genes) necessary for adaptation.

[0038] Therefore, we hypothesized that transcription factors are involved in soil survival rates through the regulation of gene expression, and we investigated the relationship between transcription factors and soil survival rates. Another reason for focusing on transcription factors was that it eliminated the need to analyze all approximately 4,700 genes in the genome.

[0039] <Materials and Methods> 1. Soil used and pretreatment Black soil (sieved) was used. The maximum water content of this soil is 144g (water) / 100g (soil). This soil was further sieved through a 2 mm sieve and dried (the moisture saturation at the time of drying was set to 0%). The soil was transferred to a medium bottle, its net weight was measured, and it was sterilized by autoclaving at 121°C for 20 minutes. After drying on a drying rack at 50°C, the weight was measured and confirmed to be unchanged from the weight before autoclaving. It was also confirmed that the bacteria originally from the soil were killed by the autoclaving process. The experiment was conducted with 60% water content, and the amount of bacterial suspension added to the soil was 864 μL.

[0040] 2. Container and soil volume 15 mL capacity sterilized Falcon tubes were used as containers. 1 g of soil was placed in each tube.

[0041] 3. Method for preparing E. coli applied to soil. First, each E. coli strain lacking one of the approximately 300 transcription factor genes was cultured in LB liquid medium for 24 hours. The cells were collected by centrifugation, the supernatant was removed, and the cells were resuspended in 864 μL of PBS buffer. 3 x 10⁶ solutions were then applied to 1 g of soil. 7 CFU (OD 600 = 1 to 3 x 10 8 The mixture was applied to the soil in an amount equivalent to CFU. After that, it was thoroughly mixed by inverting and stirring, and then cultured statically in a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 60%.

[0042] 4. Measurement of viable cell count (dilution plate method or colony count method) Viable cell counts were measured over time on days 0, 3, 7, 21, and 42. 10 mL of sterile water was added to 1 g of soil in a tube (10-fold dilution), followed by 5 inversions and 2 20-second vortexings. The mixture was then allowed to stand for 2 minutes until the soil naturally fell to the bottom of the tube. The supernatant was then serially diluted and spread onto LB agar plates. The cells were then incubated overnight at 37°C, and the number of colonies was counted to determine the viable cell count. Colony counts were automatically measured using a colony counter.

[0043] <Results and Discussion> Significant changes in soil viability were observed for deletions of seven transcription factor genes. Three independent experiments were conducted, in which the percentage of viable E. coli strains with each gene deletion compared to the viable number of wild-type strains (viability) was analyzed over a 42-day period.

[0044] Figure 1 and Table 1 summarize the results of the above experiment. Figure 1 shows the soil survival rate of the gene-deficient strain, with the soil survival rate of the wild strain at 42 days set to 100%. Three independent measurements were performed, and the survival rate for each measurement is shown.

[0045] Table 1 shows the survival rate values ​​for each trial and the NCBI gene IDs for each gene, as shown in Figure 1. In all three experiments, genes whose soil survival rate was 5 times or more that of the wild type were classified as having a "large" influence, while those whose survival rate was between 2 and 5 times the wild type were classified as having a "small" influence.

[0046] [Table 1]

[0047] For the four genes cbl, nhaR, crp, and cadC, deletion of each gene resulted in a remarkably increased soil survival rate, more than five times higher than that of the wild type. Furthermore, in cells lacking the three genes nemR, citR, and csuR, respectively, soil survival rates were observed to be between two and five times higher than that of the wild type.

[0048] These results illustrate that by identifying proteins (or genes encoding them) involved in the survival of soil microorganisms and modifying their function, the survival of target microorganisms in the soil can be individually regulated. The genes identified in this example suggest that other microorganisms may have similar functions.

[0049] This disclosure includes the following embodiments. (Section 1) A method for regulating the viability of microorganisms in soil, This includes regulating gene expression in the aforementioned microorganism. The gene is one or more genes selected from cbl, nhaR, crp, cadC, nemR, citR, and csuR. method. (Section 2) The method according to claim 1, wherein the method for regulating the viability of the microorganism in soil is a method for increasing the viability of the microorganism in soil, and regulating the expression of the gene includes decreasing the expression of the gene. (Section 3) The method according to claim 2, wherein the gene is an endogenous gene of the microorganism, and reducing the expression of the gene includes deleting the endogenous gene of the microorganism, or introducing an antisense nucleic acid complementary to the transcript of the endogenous gene of the microorganism. (Section 4) The method according to claim 1, wherein the method for regulating the viability of the microorganism in soil is a method for reducing the viability of the microorganism in soil, and regulating the expression of the gene is a method for increasing the expression of the gene. (Section 5) The method according to claim 4, wherein increasing the expression of the gene comprises transforming the microorganism or transducing the microorganism. (Section 6) A method for producing microorganisms with increased viability in soil, This includes reducing gene expression in the aforementioned microorganism. The aforementioned gene is one or more genes selected from cbl, nhaR, crp, cadC, nemR, citR, and csuR. Creation method. (Section 7) The method for producing the product according to item 6, wherein the microorganism is a non-E. coli microorganism. (Section 8) A soil composition containing microorganisms in which gene expression is reduced, The aforementioned gene is one or more genes selected from cbl, nhaR, crp, cadC, nemR, citR, and csuR. Soil composition. (Section 9) The method according to any one of claims 1 to 5, wherein the microorganism is a non-Escherichia coli microorganism. (Section 10) The method for producing the microorganism described in item 8, wherein the microorganism is a non-E. coli microorganism.

[0050] While this disclosure has been described with reference to some embodiments described above, it is not limited to these embodiments. Various modifications can be made to the structure and details of the present invention within the scope of this disclosure.

Claims

1. A method for regulating the viability of microorganisms in soil, This includes regulating gene expression in the aforementioned microorganism. The gene is one or more genes selected from cbl, nhaR, crp, cadC, nemR, citR, and csuR. method.

2. The method according to claim 1, wherein the method for regulating the viability of the microorganism in soil is a method for increasing the viability of the microorganism in soil, and regulating the expression of the gene includes decreasing the expression of the gene.

3. The method according to claim 2, wherein the gene is an endogenous gene of the microorganism, and reducing the expression of the gene includes deleting the endogenous gene of the microorganism, or introducing an antisense nucleic acid complementary to the transcript of the endogenous gene of the microorganism.

4. The method according to claim 1, wherein the method for regulating the viability of the microorganism in soil is a method for reducing the viability of the microorganism in soil, and regulating the expression of the gene includes increasing the expression of the gene.

5. The method according to claim 4, wherein increasing the expression of the gene includes transforming the microorganism or transducing the microorganism.

6. A method for producing microorganisms with increased viability in soil, This includes reducing gene expression in the aforementioned microorganism. The aforementioned gene is one or more genes selected from cbl, nhaR, crp, cadC, nemR, citR, and csuR. Creation method.

7. The method for producing the microorganism according to claim 6, wherein the microorganism is a non-E. coli microorganism.

8. A soil composition containing microorganisms in which gene expression is reduced, The aforementioned gene is one or more genes selected from cbl, nhaR, crp, cadC, nemR, citR, and csuR. Soil composition.

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