Microbial survival rate control method and microbial survival rate control agent

Abstract

To provide a microbial survival rate control method and a microbial survival rate control agent which enable the selective increase or decrease of only the target microbial species in soil, as well as a microbial survival rate control agent.SOLUTION: The present invention provides a microbial survival rate control method that includes step s3 of supplying soil with a microorganism lacking at least one gene selected from the genes ihfA, leuO, and dps, which encode transcription factors, and genes with high sequence homology to these genes, as well as a survival rate control agent comprising the above-mentioned microorganism.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for controlling the viability of microorganisms contained in soil and an agent for controlling the viability of microorganisms. [Background technology]

[0002] In recent years, in order to mitigate climate change, many attempts have been made to reduce emissions of greenhouse gases such as carbon dioxide through the introduction of electric vehicles and clean energy sources.

[0003] However, for example, emissions from human activities account for only about 4.8% of total carbon dioxide emissions, while emissions from the ocean account for about 33.7% and emissions from soil account for about 61.3%. Most emissions of greenhouse gases such as carbon dioxide from soil are caused by microorganisms in the soil.

[0004] Therefore, it may be possible to significantly mitigate climate change by reducing microbial species in the soil that emit large amounts of greenhouse gases and increasing microbial species that are useful for reducing greenhouse gases.

[0005] One method for reducing greenhouse gas emissions is to promote plant growth and increase the amount of carbon dioxide fixed. In order to promote plant growth, it is common to supply fertilizers containing the three major nutrients for plants, nitrogen, phosphorus, and potassium, to the soil.

[0006] However, supplying nutrients to the soil in this way may increase the overall activity of the microorganisms in the soil, which may result in increased emissions of greenhouse gases such as carbon dioxide and nitrous oxide from the soil.

[0007] Furthermore, indiscriminately killing or reducing the activity of microorganisms in the soil may also affect beneficial microorganisms that supply nitrogen sources essential for plant growth, thereby posing a risk of hindering greenhouse gas reduction.

[0008] On the other hand, Non-Patent Document 1 discloses a method for modifying the microbial species growing in soil by changing the nutrients added to the soil. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Noah, F., et al. Comparative metagenomic, phylogenetic and physiological analyzes of soil microbial communities across nitrogen gradients. The ISME Journal volume 6, pages1007-1017, 2012. Summary of the Invention [Problem to be solved by the invention]

[0010] However, even with the modification method disclosed in Non-Patent Document 1, it is difficult to reduce only microbial species that emit large amounts of greenhouse gases or to increase only microbial species that are useful for reducing greenhouse gases.

[0011] The present invention has been made in consideration of the above circumstances, and aims to provide a method for controlling microbial viability and a microbial viability controller that are capable of increasing or decreasing only the target microbial species in soil. [Means for solving the problem]

[0012] The present inventors investigated the effect of each transcription factor on the survival rate of Escherichia coli, a model microorganism, by deleting the functions of some of the genes encoding about 300 transcription factors that control about 4,700 genes in Escherichia coli. As a result, the present inventors found that ihfA, leuO, and dps are each significantly involved in the survival rate of microorganisms in soil, and thus completed the present invention.

[0013] One aspect of the present invention includes the following aspects.

[0014] In one embodiment, the present invention provides a method for controlling microbial viability, comprising the step of supplying to soil a microorganism lacking the function of at least one of the following genes (a) to (i): (a) ihfA gene; (b) leuO gene; (c) dps gene; (d) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (f) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (h) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability; (i) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability.

[0015] In one embodiment, the present invention provides a method for controlling the viability of a microorganism, comprising the step of supplying to soil a microorganism in which a promoter controlling the transcription of at least one of the following genes (a) to (i) has been modified to a promoter that has higher expression than an endogenous promoter: (a) ihfA gene; (b) leuO gene; (c) dps gene; (d) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (f) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (h) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability; (i) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability.

[0016] In one embodiment, the present invention provides an agent for controlling the viability of a microorganism, comprising a microorganism defective in the function of at least one of the following genes (a) to (i): (a) ihfA gene; (b) leuO gene; (c) dps gene; (d) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (f) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (h) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability; (i) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability.

[0017] In one embodiment, the present invention provides a microbial viability regulator comprising a microorganism in which a promoter controlling the transcription of at least one of the following genes (a) to (i) has been modified to a promoter that has higher expression than the endogenous promoter: (a) ihfA gene; (b) leuO gene; (c) dps gene; (d) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having a function of regulating viability; (f) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and having a function of regulating viability; (h) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability; (i) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 3, and having a function of regulating viability. Effect of the Invention

[0018] According to the present invention, it is possible to provide a method for controlling microbial viability and a viability controller capable of increasing or decreasing only a target microbial species in soil. [Brief description of the drawings]

[0019] [Figure 1] 1 is a flow chart showing a method for controlling the survival rate of microorganisms according to a preferred embodiment of the present invention. [Diagram 2] 1 is a graph showing the transition of viable cell counts in soil for a wild-type Escherichia coli strain and strains lacking each of the transcription factors ihfA, leuO, dps, etc. [Diagram 3]1 is a graph showing the survival rates of a wild-type Escherichia coli strain and strains lacking each of the transcription factors, such as ihfA, leuO, and dps, in soil. [Figure 4] 1 is a graph showing the survival ratio of Escherichia coli wild-type strains in soil lacking each of the transcription factors ihfA, leuO, dps, etc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings where necessary.

[0021] FIG. 1 is a flow chart showing a method for controlling the survival rate of microorganisms according to a preferred embodiment of the present invention.

[0022] The method for controlling the survival rate of microorganisms according to this embodiment includes the steps of preparing a strain of a microorganism (hereinafter referred to as a "target microorganism") to be increased or decreased in soil, in which the expression level of a transcription factor that is significantly related to the survival rate has been changed from that of a wild-type strain (strain preparation step s1), culturing and growing the target microorganism strain prepared in the strain preparation step s1 (growing step s2), and supplying the grown microbial strain to soil (soil supply step s3). Note that it is not always necessary to provide the growing step s2, and the target microorganism strain prepared in the strain preparation step s1 may be supplied to soil without going through the growing step s2.

[0023] Target microorganisms to be reduced in soil include, but are not limited to, microbial species that emit large amounts of greenhouse gases and microbial species that inhibit plant growth.

[0024] Examples of target microorganisms that one wishes to increase in soil include, but are not limited to, microorganisms that contribute to greenhouse gas reduction and microorganisms that compete with the target microorganisms one wishes to decrease.

[0025] Examples of microorganisms that contribute to reducing greenhouse gases include microorganisms that are useful for plant growth and microorganisms that have the ability to convert greenhouse gases into other substances.

[0026] Microorganisms that are beneficial to plant growth include nitrite bacteria, which oxidize ammonia and organic nitrogen in the soil to nitrite, and nitrate bacteria, which oxidize nitrite to nitrate. Increasing the number of these bacteria in the soil can increase the amount of nitrate nitrogen available to plants.

[0027] Examples of microorganisms that have the ability to convert greenhouse gases into other substances include rhizobia, which convert nitrous oxide (N2O), which has a greater greenhouse effect than carbon dioxide, into nitrogen (N2), and nitrous oxide-reducing bacteria of the Herbaspirillum genus.

[0028] As microorganisms that compete with the target microorganisms to be reduced, non-pathogenic strains of the same species as the bacterial wilt bacterium Ralstonia solanacearum, or bacteria of the genus Bacillus or Pseudomonas, can be used as so-called biological control. By increasing the number of such microorganisms in the soil, the target microorganisms to be reduced can be reduced.

[0029] In the following, among the target microorganisms, the target microorganisms that one wishes to increase in the soil are also referred to as "increased target microorganisms", and the target microorganisms that one wishes to decrease in the soil are also referred to as "reduced target microorganisms".

[0030] <Stock preparation process s1> In the strain preparation step s1, a strain is prepared in which the expression level of at least one of (a) the ihfA gene, (b) the leuO gene, and (c) the dps gene, which code for a transcription factor that is significantly involved in the survival rate of the target microorganism, and a gene that is highly homologous (sequence identical) to these genes, is altered from that of a wild-type strain.

[0031] (a) ihfA gene refers to the ihfA gene in each target microorganism, and its base sequence differs depending on the type of microorganism.

[0032] (a) Genes highly homologous to the ihfA gene are (d) genes comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 and encoding a protein having the function of regulating viability, (e) genes comprising an amino acid sequence having 60% or more identity (preferably 80% or more, more preferably 90% or more, and most preferably 95% or more) with the amino acid sequence shown in SEQ ID NO: 1 and encoding a protein having the function of regulating viability, and genes encoding a protein having an E-value of less than 0.001 (preferably 0.0001) in the BLAST program between the amino acid sequence shown in SEQ ID NO: 1.

[0033] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the transcription factor IhfA expressed from the ihfA gene (see NCBI GenBank Gene ID: 945472, SEQ ID NO: 4) which is deeply involved in the survival rate of E. coli.

[0034] (b) leuO gene refers to the leuO gene in each target microorganism, and its base sequence differs depending on the type of microorganism.

[0035] (b) Genes highly homologous to the leuO gene are: (f) a gene comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having the function of regulating viability; (g) a gene comprising an amino acid sequence having 60% or more identity (preferably 80% or more, more preferably 90% or more, and most preferably 95% or more) with the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having the function of regulating viability; and a gene encoding a protein having an E-value of less than 0.001 (more preferably 0.0001) in the BLAST program between the amino acid sequence shown in SEQ ID NO: 2.

[0036] The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of the transcription factor LeuO expressed from the leuO gene (see NCBI GenBank Gene ID: 949034, SEQ ID NO: 5) which is closely related to the survival rate of E. coli.

[0037] (c) The dps gene refers to the dps gene in each target microorganism, and its base sequence differs depending on the type of microorganism.

[0038] (c) Genes highly homologous to the dps gene are: (h) genes comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3 and encoding a protein having the function of regulating viability; (i) genes comprising an amino acid sequence having 60% or more identity (preferably 80% or more, more preferably 90% or more, and most preferably 95% or more) with the amino acid sequence shown in SEQ ID NO: 3 and encoding a protein having the function of regulating viability; and genes encoding a protein having an E-value of less than 0.001 (preferably 0.0001) in the BLAST program between the amino acid sequence shown in SEQ ID NO: 3.

[0039] The amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of the transcription factor Dps expressed from the dps gene (see NCBI GenBank Gene ID: 945101, SEQ ID NO: 6) which is closely related to the survival rate of E. coli.

[0040] As used herein, "an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted" refers to an amino acid sequence in which one or more and ten or less, preferably one or more and eight or less, more preferably one or more and five or less, and even more preferably one or more and three or less amino acids have been deleted, substituted, added, or inserted.

[0041] In addition, in the present specification, the sequence identity of a subject amino acid sequence to a reference amino acid sequence (for example, the amino acid sequences shown in SEQ ID NOs: 1 to 3 above) can be determined, for example, as follows. First, the reference amino acid sequence and the subject amino acid sequence are aligned. Each amino acid sequence may contain gaps so as to maximize the sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (1). Sequence identity (%) = number of matching amino acids / total number of amino acids in the target amino acid sequence × 100… (1)

[0042] ihfA is a gene encoding the transcription factor IhfA, which is involved in the control of genome structure.

[0043] leuO is a gene encoding the transcription factor LeuO, which is involved in the antisilencer of the nucleoid protein H-NS.

[0044] dps is a gene that encodes the transcription factor Dps, which is involved in maintaining genome structure.

[0045] In other words, ihfA, leuO, and dps are all nucleoid-associated genes, and the present inventors discovered that the transcription factors IhfA, LeuO, and Dps expressed from these genes are proteins that have the function of regulating the viability of microorganisms.

[0046] ihfA, leuO, and dps are conserved in a wide variety of microorganisms, including the above-mentioned nitrite bacteria, nitrate bacteria, and nitrous oxide-reducing bacteria, as well as Acinetobacter, Cyanobacteria, Firmicutes, and Proteobacteria. In the following, unless otherwise specified, "ihfA" includes genes highly homologous to ihfA, "leuO" includes genes highly homologous to leuO, and "dps" includes genes highly homologous to dps.

[0047] Strains in which the expression levels of the above three transcription factors have been altered from the wild-type strains include, for example, strains lacking one or more of the three genes (including genes highly homologous to the three genes) that code for the above transcription factors (strains that do not express transcription factors) and strains in which one or more of the promoters controlling the transcription of the above three genes have been modified to have higher expression levels than the endogenous promoters in the wild-type strains of each microbial species (strains in which the expression levels of transcription factors are higher than those of the wild-type strains).

[0048] By supplying soil with a strain in which at least one of the ihfA, leuO, and dps genes is defective, the survival rate of the target microorganisms in the soil can be reduced. As a result, the number of the target microorganisms in the soil is reduced. The reduction effect of the target microorganisms is particularly remarkable in the strain in which the ihfA gene function is defective, and is particularly remarkable in the strain in which the leuO gene function is defective.

[0049] The method for preparing a strain lacking the function of one or more of the above three genes (including genes highly homologous thereto) is not particularly limited. For example, a commercially available lacking strain such as the Keio collection may be obtained, but it can also be created by deleting the target gene by genome editing a wild strain. A commercially available wild strain may be used, but one isolated from soil or the like and cultured may also be used. An example of a method for creating a lacking strain is described below, but the genome editing method is not particularly limited, and various methods can be used.

[0050] First, the upstream and downstream regions of the target gene encoding a transcription factor are amplified using PCR (Polymerase Chain Reaction) or other methods, and then the upstream and downstream regions are joined together to circularize the gene.

[0051] This nucleic acid fragment is then transformed into a wild-type strain, thereby causing a deletion of the targeted gene encoding the transcription factor.

[0052] As mentioned above, the transcription factors IhfA, LeuO, and Dps are all closely related to the survival rate of microorganisms, and mutants lacking these transcription factors have significantly reduced survival rates in soil.

[0053] Therefore, by modifying one or more promoters that control the transcription of the ihfA, leuO, and dps genes to promoters that are more highly expressed than the endogenous promoters of the wild strain, the expression level of one or more transcription factors among IhfA, LeuO, and Dps can be increased, and the survival rate of the target microorganisms in soil can be improved. By creating such a promoter-modified strain of the target microorganisms and supplying it to soil, the number of the target microorganisms in soil can be increased.

[0054] The type of promoter that has higher expression than the endogenous promoter is not particularly limited, and may be replaced with a commercially available promoter such as the pET system (Novagen), or with a well-known strong promoter such as the tac promoter, PhoA, lac promoter, PL promoter, T5 promoter, T7 promoter, or SP6 promoter.

[0055] Instead of replacing the endogenous promoter region entirely, the expression levels of the above three types of transcription factors may be increased by modifying the promoter region with partial base deletion, addition, substitution, or insertion. Also, by adding a mutation to the gene encoding the transcription factor, the activity of the transcription factor can be increased more than that of the wild type, thereby improving the survival rate of microorganisms in soil.

[0056] <Proliferation step s2> In the propagation step s2, the strain obtained in the strain preparation step s1 is cultured and propagated for the target microorganism. By propagating the target microorganism and then supplying it to the soil in the soil supply step s3, the effect of increasing or decreasing the target microorganism can be enhanced. For the culture in the propagation step s2, an appropriate medium and culture method can be selected depending on the type of microorganism.

[0057] In addition, in the strain preparation step s1, when a large number of strains in which the expression level of the transcription factor has been altered from that of the wild-type strain are already available, such as when a commercially available product is obtained, the propagation step s2 is not necessarily required.

[0058] <Soil supply process s3> In the soil supplying step s3, the target microorganisms cultured in the propagation step s2 are supplied to the soil in which it is desired to increase or decrease the number of target microorganisms. Hereinafter, the soil in which it is desired to increase or decrease the number of target microorganisms is referred to as the "target soil." When the propagation step s2 is not performed, the strains of the target microorganisms prepared in the strain preparation step s1 are supplied to the target soil.

[0059] In this way, by adding a strain in which the expression level of at least one of the ihfA gene, leuO gene, or dps gene encoding transcription factors has been altered from that of the wild-type strain to the target soil, it is possible to increase the number of increased target microorganisms or decrease the number of decreased target microorganisms in the target soil.

[0060] It is possible to directly reduce greenhouse gas emissions from the target soil, or indirectly reduce greenhouse gas emissions by promoting the growth of plants cultivated or native to the target soil.

[0061] For example, when a strain of an enhanced target microorganism with a promoter-modified high expression of one or more of ihfA, leuO, and dps is supplied to a target soil, the ratio of the promoter-modified strain to the wild-type strain gradually increases, resulting in a reduction in greenhouse gases.

[0062] In addition, when supplying to the soil a strain whose survival rate has been reduced compared to that of a wild-type strain in order to reduce the target microorganisms in the soil, the effect of reducing the target microorganisms in the target soil can be enhanced by carrying out a step of reducing (sterilizing) or sterilizing the bacteria in the soil prior to the soil supply step s3.

[0063] Furthermore, when a strain with a lower survival rate than the wild-type strain is supplied to soil, it is preferable to continuously supply the strain to the soil until the strain becomes established in the soil and replaces the wild-type strain.

[0064] Methods for sterilizing or disinfecting soil include, for example, heating the soil or irradiating it with ultraviolet light, but are not limited to these.

[0065] For example, soil can be sterilized or sterilized by drying it in the sun, placing it in a black bag and exposing it to high heat, or autoclaving it.

[0066] [Other embodiments] In one embodiment, the present invention provides a viability control agent comprising a microorganism lacking the function of at least one of the ihfA gene, the leuO gene, and the dps gene (in this paragraph, "ihfA," "leuO," and "dps" do not include highly homologous genes), which encode transcription factors, and (d)(e) a gene highly homologous to ihfA, (f)(g) a gene highly homologous to the leuO gene, and (h)(i) a gene highly homologous to the dps gene.

[0067] Such a microorganism may be a deletion strain in which one or more of the ihfA, leuO, and dps genes of the reduction target microorganism are deleted, as prepared in the strain preparation step s1 in the embodiment shown in FIG.

[0068] Furthermore, in one embodiment, the present invention provides a viability control agent comprising a microorganism in which a promoter controlling the transcription of at least one of the ihfA gene, leuO gene, and dps gene (in this paragraph, "ihfA," "leuO," and "dps" do not include genes with high homology), which encode transcription factors, and (d)(e) a gene with high homology to the ihfA gene, (f)(g) a gene with high homology to the leuO gene, and (h)(i) a gene with high homology to the dps gene, has been modified to a promoter with higher expression than the endogenous promoter.

[0069] As for such microorganisms, similar to the target microorganisms for reduction prepared in the strain preparation step s1 in the embodiment shown in Figure 1, promoter-modified strains in which the promoters controlling the transcription of the ihfA, leuO, and dps genes have been replaced with commercially available products such as the pET system (manufactured by Novagen), or with stronger promoters than endogenous promoters such as the tac promoter, PhoA, lac promoter, PL promoter, T5 promoter, T7 promoter, and SP6 promoter, as well as promoter-modified strains in which the expression level of transcription factors has been increased by modifying the endogenous promoter region with a mutation can be used.

[0070] Furthermore, in one embodiment, the present invention provides a viability control agent comprising a microorganism in which a mutation has been introduced into the base sequence of at least one of the ihfA gene, leuO gene, and dps gene (in this paragraph, "ihfA," "leuO," and "dps" do not include genes with high homology), which encode transcription factors, and (d)(e) a gene with high homology to the ihfA gene, (f)(g) a gene with high homology to the leuO gene, and (h)(i) a gene with high homology to the dps gene, thereby improving the activity of the transcription factor compared to that of an endogenous transcription factor.

[0071] Each of these viability control agents can be applied to a target soil to increase or decrease the number of target microorganisms in the target soil.

[0072] Furthermore, these viability control agents may contain, in addition to the microorganism, a culture medium such as a culture solution for the microorganism, water, or soil, and may further contain other substances.

[0073] Additionally, these viability regulators may be freeze-dried soil containing microorganisms in which one or more of the ihfA, leuO, and dps genes have been deleted or one or more of their promoters have been modified.

[0074] In another embodiment, the present invention provides a method for controlling the viability of a microorganism, comprising the step of supplying to soil a microorganism lacking a gene encoding a protein having an E-value of less than 0.0001 in a BLAST program relative to a protein expressed from a gene comprising any one of the base sequences of SEQ ID NOs: 4 to 6.

[0075] In another embodiment, the present invention provides a method for controlling the viability of a microorganism, comprising the step of supplying to soil a microorganism in which a promoter controlling the transcription of a gene encoding a protein having an E-value of less than 0.0001 in a BLAST program for a protein expressed from a gene comprising any one of the base sequences of SEQ ID NOs: 4 to 6 has been modified to a promoter with higher expression than an endogenous promoter.

[0076] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included in the scope of the present invention.

[0077] In the following, unless otherwise specified, ihfA, leuO, and dps are not considered to include genes highly homologous to each other. EXAMPLES

[0078] In this example, a wild-type strain (K-12 strain) of Escherichia coli, which is a model microorganism, and a deletion strain in which one gene encoding a transcription factor such as ihfA, leuO, or dps was deleted were supplied to separate sterilized soils, and the survival ratio of the wild-type strain to the deletion strain, such as the viable cell count and survival rate of each strain, was measured. Then, from the survival ratio of each deletion strain to the wild-type strain, the extent to which each deleted gene is involved in the survival rate of the microorganism in soil was clarified.

[0079] (Soil used and pretreatment) The soil used was sieved black soil. The maximum water capacity of this soil was 144g (water) / 100g (soil). This soil was then sieved through a 2mm sieve and dried. The moisture saturation level after drying was set to 0%.

[0080] The soil was then transferred to a medium bottle, the net weight of the soil was measured, and sterilization was performed by autoclaving at 121°C for 20 minutes.

[0081] After that, it was dried in a drying rack at 50℃, and the weight of the soil was measured to confirm that it had not changed from the weight before autoclaving. It was confirmed that the autoclaving process had killed the original bacteria originating from the soil.

[0082] (Container and capacity) The containers used were sterile Falcon tubes with a capacity of 15 mL, and each tube contained 1 g of soil.

[0083] (Proliferation step s2) A wild-type strain of E. coli and mutant strains lacking each gene encoding a transcription factor were cultured in LB medium for 24 hours. The mutant strains were obtained from the Keio collection.

[0084] (Preparation process of E. coli) First, the cells that had undergone the growth step s2 were harvested by centrifugation, and the supernatant was removed.

[0085] In this example, since E. coli was statically cultured in soil with a water capacity of 60% in the bacterial cell culture step, after removing the supernatant, the bacterial cells were resuspended in 864 uL of PBS buffer to obtain a bacterial cell suspension.

[0086] (Soil supply process s3) The bacterial cell suspension was diluted 1x10 per gram of soil in separate tubes. 7 CFU (Colony Forming Unit) (OD600 = 1 x 3 x 10 8 After that, each tube was thoroughly mixed by inversion.

[0087] (Bacterial cell culture) Each strain was statically cultured in a thermo-hygrostat at a temperature of 25°C and a humidity of 25%.

[0088] (Measurement of viable bacteria count) For each tube in which E. coli strains were statically cultured, the viable cell count was measured over time on days 0, 3, 7, 21, and 42 after the start of culture.

[0089] In detail, first, 10 mL of sterile water was added to 1 g of soil in each tube (10-fold dilution).

[0090] The tube was then mixed by inversion five times and vortexed twice for 20 seconds each, after which it was left to stand for 2 minutes until the soil naturally fell down the tube.

[0091] The supernatant in the tube was then serially diluted and plated on LB agar medium.

[0092] The resulting colonies were then counted overnight at 37°C, and the viable cell counts of each strain on days 0, 3, 7, 21, and 42 after the start of culture were calculated based on the colony counts. The colony counts were automatically performed using a colony counter.

[0093] In addition, the survival rates of the wild-type strain and each transcription factor-deficient strain were calculated on days 0, 3, 7, 21, and 42 after the start of culture. The survival rate of each strain is shown as the survival rate on each measurement day, with the viable cell count on day 0 after the start of culture set at 1.

[0094] Furthermore, the relative numbers of each transcription factor-deficient strain, when the number of wild-type strains on days 0, 3, 7, 21, and 42 after the start of culture was set to 1, were calculated as survival ratios.

[0095] FIG. 2 is a graph showing the transitional changes in viable cell counts of wild-type E. coli strains and strains lacking each of the transcription factors, such as ihfA, leuO, and dps, in soil, and FIG. 3 is a graph showing the survival rates of wild-type E. coli strains and strains lacking each of the transcription factors, such as ihfA, leuO, and dps, in soil.

[0096] In addition, Figure 4 is a graph showing the survival ratio of each transcription factor-deficient strain, such as ihfA, leuO, or dps, to the wild-type strain of E. coli in soil. Figure 4 shows the relative viable cell count of each transcription factor-deficient strain when the wild-type strain count on each measurement day is set to 1.

[0097] In addition, Table 1 below shows the ratio of viable cell counts of various transcription factor-deficient strains, including ihfA, leuO, and dps, when the viable cell count of the wild-type strain on the 42nd day of static culture in soil is set to 1.

[0098] [Table 1] Table 1 lists only genes for which the "ratio" values, which indicate the survival ratio of each transcription factor-deficient strain to the wild-type strain on day 42 of static culture, were significant, as a result of the inventors of the present application measuring the survival rate by deleting some of the approximately 300 transcription factor-encoding genes that control all of the approximately 4,700 genes in E. coli. The "ratio" values ​​for ihfA, leuO, and dps in Table 1 are the same as the values ​​on day 42 shown in Figure 4.

[0099] The ihfA gene knockout strain, indicated as "down" in the "Effect" column in Table 1, showed that on the 42nd day after the start of static culture, the number of live bacteria was 1 / 247 of the wild-type strain.

[0100] Similarly, it has been shown that the leuO gene-deficient strain had 1 / 112 the number of live bacteria of the wild-type strain 42 days after the start of static culture, and the dps gene-deficient strain had 1 / 11 the number of live bacteria of the wild-type strain 42 days after the start of static culture.

[0101] As shown in Figs. 2 to 4 and Table 1, the ihfA-deficient strain, the leuO-deficient strain, and the dps-deficient strain each had a significantly lower survival rate in soil than the wild-type strain.

[0102] Therefore, the number of target microorganisms in the target soil can be reduced by supplying to the target soil a defective strain of the target microorganism in which at least one of the ihfA, leuO and dps genes, (d)(e) a gene highly homologous to the ihfA gene, (f)(g) a gene highly homologous to the leuO gene, and (h)(i) a gene highly homologous to the dps gene has been deleted.

[0103] In addition, the survival rate of the target microorganisms to be amplified in soil can be increased by modifying the promoter of at least one of the ihfA, leuO and dps genes, (d)(e) a gene highly homologous to the ihfA gene, (f)(g) a gene highly homologous to the leuO gene, and (h)(i) a gene highly homologous to the dps gene, which contribute greatly to the survival rate of the target microorganisms to be amplified, to a promoter with higher expression than the endogenous promoter.

[0104] In addition, by supplying a promoter-modified strain that has been modified to have a highly expressing promoter to the target soil, the number of increased target microorganisms in the target soil can be increased in the long term, and greenhouse gas emissions from the target soil can be reduced. [Explanation of symbols]

[0105] s1 Stock preparation process s2 Propagation process s3 Soil supply process

Claims

1. A method for controlling the viability of a microorganism, comprising the step of supplying to soil a microorganism lacking the function of at least one of the following genes (a) to (i): (a) ihfA gene (b) leuO gene (c) dps gene (d) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (f) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (h) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3 and having a function of regulating survival rate; (i) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 3 and having a function of regulating survival rate;

2. A method for controlling the viability of a microorganism, comprising the step of supplying to soil a microorganism in which a promoter controlling the transcription of at least one of the following genes (a) to (i) has been modified to a promoter that has higher expression than an endogenous promoter: (a) ihfA gene (b) leuO gene (c) dps gene (d) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (f) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (h) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3 and having a function of regulating survival rate; (i) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 3 and having a function of regulating survival rate;

3. 3. The method for controlling the survival rate of microorganisms according to claim 1, further comprising a step of culturing the microorganisms prior to the step of supplying the microorganisms to soil.

4. 2. The method for controlling the survival rate of microorganisms according to claim 1, further comprising a step of sterilizing or sterilizing the soil prior to the step of supplying the microorganisms to the soil.

5. The method for controlling the viability of a microorganism according to claim 1 , wherein the identity in each of (e), (g), and (i) is 95% or more.

6. An agent for controlling the viability of a microorganism, comprising a microorganism lacking the function of at least one of the following genes (a) to (i): (a) ihfA gene (b) leuO gene (c) dps gene (d) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (f) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (h) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3 and having a function of regulating survival rate; (i) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 3 and having a function of regulating survival rate;

7. A microbial viability regulator comprising a microorganism in which a promoter controlling the transcription of at least one of the following genes (a) to (i) has been modified to a promoter that has higher expression than an endogenous promoter: (a) ihfA gene (b) leuO gene (c) dps gene (d) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (e) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having a function of regulating survival rate; (f) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (g) a gene encoding a protein having an amino acid sequence having 60% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having a function of regulating survival rate; (h) a gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3 and having a function of regulating survival rate;

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