Additive compositions and methods for improving ionic tolerance of microorganisms

Abstract

To provide a composition and method for improving the resistance of a microorganism to cesium and / or lithium ions.SOLUTION: Provided is an additive composition for improving the resistance of a microorganism to cesium and / or lithium ions. The additive composition comprises an ionic compound composed of magnesium ions. Also provided is a method of improving the resistance of a microorganism to cesium and / or lithium ions by: introducing magnesium ions into a culture medium containing the microorganism and cesium and / or lithium ions by adding an additive composition to the culture medium; or expressing a magnesium ion transporter in the microorganism.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present disclosure relates to microbial ion tolerance, particularly to cesium and lithium ions. In particular, the present disclosure relates to compositions and methods for improving microbial tolerance to these ions. [Background technology]

[0002] Cesium is practically nonexistent in the normal environment, i.e., not present in quantities that would cause problems. However, it is known that large amounts of radioactive cesium can be released into the environment as a result of accidents at nuclear power plants, and serious environmental contamination by radioactive cesium can also occur when nuclear weapons are used (including in tests). Cesium, including those that are not necessarily radioactive, is also used in the oil extraction, chemical, biological and medical industries, and has the potential to be released into the environment from these sources.

[0003] Apart from the radiation of radioactive cesium, cesium itself is toxic to microorganisms (Non-Patent Document 1), and is presumed to be toxic to the cells of other organisms, such as mammals. The mechanism of cesium toxicity in Escherichia coli is understood to be as follows. That is, as can be seen from its position on the periodic table, cesium is physically and chemically similar to potassium, and cesium compounds dissolved in water are converted into cesium ions (Cs + ) and potassium ions (K + ) through the transport system + However, the K + / H + Antiporter is Cs + Without discharging K + Only Cs is excreted from the cell and high concentrations of Cs are released into the cell. + is K + It also inhibits new uptake of K + There will be a shortage of K +Since is an ion that plays a role in the synthesis of nucleic acids and the stabilization of ribosomes, if there is a shortage of it within a cell, the normal function and growth of the cell will be inhibited.

[0004] Cs + The existence of bacteria that exhibit resistance has also been reported (Non-Patent Documents 2 and 3). + To date, the specific molecular mechanisms of resistance remain largely unknown.

[0005] In another background study, in which no connection with cesium was discussed, Microbacterium sp. TS-1 strain was isolated from a jumping spider (Non-Patent Document 4). This TS-1 strain is an alkaliphilic bacterium with an optimum growth pH of 9.0, and is thought to inhabit the intestines of jumping spiders.

[0006] On the other hand, lithium (lithium ions) is an element that is ubiquitous in seawater and the like. However, it is known that lithium can also be toxic to microorganisms when its concentration reaches a certain level (Non-Patent Document 1). In modern society, lithium is used in large quantities industrially, including as a material for batteries, and it is believed that high concentrations of lithium ions are frequently leaking into the environment.

[0007] As the importance of efficient use of energy and resources increases, bioprocessing, a technology that utilizes the capabilities of microorganisms for the synthesis, conversion, decomposition, purification, etc. of various substances, is attracting increasing attention. If it is possible to activate microorganisms in the presence of toxic ions, the scope of application of bioprocessing can be further expanded. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Wackett et al. (2004) Appl. Environ. Microbiol. Vol. 70, No. 2, 647-655 [Non-Patent Document 2] Kato et al. (2016) Scientific Reports 6:20041 [Non-Patent Document 3] Dekker et al. (2014) FEMS Microbiol. Lett. 359, 81-84 [Non-Patent Document 4] Fujinami et al. (2013) Genome Announc. 1(6). pii: e01043-13 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present disclosure is to improve the resistance of microorganisms to cesium ions and / or lithium ions. [Means for solving the problem]

[0010] The inventors have demonstrated that Microbacterium sp. TS-1 can grow in the presence of high concentrations of cesium, Cs + It was found that this bacterium was resistant to Cs + Using the resistance as a clue, various genetic and microbiological experiments were conducted, and it was found that magnesium ion transporters and magnesium ions are involved in the Cs + The present invention has been completed based on the discovery that this can contribute to resistance.

[0011] The present disclosure encompasses at least the following embodiments. [1] 1. An additive composition for improving resistance of a microorganism to cesium ions and / or lithium ions, comprising an ionic compound composed of magnesium ions. [2] The additive composition according to [1], wherein the ionic compound comprises magnesium chloride, magnesium bromide, magnesium sulfate, or magnesium nitrate. [3] The additive composition according to [2], wherein the additive composition contains magnesium chloride or magnesium sulfate, and is added to a culture medium of the microorganism so that the magnesium chloride or magnesium sulfate has a concentration of 10 to 250 mM. [4] A method for improving resistance of a microorganism to cesium ions and / or lithium ions, comprising providing a mixture of a culture medium, the microorganism, cesium ions and / or lithium ions, and the additive composition according to any one of [1] to [3]. [5] A method for improving the resistance of a microorganism to cesium ions and / or lithium ions, the method comprising expressing a magnesium ion transporter having the amino acid sequence of SEQ ID NO:1 in the microorganism. [6] An Escherichia coli cell expressing a magnesium ion transporter having the amino acid sequence of SEQ ID NO:1. Effect of the Invention

[0012] According to the embodiments of the present disclosure, it is possible to easily confer resistance to cesium ions and / or lithium ions to various microorganisms without relying on rare resistant strains that may exist in nature, thereby expanding the applicability of bioprocesses using these microorganisms. [Brief description of the drawings]

[0013] [Figure 1] 1 shows the amino acid sequence (SEQ ID NO: 1) of the magnesium ion transporter encoded by the MTS1_03028 gene. The underlined portion indicates the predicted transmembrane domain, and the italicized bolded characters indicate the amino acids that were mutated in the RM1 and TM17 strains. [Diagram 2]FIG. 2 is a graph showing the relationship between the cesium concentration in the medium and the growth ability (medium turbidity expressed as absorbance OD600 after inoculation into a liquid natural medium and culturing at 30°C for 18 hours) of the wild-type Microbacterium sp. TS-1 strain, as well as the Cs+-sensitive mutants RM1 (A) and TM17 (B), and their revertant strains. [Diagram 3] Figure 3 shows a part of the amino acid sequence of the MTS1_03028 gene product of the wild type TS-1 strain (Wild Type), Cs+ sensitive mutant strains (RM1, TM17), and revertant strains (RM1R, TM17R). In RM1 (top) and TM17 (bottom), amino acid substitutions exist at different positions (see amino acid numbers and arrows) in the same MTS1_03028 gene product, and the wild type amino acids are restored in each revertant strain. [Figure 4] FIG. 4 shows a molecular phylogenetic tree of Mgt magnesium ion transporters. [Diagram 5] FIG. 5 shows the growth ability of RM1 and the revertant strain RM1R (A), and TM17 and the revertant strain TM17R (B), compared with that of the wild-type TS-1 strain (Wild type), when magnesium ions (100 mM MgCl2) were added to the medium. [Figure 6] Figure 6A shows the results of an experiment demonstrating that adding magnesium ions to culture media can confer cesium resistance to various microorganisms. The vertical axis shows the absorbance (OD600) of the culture media, and the horizontal axis shows the concentration of CsCl (mM) in the media. The grey line shows the case without the addition of magnesium ions, and the black line shows the case with the addition of magnesium ions. Figure 6B shows that similar results are obtained when the counter ion is changed from chloride ions to sulfate ions. In this experiment, LB medium contains CsCl, and modified LB medium contains Cs2SO4. [Figure 7] Figure 7, A to D, show the relationship between the addition of different concentrations of magnesium ion compounds and cesium resistance in Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus (S. aureus), respectively. [Figure 8]FIG. 8 shows the relationship between the addition of different concentrations of magnesium ion compounds and cesium resistance in yeast (S. cerevisiae). [Figure 9] Figure 9 shows the experimental results indicating that the addition of magnesium ions can confer lithium tolerance to microorganisms. The vertical axis indicates the absorbance (OD600) of the culture medium, and the horizontal axis indicates the concentration of LiCl (mM) in the medium. The gray line indicates the case without the addition of magnesium ions, and the black line indicates the case with the addition of magnesium ions. [Figure 10] FIG. 10 shows that heterologous expression of a magnesium ion transporter confers cesium resistance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In one aspect, the present disclosure provides a method for producing a cesium ion (Cs + ) and / or Lithium-ion (Li + The present invention provides an additive composition for improving the resistance of microorganisms to magnesium ions. The composition is characterized by including an ionic compound composed of magnesium ions.

[0015] An ionic compound is a compound in which atoms exchange electrons between each other to form ions and are stable due to ionic bonds (Tokyo Kagaku Dojin Chemical Dictionary, 1st Edition). In this disclosure, an "ionic compound composed of magnesium ions" refers to a compound that, when dissolved in water, forms magnesium ions (Mg 2+ ) and can be regarded as a magnesium ion supply source. In the present disclosure, an ionic compound composed of magnesium ions is also simply referred to as a magnesium ion compound. The ionic compound composed of magnesium ions may be included in the composition in the form of a solution, particularly an aqueous solution, that is, in an ionized state.

[0016] Specific examples of magnesium ion compounds include, but are not limited to, magnesium chloride, magnesium bromide, magnesium sulfate, magnesium nitrate, etc. Magnesium chloride or magnesium sulfate is particularly preferred. When the magnesium ion compound is added to a culture medium, the magnesium ion compound dissolves. The solubility of the magnesium ion compound in water at 25°C is preferably 1 g / 100 mL or more, more preferably 5 g / 100 mL or more, even more preferably 10 g / 100 mL or more, and particularly preferably 20 g / 100 mL or more.

[0017] In one embodiment, the additive composition contains magnesium chloride or magnesium sulfate, and is added to the microbial culture medium so that the magnesium chloride or magnesium sulfate has a concentration of 10 to 250 mM or 25 to 200 mM. This concentration is more preferably 25 to 150 mM, and even more preferably 50 to 125 mM. A particularly suitable concentration is 90 to 110 mM. When the additive composition contains magnesium sulfate, it may be more preferable that magnesium sulfate is added to the microbial culture medium at half these concentrations.

[0018] Even if the magnesium ion compound is a magnesium ion source other than magnesium chloride or magnesium sulfate, the ion compound is considered to have the same Mg concentration as the above-mentioned magnesium chloride added to water or LB medium (pH 7.0) at 37°C. 2+ The composition of LB medium is as follows: 10 g of tryptone (tryptic hydrolysate of casein), 5 g of yeast extract, 10 g of sodium chloride, and the remainder water per liter. In one embodiment, the additive composition contains preferably 10 to 250 mM or 25 to 200 mM, more preferably 25 to 150 mM, even more preferably 50 to 125 mM, and particularly preferably 90 to 110 mM of Mg. 2+ The amount of the agent that can be added to the microbial culture medium is preferably 0.1 to 100 μg / mL.

[0019] In the present disclosure, a microbial medium refers to an aqueous liquid-based composition that is an environment for growing microorganisms. This is not limited to laboratory-prepared experimental media, but may also include liquids found in the natural environment, industrial facilities, residential facilities, waste sites, etc. The term "aqueous liquid-based environment" as used herein may include, for example, aqueous liquids impregnated in matrix-like solids, and even colloidal liquid dispersion media such as agar gel. Those skilled in the art will understand that the medium contains at least water, and may also contain carbon sources (especially sugars), nitrogen sources (amino acids, etc.), vitamins, inorganic salts, etc.

[0020] A microorganism in the present disclosure can be defined as a single-celled organism with a size (maximum diameter) of 0.1 mm or less. Bacteria and yeast are typical microorganisms, and the bacteria may be either gram-positive or gram-negative. Specific examples of microorganisms include, but are not limited to, the genus Microbacterium (including TS-1 and its mutants), the genus Escherichia (e.g., E. coli), the genus Bacillus (e.g., B. subtilis), the genus Pseudomonas (e.g., P. aeruginosa), the genus Staphylococcus (e.g., S. aureus), and the genus Saccharomyces. As described below, the microorganism in the present disclosure may be one in which a gene for a magnesium ion transporter having the amino acid sequence of SEQ ID NO: 1 is introduced from outside and the gene product is expressed.

[0021] The additive composition may contain ingredients other than the magnesium ion compound. Examples of such ingredients may include, but are not limited to, water, glucose, a pH adjuster, a colorant, etc.

[0022] When cesium and lithium compounds are dissolved in water, they turn into cesium ions (Cs + ) and lithium-ion (Li + In the present disclosure, resistance to cesium ions and resistance to lithium ions are also simply referred to as cesium resistance and lithium resistance, respectively.

[0023] By adding the additive composition to a culture medium, the cesium tolerance and / or lithium tolerance of a microorganism can be improved. This means that the growth rate of a microorganism increases in a culture medium containing cesium ions and / or lithium ions, compared to a culture medium in which the additive is not added. In this case, the concentration of cesium ions may be, for example, 10 to 700 mM, 50 to 500 mM, or 100 to 300 mM in terms of CsCl. The term "CsCl equivalent" refers to the amount of Cs supplied by CsCl of the corresponding concentration added to water or LB medium (pH 7.0) at 37°C. - Similarly, the concentration of lithium ions may be, for example, 100 to 1500 mM, 200 to 1000 mM, or 400 to 800 mM in terms of LiCl. The term "LiCl equivalent" refers to the amount of Li that is equivalent to that supplied by LiCl of the corresponding concentration added to water or LB medium (pH 7.0) at 30°C. - The term "caesium ion concentration" refers to a concentration of cesium ions in the medium. The caesium ion concentration in the medium may be 10 to 700 mM, alternatively 50 to 500 mM, alternatively 100 to 300 mM. The lithium ion concentration in the medium may be 100 to 1500 mM, alternatively 200 to 1000 mM, alternatively 400 to 800 mM. It should be understood that sensitivity to caesium ions and lithium ions may vary depending on the species and strain of the microorganism.

[0024] Thus, in another aspect, the present disclosure provides a method for improving the resistance of a microorganism to cesium ions and / or lithium ions, the method comprising providing a mixture of a culture medium, a microorganism, cesium ions and / or lithium ions, and the additive composition described above. In one embodiment, the method comprises introducing magnesium ions into a culture medium containing a microorganism and cesium ions and / or lithium ions by adding the additive composition described above. However, the culture medium, the microorganism, the cesium ions and / or lithium ions, and the additive composition may be mixed in another order. For example, the mixture of the culture medium, the cesium ions and / or lithium ions, and the additive composition may be present first, and the microorganism may be introduced thereto, or the mixture of the culture medium, the microorganism, and the additive composition may be present first, and the cesium ions and / or lithium ions may be introduced thereto.

[0025] In another aspect, the present disclosure provides a medium composition comprising a microorganism, cesium ions and / or lithium ions, water, and the additive composition described above.

[0026] The present application discloses various embodiments for improving cesium tolerance and / or lithium tolerance of microorganisms through a common element, magnesium ions. In one such embodiment, the present disclosure provides a method for improving the tolerance of a microorganism to cesium ions and / or lithium ions by expressing a magnesium ion transporter having the amino acid sequence of SEQ ID NO:1 in the microorganism.

[0027] The magnesium ion transporter having the amino acid sequence of SEQ ID NO: 1 is encoded in the genome of Microbacterium sp. strain TS-1 (Non-Patent Document 4) isolated from a jumping spider, and is a protein endogenously expressed in the strain.

[0028] The inventors found that the TS-1 strain had high Cs + The results showed that the β-Cs in the 100-kDa mice were resistant to the β-Cs +Cs capable of growing resistant bacteria + The upper limit concentration was 200 mM and 300 mM for Flavobacterium sp. 200Cs-4 strain (Non-Patent Document 2) and Serratia sp. Cs60-2 strain (Non-Patent Document 3), respectively. At the highest, Yersinia sp. Cs67-2 strain (Non-Patent Document 2) could only tolerate up to 500 mM CsCl. In contrast, the maximum CsCl concentration at which Microbacterium sp. TS-1 strain could grow was 100 mM. + The upper limit of the concentration was found to be extremely high, about 1200 mM. + It was speculated that the strain may have a specific resistance mechanism against the virus.

[0029] The inventors have demonstrated that the TS-1 strain of Cs + In order to elucidate the molecular mechanism of resistance, a series of experiments and studies exemplified in Example 1 were carried out, and as a result, a protein having the amino acid sequence of SEQ ID NO: 1, which is responsible for at least part of the above-mentioned molecular mechanism, was successfully identified.

[0030] The amino acid sequence of SEQ ID NO: 1 is shown in Figure 1. The amino acid sequence of SEQ ID NO: 1 is the product of a gene tentatively designated MTS1_03028, which can also be found in the GenBank database under the accession number GAD35185.1.

[0031] In the amino acid sequence shown in FIG. 1, the underlined portion is a predicted transmembrane domain by a method commonly used by those skilled in the art. Based on the homology to known amino acid sequences, this protein is an evolutionarily conserved Mg protein among various microbial species. 2+ It is understood by those skilled in the art that the magnesium ion transporter is a Cs transporter (Mgt). The invention of the additive containing the above-mentioned magnesium ion compound also relates to the magnesium ion transporter. + This is based on the discovery that it confers resistance.

[0032] Techniques for expressing foreign gene products in microorganisms are well known to those skilled in the art, and such known techniques can be used to artificially introduce the magnesium ion transporter of SEQ ID NO: 1 into a microorganism and express it. For example, a gene nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 can be synthesized and cloned into an expression vector such as a plasmid. Expression can be achieved by introducing such an expression vector into a target microorganism.

[0033] The magnesium ion transporter used in this embodiment may consist of only the amino acid sequence of SEQ ID NO: 1, but may also have a peptide tag added to the N-terminus or C-terminus or both in addition to the sequence of SEQ ID NO: 1. Peptide tags are well known to those skilled in the art, and examples include, but are not limited to, His tags (HHHHHH, etc.), HA tags, FLAG tags, Myc tags, etc. The length of one peptide tag is preferably 25 amino acids or less, more preferably 20 amino acids or less, even more preferably 15 amino acids or less, and particularly preferably 10 amino acids or less. As is known to those skilled in the art, these peptide tags are usually added to facilitate purification and detection of proteins (although there may be cases where such a specific use is not intended for the peptide tag), and do not impair the function of the protein.

[0034] It is preferable that the coding region of a gene on an exogenously introduced nucleic acid is artificially linked to a promoter other than the original promoter of the gene, i.e., a heterologous promoter. Such a promoter can be appropriately selected by a person skilled in the art according to the host microorganism, and examples thereof include, but are not limited to, the PBAD promoter, the tac promoter, and the T7 promoter. The promoter does not necessarily have to be a constitutive promoter, and may be an inducible promoter. That is, in this embodiment, a microorganism that "expresses a protein" does not necessarily mean a microorganism that constantly expresses the protein, but should be understood to mean a microorganism that has the ability to express the protein.

[0035] It has been found that by expressing a magnesium ion transporter having the amino acid sequence of SEQ ID NO:1 in a microorganism, the same effect as that obtained by adding magnesium ions to the medium can be obtained. This effect may include imparting not only cesium resistance but also lithium resistance. It is strongly suggested that the expression of this magnesium ion transporter and the addition of magnesium ions affect the microorganism through a common or closely related mechanism. In fact, it has been found that the combined use of both the expression of the magnesium ion transporter and the addition of magnesium ions can significantly improve ion resistance in a synergistic manner.

[0036] E. coli is particularly suitable in this embodiment because it is a microorganism for which transformation techniques are particularly well developed. Thus, in one aspect, the present disclosure provides an E. coli cell expressing a magnesium ion transporter having the amino acid sequence of SEQ ID NO: 1. However, those skilled in the art will appreciate that transformation techniques can also be applied to other microorganisms, and E. coli cells are merely an example.

[0037] In the present disclosure, the symbol "~" indicating a numerical range means that the numerical values ​​before and after it are included as the lower and upper limits, respectively. When multiple possible numerical ranges are separately described, such as "A~B" and "C~D", it should be understood that the numerical range combining one lower limit or upper limit with the other upper limit or lower limit (e.g., "A~D" and "C~B") is also disclosed in this specification. In addition, when a combination of a first element and a second element is described, and multiple possible options are described for each of the first element and the second element, it should be understood that all possible combinations of each option of the first element and each option of the second element are disclosed in this specification. EXAMPLES

[0038] Hereinafter, the embodiments of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these specific examples.

[0039] [Example 1: Isolation and genetic analysis of cesium-sensitive mutants and revertants derived from cesium-resistant Microbacterium sp. TS-1] To obtain cesium-sensitive mutants from the originally cesium-resistant TS-1 strain, the culture solution of the TS-1 strain in the logarithmic growth phase was treated with ethyl methanesulfonate, a chemical mutagen, at a final concentration of 3% for 120 minutes. The mutants were identified by replica plate method. That is, colonies were formed on an agar medium using the culture solution after the above-mentioned chemical mutagen treatment, and the colonies were then transferred to velvet cloth. The velvet cloth was contacted with a normal solid natural medium and a solid natural medium containing 100 mM CsCl, and the colonies were transferred to these media, respectively. By comparing the growth on both plates, i.e., by finding the positions of colonies that grew on the normal medium but not on the CsCl medium, it was possible to identify the CsCl-sensitive mutants. + Two susceptible mutant strains were identified and obtained, and these two strains were designated RM1 and TM17, respectively.

[0040] The whole genome sequence of the obtained mutant strain was analyzed by next-generation sequencer. + Cs was spontaneously produced from the susceptible mutants (i.e., without further treatment with chemical mutagens). + We were able to obtain revertant mutants that had regained resistance. The revertant from the RM1 strain was named RM1R, and the revertant from the TM17 strain was named TM17R. The whole genome sequences of these revertant mutants were also analyzed using a next-generation sequencer.

[0041] FIG. 2 shows data indicating that the wild-type TS-1 strain is cesium resistant, whereas the RM1 (A) and TM17 (B) strains are cesium sensitive, and that their revertants regained cesium resistance.

[0042] From the whole genome sequences obtained, the nucleic acid sequence mutation sites in the cesium-sensitive mutants and revertant mutants were identified in comparison with the wild-type TS-1 strain. As a result, based on the whole genome sequence of the wild-type TS-1 strain, 177 mutations were found in the RM1 strain and 165 mutations in the TM17 strain. Based on the whole genome sequence of the RM1 strain, the RM1R strain had 10 mutations, and the TM17R strain had 2 mutations based on the whole genome sequence of the TM17 strain.

[0043] As shown in FIG. 3, it was found that both the RM1 and TM17 strains had mutations that caused different amino acid substitutions in the coding sequence of the MTS1_03028 gene that encodes the polypeptide of SEQ ID NO:1 (FIG. 1). Moreover, it was found that in the two revertant strains, each of the substituted amino acids had reverted to the wild-type amino acid. These results indicate that the MTS1_03028 gene is a Cs + In addition, the absence of normal expression of the MTS1_03028 gene product inhibited Cs + Expression of this gene product results in sensitivity to Cs + It has been shown to confer resistance.

[0044] The amino acid sequence of the polypeptide encoded by the MTS1_03028 gene (SEQ ID NO: 1) can be found under GenBank accession number GAD35185.1. The product of the MTS1_03028 gene ("M. TS-1" in FIG. 4) is a member of the Mg protein, which is evolutionarily conserved among various microbial species. 2+ It is a member of the transporter (Mgt) family (Figure 4).

[0045] Example 2: Effect of Mg on cesium resistance of Microbacterium sp. TS-1 strain 2+ Effects of Addition Since the mutations in both the RM1 and TM17 strains are located in or near the transmembrane domain, rather than in the long N-terminal domain on the cytoplasmic side of Mgt (Figs. 1 and 3), it was suggested that the magnesium ion transport event itself across the cell membrane, rather than some kind of signal transduction, affects cesium resistance. Therefore, we performed experiments in which the wild-type TS-1 strain, as well as the RM1 and TM17 strains and their revertant strains, were cultured in the presence of different concentrations of cesium and 100 mM of a magnesium ion compound (MgCl2).

[0046] Specifically, the above strains were pre-cultured in 30 mM Tris medium for 18 hours (200 rpm, 28°C), and then inoculated at 0.5% by volume into 30 mM Tris medium containing different concentrations of CsCl and with or without 100 mM MgCl2 as shown in Figure 5, and main culture was carried out for 18 hours (200 rpm, 28°C). The optical density (OD) of the culture medium at a wavelength of 600 nm was 600 The cell growth rate was quantified as the cell viability (Mg / L). The composition of 30 mM Tris medium is as follows: 3.63 g Tris base, 1.47 g citric acid monohydrate, 5 mL 10% yeast extract, 10 mL STS, 50 mL 1 M glucose, the remainder water, pH 8.0. Yeast extract, which is a natural ingredient, is thought to contain a trace amount of magnesium. In the above medium composition, STS is a mixture of the following trace ingredients. [Table 1] [Table 2]

[0047] Surprisingly, the addition of 100 mM MgCl2 conferred significant cesium tolerance to all strains, including wild-type, mutant, and revertant strains (Fig. 5). 2+ The presence or absence of transporters 2+ Although high concentrations of Mg affect the sufficient uptake of cesium and cesium tolerance, 2+ When Mg was added to the medium, Mg was increased regardless of the presence or absence of the MTS1_03028 gene product. 2+ It was thought that this flowed into the cells and provided cesium resistance.

[0048] [Example 3: Mg 2+ [Addition of cesium to various microorganisms to give them resistance to cesium] To verify the possibility that the above phenomenon is not specific to the TS-1 strain but occurs universally in other microorganisms, the following experiment was performed. Specifically, E. coli W3110 (gram-negative bacillus), B. subtilis Marburg 168 MR151MA (gram-positive bacillus), P. aeruginosa IFO13275 (gram-negative bacillus), and S. aureus IAM12544 (gram-positive cocci) were precultured in 2X TY medium at 37°C for 7 hours, and then the preculture was inoculated into normal LB medium (pH 7.5) at 0.5% by volume and main cultured at 37°C for 16 hours. The composition of LB medium is as follows: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl per liter, the balance water, pH 7.5. During the main culture, different concentrations of CsCl and 100 mM MgCl2 were added to the medium as described above. As shown in Figure 6A, it was confirmed that the addition of MgCl2 to the medium conferred cesium resistance to all microorganisms (black line). Furthermore, as exemplified in Example 5, the addition of MgCl2 to the medium conferred cesium resistance to all microorganisms.2+ It was found that the addition of cesium conferred resistance not only to prokaryotes but also to eukaryotic microorganisms.

[0049] [Example 4: Consideration of counter ions] Next, an experiment similar to that in Example 3 above was carried out with E. coli, using Cs2SO4 instead of CsCl, and adding 50 mM Mg2SO4 instead of 100 mM MgCl2. In this experiment, NaCl in the composition of the LB medium was also replaced with Na2SO4. The composition of the modified LB medium used here is as follows: 10 g of Tryptone, 5 g of Yeast Extract, 12 g of Na2SO4 per liter, the remainder water, pH 7.5. The results are shown in Figure 6B. This figure shows the LB medium and the cesium counterion as Cl. - The comparison is shown with a control experiment in which the magnesium was added in the form of 50 mM Mg2SO4, but in this control experiment too. In conclusion, essentially the same results as in Example 3 were obtained when the counter ion was changed from chloride to sulfate. Therefore, it was the Cs + ions and Mg 2+ It was determined that the ions contribute to toxicity and resistance, respectively.

[0050] [Example 5: Examination of the concentration of magnesium ion compound added] Next, the relationship between the concentration of MgCl2 and cesium resistance was investigated. In this experiment, the strain was cultured at 37°C using LB medium with a pH of 7.0 to 7.5. As shown in Figure 7, it was confirmed that cesium resistance was clearly conferred when the MgCl2 concentration was 10 mM or more.

[0051] Figure 8 shows the results of a similar experiment using the yeast Saccharomyces cerevisiae, a unicellular eukaryote. In this experiment, Saccharomyces cerevisiae (JCM1499 strain) was pre-cultured in YM medium for 24 hours (200 rpm, 25°C), and the pre-cultured liquid was inoculated into fresh YM medium at 0.5% by volume and further cultured for 24 hours (200 rpm, 25°C). The OD600 The cell growth rate was quantified as a function of the concentration of cesium. CsCl and MgCl2 were added to the YM medium for the main culture at different concentrations as shown in Figure 8. The composition of the YM medium was 10 g glucose, 5 g peptone, 3 g yeast extract, 3 g malt extract, and the remainder water per liter, with the pH adjusted to 6.2 with H2SO4. Thus, similar results were obtained in a variety of microorganisms that are widely separated phylogenetically, and the generality of cesium resistance conferred by magnesium ion compounds can be recognized.

[0052] [Example 6: Mg 2+ Imparting lithium resistance to various microorganisms by adding To verify the possibility that the addition of magnesium ions is also effective for resistance to ions other than cesium ions, we focused on lithium, another Group 1 element known to be toxic to microorganisms. Therefore, we replaced CsCl with LiCl and conducted experiments under the same conditions as in Example 3, except that the culture temperature was 30°C. As shown in Figure 9, it was revealed that when 100 mM MgCl2 was added to the medium (black line), growth of each microorganism could be maintained in the presence of LiCl at a concentration that would normally significantly inhibit growth.

[0053] [Example 7: Imparting cesium resistance to microorganisms by expression of MTS1_03028 magnesium ion transporter] The MTS1_03028 gene identified in Example 1 was cloned into the plasmid vector pGEM7zf(+) to obtain the pGEM7-Mgt construct. This gene construct was introduced into Escherichia coli (E. coli DH5α strain) by conventional transformation. In this way, the MTS1_03028 gene product, a magnesium ion transporter, was expressed in the E. coli cells via a heterologous promoter (T7 promoter).

[0054] The transformed E. coli was precultured in LB medium (pH 7.0) at 37°C for 8 hours, then inoculated into the same medium containing different concentrations of CsCl and with or without the addition of 50 mM MgCl2, and cultured at 37°C for 16 hours. Growth was quantified as absorbance at 600 nm. Figure 10 summarizes the results of three independent replica experiments. In the figure, pGEM7zf(+) represents E. coli transformed with an empty plasmid vector, i.e., not expressing the MTS1_03028 gene product.

[0055] The results shown in Figure 10 clearly indicate that expressing the MTS1_03028 gene product, a magnesium ion transporter, in a heterologous microorganism has the same effect as adding magnesium ions to the medium, that is, imparting cesium resistance to the microorganism. In fact, the combined use of magnesium ion transporter expression and addition of magnesium ions to the medium was found to have a synergistic effect on significantly improving cesium resistance.

[0056] [Summary of the Examples] Analysis of the cesium-resistant Microbacterium sp. TS-1 strain and its derived cesium-sensitive mutants and cesium-resistant revertant strains revealed that magnesium ions are important for cesium resistance in microorganisms. In particular, it was discovered that cesium resistance is conferred by the expression of the magnesium ion transporter of sequence number 1, and that cesium resistance can be conferred simply by adding relatively high concentrations of magnesium ions to the growth environment. These effects were confirmed not only in Microbacterium sp. TS-1 strain but also in other microbial species. [Industrial Applicability]

[0057] The present invention makes it possible to create highly functional microorganisms that are resistant to toxic ions, and therefore has potential applicability in all industries that use bioprocesses. As an example, by imparting cesium resistance to a microorganism that originally has the ability to accumulate cesium but has low cesium resistance (i.e., the ability to grow in the presence of cesium), purification of cesium-contaminated liquid by bioprocesses can be realized. In other words, by allowing the microorganism to grow in a cesium-contaminated liquid and accumulating water-soluble cesium in the bacterial cells, and then recovering the bacterial cells from the liquid after a certain period of time, cesium can be concentrated and separated as a solid substance called the bacterial cells, and purification or reduction of the cesium-contaminated liquid can be achieved.

Claims

1. An additive composition for improving the resistance of a microorganism to cesium ions, the additive composition comprising an ionic compound composed of magnesium ions, the additive composition being added to a culture medium of the microorganism in an amount that provides the culture medium with a magnesium ion concentration of 25 to 250 mM, the composition improving the resistance of the microorganism to cesium ions; The microorganism is a bacterium or a yeast. Additive composition.

2. 2. The additive composition of claim 1, wherein the ionic compound comprises magnesium chloride, magnesium bromide, magnesium sulfate, or magnesium nitrate.

3. 1. A method for improving the resistance of a microorganism to cesium ions, comprising providing a mixture of a culture medium, the microorganism, cesium ions, and an additive composition; the additive composition comprises an ionic compound comprised of magnesium ions, and is added to the mixture in an amount to provide a magnesium ion concentration of 25 to 250 mM to enhance the resistance of the microorganism to cesium ions; The microorganism is a bacterium or a yeast. method.

4. The method according to claim 3, wherein the microorganism is of the genus Microbacterium, Escherichia, Bacillus, Pseudomonas, Staphylococcus, or Saccharomyces.

5. 1. A method for improving resistance of a bacterium to cesium ions, comprising expressing in the bacterium a magnesium ion transporter having the amino acid sequence of SEQ ID NO:

1.

6. An Escherichia coli cell expressing a magnesium ion transporter having the amino acid sequence of SEQ ID NO:1.

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