JP4085523B2 - Natto bacillus with natural transformation ability - Google Patents

Description

【００３２】
（３）形質転換株のβ−ガラクトシダーゼ活性の測定
168［amyE::comG-lacZ(cat)］及びOK2［amyE::comG-lacZ(cat)］は、５μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地で、また、OK2［mecAΔ(spc)amyE::comG-lacZ］及びOK2［mecBΔ(spc)amyE::comG-lacZ］は、５μｇ／ｍｌのクロラムフェニコール及び１００μｇ／ｍｌのスペクチノマイシンを含むＬＢ寒天培地で、それぞれ３７℃で一晩培養し、ビオチン０．１μｇ／ｍｌと５μｇ／ｍｌのクロラムフェニコールを含むＣＩ培地（mecA変異株及びmecB変異株はさらに５０μｇ／ｍｌのスペクチノマイシンを含む）５ｍｌに、ＯＤ₆₆₀が約０．１になるように植菌し、３７℃で振盪培養した。培養開始から１時間毎にＯＤ₆₆₀を測定し、同時に１５０００rpm、２分間の遠心で集菌し、活性測定まで−３０℃に保存した。０、１、２時間目は１ｍｌ、３、４、５、６、７時間目は０．５ｍｌの培養液を分取した。
【００３３】
上記のようにして集菌した細胞をＺバッファー（60mM Na₂HPO₄, 40mM NaHPO₄・2H₂O, 10mM KCl, 1mM MgSO₄・7H₂O, 50mM 2-メルカプトエタノール）５００μｌに懸濁し、トルエンを４滴加え、２０秒間ボルテックスし、２８℃の恒温槽で２分間プレインキュベートし、反応液の温度を２８℃とした。次に、基質として、分解されると黄色の生成物を生じるＯＮＰＧ（Ｏ−ニトロフェニル−β−Ｄ−ガラクトピラノシド）を４ｍｇ／ｍｌ含むＺバッファーを２００μｌ加えて混合し、２８℃で反応を開始させた。薄く黄色く色づいたら、１Ｍの炭酸ナトリウム溶液を５００μｌ加え、反応を停止させ、氷中に保存した。反応開始から停止までの時間をＴ（分）とする。尚、色づかなかったサンプルは、最大２０分間２８℃で反応させてから、反応を停止させた。反応溶液を１５０００rpm、４℃で５分遠心した後、上清をキュベットに移し、分光光度計でＡ₄₂₀を測定した。β−ガラクトシダーゼ活性は、次のようにして算出した（Wang, P-Z and Doi, R.H., J. Biol. Chem., 259, 8619-8625 (1984)）。結果を、図１に示す。図中、活性を縦軸に、サンプリングした時間を横軸に示す。
【００３４】
【数１】
活性（unit）＝A₄₂₀×1000／Ｔ(分)×ＯＤ₆₆₀×Ｖ(ml)
【００３５】
＜４＞形質転換可能な納豆菌の形質転換能の評価
納豆菌自身の薬剤耐性マーカー遺伝子を得るために、納豆菌より自然突然変異によるリファンピシン耐性変異（rif^r）株をまず単離し、OK2SR21株と命名した。このリファンピシン耐性変異が、ＲＮＡポリメラーゼβサブユニット遺伝子の変異であることは、３０ＳリボゾームＳ１２タンパク質遺伝子（rpsL）内のストレプトマイシン耐性変異（strA）との同時形質転換（co-transformation）頻度により確認した。
【００３６】
バチルス・サブチリス168株のstrA47変異は、rpsL遺伝子内の変異であり、同株でたった一つのコドンの変異のみが知られている。また、rif1728は、168株のＲＮＡポリメラーゼβサブユニット遺伝子内の変異であり、多くのrif^rが同じ変異であることが知られている。OK2株で取得した自然突然変異（rif^r）が、既知のこれらの遺伝子内の変異であるか否かを、互いにマッピングすることにより推定した。その結果、OK2で得られたrif^r（rif21と命名した）はrpoBの変異であり、strAもrpsL（strA）の変異であることがわかった。
【００３７】
OK2SR21株（rif21）及び168株のrif^r株（rif1728）から調製したＤＮＡを用いて、＜３＞で得た形質転換可能な納豆菌OK2 mecAΔ(spc)amyE::comG-lacZ株、OK2 mecBΔ(spc)amyE::comG-lacZ、及び168株を、通常の形質転換法により形質転換した。OK2SR21株由来のＤＮＡは２３μｇ／ｍｌ、168株のrif^r株由来のＤＮＡは４３μｇ／ｍｌのＤＮＡ溶液を、それぞれ細胞懸濁液０．１ｍｌに対して３μｌ加えた。形質転換株の出現頻度は、表２に示すとおりであった。
【００３８】
【表２】

【００３９】
＜５＞OK2誘導株の制限・修飾系の有無の確認
バチルス・サブチリスには多くの制限・修飾系が知られているので、プラスミドpUB110（カナマイシン耐性（Kmr）マーカーを含む）を用いて、OK2 mecAΔ(spc)amyE::comG-lacZ、OK2 mecBΔ(spc)amyE::comG-lacZの制限・修飾系の有無を調べた。
【００４０】
168株を用いて増殖させたpUB110、及びOKSR21株由来のＤＮＡを用いて、OK2 mecAΔ(spc)amyE::comG-lacZ株、OK2 mecBΔ(spc)amyE::comG-lacZ168株、OK2株、及び168株を、通常の形質転換法により形質転換した。結果を表３に示す。この結果から、納豆菌には、168株と異なる制限・修飾系は存在しないことがわかる。
【００４１】
【表３】

【００４２】
＜６＞comK遺伝子の発現強化によるコンピテンス遺伝子の発現回復
枯草菌のファージSPO1の強力なプロモーター及びエシェリヒア・コリのlacプロモーターとの融合プロモーターであるPspac（Methods in Enzymology, vol.185, 185- (1990)）を含むプラスミドpAG58をEcoRI及びBamHIで切断し、Pspacを含む断片を切り出した。バチルス属細菌のプラスミドpUB110をEcoRI及びBamHIで切断し、前記断片と連結し、プラスミドpULI7を得た（図２）。
【００４３】
一方、バチルス・ズブチリス168株の染色体ＤＮＡを鋳型とし、配列番号１及び２に示す塩基配列を有し、末端にXbaIの認識配列を有するオリゴヌクレオチドをプライマーとするPCRにより、comK遺伝子を含むＤＮＡ断片を増幅した。この断片をXbaIで消化し、XbaIで消化したpUL17に連結し、プラスミドpULI7SK27を得た（図２）。同プラスミドは、Pspacの制御下で発現するcomK遺伝子を有しており、同遺伝子はIPTGにより誘導され得る。尚、上記の各プラスミドの構築は、バチルス・ズブチリス168株を用いて行った。
【００４４】
OK2［amyE::comG-lacZ(cat)］を上記プラスミドpULI7SK27で形質転換した。形質転換株を、カナマイシン7.5μg/mlを含むLB培地で培養し、各種濃度のIPTGを添加してcomK遺伝子の誘導を行い、β−ガラクトシダーゼ活性を測定した。結果を図３に示す。また、同様にして培養した形質転換株を、OK2SR21株の染色体ＤＮＡで形質転換し、rif^rを指標として形質転換効率を評価した。結果を表４に示す。この結果から、comK遺伝子の発現を増強することによって、納豆菌の形質転換能を示すことがわかる。
【００４５】
【表４】

【００４６】
【発明の効果】
本発明により、納豆菌等の、野生株が実質的に自然形質転換能を有しないバチルス属細菌の形質転換を可能にすることができる。本発明によれば、自然突然変異に比べて有意に高い頻度で納豆菌等の形質転換を行うことが可能となるので、納豆菌等の分子育種に好適に利用することができる。
【配列表】

【００４７】

【００４８】

【００４９】

【図面の簡単な説明】
【図１】 168［amyE::comG-lacZ(cat)］、OK2［amyE::comG-lacZ(cat)］、OK2［mecAΔ(spc)amyE::comG-lacZ］及びOK2［mecBΔ(spc)amyE::comG-lacZ］のβ−ガラクトシダーゼ活性を示す図。活性を縦軸に、サンプリングした時間を横軸に示す。
【図２】 IPTGで発現が誘導されるcomK遺伝子を有するプラスミドpULI7SK27の構築過程を示す図。
【図３】 IPTGで発現が誘導されるcomK遺伝子を有するプラスミドpULI7SK27を含む納豆菌形質転換体のβ−ガラクトスシダーゼ活性のIPTGによる誘導を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a bacterium belonging to the genus Bacillus that has acquired the ability to be naturally transformed, and in particular, a mutant or recombinant strain of a bacterium belonging to the genus Bacillus whose wild strain does not have a natural transformation ability, and has a natural transformation ability. It relates to the genus bacteria.
[0002]
[Prior art]
Bacillus subtilis bacteria such as Bacillus subtilis are not only industrially used as bacteria producing extracellular enzymes, but are also old in food production like Bacillus subtilis natto. Some are used from. In recent years, focusing on the strong secretory production ability of Bacillus bacteria, it has been used as a host for genetic recombination along with E. coli and yeast.
[0003]
As a method for transforming Bacillus subtilis, a natural transformation method (competent cell method), a protoplast method, and an electroporation method are mainly used. Among these methods, the natural transformation method has an advantage that a relatively large DNA molecule can be introduced, but the natural transformation is a strain of Bacillus subtilis Marburg (168, 166, 160 strains). It is said that the frequency of natural transformation is very low in Bacillus natto and the like, and it is said that it does not have transformation ability (competency). The protoplast method and the electroporation method can be applied to many bacterial species, while the protoplast method is unstable and easily burst, has low reproducibility, and uses a complete medium for protoplast regeneration. Therefore, there are drawbacks such as the inability to use an auxotrophic marker. Furthermore, the electroporation method has a problem of low transformation efficiency (Molecular Biological Method for Bacillus, Edited by Harwood, CR et al., John Wiley & Sons, p. 98-103).
[0004]
In Bacillus subtilis Marburg strain, competence is known to be induced by early and late competence genes (Solomon, JM, et al., Genes & Development, 10, 2014-2024 (1996) Lazazzera, BA, et al., Genes & Development, 19, 455-458 (1997), Dubnau, D., Gene, 192, 191-198 (1997)). The expression of the late competence gene group requires the comK product (ComK) which is an early competence gene. Although the comK gene is constitutively expressed, under normal growth conditions, the ComK protein is degraded by the gene products of mec (medium independent competence) A, mecB (clpC), and clpP, which are also early competence genes. Competence genes are not expressed. On the other hand, when the culture is performed under conditions in which competence is induced, the expression of the comK gene is promoted, so that active comK induces the expression of the late-stage competence gene group. As a result, competence is induced.
[0005]
However, it is not known whether Bacillus bacteria having no natural transformation ability such as Bacillus natto have the same competence gene group or can be expressed even if they have.
[0006]
[Problems to be solved by the invention]
The present invention has been made from the above viewpoint, and it is an object of the present invention to provide a technique that enables transformation of a Bacillus bacterium, such as Bacillus natto, in which a wild strain has substantially no natural transformation ability. .
[0007]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor has no ability to induce late competence genes because of the absence of ComK protein activation in Bacillus natto. I found out. And when the mecA gene or mecB gene of Bacillus natto was disrupted, it succeeded in constructing Bacillus natto which acquired natural transformation ability, and came to complete this invention.
That is, the present invention is as follows.
[0008]
(1) A Bacillus that is a mutant or recombinant strain of a bacterium belonging to the genus Bacillus that does not substantially have a natural transformation ability, can express a late competence gene group, and has acquired the natural transformation ability Genus bacteria.
(2) The bacterium according to (1), which is Bacillus subtilis.
(3) The bacterium according to (2), which is Bacillus subtilis natto.
(4) The bacterium according to (1), wherein the late competence gene group is selected from a comC operon, a comE operon, a comF operon, and a comG operon.
(5) The bacterium according to (1), which retains an active ComK protein.
(6) The bacterium according to claim 5, which retains an active ComK protein due to enhanced expression of the comK gene.
(7) The bacterium according to (5), which retains an active ComK protein by disrupting one or both of the mecA gene and the mecB gene.
(8) A method for imparting natural transformation ability to a Bacillus bacterium, characterized by imparting late competence gene expression ability to a Bacillus bacterium having substantially no natural transformation ability.
[0009]
In the present invention, natural transformation means that when DNA is added to a culture medium of bacteria cultured under specific conditions and cultured, the DNA is taken up and retained in bacterial cells. “Substantially not having natural transformation ability” means that the expression frequency of a phenotype such as drug resistance is transformed with DNA containing a gene corresponding to the phenotype, and spontaneous mutation It means that there is no significant difference. Furthermore, imparting natural transformation ability means imparting natural transformation ability to a bacterium not having natural transformation ability, and enhancing natural transformation ability in a bacterium having very low natural transformation ability. Say.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The bacterium of the present invention is a mutant or recombinant strain of a bacterium belonging to the genus Bacillus whose wild strain has substantially no natural transformation ability, can express a late competence gene group, and has a natural transformation ability. Acquired Bacillus bacteria.
[0011]
As bacteria belonging to the genus Bacillus, Bacillus subtilis, Bacillus stearothermophilus, Bacillus methanolicus, Bacillus amiloliquefaciens, specifically Bacillus amiloliquefaciens. (Bacillus subtilis natto). It is said that natural transformation occurs only in Bacillus subtilis Marburg strains in Bacillus bacteria, and Bacillus bacteria other than those strains apply natural transformation ability by applying the present invention. Can be granted. Even in the case of a Marburg strain, the natural transformation ability can be improved in the same manner.
[0012]
When strains of Marburg are cultured under specific culture conditions, competent genes are induced and natural transformation ability is expressed, but Bacillus bacteria (hereinafter referred to simply as “Natto”) that do not have natural transformation ability, such as Bacillus natto. Even when cultured under such conditions, the expression of the late-competence genetic group is not induced. Therefore, natural transformation ability can be imparted to Bacillus natto and the like by enabling expression of the late-stage competence gene group.
[0013]
In order to enable expression of late-stage competence genes, the expression control system is modified so that late-stage competence genes originally retained by Bacillus natto, etc. are expressed, or the late-stage competence genes that can be expressed are changed to Bacillus natto, etc. What is necessary is just to introduce. Specifically, for example, the active ComK protein may be retained in the cell in an amount that induces the expression of the late-stage competence gene group. In order to retain the active ComK protein in the cell, the expression level of the comK gene may be increased, or the mecA gene or mecB gene may be disrupted or mutated so that the MecA protein or MecB protein does not function normally. . Either one or both of the mecA gene and the mecB gene may be disrupted.
[0014]
The mecA gene or mecB gene is destroyed by adding a mecA gene or mecB gene into which another DNA sequence has been inserted, or a DNA fragment containing the mecA gene or mecB gene from which the inside has been deleted, to a culture solution such as Bacillus natto. It can be performed by transforming cells such as Bacillus natto by culturing and causing homologous recombination with the mecA gene or mecB gene on the chromosome.
[0015]
The mecA gene (Genbank / EMBL / DDBJ accetion No. L06059) or mecB gene (Genbank / EMBL / DDBJ accetion No. U02604) of Bacillus subtilis has been reported and synthesized based on the base sequence. It can be isolated from Bacillus bacterial chromosomal DNA by the polymerase chain reaction method (PCR: Polymerase chain reaction; see White, TJ et al., Trends Genet., 5,185 (1989)) using the prepared oligonucleotide as a primer.
[0016]
Transformation of natto, etc. is the same as the natural transformation method (competent cell method) normally used in Bacillus subtilis (J. Spizizen, Proc, Natl. Acad. Sci. USA, 44, 1072 (1958)) Thus, DNA can be added to the culture medium of cells cultured under specific conditions and cultured. Bacillus natto and the like have increased transformation ability in the growth phase at the time of transition from the logarithmic growth phase to the stationary phase. Specifically, for example, transformation can be performed as follows. That is, natto bacteria and the like were inoculated into the competence induction medium shown in the Examples so that the OD ₆₆₀ was about 0.05, and after shaking culture at 37 ° C. for 5 hours, the cells were suspended in the competence induction medium. After turbidity, DNA was added to this cell suspension, and further cultured with shaking at 37 ° C. for 1.5 hours. After that, LB liquid medium was added, and osmotic culture was continued at 37 ° C. for 1 hour, and then applied to LB agar medium. Incubate overnight at 37 ° C.
[0017]
Selection of the transformed strain can be performed by holding a marker such as a drug resistance gene in the DNA to be introduced and using the marker trait of the cell after transformation treatment as an index. The mecA gene or mecB gene containing the marker gene can be suitably used for disrupting these genes. As a Bacillus subtilis carrying mecA gene or mecB gene containing such marker gene, mecA-deficient strain BD2123 [hisB2 leu-8 metB5 mecAΔ (spc) amyE :: comG-lacZ (cat)], and mecB-deficient Strain BD2243 [hisB2 leu-8 metB5 mecBΔ (spc) amyE :: comG-lacZ (cat)] (see Dubnau, D., Gene, 192, 191-198 (1997)). Chromosomal DNA fragments of these strains can be used as they are for disrupting the mecA gene or mecB gene.
[0018]
The occurrence of transformation can be confirmed by preparing DNA from the transformant, transforming a Bacillus subtilis Marburg strain with the DNA, and obtaining a transformant carrying the marker gene. .
[0019]
In addition, the mecA-deficient strain or mecB-deficient strain obtained as described above retains the active ComK protein in the cell and is capable of expressing the late-competence gene group. This is because the operon promoter of the late-competence gene group It can be confirmed by holding a fusion gene linked to a reporter gene such as β-galactosidase gene in a mecA-deficient strain or mecB-deficient strain and expressing the β-galactosidase activity of these strains. Examples of the fusion gene include BD2123 strain [hisB2 leu-8 metB5 mecAΔ (spc) amyE :: comG-lacZ (cat)] (Liyun Kong and David Dubnau, Proc, Natl. Acad. Sci. USA, 91, 5793 -5797 (1994)) is a fusion gene (PcomG-lacZ) of the transcriptional regulatory region of the Bacillus subtilis comG gene and the lacZ gene encoding β-galactosidase derived from Escherichia coli K12 strain, which is retained on the chromosomal DNA. It is done.
[0020]
In addition, by creating a fusion gene in which the promoter of the operon of the late-competence gene group is replaced with a promoter that does not require the ComK protein for expression, and introducing the fusion gene into Bacillus natto, etc., natural transformation ability is also imparted. can do. Natural transformation ability can also be imparted by substituting the promoter of the late competence gene group operon on the chromosome such as Bacillus natto with a promoter that does not require the ComK protein for expression. A technique for replacing a promoter of a gene on a chromosome is disclosed in JP-A-1-215280.
[0021]
The late competence gene group includes one or more selected from the comC gene, the comE operon, the comF operon, and the comG operon.
[0022]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
<1> Isolation of Bacillus natto Bacillus natto was isolated from Natto ("Okame Natto" (registered trademark)) of Takano Foods Co., Ltd. The diluted natto solution was applied to an LB agar medium to separate a single colony, and two kinds of natto bacteria were isolated. One species forms a very rough colony, named Bacillus subtilis natto OK1, and the other species forms a colony similar to Bacillus subtilis Marburg 168, with Bacillus subtilis natto OK2. Named. These strains retained two plasmids of several tens of kb (70 kb or less) and about 6 kb, and required biotin (vitamin H) for their growth.
[0023]
<2> Construction of Bacillus subtilis OK2 strain with PcomG-lacZ fusion gene
DNA in which the transcriptional regulatory region of the comG gene and the lacZ gene encoding β-galactosidase derived from Escherichia coli K12 strain (PcomG-lacZ) and the chloramphenicol acetyltransferase gene are inserted into the amylase gene (amyE) The BD2123 strain [hisB2 leu-8 metB5 mecAΔ (spc) amyE :: comG-lacZ (cat) in which the fragment (amyE :: comG-lacZ (cat) is inserted into the amyE region on the chromosome of the Bacillus subtilis 168 strain ] (Liyun Kong and David Dubnau, Proc, Natl. Acad. Sci. USA, 91, 5793-5797 (1994)), the chromosomal DNA was obtained from the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963). )), And introduced into the Bacillus subtilis 168 strain by the usual transformation method (J. Spizizen, Proc, Natl. Acad. Sci. USA, 44, 1072 (1958)) using the obtained DNA. .
[0024]
A strain in which a DNA fragment containing the target fusion gene was inserted between amyE genes was obtained by using an LB agar medium containing 5 μg / ml of chloramphenicol (Luria-Bertani medium: Sambrook, J., Fritsch, EF, and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)), and colonies that appeared were selected.
[0025]
Furthermore, a strain in which the above PcomG-lacZ fusion gene was introduced into the OK2 strain was constructed. The chromosomal DNA of Bacillus subtilis 168 strain having the PcomG-lacZ fusion gene obtained above was extracted in the same manner as described above. On the other hand, the OK2 strain was added to a competence induction medium supplemented with 0.1 μg / ml biotin [CI liquid medium: Spizizen minimal medium (J. Spizizen, Proc, Natl. Acad. Sci. USA, 44, 1072 (1958), 0.05% Yeast Extract (Difco) and 0.1 μg / ml biotin added medium] was inoculated so that OD ₆₆₀ was about 0.05, and after 5 hours of shaking culture at 37 ° C., 0.5 ml The culture solution is collected, collected by centrifugation at 8000 rpm, 4 ° C. for 2 minutes, and 1 ml of CI medium [Spizizen minimal medium added with 0.025% Yeast Extract (Difco) and 0.1 μg / ml biotin] To 0.1 ml of this cell suspension was added 0.2 μg of chromosomal DNA of Bacillus subtilis 168 strain having the PcomG-lacZ fusion gene, and the mixture was further cultured with shaking at 37 ° C. for 1.5 hours. Add 0.3 ml of LB liquid medium and continue osmotic culture at 37 ° C for 1 hour The LB agar medium containing 5 μg / ml chloramphenicol was spread and cultured overnight at 37 ° C. As a result, two colonies were obtained.
[0026]
Using the above two strains, the expression of the later mentioned competence gene in the CI medium was examined using lacZ activity (β-galactosidase activity) as a reporter, and no expression was observed. This suggests that the complete PcomG-lacZ fusion gene has not been introduced into these transformed strains, or that the competence gene induction system described below has some defects in Bacillus natto. In order to confirm this, the Bacillus subtilis 168 strain was transformed with the DNA extracted from the above transformant by a conventional transformation method, and the obtained Cm ^r transformant was cultured in a CI medium to obtain lacZ activity. I investigated. As a result, the expression of PcomG-lacZ was observed in all of the Cm ^r stock (50 shares in 50 shares). From the above, it was confirmed that the PcomG-lacZ fusion gene introduced into the OK2 strain had the intended structure, suggesting that the competence gene induction system described below is defective.
[0027]
<3> Recovery of expression of competence genes by introduction of mecA mutation or mecB mutation (1) Introduction of mecA and mecB mutations into Bacillus subtilis 168 strain having amyE :: comG-lacZ (cat) The comG operon is not induced above This was thought to be because the ComK protein, which is a positive transcription factor of this operon, was not activated, and the mecA and mecB mutations of the Bacillus subtilis 168 strain were introduced into the OK2 strain. 168 strains of Bacillus subtilis mecA deficient BD2123 [hisB2 leu-8 metB5 mecAΔ (spc) amyE :: comG-lacZ (cat)] and mecB deficient strain BD2243 [hisB2 leu-8 metB5 mecBΔ (spc) amyE :: comG -lacZ (cat)] was extracted from the DNA and introduced into the Bacillus subtilis 168 (trpC2) strain having amyE :: comG-lacZ (cat) by a conventional transformation method. In BD2123 strain and BD2243, the spectinomycin resistance gene is inserted into the mecA gene and mecB gene, respectively. In these mutant strains, it has been reported that the ComK protein is not degraded and the late competence genes are constitutively expressed (Dubnau, D., Gene, 192, 191-198 (1997)).
[0028]
As described above, mecA mutant Bacillus subtilis [mecAΔ (spc) amyE :: comG-lacZ] with PcomG-lacZ and mecB mutant Bacillus subtilis [mecBΔ (spc) amyE: with PcomG-lacZ :: comG-lacZ]. These transformants were selected on LB agar medium containing 100 μg / ml spectinomycin and 5 μg / ml chloramphenicol, and X-gal [5-bromo-4- (chloro-3-indolyl) -Β-D-galactoside)] in blue on an LB agar medium containing 100 μg / ml, that is, the constitutive expression of the PcomG-lacZ gene confirmed that the target DNA was introduced. .
[0029]
(2) Construction of mecA and mecB deficient strains of OK2 strain mecA mutant [mecAΔ (spc) amyE :: comG-lacZ] and mecB mutant [mecBΔ (spc) amyE: : comG-lacZ] was prepared and introduced into the OK2 strain having PcomG-lacZ constructed in <2>. Specifically, transformation was performed in the same manner as described in <2>. The DNA derived from the mecA mutant [mecAΔ (spc) amyE :: comG-lacZ] is 23 μg / ml, and the DNA derived from the mecB mutant [mecBΔ (spc) amyE :: comG-lacZ] is 43 μg / ml. 3 μl of the solution was added to each 0.1 ml of cell suspension.
[0030]
Transformants are selected by applying to LB agar medium containing 100 μg / ml spectinomycin and 5 μg / ml chloramphenicol, and resistant to spectinomycin and chloramphenicol (Spc ^r , Cm ^r ) Acquired shares. In this way, transformable natto bacteria OK2 mecAΔ (spc) amyE :: comG-lacZ and OK2 mecBΔ (spc) amyE :: comG-lacZ were obtained. The frequency of appearance of the transformant was as shown in Table 1.
[0031]
[Table 1]

[0032]
(3) Measurement of β-galactosidase activity of transformed strain
168 [amyE :: comG-lacZ (cat)] and OK2 [amyE :: comG-lacZ (cat)] are LB agar media containing 5 μg / ml chloramphenicol, and OK2 [mecAΔ (spc) amyE :: comG-lacZ] and OK2 [mecBΔ (spc) amyE :: comG-lacZ] are LB agar media containing 5 μg / ml chloramphenicol and 100 μg / ml spectinomycin, each at 37 ° C. Cultivate overnight, 5 ml of CI medium containing 0.1 μg / ml biotin and 5 μg / ml chloramphenicol (mecA mutant and mecB mutant further contain 50 μg / ml spectinomycin) and OD ₆₆₀ Inoculated to about 0.1, and cultured at 37 ° C. with shaking. OD ₆₆₀ was measured every hour from the start of culture, simultaneously collected at 15000 rpm for 2 minutes, and stored at −30 ° C. until activity measurement. At 0, 1, 2 hours, 1 ml, 3, 4, 5, 6, 7 hours, 0.5 ml of the culture solution was collected.
[0033]
Cells collected as described above are suspended in 500 μl of Z buffer (60 mM Na ₂ HPO ₄ , 40 mM NaHPO ₄ .2H ₂ O, 10 mM KCl, 1 mM MgSO ₄ .7H ₂ O, 50 mM 2-mercaptoethanol) and toluene. 4 drops were added, vortexed for 20 seconds, pre-incubated for 2 minutes in a constant temperature bath at 28 ° C., and the temperature of the reaction solution was adjusted to 28 ° C. Next, 200 μl of Z buffer containing 4 mg / ml of ONPG (O-nitrophenyl-β-D-galactopyranoside), which yields a yellow product when decomposed, is added as a substrate, mixed, and reacted at 28 ° C. Was started. When it turned pale yellow, 500 μl of 1M sodium carbonate solution was added to stop the reaction and stored in ice. The time from the start to the end of the reaction is T (minutes). In addition, the sample which was not colored was made to react at 28 degreeC for a maximum of 20 minutes, Then, reaction was stopped. The reaction solution was centrifuged at 15000 rpm and 4 ° C. for 5 minutes, then the supernatant was transferred to a cuvette and A ₄₂₀ was measured with a spectrophotometer. β-galactosidase activity was calculated as follows (Wang, PZ and Doi, RH, J. Biol. Chem., 259, 8619-8625 (1984)). The results are shown in FIG. In the figure, the activity is plotted on the vertical axis and the sampling time is plotted on the horizontal axis.
[0034]
[Expression 1]
Activity (unit) = A ₄₂₀ x 1000 / T (min) x OD ₆₆₀ x V (ml)
[0035]
<4> Evaluation of transformability of transformable Bacillus natto In order to obtain the drug resistance marker gene of Bacillus natto itself, a rifampicin resistant mutant (rif ^r ) strain by natural mutation was first isolated from Bacillus natto, and OK2SR21 strain Named. This rifampicin resistance mutation was confirmed to be a mutation in the RNA polymerase β subunit gene by the co-transformation frequency with the streptomycin resistance mutation (strA) in the 30S ribosomal S12 protein gene (rpsL).
[0036]
The strA47 mutation in Bacillus subtilis strain 168 is a mutation in the rpsL gene, and only one codon mutation is known in the same strain. Further, rif1728 is a mutation in the RNA polymerase β subunit gene of 168 strains, and it is known that many rif ^r are the same mutation. It was estimated by mapping each other whether the natural mutation (rif ^r ) obtained in the OK2 strain was a known mutation in these genes. As a result, it was found that rif ^r (named rif21) obtained with OK2 was a mutation of rpoB, and strA was also a mutation of rpsL (strA).
[0037]
Using DNA prepared from OK2SR21 strain (rif21) rif ^r strains and 168 strain (rif1728), <3> transformable obtained in a natto OK2 mecAΔ (spc) amyE :: comG -lacZ strain, OK2 mecBΔ (spc) amyE :: comG-lacZ and 168 strains were transformed by a conventional transformation method. DNA from OK2SR21 strain DNA from rif ^r strain 23μg / ml, 168 strain a DNA solution 43μg / ml, were added 3μl respectively cell suspension 0.1 ml. The frequency of appearance of the transformant was as shown in Table 2.
[0038]
[Table 2]

[0039]
<5> Confirmation of the presence or absence of restriction / modification systems in the OK2 derivative strain Since many restriction / modification systems are known for Bacillus subtilis, the plasmid pUB110 (including the kanamycin resistance (Kmr) marker) can be used. The presence or absence of a restriction / modification system of mecAΔ (spc) amyE :: comG-lacZ, OK2 mecBΔ (spc) amyE :: comG-lacZ was examined.
[0040]
PUB110 grown using 168 strain, and DNA derived from OKSR21 strain, OK2 mecAΔ (spc) amyE :: comG-lacZ strain, OK2 mecBΔ (spc) amyE :: comG-lacZ168 strain, OK2 strain, and 168 strains were transformed by a conventional transformation method. The results are shown in Table 3. From this result, it can be seen that there are no restriction / modification systems different from 168 strains in Bacillus natto.
[0041]
[Table 3]

[0042]
<6> Recovering Competence Gene Expression by Enhancing Expression of comK Gene Pspac (Methods in Enzymology, vol. 185, 185- (1990), a fusion promoter of Bacillus subtilis phage SPO1 and the Escherichia coli lac promoter )) Plasmid pAG58 was digested with EcoRI and BamHI, and a fragment containing Pspac was excised. The plasmid pUB110 of the bacterium belonging to the genus Bacillus was cleaved with EcoRI and BamHI and ligated with the fragment to obtain a plasmid pULI7 (FIG. 2).
[0043]
On the other hand, a DNA fragment containing the comK gene by PCR using the chromosomal DNA of Bacillus subtilis strain 168 as a template, the base sequence shown in SEQ ID NOs: 1 and 2, and the oligonucleotide having the XbaI recognition sequence at the end as a primer Was amplified. This fragment was digested with XbaI and ligated to pUL17 digested with XbaI to obtain plasmid pULI7SK27 (FIG. 2). This plasmid has a comK gene that is expressed under the control of Pspac and can be induced by IPTG. Each plasmid was constructed using Bacillus subtilis 168 strain.
[0044]
OK2 [amyE :: comG-lacZ (cat)] was transformed with the plasmid pULI7SK27. The transformed strain was cultured in LB medium containing 7.5 μg / ml kanamycin, various concentrations of IPTG were added to induce the comK gene, and β-galactosidase activity was measured. The results are shown in FIG. In addition, the transformed strain cultured in the same manner was transformed with the chromosomal DNA of the OK2SR21 strain, and the transformation efficiency was evaluated using rif ^r as an index. The results are shown in Table 4. From this result, it can be seen that by enhancing the expression of the comK gene, the transformation ability of Bacillus natto is shown.
[0045]
[Table 4]

[0046]
【The invention's effect】
According to the present invention, it is possible to transform a Bacillus bacterium, such as Bacillus natto, in which a wild strain has substantially no natural transformation ability. According to the present invention, since it becomes possible to transform natto bacteria and the like with a frequency significantly higher than that of natural mutation, it can be suitably used for molecular breeding of natto bacteria and the like.
[Sequence Listing]

[0047]

[0048]

[0049]

[Brief description of the drawings]
[Fig. 1] 168 [amyE :: comG-lacZ (cat)], OK2 [amyE :: comG-lacZ (cat)], OK2 [mecAΔ (spc) amyE :: comG-lacZ] and OK2 [mecBΔ (spc) The figure which shows (beta) -galactosidase activity of amyE :: comG-lacZ]. Activity is plotted on the vertical axis, and sampled time is plotted on the horizontal axis.
FIG. 2 shows the construction process of plasmid pULI7SK27 having a comK gene whose expression is induced by IPTG.
FIG. 3 shows the induction of β-galactosidase activity by IPTG in a Bacillus natto transformant containing plasmid pULI7SK27 having a comK gene whose expression is induced by IPTG.

Claims (5)

Expression of comK gene is enhanced, COMC operon, COME operon, a late competence genes selected from comF operon or comG operon expressible, and, natto (Bacillus subtilis natto) that won natural transformation capability. One or both of the mecA gene and the mecB gene are disrupted, a late competence gene group selected from the comC operon , the comE operon, the comF operon and the comG operon can be expressed, and Bacillus natto ( Bacillus) has acquired natural transformation ability. subtilis natto ) . The Bacillus natto according to claim 1 or 2 , which retains an active ComK protein. By enhancing the expression of the comK gene, COMC operon, COME operon, characterized by imparting ability to express late competence genes selected from comF operon or comG operon Bacillus natto, a natural transformation ability to Bacillus natto How to grant. by disrupting one or both of the mecA gene and mecB gene, COMC operon, COME operon, characterized by imparting ability to express late competence genes selected from comF operon or comG operon Bacillus natto, Bacillus natto A method of imparting natural transformation ability.

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