JP2001008686A - Bacillus subtilis natto having ability of being naturally transformed - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new bacterium which is a mutant or recombinant of a bacterium belonging to the genus Bacillus whose wild type does not substantially have an ability of being naturally transformed, has late-stage competence genes which can express, and acquired an ability of being naturally transformed. SOLUTION: This is a new bacterium which is a mutant or recombinant of a bacterium belonging to the genus Bacillus whose wild type does not substantially have an ability of being naturally transformed, has late-stage competence genes which can express, and acquired an ability of being naturally transformed. This bacterium can be transformed at a significantly high frequency compared with the natural mutation, so that it can be preferably used for the molecular breeding. This bacterium is obtained by keeping active ComK protein by destroying mecA gene and/or mecB gene of Bacillus subtilis natto, followed by constructing B. subtilis natto which acquired an ability of being naturally transformed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a bacterium belonging to the genus Bacillus which has acquired natural transformation capability. More specifically, the present invention relates to a mutant or recombinant strain of a bacterium belonging to the genus Bacillus in which a wild-type strain has no natural transformation ability. The present invention relates to a Bacillus bacterium capable of natural transformation.

[0002]

2. Description of the Related Art Bacillus bacteria such as Bacillus subtilis are not only industrially used as bacterium producing extracellular enzymes but also Bacillus subtilis (Bacillus subtilis).
Some of them have long been used in food production, such as us subtilis natto). In recent years, attention has been paid to the strong secretory production ability of Bacillus bacteria, and they are used as hosts for genetic recombination along with Escherichia 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. However, the natural transformation method is based on Bacillus subtilis Marburg strains (168, 166, 160 strains). It is said that the frequency of natural transformation is extremely low in Bacillus natto and the like, and that it does not have transformation ability (competency). In addition, the protoplast method and the electroporation method can be applied to many strains, while the protoplast method is unstable and easily bursts, has low reproducibility, and further uses a complete medium for the regeneration of protoplasts. Therefore, there is a drawback that a nutritional requirement marker cannot be used. In addition, the electroporation method has a problem that the transformation efficiency is low.
cularBiological Method for Bacillus, Edited by Har
wood, CR et al., John Wiley & Sons, p.98-10
3).

[0004] In Bacillus subtilis Marburg strains, competence is known to be induced by early and late competence genes (Solomon, JM, et al., Genes & Developm).
ent, 10, 2014-2024 (1996), Lazazzera, BA, et a
l., Genes & Development, 19, 455-458 (1997), Dubna
u, D., Gene, 192, 191-198 (1997)). Expression of the late competence genes requires a comK product (ComK), which is an early competence gene. Although the comK gene is constitutively expressed, under normal growth conditions,
The K protein is also an early competence gene
ec (medium independent competence) A, mecB
Since it is degraded by each of the (clpC) and clpP gene products, the late competence genes are not expressed. On the other hand, when cultured under conditions in which competence is induced, com
Since the expression of the K gene is promoted, the active form of comK induces the expression of a group of late competence genes. as a result,
Competence is induced.

[0005] However, it is not known whether Bacillus bacteria having no natural transforming ability such as Bacillus natto have the same group of competence genes or can be expressed even if they do.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made from the above viewpoint, and enables the transformation of a Bacillus bacterium, such as Bacillus natto, whose wild strain has substantially no natural transformation ability. The task is to provide technology.

[0007]

Means for Solving the Problems The present inventor has conducted intensive studies in order to solve the above-mentioned problems.
It was found that the late competence gene was not induced because the mK protein was not activated, and as a result, it did not have a natural transformation ability. And mec of natto bacteria
Disruption of the A gene or the mecB gene resulted in the successful construction of Bacillus natto having acquired the natural transformation ability, thereby completing the present invention. That is, the present invention is as follows.

(1) A wild-type strain is a mutant or recombinant strain of a bacterium belonging to the genus Bacillus having substantially no natural transformation ability, capable of expressing a late-competence gene group, and having a natural transformation ability. Bacillus bacteria acquired. (2) The bacterium according to (1), which is Bacillus subtilis. (3) The bacterium according to (2), which is Bacillus subtilis natto. (4) The late competence gene group is a comC operon, a comE operon, a comF operon, or a com operon.
(1) The bacterium selected from the G operon. (5) The bacterium according to (1), which retains an active form of the ComK protein. (6) The bacterium according to (5), wherein the bacterium retains an active ComK protein due to enhanced expression of the comK gene. (7) The bacterium according to (5), wherein one or both of the mecA gene and the mecB gene are disrupted, thereby retaining the active ComK protein. (8) A method for conferring natural transformation ability on a Bacillus bacterium, which comprises imparting the ability to express a late competence gene to a Bacillus bacterium having substantially no natural transformation ability.

[0009] In the present invention, natural transformation refers to
When DNA is added to a culture solution of bacteria cultured under specific conditions and cultured, the DNA is taken up and retained by the bacterial cells. "Substantially does not have natural transformation ability" means that the frequency of expression of a phenotype such as drug resistance is the case where transformation is carried out using DNA containing a gene corresponding to the phenotype, Means that there is no significant difference between In addition, to confer natural transformation ability, to confer natural transformation ability to bacteria that do not have natural transformation ability,
In addition, it refers to enhancing the natural transformation ability of a bacterium having a very low natural transformation ability.

[0010]

BEST MODE FOR CARRYING OUT 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 in which a wild strain has substantially no natural transformation ability,
It is a bacterium belonging to the genus Bacillus that can express a group of late competence genes and has acquired a natural transformation ability.

[0011] Examples of Bacillus bacteria include Bacillus subtilis, Bacillus stearothermophilus, Bacillus methanolicus, and Bacillus amiloliquefaciens.
aciens), specifically Bacillus subtilis natt
o). Natural transformation of Bacillus sp. Occurs with Bacillus subtilis Marburg
It is said that these strains are only g) strains, and Bacillus bacteria other than those strains can be imparted with natural transformation ability by applying the present invention. Even in the case of Marburg strains, the natural transformation ability can be improved in the same manner.

Bacterial strains of the Bacillus genus which do not have natural transforming ability such as Bacillus natto (hereinafter referred to as "natto bacterium") are derived from the Marburg strain when they are cultured under specific culturing conditions. (Simply referred to as "natto bacteria") does not induce expression of the late competence gene group even when cultured under such conditions. Therefore, by allowing the expression of late competence genes,
Natural transforming ability can be imparted to Bacillus natto or the like.

In order to enable expression of the late competence gene group, the expression control system is modified so that the late competence gene group originally retained by Bacillus natto or the like, or the late competence gene group that can be expressed is converted to natto. What is necessary is just to introduce into bacteria. Specifically, for example, the active ComK protein may be retained in a cell in such an amount as to induce the expression of the late competence gene group. In order to retain the activated ComK protein in the cell, the expression level of the comK gene is increased or the mecA gene or mec
The B gene may be disrupted or mutated so that the MecA protein or MecB protein does not function normally. Disruption of the mecA gene or the mecB gene may be either one or both.

Disruption of the mecA gene or the mecB gene can be accomplished by adding a mecA gene or a mecB gene into which another DNA sequence has been inserted, or a DNA fragment containing a mecA gene or a mecB gene having an internally deleted gene, to a culture solution of Bacillus natto or the like. By transforming cells such as Bacillus natto by culturing in addition to the above, the homologous recombination with the mecA gene or the mecB gene on the chromosome can be performed.

Bacillus subtilis mecA gene (Genbank / EMBL / DDBJ accetion No. L06059) or me
cB gene (Genbank / EMBL / DDBJ accetion No. U0260
4) reports their base sequences, and uses a polymerase chain reaction method (PCR: PCR) using an oligonucleotide synthesized based on the base sequence as a primer.
polymerase chain reaction; White, TJ et al., Tre
nds Genet., 5,185 (1989)).

Transformation of Bacillus natto and the like can be carried out by a natural transformation method (competent cell method) usually used for Bacillus subtilis (J.
Spizizen, Proc, Natl. Acad. Sci. USA, 44, 1072 (1
958)), DNA can be added to a culture solution of cells cultured under specific conditions and cultured. Bacillus natto and the like have an increased transforming ability in the growth phase when the phase shifts from the logarithmic growth phase to the stationary phase. Specifically, for example, the transformation can be performed as follows. That is, natto or the like is inoculated into the competence induction medium described in the Examples so that the OD ₆₆₀ is around 0.05, and cultured with shaking at 37 ° C. for 5 hours, and then the cells are suspended in the competence induction medium. After turbidity, DNA was added to the cell suspension, followed by shaking culture at 37 ° C. for 1.5 hours, LB liquid medium was added, culture was continued for 1 hour at 37 ° C., and then applied to LB agar medium. Incubate at 37 ° C overnight.

The selection of the transformant depends on the D to be introduced.
A marker such as a drug resistance gene can be retained in NA, and the marker trait of the transformed cell can be used as an index. The mecA gene or mecB gene containing a marker gene therein can be suitably used for disruption of these genes. As a Bacillus subtilis carrying a mecA gene or a mecB gene containing such a marker gene therein, a mecA-deficient strain BD2123 [hisB2 le
u-8 metB5 mecAΔ (spc) amyE :: comG-lacZ (cat)], and
mecB deficient strain BD2243 [hisB2 leu-8 metB5 mecBΔ (spc) am
yE :: comG-lacZ (cat)] (Dubnau, D., Gene, 192, 191-1
98 (1997)). The chromosomal DNA fragments of these strains can be directly used for disruption of the mecA gene or the mecB gene.

[0018] The fact that the transformation has occurred is as follows. DNA is prepared from the transformed strain, and B. subtilis is used for the DNA.
It can be confirmed by transforming a Marburg strain and obtaining a transformant having the marker gene.

Further, mecA obtained as described above is used.
The fact that the deficient strain or the mecB deficient strain retains the active form of the ComK protein in the cell and the late competence gene group can be expressed means that the reporter gene such as the β-galactosidase gene is linked to the promoter of the operon of the late competence gene group. The fusion gene obtained can be retained in a mecA-deficient strain or a mecB-deficient strain, and the β-galactosidase activity of these strains can be confirmed by expression. Examples of the fusion gene include BD2123 strain [hisB2 leu-8 metB5 mecAΔ (spc) amyE :: comG-lacZ (ca
t)] (Liyun Kong and David Dubnau, Proc, Natl. Aca
d. Sci. USA, 91, 5793-5797 (1994))
And a fusion gene (PcomG-lacZ) of the transcription regulatory region of the Bacillus subtilis comG gene and the lacZ gene encoding β-galactosidase derived from Escherichia coli K12 strain, which is retained above.

Further, a fusion gene is prepared by replacing the promoter of the operon of the late competence gene group with a promoter that does not require a ComK protein for expression.
Natural transformation ability can also be imparted by introducing the fusion gene into Bacillus natto or the like. Natural transformation ability can also be imparted by replacing the promoter of the operon of the late competence genes on the chromosome such as Bacillus natto with a promoter that does not require a ComK protein for expression. A technique for replacing a promoter of a gene on a chromosome is disclosed in JP-A-1-215280.

The late competence genes include co
One or more selected from the mC gene, the comE operon, the comF operon, and the comG operon.

[0022]

EXAMPLES 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 solution of natto was spread on an LB agar medium to separate a single colony.
The species Bacillus natto was isolated. One species forms a very rough colony, and Bacillus subtilis natto (Bacillus s.
ubtilis natto) Named OK1 and the other is Bacillus
Form a colony similar to Subtilis Marburg 168,
Bacillus subtilis Natto named OK2. These strains maintained two plasmids of several tens kb (70 kb or less) and about 6 kb, and required biotin (vitamin H) for their growth.

<2> Construction of Bacillus subtilis OK2 strain having PcomG-lacZ fusion gene Transcriptional regulatory region of comG gene and Escherichia coli K12
A fusion gene (PcomG-lacZ) with a lacZ gene encoding β-galactosidase derived from a strain and a DNA fragment (amyE :: comG-lacZ) having a chloramphenicol acetyltransferase gene inserted into an amylase gene (amyE)
(cat) on the chromosome of Bacillus subtilis 168 strain
BD2123 strain inserted into the amyE region [hisB2 leu-8 metB5 me
cAΔ (spc) amyE :: comG-lacZ (cat)] (Liyun Kong and D
avid Dubnau, Proc, Natl. Acad. Sci. USA, 91, 5793-5
797 (1994)), extracted by the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)), and using the obtained DNA, a conventional transformation method (J. Spi
zizen, Proc, Natl. Acad. Sci. USA, 44, 1072 (195
8)), and introduced into 168 strains of Bacillus subtilis.

The DNA fragment containing the desired fusion gene is
The strain inserted between the amyE genes was 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 (19
89)) and obtained by selecting colonies that appeared.

Further, a strain in which the above PcomG-lacZ fusion gene was introduced into the OK2 strain was constructed. PcomG- obtained above
The chromosomal DNA of Bacillus subtilis strain 168 having the lacZ fusion gene was extracted in the same manner as described above. On the other hand, OK2
The strain was added to a competence induction medium to which 0.1 μg / ml biotin was added [CI liquid medium: Spizizen minimal medium (J. Spizi
zen, Proc, Natl. Acad. Sci. USA, 44, 1072 (1958)
After 0.05% Yeast Extract (Difco) and 0.1 [mu] g / ml of OD ₆₆₀ in culture medium] plus biotin was inoculated so that the front and rear 0.05, was carried out for 5 hours by shaking culture at 37 ° C., the
An aliquot of 0.5 ml of the culture solution was collected and subjected to 8000 rpm at 4 ° C for 2 hours.
After centrifugation to collect cells, 1 ml of CI medium [0.025% yeast extract (Difco) and 0.1 μg
/ Media supplemented with biotin / ml). To 0.1 ml of this cell suspension, 0.2 μm of chromosomal DNA of Bacillus subtilis strain 168 having the PcomG-lacZ fusion gene was added.
g, followed by shaking culture at 37 ° C. for 1.5 hours.
0.3 ml of LB liquid medium was added, culture was continued for 1 hour at 37 ° C., applied to LB agar medium containing 5 μg / ml chloramphenicol, and cultured at 37 ° C. overnight. As a result, two colonies were obtained.

Using the above two strains, the expression of the competence gene described below in a CI medium was examined using lacZ activity (β-galactosidase activity) as a reporter. As a result, no expression was observed. This suggests that the complete PcomG-lacZ fusion gene was not introduced into these transformants, or that there was some defect in the competence gene induction system described later in Bacillus natto. To confirm this, the Bacillus subtilis 168 strain by the DNA extracted from the transformant transformed by conventional transformation method, the resulting transformant of Cm ^r cultured in CI medium, l
The acZ activity was examined. 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 desired structure, suggesting that the competence gene induction system described below was defective.

<3> Recovery of competence gene expression 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) It is thought that ComK protein, which is a positive transcription factor of this operon, was not activated. Therefore, the mecA mutation and the mecB mutation of Bacillus subtilis 168 strain were introduced into the OK2 strain. Bacillus subtilis strain 168 mecA-deficient strain BD2123 [hisB2 leu
-8 metB5 mecAΔ (spc) amyE :: comG-lacZ (cat)] and m
ecB deficient strain BD2243 (hisB2 leu-8 metB5 mecBΔ (spc) amy
DNA was extracted from E.:comG-lacZ(cat)] and introduced into a Bacillus subtilis 168 (trpC2) strain having amyE :: comG-lacZ (cat) by a usual transformation method. BD2123 strain and BD22
No. 43 has a spectinomycin resistance gene inserted inside the mecA gene and the mecB gene, respectively. In these mutants, 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 (199
7)).

As described above, mecA having PcomG-lacZ
Mutant Bacillus subtilis [mecAΔ (spc) amyE :: comG-
lacZ] and a mecB mutant Bacillus subtilis having PcomG-lacZ [mecBΔ (spc) amyE :: comG-lacZ] were constructed.
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)] on an LB agar medium containing 100 μg / ml, that is, since the PcomG-lacZ gene was constitutively expressed, it was confirmed that the target DNA had been introduced. .

(2) Construction of mecA and mecB deficient strains of OK2 strain mecA mutant [mecAΔ (spc) amyE :: comG-lacZ] and mecB mutant of 168 Bacillus subtilis strain obtained above
DNA was prepared from cBΔ (spc) amyE :: comG-lacZ], and <2
And introduced into the OK2 strain having PcomG-lacZ constructed in>. Specifically, transformation was performed in the same manner as described in <2>.
The DNA derived from the mecA mutant [mecAΔ (spc) amyE :: comG-lacZ] was 23 μg / ml, and the mecB mutant [mecBΔ (sp
c) For DNA derived from amyE :: comG-lacZ], 3 μl of a 43 μg / ml DNA solution was added to 0.1 ml of the cell suspension.

Transformants were selected by plating on LB agar medium containing 100 μg / ml spectinomycin and 5 μg / ml chloramphenicol, and resistant to spectinomycin and chloramphenicol (Spc
^r , Cm ^r ) strain was obtained. Thus, the transformable Bacillus natto OK2 mecAΔ (spc) amyE :: comG-lacZ and OK2 mecBΔ (s
pc) amyE :: comG-lacZ was obtained. The appearance frequency of the transformant is
As shown in Table 1.

[0031]

[Table 1] Table 1 ──────────────────────────────────── donor DNA recipient bacterium Spc ^r, Cm ^r strain ──────────────────────────────────── 168 mecAΔ (spc) OK2 2.2 × 10 ² / ml amyE :: comG-lacZ amyE :: comG-lacZ ──────────────────────────────────── 168 mecBΔ (spc) OK2 4.6 × 10 ² / ml amyE :: comG-lacZ amyE :: comG-lacZ ──────────────────────────── ────────

(3) Measurement of β-galactosidase activity of the transformant 168 [amyE :: comG-lacZ (cat)] and OK2 [amyE :: comG-lac
Z (cat)] is an LB agar medium containing 5 μg / ml chloramphenicol, and OK2 [mecAΔ (spc) amyE :: comG
-lacZ] and OK2 [mecBΔ (spc) amyE :: comG-lacZ]
Chloramphenicol at 100 μg / ml and 100 μg / m
Each of the cells was cultured overnight at 37 ° C. on an LB agar medium containing 1 μl of spectinomycin.
CI medium containing μg / ml chloramphenicol (me
cA mutants and mecB mutant further comprises spectinomycin 50μg / ml) 5ml, OD ₆₆₀ is about 0.1
And cultured at 37 ° C. with shaking. OD ₆₆₀ is measured every hour from the start of culture, and 15,000 rp
The cells were collected by centrifugation for 2 minutes and stored at -30 ° C until activity measurement. At 0, 1, 2 hours, 1 ml, 3, 4, 5, 6,
At the 7th hour, 0.5 ml of the culture solution was collected.

[0033] Cells were harvested as described above Z buffer _{(60mM Na 2 HPO 4, 40mM} NaHPO 4 · 2H 2 O, 10mM KCl, 1m
_{M MgSO 4 · 7H 2 O,} 50mM 2- mercaptoethanol) 500
The mixture was suspended in μl, 4 drops of toluene were added, vortexed for 20 seconds, and pre-incubated for 2 minutes in a thermostat at 28 ° C., and the temperature of the reaction solution was set to 28 ° C. Next, as a substrate,
4 mg ONPG (O-nitrophenyl-β-D-galactopyranoside), which gives a yellow product when decomposed
Add 200 μl of Z buffer containing 50 ml, and mix.
The reaction was started at ° C. When it turned pale yellow, 500 μl of 1 M sodium carbonate solution was added to stop the reaction, and stored on ice. The time from reaction start to stop is T
(Minutes). Samples that were not colored were up to 2
After reacting at 28 ° C. for 0 minutes, the reaction was stopped. The reaction solution was centrifuged for 5 minutes at 15000 rpm, 4 ° C., the supernatant was transferred to a cuvette and measured A ₄₂₀ in a spectrophotometer.
β-galactosidase activity was calculated as follows (Wang, PZ and Doi, RH, J. Biol. Chem., 259, 86).
19-8625 (1984)). The results are shown in FIG. In the figure, the activity is shown on the vertical axis, and the sampling time is shown on the horizontal axis.

[0034]

## EQU1 ## Activity (unit) = A ₄₂₀ × 1000 / T (min) × OD ₆₆₀
× V (ml)

<4> Evaluation of Transformation Ability of Bacillus natto Transformable In order to obtain a drug resistance marker gene of Bacillus natto, a rifampicin resistant mutant (rif ^r ) strain by natural mutation was first isolated from Bacillus natto. The strain was named OK2SR21 strain. The fact that the rifampicin resistance mutation is a mutation of the RNA polymerase β subunit gene means that the rifampicin resistance mutation is a mutation of the streptomycin resistance mutation (strA) in the 30S ribosomal S12 protein gene (rpsL) (co-transformaton).
ion) frequency.

The strA47 mutation in Bacillus subtilis strain 168 is a mutation in the rpsL gene, and only one codon mutation is known in the strain. Also, rif1728 is 168
Strains are mutant RNA polymerase β subunit in a gene, it is known that many rif ^r is the same mutation. Whether the spontaneous mutation (rif ^r ) obtained in the OK2 strain was a known mutation in these genes was estimated by mapping each other. As a result, the rif ^r (named rif21) obtained in OK2 was a mutation of rpoB and strA
Was also found to be a mutation in rpsL (strA).

[0037] of OK2SR21 shares (rif21) and 168 shares rif ^r strain (r
using the DNA prepared from if1728), the transformable Bacillus natto OK2 mecAΔ (spc) amyE :: comG-lacZ obtained in <3>.
Strain, OK2 mecBΔ (spc) amyE :: comG-lacZ, and 168 strains,
Transformation was performed by the usual transformation method. DNA from OK2SR21 share 23μg / ml, 168 shares DN derived from the rif ^r strains of
In A, 3 μl of a 43 μg / ml DNA solution was added to 0.1 ml of the cell suspension. The frequency of appearance of the transformed strain was as shown in Table 2.

[0038]

[Table 2] Table 2 ──────────────────────────────────── donor DNA recipient bacterium Spc ^r, Cm ^r strain ──────────────────────────────────── OKSR21 rif21 OK2 mecAΔ (spc) 2.46 × 10 ⁴ / ml amyE :: comG-lacZ ──────────────────────────────────── OKSR21 rif21 OK2 mecBΔ (spc) 2.00 × 10 ⁴ / ml amyE :: comG-lacZ ──────────────────────────────────── OKSR21 rif21 168 2.91 × 10 ⁴ / ml ──────────────────────────────────── 168 rif1728 168 1.98 × 10 ⁴ / ml ────────────────────────────────────

<5> Confirmation of Presence or Absence of Restriction / Modification System of OK2 Derivative Strain Since many restriction / modification systems are known in Bacillus subtilis, plasmid pUB110 (kanamycin resistance (Km
r) Including OK2 mecAΔ (spc) amyE ::
comG-lacZ, OK2 mecBΔ (spc) amyE :: comG-lacZ restrictions
The presence or absence of the modification system was examined.

PUB110 grown using strain 168, and OKS
Using DNA from the R21 strain, OK2 mecAΔ (spc) amyE :: co
mG-lacZ strain, OK2 mecBΔ (spc) amyE :: comG-lacZ168 strain, OK
Two strains and 168 strains were transformed by a usual transformation method. Table 3 shows the results. From these results, it was found that Bacillus natto
It can be seen that there is no restriction / modification system different from the eight strains.

[0041]

Table 3 ──────────────────────────────────── Donor DNA recipient Rif ^r strain Cm ^r strain ──────────────────────────────────── OKSR21 rif21 168 1.28 × 10 ⁵ / ml pUB110 (5 μg / ml) 168 1.05 × 10 ⁴ / ml pUB110 (10 μg / ml) 168 3.32 × 10 ⁴ / ml ─────────────────────────── ───────── OKSR21 rif21 OK2 3.5 × 10 ² / ml pUB110 (5 μg / ml) OK2 1.0 × 10 ² / ml pUB110 (10 μg / ml) OK2 9.5 × 10 / ml ─────── ───────────────────────────── OKSR21 rif21 OK2 mecAΔ 3.90 × 10 ⁴ / ml pUB110 (5μg / ml) OK2 mecAΔ − pUB110 ( (10μg / ml) OK2 mecAΔ 1.95 × 10 ⁴ / ml ──────────────────────────────────── OKSR21 rif21 OK2 mecBΔ 2.13 × 10 ⁴ / ml pUB110 (5μg / ml) OK2 mecBΔ 6.8 × 1 0 ² / ml pUB110 (10 μg / ml) OK2 mecBΔ 1.1 × 10 ³ / ml ──────────────────────────────── ────

<6> Recovery of Competence Gene Expression by Enhancing Expression of comK Gene Pspac (Methods in Enzymology, vol. -
(1990)) was cut with EcoRI and BamHI, and a fragment containing Pspac was cut out. Bacillus bacterium plasmid pUB110 was digested with EcoRI and BamHI, and ligated with the fragment to obtain plasmid pULI7 (FIG. 2).

On the other hand, the comK gene was subjected to PCR using the chromosomal DNA of Bacillus subtilis strain 168 as a template, an oligonucleotide having the nucleotide sequence shown in SEQ ID NOS: 1 and 2 and an end having an XbaI recognition sequence as a primer. The containing DNA fragment was amplified. This fragment is digested with XbaI and XbaI
The plasmid was ligated to pUL17 digested with, to obtain plasmid pULI7SK27 (FIG. 2). This plasmid expresses c under the control of Pspac.
It has the omK gene, which can be induced by IPTG. The construction of each of the above plasmids was carried out by Bacillus
Subtilis 168 strain was used.

OK2 [amyE :: comG-lacZ (cat)] was transformed with the above plasmid pULI7SK27. The transformed strain was cultured in an LB medium containing 7.5 μg / ml of kanamycin, and various concentrations of I
The comK gene was induced by adding PTG, and β-galactosidase activity was measured. The results are shown in FIG. In addition, the transformant cultured in the same manner was transformed into chromosome D of OK2SR21 strain.
Was transformed with NA, it was evaluated transformation efficiency as an indicator rif ^r. Table 4 shows the results. From these results, it can be seen that by enhancing the expression of the comK gene, Bacillus natto can be transformed.

[0045]

[Table 4] ────────────────────────────── recipient bacteria IPTG concentration (mM) rif ^r stock ────── ──────────────────────── 0 2.0 × 10 / ml OK2 [amyE :: comG-lacZ (cat)] 0.1 8.5 × 10 / ml 1 2.35 × 10 ³ / ml ──────────────────────────────

[0046]

Industrial Applicability According to the present invention, it is possible to transform a Bacillus bacterium, such as Bacillus natto, whose wild strain has substantially no natural transformation ability. According to the present invention, transformation of Bacillus natto and the like can be performed at a significantly higher frequency than spontaneous mutation, so that it can be suitably used for molecular breeding of Bacillus natto and the like.

[Sequence List] SEQUENCE LISTING

<110> Ajinomoto Co., Inc. <120> Bacillus natto having natural transformation ability <130> P-6557 <141> 1999-06-25 <160> 2 <170> PatentIn Ver. 2.0

<210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Bacillus subtilis comK gene <400> 1 catctctaga gatggaggcc ataatatgag tcag 34

<210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Bacillus subtilis comK gene <400> 2 ttagtctaga ctaataccgt tccccgagct cacg 34

[Brief description of the drawings]

[Figure 1] 168 [amyE :: comG-lacZ (cat)], OK2 [amyE ::
comG-lacZ (cat)], OK2 [mecAΔ (spc) amyE :: comG-lac
The figure which shows (beta) -galactosidase activity of Z] and OK2 [mecB (spc) amyE :: comG-lacZ]. The activity is shown on the vertical axis and the sampling time is shown on the horizontal axis.

FIG. 2 is a view showing a process of constructing a plasmid pULI7SK27 having a comK gene whose expression is induced by IPTG.

FIG. 3 is a diagram showing the induction of β-galactosidase activity of a Bacillus natto transformant containing plasmid pULI7SK27 having a comK gene whose expression is induced by IPTG by IPTG.

Claims (8)

[Claims] 1. A wild-type strain which is a mutant or recombinant strain of a bacterium belonging to the genus Bacillus which has substantially no natural transformation ability, is capable of expressing a late-competence gene group, and has a natural transformation ability. Bacillus bacteria. 2. Bacillus subtiris (Bacillus subtiris)
The bacterium according to claim 1, which is lis). 3. The bacterium according to claim 2, which is Bacillus subtilis natto. 4. The late competence gene group comprises a co
The bacterium according to claim 1, which is selected from mC operon, comE operon, comF operon, and comG operon. 5. The bacterium according to claim 1, wherein the bacterium retains an active ComK protein. 6. The bacterium according to claim 5, wherein the bacterium retains an active ComK protein due to enhanced expression of the comK gene. 7. The bacterium according to claim 5, wherein one or both of the mecA gene and the mecB gene is disrupted to retain the active ComK protein. 8. A method for imparting a natural transforming ability to a Bacillus bacterium, which comprises imparting a late competence gene expression ability to a Bacillus bacterium having substantially no natural transformation ability.