JPS5945358B2 - Novel plasmid with neomycin and kanamycin resistance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel plasmid useful as a vector for recombinant DNA experiments using thermophilic bacteria as a host.
More specifically, the present invention relates to a novel plasmid that contains neomycin and kanamycin resistance genes, has a molecular weight of approximately 3.0 megadaltons, and is characterized by the restriction enzyme cleavage map shown in the figure.

Conventionally, recombinant DNA experiments have been widely conducted mainly in systems using E. coli as a host, and great results have been achieved, such as the mass production of insulin, interferon, human growth hormone, etc. using E. coli.

The host-vector system for Escherichia coli has almost been completed, and host-vector systems have been developed for yeast, Bacillus subtilis, etc. in addition to Escherichia coli, and avenues for application are being considered.

However, all of the above bacteria have a growth temperature of 30°C to 37°C.
The problem is that it is a mesophilic bacterium.

On the other hand, thermophilic bacteria are broadly divided into moderate thermophiles, whose upper limit temperature for growth is between 55°C and 75°C, and highly thermophilic bacteria, whose growth temperature i>1 is 75°C or higher. Regarding,
It is known that the enzymes and biological components that it contains are excellent in heat resistance and solvent resistance, especially in terms of the application of thermostable enzymes derived from thermophilic bacteria and thermostable biological functions to industrial processes such as bioreactors. It is attracting attention from

Therefore, the breeding of thermophilic bacteria is considered to be important, and research on the development of host-vector systems for thermophilic bacteria, which is considered to be one of the most effective means for this purpose, is necessary. Even after searching, only the following reports are known.

(1) Isolation of extrachromosomal DNA from highly thermophilic bacteria Hishinuma, F., Tanaka, T., and Sakaguchi, K.

J. Gen. Microb, 104. 193-
199 (1978) (2) Isolation of four types of plasmids from drug-resistant thermophilic Bacillus bacteria and partial analysis of their properties Pingham 1. A, H, A, Bourdon, C, J-
and Atkinnon, T.

J, Gen, Mjcrob-, 114,401-
408 (1979) (3) Analysis of plasmid PAB124 of Bacillus stearothermophilus and creation of deletion derivatives Pingham, A. Hl., Bourdon, C. J.
.

and Atkinson, T.

J, Gen, Microb, , 119.109-
115 (1980) (4) Isolation and analysis of drug-resistant plasmids from thermophilic Bacillus bacteria and creation of partially deleted plasmids Imanaka, T., Fujii 0M, and I μ, S.

J, Bact, 146(3), 1091-109.
7 (1981) (5) Physical properties of a plasmid (PTT 1 ) isolated from Thermus thermophilus Eberhard, M.D., Vasquez, C. Valenzuela, P., Vicuna, R., Andyudelevik. ,A.

Plasmjd, 6.1-6 (1981) (6) Transformation of Bacillus stearothermophilus with plasmid DNA and properties of shared vectors between Bacillus stearothermophilus and Bacillus zugutilus Imanaka, T., Fujii 2M, . Aramori.

■, and i μ, S.

J.Bact., 149(3), 824
-830 (1982) Therefore, the present inventors searched the natural world for microorganisms whose hosts are thermophilic microorganisms and which have genes for neomycin and kanamycin resistance, and as a result, found a strain belonging to the genus Bacillus. We succeeded in obtaining a new plasmid from.

This plasmid is shown in the above-mentioned restriction enzyme cleavage map, has a small molecular weight, has specific cleavage points by various restriction enzymes, and has a resistance gene for neomycin and kanamycin on the plasmid DNA.

(Hereinafter, this plasmid will be abbreviated as IPTHNIJ.

) The abbreviations of the restriction enzymes shown in the figure are as follows.

(υ AccI is an enzyme derived from Acinetobacter calcoaceticus (2) Bglm is an enzyme derived from Bacillus globigii (3) EcoRI is an enzyme derived from Escherichia coli (4) HhaI is an enzyme derived from Haemophilus haemoridicus (5) Hpall is an enzyme derived from Haemophilus parainfluenzae (6), PstI is an enzyme derived from Providencia stoarteii (7), and TaqI is an enzyme derived from Thermus aquaticus.

Differences from known thermophile-derived plasmids having resistance to neomycin and kanamycin are shown in the table below.

As is clear from the table, PTHNl is clearly different from known plasmids in molecular weight and restriction enzyme cleavage pattern, and is recognized as a novel plasmid.

In order for plasmid DNA to be used as a vector, the plasmid must have the ability to autonomously reproduce within the host and a selection marker (
It is essential that the plasmid has a marker indicating that it is present in the host, but PTHNl has the ability to autonomously grow in thermophilic bacteria and Bacillus subtilis, and neomycin,
It has kanamycin resistance, a marker that is extremely advantageous for selection.

Furthermore, as is clear from the figure, PTHNl is Accl,
Bgll, EcoRI, HhaI, Ps
It has a cleavage site by a restriction enzyme such as tI at a specific and limited position.

This means that when using PTHNI as a vector,
This is advantageous in that the introduction site for the heterologous gene to be inserted can be significantly retained.

Furthermore, PTHNl is advantageous in that it can be used as a vector for both Bacillus subtilis and thermophilic bacteria.

Therefore, by using PTHNI as a vector, it is also possible to simultaneously clone a heterologous gene into Bacillus subtilis and a thermophilic bacterium.

In addition, since Bacillus stearothermophilus, a thermophilic bacterium from which Bacillus subtilis was isolated, has a common feature that they belong to the same genus Bacillus, due to their close kinship, the heterologous genes already expressed in Bacillus subtilis are , it is thought that there is a high possibility that it is also expressed in thermophilic bacteria.

Therefore, the genes that had already been cloned into Bacillus subtilis were
By using this plasmid to transfer into thermophilic bacteria, if the gene product has heat resistance around 55°C, reduction of cooling costs in the fermentation industry can be achieved by fermentative production by thermophilic bacteria. It happens.

In addition, by cloning the enzyme gene of thermophilic bacteria, which has excellent properties such as heat resistance and solvent resistance, into a thermophilic bacterial host using this plasmid as a vector and mass producing it, it will be possible to apply it to bioreactors, etc. This is possible and is expected to be applied to industrial processes.

PTHNl was obtained by growing one strain of Bacillus stearothermophilus, a moderately thermophilic bacterium newly isolated by the present inventors from soil, in TYS medium until late logarithmic growth. , achieved by lysing the bacteria by SDS treatment.

Bacillus stearothermo'hui/L's NI strain is an aerobic sporobacillus, positive for Durham staining, and has an optimal growth temperature of approximately 55°C and does not grow at 37°C, but is resistant to PTHNI and tetracycline. It is a novel microorganism that has not been previously recognized in that it possesses the plasmid PTHT15.

This strain was initially isolated from soil as a neomycin-resistant strain, but it was resistant not only to neomycin, but also to kanamycin, ampicillin, and tetracycline, and to erythromycin, chloramphenicol, and streptomycin. It was sensitivity.

In addition, this strain has been deposited as Microtechnical Research Institute No. 6660.

The present invention will be described in more detail below from Example 1.

Example 1 Screening of bacterial strains Approximately 11 soil samples from Yatabe Town, Tsukuba District, Ibaraki Prefecture were grown in TYS medium (Difco Tryptone 2%, Difco Yeast
Extract 1%, NaC 11%) 100#! After shaking culture at 55°C for about 8 hours, neomycin (20μ?
Bacillus stearothermophilus strain N1 (Feikoken Bacterial Serial No. 6660) was obtained from one of the colonies grown on a TYS agar plate containing 100% Bacillus stearothermophilus.

Example 2 Isolation of plasmid PTHN1 from Bacillus stearothermophilus strain N1.
21 neomycin 20μf? /ml was inoculated into TYS medium (2 ml of Difco band tryptone, 1% Difco yeast extract, 11% NaC) and cultured with shaking at 55°C for 16 to 18 hours.

Add 1 t of this culture solution to 20μ' of tetracycline. /71
11 and cultured at 55° C. for 5 hours.

The bacterial cells were collected by centrifugation and added to TBS (20mM Trjs-
HCl, 5mM EDTA.

After washing with 100mM NaCl PH7.5), per 41 bacterial cell wet weight, 10772# (7) 25% sucrose-containing TE
Suspend in S.

2 ml of lysozyme (10 μg/me), 0.025 M
-Add 4 ml of BDTA (PH8,0), leave at 0°C for 10 minutes, and then incubate at 37°C for 10 minutes.

Add 2 ml of 10% SD to this cell mixture, 815 ml of 5M
-Add NaCl and leave at 4°C for 15-18 hours.

This is centrifuged by ultracentrifugation at 2800 rpm for 1 hour to obtain a supernatant.

To this supernatant, add 10 parts of polyethylene glycol 6000 (
w/v) and left at 2200 rpm for 2 to 3 hours.
, obtain a precipitate by centrifugation for 2 minutes.

This precipitate was dissolved in 151111 TBS, and C3Ct and ethidium bromide were added to adjust the density to 1.61-1.
Adjust to 62.

This sample is subjected to density gradient centrifugation at 3800 rpm for 30-40 hours.

The resulting plasmid DNA bands were collected, ethidium bromide was removed with isoamyl alcohol, and then TEN
(20mM Tris-HCL, 1mM EDTA, 20mM Tris-HCL, 1mM EDTA,
mMNaC! ) by dialysis against plasmid DNA.
Solution A is obtained.

This plasmid DNA solution contains PTHNI and PTHT15.
This solution is subjected to electrophoresis using 0.7% low-melting-point agarose (manufactured by BRI), and the portion corresponding to PTHNl is cut out from the two bands generated and eluted from the gel to obtain pure PTHNl. is obtained.

DN4 was recovered from the low melting point agarose gel according to the following procedure.

The cut out gel slices were kept warm at 65°C to thaw, and then added with 50 mMT containing twice the amount of 0.5 m MEDT A.
Add p i s HC7 buffer (PH8,0),
Transfer to 37℃ and keep warm.

Phenol saturated with an equal amount of 0.1 M Tris-HCIH solution (PH80) is added to this, mixed, and after centrifugation (3000 to 5000 rpm, 5 minutes), the upper aqueous layer is separated.

After performing phenol extraction once more and removing phenol from the aqueous layer with ether, 1/10 volume of 3M acetic acid amuniram solution is added, and ethanol precipitation is performed with 3 volumes of ethanol.

The obtained precipitate was dissolved in TEN to prepare a plasmid solution.

Procedure for characterizing PTHNI The molecular weight of PTHNI is determined by its superhelical structure.
cciled 5structure) and a fragment cut with a restriction enzyme by agarose gel electrophoresis.

The molecular weight marker at this time was PBR322DNA (2,6
7md), Co1EI Otori (4, 2md) A and Lambda DN
Hin d N degradation fragments of A (14, 6, 5, 84,
4,05, 2,67, 1,04.

1.21, 0.34md), Lambda DNA (7) Ec
oRI degradation fragment (13,7,4,74,3,73,3,
48,3,02°2.13md) was used.

Cleavage with restriction enzymes was carried out by precipitating DNA from a plasmid DNA solution by ethanol precipitation and dissolving it in an appropriate buffer.

A commercially available restriction enzyme from Zenshuzo was used. Agarose gel electrophoresis was carried out using Secum's agarose at a concentration of 0.5% or 0.7% in a horizontal gel electrophoresis chamber at a constant voltage of 1.5 V per 1 hour of gel length for 15 to 17 hours.

It was confirmed by a transformation experiment into Bacillus subtilis RM125 strain protoplasts that PTHNI has neomycin and kanamycin resistance genes on its DNA.

The procedures for the synthetic transformation of protoplasts of Bacillus subtilis strain RM125 and the regeneration of protoplasts are as follows:
G &Cohen's method (Molec, Gen.

Genet, 168, 111-115 (1979).

The outline of this method is to convert logarithmically grown bacterial cells of the RM125 strain into protoplasts by treating with lysozyme in a given solution, add a plasmid DNA solution to this protoplast suspension, and
After promoting the uptake of DNA into protoplasts using polyethylene glycol 6000 FC, the protoplasts are regenerated into vegetative cells using a regeneration medium.

Approximately 1 μg of PTHNI DNA was used to transform 0.5 mg of protoplast suspension of RM125 strain (2x), Os protoplast/7721), and the appearance of neomycin- and kanamycin-resistant strains was examined on regeneration medium. , the regeneration rate of protoplasts at this time is 10
IX 103/m for % (2X108/772β)
Neomycin- and kanamycin-resistant strains occurred at a frequency of e.

On the other hand, in the system to which no plasmid DNA solution was added (control), neomycin- and kanamycin-resistant strains were treated with 2×
Even after 10+ protoplasts were produced, no protoplasts were produced.

Plasmid DNA was extracted from the neomycin- and kanamycin-resistant RM125 strain obtained here, using the same method as in the extraction of plasmid DNA from Bacillus stearothermophilus N1 strain (the temperature and time of lysozyme treatment were 37°C and 30°C).
The difference is that it is 0 minutes.

), plasmid DNA was recovered that had the same molecular weight as PTHNl and had exactly the same restriction enzyme cleavage pattern.

This fact indicates that PTHNI has neomycin and kanamycin resistance genes, and this plasmid
This proves that the RM125 strain has become resistant to neomycin and kanamycin.

At the same time, this study reveals that PTHNl is a plasmid capable of autonomous growth and expression of traits in thermophilic bacteria and Bacillus subtilis, that is, the fact that this plasmid can be used as a vector in both strains.

Plasmids for thermophilic bacteria resistant to neomycin and kanamycin are shown in the table above, but PT
As mentioned above, HNl and other plasmids are clearly different, and PTHNl is a novel plasmid that has not been previously recognized.

[Brief explanation of drawings]

The figure shows the restriction enzyme cleavage map of PTHNl, and A in the figure shows the restriction enzyme cleavage map of PTHNl.
ccl is an enzyme derived from Acinetobacter calcoaceticus, Bglff is an enzyme derived from Bacillus globigii,
EcoRI is an enzyme derived from Escherichia coli, HhaI
is an enzyme derived from Haemophilus haemolyticus, Hp
aI represents an enzyme derived from Haemophilus rosea influenzae, PstI represents an enzyme derived from Providencia stoarteii, and Taq■ represents an enzyme derived from Thermus aquaticus.

Claims (1)

[Claims] 1. A novel plasmid with neomycin and kanamycin resistance, which internally carries neomycin and kanamycin resistance genes, has a molecular weight of approximately 3.0 megadaltons, and is characterized by the restriction enzyme map shown in the figure.