JPS5978685A - Novel bacterium containing novel plasmid - Google Patents

Abstract

PURPOSE:To obtain a bacterium containing a novel plasmid useful as a vector of a recombinant DNA experiment using a highly thermophilic bacterium as a host. CONSTITUTION:Water of hot spring is added to a thermophilic medium(0.4wt% Defico yeast extract, 0.8wt% polypeptone(nutrition), and 0.2wt% NaCl), subjected to shaking culture, and Thermus flavus IS21 strain(FERM-P 6752) is obtained from one of colonies grown in a thermophilic agar flat plate containing streptomycin. This strain has about 3.1 megadalton molecular weight, and contains a plasmid characterized by a restriction enzyme map shown by the figure. Xbal in the figure is an enzyme derived from Xanthomonas badrii, Accl is an enzyme derived from Acinetobacter calcoaceticus, Kpnl is an enzyme derived from Klebsiella pneumoniae, and Hincll is an enzyme derived from Haemophilus influenza.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism possessing a novel plasmid useful as a vector for recombinant DNA experiments using hyperthermophilic bacteria as a host, and more specifically, the present invention relates to a novel microorganism having a novel plasmid having a molecular weight of 3.1. Thermus flavus is a megadalton and carries a novel plasmid 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 in 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
The problem is that it is a mesophilic bacterium with a temperature of ℃ to 37℃.

On the other hand, thermophilic bacteria are broadly divided into moderate thermophiles, which have an upper limit of growth temperature between 55°C and 75°C, and highly thermophilic bacteria, which have an upper limit of growth temperature of 75°C or higher. 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, breeding of thermophilic bacteria is considered to be important, and one of the
Moreover, research on the development of a host/vector system for thermophilic bacteria, which is considered to be a powerful means, and in particular, research on the development of a host/vector system for highly thermophilic bacteria, has not been conducted at all to date. Moreover, regarding the search for plasmid DNA, which should form the basis of vector development research, only the following two reports are known of research using hyperthermophilic bacteria as a material.

(1) Isolation of extrachromosomal DNA from highly thermophilic bacteria F. , Tanaka, T. and Sakaguchi, K. J. Ge
n. Microb. , 104, 193-199 (197
8) (2) Physical properties of the plasmid (pTT1) isolated from Thermus thermophilus Eberhard, M.; D. , Vasquez, C. , Valenzuela, P. , Vicuna, R. and Yudelevik, A.
．． Plasmid, 6, 1-6 (1981) The plasmids described in the above two reports are all so-called cryptic plasmids whose properties are unknown, and their molecular weights are also somewhat large, about 6 megadaltons. Therefore, it would be extremely difficult to use them as vectors or to develop vectors using them as materials. Therefore, the present inventors searched for a plasmid that has a selection marker (a marker indicating that the plasmid is present in the host) and has a small molecular weight from highly thermophilic bacteria. As a result, we succeeded in isolating a plasmid with a molecular weight of approximately 3.1 megadaltons from Thermus flavus that showed streptomycin resistance.

As shown in the above-mentioned restriction enzyme cleavage map, this plasmid has a small molecular weight and has specific cleavage points for several types of restriction enzymes (hereinafter, this plasmid is referred to as pNH
(abbreviated as S211).

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

Xba■ is an enzyme derived from Xanthomonas badorii, Ac
c■ represents an enzyme derived from Acinetobacter calcoaceticus, Kpn■ represents an enzyme derived from Klebsiella pneumoniae, and Hinc■ represents an enzyme derived from Haemophilus influenzae.

Differences from plasmids derived from Thermus bacteria, that is, extreme thermophiles, that have been reported so far are shown in the table below.

As is clear from the table, pNHS211 is clearly different from known plasmids in molecular volume 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. However, when considering bacteria such as highly thermophilic bacteria whose growth environment is hot springs with poor nutritional sources and no antibiotics, it is not easy to obtain plasmids containing drug-resistant genes. Therefore, vector development will have to be carried out by providing a marker derived from the host chromosome to a so-called cryptic plasmid whose properties are unknown. It would be extremely convenient to use pNHS211 in this case.

This is because, firstly, pNHS211 is a plasmid that can replicate in hyperthermophiles, and secondly, it has a small molecular weight compared to known cryptotic plasmids derived from other hyperthermophiles. This is because it has the advantage that analysis of essential regions of this plasmid, such as the replication origin region, genes involved in replication, etc., can be performed more easily than with other plasmids with larger molecular weights.

Furthermore, pNHS211 is Acc
It has a cleavage site by restriction enzymes such as (1) and Xba (2) at specific and limited positions.

This is advantageous in that when pNHS211 is used as a vector, a site for introducing a heterologous gene to be inserted can be significantly retained.

Now, if a different type of heat-resistant gene is introduced into a thermophilic bacterium using this plasmid as a vector, a reduction in cooling costs in the enzyme industry will be achieved.

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.

pNHS211 was produced using Thermus flavus TS21, a highly thermophilic bacterium newly isolated by the present inventors from hot spring water.
The strain was placed in Thermus medium (Difco Yeast Extract 0.4%, Polypeptone (Five Major Nutrients) 0.8%, NaC
This can be achieved by lysing the bacterial cells obtained by growing them to the late logarithmic stage using 10.2%) by treating them with lysozyme and SDS, but this strain is novel in that it possesses this plasmid.

In addition, Thermus flavus strain TS21 is an aerobic Gram stain-negative bacillus that produces yellow pigment, has a DNA GC content of approximately 70%, and has an optimal growth temperature of 70°C.
It is a novel microorganism that has not been previously recognized as possessing HS211. This bacterial strain was isolated from hot spring water as a streptomycin-resistant strain.

In addition to the above-mentioned plasmid (pNHS211), this strain also possesses a novel plasmid with a molecular weight of approximately 5.4 megadaltons, which is characterized by the restriction enzyme cleavage map shown in Figure 2. Admitted.

This strain has been deposited as Microtechnical Research Institute No. 6752.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (Screening of bacterial strains) Approximately 1 ml of hot spring water from Atagawa Onsen in Shizuoka Prefecture was added to 100 m of Thermus medium (Difco yeast extract 0.4%, polypeptone (five major nutrients) 0.8%, NaCl 0.2%).
Thermus flavus TS2 was extracted from one of the colonies grown on a Thermus agar plate containing streptomycin (20 μg/ml) after shaking culture at 70°C for about 18 hours.
One strain (Feikoken Bacillus No. 6752) was obtained.

Example 2 Plasmid pNHS211 of Thermus flavus TS2
Isolated from 1 strain Thermus flavus TS21 strain (Feikoken Bacterium No. 6752
100 ml of Thermus medium (Difco yeast extract 0.4%,
Polypeptone (five major nutrients) 0.8%, NaCl 0.2%
, pH 7.5) and cultured with shaking at 70°C for 16 to 18 hours.

Add 1 liter of this culture solution to 20 μg/ml of streptomycin.
Thermus medium containing the following was inoculated and cultured at 70°C for 5 hours. The bacterial cells were collected by centrifugation, and TES (20mMTr
is-HCl, 5mM FDTA, 100mM NaClp
After washing with H7.5), 10 ml of 2
Suspend in TES containing 5% sucrose. Lysozyme (10m
g/ml), 2 ml of 0.25 M-EDTA (pH 8.
0) Add 4 ml and leave at 0°C for 10 minutes, then at 37°C.
Keep warm for 10 minutes. Add 2 ml of 10% to this cell mixture.
Add SDS, 5 ml of 5M NaCl and heat to 4°C for 15-18
Let stand for a while. This is centrifuged by ultracentrifugation at 28,000 rpm for 1 hour to obtain a supernatant. Add 10% (w/v) polyethylene glycol 6000 to this supernatant and
Leave at 0°C for a time and centrifuge at 2200 rpm for 2 minutes to obtain a precipitate. This precipitate was dissolved in 15 ml of TES and CsCl
and ethidium bromide (density 1.61-1
．． Adjust to 62. This sample was run at 38,000 rpm for 30
Equilibrium density gradient centrifugation for ~40 hours. The resulting plasmid DNA bands were collected, and after removing ethidium bromide with isoamyl alcohol, TEN (20mM Tri
A plasmid solution is obtained by dialysis against 50% HCl, 1mM EDTA, 20mM NaCl). This plasmid solution is a mixture of pNHS211, pNHS212 with a molecular weight of approximately 5.4 megadaltons, and pNHS213 with a molecular weight of approximately 10 megadaltons. By subjecting it to
HS211 is obtained.

DNA 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 50mM containing twice the amount of 0.5mM EDTA was added.
Add MTris-HCl buffer (pH 8.0) and
Transfer to ℃ and keep warm. Equivalent amount of 0.1M Tris-H to this
Add phenol saturated with Cl buffer (pH 8.0), mix, and centrifuge (3000-5000 rpm, 5 minutes).
Separate the upper aqueous layer. After performing another phenol extraction and removing phenol from the aqueous layer with ether, 3
Add 1/10 volume of M acidic ammonium solution and perform ethanol precipitation with 3 volumes of ethanol. The obtained precipitate is
It was dissolved in EN to prepare a plasmid solution.

Procedure for characterizing pNHS211 The molecular weight of pNHS211 is determined by its superhelical structure (sup
ercoiled structure) and fragments cleaved with restriction enzymes by agarose gel electrophoresis and polyacrylamide gel electrophoresis.

The molecular weight marker at this time was pBR322DNA (2.6
7md), CoHTDNA (4.2md) and Lambda D
Hind■ analysis fragments of NA (14.6, 5.84, 4.
05, 2.67, 1.30, 1.17, 0.34md)
, EcoR■ degradation fragment of lambda DNA (13.7, 4.
74, 3.73, 3.48, 3.02, 2.13md)
, ae-digested fragment of φ×174 DNA (0.836, 0
．． 666, 0.539, 0.373, 0.192, 0.
174, 0.167, 0.145, 0.120, 0.0
73, 0.044 md) 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.

Restriction enzymes used were commercially available products from Takara Shuzo and Boehringer Mannheim. Agarose gel electrophoresis uses SeaChem's agarose at a concentration of 0.5% or 0.7%, and is carried out in a horizontal gel electrophoresis tank at 1.0% per cm of gel length.
The test was carried out at a low voltage of 5V for 15 to 17 hours.

For polyacrylamide gel electrophoresis, polyacrylamide/bisacrylamide manufactured by Seikagaku Kogyo Co., Ltd. was used.
The gel was run in a vertical slab gel electrophoresis chamber with a cross-linking degree of 30:1, using a constant voltage of 10 V per cm of gel length for 2-3 hours.

The plasmids of hyperthermophilic bacteria are as shown in the table above, but pNHS211 and the others are clearly different as described above, and pNHS211 is a novel plasmid that has not been previously recognized.

[Brief explanation of drawings]

Figure 1 shows the restriction enzyme cleavage map of pNHS211.
Acc■ is an enzyme derived from Acinetobacter calcoaceticus, Kpn■ is an enzyme derived from Klebsiella pneumonia, and Hinc■ is an enzyme derived from Haemophilus influenzae. Figure 2 shows the restriction enzyme cleavage map of pNHS212, and the Mlu■ represents an enzyme derived from Micrococcus luteus, Bgl■ represents an enzyme derived from Bacillus globigii, and Pst■ represents an enzyme derived from Providencia stuartii.

Claims (1)

[Claims] A novel Thermus flavus TS21 strain carrying a plasmid with a molecular weight of approximately 3.1 megadaltons and characterized by the restriction enzyme map shown in the figure.