JPS58170800A - Novel plasmid having resistance to tetracycline and novel microorganism containing it - Google Patents

Abstract

PURPOSE:Plasmid pTHT22 useful as a vector for recombination DNA operation using a thermophilic bacterium having autonomically propagating ability in a thermophilic bacterium and Bacillus subtilis and resistance to tetracycline as a host, obtained from a novel strain belonging to the genus Bacillus. CONSTITUTION:Plamid pTHT22 containing a gene with resistance to tetracycline in it, having about 5.6 megadalton molecular weight and a restricted enzyme map shown by the figure, obtained by propagating Bacillus stearothermophilus t22 strain (FERM-P 6437), aerobic spore-containing bacillus, positive in Gram staining, a thermophilic bacterium of middle grade, having about 55 deg.C fittest temperature for growth, not growing at 37 deg.C, in TYS medium to the latter period of logarithmic propagation, followed by subjectig the prepared mold to bacteriolysis by lysozyme or SDS treatment. The reduction in cooling cost in fermentation industry can be attained by entering a gene which is already cloned in Bacillus subtilis into a thermophilic bacterium using this plasmid.

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, and a novel microorganism harboring the same. A new bacillus harboring
It concerns Stearothermophilus.

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 of Escherichia coli is almost complete, and in addition to Escherichia coli, host-vector systems have been developed in yeast, Bacillus subtilis, etc., and ways to apply them 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 classified into moderate thermophiles, whose optimal growth temperature is 55°C to 75°C, and highly thermophilic bacteria, whose total optimal growth temperature IW is 75°C or higher.

It is known that the enzymes and biocomponents of Izuwa have excellent heat resistance and solvent resistance. It is attracting attention because of its application to industrial processes. Therefore, 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 effective means for this purpose, is
Even after searching for plasmids considered to be vector vector candidates, only the following reports are known.

(1) Isolation of extrachromosomal DNA from high school 1st grade thermophilic bacteria Hi/Numa, F., Tanaka, T., Antosakaguchi, K.

J, (icn, Microb, 104.193-1
99 (197B) (2) Isolation of four types of plasmids from drug-resistant thermophilic Bacillus bacteria and partial analysis of their properties Pingham, A.H.A., Bourdon.
C, J., and Atkinson, T.

J, Gen, Microb, 114.401-408
(1979) Analysis of plasmid pA, B+24 of Bacillus stearothermophilus and creation of deletion derivatives Pingham, A., HoA., Bourdon, C.J., and Atkinson, T.

J. Gen. Microb, 119, 109-11
5 (1980) <4) Isolation and analysis of drug-resistant plasmids from thermophilic Bacillus bacteria, and creation of partially deleted plasmids Imanaka, T., Fujii 2M, Andaiba, S.

J. Bact., 146(3), 1091-10.
97 (1981) Therefore, the present inventors discovered that the host is a thermophilic microorganism, has tetracycline-resistant water inside, has a small molecular weight, and has specific cleavage points by various restriction enzymes. It has a tetracycline resistance gene on its plasmid DNA. (below,
This plasmid is abbreviated as pTHT22J. ) The abbreviations of the restriction enzymes shown in the figure are as follows.

(1) 13g1l is an enzyme derived from Bacillus norovigii (2) F2coR,I is an enzyme derived from Nijieria coli (3) 1 (indlI[il, indicates an enzyme derived from Haemophilus influenzae).

Differences from known thermophilic-derived plasmids having tetracycline resistance are shown in the table below.

As is clear from the table, pTH'T22 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 marker that is extremely advantageous for selection.

Furthermore, as is clear from the figure, pTHT22 is Bgl
It has cleavage sites by restriction enzymes such as T[, EcoRI, and Hind Iff at specific and limited positions.

This is advantageous in that when pTHT22 is used as a vector, a site for introducing a heterologous gene to be inserted can be significantly retained. Furthermore, pTIT22 is advantageous in that it can be used as a vector for both Bacillus subtilis and thermophilic teeth. Therefore, by using pT((T22) as a vector, a heterologous gene can be expressed at the same time in a thermophilic bacterium due to their close relatedness. Therefore, by transferring a gene that has already been cloned into Bacillus subtilis into a thermophilic bacterium using this plasmid, it is possible to If the thermophilic bacteria have the same properties, reductions in cooling costs in the fermentation industry will be achieved through fermentation production using thermophilic bacteria.

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.

pTHT22 was created by expanding Bacillus stearothermophilus strain T22, a moderately thermophilic bacterium newly isolated from soil by the present inventors, in TYS medium to late logarithmic growth H6. ! This can be achieved by lysing the bacterial cells by treating them with lysozyme and SO8.

Bacillus stearothermophilus T22 not only showed resistance to zyclin, but also to six other antibiotics tested.

In addition, this strain has been deposited as Microtechnical Laboratory No. 1 Bacteria No. 1.

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

Example 1 (Screening of bacterial strains) Approximately 1 g of soil sample from Yatabe Town, Tsukuba District, Ibaraki Prefecture was added to TYS medium (Difco Tryptone Section 2, Difco East Co., Ltd.
Extract 1%, NaC 11%) and TY containing tetracycline (20 μg/yrl) after shaking culture at 55°C for about 8 hours.
Bacillus from one of the colonies grown on the S agar plate.
Stearothermophilus strain T22
No. 7) was obtained.

Example 2 Isolation of plasmid pTHT22 from Bacillus stearothermophilus strain T22.
0++/TYS medium (Difco Vandt Tryptone 2%, Difco Yeast Extract 1%, Na
C11%) and cultured with shaking at 55°C for 16 to 18 hours. This culture solution was treated with 12 tetracyclines at 20 μg/d.
The cells were inoculated into TYS medium containing the following, and cultured at 55°C for 5 hours. The bacterial cells were collected by centrifugation, and TE8 (20mM
Tris-HCI, 5mMEDTA, ]O
After washing with 00mM NaCl pH 7.5), the sprouts were allowed to stand for 10 minutes, and then kept at 37°C for 10 minutes.

Add to this cell mixture 2 volumes of 10% 8DS, 5 volumes of 5 M-
Add Na('l) and let stand at 4°C for 15-18 hours.

This was centrifuged by ultracentrifugation at 2800 rpm for 1 hour, and the supernatant was diluted to +4. Add 10% (w/v) polyethylene glycol 6000 to this supernatant, leave it at 0°C for 2 to 3 hours, and damage the precipitate by blowing at 2200 rpm for 2 minutes.

After collecting the NA band and removing ethidium bromide with isoamyl alcohol, TEN(2(1)
mMTris-HCI, 1 mMBDTA, 2
Pure pTHT22 is isolated by permeation with 0mM NaC1).

Procedure for characterizing p l” IIT 22 pTHT22
The molecular weight of
It was determined by agarose gel electrophoresis of the DNA of d. The molecular weight marker at this time was pBR322DNA (
2,67md), Col EIONA (4,2md) and Hindl of lambda DNA [[degradation fragment (+4..6
゜5.84, 4,05, 2.67, 1,40,
1.21, 0.34), Eco of lambda DNA
RI-digested fragments (13, 7, 4, 74.

3.73, 3,48, 3,02, 2.13) were used. Cleavage with restriction enzymes (d) DNA was precipitated from a plasmid DNA solution by ethanol precipitation and dissolved in an appropriate buffer. Restriction enzymes were commercially available from Takara Shuzo Co., Ltd. Agarose gel electrophoresis was performed using Sechem Co., Ltd. Using agarose at a concentration of 0.5 or 0.7%, 1.5 V per gel length ICrn by a horizontal gel electrophoresis chamber.
The test was carried out at a constant voltage of 15 to 17 hours.

p T HT 22 has a tetracycline resistance gene on its DNA.
This was confirmed by a transformation experiment into protoplasts of sSubtilis RM 125 strain. Performed by Bacillus subtilis. The outline of this method is RM 12 s
Logarithmic increase of the strain/1iIi bacterial cells were converted into protoplasts by lysozyme treatment in an isotonic solution, a plasmid DNA solution was added to the protoplast suspension, and the uptake of the DNA into the protoplasts was promoted with polyethylene glycol 6000, followed by regeneration. Medium F aims to regenerate protoplasts into vegetative cells.

Using approximately 0.5 μg of pTIIT 22 DNA,
RM125, protoplast suspension of strain 0.25x/(
Approximately 10° protoplasts/rd) were transformed and examined for the appearance of tetracycline-resistant strains in the regeneration medium. In this case, the regeneration rate of protoplasts was 0.
1 to 1 for 1 section (1 to 2 x 10'/me)
Atracycline-resistant strains arose at a frequency of 2×103/shoulder t. On the other hand, in the control case in which no plasmid DNA solution was added, the number of tetracycline-resistant strains was 107.
No matter how many protoplasts were seeded, no protoplasts were produced. .

Plasmid DNA was extracted from the tetracycline-resistant strain RM125 obtained here in the same manner as in the extraction of plasmid DNA from Bacillus stearothermophilus nii strain 22 (the temperature and time of lysozyme treatment were 37°C and 30 minutes). When extracted and examined using the method (different), p
Plasmid DNA with the same molecular weight as THT22 and exactly the same cleavage pattern with restriction enzymes was recovered. This fact proves that pTHT22 has a tetracycline resistance gene, and that this plasmid was introduced into the RM125 strain, which caused the RM125 strain to exhibit the tetracycline resistance trait. At the same time, pTHT
This clarifies that No. 22 is a plasmid capable of autonomous growth and expression of traits in thermophilic bacteria and Bacillus subtilis, that is, this plasmid can be used as a vector in both strains.

Plasmids for thermophilic bacteria having tetracycline resistance are shown in the table above,

[Brief explanation of the drawings] The drawing shows a restriction enzyme cleavage map of pTf (T22, in which glff is an enzyme derived from Bacillus globigii. Hindlll is an enzyme derived from Bacillus globigii.・Influenzae derived) enzymes are shown. 1986 Special construction 1
Application No. 37:) 39 No. 2 Name of the invention 3, Supplement 1 ■ Relationship with the Kutsu incident, Patent applicant (1 location, Totaku, Shimodaita-ku, Kasumigaseki 1T [L1 No. January Name (114) -1 Ko Gijutsuin Ai 71
Makoto Saka -+1? l1iII instruction 1] I・1
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Claims (1)

[Scope of Claims] 1. A novel plasmid 0 with tetracycline resistance, which internally carries a tetracycline resistance gene, has a molecular weight of approximately 5.6 megadaltons, and is characterized by the restriction enzyme map shown in the figure. 2. Novel T. stearothermophilus strain T22 carrying the above-mentioned plasmid with tetracycline resistance