JPS58170799A - Novel plasmid having resistance to tetracycline and novel microoragnism containing it - Google Patents

Abstract

PURPOSE:Plasmid pTHT9 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:Plasmid pTHT9 containing a gene with resistance to tetracycline in it, having about 5.2 megadalton molecular weight and a restricted enzyme map shown by the figure, obtained by propagating Bacillus stearothermophilus T9 strain (FERM-P 6436), 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 subjecting 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 recombinant DNA experimental D vector using thermophilic bacteria as a host, and a novel microorganism containing the same. It has a molecular weight of about 5
．． It is 2 megadaltons and concerns Rus.

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 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 have an optimal growth temperature of 55°C to 75°C.
They are broadly divided into moderate thermophiles, which have an optimal growth temperature of 75°C or higher, and highly thermophilic bacteria, which have an optimal growth temperature of 75°C or higher, but both have enzymes and biological components that make them resistant to heat and solvents. It is known to be excellent, and is attracting attention in particular from the point of view of its application to industrial processes such as thermostable enzymes derived from thermophilic bacteria and bioreactors with thermostable biological functions. 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 most effective means for this purpose, requires searching for plasmids that are considered to be strong candidates for vectors. Even including the following reports, only the following are known.

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

J, Ocn, 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, A.H.A., Bourdon, C.
, J., and Atkinson, T.

J, (tcn, Microb, 114. 401
408 (1979) (3) Furano of Bacillus stearothermophilus;''' J, Oen, Micr
ob, Su9. 109-115 (1980) (4) Isolation and analysis of drug-resistant plasmids from thermophilic Bacillus bacteria and creation of partially deleted plasmids Imanaka, T., Fujii 2M, A/Do Aino (, S.).

J, Bacs, 146(3), 1091-10
97 (1981) Therefore, the present inventors searched for microorganisms whose hosts are thermophilic microorganisms and which are equipped with a tetracycline resistance gene in the natural world. It has a specific cleavage point with a restriction enzyme, and contains a tetracycline resistance gene on its plasmid DNA. (Hereinafter, this plasmid will be abbreviated as pTHT9J.) The abbreviations of the restriction enzymes shown in the figure are as follows.

(1) BgtII enzyme derived from Habacillus globigii (
2) EcoH,l (d, enzyme derived from Nisierisia coli (3) It is recognized that the enzyme in order H+dlll is derived from Haemophilus influenzae.

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 advantageous for selection.

Furthermore, as is clear from the figure, pTHT9 has Bgt1
It has cleavage sites by restriction enzymes such as 1+EcoRI and HindIH at specific and limited positions. This is advantageous in that when pTHT9 is used as a vector, a site for introducing a heterologous gene to be inserted can be significantly retained. Furthermore, pTHT9 is advantageous in that it can be used as a vector for both Bacillus subtilis and thermophilic bacteria. Therefore, by using pTHT9 as a vector, a heterologous gene can be simultaneously expressed in Bacillus subtilis and a thermophilic bacterium.Thealothermophilus and Thealothermophilus belong to the same Bacillus group, so it has already been expressed in Bacillus subtilis due to their close kinship. It is considered that the heterologous gene is likely to be expressed also in thermophilic bacteria. Therefore, we will use genes that have already been cloned into Bacillus subtilis.

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. It is possible, and it is expected that it will be applied to industrial production.

To obtain pTHT9, we used Bacillus stearothermophila strain 219, a moderately thermophilic bacterium newly isolated from soil, in TYS medium. Yes, the optimum temperature for growth is approximately 55
Although this strain does not grow at 37°C, it is a novel microorganism that has not been previously recognized as possessing pTHT9. The strain was resistant to tetracycline, but sensitive to all six other antibiotics tested: anopicillin, erythromycin, chloramphenicol, kanamycin, neomycin, and streptomycin.

This strain has been deposited as FAIKEN Bacteria No. 6'' Tano/.

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

Example 1 (Screening of bacterial strains) Approximately 11 soil samples from Yatabe Town, Tsukuba District, Ibaraki Prefecture were placed in TTS medium (Difco
Tryptone 2T, Difco Yeast Extract 1%, NaC 11%) Lacycline (20 μP/m
19 strains of Bacillus stearothermophilus (Feikoken Bacteria 1st, No. z) were obtained from one of the colonies grown on the TYS agar plate containing Bacillus stearothermophilus.

Example 2 Isolation of plasmid pTHT9 from Bacillus stearothermophila strain 219.
0 ml of TYS medium (Difco Band Trypto 72
%, Difco Yeast Extract 1%,
(11% NaC) and cultured with shaking at 55°C for 16 to 18 hours. This culture solution was treated with 20μ of It's tetracycline.
TYS medium containing 9-/ml was inoculated and incubated at 55°C for 5 days.
Incubate for hours. The bacterial cells were collected by centrifugation and subjected to TES (
20mM Tris-110t, 5mM EDTA.

After washing with 100 mM Na04 (pH 7.5), the cells were suspended in 25 loml of trisucrose-containing 'rgs per 41 wet weight of bacterial cells.

2ml 110.25 lysozyme (10rug/ml)
M-EDTA Leave to stand at 4°C for 15-18 hours. This 2
Centrifuged by ultracentrifugation at 8,000 rpm for 1 hour,
Obtain the supernatant. This supernatant contains polyethylene glycol 6,
Add 10% (W/V) of 000 and leave it at θ℃ for 2 to 3 hours.
Equilibrium density gradient centrifugation for ~40 hours. The resulting plasmid DNA bands were collected, and after removing ethynium bromide with isoamyl alcohol, TEN (20mM
Tris-HCL.

Pure pTHT 9 is obtained by dialysis against 1 mM EDTA, 20 mM Mail).

Procedure for characterizing pTHT 9 The molecular weight of pTHT 9 was determined by its superhelical structure (5upe
rcoiledstructure) DNA and
Obtained by agarose gel electrophoresis of fragments cleaved with restriction enzymes. The molecular weight marker at this time is pBR3
22-[3N A (2,67+ud), 0otEI
DNA (4,2md) and lambda I') N
Hind III fragment of A (14, 6, 5, 84,
4,05°2.67, 1.40.1.21.0.34
), BcoRI-digested fragment of lambda DNA (13,7,
4,74,3,73,3,48,3,02,2,13)
was used. Cleavage with restriction enzymes was performed 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 uses Sechem's agarose at a concentration of 0.5% or 0.7%, and is carried out in a horizontal gel electrophoresis chamber at a concentration of 1.5% per 1 Crn of gel length.
The test was carried out at a constant voltage of V for 15 to 17 hours.

The fact that pTIIT9 has a tetracycline resistance gene on its DNA is due to the fact that pTIIT9 has a tetracycline resistance gene on its DNA.
This was confirmed by a transformation experiment into protoplasts of the blyamas RM125 strain. 5 Chirus suftilis R
The procedures for protoplast preparation, transformation, and protoplast regeneration of the M125 strain were described by Chang & (3ohen
method (Mo1cc, Gen, 0cnet, 16
8111115 (1979)) Yoni Plasmid D
Add NA solution and add polyethylene glycol 6,000
After promoting the uptake of DNA into protoplasts, the protoplasts are regenerated into vegetative cells on a regeneration medium.

p'I'HT9. DNA about 0.5μ? Using o, 2 smg of protoplast suspension of strain RM125 (
Approximately 10' protoplasts/ml) were transformed and examined for the appearance of tetracycline-resistant strains on the regeneration medium, and the regeneration rate of protoplasts was 0.61%.
(1 to 2 x 103/1 for 1 to 1 x lO'/mA
Tetracycline-resistant strains occurred at a frequency of nl. on the other hand,
In the system to which no plasmid DNA solution was added (control), no tetracycline-resistant strains were produced even when 10 7 "or more protoplasts were sown.

Plasmid DNA was extracted from the tetracycline-resistant strain of the M125 strain obtained here in the same manner as in the extraction of plasmid DNA from T. stearothermophilus T9 strain (temperature and time of lysozyme treatment at 37°C for 30 minutes). When extracted and examined using the method (different in some respects), it was found that G
Plasmid DNA with the same molecular weight as ITHT 9 and exactly the same cleavage pattern with restriction enzymes was recovered. This fact proves that pTIIT9 has a tetracycline resistance gene, and that the introduction of this plasmid into the RM125 strain caused the RM125 strain to exhibit the tetracycline resistance trait. At the same time, pTl
This study demonstrates that lT9 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.

The plasmids of thermophilic bacteria with tetracycline resistance are as shown in the table above, but pTHT9 and the others are clearly different as mentioned above, and pTHT9
9 is a novel plasmid not previously recognized.

6” gtl Iri enzyme derived from Bacillus groppigii,
EcoRI is an enzyme derived from Nijieria coli, Hindf
11 indicates an enzyme derived from No. emophilus influenzae.

Procedural amendment Natsume (spontaneous) Special Office Ryomiya 1, Table of matter 11 tJL 1982 Patent Application No. 37538 2, Name of invention New /71->Sumi 1 with resistance to heliicrin
and new charcoal microorganisms possessing the same 3, relationship with the case of the person making the amendment, patent applicant address 1, Kasumigaseki, Daita-ku, Higashiyama-bu, No. 3, No. 1 Name (114) Director of T Technology Institute Ishizaka Makoto-5, date of amendment order spontaneous 6, number of inventions increased by amendment 1)
, details inner cylinder 2 negative 201j ``Growth〒suitable temperature is j, %
"l1 unit 1 limit: Akira If5 /+'i i L" correction shima-4゜〈2), detailed reading, page 3, line 1 [Total appropriate humidity during life]
q changes 1 to [1 unit [limited supply] 1 ma (-corrects.

Claims (1)

[Claims] 1. Contains a tetracyclycide/resistance gene,
A novel plasmid with tetracyclytic/resistance whose molecular weight is approximately 5.2 megadaltons and is characterized by the restriction enzyme map shown in the figure. gtn 0 2 A novel Bacillus stearothermophilus strain 19 carrying the above-mentioned plasmid with tetracycline resistance.