JPS5928396B2 - Novel plasmid with tetracycline 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, it relates to a novel plasmid that contains a tetracycline resistance gene, has a molecular weight of approximately 2.8 megadaltons, and is characterized by the restriction enzyme cleavage map shown in the figure.

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

The host-vector system of Escherichia coli has been fully developed, and in addition to Escherichia coli, host-vector systems have been developed in yeast, Bacillus subtilis, etc., 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 with a temperature of ℃.

On the other hand, thermophilic bacteria are broadly divided into moderate thermophiles, which have an upper limit of growth humidity between 55°C and 75°C, and extreme thermophiles, which have an upper limit of growth humidity of 75°C or higher. It is known that the enzymes and biological components it contains have excellent heat resistance and solvent resistance, and are particularly useful for industrial processes such as heat-resistant fermentation derived from thermophilic bacteria and bioreactors with heat-resistant biological functions. It is attracting attention from a number of points.

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. Microt), 104, 193-19.
9 (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., & Atkinson, T.

J, Gen, Microb, 114. 401-
408 (1979) (3) Analysis of plasmid pAB124 of Barurus stearothermophilus and creation of deletion derivatives Pingham, A, H, A, Bourdon, C', J, and Atkinson, T.

J, Gen1M1crob,, 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, and Aiba, s.

J, Bact, 146(3), 1091-109.
7 (1981) (5) Berhard, M.D., Vasquez, C.1., Gilenzuela, P., Vicuna, R., and Yudelevik, on the physical properties of plasmid (pTTI) isolated from Thermus thermophilus. A.

P Iasmid, 6. 1-6 (1981) (6)
Transformation of Bacillus stearothermophilus with plasmid DNA and properties of Bacillus stearothermophilus shared vectors Imanaka, T., Fujii 2M, Aramori, ■, and Aiba, S.

J, Bact, 149(3), 824-830 (198
2) Therefore, the present inventors searched the natural world for microorganisms whose host is a thermophilic microorganism and which contains a tetracycline resistance gene, and as a result, they discovered a new plasmid from a strain belonging to the genus Bacillus. succeeded in obtaining it.

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

(Hereinafter, this plasmid will be abbreviated as IpTHTt5J.

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

(1) C1al is an enzyme derived from Caryophanon latum yume (2) E coRI is an enzyme derived from Nijieribia lily (3) Hhal is an enzyme derived from Haemophilus baemolyticus (4) HpaI is an enzyme derived from Haemophilus parainfluenza (5) Hpall indicates an enzyme derived from Haemophilus parainfluenza (6) TaqI indicates an enzyme derived from Thermus a noaticus.

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

As is clear from the table, T)THT15 is clearly different in quantity and restriction enzyme cleavage pattern compared to known plasmids, 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 pTHT15
has extremely advantageous selection markers such as the ability to grow autonomously in thermophilic bacteria and Bacillus subtilis, and resistance to tetracycline.

Furthermore, I) THT15 is C1a, as is clear from the figure.
It has cleavage sites by restriction enzymes such as 1, EcoRI, Hpal, and Hpall at specific and limited positions.

This is advantageous in that when pTHT15 is used as a vector, a site for introducing a heterologous gene to be inserted can be significantly retained.

Further, pTHT15 is advantageous in that it can be used as a vector for both Bacillus subtilis and thermophilic bacteria.

Therefore, by using pTHT15 as a vector, it is also possible to simultaneously clone a heterologous gene into Bacillus subtilis and a thermophilic bacterium.

In addition, because Bacillus subtilis and the thermophilic bacteria, Bacillus, and Stearothermophilus that are the sources of pTHT15 have in common that they belong to the genus Bacillus, the heterologous genes already expressed in Bacillus subtilis are closely related. 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.

1) THT15 was obtained by growing Bacillus stearothermophile strain T15, a moderate thermophilic bacterium newly isolated from soil by the present inventors, in TYS medium until late logarithmic growth. Lysozyme is achieved by lysing the bacteria by SDS treatment.

In addition, Bacillus and Stearothermophilus strains are aerobic sporobacilli, positive for Durham staining, and the optimal humidity for growth is approximately 55°C, although they do not grow at 37°C, but I) THT
It is a novel microorganism that has not been recognized before in that it possesses 15.

This strain was not only resistant to tetracycline, but also chloramphenicol and erythromycin, and sensitive to ampicillin, kanamycin, neomycin, and streptomycin.

This strain has been deposited as Microtechnical Research Institute No. 6661.

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

Example 1 (Screening of bacterial strains) Approximately 19 soil samples from Yatabe Town, Tsukuba District, Ibaraki Prefecture were added to 100 ml of TYS medium (Difco Tryptone 2%, Difco Yeast, Extract 19 J, NaC 11%) and incubated at 55°C to approx. After shaking culture for an hour, tetracycline (2
One of the colonies grown on a TYS agar plate containing 0μ, 9/ml) contained Bacillus, Atearosavirus TI5.
strain (Feikoken Bibori No. 6661) was obtained.

Example 2 Plasmid pT) (Bacillus T15, isolated from Stearothermophilus strain T15, Bacillus pT) isolated from a biologically pure culture medium of Stearothermophilus strain T15 ((Feikoken Bacterial Serial No. 6661))
T containing 0 ml of tetracycline 20μ9/ml
YS medium (Difco, Bacto, Tryptone Part 2, Difco, Yeast, Extract Part 1, NaC 11%)
and cultured with shaking at 55°C for 16 to 18 hours.

This culture solution is inoculated into TYS medium containing 20 μ9/ml of 11 tetracyclines and cultured at 55° C. for 5 hours.

The bacterial cells were collected by centrifugation, and TES (20mMTi
sr-HCI, 5mM EDTA, 100mM NaCl
After washing with pH 7.5), each 4 g of wet bacterial cells was suspended in 10 ml of TBS containing 25% sucrose.

2ml of lysozyme (10μ, 9/ml), 0.25M
- Add 4 ml of EDTA (PH8,0) and let stand at 0°C for 10 minutes, then incubate at 37°C for 10 minutes.

Add 2 ml of 10% SDS, 5 ml of 5M to this fine foam mixture.
-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% polyethylene glycol 6000 (
w/v) and left at 0°C for 2 to 3 hours at 220 Orpm.
Obtain a precipitate by centrifuging for 2 minutes.

This precipitate was dissolved in 15 ml of TBS and CsCl and ethidium bromide were added to bring the density to 1.61-1.62.
Adjust to.

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-HCI, 1mM EDTA, 20mM Tris-HCI, 1mM EDTA, 20mM
Pure pTHTl was obtained by dialysis against mMNaC1).
5 is obtained.

Procedure for characterizing pTHTl5 The molecular weight of pTHTl5 is determined by its superhelical structure (5up
ercoiled 5structure) DNA
and obtained by agarose gel electrophoresis of fragments cleaved with restriction enzymes.

The molecular weight marker at this time was pBR322DNA (2,6
7m d), Co1EI DNA (4,2m d) and Hind-digested fragment of lambda DNA (14,6,5,8
4, 4, 05, 2, 67.

1.04, 1.21, 0.34md), Lambda D
EcoRI-digested fragment of NA (,13,7,4,74,3
, 73.

3.48, 3.02, 2.13md) were 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 were commercially available from Zenshuzo.

For agarose gel electrophoresis, SeChem agarose was used at 0.
．． 5 or at a concentration of 0.7%, using a horizontal gel electrophoresis chamber at a constant voltage of 1.5 V per crIl of gel length.
It lasted ~17 hours.

It was confirmed that pTHTl5 has a tetracycline resistance gene on its DNA by a transformation experiment into Bacillus subtilis, Bacillus subtilis RM125 strain protoplasts.

Bacillus. Chang
&Cohen's method (Mo Ie C, Gen,
Genet.

168.111-115 (1979)).

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

Approximately 1 μ9 of pTHTl 5 DNA was used to transform 0.5 ml of protoplast suspension of RM125 strain (2 x 109 protoplasts/m), and the appearance of tetracycline-resistant strains on the regeneration medium was examined. Play rate of 10 (2X108AI
), tetracycline-resistant strains occurred at an order of 1 x 103/ml.

On the other hand, in the system to which no plasmid DNA solution was added (connarol), no tetracycline-resistant strains were generated even after passing through 2 x 108 or more protoplasts.

Plasmid DNA was extracted from the tetracycline-resistant RM125 strain obtained here in the same manner as in the extraction of plasmid DNA from Bacillus 6 stearothermophilus TI5 strain (the humidity and time of lysozyme treatment was 37°G for 30 minutes). When extracted and examined using the method (different), it was found that T
) Plasmid DNA with the same molecular weight as THT15 and exactly the same cleavage pattern with restriction enzymes was recovered.

This fact proves that pTHT15 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, T) THT15 is a plasmid capable of autonomous growth and expression of traits in thermophilic bacteria and Bacillus subtilis;
In other words, this study reveals the fact that this plasmid can be used as a vector for both strains.

The plasmids of thermophilic bacteria having tetracycline resistance are as shown in the table above, but as mentioned above, pTHT15 and the others are clearly different, and I)
THT15 is a novel plasmid that has not been previously recognized.

[Brief explanation of the drawing]

The figure shows a restriction enzyme cleavage map of pTHT15, and CIaI in the figure represents caryophanone, an enzyme derived from Latum, and E c
o RI is an enzyme derived from Escherichia coli, HhaI
is an enzyme derived from Haemophilus haemolyticus, Hp
al represents an enzyme derived from Haemophilus or parainfluenza, Hp a II represents an enzyme derived from Haemophilus or parainfluenza, and TaqI represents an enzyme derived from Thermus or Aquaticus.

Claims (1)

[Claims] 1. A novel plasmid with tetracycline resistance, which internally carries a tetracycline resistance gene, has a molecular weight of approximately 2.8 megadaltons, and is characterized by the restriction enzyme map shown in the figure.