JPS60188076A - Transformation of highly thermophilic bacterium - Google Patents

Abstract

PURPOSE:To transform a bacterial strain belonging to Thermus genus, easily and efficiently, by culturing the strain in contact with deoxyribonucleic acid. CONSTITUTION:A bacterial strain belonging to Thermus genus, e.g. Thermus thermophilus, Thermus flavus, etc. is cultured by conventional method, the obtained culture liquid is added with the objective deoxyribonucleic acid, and the cultivation is continued for several min - about 1hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides deoxyliponucleus m<rDN of Thermus sp.
My name is AJ. ) is related to the transformation method.

Bacteria of the genus Thermus grow ll1I! Belongs to highly thermophilic bacteria that exist at temperatures above 75°C, and grams l! i gender, neutral i
It is a bacteria that grows nearby. Because of its ability to grow under high temperatures, it is known that its enzymes and biological components are particularly excellent in properties such as heat resistance and solvent resistance.

Therefore, in addition to the direct use of the thermostable enzymes produced by this bacterial group, the thermostable biological functions of this bacterial group can be utilized from multiple angles to create bioreactors with high heat resistance, solvent resistance, and stability. creation is considered. In addition, this group of bacteria is a thermophilic bacteria that has the characteristic of growing near neutrality, so it is a group of bacteria suitable for fermentation production, reducing cooling costs and distillation costs, and avoiding contamination with contaminants. It is also attracting attention as a host strain for high-temperature fermentation, which is useful in terms of. From such an applied perspective as well as from the basic academic aspect of elucidating the anti-Da9 mechanism, 1゛C is my favorite.

It is desired to develop a host, Hekuku type, that can be used by Ninobe.

The one-host Hector line is a 3.11 match between a host that is amenable to genetic manipulation, capable of transformation, and a host that replicates autonomously within the host and carries a selection marker. It is composed of:

However, very little genetic research has been conducted on this bacterial group, and only a few mutant strains have been obtained, which serve as the basis for ultra-hypogenetic analysis.

In addition, Plasmi 1 and I, which can be used as Hector, are also available.
Regarding JNA, although several types of plasmids have been reported for the Thermus genus, they are all cryptic plasmids that do not have markers such as drug resistance, so the transformation conditions for introducing them into the bacterial cells are difficult. The problem was that it was not possible to examine the

Therefore, the present inventors have determined that the bacteria of the genus Nomus have characteristics such as not having a plasmid and growing on a minimal synthetic medium, and are candidate hosts for genetic manipulation. An amino acid auxotrophic strain was isolated from the Remus 4 monomorphophilus HB27 strain, which is thought to be suitable for use as an amino acid, by treatment with nitrosoguanidine. When we tried transformation with the wild-type chromosomal DNA, we were surprised to find that chromosomal DNA could be transformed into 41 bacteria without the need for CaC1 treatment, protoplast formation, and regeneration, which are performed with other bacteria.
By simply adding NA and culturing for a short period of time, all auxotrophic strains undergo recombination between the chromosomes and the added DNA, resulting in a trait in which they no longer require amino acids. It was observed that the chromosomal DNA of the streptomycin-rigid (hereinafter abbreviated as Sm') strain was used to similarly transform Remus genus bacteria other than the strains listed in Nia =: j' and r. When attempting this, it was revealed that transformants that became Sm' could be obtained with high efficiency.

Regarding plasmids, the
Cryptic plasmid pTT8 (
It was confirmed by the colony hybridization method that a strain containing the plasmid was obtained by adding DNA of 9°7khp to a strain of the genus Xermus without 1.5 plasmids and culturing for 1 hour.

The present invention was completed based on such knowledge.

That is, the present invention relates to a method for transforming a bacterium of the genus Rhymus, which is characterized in that in transforming the bacterium of the genus Rhymus, contact culture is carried out between nuclei and deoxyribonucleic acid.

As described above, the present invention has conclusively discovered that transformation can be easily carried out simply by bringing bacterial cells into contact with DNA, and has industrial utility as a simple and efficient method for transforming hyperthermophilic bacteria. is large.

The present invention will be described in more detail below.

Typical Thermus genus bacteria that can be used in the present invention include Thermus therml lyrus, Thermus flahus, Thermus caldophilus, etc. After culturing these strains by a conventional method, This is usually done by contacting the DNA of interest or adding l) NΔ to the bacterial culture solution.
Adding is done by Nino.

To avoid transformation, the bacteria were cultured using Zarmus medium (Din), yeast extract 1-0.2%, polypeptone (Daigoei a) 0.4%, NaC7!0.1%,
I) It is not necessary to use a special &+tl-forming medium before or after adding NΔ. Also, lag phase, logarithmic growth, stationary ill
No matter which of the bacteria is added DNA, the trait φj and dust efficiency hardly change. The temperature is 60-75'C, which is good for growth.
(□l It's so scary.

1) The term 5 hours after adding NΔ may be several minutes to 1 hour,
Even if J1ζS is further depleted, the ratio of transformants to the number of viable cells will not increase.

In the transformation method using the DNA of various microbial species, in view of the fact that specific l) NΔtakedena (DNA in general) is incorporated, the transformation method of trout spp. 1) NA and chromosomal DNA of the genus Thermus and other species having other markers, including but not limited to plasmid pTT8, as well as other plasmids capable of autonomous replication in the genus Thermus. It goes without saying that this method can also be applied to plasmids and phage A having markers that are expected to be divided or created in the future.

・：! Hereinafter, the present invention will be specifically explained in detail with reference to Examples.

Example 1 □:,<,11'p) Chromosome 1) Preparation of N.
Thermophilus I (strain 827 (Fl RM P-750)
2) N.

2. Zamasu Laji (Difco Yeast Extract Tone (Daigo Nutrition) 4g. NaC7!Ig, Nitrilotriacetic acid 0.1g, CaSO, -7820 0.06
g. MgSO, 7HzO O. Ig, KNO3 0.
1 05g. NaNO3 0.7g- NazHPOa
0.11 g, FcC4z 0.3 mg + MnSOa
2.2mg. , ZnSOn 0.5+n+r, H 3
130 30, 5■, CuSO-0. 0 1 6
mg, Na2MoO4 0. O25+n+rCoCn
z'6Hz0 0.046mg in pure water 11! Included in p
(H adjusted to 7.5) and harvested near the end of the logarithmic growth phase to obtain about 6 g of wet bacterial cells. This was added to lysozyme with a capacity of 12 μm [0.15 MN of im6].
acE-0. IM ED'FΔ (pH 8): peep,
After incubating at 37°C for 20 minutes, place in ethanol/dry ice refrigerant and freeze immediately. 5 Qm12 0.1'M) Lis-HCl buffer (19.011 -4-
%SDSO. After adding 1M NaC4 and stirring, add 56 mβ of phenol saturated with the above buffer, shake at 4°C, centrifuge, and separate the upper layer.

Add 2 volumes of cold alcohol to this and collect the nucleic acid fraction as a filamentous precipitate. , 11. mx O. txssc (0
．． 15M NaC6-0.015M 44 Sodium Formicate L,) 2m7! IFIXssc: Not added, ■ × 4, : l: j4, C [1) To N? Next, RNas a△ (Soge I Kaku X Shiko) and d table Ja Sc T 1 (manufactured by Sigma Corporation) were obtained at 50 trg/m7! and 30 II, respectively.
After adding H/m9 and incubating at 37°C for 30 minutes to degrade the RNA, repeat the phenol treatment and dissolve into chromosome 1)N/1'9. to do.

(2) Chromosome I) Amino acid auxotrophic strain form 1'1 due to NΔ
Transformation 1) Relationship between added DNA tail and number of transformants Proline requirement of Thermus thermophilus 11 13 2 7 (
p r o -) 41i, Thermus → Nomophilus llr327pro- (MP-7 with FE+
50 3) to 1 0+nl! After inoculating 50 pe into a 10 mρ Thermus medium and culturing at 70°C for 15 hours, the cells were incubated at 70°C for 90 minutes.

This culture solution is 0.36m7! 2, B2-mass and Mophilus I (B2
7 The chromosomal DNA of the wild strain was determined by DNA content from In8 to 10.
7 volumes of I)NA diluted with SSC to give a total concentration of 40 IIR was added, and the mixture was further cultured at 70°C for 1 hour. This was mixed with physiological saline (0.8.'+%NaC
After appropriately diluting with β), a proline-free minimal synthetic agar medium 4.1 was applied, and after culturing at 70°C for 2 days, the colonies of transformants that had grown and became proline-requiring (:) were counted. These results are shown in Table 1.

In addition, this strain is Themus cymophilus H B 2
7 T F p r o 'ni'□111. Oh MP-
75°8) Yo, 7 Deposit, I, I,.

As is clear from Table 1, the number of transformants depends on the amount of DNA, and when a saturating amount of DNA was added, 10 to 12% of transformed bacteria were observed per number of viable bacteria.

The minimum synthetic agar medium is Sicosugar 51T-K2HP 04
0.5 g, KH2P Os0.25 g, NaCff
2H1(NH4)wsOa 2.5g-Biotin 10
0 tt g, thiamine hydrochloride 1 mg, MgC6z・,6
Hz0 0.1. -25g, -Ca(J!z' 2Hz
0 25n+gXFeSOa・IHzo 6mg, Co
Cj! -611□0 0. '8m8, NiCaz・
60z0 20pg,, NaMo0z '211.0
1.2mg, VO3Os・3HzOO, 1mg, Mn(
1! ,・4H20 JIBff GO-1880713
(3) 5mL+, Zn5On・7tjzOGOIIg,
CuSOs・5HzOl 5711; and 15 g of agar (Hani Chemical) were included in pure water lβ, and the pH was adjusted to 7.0 to 7.2.
J! i! I used the prepared one. Namas 4 Normophilus II B 27! It grows in uEt, I'A'Jl medium, but amino acid auxotrophs such as proline cannot grow unless the above medium contains aminophilic 950B/R.

ii) Transformation using DNA treated with DNAse, RNAse, and pronase Transformation was performed in exactly the same manner as in I), except that DNA
In A, 1 μg of wild strain chromosomal DNA was treated as follows.

1) For NASA processing, add DNascl to DNNi8 trl
(manufactured by Sigma) and M g C7! 1 On for each Z
g/mβ, 20mM, at 37°C; 10
I kept it warm for a minute. RNase treatment and pronase treatment are DNA Lfj? (1 contains 504 g each of RNase A (manufactured by Sigma) and Pronase (Kaken Chemical).
/mff1. Force t1 so that it becomes 250μg/mff1
The mixture was kept warm at 37°C for 30 minutes. The above results are shown in Table 2.

(Hereafter blank) Table 2 1 Live bacteria 13 1.8 Xl07Cells /Samp
As is clear from the le table, DNAs that degrade DNA
With C treatment, no transformants appeared, but with RNase treatment and pronase treatment, which do not act on DNA, there was almost no change compared to no treatment.

Leucine, lysine, methionine, and tryptophan in the traits of each auxotroph strain l-1 Mulberry i), ii) By exactly the same method as above, leucine,
1μg of wild-type chromosomal DNA as a recipient strain for each amino acid auxotroph for lysine, methionine, and tryptophan
Transformation was performed using

These results are shown in Table 3.

(below margin) Table 3 All 15 strains had mutations of 10-6 or less.

The recipient strain and transformant used in Example 1-(2) were imitated/1-Anagi Industrial Technology (4・I Research Institute!j'l:
C and has the accession number shown in Table 4.

Table 4 (3) Sm' strain Transformation with chromosomal DNA Two normal mass thermosoi:>Sm1 strain 3275m' (FERM
I)-7513: Thermus thermophilus HB''/127', originally susceptible to Strebutoma fnon, strain 911 to 7112, chromosome 1) Due to a mutation in NA, streptomycin, Vera 151'l Oμ8/n
This is a mutant strain that can grow in 18 areas of 1β3-bearing trout. Chromosome 1) NA was prepared from this Hayashi using the same method as in (1).

Suwamas Suwako Philas on27a, Thermas
Furahas Δ1゛62 strain (FEI?MP-7515),
Thermus cardophilus GK24 strain <+;a RM
P-7517) respectively 1'O++/! Zamasu Culture Il! ! 70'CteI 5 l"+1i
ill city? l-? Two. 'c reso h(D +3 'm'
/(k 50 μm, 10 m/! Nori-mas +, 75 + 1!!) was inoculated, cultured for 90 minutes, and the chromosomal DNA of the Sm' strain (n:1) was inoculated into (1,72++l).
8 (11! (1.6 μg DNA)) and cultured for an additional 2 hours. After diluting the culture solution with appropriate physiological saline,
602&ml; applied to a mass agar medium containing S. I. leptomycin (50/J g/nu) and heated at 70°C for 20 hours.
One of the second transformants that became resistant to streptomycin was IVJ. The above results are shown in Table 5.

(The above is blank) Table 5 9 natural 611 sex strains are 10-" or less.

The recipient strain and transformed bacteria used in Examples]-(3) were entrusted to the Nigyo Technical Research Institute C Tan and have the deposit numbers shown in Table 6.

"TFSm'" in Table 6 indicates that it became Smr through transformation.

4r℃! Example 2: Isolation of ,'""l'(,1)p'r'+'13 Blasmi I"DNA・"l:p,TT8 plasmid was isolated from Thermus thermophilus strain 1188 (FE
This is a plasmid carried by RMI/mouth τ)+-'l-7519). This Thermus Thermophilus 14B8
100 mρ from a biologically pure culture medium of the strain Thermas 1: SIL! ! and culture with shaking at 70°C for 16-18 hours. This culture solution was inoculated onto 11 strawberry and trout la fields and cultured at 70°C for 5 hours. The bacterial cells were collected by centrifugation and treated with TIES (20mM Tris-C1l, 5mM
EDTA.

After washing with 10f1mM N, 1Cp p147.5), suspend in 10ml+ TES containing 25% sucrose per 4g. Rizochi 1. (10mg/m
7ri 2m/! , 0.25M-EDTA (pi-18
) was added to 4mp and left at 0°C for 10 minutes, followed by j37
Incubate at °C for 10 minutes. 2 rhe in this cell mixture night
A 1-e-SQ D S, 5 mn of 5MNac++ was heated to 11° C. for 15 to 18 hours. This was subjected to ultracentrifugation at 2800 rpm for 1 hour, and then heated to 1 ilr. Add 10% (W/V) of polyethylene glycol 6000 to this 2
Centrifuge the apparatus at 0'C for ~3 hours, 2200 rpm, 2 minutes to obtain the pellet. This precipitate was dissolved in 15% TBS and C
Add sC1 and ethidium bromide to bring the density to 1.6
Adjust to 1 to 1.62.

The sample is centrifuged in an equilibrium density gradient for 30-40 hours at 38,000 rpm.

The resulting whitebait I''DN hands were collected, and after removing the edges with isoamyl alcohol, T ImiN (20mM Tri 5-Cj!
1mM! T! l) Plasmid hin1'l by injecting i3 into rΔ, 20mMN, IC7.

1 son was beaten by 1 person.

d (2) Introduction of plasmid (transformation) Thermus sumophilus, which does not originally have a plasmid
111 27pro-(FERM P-7503) strain overnight + 6 fJ solution 50 μ4 was inoculated into] Omρ Thermus medium, and after culturing at 70°C for 90 minutes, 50 μC of (1
pTT8 plaso, mil prepared by the method described in )
"I), 'Add 2 μg of J△ and incubate at 70°C for 1 hour.
I did Δ■. Appropriately dilute with physiological saline and add 4 degrees of
When it was spread on a trout agar medium and cultured at 70°C for 15 hours, the number of viable bacteria was 6 x 101'. The grown colonies were randomly transferred to G900 strain Oζ, sterilized, and replicated onto a membrane filter (mm 7j Mia IIAWP) placed in a 4-normal agar pond and cultured at 70°C for 15 hours. . Using the filter, the L32p-labeled p'FT8 plasmid DN8 was added to Amarjam Two Translation 4-I, and the two lI+-:'--high Bridization was carried out in a conventional manner.

Nine of the 900 strains hybridized with labeled pTT8. When Shirasumi I was prepared using the method described in (1) from 9 strains including II'1゜, it contained a plasmid of the same size as Suba ζpTT8, and the restriction enzyme G.
It was confirmed by agarose gel electrophoresis that all cleavage patterns were the same. In addition, all nine strains had proline auxotrophic markers, and were confirmed to be derived from recipient bacteria.

As mentioned above, 6 of the Thermus thermophilus tl 1327 p r O- strains that did not have the plasmid
Plasmi F p T
It was confirmed that m T8s were killed.

Thermus thermophilus [(B27pro-/pTT8) into which the plasmid was introduced has been deposited at the Microbial Technology Research Institute.

(FEI? MP-7520> Hair Material ε Line 1i Orthographic (Method) % Formula % 1, Incident Display Patent Application No. 44895 of 1982 2, Title of Invention Altitude (11' 15 Bacterial Transformation Method 3 , Person making amendment Office application procedure amendment dated October 24, 1980 l, Indication of case Patent Application No. 44895 of 1982 2, Title of invention Transformation method of highly thermophilic bacteria i (b) Horse; W , C 4, Designated Agent - Overturn m: Translation 7, Subject of amendment Correct the "detailed description of the invention" in the specification.

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Claims (1)

[Claims] A method for transforming Thermus genus bacteria, which comprises bringing the microorganism into contact with deoxyliponucleic acid and culturing the Thermus genus bacteria.