JPS61146186A - Dna chain containing streptomycin-resistant gene - Google Patents

Abstract

PURPOSE:To obtain a DNA chain having a structure characterized in that an incised DNA fragment containing the stretomycin (SM) of the DNA of Streptomyces griseus (SG) therein is integrated in another specific DNA fragment, and useful for the transduction, etc., to impart SM-resistance, etc. CONSTITUTION:DNA of SG is incised with a restriction enzyme BglII to obtain an incised DNA fragment containing the SM-resistance gene and having a molecular weight of about 7.0Kb. Hybrid plasmid PST141 is created by integrating the DNA fragment into a DNA fragment obtained by incising the DNA of known plasmid PIJ702 with restriction enzyme BglII. The plasmid is introduced into the cell of actinomycete to obtain the objective substane utilizable for the transformation to impart and promote the SM-resistance and as a vector having SM-resistance as a selected marker.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antibiotic resistance gene of actinomycetes, specifically a novel DNA strand equivalent to a streptomycin resistance gene. If recombinant DNA technology could be used to breed actinomycetes or elucidate their genetic properties, they are the most important fermenting microorganisms that are widely used in the microbial industry as producers of useful substances such as antibiotics.・In particular, the antibiotic resistance gene of Streptomyces or its equivalent DNA chain is industrially useful as it can serve as a useful selective marker for constructing host-vector systems in Streptomyces. 11 also.

Antibiotic-producing bacteria have self-resistance to the antibiotics they produce, but if the above-mentioned resistance gene or an equivalent DNA strand is used, a higher degree of resistance can be artificially imparted to the producing bacteria. In addition, in the biosynthesis of streptomycin, streptomycin 14-syringe kinase encoded by the streptomycin resistance gene (rAgricultural an
d Biological Chemlstry j 3
5.848-861 (1971)) is thought to be involved in the biosynthetic process (rJourn&1
of Bacteriology J 9L 401~
405. Cl969)).

From the above background, the present inventors have developed Streptomyces griseus I8P 5236 (Streptomyces
griseua ISP 5236), and as a result of experiments, we succeeded in isolating the streptomycin resistance gene and elucidating its properties.

The streptomycin resistance gene discovered by the present inventors has the following properties.(f) This gene is located on chromosome D of Streptomyces griseus.
Contained in the DNA fragment corresponding to the molecular weight 7.0 kilobase fragment obtained by cutting with NA1 restriction enzyme Bgl II (+:9 The above 7.0 kilobase fragment containing this gene is the DNA fragment of The restriction enzyme cleavage map of this gene is shown in Figure 1 of the attached drawings.
The present inventors obtained Streptomyces griseus, which encodes an enzyme that phosphorylates -OH groups, by cleaving the DNA of its mutant strain with the restriction enzyme Bgl I. A molecular weight of 7.0 kilograms containing a streptomycin resistance gene is a DNA fragment containing a streptomycin resistance gene, and a restriction enzyme i sph I and/or
We found that the streptomycin resistance gene was found in the 'fcDNA fragment obtained by cutting with Pst I. As mentioned above, the present inventors digested the chromosomal DNA of Streptomyces griseus with the restriction enzyme Bgl I [ It can be obtained by cutting with streptomycin (hereinafter referred to as "streptomycin").

Using a DNA cleavage fragment with a molecular weight of approximately 7.0 kilopaces (kb) that contains a resistance gene (abbreviated as SM), this is a known method. DNA obtained by cutting the DNA of lasmid pIJ702 with the restriction enzyme Bgl I [
A new hybrid plasmid was successfully created by integrating it into the A fragment using known genetic recombination technology, and this new hybrid plasmid f ps
We named the plasmid p8TI41 as p8TI41, and found that the molecular weight of the plasmid p8TI41 was 12.6 kb, and that the restriction enzyme cleavage map of the hybrid plasmid psT141 was as shown in Figure 2 of the attached drawings. The hybrid plasmid psT 141, which was created by the present inventors in 1999, is a vector that has SM resistance as a selection marker and can be used in transduction or transformation techniques to confer or enhance 8M resistance by introducing it into actinomycete cells. It has been confirmed that it can be used as The present invention has been completed based on the above-mentioned findings, and the gist of the present invention is to obtain a Bgl cleavage fragment of Streptomyces griseus DNA, pIJ 702f, and a Bgl cleavage fragment of lasmid DNA. Stregutomyces Griseus DNA in a hybrid plasmid p8T 141 which has the restriction enzyme cleavage cytoplasm shown in the restriction enzyme map in FIG. 2 of the attached drawings and has a molecular weight of approximately 12.6 kb = i. It is characterized by being equivalent to the derived DNA fragment. Streptomycin resistance gene is found in the DNA strand held within the chain.

Here, ``a DNA strand that is equivalent to a DNA fragment derived from Streptomyces griseus DNA'' refers to a DNA strand that has exactly the same base sequence as the DNA fragment derived from Streptomyces griseus DNA. DNAs that have different base sequences due to the degeneracy of the DNA codon, but are recognized as the same in dienotig.
This term also includes Afil, and in particular, DNA encoding an enzyme that phosphorylates the 6-OH group of aminoglycoside antibiotics.
＠Substantially, 7.0kb in Figure 1
This means that it is a DNA strand corresponding to a DNA fragment obtained by further cleaving the DNA fragment of , abbreviated as SM-resistant DNA strand) is a DNA corresponding to a fragment obtained by cutting with the chromosomal DNA'i restriction enzyme Bgl II of Streptomyces ° griseus, that is, a cleavage fragment, and is shown in FIG. It can be a DNA strand characterized by a restriction enzyme cleavage map.

Here, [chromosomal DN of Streptomyces griseus]
The DNA AJ corresponding to the fragment obtained by cutting with the At-restriction enzyme Bgl ff, that is, the cleavage fragment, is
The chromosomal DNA At-7, O cut with the enzyme Bgl I [
In addition to meaning the fragment of kb itself, it also includes the DNA strand with the same base sequence as this.The latter DNA
A specific example of the A chain is the DNA of a Bgl II cleavage fragment of Streptomyces griseus or its mutant strain.
strand, as well as its equivalent synthetic NA.

A preferred specific example of the SM-resistant DNA strand according to the present invention is the chromosomal DNA 1 of a mutant strain of Streptomyces griseus maku obtained by cleaving it with the restriction enzyme Bgl I [7].
, which is included in the 0 kilobase bear fragment itself. The SM-resistant DNA strand of the present invention can be a DNA fragment of this 7.0 kilobase pair, and can also contain DNAfe corresponding to the fragment of the 7.0 kilobase pair, as well as restriction enzymes sph I and pst.
Another preferred embodiment of the 8M resistant DNA strand according to the invention is Streptomyces bricella.

or its mutant strain DNAt-restriction enzyme Bgl It
A fragment of 7.0 km Pacebear obtained by cutting it with.

More restriction enzymes! iph I and/or Pat
It is a DNA strand obtained by cutting with BglI[, 8phI and (
This does not exclude the case where the DNA is obtained by simultaneously acting simultaneously with pst I on chromosomal DNA derived from Streptomyces griseus t or its mutants to cleave it.

The 8M resistant DN chain of the present invention is an aminoglycoside antibiotic.
This means that a host actinomycete can be transformed with a hybrid plasmid containing the 8M resistant DNA strand of the present invention, such as ps'r 141 described above, to generate an 8M resistant strain. The SM-resistant DNA strand according to the present invention is usually obtained from Streptomyces griseus or its mutants, but it can be obtained artificially. (1) Production bacterium Chromosomal DNA from which the SM-resistant DNA strand of the present invention is isolated
The streptomycin-producing bacterium used as a donor is Streptomyces griseus te, a mutant thereof.

For example, Streptomyces griseus.

Known bacterium Streptomyces griseus ISP 523
6 @This strain is stored in the American Type Culture Collection (ATC) in Maryland, USA.
It has been deposited in a releasable condition with deposit number ATCC 23345, and is listed in the ATCC catalog. Its mycological properties are r International
ional Journal of Systemat
icBacteriology J IL A 4.3
32-334 (1968). From the nature of the present invention, one mutant strain of Streptomyces griseus that can be used is a DNA 7 fragment having the restriction enzyme cleavage map shown in FIG. DN that gives
It should be a strain with At-.

(2) Cloning of SM-resistant DNA strand from selected streptomycin-producing bacteria.

Total DNAt-f9D8-phenol method (M, G, S
m1thet al r Methods in In
zymology J 12.545 (+967) )
Extract using a known DNA extraction method such as When the extracted total DNA is cut with the restriction enzyme BgllI, a DNA fragment containing the desired 8M resistance gene, that is, a DNA fragment carrying the desired 8M resistance DNA strand, is obtained from various DNAs.
It is obtained in a mixed state with fragments.

In order to extract all the fragments containing the desired 8M resistant DNA strand from the DNA mixture thus obtained and to obtain it in large quantities, the DNA strand must be integrated into a suitable viral or circular plasmid as a vector, And the Hyzolid thus generated! Genetic recombination techniques including transformation, transduction or transfection of hosts with lasmids are available. Examples of such recombinant techniques include the following: When cutting the chromosomal DNA with Bgl [, a vector plasmid is allowed to coexist and this vector plasmid is also cut with Bgl m at the same time, or this vector plasmid is cut with another K Bgl m and then the above chromosomal DNA is cut with Bgl m. mixed with the Bgl It cleavage of
Streptomyces griseus I.
Obtaining a plasmid mixture containing a hybrid plasmid incorporating a DNA fragment of SP 5236. In general, vector plasmids that can be used in this case include:

Vector geramides which have a Bgl [cleavage site (and optionally Pst I and (i or) Sph I sites) and which when cut with 1tgl [ give a propagable fragment are suitable; The origin of the vector plasmid may be actinomycetes, Escherichia coli, Bacillus subtilis, or other specific microorganisms.

A typical vector plasmid is pIJ 702, a 5.65 kilobase vector derived from actinomycetes.

Plasmid pIJ 702 is a well-known actinomycete plasmid (r Journal of Genera
lMierobology J 129.2703
(1983) ), and the desired plasmid pIJ702 can be obtained from the host bacterium by following the procedures commonly used for genetic recombination. Next, the host 11 is transformed with the plasmid mixture obtained as described above.The host bacteria include actinomycetes, Escherichia coli, subtilis 1, and other microorganisms, depending on the type of vector used. All you have to do is choose the appropriate one. When the vector is an actinomycete plasmid, actinomycetes are used as the host bacteria.

A specific example of this case is Stregutomyces lividans 66, which was also used in the present invention to clone the hybrid plasmid psT 141. Stregutomyces lividans 66 is a known strain, [Journal of Virology J
I, 258 (1972) K: Described, and its mycological properties are disclosed in Japanese Patent Publication No. 75889/1989. Obtained by incorporating an 8M-resistant DNA fragment derived from an 8M-producing bacterium into a vector. In order to introduce the resulting hybrid plasmid into a host bacterium, the most efficient method is generally selected depending on the host bacterium and the type of vector. However, when using actinomycete plasmid vectors,
Since the most suitable method is to transform the host bacterium with Protogellast 2, the present inventor adopted this method. Genetic indicators possessed by the vector used, such as antibiotic resistance, pock formation, etc., can be used. To select cells containing the 8M resistant DNA of the present invention from the recombinants thus obtained, - It is most efficient to select 9SM-resistant strains generated by the expression of 8M resistance. for that purpose.

First, determine the minimum inhibitory concentration of 8M against the host bacteria, select clones that grow in a medium containing SM at or above that concentration, culture the obtained 95Fllll-resistant clones, and extract the plasmid from the 8M-resistant bacterial cells according to a known method. The inserted DNA fragment was isolated by restriction enzyme treatment, and the DNA containing the 8M resistance gene was extracted by agarose gel electrophoresis.
The 7t DNA fragment, ie, the BM-resistant DNA human strand thus obtained, was further cleaved with various restriction enzymes.

By performing agarose gel electrophoresis and analyzing the size of each fragment, a restriction enzyme cleavage map of the above-mentioned DNA fragments is created.Furthermore, each of the above-mentioned fragments is inserted into an appropriate vector and recloned. We conducted a structural analysis of the 8M resistance gene and determined its characteristics '9: FIi4.
Rakashi friend. It was found that the restriction enzyme cleavage map of the SM resistance DNA strand containing the 8M resistance gene according to the present invention is as shown in FIG.

The above-mentioned Hyturidozolasmid ps7141k was created by the present inventor by incorporating the SM-resistant DNA strand of the present invention.
Streptomyces lividans 4-1, a new strain obtained by transformation by introducing it into Streptomyces lividans 66, was deposited with the Microtech Institute on December 6, 1980, and its deposit number is the 7th microtechnical laboratory
No. 984 6 Therefore, the hybrid plasmid psT
To obtain 141, Streptomyces lividans 4
-1 (Feikoken Bacteria No. 7984) was used as lysozyme.

0, which can be achieved by lysis by SDS treatment.
And its isolated next hybrid grass 2 de p8T
The 8M resistant DNA of the present invention can also be obtained by cleaving 141 with the restriction enzyme Bgl■ Example + 11 SM resistant DNA of the present invention containing the 8M resistant gene
Strand Cloning (U) Hybrid Plasmid Formation Streptomyces griseus ISP 5236 (ATCC 2334
5) f, I % l s −x, 0.3'
4 East Nigosu (Difco), 0.5-=
Cultured at 27°C for 2 days in a 500-erlenmeyer flask containing 100gt of a medium containing 0.39Gw rut extract (Oxoid),
The culture solution was centrifuged at 9,000 rpm for 10 minutes to collect the bacterial cells.Next, the bacterial cells were diluted with f:2XT)l buffer (50mM
Tri8-HCJ (pH 7,4), 50 rrMI
Wash with DTA, 50 mM NaCl) and remove the DNA.
1 Known method Chater et al. Current To
ples in Microbiology and
Imnuno-1ogyj qb, 69 <violation 981
) ) to obtain donor DNA. In this way, 8 μt of donor DNA and 1. 4 μt of actinomycete vector plasmid pIJ 702 was mixed with 4 μt of actinomycete vector plasmid pIJ 702, and 20 units of restriction enzyme Bgl II was added to the mixture to obtain + OmM Trim -HCJI) H7,5.
, 7mM MgO,.

The mixture was reacted for 1.5 hours at 37'C in 50μ of a buffer consisting of 100mM NaCl and 7mM Mercagtethanol. After the reaction, heat treatment was performed at 68°C for 19 minutes. -0, I M Tr
im-Ht, :z (pH 8,0), an equal volume of phenol solution saturated with a buffer consisting of 0.2% Mercagtoethanol was added, shaken, and then centrifuged at 15,000 rpm for 7 ha. Extract the layer twice with ethyl ether to remove phenol, add twice the amount of cold ethanol, and leave at -20'C overnight.
The DNAt-precipitate was recovered by centrifugation on rotation for 10 minutes.

The recovered DNA'i was dried under reduced pressure and dissolved in 40 μt of pure water.
-HCA! ([)H7,6) , 66 mM M
gCl2.0. I M dithiothreitol, I O
5 μt of buffer consisting of mMAT P and T4 DNA
Process 15 units (5 μL) of ligase and react overnight at 4°C. In this way, the actinobacterial plasmid pIJ
Bgl of 702 [cleavage-to fragment, chromosomal DNA of Streptomyces griseus I8P 5236
A mixture of various hybrid plasmids containing the hybrid plasmid p8T 141 t- which incorporated the Bgl 1 cut DNA fragment of A was obtained.

-) Cloning Using the hybrid plasmid mixture obtained in this way, transform the host Streptomyces lividans 66 daydoplasts, select 8M-resistant recombinants, and extract the hybrid plasmid from the cells. p8T
141, the 8M resistance gene and therefore the SM resistance DNA strand of the present invention was cloned.

Grotograst of Streptomyces lividans 66 was prepared as follows. Streptomyces
Lividance 661! I: lfk glucose.

0.3% Yeast x Ki x (DlfcO), 0.5
%Af) (Dlfeo), 0.3 Ls malt extract (Oxo1d).

It was inoculated into a medium consisting of 0.34 Tisciformes sugar and 0.005 M C12,0.5 tiglycine, and cultured for 1.2 days.
Collect bacterial cells by centrifugation for 10 minutes, 10.3-
Wash with sucrose solution/Washed bacterial cells are 1 cape/vIl
P medium containing egg white lysozyme (10.3% cy=i 1
1. 0.025% K2SO3,0,2* MgC
l2-2H20.

0-000008 'l'zn C42, O-000
04% “C!s, 6 H2O-0.000002
Yu CuCl2.0.000002chi Mic7!2-4
H2O.

0.000002% Na2B40. 'Ion20.0.
000002% (NH4)6Mo, 2 to 024'4
0.0.005%KH2PO4,0,371b Ca(
J2 "2 H2O, 25mM TE 8 buffer - m7.2) 16- Suspend in 16- and incubate at 32°C for 60-90 minutes to form protoplasts. Bacterial cells that have not become protoplasts are placed on cotton and pore size 3. Remove by passing through a 0 μm filter and collect the 29-year-old Togellast gold by centrifugation at 2500 rpm for 10 minutes.

The collected zolotogerast was washed once with P medium, and then
Resuspend in P medium at 10 cells/-.

For the above fcf doplast suspension, the Bgl ft truncated DN of the above-mentioned Streptomyces plus, midopIJ 702
A fragment and Streptomycen griseus IS
A hybrid plasmid psT 14 consisting of a Bgl ft cut DNA fragment of P 5236 DNA.
Immediately after adding 50 µt of a solution of a hyturide plasmid mixture containing 1 h41oo containing polyethylene glycol-1
Add O solution (P medium containing polyethylene glycol Luna 100033) and mix for 60 seconds.
The protoplasts were diluted with P medium 5-5 and centrifuged at 2,500 rpm for 10 minutes to suspend the protoplasts t-ti7Ca in the P medium of protoplasts 1-t-1.

A medium (10,3-sucrose, 0.025-2So4
゜1 T MgC42・6H20, L Tiglucose, 0.0
1 Ticademinoic acid (Dlfco), 0.00000
8 * ZnC/2.0.00004fb Fetl!
,”6H20,0,000002511Cu(J2.0
．． 000002 %1itn(,:j2・4HzO-Q
,000002*Na,B2O,-10H20.

0.0000021 (NH4)6M0.024-4H
20,0,005%KH2PO4゜0.3%CaC/2
・2H20,0,3% K, -Zorori=z, 25mMT
E8 Buff 7-(p87.2), 0.005
MNaOH.

0.5% yeast essence (Dlfao).
After applying one cell at a time and culturing overnight at 20C, 1/4 of the same soft agar medium containing 100 μt/ydf of teozgutin was overlaid, and culture was continued for an additional 4 days to regenerate protoplasts.

Regenerated clones were grown in I8P A4 medium and 8M20μ?
27°C
Cultivate for 3 days and select 8M resistant clones grown simultaneously in medium containing and without 8Mt. The resulting 8M resistant hybrid plasmid 8T 14f t? , pIJ 702 used as a vector and pIJ 702 used for the vector and 7.
0* Robase was detected as a DNA fragment of A1.0 The molecular weight of approximately 12.6 kilobases was obtained by integrating the SM-resistant DNA of 7.0 kilobases obtained as described above into the actinomycete plasmid pIJ 702. This hybrid plasmid was designated psT141 by the present inventor. The above-mentioned 7.0Φ rope strip containing 8M resistance gene gold was prepared by known methods using various restriction enzymes.
From the results of analyzing the DNA fragments of
Then, Stregutomyces °lividans 661FI was transformed again using the method described above, and 8M
A resistant clone, namely Streptomyces lividans 4-1, was obtained. (2) Genetic analysis Streptomyces lividans 4-1 transformed with plasmid psT 141 was transformed with SM5 μf/mtt containing tr-IS glucose, -0.3% yeast extract (D
irco ) + O-596'deton (Dlfco
), inoculated into a medium consisting of 0.3 timaltoacetic acid (Oxoid).

Culture was performed at 27°C for 4 days. The bacterial cells were collected by centrifugation at 9,000 rpm for 10 minutes, and after washing the bacterial cells with physiological saline, 10 g of bacterial cells were added to t-125mM Tria-wal.
ata, 12.5mM Mg504pH7,0
The bacterial cells were suspended in a buffer solution consisting of 40-ml of the following and disrupted by ultrasonication in ice water.

Centrifugation was performed at 16,000 rpm for 10 minutes to obtain a cell-free enzyme extract.

6001+IPOA TP in cell-free enzyme extract,
Add I wte toluene and 2001!9 SM 3
The reaction solution was incubated at 7°C for 16 hours and centrifuged at 16,000 rpm for 10 minutes.

CM-Sephadex C-25CO, I MNILCJ
Chromatography was performed on a column with an equilibrium ratio of 100.

After washing with water, the @Sakaguchi reaction positive area eluted with 0.1M to IM NHCl was applied to a column of activated carbon 50-, and after conditioning with water, 40 thiacetone, and 80 thiacetone, 0.04 NHC
All the Sakaguchi reaction positive areas eluted with 80% acetone containing l were collected, IIm under reduced pressure and lyophilized to obtain a powder of lQ3++y. The mixture was developed with water (1:I), and the Sakaguchi reaction-positive sections were collected, condensed under reduced pressure, and lyophilized to obtain a powder of 20.99%.

When this powder was analyzed by SIMS and y'Mb/m, a five-molecular ion peak was found at 662, and m-streptomycin was confirmed by phosphoric acid ratio at the 6th position.

That is, the SM-resistant DNA strand of the present invention cloned from Streptomyces griseus is inactive [
It was revealed that it encodes a phosphotransferase (APR+61t-) that

[Brief explanation of drawings]

Figure 1 shows the molecular weight of 7.0k obtained by cutting Streptomyces griseus DNA with the restriction enzyme Bgl II.
b Restriction enzyme cleavage map of the 8M resistant DNA strand of
Figure 2 shows the entire SM-resistant DNA strand vector of Figure 1! This is a restriction enzyme map of the hybrid plasmid p8T141 which has been integrated into lasmid pIJ 702 and Qd'd. 19th of the month

Claims (1)

[Claims] 1. Bgl of Streptomyces griseus DNA
A hybrid plasmid consisting of a BglII cleavage fragment and a BglII cleavage fragment of pIJ702 plasmid DNA, which has the restriction enzyme cleavage site shown in the restriction enzyme map in Figure 2 of the attached drawings and has a molecular weight of approximately 12.6k.
DNA from Streptomyces griseus DNA in hybrid plasmid pST141 with b.
A DNA strand carrying a streptomycin resistance gene internally, characterized in that it is equivalent to the A fragment.