JPS59175887A - Gene resistant to kenamycin and plasmid - Google Patents

Abstract

PURPOSE:To prepare a plasmid having genetic index of Kanamycin resistance from DNA of chromosome of Streptomyces Kanamyceticus in a system using a plasmid derived from ray fungus. CONSTITUTION:A gene -I resistant to Kanamycin contained in DNA corresponding to a fragment having 1.3 megadalton obtained by scissoring DNA of Streptomyces Kanamyceticus with restricted enzyme BclI, coding aminoacetyltransferase to acetylate 6'-amino group of aminosugar antibiotic or a gene-II resistant to Kanamycin contained in DNA corresponding to a fragment having 5.2 megadalton obtained by scissoring DNA of Streptomyces Kanamyceticus with restricted enzyme PstI. The gane resistant to Kanamycin selected from the two groups is integrated into a vector plasmid to give a hybrid plasmid.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to an antibiotic resistance gene of actinomycetes and a hybrid plasmid incorporating the same.

Actinomycetes are the most important fermentation microorganisms that are widely used in the microbial industry as a producer of useful substances such as antibiotics.

If recombinant DNA technology can be used to breed actinomycetes or to elucidate their genetic properties, we should be able to expect extremely valuable effects on production channels.

Summary of the Invention The present inventors have developed DNA fragments containing two types of kanamycin rigidity genes from Streptomyces kanamyceticus chromosomal DNA using a system using Streptomyces as a host and Streptomyces-derived plasmids. ) We have succeeded in constructing plasmid P that has a genetic indicator of kanamycin resistance as well as release.

Streptomyces kanamyceteicus produces several types of aminoglycoside antibiotics, including kanamycin A and B, and naturally this bacterium has self-resistance to these antibiotics. Furthermore, as a result of applying artificial means such as mutagenesis to this bacterium, strains with even higher levels of resistance have been obtained.

The present invention is based on the above research results of the present inventors.

Therefore, the kanamycin resistance gene according to the present invention is one selected from the group consisting of (I) and (II) below;
It is.

(I) Kanamycin resistance gene - ■ (A) This gene is the DNA of Streptomyces kanamyceteicus.
It is obtained by cutting A with the restriction enzyme BclI. L, contained in the DNA corresponding to the 3 megadalton fragment.

(b) The restriction enzyme cleavage map of this gene is shown in Figure 1 (4). It is as follows.

(c) This gene is an aminoacetyl gene that acetylates the 6'-amino group of mino-glycoside antibiotics. (a) This gene is a D
It is contained in DNA corresponding to a 5.2 megadalton fragment obtained by cutting NA with the restriction enzyme pstI.

(b) The restriction enzyme cleavage map of this gene is shown in Figure 3.

Furthermore, the DNA fragment according to the present invention is derived from Streptomyces kanamyceteicus or a mutant strain thereof selected from the group consisting of (1) and (2) below.

(1) DNA of Streptomyces kanamycetacus or its mutant strain was digested with restriction enzyme BCII 1, 3 containing kanamycin resistance gene-■
Megadalton DNA fragment.

(2) Cutting the DNA of Streptomyces kanamyceteicus or its mutant strain with the restriction enzyme Pst I at 1-℃
The obtained kanamycin resistance gene-■ containing 5,
2 megadalton DNA fragment.

In addition, the hybrid P plasmid P according to the present invention is constructed by incorporating the kanamycin resistance gene selected from the group consisting of (Il and (IT)) into a vector plasmid.

(I) Kanamycin resistance gene -■ (A) This gene is the D of Streptomyces kanamyceteicus.
1. Cut NA with restriction enzyme Bcl I. 1 obtained by
It is contained in the DNA corresponding to a 3 megadalton fragment.

(b) The restriction enzyme cleavage map of this gene is shown in Figure 1.

(c) This gene encodes aminoacetyltransferase, which acetylates the 6'-amino group of aminoglycoside antibiotics.

(TT) Kanamyces also has the W gene - (a) This gene corresponds to a 5.2 megadalton fragment obtained by cutting the DNA of Streptomyces kanamyceticus with the restriction enzyme Pst I. contained within.

(b) The restriction enzyme cleavage map of this gene is shown in Figure 3.

The plasmid having a kanamycin resistance marker according to the present invention is particularly suitable for use with a recombinant DNA vector whose host is actinomycetes, which are producing bacteria of useful substances such as antibiotics. It can be used. Extremely valuable results can be expected by breeding or genetic analysis of actinomycetes through recombinant DNA experiments using such an actinomycete host-vector system.

The kanamycin (hereinafter referred to as KM and t,'5) resistance gene according to the present invention can be used in Streptomyces (S).
It is contained in the DNA corresponding to the 1, 3 megadalton (Md) or 5.2Md fragment obtained by cutting (-) DNA of C.ceticus) with the restriction enzyme BclIf: or pstI, and is used (7). There are two types depending on the type of restriction enzyme used:

fil KM resistance gene-I The restriction enzyme used was BCI I, and the cleavage fragment was 1.3
It is the size of Md. The restriction enzyme cleavage map of the KM resistance gene-■ is shown in Figure 1, and this enzyme is an aminoacetyltransferase [AAC (6'):) that acetylates the 6'-amino group of aminoglycoside antibiotics. Co-P.

+21 KM resistance gene - ■ The restriction enzyme used was Pst I and the cleavage fragment was 5.2°C.
It is the size of Md. The restriction enzyme map of the KM resistance gene-■ is shown in FIG.

Here, the DNA of Kanamyceteichus was digested with restriction enzyme B.
DNA J, which corresponds to the 1 to 3 megadalton fragment obtained by cll cleavage, refers to not only the Bcl I cleaved fragment itself but also DNA having the same base sequence as the Bcl I cleaved fragment. Specific examples of the latter are BclI cleavage fragments of mutant strains of S. kanamyceteichus and synthetic NAs. The situation is exactly the same when the restriction enzyme is PstI.

(8) Preference for the KMid sex gene according to the present invention (7 specific examples include:
What is contained in the 1,3 Md fragment obtained by cleaving the DNA of S. kanamycetacus or its mutants with the restriction enzyme BclI and the DNA of S. kanamycetacus or its mutants with the restriction enzyme PstI. This is contained in the 5,2Md fragment obtained by cleavage 1-1. The present invention also provides these 1.3 Md fragments and 5.
It also concerns fragments of 2Md.

K M resistance gene-■ is the 6' of aminoglycoside antibiotic
-Has the ability to copy aminoacetyltransferase [AAC(6')), which acetylates amino groups. This Ikuriki is an actinomycete hybrid P containing this gene.
This is confirmed by transforming a host actinomycete with Plasmir r to obtain a KM-resistant recombinant, and examining the antibacterial spectrum of this recombinant against various aminoglycoside antibiotics (details will be described later).

Gene Production The KM resistance gene according to the present invention may be artificially synthesized, but is usually obtained from S. kanamyceteicus or its mutants.

(1) Production bacterium DNA donor (7) The KM producing bacterium is Streptomyces kanamyceteicus or its fJa.

Examples of S. kanamiseticus include "S. kanamiseticus (parent stock)." This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feikoken) under accession number 3363, and is available for sale. Examples of other strains include S. kanamai superiorticus ATCC strain 21252.

In view of the nature of the present invention, mutant strains of S. kanamyceteicus should have DNA that provides the restriction enzyme cleavage map shown in FIG. 1 or FIG. 3.

(2) Gene cloning Total DNA was extracted from the cells of the selected KM-producing bacteria using the SDS-phenol method (M, G, Sm1th et al.
methods in Enzymology, 12.5
45 (1967)). By cutting the extracted DNA with restriction enzymes BCII or PstI, DNA fragments containing the gene of interest are converted into various DNA fragments.
Obtained with fragments.

In this way, to extract the desired gene-containing fragment from the DNA fragment mixture obtained in (-), in order to obtain the desired gene-containing fragment 1 to (-), insert it into an appropriate viral or circular plasmid P as a vector. Genetic recombination techniques including transformation, transduction, or transfection of host bacteria by the resulting hybrid plasmids can be utilized.General genetic recombination techniques are discussed in Section 1-1, edited by Yasutaka Takagi, You can refer to "Manipulative Experiment Method" (Kodansha).

An example of such a recombinant technique is as follows. That is, BclI or P of the above donor DNA
When cleaved with stI, vector plasmid P is allowed to coexist, and this vector plasmid is also cleaved with BclI.
or at the Pst I site, or this vector plasmid is separately cut with Bcl I or Pst I and mixed with the Bcl I or Pst I cut of the donor DNA.

Treat with appropriate gauze, such as T4 ligase.

S. kanamyceteicus DNA in the vector plasmid
A plasmid mixture containing chimeric plasmid P into which the fragment has been incorporated is obtained. Used in this case 1. Uru Vector Plasmi r
Generally suitable are those which have a BCII or pstI cleavage site and which give a propagable fragment when cut with nclI or pstI. Its origin is actinomycetes and Escherichia coli.

Bacillus subtilis may be a specific microorganism, but some specific examples include those derived from actinomycetes (-℃PSF68
9. pSF619 and psF7651m and above, both of which were published in Japanese Patent Application Laid-Open No. 57-1. See FH'3600 publication. Also, see the same publication for the mycological properties of each host fungus], and others. To obtain the target plasmid from the host bacterium, follow the technique described in the above-mentioned publication and other methods commonly used for genetic recombination ( 9.1 Md psF 689.

Its host bacterium, Streptomyces platensis 5
F6R9C Kaikoken Article No. 121) (and the mycological properties of this bacterium) are described in JP-A-57-188600 and JP-B-45-6076 as mentioned above, and are available for sale. It is in.

Another representative example of a vector plasmid (plasmid) 4 derived from Streptomyces is pSF 765 of 4.8 Md. The host bacterium S. fradiace 5F765 (Feikoken Joyori No. 124
No.) (and the mycological properties of this bacterium) are described in JP-A-57-188600 as mentioned above and are available for sale.

Next, host bacteria are transformed with the obtained plasmid mixture. Depending on the type of vector used, select a suitable host bacterium from among actinomycetes, Escherichia coli, Bacillus subtilis, and other microorganisms, especially those with high transformation efficiency and high sensitivity to KM. Bye. When the vector is an actinomycete plasmid, an actinomycete is used as the host bacterium, and a specific example in this case is S. lividans 66 (
6982).

In order to introduce a vector incorporating a DNA fragment of a KM-producing bacterium into a host bacterium, the most efficient method is selected depending on the host bacterium and the type of vector. The most common method is to transform bacterial protoplasts.

For selection of recombinants obtained by transformation.

For example, genetic index 1 carried by the vector used.

Antibiotic resistance, pock formation (M, J, Bibh e
tal: Mo1ec, gen, Genet, 154
, 155 (1977), Nature274,
39R (1978)) etc. can be used.

In this way 1. In order to select those containing the KMi- sex gene from the recombinants obtained, it is most efficient to select KMM-prone strains produced by the expression of KM resistance. To do this, first, the minimum inhibitory concentration of KM against the host bacteria is determined, and clones that grow in a selective medium containing KM at or above the determined concentration are selected.

The obtained KMM clone was cultured, and the bacterial cells were cultured using a known method [Hansen et al: J, Bacter.
iol, 5135°227, 1.978], and the inserted DNA was isolated by restriction enzyme treatment.
Take out the fragments1. , K by agarose gel electrophoresis
A DNA fragment containing the MM gene can be isolated. In addition, to confirm that it is a KMM gene clone, reintroduce the isolated (~da plasmid) 4 into the host bacterium (-teK).
This can be done by examining the expression of M resistance.

The obtained DNA fragments are further cut with various restriction enzymes (
- By performing agarose gel electrophoresis and analyzing the size of each fragment, a restriction enzyme cleavage map can be created, and by incorporating each fragment into an appropriate vector and recloning. By performing structural analysis of the KMM gene, its characteristics can be clarified. As described above, the restriction enzyme cleavage map of the DNA containing the KMM gene of the present invention is as shown in FIGS. 1 and 3.

Hybrid r plasmid P The hybrid r plasmid P according to the present invention contains the KMM gene described in 1- above.

Vibe of the present invention when the vector plasmid P into which the KM resistance gene is to be incorporated is actinomycete plasmid P (15) A specific example of plasmid plasmid P related to cloning] is already mentioned in 1 to 1 above.

Hybrid P Plasmi according to the present invention? Some specific examples are as follows.

(a) pMs 18 pMS 18 is the actinomycete plasmid pSF 765 (
1.3 Md containing KMM sex gene-■ in 4.RMd)
The actinomycete plasmid (6,
1Md) (Fig. 2).

(b) pMs21 pMS 21 is the actinomycete plasmid psF689 (9
, 1Md) containing the KMM sex gene-■
Ligate NA fragments 1. actinomycete plasmid Y (14,
3Md) Yes (Fig. 4).

Note that these plasmids P and the bacteria transformed by them are 1. It has been deposited at the National Institute of Fine Technology (details below).

When the corresponding host bacteria are transformed with these KM-resistant white locusts, the host bacteria can express KM resistance.

(16) The KM plasmid can be used to investigate the genetic properties of this KMM gene. That is,
KMM gene of the present invention - DNA fragment (1,3
When S. lividans 66 was transformed with plasmid pM818 (Fig. 2), in which Md) was integrated into Streptomyces plasmid psF765, 100μ of kanamycin A was transformed.
A recombinant resistant to g/ml was obtained, but the cell-free extract prepared from the cultured cells was infused with aminoacetyltransferase CAAC (
6′)] activity was significantly observed, whereas nucleotidylation and phosphorylation activities were not detected. Therefore, it is clear that the KMM gene-■ encodes aminoacetyltransferase. on the other hand.

A plasmid yI in which a DNA fragment (5,2Md) containing the KMM gene-■ was incorporated into actinomycete plasmid F″psF689
) When S. lividans 66 was transformed with MS21 (Fig. 4), a recombinant resistant to kanamycin A at 250 μg/m+ was obtained, but acetylated and nucleotidyl K such as oxidation and phosphorylation
Since no M-inactivating enzyme activity was detected, it was shown that the KM resistance gene-■ has a different resistance mechanism from that of the KM resistance gene-■.

Examples Example 1 [Preparation method of pMs 18 and properties of KM resistance gene-■] Streptomyces kanamyceteicus (Feikoken Bacterial Serial No. 3363) was cultured in MYG medium (1% malt extract,
4% yeast extract, 0.5% glucose.

p) (7,0) 80ml was inoculated and cultured with shaking at 4°C for 2 days, and the bacteria were collected by centrifugation at 10,000 xg/10 minutes. From this bacterial cell, a known method (Methods In Enzymology) by Smith et al.
=, 12.545.1967) and purified with TESL buffer (10mM Tris-HCl).
, P+H8,0,1mM EDTA, 2.5mMNa
It was dialyzed against C1) to obtain donor DNA.

In this way, 6 μg of the donor DNA obtained in (-) and 1.5 μg of plasmid PpSF765 were mixed, and the total volume was 100 μl.
10mM Tris-HCI, pH 7, 4, 6mM dithiothreitol.

], 00 mM MgCI, 250 mM NaCl, 5 units of restriction enzyme Bcl I was added to a 1 tll reaction solution, and 50
The reaction was carried out at ℃ for 2 hours.

Add TESH buffer (0, 2M Tris-H
CI, pH 8, 0, 2 fimM dithiothreitol, 50 mM MgCl) was added to the 1st volume (-10 μl of phenol solution and shaken for 1 minute).
After centrifugation in Ox g for 15 min, the upper layer (aqueous layer) was removed with an A stool pipette.

This aqueous layer containing DNA was extracted with ethyl ether.
l' l, , 300μI after removing phenol
of ethanol and left at -80℃ for 2 hours, 10,
The DNA was precipitated by centrifugation at 000xg for 15 minutes.

The DNA thus obtained was washed with 500 μm of ethanol, dried under reduced pressure, and then dissolved in 40 μl of heat-sterilized pure water. Ta. This DNA solution was heated to 70°C for 1
Heat treated for 0 minutes, then gradually cooled to room temperature, and added buffer solution (0
, 66MTris-HCI, pH 7,6,66mMM
gCI2゜0.1M dithiothrei, 1. OmMATP
) and 5 μm (1 unit) of T4 ligase were added and reacted at 22°C for 2 hours to prepare recombinant DNA of psF765 and Streptomyces mosquito (]9] Namyceteicus DNA (also.

This DNA was used to create a host bacterium Streptomyces lividans 66 (Lomovskaya et al: G
Genetics, 68° 341, 1971) was transformed using the known method of Cheyter et al. (Curr, Top
ics Microblol, Immunol,
96°69.1981). That is,
80ml YEME medium (34% sucrose, 0.3% yeast extract.

0.5% Nosectopezotone 5063% Malto Extract, 1
% glucose, 5mM MgCI2.0.5% glycine, pH 7.0) was inoculated with Streptomyces lividans 66 and cultured with shaking at 32°C for 36 hours.
The mycelia were collected by centrifugation at 10,000
M sucrose, 1.4mM K2SO4, IQrnMMgCI
□, 0.4mM KH2PO4゜2.5mM CaC+
2.25mM TES buffer, PH7,2,11500
It was suspended in a trace amount of the original accumulated liquid. Then a final concentration of 1 m
Egg white lysozyme was added to the mixture at a concentration of 1-g/ml and kept at 32°C for 60 minutes to form protoplasts, and mycelium that did not form into protoplasts was removed by cotton filtration (20). Protoplasts were collected by centrifugation at 800×g/7 min, washed once with P medium (then diluted with 2 ml P
It was suspended in a medium. (Composition of fine matrix liquid (in 1 liter): 40mgZnc12.200mgFec13
゜6H20, 10mg CIIC+2 War 2H20, 1
0rng MnC12・4H20.

10mgNa2B4O7・10H20,10mg (N
T(,)6MO7024-4H20 human protoplast solution 100 μm, P-maleic acid buffer prepared to 3/2 concentration (2.5% sucrose, 1.4rnM K2SO4, 11
500k micromouth/Yuanju Ding liquid, 100rnM100rn
, 50ml Tris-maleic IL pH8,o)
100 μm.

Recombinant DNA solution 50 μm and 375 μm polyethylene glycol I solution (PolJ ethylene glycol +1
．． 00033%/p-maleic acid buffer) and 1
After standing at room temperature for a minute, dilute with 5 ml of P medium and incubate at 800 x
Protoplasts were collected by centrifugation at g/10 min and 2 m
1 of P medium. This protoplast solution was added to 0.
Add 1ml of R2YE agar medium (0.3M sucrose) to a 9cm diameter circular plastic plate.

1.4mM K2SO4, 50mM MgCl. , 1% glucose.

0.01% casamino acids, 1150 mass element solution,
0.4mMKH2P04.20mMCaCl2.0.3
% proline, 25mM TES buffer, pH 7,2,5
(mM NaOH, 0.5% yeast extract, 2.2% agar) and cultured at 31°C for 4 days. Ta. Bacteria regenerated from protoplasts on an agar medium were grown in kanamycin-added μg/ml' and tr-benzene medium (1% glucose, 0.2% pezotone).

0.1% meat extract, 0.1% yeast extract, 2% agar, pH 7.0) were replicated using velvet cloth and cultured at 31°C for 2 days. As a result, 7 kanamycin-resistant clones of 5 types of Streptococcus were obtained, and each of these colonies on the selective medium was scraped off with Aze and mixed with 0.1 to 0.2 ml of sterile water using a glass Botter homogenizer.
ml, apply this on R2YE medium and incubate at 31°C.
The cells were cultured for 5 days.

The five obtained kanamycin-resistant Streptomyces lividans 66 strains were each inoculated into 80 ml of YEME medium.
．． From cultured bacterial cells shaken at 31°C for 2 days, a well-known method by Hansen et al. (J, Bacteriol, 135
222, 1978). When Streptomyces lividans 66 was transformed again with these Shirasumi P, kanamycin resistance was expressed in all of them (. When examined by electrophoresis, both psF 765 were 4.8Md.
In addition, a common NA fragment of 1.3Md was observed. Furthermore, these Shirasumi r were 5alI.

When the fragment was cut with restriction enzymes Ava I and Pvu H and examined in detail, the direction in which the 1.3M dDNA fragment was integrated into plasmid psF765 was not constant. However, the resistance level of Streptomyces lividans 66 to kanamycin A was 100 μg/ml when transformed with two types of Cillasmi r with different insertion directions of 1.3M dDNA.

There was no change in resistance depending on the insertion direction.

The kanamycin resistance gene encoded by the 1.3M dDNA is shown in Figure 1 as kanamycin resistance gene-I.
The restriction enzyme map of the fragment and the restriction enzyme cleavage map of pSF765, which has incorporated this DNA fragment and is named 9MS18, are shown in FIG. Ta.

-1:) To investigate the kanamycin resistance (23) resistance mechanism of fma/(sin resistance gene-I), we
3 g of wet bacterial cells were obtained by culturing Su66 (pMs 18) in 80 ml of MYG medium at 31-C for 2 days with shaking and centrifugation. This bacterial buffer solution (20m
M sodium phosphate buffer, pH 7-0, 10rnM
(CHaCOO) 2Mg, 60mM KCl, 1mM
Wash once with dithiothreitol (-1 same buffer 7.5
1-13 After suspending in
The supernatant obtained by centrifugation at 0,000 x g/30 minutes was mixed with a crude enzyme solution (-).The crude enzyme solution was tested for the activity of kanamycin inactivating enzyme using kanamycin A. Namely, 100mM phosphate (-Na) buffer, p+(7,0,60mM KCl.

10mM (CH3COO)2Mg, 1mM dithiothreitol.

0.5mM Kanamaishi yA, 4mMATP, 0.1m
1 crude fermentation enzyme total ft0.2ml reaction system at 37℃/2
The remaining activity to kanamycin was determined by Bacillus subtilis.
s) was assayed by the Peno R-disc method using assay bacteria, and the enzymatic activities of kanamycin A phosphorylation and nucleotidylation were investigated. Among the above reaction systems, the acetylation reaction is AT
The same procedure was performed by excluding P(24) (- and adding 2mM acetyl-CoA. As a result, kanamycin A was phosphorylated.

It was not inactivated by the adenylation system, but was inactivated by the acetylation reaction.

Furthermore, when we examined the acetyltransferase activity using other aminoglycoside antibiotics as substrates, we found that it was particularly strongly inactivated by kanamycin A, dipekacin, and ri-sho-stamycin (-but not by gentamicin and ribi-P-mycin). Only a relatively low degree of inactivation was observed. Therefore, the kanamycin resistance gene-■ was found to be aminoglycocyanyl P acetyltransferase AAC (6).

Example 2 [Preparation method of pMS21 and kanamycin resistance gene-■
[Characteristics] 6 μg of DNA of Streptomyces kanamyceticus (Feikoken Bacillus No. 3363) and plasmid psF68
92/! jg and 10mM Tris-HCI
, pH 7,2°, 10 mM MgCl, 6 mM dithiothreitol in a total volume of 100 μl.
Add 5 units of I and react at 37°C for 2 hours to form D
Cut the NA1. Ta.

Recombinant DNA was prepared in the same manner as in Example 1.

Streptomyces lividans 66 was transformed with the recombinant DNA of psF689 and Streptomyces kanamyceteicus DNA in exactly the same manner as in Example 1, and screened with a selection medium containing added μg/ml kanamycin. As a result, 7 kanamycin-resistant cloe strains were obtained. Plasmids were extracted from 7 strains of bacteria (2. After being cut with various restriction enzymes and examined by agarose gel electrophoresis, 7
Plasmi P of three of the strains was 1.
3Md DNA fragments were generated, all of which matched the DNA fragment of kanamycin resistance gene-I. remaining 4
When the plasmid of the strain is cut with PstI, the common 5.2
MdDNA fragment is integrated into psF689, 5.
Although the direction of insertion of the 2M dDNA fragment into psF689 was not constant, no matter which direction the 2M dDNA fragment was inserted, these plasmids were transformed into 1. The resistance of Streptomyces Q lividans 66 to kanamycin A was 250 μg/ml, with no change observed.

The gene that is different from this) kanamycin resistance gene -■ is named kanamycin resistance gene -■, and the 5 genes containing this gene are named kanamycin resistance gene -■.
．． The restriction enzyme cleavage map of the 2M dDNA fragment is shown in Figure 3.
7. In addition, psF6 incorporating a 5.2M dDNA fragment
89 was designated as plasmid)' pMS21.

Figure 4 shows the restriction enzyme cleavage map.

A crude enzyme solution was extracted from Streptomyces lividans 66 cells transformed with pMS 21 using the method described in Example 1, and the activity of the kanamycin A inactivating enzyme was examined. No enzyme activity of zylation was detected.

pMs 18 with kanamycin resistance gene-■ and this p
The susceptibility of Streptomyces lividans 66 transformed with Ms 21 and Streptomyces lividans 66 to various aminoglycoside antibiotics was investigated. Streptomyces lividans 66 (pMs1) is resistant to a wide range of aminoglycoside antibiotics.
8), whereas Streptomyces (27) lividans 66 (pMs 21)
It was specifically resistant to , but weakly resistant to other antibiotics, showing completely different resistance, 6 turns. Therefore, this kanamycin resistance gene -■ is the resistance gene -■ (AAC(6'
)) It has become clear that it has a different resistance mechanism.

1) Accession No. 3363 (June 11, 1976) 2)
Mycological properties (1) Microscopic morphology Aerial hyphae are produced from branched basal aerial hyphae with a width of about 1 micron. Aerial mycelium is relatively not densely divided. Spiral threads and circular threads are usually not recognized. Spores are formed from the ends of aerial hyphae, and the spores are usually oval or cylindrical.

(2) Glycerin Czapek agar medium (cultured at 2'7°C) The growth is colorless at first, but later becomes lemon yellow, and the underside is grass yellow. Aerial mycelium is white to yellow, sometimes tinged with green or pale pink (28). No soluble pigment is produced, or a brownish soluble pigment may be produced.

(3) Kleinsky's Agar medium (cultured at 27°C): Growth is white to yellow. The underside is white, pale pinkish-white, yellow or hay-colored Mycelia are usually few and occur from the center of the colony, and are white, pale pinkish-white, greenish-yellow, or yellow. Soluble dyes are not normally produced.

(4) Calcium malate agar medium (cultured at 27°C)
: The properties are almost the same as those on Kleinsky's Bructrose iR Ragin agar medium, but the growth is weak and sometimes the growth is far apart.

(5) Starch agar plate medium (cultured at 27°C): No localized growth (-, exhibits almost the same properties as on Kleinsky's glucose agar medium, but usually no aerial mycelia are observed. Starch ice-melting ability (16mm/7mm).

(6) Potato plugs (cultured at 37°C) Rainbow-like growth 121 The surface is granular and light tan to yellow in color. No aerial hyphae are produced, or a small amount of white aerial hyphae can be heard. Soluble pigments are usually not observed. Potato plugs may turn pale and dark at the point of growth.

(Cultured at 2'7℃): Normally, growth is poor.In addition, the findings are almost the same as those of potato stems.

(8) Peptone water (0.2% sodium nitrate, cultured at 27°C)
: Forms a white precipitate or forms colorless to yellow annular growths on the surface. White aerial mycelium may be observed. Forms nitrate-free.

Does not produce soluble dyes.

(9) Pezoton meat extract agar medium (cultured at 37° C.): 1-1 Aerial mycelia and lytic pigments are not usually produced, with wrinkled white to yellowish growth.

00) Blood agar medium (cultured at 37°C) Rainbow-like gray to brown growth is observed, and aerial mycelium-soluble pigments are usually not observed.

Ql) Milk medium (cultured at 37°C): Almost no change is observed, and no growth is usually observed on the surface.

αri Egg culture (cultured at 37°C) shows rainbow-like yellow growth (-1 aerial hyphae are not observed).

(13) Leffler clotted serum medium (cultured at 37°C): Localized white to lemon yellow growth, no soluble pigment observed. No liquefaction is observed.

α荀 Gelatin medium: Liquefaction is observed. Usually soluble dyes are not washed away.

(Usage of carbohydrates) The results of testing the utilization of various carbohydrates using Tuapek salt as a basal medium are as follows.

Commonly used carbohydrates: arapinose, dextrin, fructose, galactose. Glucose, glycerin, maltose, mannitolmannose, raffinose, starch, sucrose, sodium succinate.

Used and miscellaneous: Inojit. inulin, lactose,
Rhamnose, turdace, gycylose, sodium acetate.

Rarely used 1 - None: Aesculin, salicin, sorbitol, sodium citrate.

S. platensis 5F689 1) Accession number (31) FIKEN Article No. 121 (May 1, 1980) 2) Mycological properties JP-A-57-188600 and JP-B-Sho 46-6
See Publication No. 076 S. Fraday Ryoko 5F765 1) Accession No. Meikoken Meiyori No. 124 (May 1, 1981) 2) Mycological properties See Japanese Patent Application Laid-open No. 188600/1983.

S @ Lividance 1) Accession number: Microtechnical Research Institute Bacteria No. 6982 (March 10, 1947) 2) Dutch properties (1) Forms a spiral spore chain L, mature [-] The spore chain is medium moderately long, - consisting of 10 or more spores per chain.

This form is compatible with yeast malt agar, oatmeal agar, starch mineral salt agar and glycerol.
Found on As/ξlagin agar medium, the spore surface is smooth.

(2) The color of aerial mycelia is yeast malt agar medium.

(32) Gray color 1 on oatmeal agar, starch inorganic salt agar, and glycerol/snosoragin agar.
．． It exhibits a reddish color.

(3) The color of the back side of the bacterial cells shows a characteristic pale to dark gray purple or Daleish purple ξ-pur on yeast malt agar, oatmeal agar, inorganic salt agar, and glycerol-asparagine agar. . The pigment on the back side of the bacterial cell is 0.
The color changes from reddish or iolet to blue by adding 05N caustic soda. Moreover, violet or blue changes to red by addition of 0.05N hydrochloric acid.

(4) Soluble pigments: Melanin-like pigments are not formed in pezotone-yeast iron agar, tyrosine agar or tryptone-yeast liquid media. A blue or iolet coloration is observed in yeast malt agar, oatmeal agar, starch inorganic salt agar, and glycerol-asparagine agar. This pigment is PHK sensitive and undergoes the same changes as described for the pigment on the back side of the bacterial cell.

(5) Sugar assimilation: D-glucose, L-arabinose, D-xylose. l-inositol.

Assimilates all of D-mannitol, D-fructose, rhamnose, sucrose and raffinose. Just 17. Bacterial growth on sucrose and raffinose is poor compared to other sugars.

S. lividans 66 (pMS 18) 1) Accession No. 6946 (March 2, 1980) 2) Mycological properties Except for the properties introduced by Plasmi rpMS18.

It is the same as that of S. Lividans 66 (Feikoken Bibori No. 6982).

S. lividans 66 (pMS 21) 1) Accession No. 6947 (March 2, Showa Leap Year) 2) Mycological properties Other than the properties introduced by plasmid pMs 21,
S, IJ Bidans 66 (Feikoken Bibori No. 6982)
It is the same as that of

[Brief explanation of the drawing]

FIG. 1 and FIG. 3 are restriction enzyme cleavage maps of the KM resistance genes of the present invention -■ and ■, respectively. Figures 2 and 4 show the plasmid pMs of the present invention, respectively.
Restriction enzyme cleavage maps of pMS 18 and pMS 21. Applicant's agent Seiyoshi Inomata 1 Figure 2 Figure 53 Figure 64 Amendment to figure procedure 1982 K, October-2/] Commissioner of the Patent Office Kazuo Wakasugi 1, Indication of case 1988 Patent Application No. 52275 No. 2, Name of the invention Kanamycin resistance gene and plasmid 3, Person making the amendment Relationship to the case Patent applicant (609) Meiji Seika Co., Ltd. 7, Subject of the amendment Meika's "Detailed Description of the Invention" column 8. The statement of contents of the amendment shall be amended as follows. Takayuki Before correction After correction 146 Fradiae Fradiae 205
Dithiothlate-EDTA 2310 Class 51 Same number 5 pieces 283-4 Different Different 2913
Basal Silver Thread Basal Mycelium (1) Procedural Amendment 1980 2/Otsu [1 Commissioner of the Patent Office Kazuo Wakasugi 1, Indication of the Case 1988 Patent Application No. 52275 Shirasmid 3, Person Making Amendment Case and Relationship with Patent Applicant (609) Meiji Seika Co., Ltd. [Telephone Tokyo (21]) 2321 Main Representative] 6 Subject of Amendment "Contents of Amendment" of the Procedural Amendment Officer dated October 21, 1981
Column (2) 7. Contents of the amendment Written amendment dated October 21, 1982, page 2, lines 9-10, 1-29 8 8. Kanamisex S, Myceteika (parent stock) S" was deleted,
The statement on page 4, line 8 of the specification is restored to what it was at the time of filing.

Claims (1)

[Claims] 1. A kanamycin resistance gene selected from the group consisting of (I) and (■) below. (Il Kanamycin resistance gene - ■ (a) This gene is the DNA of Streptomyces kanamyceteicus.
It is contained in the DNA corresponding to the 1 to 3 megadalton fragment obtained by cleaving A with the restriction enzyme BCI■. (b) The restriction enzyme cleavage map of this gene is shown in Figure 1. (c) This gene is an aminoacetic acid that acetylates the 62-amino group of aminoglycoside antibiotics. (b) This gene is a DNA of Streptomyces kanamyceticus.
It is contained in the DNA corresponding to the 5.2 megadalton fragment obtained by cutting with the restriction enzyme Pst I. (b) The restriction enzyme cleavage map of this gene is shown in Figure 3. 2. The kanamycin gene according to claim 1, wherein the DNA containing the gene is derived from Streptomyces kanamyceteicus or a mutant strain thereof. 3. A DNA fragment derived from Streptomyces kanamyceticus or a mutant strain thereof selected from the group consisting of (1) and (2) below. (1) Cut the DNA of Streptomyces [phase] Kanamycetacus or its mutant strain with the restriction enzyme BCI I [
A DNA fragment of 1 to 3 megadaltons containing the kanamycin resistance gene -■ obtained by -. (2) 5,2 containing the kanamycin resistance gene -■ obtained by cleaving the DNA of Streptococcus kanamyceticus or its mutant strain with the restriction enzyme pst I
Mega Dalton's DNA II'Jj piece. 4. A hybrid plasmid obtained by incorporating a kanamycin resistance gene selected from the group consisting of (1) and (IT) below into a vector plasmid. (I) Kanamycin resistance gene -■ (A) This gene is the D of Streptomyces kanamyceteicus.
It is contained in the DNA corresponding to a 1 to 3 megadalton fragment obtained by cutting NA with the restriction enzyme BclI. (b) The restriction enzyme cleavage map of this gene is shown in Figure 1. (c) This gene is an aminoase that acetylates the 6'-amino group of aminoglycoside antibiotics. (b) This gene is a DNA of Streptomyces kanamyceteicus.
It is contained in the DNA corresponding to the 5.2 megadalton fragment obtained by cutting the 5.2 megadalton fragment with the restriction enzyme PstI. (b) The restriction enzyme cleavage map of this gene is shown in Figure 3. 5. Vector plasmid P0 according to claim 4, wherein vector plasmid P is an actinomycete plasmid.