JP2986338B2 - Double-stranded DNA phage φIN93 that can be induced from hyperthermophilic bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel double-stranded DNA phage φIN93 which can be derived from a highly thermophilic bacterium of the genus Thermus.

[0002]

BACKGROUND OF THE INVENTION Many researchers are infected with thermophilic bacteria
Report on bacteriophage. These good
Many thermophilic bacteriophages are moderately thermophilic bacteria
Infect Bacillus spp. [On
odera, N. (1961)J. Electronmicroscopy Ten, 91-102; S
hafia, F. and Thompson, T.L. (1964)J. Bacteriol. 89,
999-1002; Welker, N.E. and Campbell, L.L. (1965)J.
Bacteriol. 89, 175-184; Sharp, R.J. et al. (1986)J.Ge
n.Microbiol. 132, 1709-1722]. On the other hand, high thermophilic fineness
Very few reports of bacteriophage infecting bacteria
Oshima et al. Are thermus sir, a highly thermophilic bacterium.
Infects the morphophilus (Thermus thermophilus) HB8
Phage φYS40 was first isolated from soil.
Sakaki, Y. and Oshima, T. (1975)J. Viro
logy. Fifteen, 1449-1453].

[0003] Also, many vector systems using Escherichia coli as a host useful for gene recombination techniques have been reported. Although these vector systems are effective in producing various useful substances in E. coli, they are not suitable for producing substances produced by thermophiles. The production of a thermostable protein requires a vector system using a thermophilic bacterium as a host. Recently, Hoshino et al. Developed a drug-resistant plasmid vector for the Bacillus stearothermophilus, a moderately thermophilic bacterium [Hoshino, T. et al. (1985) C
an.J. Microb. 31 , 339-341]. On the other hand, for highly thermophilic bacteria, the above-mentioned phage φYS40 of Oshima et al. Is known, and Hishinuma et al. Are searching for a plasmid that can be a vector [Hishinuma, F. et al. (1978)
J. Gen. Microb. 104 , 193-199], a useful host vector system utilizing these phages or plasmids has not yet been developed.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have obtained a strain of about 19.4 kb (size: 19.4 kb) from a bacterium belonging to the genus Thermus (Thermus aquaticus TZ2 strain) obtained by newly screening from soil. Phage φIN93 using double-stranded DNA (about 1.3 × 10 ⁷ daltons) as a nucleic acid was induced. The phage φIN93 is clearly different from the phage φYS40 because the DNA size of the phage φYS40 listed above is 1.36 × 10 ⁸ daltons. That is, the present invention provides a novel bacteriophage φIN93 that infects highly thermophilic bacteria, and a Thermus aquaticus TZ that lysogenizes this phage.
It offers two strains. Such hyperthermophilic bacteriophages are useful in characterizing the biological properties of phage and in creating vector systems for hyperthermophilic bacteria.

[0005]

Means for Solving the Problems The phage of the present invention can be obtained as described in Examples below. Briefly, mitomycin C (a phage inducing agent) is added to the Thermus aquaticus TZ2 strain and cultured to induce phage outside the cells. After diluting the phage of the culture supernatant, the phage is plated on an agar plate together with the indicator bacteria to form plaques. The resulting plaque is scraped off and cultured with the indicator bacteria to amplify the phage. Phage can be isolated and purified from a cell culture supernatant containing the phage by a conventional method.

[0006]

The present invention will be described in more detail with reference to the following examples. A. Bacteria and culture medium Thermus aquaticus TZ2 strain was isolated from soil near the source of Fukuchi Onsenkyo in Gifu Prefecture. Thermus aquaticus (ATCC 27634) used for comparative analysis with the isolated bacteria was Ameri
purchased from can Type Culture Collection. A2 medium consists of 0.1% tryptone (Difco) and 0.1% yeast extract
(Difco) containing a basic salt solution of Castenholtz [Brock, T .;
D. and Freeze, H. (1969) J. Bacteriol . 98 , 289-297]. C3 medium is a basal salt solution (pH 7.5) containing 0.8% tryptone and 0.4% yeast extract. Casten
The holtz 10 × basal salt storage solution is composed of nitrilotriacetic acid (1 g) and CaSO ₄ .2H ₂ O (0.6 g / L of distilled water).
_{g), MgSO 4 · 7H 2} O (1.0g), NaCl (0.08g),
KNO ₃ (1.03 g), NaNO ₃ (6.89 g), Na ₂ HPO ₄
(1.11 g), FeCl ₃ solution (0.28 g per liter of distilled water)
(10 ml), containing Nitsch trace element solution (10 ml). This was adjusted to pH 8.2 with 1N NaOH,
Autoclaved. Nitsch trace element solutions
H ₂ SO ₄ (0.5 ml), MnSO _{4.
H 2 O (2.2g), ZnSO} 4 · 7H 2 O (0.5g), H 3 BO 3
(0.5 g), CuSO ₄ (0.016 g), Na ₂ MoO ₄ .2H ₂
O (0.025 g) and CoCl ₂ .6H ₂ O (0.046 g). The agar medium was prepared by adding 3% agar [Wako Pure Chemical Industries, Osaka] to A2 medium. The soft agar medium was prepared by adding 1.5% agar to A2 medium.

B.Bacterial culture characteristics Shaking culture of Thermus aquaticus TZ2 strain in A2 medium
did. This strain can grow in the temperature range of 45 ° C to 85 ° C
And exhibited the characteristics of highly thermophilic bacteria. Also, this strain
Gram-negative rod, 0.5 μm in diameter and 3-5 μm in length
It was a fungus and no sporulation was observed. Furthermore, this
During the cessation period of growth, the cells are rod-shaped or radial.
Phenomena such as clustering and arranging of cells in a circle were observed. this
The morphological characteristics of such a TZ2 strain are described by Brock et al.J.Bacterio
l. 98, 289-297 (1969)]
This was in good agreement with the culture characteristics observed during the arrest period.

C. Bacterial Chemical Characteristics Thermus aquaticus TZ2 strain was transformed into A2 medium (5 L).
The medium was shake-cultured at 75 ° C. for 15 hours and centrifuged at 1500 × g to collect the cells. Next, the cells were washed with distilled water, freeze-dried and used as the following analysis samples. Analysis of isoprenoid quinone was performed using chloroform-methanol (2: 1, v / v).
After extracting quinone from the dried cells using v), analysis was performed by reversed-phase high-performance liquid chromatography using methanol-isopropanol (1: 1, v / v) as a developing solvent.
For the analysis of GC content, DNA was extracted from dried cells using Tris-SDS and phenol, treated with nuclease P1, and then analyzed by reversed-phase high performance liquid chromatography. Analysis of fatty acid composition was performed using 6 dried cells.
After hydrolyzing with N hydrochloric acid and methylating the fatty acid extracted with hexane, it was analyzed by gas chromatography. The analysis of the cell wall amino acid composition was carried out by pulverizing the dried cells with an ultrasonic grinder and hydrolyzing the cell wall prepared by SDS and Pronase E with 6N hydrochloric acid.
Analysis was performed by an automatic amino acid analyzer. As a result, TZ
Two strains contained menaquinone-8, ornithine was found as a diamino acid of cell wall peptidoglycan, and diaminopimelic acid was not found. High concentrations of alanine and glycine were observed in peptidoglycan.
The GC content of the TZ2 strain was 65%. The chemical properties of these TZ2 strains were clearly different from those of Bacillus spp. And were in good agreement with the properties of Thermus spp. Furthermore, assimilation of the TZ2 strain with respect to glycine, glutamine, pyruvic acid and 17 types of sugars was examined. The TZ2 strain is B
Rock et al . [ J. Bacteriol. 98 , 289-297 (1969)] separated Thermus aquaticus YT-1 and Oshima et al . [ J. Virology.
15 , 1449-1453 (1975)], unlike the isolated Thermos thermophilus HB8, in which no pigment was observed.
Robert et al. [Adams, MH (1950) Methods Med. Res. 2 , 1-7
Dyeless type Thermos aquaticus X reported by 3]
Probably the same as -1.

The results are summarized in Table 1 below.

TABLE 1 thermophilic T. aquaticus TZ2 strain taxonomic characteristics identified item TZ2 strain properties Gram stain Negative Spore formation without motility without colony color white oxidase + catalase + Growth temperature (° C.) 45 to 85 ° C. Growth pH 5.7 to 10.2 Isoprenoid quinone MK-8 G + C content (mol%) 65 Intracellular fatty acid: Non-polar fatty acid i15: 0 + i17: 0 * Hydroxy fatty acid Not detected Cell wall amino acid (molar ratio): Glutamate 1.00 Glycine 2.89 alanine 4.28 ornithine 0.91 lysine 0.23 meso-diaminopimelic acid 0.000 *: i indicates isoform branching, the first number indicates the number of carbon atoms, and the last number indicates the number of double bonds. The highly thermophilic bacterium Thermos aquaticus TZ2 strain according to the present invention has been deposited with the National Institute of Bioscience and Human Technology as FERM P-13713 (deposit date: June 29, 1993).

D. Induction of phage The Thermus aquaticus TZ2 strain was cultured in a C3 medium (2 ml) containing 0.5 μg / ml mitomycin C at 75 ° C. for 2 hours with shaking, and then centrifuged at 1500 × g for 10 minutes to remove the supernatant. A fresh medium (2 ml) was added and the cells were further cultured for 15 hours. The resulting culture was centrifuged at 1500 × g for 10 minutes, and the supernatant was used as a phage solution. Phage φIN
93 is transmitted by the method of Adams et al . [ Methods Med. Res.
2 , 1-73 (1950)]. That is, a soft agar medium (5 μl) in which the A2 medium culture (100 μl) of the Thermus aquaticus TZ2 strain and a diluted phage solution (100 μl) were maintained at 55 ° C.
ml) and inoculated on an agar medium. 15 at 75 ° C
After culturing for 1 hour, the resulting clear plaque having a diameter of 1 to 3 mm was scraped off with an agar with a Pasteur pipette, added with an A2 medium culture solution (2 ml) of the S. aquaticus TZ2 strain, and cultured at 75 ° C. for 15 hours. Was amplified. When the TZ2 strain cultured in C3 medium was treated with 0.5 μg / ml mitomycin C, 10 ⁵ PFU / ml φIN93
Was triggered. On the other hand, phage induction was not observed from the TZ2 strain cultured in the A2 medium even when treated with mitomycin C. Thus, induction of φIN93 was found to be medium-dependent.

E. Purified φIN93 particles of phage φIN93 were prepared according to the method of Yamamoto et al. [ Virology 40 , 734].
-744 (1970)]. That is, 2.5 M NaCl (100 ml) containing 25% polyethylene glycol 6000 was added to the culture supernatant (500 ml) containing the φIN93 particles, left overnight at 4 ° C., and centrifuged at 11,000 × g for 30 minutes. . The precipitated φIN93 particles were mixed with SM buffer [0.1
M NaCl, was dissolved in 50mM Tris -HCl (pH7.5)] (2ml) containing 7.5 mM MgSO ₄ and 2% gelatin,
Density gradient centrifugation with 1.70 to 1.15 g / ml CsCl (3
ΦIN93 particles were separated at 2,000 rpm (1 hour). After separating the white band of φIN93 particles, SM
It was dialyzed overnight against a buffer to obtain purified φIN93 particles.

F. Properties of φIN93 DNA φIN93 DNA was purified by extracting the above purified φIN93 particles with phenol / chloroform.
This purified φIN93 DNA was used for base composition analysis, cleavage experiments with various enzymes, and size measurement. φI
When the base composition analysis of N93 DNA is performed, G: C: A:
T = 31: 34: 17: 18, and the molar ratios of G and C and A and T showed almost the same value. The result is φ
This shows that IN93 DNA is double-stranded DNA. The GC content of φIN93 DNA is 65%.
And showed a high value that was almost the same as the GC content of the DNA of the host cell, and no modified base was detected. When cutting experiments were performed using various restriction enzymes, φIN93
DNA was found to be cleaved by several types of restriction enzymes as described below. From this, φIN93 DN
A was shown to be double-stranded. φIN93 DNA
Was subjected to 1.0% agarose gel electrophoresis [McDonell, MW et al.
77) J. Mol . Biol. 110 , 119-146] (FIG. 1). In this figure, lane 1 is φIN93 DNA, and lane 2 is HindIII digested fragment of λ phage DNA (molecular size 23,130, 9,416, 6,557,4,36).
1, 2,322, 2,027, and 564 bp).
From this result, it was found that the length of φIN93 DNA was about 20 kb.

G. Restriction enzyme digest of φIN93 DNA
The double-stranded FaiIN93 DNA was analyzed purified was cleaved with various restriction enzymes. The restriction enzymes used were BamHI, PstI, Kpn
I, XhoI, DraI and EcoRI (Takara Shuzo), and AccI, SphI, HindIII and XbaI (Nippon Gene). Double-stranded DNA (1 μg) was reacted with restriction enzymes (10 units each) at 37 ° C. for 2 hours in an appropriate buffer (20 μl). The digested sample is subjected to a 1.2% agarose gel or a 6% polyacrylamide gel,
Electrophoresis was performed at a voltage of 10 V per 1 cm width for 2 hours. After the electrophoresis, the gel was stained with a 1 μg / ml ethidium bromide solution, and the gel was irradiated with ultraviolet rays to determine the number of generated fragments.
HindIII digested fragment of NA or φX174 phage DN
The molecular weight of each fragment was calculated based on the known molecular weight of the HincII digested fragment of A. These results are summarized in Table 2 below.

Table 2 Restriction enzyme cleavage analysis of double-stranded φIN93 DNA Restriction enzyme having cleavage sensitivity AccI BamHI PstI SphI HindIII KpnI Number of cleavage sites 68 6 2 3 4 Length of each generated fragment (kb) 12.0 5.4 12.0 15.0 19.0 17.3 2.3 4.1 3.3 7.0 1.5 2.0 2.1 2.9 1.6 1.0 0.9 1.4 2.7 1.3 0.8 1.2 1.4 1.2 0.3 1.3 0.6 0.5 0.4 φIN93 DNA 19.3 18.7 22.0 22.0 21.5 21.0 length (kb) cleavage-insensitive restriction enzyme XhoI, XbaI, DraI, EcoRI

H. Restriction enzyme map of φIN93 DNA To prepare a restriction enzyme map, purified double-stranded φI
N93 DNA was cut with various restriction enzymes. The restriction enzymes used were BssHII, HindIII, NotI, NruI, Sph
I and XbaI (Nippon Gene). The same sample was cut simultaneously using two or three types of restriction enzymes. DNA cleavage was performed using φIN9
3 DNA (1 μg) was reacted with restriction enzymes (10 units each) in the appropriate buffer (20 μl) at the appropriate temperature for 2 hours. The digested sample was applied to a 1% agarose gel, and electrophoresed at a voltage of 10 V per 1 cm width for 2 hours. After the electrophoresis, the DNA was stained with a 1 μg / ml ethidium bromide solution, and the gel was irradiated with ultraviolet rays to detect DNA. The molecular weight (kb) of the obtained cleavage fragment was calculated based on the known molecular weight of the HindIII digested fragment of λ phage DNA, and the cleavage sites of various restriction enzymes were analyzed. As a result, it was found that the length of φIN93 DNA was about 19.4 kb, and the restriction enzyme map shown in FIG. 2 was obtained.

[Brief description of the drawings]

FIG. 1. The size of phage φIN93 DNA was 1.0
5 is a photograph instead of a drawing showing the result of examination by% agarose gel electrophoresis.

FIG. 2 is a schematic diagram of a restriction enzyme map of phage φIN93 DNA.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 C12N 1/00-7/08 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A phage which can be derived from a bacterium belonging to the genus Thermus, wherein the nucleic acid is a double-stranded DNA of about 19.4 kb in length having the following restriction enzyme map: 1) 2. The phage φIN9 according to claim 1, wherein the bacterium belonging to the genus Thermus is Thermus aquaticus TZ2 strain.
3. 3. The phage φIN93 according to claim 1, which can be induced by treating a Thermus bacterium with mitomycin C. 4. A Thermus aquaticus TZ2 strain which lysogenizes phage φIN93.