JPS5945877A - Novel microorganism having tetracycline-resistant plasmid - Google Patents
Novel microorganism having tetracycline-resistant plasmidInfo
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- JPS5945877A JPS5945877A JP57157367A JP15736782A JPS5945877A JP S5945877 A JPS5945877 A JP S5945877A JP 57157367 A JP57157367 A JP 57157367A JP 15736782 A JP15736782 A JP 15736782A JP S5945877 A JPS5945877 A JP S5945877A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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Abstract
Description
【発明の詳細な説明】
本発明は好熱菌を宿主とする組換えＤＮＡ実験のベクタ
ーとして有用す新規なプラスミドを保有する新規な微生
物に関り−るーｂのであり、より詳しくはテトラサイク
リン耐性の遺伝子を内部に備え、ぞの分子量が約２．８
メガゲノ１朴ンであり、図に示される制限醇索聞裂地図
に。１：り特徴づ（）られる新規なプラスミドを保有す
る新規なバｆルス・スデアｒｉｌ＋−−モフィルスに関
するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism harboring a novel plasmid useful as a vector for recombinant DNA experiments using thermophilic bacteria as a host, and more specifically relates to It has a gene inside and its molecular weight is about 2.8.
It is Mega Geno 1 Park, and the restriction map shown in the figure. 1: Concerning a new B. suda ril+--morphillus carrying a novel plasmid that has been characterized.
従来、組換えＤＮＡ実験は主どじで大腸菌を宿１謀！づ
−る系了・広（１ｉｌｌ究がおこなわれインシー１リン
、インターフｒロン、に１〜成長ホルモン等が大腸菌で
１産されるなど大きな成果を挙げている。大腸菌の宿主
−ベクター系はほぼ完成されており、また大腸菌以外に
、ｂ酵母、枯草菌などで宿主−ベクター系が開発され応
用への道が検バ（１されつつある。しかし、１配の菌は
いずれも生育温度が３０℃〜３７℃の中温菌である点に
問題がある。Traditionally, recombinant DNA experiments have been carried out mainly because of E. coli! Research has been conducted on the Zuru system, and great results have been achieved, including the production of incy-1 phosphorus, interfuron, and ni-1-growth hormone in Escherichia coli.The host-vector system of Escherichia coli is approximately In addition to Escherichia coli, host-vector systems have been developed using B yeast, Bacillus subtilis, etc., and the path to application is being investigated. However, all of the first strains have a growth temperature of 30 The problem is that it is a mesophilic bacterium with a temperature of ℃ to 37℃.
一方、好熱↑１１細菌は、生育−に限温麻が５５℃〜７
５℃にある中等痕好熱菌と、生育上限温度が７５℃以上
である高度好熱菌とに大別されるが、０１ｒれについて
も、その有する酵素、生体成分が耐熱性、耐溶媒１／目
こ優れ−（いる事が知られており、とりねり好熱菌由来
の耐熱ｆ１酵素及び耐熱＋４１牛体機能のバイオリアク
ター等の工業プロレスへの応用という点から注目を集め
７いる。従−）で、好熱性細菌の育種が重要と考えられ
るが、その為の一つの、しかも有力な手段ど考えられる
好熱ｖ１細菌の宿主−ベクター系の開発研究については
、ベクターの有力１侯補と考えられるプラ・ツミドの検
索を含めてｂ以下の報告しか知られていない。On the other hand, thermophilic ↑11 bacteria grow at 55°C to 7°C.
They are broadly divided into moderate thermophiles, which live at 5°C, and highly thermophilic bacteria, which have an upper limit of growth temperature of 75°C or higher. It is known that there is a heat-resistant f1 enzyme derived from Toneri thermophile and the heat-resistant +41 cow body function is being applied to industrial wrestling, such as bioreactors. -), breeding of thermophilic bacteria is considered to be important, and research on the development of a host-vector system for thermophilic v1 bacteria, which is considered to be one of the most effective methods for this purpose, is one of the leading candidates for vectors. Only reports below B are known, including the search for Plathumid, which is thought to be.
、Ｊ、 Ｇｅｎ、 Ｍｉｃｒｏｂ 、 、 １０４．
１！１３−１９！１ （１９７８）（２） 薬剤耐ｆ
ｌ［の好熱↑」バチルス属細菌Ｊ５りの４種類のプラス
ミドの分１ｌｌ１１どでの性質の部分解析
ヒングハム、△、Ｈ，Ａ、、 １ルトン、Ｃ，Ｊ、
アンドアトキンソン、王。, J. Gen. Microb, , 104.
1!13-19!1 (1978) (2) Chemical resistance f
Partial analysis of the properties of four types of plasmids of Bacillus bacteria J5 Hingham, △, H, A, 1 Luton, C, J,
And Atkinson, King.
、Ｊ、 （’、０ｎ、 Ｍｉｃｒｏｂ 、 、
１１４． ４（１１−４０８（１９７り）（３） バ
゛１ルス・スｊアロザー［フィルスのプラスミドｐＡｒ
３１２４の解析及び欠失誘導体の創製
ヒングハム、△、 １１． Ａ、 、プル１〜ン、 Ｃ
，Ｊ、 アンドア１〜１−ンソン、王。,J,(',0n,Microb, ,
114. 4 (11-408 (197ri) (3) Virus plasmid pAr
Analysis of 3124 and creation of deletion derivatives Hingham, Δ, 11. A, , pull 1~n, C
, J., Andor 1-1-Nson, Wang.
Ｊ、 Ｇｅｎ、 １ｖｌｉｃｒｏｂ 、 、 １１０
． １０９−１１！ｉ （１９８０）（４） 好熱４！
＋バブルス属■１菌Ｊ：りの薬剤耐ｆ！＋プラスミドの
分−１と解析、及び部分欠失プラスミドの創製
イマナカ、■、、フジイ１Ｍ、 アンド アイバ、Ｓ。J, Gen, 1vliclob, , 110
．． 109-11! i (1980) (4) Thermophilic 4!
+ Bubbles genus ■1 Bacteria J: Rino's drug resistance f! + Analysis of plasmids and creation of partially deleted plasmids Imanaka, ■, Fujii 1M, and Aiba, S.
、１． ｒ３ａｃｔ 、 、 １／１Ｇ （３） 、
１０９１−１０９７ （１９８１）（ｊ３） リー
マス・リーモーフイルスから＋＋禰ｔされたプラスミド
（ｐ −ｒ １−１）の物理的性状
「ベルハード、Ｍ、Ｄ、、ハスク１゛ズ、Ｃ０，バレン
ズ■う、Ｐ。, 1. r3act, , 1/1G (3),
1091-1097 (1981) (j3) Physical properties of the plasmid (p-r 1-1) isolated from Remus remorophilus "Berhard, M.D., Husk1's, C0, Barens. ■Uh, P.
、ビキｑｔ、Ｒ，アンド コテ゛ｌノビツク、△。, Bikiqt, R., and Kote Novick, △.
Ｐｌａｓｍｉｄ、 ６．１−６ （１９８１）（６）
プラスミドＤＮＡによるバチルス・ステアロリーモーノ
イルスの形￥ｇ＋ｒ＋＝ｉ負及びバー１ルス・ステアロ
リー−［フィルス、バチルス・ズブヂルス間旦用ベクタ
ーの性質
イマナカ、■、、フジイ１Ｍ、、アラ−しり、■、 ア
ンドアイバ、Ｓ
、１． ＢａＣｌ 、 、 １４９ （３） 、
８２４−８３０ （１９８２）ぞこτパ、本発明者らは
、その宿主が好熱性の微生物であって、その内部に７１
−ラリイクリン耐１１の遵伝Ｔを備えた微生物を自然界
より検索した結果、バチルス属に属する一菌株から新規
なプラスミドを１＋７にとに成功しノだ。Plasmid, 6.1-6 (1981) (6)
Form of Bacillus stearolimonoirus by plasmid DNA g + r + = i negative and bar 1 Bacillus stearoli - [Filus, properties of vector for Bacillus subtilis , Andaiba, S., 1. BaCl, , 149 (3),
824-830 (1982), the present inventors discovered that the host is a thermophilic microorganism and that 71
- As a result of searching for microorganisms in the natural world with a tolerance T of 11 for lariicrin resistance, we succeeded in generating 1+7 new plasmids from a strain belonging to the genus Bacillus.
このプラスミドは前記の制限酵素開裂地図に示され、分
子種は小さく、また種々の制限酵素による特異的な切断
点を有し、テ１ヘラ１ノーイクリンに対Ｉする耐性渭伝
了をプラスミドＤＮＡ上に有し−Ｃいる。、（以下、本
プラスミドをｒ ｒｌＴＩＩＴ １５ 、］ど略称する
。）イｌ−１１３、図に示されている制限酵素の略称は
次のとａ３りである。This plasmid is shown in the above-mentioned restriction enzyme cleavage map, has a small molecular species, has specific cleavage points by various restriction enzymes, and has been shown to be resistant to Te1hera1noicrin on the plasmid DNA. It has -C. (Hereinafter, this plasmid will be abbreviated as rrlTIIT15.) Il-113. The abbreviations of the restriction enzymes shown in the figure are as follows.
（１） Ｃ１ａ）はカリＡ−ファノン・ラツム由来の
酵素（２） ｒｃｏＲｒは−ｒシエリヒア・ニー１り
山ヌ（のＭ’を素−３＝
１３ ） Ｈｈａ Ｉ ＋、＋バー「上フィルス・ハ
エモリティカス由来の酵素（６） １−ａｑｌはり一
−マス・アクアディカス由来の酵素を示ず。(1) C1a) is an enzyme derived from Kali A-Phanon Ratum (2) rcoRr is -r Schierichia knee 1 Riyama Nu (prime M' of -3 = 13) Hha I +, Enzyme derived from H. aquadicus (6) 1-aql Enzyme derived from H. aquadicus is not shown.
以下、テ１−ラリイ′ノリン耐＋１を右する既知の好熱
菌由来のプラスミドとの相違点を表に示す。Differences from known thermophilic bacterium-derived plasmids with teralii'norrin resistance +1 are shown in the table below.
以下余白
４−
人から明Ｉ）かな３１．・うに、ｌ’）ＴｌｌＴ１５は
既知のプラスミドに較べ、分ブンスミドｒ）Ｎ△がベク
ターたり得る為には、ぞのプラスミドが宿主内γ・・の
自？＋’的増殖能、及び選択マーカー（イのプラスミド
が宿主内に存（Ｆ １．／ ”Ｃいることを示ずマーカ
ー）を有していることが必須であるが、「）旧ｌＴ１５
は好熱菌及び枯草菌ｒの自什的！曽殖能及びテトラリイ
クリン耐１ノ１という極めて選択に右利なマーカーを有
している。Margin below 4-person to clear I) Kana 31.・In comparison with known plasmids, sea urchin, l') TllT15 is different from other known plasmids.In order for Bunsumid r)N△ to be able to serve as a vector, is it necessary for the plasmid to be γ...in the host? It is essential that the plasmid has positive growth ability and a selection marker (a marker indicating that the plasmid is present in the host (F1./"C");
is independent of thermophilic bacteria and Bacillus subtilis r! It has excellent reproductive ability and tetraliicrine resistance of 1 in 1, making it an extremely selective marker.
更ニｒ）ＴＩ−１’Ｔ １５は図からも明らかなように
、Ｃ１ａｌ、ＥｃｏＲＴ。As is clear from the figure, TI-1'T 15 is C1al and EcoRT.
１−１１１．Ｉ Ｉ、ｌ−１ｐａ Ｉ’Ｉなどの制限酵
素による開裂部位を特定のしかも限られた位置に右Ｉノ
゛Ｃいる。このことはｒ）ＴＩ−（Ｔ１５をベクターと
して利用する際に、挿入すべき前杆遺伝子の導入部位を
有意に保持できるという点で右利である。また、ｐＴｌ
−ｌＴ１５は枯草菌でも好熱菌でもベクターどして利用
できる点で有利である。従って、ｒ）ＴＮＴ１５をベク
ターとして用いることにＪ二り箕種遺伝子を同時に枯草
菌と好熱菌にクローン化ザることら可能である。また、
枯草菌と１）刊〜ｌＴ１５の分離源である好熱菌、バチ
ルス・ステア［］リーモフィルスとは同じバチルス属に
屈５」るという共通点を右するためその近縁性から既に
枯草菌で発現１ノでいる異種遺伝子は、好熱菌でも発現
される可能１ノ１が高いものど考えられる。そこで、既
に枯草菌にり【１−ン化されている遺伝子を、本プラス
ミドを用いて好熱菌（９−移入ンＪる事によ−）て、も
しぞの遺伝子産物／ｌ＜ り ！；°（ニド１近（・（
ｈ耐熱（／ｌを右・Ｊる＜７らば、発酵Ｔ業にお【Ｊる
冷却−１スＩ・の節減が、θｒ熱菌ＩＪ、Ｊ、イ）発酵
戸［１わ・−」ご）て達成される事とイ「る３゜、４、
ｌ、：１耐熱性、耐溶媒性等の性質に優れた好熱菌の酵
素の萌仏了を、木シラスミドをベクターとして好熱菌宿
主にり［］−ン化し、ぞの吊片を図る事Ｌ−ｊニー）−
（、バイオリアクター等への応用が可能（′″゛あ（′
）、１−業プ「］（ツノへの１１ｂ用が期待される。1-111. The cleavage site by restriction enzymes such as II, l-1pa I'I, etc. is located at a specific and limited position. This is advantageous in that when using r)TI-(T15 as a vector, the introduction site of the pro-rod gene to be inserted can be significantly retained.
-IT15 is advantageous in that it can be used as a vector for both Bacillus subtilis and thermophilic bacteria. Therefore, by using r) TNT15 as a vector, it is possible to simultaneously clone the J.subtilis gene into Bacillus subtilis and a thermophilic bacterium. Also,
Bacillus subtilis and the thermophilic bacterium Bacillus stare [] leumophilus from which 1) T15 was isolated have a common feature that they belong to the same genus Bacillus. It is conceivable that the heterologous genes listed in 1.1 are likely to be expressed in thermophilic bacteria as well. Therefore, by transfecting the gene that has already been transferred into Bacillus subtilis into a thermophilic bacterium using this plasmid, we can create the desired gene product. ;°(Near 1 (・(
h heat resistant (/l to the right, Jru <7, for the fermentation industry [Jru cooling - 1 s I, savings, θr fever bacteria IJ, J, I) fermentation door [1wa...] What will be achieved by
1. The enzyme moebutsu-ryo of thermophilic bacteria, which has excellent properties such as heat resistance and solvent resistance, is transformed into a thermophilic bacterial host using wood shirasmid as a vector, and the enzyme is suspended. thing L-j knee)-
(Can be applied to bioreactors, etc.)
), 1-work ``] (11b use for horns is expected.
ｐｉ−１１Ｔ１５の八ｆは、本発明者らが土壌中から新
たに分離した中等度好熱菌、バアルＳλ・ステアロ１−
モフイルスＴ １５株を−ｒＹｓｌＱ地ｃ、＝、Ｊ、り
対数１件殖後明迄増殖ざＵて得た菌体を、リゾチー１１
．５ＩＩＳ処理にＪ、っＣ溶菌さｌる事によ−〕て達け
られる。8f of pi-11T15 is a moderate thermophilic bacterium, Baal Sλ stearo1-, which the present inventors newly isolated from soil.
Mophilus T 15 strain was grown until 1 day after incubation, and the resulting bacterial cells were grown until dawn.
．． This can be achieved by lysis of bacteriolytes by 5IIS treatment.
また、バグーラス・ステアロ１ナーモフイルス株は好気
ｆ１のイ１胞子稈菌、グラｌＸ染色用Ｊｒ！［ぐあり、
十台至適渇度が約５５℃で３７℃で１．１生育しイｊい
菌株τ′あるがＬ’１ＴｌｌＴ１５を採石する点では従
来には認められない新規へ一像１１物Ｔ’ Ａする１、
不菌株はブトつりでタリン（に対しく耐ｆＩｌを示した
のみイｔらずクロラムフ「二Ｔ］−ル、１−リスロン−
ｒシンに対１ノで１１・１性を示し、アンピシリン、カ
リマイシン、ネΔマイシン、スト１ノブ１〜マイシンに
は感・乎（／ｌを示しＬ−１゜
なお、不菌株は微］研菌奇第６６６１月と１１６台託さ
：Ｐ１．−ｉいる、。In addition, the Bagurus stearo1 nermophilus strain is an aerobic f1 I1 spore culm fungus, and Jr. for Gla lX staining. [Guari,
There is a good strain τ' that grows at an optimum thirst of about 55℃ and 1.1 at 37℃, but it is a novel strain that has not been recognized in the past in terms of quarrying L'1TllT15. Do 1,
The sterile strain showed resistance not only to talin (fIl) but also to chlorampfyl, 1-lythron-
It shows 11.1 susceptibility to r-syn, and is sensitive to ampicillin, carimycin, neΔmycin, and strinomycin. The 116th month of the 666th month of fungi was entrusted with: P1.-i.
１、／Ｆ、実施例に３１ζり本発明をより具体的に詳述
１する３□７−
１戸施例１（菌株のスクリーニン／７”１ｊ茨城１ル筑
波郡谷ロ１部町の１川１ｊンーｌル約１０をＴＹＳ培曲
（ディフ］゛゛・−１〜１１１〜２２％、ｊイフ］・イ
ース１−・］−４７１１〜９１１〜１％Ｎａ Ｃ１１％
）１００ｍｌに加え５５℃で約８時間振盪培養後、テ１
〜ラリイクリン（２０μ（Ｊ／ｍ＋＞を含む−ｒＹＳ寒
天平板上で４Ｖ育したτ１０ニーの一−一）からバーＴ
ルス・ステア［１リ一モフイルス７１５株（微■研菌奇
第６６６１月）が得られた。1, /F, Example 31ζ Detailed description of the present invention in more detail 3□ 7-1 Example 1 (screening of bacterial strains / 7" 1 j Ibaraki 1 Le Tsukuba-gun Yaro 1 Be-cho) 1 river 1j - l about 10 TYS culture music (diff]゛゛・-1~111~22%, jif]・ys 1-・] -4711~911~1% Na C11%
), and after shaking culture at 55℃ for about 8 hours,
~ Lariiculin (1-1 of τ10 knees grown for 4V on −rYS agar plates containing 20 μ(J/m+)) to bar T
A strain of 715 limoviruses (microorganisms 6661 months) was obtained.
実施例２
プラスミドｐＴｌ−ｌＴ１５のバチルス・ステアロリー
モフィルスＴ１５株からの分−１
バチルス・ステア１：１サーモフィルス１−１５株（微
工研菌奇第６６６１号）の生物学的に純粋な培養基から
１００ｍ１のテトラ→ノイクリン２０１１ Ｑ ／ｍ
ｌを含むＴＹＳ培地（ディフコ・バク１−・［・リプ１
〜ン２％、ディフコ・イース１〜・エキス１−ラフ１−
１％、Ｎａ Ｃ１１％）に接種し５５℃′Ｃ：１６〜１
８時間振盪培養−ＣＪる１、この培養液を１Ｑ、のテ１
〜ラリイクリン２０７、ｚｇ／ｎｕを含有づるＴ Ｙ
Ｓ ｔａ地に接種し、５５℃で５時間培養する。Example 2 Plasmid pTl-lT15 from Bacillus stearolymophilus strain T15-1 Biologically pure preparation of Bacillus stear 1:1 thermophilus strain 1-15 (Feikoken Bacteria No. 6661) 100ml of tetra from culture medium → Neuclin 2011 Q /m
TYS medium containing L (Difco Bac1-[・Lip1
～2%, Difco Ease 1～・Extract 1-Rough 1-
1%, Na C 11%) at 55°C: 16-1
8-hour shaking culture - CJru1, this culture solution in 1Q, Te1
~ Contains Rariikrin 207, zg/nu T Y
It is inoculated onto Sta soil and cultured at 55°C for 5 hours.
菌体を）重心にＪ、−）で集め、Ｔ’ＥＳ （２０ｍＭ
−ｒｒｉｓ −Ｌｌｃ＋ 、５ｍＭｒ：ｌ）ＴΔ、１０
０ｍＭＮａ Ｃｌ ｐｌ−１７，５＞ｒ洗浄後閑体湿
重吊４Ｑ当り、１０ｍ１の２５％シ丑１糖含右ＴＦＳに
懸濁する。リゾチーム（１０ｚｚａ／ｍｌ）を２ｍｌ、
０．２５Ｍ−ＥＤＴΔ（ＤＩ−１８，０）４ｍｌを加え
、８−
心し、Ｊ清を得る。この上清にポリ下チレングリ］−ル
６０００を１０％（Ｗ ／Ｖ ”）加え、２・〜３時間
Ｏ℃に静Ｆ、７２２０Ｏｒｐｍ 、２分の遠心で沈澱を
得る。この沈澱を１５ｍ１のＴＦＳに溶解１．／ 、
□ｓ Ｃｌ ７ｋ（Ｊ’ １． ｆ シラノ、ハＩマイ
トを加λて密麻を１．６１−・１．６２に調整子するｌ
ｌ にの試別を３８０（１（１ｐｐｍで３０〜４０時間
、平衝密庶勾配連心゛りる。１じたｌラスミドＤＮＡの
バンドを集め、イソアミル−１ル］−ルで一丁チジ「ジ
ノ、７１−ｌ’ｖ’ （トラ除去した後、Ｔ［Ｎ （２
（１ｍＭＴｒｉｓ −１−ＩＣＩ 、１ｍＭ［ｒ′１丁
△、２０ｒｎＭＮａ Ｃ１）に透析する事によって純粋
なｐ ’１’ １１”Ｉ’ １５が得られる。Collect the bacterial cells at the center of gravity with J, -) and add T'ES (20mM
-rris-Llc+, 5mMr:l)TΔ, 10
0mM NaCl pl-17,5>r After washing, suspend in 10 ml of 25% monosaccharide-containing TFS per 4Q of dry suspension. 2 ml of lysozyme (10zza/ml),
Add 4 ml of 0.25M-EDTΔ(DI-18,0) and 8-centigrade to obtain J serum. Add 10% (W/V'') of polyethylene glycol 6000 to this supernatant and centrifuge at 7220 rpm for 2-3 hours at 0°C for 2 minutes to obtain a precipitate. Dissolved in 1./,
□s Cl 7k (J'1.
The lasmid DNA band was collected at 380 μl (1 ppm for 30 to 40 hours), and the lasmid DNA band was collected and diluted with isoamyl-1 μl. "Jino, 71-l'v' (After removing the tiger, T[N (2
Pure p'1'11''I'15 is obtained by dialysis against (1mM Tris-1-ICI, 1mM [r'1t△, 20rnMNa C1).
ｐＴＩ−ｌＴ１５の特性決定の手順
ｐＴＨＴ１５の分子量は、その超らせん構造（５ｕｐｅ
ｒｃｏｉｌｅｄｓ＋ ｒｌｌｃｌｌｌｒｏ ）のＤＮＡ
及び制限酵素によって切断された断片のアガ［１−スゲ
ルミ気泳動より得られた。この際の分子量マーカーはｐ
ＢＲ３２２１）Ｎ△（２，ｆ’） ７ｍ（１） 、Ｃｏ
１Ｅ ＴＤＮＡ（４，２ｍｒｌ＞及びラムダ１）ＮΔの
１１ｉｎｒＩ１１１分解断Ｉｓ’＜１４．６．５．８１
．４．０５．２．６７．１．０４．１．２１．０．３旬
１ｄ）、うｌ＼ダ［）ＮΔのＥＣｏＲＩ分解断；１（１
３，７，１１，７１１，３，７３，３，４８，３，０２
，２，１３ｍ（１）−ケム社の１７ガロースを０．５％
又は０．７％の濃度で用い、水平ゲル電気泳動槽にＪ：
つてゲル長さ１ｃｍ当り１．５Ｖの定電に’（：’ １
５・〜・１７時間行なった。Procedure for characterizing pTI-IT15 The molecular weight of pTHT15 is determined by its supercoiled structure (5upe
rcoileds+ rllcllllro) DNA
and fragments cleaved with restriction enzymes were obtained by Aga[1-Sgermi electrophoresis. The molecular weight marker at this time is p
BR3221)N△(2,f') 7m(1), Co
1E TDNA (4,2mrl> and lambda 1) NΔ 11inrI111 decomposition Is'<14.6.5.81
．． 4.05.2.67.1.04.1.21.0.3 ECoRI decomposition of 1d), ul\da[)NΔ; 1(1
3,7,11,711,3,73,3,48,3,02
,2,13m(1)-0.5% of 17 galose from Chem Co.
or J at a concentration of 0.7% in a horizontal gel electrophoresis chamber:
'(:' 1
It took 5 to 17 hours.
ｌ’） ’ｌ−ＨＴ’ １５が、そのＤＮＡ」にテ１〜
ラリイクリン耐性遺伝子を右し−（いる事は、枯華菌バ
ｆ−ルス・ズブチルスＲＭ１２５株プ叫−プラストへの
形Ｖ（転換実験にＪ： −）で確かめられた。バチルス
・スブチルスＲＭＩ２！：ｉ株のプロミルプラス１〜の
調製、形質転換、プロトプラス１−の再生の手順はＣｈ
ａ ｎＣ１＆ Ｃ０ｆＩｅｎの方法（Ｍ（ｌｌｅｆｊ、
ＱＯｎ、 Ｇｅｎｅ＋、 、 １６８． １１１−
１１５（＋９７９） ）によって行なった。この方法の
概略はＲＭ１２５株の月数増殖国体を等張液中でリゾチ
ーム処理によってプロトプラス１〜化し、このプロ１−
ブラスト懸濁液にプラスミドＤＮＡ溶液を加え、ポリエ
チレングリ′」−ル６０００によつ−ＵＤＮΔのプ［月
〜プラス１〜内への取込みを促した後、再生培地１でブ
ｒ’ｌ ｌ〜プラス１〜から栄養細胞への再生を図ると
いうものである。l') 'l-HT' 15 is Te1~ in that DNA'
The presence of the laryculin resistance gene was confirmed in Bacillus subtilis RM125 strain Plast to form V (conversion experiment J: -). Bacillus subtilis RMI2!: Procedures for preparing i strain Promilplus 1~, transformation, and regeneration of Protopulus 1~ are described in Ch.
a nC1&C0fIen's method (M(llefj,
QOn, Gene+, , 168. 111-
115 (+979) ). The outline of this method is that RM125 strain Kokutai, which has been grown for several months, is treated with lysozyme in an isotonic solution to convert it to protoplast 1.
After adding a plasmid DNA solution to the blast suspension and promoting the uptake of UDNΔ into the polyethylene glycol 6000, it was mixed with regeneration medium 1. The aim is to regenerate vegetative cells from plus 1.
ＤＴＨＴ１５ＤＮＡ約１ｔｌＱを用いて、ＲｔＶ１１２
５株のプ叫〜プラス１−懸濁液０．５ｍｌ （２Ｘ１０
９プ［旧・プラス１−／ｍｌ）に対して形質転換を行な
い、再生培地４−Ｃテ１〜う→ノイクリン耐性株の出現
を検問１ノだとこ軍、この際の１１月〜ｌ）スｌ〜の再
生率１０％（２ｘ １０” 、’ｎｕ ＞に対１・・１
５
、′（（て１ ｘ １０シ′＋＋＋！の頻度でテ１〜ラ
リイクリン耐１テ１株が７１じ！、−８−ｆ）、Ｊ’
ノ’ｙ Ｅ、ドＤＮΔ溶液を加えなか゛）た系（−１
ン１〜］］−ル）’ｒ−は一７ｌ−−ノリイクリン耐性
株は２Ｘ１０”個以上のプ１゛目・プラストを徹いてノ
）生じては、二４Ｉ−か−）た５゜
ここ′ｒ″ｉｑられたＲＭ１２５株のデＩ−ラリイクリ
ン耐十１１株、１．１つグラスミドＤＮＡを、バチルス
・ステア１］サーモフイルスＴ１５株、にりのプラスミ
ド「）Ｎ△の抽出の際と同様（リゾチーム処理の温１σ
涜び時間／）＜３７°Ｃ３０分間７パある点が異なる。Using approximately 1tlQ of DTHT15 DNA, RtV112
0.5ml suspension of 5 strains (2X10
Transformation was performed on the regeneration medium 4-Cte1~u → the emergence of neuclin-resistant strains was carried out on the regeneration medium 4-Cte1~u → the emergence of neuclin-resistant strains was carried out in November~l). Reproduction rate of 10% (2x 10", 'nu > 1...1
5,'((Te1 x 10shi'+++! frequency of Te1 to Rariicrin resistant 1Te1 strain is 71 days!, -8-f), J'
No'y E, the system without adding DNAΔ solution (-1
1~]]-ru)'r-is 17l--Noriiculin-resistant strains are 2x10'' or more, passing through the 1st plast. 'r''iq'ed RM125 strain DeI-rariicrin resistant 11 strains, 1. Grasmid DNA was extracted from Bacillus stare 1] thermophilus T15 strain, the same way as in the extraction of nicotine plasmid ')N△ ( Temperature 1σ of lysozyme treatment
The difference is that the heating time is <37°C for 30 minutes.
）の方法で抽出ｌノ検ｉ’ｌ ｌノたどころ、ｌ） Ｔ
１１Ｔ１Ｆｉと同 分子量で、制限酵素による切断パ
ターン０全＜ＩＩ″ｉｌ−イｊブうスミドＤ Ｎ Ａが
回収された３、この事実は、ＩＩＴＩＩＴ １ ！′１
がロ〜ラリイクリン耐Ｍ ？ｌｆｆ伝子を右ｌノーＣお
り、このプラスミドがＲＭ ”Ｉ ２５１１、に人つ１
５：１ｓＬ−、Ｊ−＊”ｌ？ｆｖｌ １２５’ｒ’Ｂ＜
−’Ｆ Ｉ”５−’）イク’、）ン耐１’ｌノｌｌＪ’
Ｊ−’、（４’ 示ηニ到−）／、−巾を証明り”る：
１）のである。同時に、ｒ）ＴＩＩＴ１！’ｉが、好熱
１′４１及び枯草菌Ｃ１白仲的ＩＰ？殖能及び形質の発
現が可能なシラスミドである事、つまり不グシスミドが
両菌株−て゛ベクターどじＣ利用し１！する事実を明ら
か（Ｊ（するものである５、
７寸・ノリ、ｒクリン耐ｆ〕１を右覆る好熱菌のプラス
ミドと１ノｒ ｔ；：Ｉ、前記の表に示ｊ、／−と１１
ミ（］で（ｂるが、Ｉ）Ｔｌ−ＩＴ１５と他のものでは
前述のＪ、うに明らか（Ｊ責イ１つており、Ｄ Ｔ ｌ
−ｌ Ｔ−１！’ｉはｉＫ来認めらＪｌない新規なプラ
スト１１−
Ａハエノン・ラツム由來の酵素、ｌＴｃ０ＲＩは１−シ
Ｔリヒア・＝１り由来の酵素、１１１＋ａｌはハエ七フ
ィルス・ハエモリティカス由来の酵素、ｌ−１１１，’
ｌｌはハエ１フイルス・バラインフルエンＩＪ” １−
由来の酵素、ｌ−１ｐａ ＨはハＴモノイルλ・バライ
ンフルＪンリ″−１山来の酵素、１−ａ［はり一ンス・
アク１’ ｒ （ｊＪス由東の酵素をそれぞれ示してい
る。) Extract by the method of T
A DNA with the same molecular weight as 11T1Fi and a restriction enzyme cleavage pattern of 0 was recovered.
Is it lolly clean resistant M? The lff gene is present in the right lnoC, and this plasmid is present in RM"I 2511, one person
5:1sL-, J-*"l?fvl 125'r'B<
-'F I"5-') Iku',)'n 1'l noll J'
J-', (4' shows η ni-)/, - prove the width:
1). At the same time, r) TIIT1! 'i is thermophilic 1'41 and Bacillus subtilis C1 white middle IP? It is a cilasmid that can express reproductive ability and traits, that is, it is a cilasmid that can express both bacterial strains and vector DojiC. It is clear that the plasmids of thermophilic bacteria that cover 1 and 1 nor rt;: I, as shown in the above table, and 11
In Mi (] (bru, I) Tl-IT15 and others, the above-mentioned J, there is clearly one (J responsibility I), and D
-l T-1! 'i is a novel enzyme derived from Plasto11-Ahaenon ratum, which has not been recognized since, lTc0RI is an enzyme derived from 1-SiTlichia, 111+al is an enzyme derived from the fly Heptophilus haemolyticus, l- 111,'
ll is fly 1 philus balainfluen IJ” 1-
Enzyme derived from 1-a
Aku1' r (jJ Suyuto's enzymes are shown respectively.
１２−12-
Claims (1)
量が約２．８メガダルトンであり、図に示される制限酵
素地図で特徴づけられるプラスミドを保有する新規なバ
チルス・ステアロサーモフィルスＴ１５株。A novel Bacillus stearothermophilus T15 strain that contains a tetracycline resistance gene, has a molecular weight of approximately 2.8 megadaltons, and carries a plasmid characterized by the restriction enzyme map shown in the figure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57157367A JPS5928392B2 (en) | 1982-09-09 | 1982-09-09 | Novel microorganism harboring tetracycline resistance plasmid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57157367A JPS5928392B2 (en) | 1982-09-09 | 1982-09-09 | Novel microorganism harboring tetracycline resistance plasmid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5945877A true JPS5945877A (en) | 1984-03-14 |
| JPS5928392B2 JPS5928392B2 (en) | 1984-07-12 |
Family
ID=15648100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57157367A Expired JPS5928392B2 (en) | 1982-09-09 | 1982-09-09 | Novel microorganism harboring tetracycline resistance plasmid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5928392B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4894337A (en) * | 1989-01-17 | 1990-01-16 | Board Of Trustees Operating Michigan State University | Process for the bioproduction of cyclic hydroxides |
-
1982
- 1982-09-09 JP JP57157367A patent/JPS5928392B2/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4894337A (en) * | 1989-01-17 | 1990-01-16 | Board Of Trustees Operating Michigan State University | Process for the bioproduction of cyclic hydroxides |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5928392B2 (en) | 1984-07-12 |
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