1、細(xì)胞生化學(xué)研究室奧田芳子大腸菌酸性膜脂質(zhì)欠損株低浸透圧感受性対機(jī)械刺激受容影響pgsA 欠損株(Kikuchi et al. 2000)Phosphatidyl-glycerol phosphateGlycerol 3-phosphatePhosphatidic acidCDP-diacylglycerolPhosphatidylserinePhosphatidyl- ethanolamine(PE)PGP synthase (pgsA)Phosphatidylglycerol (PG)Cardiolipin (CL)0%50%100%p gsAp gsAwild typewild typeP

2、EPEPGPGCLCLothersothers0501001502002503003504000100200300400minutesklett unitpgsA 欠損株低浸透圧培地中生育悪。wild type0501001502002503003504000100200300400minutesklett unitLB medium (565 mosM)LB medium minus NaCl (267.5 mosM)Null pgsA mutant(250 mosM 125 mosM)(%)Wild typeNull pgsA mutant1 min5 min(有山修士論文 2003)低浸

3、透圧後pgsA欠損株細(xì)胞質(zhì)體積縮小。細(xì)胞質(zhì)大腸菌低浸透圧応答外膜細(xì)胞質(zhì)膜細(xì)胞質(zhì)膜 (內(nèi)膜內(nèi)膜) MscL (large conductance)MscS (small conductance)MscM (mini conductance) MscK (interaction with K+)H2OMechanosensitive channel(s) (MSC)速開口速開口目的 大腸菌3MSC遺伝子mscL, mscS, mscK組合破壊株作成。 MSC破壊低浸透圧耐性影響調(diào)。予想： pgsA欠損株MSC開閉調(diào)節(jié)異常実験使用大腸菌pgsA+pgsA-Wild typeS301RS330Rmsc

4、LS301RLS330RLmscSS301RSS330RSmscKS301RKS330RKmscL, mscSS301RLSS330RLSmscL, mscKS301RLKS330RLKmscS, mscKS301RSKS330RSKmscL, mscS, mscK S301RLSKS330RLSK株rcsA,lacY遺伝子破壊低浸透圧後生存率測定LB培地Klett40培養(yǎng)培養(yǎng)液等量830 mM NaCl含LB培地混合500 mM NaCl環(huán)境Klett120培養(yǎng)NaCl含LB培地500倍希釈低浸透圧適宜希釈LB寒天培地37一晩培養(yǎng)計測生存率求020406080100wild type-L-S

5、-K-LS-LK-SK-LSKsurvival (%)強(qiáng)低浸透圧後生存率 pgsA+pgsA-pgsA-株MSC破壊生存率回復(fù)L: mscLS: mscSK: mscK(1142.5 mosM 270 mosM)000前生存率100%pgsA-株MscL存在、低浸透圧後生存非常有利pgsA-株低浸透圧後細(xì)胞質(zhì)體積減少M(fèi)SC破壊防pgsA-株一MSC放出多濾過遠(yuǎn)心法大腸菌懸濁液大腸菌懸濁液過塩素酸過塩素酸D.W. (3 H2O 14C-混合液混合液)遠(yuǎn)心遠(yuǎn)心細(xì)胞取込細(xì)胞取込3H 14C放射能放射能測定測定大腸菌層通過、過塩素酸層固定細(xì)胞質(zhì)外膜細(xì)胞質(zhì)膜H2O 透過透過透過細(xì)胞質(zhì)體積細(xì)胞質(zhì)體積 =

6、(3H2O透過體積透過體積) (14C透過體積透過體積)細(xì)胞質(zhì)體積算出方法実験使用株実験使用株lacY lacY 破壊。破壊。細(xì)胞質(zhì)外膜細(xì)胞質(zhì)膜H2O 透過透過細(xì)胞質(zhì)體積細(xì)胞質(zhì)體積 = (3H2O透過體積透過體積) (14C透過體積透過體積)細(xì)胞質(zhì)體積算出方法実験使用株lacY 破壊。條件前後細(xì)胞數(shù)変化細(xì)胞質(zhì)內(nèi)取020406080100120wildwildtypetype-L-L-S-S-K-K-LS-LSsurvival (%)pgsA+pgsA-生存率生存率90%以下変異株細(xì)胞質(zhì)體積測定実験不適當(dāng)以下変異株細(xì)胞質(zhì)體積測定実験不適當(dāng)考、用。考、用。條件1細(xì)胞數(shù)低浸透圧前後変化細(xì)胞質(zhì)體積測定

7、時生存率前生存率100%放射線添加検討pgsA-株株低浸透圧同時放射線添加、低浸透圧同時放射線添加、細(xì)胞質(zhì)內(nèi)取、透過可能體積大測定細(xì)胞質(zhì)內(nèi)取、透過可能體積大測定條件2細(xì)胞質(zhì)內(nèi)取00.511.522.530 min3 min4 min後添加volume (ul)00.511.522.530 min1 min2 min3 min4 min 後添加volume (ul)3H2O透過可能體積透過可能體積14C透過可能體積透過可能體積pgsA+pgsA-pgsA株MscS, MscK欠損細(xì)胞質(zhì)體積減少緩和(250 mosM 125 mosM)020406080100120wildwildtypetype-L -L -K-Kwildwildtypetype-L-L-S-S-K-Krelative volume of