多細菌鞭毛馬達的同步轉動量測; Simultaneously monitoring the rotation of multiple bacterial flagellar motors

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題名:	多細菌鞭毛馬達的同步轉動量測;Simultaneously monitoring the rotation of multiple bacterial flagellar motors
作者:	陳修博;Chen,Hsiu-Po
貢獻者:	物理學系
關鍵詞:	大腸桿菌;鞭毛馬達;反轉;高速攝影機;暗視野顯微鏡;E coli;flagellar motor;switching;high speed camera;dark field microscope
日期:	2013-07-25
上傳時間:	2013-08-22 11:46:57 (UTC+8)
出版者:	國立中央大學
摘要:	大腸桿菌是用它們的鞭毛馬達來做游泳的運動。這一個微小的分子馬達利用氫離子流與氫離子驅動力來帶動。一隻大腸桿菌擁有4-8根左旋的鞭毛。當它們的所有鞭毛同時做逆時針的旋轉時，鞭毛會形成一束並推進細菌。而當有一根以上的鞭毛反轉，也就是做順時針旋轉的話，反轉的鞭毛就會脫離原本整束的鞭毛，並且會改變細菌的前進方向。而造成鞭毛馬達反轉的原因，是因為一個訊號傳遞的蛋白質CheY造成的。然而我們也發現，反轉的頻率會隨著鞭毛上的負載重量而改變。然而，每隻細菌的CheY的基準濃度與氫離子驅動力都不同。為了消除這兩項變因，我們使用高速攝影機(~957 fps)來拍攝細菌，並且我們讓一隻細菌身上，兩根鞭毛上各自負載了不同的重量(不同大小的珠子)。討論其同一隻菌身上，不同的負載與速度，反轉的頻率會有什麼樣的改變。在我們有限的數據之中，我們發現在高負載的區域，轉動切換的速率並沒有很大的變化。另外，我們用高速攝影機來觀察相鄰不同細菌鞭毛馬達可能透過流場造成協同轉動的可能性。與之前的間接實驗不同，我們直接觀察同一細菌相近的細菌鞭毛轉動，我們並沒有發現高度協同的現象。我們也利用相同的實驗方法來一次觀察多隻大腸桿菌的轉動情況，我們在全畫面下，能夠一次觀察多隻細菌，並且我們打入一些化學物質如酒精或是離子穿透劑等，當濃度控制得宜，可以觀察細菌逐漸降低氫離子驅動力時與分子馬達轉速的變化，大腸桿菌的鞭毛馬達的轉動，跟質子通量與體內外的膜電位成正相關，所以我們可以藉由觀測其轉速的變化，來得知其質子通量的變化，而藉由全畫面下的高速拍攝，我們可以一次得到大量的數據，可以更快速的分析。 Escherichia coli use flagellar motors to swim. This tiny molecular machine is powered by proton flux through proton-motive force. The cells can be propelled by 4-8 left-handed helical flagellar filaments in one cell. When all of the motors rotate counterclockwise (CCW), the filaments can form a bundle to propel the cells forward. When one or more motors switch to clockwise (CW), their filaments will move out of the bundle and change the direction of the cells. The switching rate of the motor is modulated by the signal transduction molecules CheY binding to the motor. However, the external loading will affect the switching rate. For large number cell average, the CCW/CW switching rates depend on load. However, the CheY concentration and motor driving are different from cell to cell. Here, we examine the two motor switching rates at different external loads in one cell to eliminate the cellular CheY concentration and proton-motive force variations. We use high speed camera (~957 fps) to monitor two different size of beads attached to the motors in single cell. In our limited data, the switching rate is independent on the external load in high load region. Appling the high speed camera method to monitoring the rotation of multiple flagellar motors, we can also test possibility of coordinated switching of bacterial flagellar motors. On contrary to the previous report, we directly measure the rotation of two flagellar motors. We did not find high rotational correlation between neighboring motors. We also use the same method, high speed camera, to observe the proton-motive force of multiple cells, we add the ethanol and the ionophore (CCCP) to the motility medium and observed cells how fast did cells died. We can record rotational speed of the tethered cells and we can know the relationship between the rotational speed and the times. The rotation of bacterial flagellar motor is a direct indication of proton motive force.
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