POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
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<strong>BLAST</strong> X Tue. Morning Session<br />
DO INDIVIDUAL BACTERIAL FLAGELLAR MOTORS USE HYSTERESIS TO MAINTAIN A<br />
ROBUST OUTPUT IN A NOISY ENVIRONMENT? – AN EXPERIMENTAL STUDY<br />
Peter Reuven 1 , Oleg Krichevsky 2 , Michael Eisenbach 1<br />
1 Department <strong>of</strong> Biological Chemistry, Weizmann Institute <strong>of</strong> Science, 76 100, Israel<br />
2 Department <strong>of</strong> Physics, Ben-Gurion <strong>University</strong>, Beer Sheva, 84 105, Israel<br />
It is known that despite imperfections <strong>of</strong> intracellular environment, flagellar motor outputs<br />
are robust against stochastic fluctuations <strong>of</strong> CheY signal. Indirect evidence from our lab<br />
suggests that, to maintain stability, the motor complex might damp out fluctuations in the<br />
intracellular level <strong>of</strong> CheY by having a hysteresis feature - two different thresholds for switching.<br />
In this case, hysteresis means that the default-state motor switches at a higher<br />
threshold from counterclockwise to clockwise state compared to a lower threshold when<br />
switching back. Such behavior will produce hysteretic loop in input/output characteristics <strong>of</strong><br />
flagellar motor.<br />
Our aim is to pin-point the level at which the noise-filtering (via hysteresis) occurs in the<br />
chemotactic network. We are studying flagellar rotation <strong>of</strong> single cells as a function <strong>of</strong> their<br />
intracellular CheY-P concentration, changing the concentration up and down in order to cover<br />
both - counterclockwise to clockwise and clockwise to counterclockwise - switching routes.<br />
Since it is impossible to distinguish CheY from CheY-P in vivo, one has to work under<br />
conditions that maintain CheY constantly fully phosphorylated. The challenge was how to<br />
effectively decrease the concentration <strong>of</strong> the phosphorylated signal.<br />
Knowing that we cannot play with the level <strong>of</strong> phosphorylation we opt to play with the<br />
level <strong>of</strong> the CheY-P protein instead. While to increase protein concentration is a routine task, to<br />
decrease it is less obvious. We cloned a bi-modal, inducible plasmid expressing a CheY fused<br />
to yellow fluorescent protein (YFP) and ssrA degradation tag. YFP is used for quantifying<br />
the signal whereas the degradation tag makes it possible to shorten the lifetime <strong>of</strong> CheY-YFP.<br />
The core assumption <strong>of</strong> our approach is that heat-shock-induced proteases accelerate the<br />
degradation <strong>of</strong> the ssrA-targeted CheY-YFP protein. We have verified this assumption<br />
experimentally.<br />
We monitor the degradation <strong>of</strong> CheY-YFP by a decrease in fluorescence intensity and<br />
this decrease is correlated with the change <strong>of</strong> the direction <strong>of</strong> motor rotation. Input/output<br />
characteristics <strong>of</strong> individual flagellar motors is build from these correlations.<br />
Advancing this study will, hopefully, enable us to deeper understand how the<br />
mechanisms <strong>of</strong> intracellular interactions affect the logic <strong>of</strong> cell's behavior.<br />
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