POSTERS - BLAST X - University of Utah

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