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Gating the bacterial mechanosensitive channel MscL invivo
Ann Finney Batiza1, Mario Meng-Chiang Kuo, Kenjiro Yoshimura
1Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706, USA. afbatiza@facstaff.wisc.edu
Summary
Mechanosensitive channel of large conductance (MscL) opens in Escherichia coli during osmotic downshock, even with YggB present. YggB acts as a buffer, influencing MscL gating in vivo.
Area of Science:
- Microbiology
- Biophysics
- Molecular Biology
Background:
- Mechanosensitive channels like YggB and MscL are crucial for bacterial survival during osmotic stress.
- The precise role of MscL in wild-type Escherichia coli under osmotic downshock remains debated due to its higher gating threshold compared to YggB.
Purpose of the Study:
- To investigate whether MscL is gated in vivo during osmotic downshock in the presence of YggB.
- To elucidate the buffering role of YggB in MscL channel gating.
Main Methods:
- Engineering a modified MscL channel (Leu-19-->Cys MscL) for in vivo structural modification and monitoring.
- Utilizing osmotic downshock and a charged reagent (MTSET+) to functionalize the engineered channel.
- Assessing channel opening by monitoring the loss of colony-forming units.
Main Results:
- An osmotic downshock of approximately 800 mmol/kg from 1,200 mmol/kg medium was sufficient to open Leu-19-->Cys MscL in the presence of YggB.
- A significantly lower downshock of approximately 400 mmol/kg was required to open the channel in the absence of YggB.
- In vitro experiments showed that MTSET+ attachment to Leu-19-->Cys MscL, after stretching, led to channel flickering with minimal suction.
Conclusions:
- MscL gating is triggered by specific osmotic downshocks in vivo, even when YggB is present.
- YggB acts as a buffer, modulating MscL gating dynamics in living bacteria.
- The study confirms that residue 19 of MscL is exposed upon channel opening.