Title: Connecting the dots between mechanosensitive channel abundance, osmotic shock, and survival at single-cell resolution,
Abstract: Rapid changes in extracellular osmolarity are one of many insults microbial
cells face on a daily basis. To protect against such shocks, Escherichia coli
and other microbes express several types of transmembrane channels which open
and close in response to changes in membrane tension. In E. coli, one of the
most abundant channels is the mechanosensitive channel of large conductance
(MscL). While this channel has been heavily characterized through structural
methods, electrophysiology, and theoretical modeling, our understanding of its
physiological role in preventing cell death by alleviating high membrane
tension remains tenuous. In this work, we examine the contribution of MscL
alone to cell survival after osmotic shock at single cell resolution using
quantitative fluorescence microscopy. We conduct these experiments in an E.
coli strain which is lacking all mechanosensitive channel genes save for MscL
whose expression is tuned across three orders of magnitude through
modifications of the Shine-Dalgarno sequence. While theoretical models suggest
that only a few MscL channels would be needed to alleviate even large changes
in osmotic pressure, we find that between 500 and 700 channels per cell are
needed to convey upwards of 80% survival. This number agrees with the average
MscL copy number measured in wild-type E. coli cells through proteomic studies
and quantitative Western blotting. Furthermore, we observe zero survival events
in cells with less than 100 channels per cell. This work opens new questions
concerning the contribution of other mechanosensitive channels to survival as
well as regulation of their activity.