Monitoring bacterial mechanosensitive channel gating using a ratiometric GFP

My project is part of a larger Marie Skłodowska-Curie innovative training network consortium project (called SynCrop) investigating means to make microorganisms more robust during production of food additives using synthetic constructs. Mechanosensitive (MS) channels, found in bacterial plasma membranes, detect changes in membrane tension and gate to release solutes during osmotic downshocks to prevent cells from lysing. In my project, I am investigating if the gating of MS channels can be monitored fluorescently through cytoplasmic pH (pHcyto) shifts. pHluorin, a pH-sensitive, ratiometric GFP, was chosen as a cytoplasmic pH probe. Escherichia coli K-12 strains Frag 1 (with all seven MS channels) and MJF641 (MS channel-less strain) were transformed with a plasmid expressing pHluorin. Combined osmotic and pH downshock assays were performed and cell survival determined through colony counting. Fluorescence microscopy was used to confirm shifts in fluorescence from green at neutral pH to blue at acidic pH. Fluorescence microscopy revealed a shift in fluorescence from green to blue upon downshock for Frag1, while almost 100% of MJF641 did not survive the downshock. These data suggest a proton influx during combined osmotic and pH downshock in Frag1, decreasing pHcyto. I will investigate if pHluorin can be utilised to confirm MS channel gating in other bacteria.

Recommended Articles