Original Paper

Archives of Microbiology

, 191:403

First online:

Levels and localization of mechanosensitive channel proteins in Bacillus subtilis

  • Paul G. WahomeAffiliated withDepartment of Molecular, Microbial and Structural Biology, University of Connecticut Health Center
  • , Ann E. CowanAffiliated withDepartment of Molecular, Microbial and Structural Biology, University of Connecticut Health CenterCenter for Cell Analysis and Modeling, University of Connecticut Health Center
  • , Barbara SetlowAffiliated withDepartment of Molecular, Microbial and Structural Biology, University of Connecticut Health Center
  • , Peter SetlowAffiliated withDepartment of Molecular, Microbial and Structural Biology, University of Connecticut Health Center Email author 

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Abstract

Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock ≥0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is partially dependent on three likely MS channel proteins of small conductance (MscS), YfkC, YhdY, and YkuT. Bacillus subtilis MS proteins were fused with green fluorescent protein (GFP) at their C termini; at least the MscL-, YfkC-, and YkuT-GFP fusions were functional and overexpression of YkuT-GFP, or YkuT alone abolished log phase mscL cells’ osmotic downshock sensitivity. Western blot analysis found high levels of MscL-GFP in early exponential phase cells with levels subsequently decreasing greatly. MscS-GFP proteins were present in exponential phase cells, but again disappeared almost completely in stationary phase cells and these proteins were not detected in spores. Western blot analyses further showed that MS-GFP proteins were associated with the plasma membrane, as expected. Fluorescence microscopy confirmed the localization of MscL-GFP and YhdY-GFP to the plasma membrane, with non-uniform distribution of these proteins along this membrane consistent with but by no means proving that these proteins are present in a helical array.

Keywords

Bacillus Mechanosensitive channels Osmoregulation Osmotic downshock Sporulation Turgor pressure