Archives of Microbiology

, 191:403

Levels and localization of mechanosensitive channel proteins in Bacillus subtilis

  • Paul G. Wahome
  • Ann E. Cowan
  • Barbara Setlow
  • Peter Setlow
Original Paper

DOI: 10.1007/s00203-009-0465-z

Cite this article as:
Wahome, P.G., Cowan, A.E., Setlow, B. et al. Arch Microbiol (2009) 191: 403. doi:10.1007/s00203-009-0465-z

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. subtilismscL 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

BacillusMechanosensitive channelsOsmoregulationOsmotic downshockSporulationTurgor pressure

Abbreviations

DTT

Dithiothreitol

GFP

Green fluorescent protein

LB

Luria-Bertani medium

LSM

Laser scanning microscope

MS

Mechanosensitive

MscL

Mechanosensitive channel of large conductance

MscS

Mechanosensitive channel of small conductance

MscM

Mechanosensitive channel of mini conductance

O/D

Osmotic downshock

ORF

Open reading frame

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate buffered saline

RBS

Ribosome binding site

PMSF

Phenylmethanesulphonyl fluoride

SDS

Sodium dodecyl sulfate

2× SG

Schaeffer’s plus glucose medium

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Paul G. Wahome
    • 1
  • Ann E. Cowan
    • 1
    • 2
  • Barbara Setlow
    • 1
  • Peter Setlow
    • 1
  1. 1.Department of Molecular, Microbial and Structural BiologyUniversity of Connecticut Health CenterFarmingtonUSA
  2. 2.Center for Cell Analysis and ModelingUniversity of Connecticut Health CenterFarmingtonUSA