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Levels and localization of mechanosensitive channel proteins in Bacillus subtilis

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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.

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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

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Acknowledgments

This work was supported by a grant from the Army Research Office to PS. We are grateful to F. Arigoni for plasmid pRDC9. The Center for Cell Analysis and Modeling at the University of Connecticut Health Center is supported by NIH grant RR022232.

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Authors and Affiliations

  1. Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3305, USA

    Paul G. Wahome, Ann E. Cowan, Barbara Setlow & Peter Setlow

  2. Center for Cell Analysis and Modeling, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3305, USA

    Ann E. Cowan

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  1. Paul G. Wahome
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  2. Ann E. Cowan
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  3. Barbara Setlow
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  4. Peter Setlow
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Correspondence to Peter Setlow.

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Communicated by Jorge Membrillo-Hernández.

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Wahome, P.G., Cowan, A.E., Setlow, B. et al. Levels and localization of mechanosensitive channel proteins in Bacillus subtilis . Arch Microbiol 191, 403–414 (2009). https://doi.org/10.1007/s00203-009-0465-z

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