The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus su
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O RI G I NAL PAPE R
The synthesis and role of the mechanosensitive channel of large conductance in growth and diVerentiation of Bacillus subtilis Paul G. Wahome · Peter Setlow
Received: 17 May 2006 / Revised: 29 June 2006 / Accepted: 10 July 2006 / Published online: 5 August 2006 © Springer-Verlag 2006
Abstract A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at signiWcant levels during log phase growth of B. subtilis, and the level of mscL–lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL–lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a signiWcant amount of -galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no eVect on B. subtilis growth, sporulation or subsequent spore germination. The mscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase mscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, mscL cells had become resistant to a 0.9 M osmotic downshift. Keywords Bacillus · Sporulation · Spore germination · Mechanosensitive channels · Osmoregulation
P. G. Wahome · P. Setlow (&) Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3305, USA e-mail: [email protected]
Abbreviations McsL The mechanosensitive channel of large conductance MS Mechanosensitive DPA Dipicolinic acid Ca–DPA a 1:1 chelate of Ca2+ and DPA MPa MegaPascals
Introduction Mechanosensitive (MS) channels are integral membrane proteins that detect mechanical force on the cell membrane, and MS proteins and channels have been identiWed in archaea, prokaryotes and eukaryotes (Pivetti et al. 2003; Edwards et al. 2004; Martinac 2004). Results of electrophysiological, genetic and biochemical studies indicate that MS channels are involved in the eZux of molecules from cells subjected to an osmotic downshift. Bacteria accumulate ions and osmolytes in media of high osmolality to counteract cell dehydration and to maintain the outwardly directed cytoplasmic turgor pressure that is essential for growth. However, upon a rapid large osmotic downshift in the external medium, a rapid inXux of water into bacterial cells can increase turgor pressure to dangerous levels unless osmolytes are extruded, and too great a turgor pressure can result in cell lysis. It is MS proteins that form the channels through which accumulated osmolytes and ions are jettisoned in response to a rapid external osmotic downshift (Schleyer et al. 1993). In Escherichia coli three classes of MS channels have been identiWed, MscL, MscS and MscM, that di
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