Growth, osmotic downshock resistance and differentiation of Bacillus subtilis strains lacking mechanosensitive channels
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ORIGINAL PAPER
Growth, osmotic downshock resistance and diVerentiation of Bacillus subtilis strains lacking mechanosensitive channels Paul G. Wahome · Peter Setlow
Received: 8 April 2007 / Revised: 8 June 2007 / Accepted: 12 July 2007 / Published online: 31 July 2007 © Springer-Verlag 2007
Abstract Previous work has shown that the mechanosensitive (MS) channel of large conductance (MscL) is essential for preventing lysis of Bacillus subtilis log phase cells upon a rapid, severe osmotic downshock. Growing cells of B. subtilis strains lacking MscL and one or more putative MS channel proteins of small conductance (YhdY, YkuT and YfkC) showed even higher sensitivity to an osmotic downshock. The eVect was greatest for a strain lacking MscL and YkuT, and a strain lacking all four MS channel proteins had a similar phenotype. These defects were complemented by expression of either MscL or YkuT in trans. All MS channel mutant strains ultimately became resistant to osmotic downshock in stationary phase but at varying times, with mscL ykuT strains taking the longest time to become resistant. Expression of -galactosidase from gene fusions to lacZ showed modest expression of ykuT and lower levels of expression of yhdY and yfkC when strains were grown in medium containing high salt. Sporulation of all MS channel mutant strains was normal, and the mutant spores germinated normally with L-alanine or dodecylamine. Keywords Bacillus · Mechanosensitive channels · Osmoregulation · Osmotic downshock · Sporulation · Spore germination
Communicated by Erko Stackebrandt. 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 DPA Pyridine-2-6-dicarboxylic acid or dipicolinic acid LB Luria-Bertani medium MS Mechanosensitive MscL Mechanosensitive channel of large conductance MscS Mechanosensitive channel of small conductance OD600 nm Optical density at 600 nm
Introduction Bacillus subtilis is a bacterium that plays a pivotal role in the decomposition of organic matter and cycling of nutrients in the soil. This organism is a model for gram-positive bacteria, which possess a thick peptidoglycan layer surrounding the plasma membrane that provides signiWcant protection against membrane rupture (Priest 1993; Wipat and Harwood 1999). The solute and moisture content of this bacterium’s environment frequently Xuctuates due to desiccation or to rain. Consequently, the bacterium must adjust levels of intracellular solute and water to avoid swelling or shrinkage due to excessive or minimal soil moisture (Bremer 2001; Holtman and Bremer 2004). In situations with low soil moisture, the bacterium accumulates high levels of compatible solutes such as glycinebetaine to prevent dehydration of the cytoplasm. However, the cytoplasm may take up water when soil water content increases, with an attendant increase in cell turgor pressure to levels that can cause cell lysis. In this situation,
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