Levels and localization of mechanosensitive channel proteins in Bacillus subtilis.
ABSTRACT Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock > or =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.
SourceAvailable from: Erhard Bremer[Show abstract] [Hide abstract]
ABSTRACT: Glycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the build-up of large pools, whose size was sensitively determined by the degree of the imposed osmotic stress. The amassing of glycine betaine caused a repression in the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was up-regulated in response to both sudden and sustained increases in the external osmolarity. Non-ionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. Expression of opuA was down regulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless whether this osmoprotectant was imported or newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was up-regulated in a B. subtilis mutant unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.Journal of bacteriology 11/2012; DOI:10.1128/JB.01505-12 · 2.69 Impact Factor
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ABSTRACT: Over the last 10 to 15 years, fluorescence methods have become frequently used for studies of the conformational changes of various membrane proteins including ion channels that function as gated membrane pores in numerous physiological processes essential for the normal function of biological cells. In combination with molecular biological methods that allow introduction of fluorescent labeling in vivo and in vitro, and functional electrophysiological voltage- and patch-clamp recording techniques, fluorescence spectroscopy methods, such as Förster resonance energy transfer (FRET), have made ion channel proteins accessible to studies of protein structure and function relationship. Here we focus on the use of these methods in studies of structural dynamics of bacterial mechanosensitive channels, which to date are among the best studied membrane channels functioning in mechanosensory transduction in living cells.Handbook of Imaging in Biological Mechanics, Edited by Corey P. Neu and Guy M. Genin, 11/2014: chapter 34: pages 425–432; CRC Press., ISBN: 978-1-4665-8813-4
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ABSTRACT: A critical event during spore germination is the release of Ca-DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca-DPA through the inner membrane of the spore is not clear, but proteins encoded by the Bacillus subtilis spoVA operon are involved in the process. We cloned and expressed the spoVAC gene in Escherichia coli and characterized the SpoVAC protein. We show that SpoVAC protects E. coli against osmotic downshift, suggesting that it might act as a mechanosensitive channel. Purified SpoVAC was reconstituted in unilamellar lipid vesicles to determine the gating mechanism and pore properties of the protein. By means of a fluorescence-dequenching assay, we show that SpoVAC is activated upon insertion into the membrane of the amphiphiles lysoPC and dodecylamine. Patch clamp experiments on E.coli giant spheroplast as well as giant unilamellar vesicles (GUVs) containing SpoVAC show that the protein forms transient pores with main conductance values of about 0.15 and 0.1nS respectively. Overall, our data indicates that SpoVAC acts as a mechanosensitive channel and has properties that would allow the release of Ca-DPA and amino acids during germination of the spore.Molecular Microbiology 03/2014; DOI:10.1111/mmi.12591 · 5.03 Impact Factor