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 0.1 nS respectively. Overall, our data indicate 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.
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