Identification of SpoVID as a putative new substrate of the sporulation-specific protease YabG in Bacillus subtilis

Abstract

Bacillus subtilis YabG, a cysteine protease expressed in mother cells during sporulation, is involved in the processing of spore coat proteins. However, the full spectrum of YabG substrates and the physiological significance of its proteolytic activity during sporulation remain poorly understood. A combination of SDS–PAGE and LC–MS/MS analysis revealed 83 proteins in yabG-deficient spores. Among these, 29 proteins were absent from wild-type spores and considered YabG-specific, including known substrates (such as SafA and SpoIVA) and newly identified candidate substrates, such as SpoVID. To elucidate the role of the YabG protease in spore coat formation in B. subtilis, we investigated the assembly of SpoVID onto the spore coat using immunoblotting with an anti-SpoVID antibody and fluorescence microscopy with a SpoVID–GFP fusion protein. Immunoblotting showed that SpoVID exists as a full-length 70-kDa protein in wild-type cells 5 h after sporulation, but was absent from or undetected in wild-type mature spores; however, it was detected as multiple bands in yabG-deficient spores. Treatment with a protease inhibitor blocked SpoVID processing in wild-type cells, indicating that YabG and other proteases are required for proteolytic processing during spore maturation. Fluorescence microscopy of GFP fusion proteins demonstrated that YabG was involved in precise SpoVID–GFP localization to the spore coat during spore maturation. Observation of YabG–GFP with 2-(4-aminophenyl) benzothiazole, a membrane stain, from sporulation to the mature spore stage showed that YabG was expressed under the control of SigK and then localized to the inner spore coat after the assembly of the SigE-dependent coat protein was completed. Additionally, in spoVID-deficient spores, the YabG–GFP signal at the forespore surface was markedly reduced, likely reflecting a general consequence of impaired spore coat encasement. Finally, our findings suggest that the YabG substrates include the basement layer and inner coat proteins and YabG processes these substrates after localizing to the basement layer and inner coat. We propose that YabG regulates spore coat formation both temporally and spatially by controlling coat protein localization and remodeling the coat structure.