Autoregulation of SafA Assembly through Recruitment of a Protein Cross-Linking Enzyme.

The coat of Bacillus subtilis spores is a multiprotein protective structure that also arbitrates many of the environmental interactions of the spore. The coat assembles around the cortex peptidoglycan layer and is differentiated into an inner and an outer layer and a crust. SafA governs assembly of the inner coat, whereas CotE drives outer coat assembly. SafA localizes to the cortex-coat interface. Both SafA and its short form C30 are substrates for Tgl, a coat-associated transglutaminase that cross-links proteins through ε-(γ-glutamyl)lysyl isopeptide bonds. We show that SafA and C30 are distributed between the coat and cortex layers. The deletion of tgl increases the extractability of SafA, mainly from the cortex. Tgl itself is mostly located in the inner coat and cortex. The localization of Tgl-cyan fluorescent protein (Tgl-CFP) is strongly, but not exclusively, dependent on safA However, the association of Tgl with the cortex requires safA Together, our results suggest an assembly pathway in which Tgl is first recruited to the forming spore in a manner that is only partially dependent on SafA and then is drafted to the cortex by SafA. Tgl, in turn, promotes the conversion of coat- and cortex-associated SafA into forms that resist extraction, possibly by catalyzing the cross-linking of SafA to other coat proteins, to the cortex, and/or to cortex-associated proteins. Therefore, the final assembly state of SafA relies on an autoregulatory pathway that requires the subcellular localization of a protein cross-linking enzyme. Tgl most likely exerts a "spotwelding" activity, cross-linking preformed complexes in the cortex and inner coat layers of spores.IMPORTANCE In this work, we show how two proteins work together to determine their subcellular location within the coat of bacterial endospores. Bacillus subtilis endospores are surrounded by a multilayer protein coat composed of over 80 proteins, which surrounds an underlying peptidoglycan layer (the spore cortex) protecting it from lytic enzymes. How specific coat proteins are targeted to specific layers of the coat is not well understood. We found that the protein SafA recruits a protein-cross-linking enzyme (a transglutaminase) to the cortex and inner layers of the coat, where both are cemented, by cross-linking, into macromolecular complexes.