"A La Recherche" of Functions for the Spore Protein SASP-E from Bacillus subtilis

We previously observed that Bacillus subtilis spores from sspE mutants presented a lower germination capacity in media containing high salt concentrations (0.9M NaCl). This deficiency was attributed to the absence of SASP-E (gamma-type small-acid-soluble protein), rich in osmocompatible amino acids released by degradation. Herein we observed that, in addition, this mutant spore presented a reduced capacity to use L-alanine as germinant (L-ala pathway), required longer times to germinate in calcium dipicolinate ($Ca^{2+}$-DPA), but germinated well in asparagine, glucose, fructose, and potassium chloride (AGFK pathway). Moreover, mild sonic treatment of mutant spores partially recovered their germination capacity in L-ala. Spore qualities were also altered, since sporulating colonies from the sspE mutant showed a pale brownish color, a higher adherence to agar plates, and lower autofluorescence, properties related to their spore coat content. Furthermore, biochemical analysis showed a reduced partition in hexadecane and a higher content of $Ca^{2+}$-DPA when compared with its isogenic wild-type control. Coat protein preparations showed a different electrophoretic pattern, in particular when detected with antibodies against CotG and CotE. The complementation with a wild-type sspE gene in a plasmid allowed for recovering the wild-type coat phenotype. This is the first report of a direct involvement of SASP-E in the spore coat assembly during the differentiation program of sporulation.