Aims:  To compare the properties of the spores of Bacillus subtilis prepared in liquid and on plates. Methods and Results:  The spores of B. subtilis were prepared at 37°C using a nutrient exhaustion medium either in liquid or on agar plates. The levels of core water, dipicolinic acid (DPA) and small, acid‐soluble spore proteins (SASP) were essentially identical in spores made in liquid or on plates. Spores prepared in liquid were killed ∼threefold more rapidly at 90°C in water than the spores prepared on plates, and the spores prepared in liquid were more sensitive to nitrous acid and a diluted stable superoxidized water. Spores prepared in liquid also germinated more rapidly with several agents than those prepared on plates. Pellets of spores prepared on plates were darker than spores prepared in liquid, and spores prepared in liquid had more readily extracted coat protein. However, there were no major differences in the relative levels of individual coat proteins or the cross‐linking of the coat protein GerQ in the two types of spores, although the inner membrane of spores prepared on plates had a higher ratio of anteiso‐ to iso‐fatty acids. Conclusions:  The preparation in liquid yielded spores with some different properties than those made on agar plates. Spores made in liquid had lower resistance to heat and several chemicals, and germinated more readily with several agents. There were also differences in the composition of the inner membrane of spores prepared under these two conditions. However, there were no major differences in the levels of DPA, core water, SASP and individual coat proteins or the cross‐linking of a coat protein in spores made in liquid and on plates. Significance and Impact of the Study:  This work demonstrates that the preparation method can affect the resistance and germination properties of bacterial spores, even if an identical medium and temperature are used. Evidence was also obtained consistent with the role of the inner membrane in spore resistance and germination, and that some factor in addition to core water, DPA and SASP content plays a role in spore resistance to wet heat.

The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an -(␥-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.

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