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Aims: To determine the effect of sporulation temperature on Bacillus subtilis spore resistanceand spore composition.Methods and Results: Bacillus subtilis spores prepared at temperatures from 22 to 48 C hadidentical amounts of dipicolinic acid and small, acid-soluble proteins but the core water contentwas lower in spores prepared at higher temperatures. As expected from this latter finding,spores prepared at higher temperatures were more resistant to wet heat than were sporesprepared at lower temperatures. Spores prepared at higher temperatures were also moreresistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidizedwater, Sterilox. However, spores prepared at high and low temperatures exhibited nearlyidentical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores preparedat different temperatures showed very little difference in structure with only a small, albeitsignificant, increase in the percentage of muramic acid with a crosslink in spores prepared athigher temperatures. In contrast, there were readily detectable differences in the levels of coatproteins in spores prepared at different temperatures and the levels of at least one coat protein,CotA, fell significantly as the sporulation temperature increased. However, this latter changewas not due to a reduction in cotA gene expression at higher temperatures.Conclusions: The temperature of sporulation affects a number of spore properties, includingresistance to many different stress factors, and also results in significant alterations in the sporecoat and cortex composition.Significance and Impact of the Study: The precise conditions for the formation ofB. subtilis spores have a large effect on many spore properties.

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 degrees 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 approximately threefold more rapidly at 90 degrees 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.