Bacterial spores of various Bacillus species are impermeable or exhibit low permeability to many compounds that readily penetrate germinated spores, including methylamine. We now show that a lipid probe in the inner membrane of dormant spores of Bacillus megaterium and Bacillus subtilis is largely immobile, as measured by fluorescence redistribution after photobleaching, but becomes free to diffuse laterally upon spore germination. The lipid immobility in and the slow permeation of methylamine through the inner membrane of dormant spores may be due to a significant (1.3-to 1.6-fold) apparent reduction of the membrane surface area in the dormant spore relative to that in the germinated spore, but is not due to the dormant spore's high levels of dipicolinic acid and divalent cations.D ormant bacterial spores of various Bacillus species are extremely resistant to chemical agents that readily kill germinated spores or growing cells (1, 2). One factor important in dormant spore resistance to such chemicals appears to be their slow permeation into the spore core or protoplast. A variety of data indicate that it is the spore's inner membrane that is the major permeability barrier restricting the passage of small molecules into the spore core (3-7), although the lipid composition of the inner membrane exhibits no anomalies that might explain its unusual properties (8)(9)(10)(11)(12). In addition, the dormant spore's inner membrane has the potential to expand significantly, because electron microscopy indicates that the volume this membrane encompasses (the spore core): (i) decreases as much as 2-fold late in sporulation (13); and (ii) increases up to 2-fold in the first minute of spore germination, when the spore's large peptidoglycan cortex is degraded and the germ cell wall expands (13)(14)(15). This increase in core volume during spore germination takes place without new membrane synthesis, because ATP production is not required, and restores relatively normal permeability to the germinated spore's plasma membrane (4,5,16,17).Whereas dyes that stain growing cells, including fluorescent lipid probes, do not stain dormant spores (B.S. and P.S., unpublished results) as expected (15, 18), lipid probes can be incorporated into the developing spore's membranes during sporulation (19). Given the latter finding, we decided to add fluorescent lipid probes during sporulation to prepare spores with labeled membranes, and then use the technique of fluorescence redistribution after photobleaching (FRAP) to directly analyze the fluidity of the spore's inner membrane, because this technique has been used successfully in analyzing the fluidity and organization of molecules in a number of other systems, including spores of Bacillus species (20-25). MethodsStrains and Spore and Cell Preparation. The iosgenic Bacillus subtilis strains used in this work are derivatives of strain 168 and were: PS832, a prototrophic derivative of strain 168; PS3207, cwlD::cam, in which the cwlD gene responsible for production of muramic acid-␦-lactam in ...