A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

Abstract

Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased Ϸ2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species. The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.S pores of various Bacillus species are formed in the process of sporulation, and these spores are adapted for long-term survival because they are resistant to many environmental stresses and are metabolically dormant (1-4). However, given the proper stimulus, generally the appearance of specific nutrients in the environment, the dormant spores can return to life through the process of spore germination and then outgrowth (2). A major factor in spore dormancy and resistance is the low level of water in the spore cytoplasm or core [25-55% of the mass of the hydrated dormant spore core depending on the species (1, 3-5)]; another factor may be the high level of pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] (Ϸ10% of the mass of the hydrated stage II-germinated spore core) in the spore core, likely present as a chelate with divalent cations, predominantly Ca 2ϩ (1,6). DPA is released in the first minutes of spore germination and is replaced with water as the spore-core water content rises slightly; these spores are said to have completed stage I of germination (2, 7). Subsequently, the large peptidoglycan cortex around the spore core is degraded, and removal of this restraint allows the spore core to expand rapidly by uptake of water and thus complete stage II of germination (2, 7). This latter event results in a level of core water (75-80% of wet weight) that is similar to that in growing cells (8). As noted above, there is neither metabolism nor enzyme action in the cytoplasm of dormant spores, although there are several enzyme-substrate pairs located in this region of the spore (3). There is also no detectable metabolism or enzyme action in the core of stage I-germinated spores that have excreted their DPA but have not degraded their cortex (7). However, in fully germinated (stage II) spores in which the cortex has been degraded and the cytoplasm is fully hydrated, enzyme action and metabolism begin rapidly (2).One explanation that has been put forward for the lack of enzyme action in dormant and stage I-germinated spores is that the level of water in these spores is too low to allow enzyme a...

The physical state of water in bacterial spores

“…In the core (at 0.6 h), proteins are thus not expected to undergo translational or rotational diffusion at measureable rates. This expectation is consistent with fluorescence studies (14) and with our analysis of the low-frequency 2 H dispersion (Fig. 2) in terms of internal water molecules in rotationally immobilized proteins.…”

“…Taking the dimensions of the assumed prolate-ellipsoidal core from Fig. 1, we find a cr ϭ 0.20 m. By using w cr ϭ 0.55 Ϯ 0.1 for the volume fraction of water in the core (14,17,32), we thus obtain P w Ͻ 1.8 Ϯ 0.6 m s Ϫ1 .…”

“…On the other hand, at least some core components appear to be immobilized. Dielectric permittivity data (11) and electron paramagnetic resonance spectra (12) suggest that most ions in the core are immobilized, 13 C NMR spectroscopy indicates that the core's large depot of pyridine-2,6-dicarboxylic acid (dipicolinic acid, DPA) is in a solid-like state (13), and fluorescence measurements show that green fluorescent protein is at least 4 orders of magnitude less mobile in the dormant core than in the cytoplasm of the vegetative B. subtilis cell (14). Although these observations are consistent with a glassy core, attempts using 1 H NMR linewidths to directly measure water mobility in spores have not been conclusive (15,16), partly because of the confounding effect of paramagnetic Mn(II) ions.…”

Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile

“…For microscopy, 10 l of spores were placed on an agarose coated slide (18,20) and a coverslip was applied and sealed with clear nail polish. Measurements of the areas of spores were obtained from epifluorescence images collected by using a 100ϫ, 1.4 N.A.…”

“…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)(21)(22)(23)(24)(25).…”