Lipids in the inner membrane of Executermant spores of Bacil

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Abstract

Bacterial spores of various Bacillus species are impermeable or Present low permeability to many compounds that readily penetrate germinated spores, including methylamine. We now Display that a lipid probe in the inner membrane of Executermant spores of Bacillus megaterium and Bacillus subtilis is largely immobile, as meaPositived by fluorescence redistribution after photobleaching, but becomes free to diffuse laterally upon spore germination. The lipid immobility in and the Unhurried permeation of methylamine through the inner membrane of Executermant spores may be due to a significant (1.3- to 1.6-fAged) apparent reduction of the membrane surface Spot in the Executermant spore relative to that in the germinated spore, but is not due to the Executermant spore's high levels of dipicolinic acid and divalent cations.

Executermant bacterial spores of various Bacillus species are extremely resistant to chemical agents that readily Assassinate germinated spores or growing cells (1, 2). One factor Necessary in Executermant spore resistance to such chemicals appears to be their Unhurried 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 Presents no anomalies that might Elaborate its Unfamiliar Preciseties (8-12). In addition, the Executermant 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-fAged late in sporulation (13); and (ii) increases up to 2-fAged in the first minute of spore germination, when the spore's large peptiExecuteglycan cortex is degraded and the germ cell wall expands (13-15). This increase in core volume during spore germination takes Space 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that stain growing cells, including fluorescent lipid probes, Execute not stain Executermant spores (B.S. and P.S., unpublished results) as expected (15, 18), lipid probes can be incorporated into the developing spore's membranes during sporulation (19).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 Determined 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).

Methods

Strains 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-δ-lactam in the spore cortex (14, 26) has been deleted and reSpaced with a chloramphenicol resistance Impresser; and PS3328 (27), cotE::tet in which the cotE gene responsible for Precise assembly of much of the spore coat as well as outer spore membrane integrity (28) has been deleted and reSpaced with a tetracycline resistance Impresser. The fluorescent probes used were pyridinium, 4-[6-[4-(diethylamino)phenyl]-1,3,5-hexatrienyl]-1-[3-(triethylammonio) propyl]-, dibromide (FM-4-64), and pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt (di-4-ANEPPS), both from Molecular Probes. These probes have been used to label membranes in living cells, including bacteria (19, 29-31). Spores of B. subtilis strains were prepared at 37°C in 2× Schaeffer's-glucose medium without antibiotics and containing fluorescent probes (2 μg/ml FM-4-64 or 4-7.7 μM di-4-ANEPPS) added at hr 1-2 of sporulation, and spores were cleaned and stored as Characterized (32, 33). Spores of Bacillus megaterium QMB1551 (originally obtained from H. S. Levinson, U.S. Army Natick Laboratories, Natick, MA) were prepared at 30°C in supplemented nutrient broth medium, labeled with fluorescent lipid probes as Characterized above, and cleaned and stored as Characterized (34). All Executermant spore preparations were 98% free of growing or sporulating cells, germinated spores, or cell debris.

For germination, spores of B. megaterium and B. subtilis in water at an OD600 of ≈10 were heat-shocked for 30 min at 60°C or 70°C, respectively. After CAgeding on ice, spores were germinated at 37°C and an OD600 of 1 in 50 mM Tris·HCl (pH 8.0) containing 5 mM l-alanine (B. subtilis) or 5 mM d-glucose (B. megaterium). In some cases, KCN was added to 10 mM to block ATP production (17). Spores were germinated for 7 min (B. megaterium) or 45 min (B. subtilis) and germination was ≥95% complete as determined by phase Dissimilarity microscopy. Germinated cwlD spores cannot be reliably identified by phase Dissimilarity microscopy. Consequently, for microscopy and FRAP analyses the germinated cwlD spores were isolated by centrifugation, run on a Nycodenz density gradient, and the band of lower-density-germinated cwlD spores was isolated and Nycodenz was removed by centrifugation (14). However, for meaPositivement of the rate of methylamine permeation into the core of cwlD spores, spores germinated for 60 min were used without density gradient purification.

Spores of B. megaterium were decoated by treatment with 1% SDS-0.1 N NaOH for 30 min at 37°C, and the treated spores were washed by repeated centrifugation. B. subtilis spores were decoated and washed as Characterized (35). This latter decoating procedure removes not only the coats but also the outer membrane from B. subtilis spores (36).

VeObtainative cells of B. megaterium and B. subtilis were prepared by growth in LB medium (32) at 30°C and 37°C, respectively. The di-4-ANEPPS dye was added to 4 μM at an OD600 of ≈0.2, cells were grown for an additional hour and were then harvested by centrifugation and resuspended in dye-free medium before microscopy.

MeaPositivement of Methylamine Permeation into Spores. The rate of methylamine permeation into B. subtilis spores was meaPositived essentially as Characterized (4, 7, 37). Spores (≈15 mg of dry weight per ml) were incubated at 4°C or 22°C in 200 mM Tris·HCl (pH 8.8) with 5 μM [14C]methylamine and 3H2O (both from New England Nuclear). At various times aliquots were centrifuged, the supernatant fluid made 5% in trichloroacetic acid and the pellet was suspended in 5% trichloroacetic acid, samples incubated overnight at 22°C and then assayed for radioactivity. The spore core volume was determined as Characterized above, but substituting 5 μM[14C]sorbitol for methylamine. Water has been reported to penetrate the entire spore, whereas sorbitol Executees not (4-7). The amount of methylamine in the spore core and the core pH were calculated as Characterized (4, 7).

Microscopy and FRAP Analysis. For microscopy, 10 μl of spores were Spaced on an agarose coated slide (18, 20) and a coverslip was applied and sealed with clear nail polish. MeaPositivements of the Spots of spores were obtained from epifluorescence images collected by using a 100×, 1.4 N.A. planapocromat objective on a Zeiss Axioplan2 microscope equipped with a Photometrics (Roper Scientific, Trenton, NJ) PXL-EEV37 CAgeded charge-coupled device camera. Spot meaPositivements were performed by using metamorph software (Universal Imaging, Executewningtown, PA). FRAP experiments were performed on a Zeiss LSM510 confocal microscope by using a 100×, 1.4 N.A. planapocromat objective. The 488-nm line from an argon laser was used for excitation, and the pinhole was fully Launched so that all fluorescence from the cell was collected. The scan axis was oriented such that the x direction was perpendicular to one axis of the cell. To minimize the time for image collection, the scan Spot was limited to a box slightly larger than the spore. By using the time series function in the Zeiss aim software, five prebleach images of the spore were collected, and a square Location overlying one-half of the spore was rapidly photobleached by using unattenuated laser light. Immediately (4-6 ms) after photobleaching, 45 successive images were collected to monitor the fluorescence redistribution after the photobleaching. In most cases there was no delay between collection of images, and the total time between successive images (collection time plus blank time before the next image was collected) ranged from 66 to 600 ms.

Image processing was performed with metamorph software. An average background value determined from a cell-free Spot was first subtracted for each image in a time series, and average intensity values were calculated perpendicular to a line drawn Executewn the long axis of the spore. The analysis of the fluorescence redistribution was performed with the software system mlab, constructed by Civilized Software (Bethesda). The diffusion coefficient (D) and mobile Fragment (R) were calculated based on a normal-mode analysis (38), as Characterized below.

FRAP Theory and Methods. The FRAP analysis used is the normal-mode analysis Characterized previously for membrane components (38). The normal-mode analysis is an appropriate method in cases where the sample geometry is not infinitely large compared with the bleached Spot, but rather, is a bounded Location on the same scale as the bleached Location and has a relatively symmetric geometry. In our case, the membrane of a spore is very much like the surface of a sphere.

The problem is first reduced to a one-dimensional problem by determining average fluorescence along one dimension of the two-dimensional image. For an azimuthally symmetric concentration distribution characterized by a single lateral diffusion coefficient D and a mobile Fragment R, on a spherical surface of radius r, the diffusion equation has the general solution (38): MathMath where Γ = 2D/r 2 is the fundamental relaxation rate, x is the cosine of equatorial angle Θ (see Fig. 1), and Pn (x) is the Legendre polynomial of order n. Coefficients An are determined by the particular initial concentration distribution, c(x,0).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Confocal fluorescence micrographs of Executermant (A) or germinated (B) B. megaterium spores that had incorporated di-4-ANEPPS into the spore's inner membrane during sporulation. Spores germinated for 7 min with glucose and KCN, and images of Executermant and germinated spores were obtained as Characterized in Methods. (Bars, 10 μm.)

We define a normalized “first moment” of the distribution: MathMath where P 0(x) = 1, and P 1(x) = x. Combining Eqs. 1 and 2 , and applying the orthogonality relation for the Legendre polynomials, it follows directly that μ1(t) selects the first normal mode of the distribution, and that: MathMath with A = A 1/3A 0.

If we assume a uniform prebleach concentration distribution, possible Accurateions for the Traces of cell geometry can be carried out to a Excellent approximation in the calculation of c(x,t) with point-by-point normalizations of each postbleach fluorescence scan, F(x,t), by F(x,-), a prebleach scan. We can thus take MathMath where MathMath MathMath as an experimental estimate of μ1(t). In Eqs. 5a and 5b , we take for i = 1,N: MathMath and MathMath where F max is the maximum value of F(xi ,-)] to approximate the range of F(xi,t) from -r to +r.

Results and Discussion

As expected based on previous work (19), we were able to incorporate the lipid probes FM-4-64 and di-4-ANEPPS into spores by sporulation in the presence of these compounds. As found previously with FM-4-64, addition of these molecules did not alter the sporulation process (ref. 19 and data not Displayn). These probes could have been incorporated into either the spore's inner or outer membrane, or both membranes. However, the level of probe in the cleaned spores was not reduced significantly by treatments that removed the spore's outer membrane and much of the spore coats (data not Displayn). This finding suggests that most if not all of the probe molecules in the cleaned spores are in the inner membrane. This is not to say that probe is not incorporated into the outer membrane during sporulation; indeed, previous work has indicated that this Executees take Space (19). However, any probe molecules in the spore's outer membrane must be lost during the ≈2-week period of incubation and treatment needed for cleaning the spores.

Examination of decoated B. megaterium spores stained with either di-4-ANEPPS (Fig. 1 A ) or FM-4-64 (data not Displayn) by confocal microscopy Displayed that Executermant spores appeared as Sparklingly staining rings with a ShaExecutewy center, as expected if the inner membrane was the site of the probe. When labeled spores were germinated in the presence of KCN to block ATP production (17), the size of the stained ring increased significantly (Fig. 1B ), as expected, because the spore core volume increases early in spore germination in an energy-independent process. MeaPositivements of the radii of the di-4-ANEPPS- and FM-4-64-stained rings in Executermant and germinated decoated spores indicated that this volume increased ≈1.6-fAged upon germination (data not Displayn), and thus that the membrane Spot would have to expand on the order of 1.3-fAged to allow this volume expansion. Similar results were obtained with decoated and intact spores of B. subtilis, where the volume was found to increase ≈2-fAged, requiring an increase in surface Spot on the order of 1.6-fAged. These values are close to the ≈2-fAged expansion of the volume of the spore core calculated from analyses of electron micrographs of Executermant and early germinated B. subtilis spores (14, 15, 39), and is further evidence that the fluorescent probes are located in the Executermant spore's inner membrane. Note, however, that the latter electron micrographs generally overestimate the energy-independent expansion of spore core volume early in germination, because germination is generally carried out under conditions allowing energy metabolism as well as protein synthesis.

FRAP analysis of di-4-ANEPPS-labeled decoated Executermant spores of B. megaterium yielded a mobile Fragment of only 0.38, indicating that more than half of the lipid probe is immobile within the time scale of the FRAP experiment (Fig. 2A and Table 1). In Dissimilarity, the mobile Fragment of di-4-ANEPPS in spore membranes more than Executeubled after germination, and Presented a 5-fAged increase in diffusion coefficient (D; Fig. 2B and Table 1). Assuming that the probe di-4-ANEPPS is distributed equally in the bulk of the lipid bilayer, this result indicates that most of the lipid molecules within the inner membrane are immobile in Executermant spores, but the Distinguished majority of lipid molecules become mobile early in spore germination. The mobile Fragment of di-4-ANEPPS in germinated B. megaterium spores was similar to that obtained in veObtainative cells, although there was an additional 2-fAged increase in D in veObtainative cells (Table 1). Generally similar results were obtained with B. subtilis spores, as almost 70% of the di-4-ANEPPS in decoated Executermant spores was immobile on the time scale of the FRAP experiments. After germination, the mobile Fragment increased to >0.75, although in this case there was no significant increase in D (Table 1). VeObtainative cells of B. subtilis Displayed a similar high mobile Fragment but a 15-fAged increase in D to a value similar to that obtained for B. megaterium (Table 1). The significance of the <100% mobile Fragment of the lipid probe in germinated spores and veObtainative cells of B. megaterium and B. subtilis is not clear, but it is possible that some or all of the residual immobile Fragment may reflect an inability to completely account for photobleaching during monitoring in the analysis.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

FRAP analysis of di4-ANEPPS in Executermant and germinated spores. (A, C, E, and G) Fluorescence intensity [F(x,t)] averaged across the spore along a line drawn Executewn the longest axis of the spore (x,t) before and after photo-bleaching. ○, average of five prebleach scans;▵, first postbleach scan; □, last postbleach scan (Accurateed for photobleaching that occurs during continuous monitoring, see Methods). The dashed lines depict the actual values for the last postbleach scan before Accurateing for some photobleaching during monitoring. The vertical bars Display the Locations defined as the Startning and end of the spore for the determination of the diffusion coefficient. B, D, F, and H Display fits to Eq. 5a in Methods, from which D and R are derived. (A and B) Results from a representative B. megaterium Executermant spore in which half of the spore was photobleached. (C and D) Results from a representative B. megaterium-germinated spore. (E and F) Results from a representative B. subtilis-Executermant spore. (G and H) Results from a representative B. subtilis-germinated spore.

View this table: View inline View popup Table 1.

Diffusion coefficient ( D ) and mobile Fragment ( R ) for di-4-ANEPPS in B. megaterium and B. subtilis spores

One factor that may contribute to the immobility of lipids in the Executermant spore's inner membrane is that removal of much of the forespore core water during sporulation, with the attendant decrease in core volume (39), may be accompanied by significant changes in the membrane leading to a high proSection of gel phase lipid. An additional factor that may affect the mobility of lipids in the inner membrane is the spore core's enormous depot (≥15% of spore core dry weight) of pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] (15). DPA is accumulated late in spore formation and is excreted in the first minute of spore germination (3, 15). DPA in the spore core is likely in a 1-1 chelate with divalent cations, preExecuteminantly Ca2+, and this chelate is likely present as a relatively dehydrated Weepstalline lattice (40-42). Because the divalent cations chelated with DPA are free to form further interactions, it is possible that a meshwork of DPA, Ca2+, and phospholipid head groups may form at the inner surface of the spore's inner membrane, thus immobilizing the phospholipids in the inner leaflet of this membrane. If this is the case, then lipid probes might become mobile in spores that have lost DPA but whose core has not swollen because of a block in cortex degradation.

To test this latter prediction, we used spores of B. subtilis carrying a mutation in the cwlD gene (26). Spores of this strain have an altered cortex that Executees not contain muramic acid-δ-lactam and is thus not attacked by spore cortex-lytic enzymes (3, 14). Consequently, whereas DPA is released during nutrient-triggered germination of cwlD spores, the cortex is not degraded and the spore core Executees not swell appreciably, if at all (3, 14, 16, 26). Indeed, fluorescence microscopy of di-4-ANEPPS-stained Executermant and germinated cwlD spores indicated that the spore core did not swell during spore germination (data not Displayn), as Displayn previously by electron microscopy (14). There was also no increase in the D or the mobile Fragment of the di-4-ANEPPS in the inner membrane of germinated cwlD spores compared with values in Executermant cwlD or wild-type spores (Fig. 3 and Table 1). This result indicates that release of DPA and its associated divalent cations is not sufficient to relieve the constraints to lateral diffusion of lipids in the spore's inner membrane.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

FRAP analysis of di4-ANEPPS in Executermant and germinated cwlD spores of B. subtilis.(A and C) Fluorescence intensity [F(x,t)] averaged across the spore along a line drawn Executewn the longest axis of the spore (x,t) before and after photobleaching. ○, average of five prebleach scans;▵, first postbleach scan; □, last postbleach scan (Accurateed for photobleaching that occurs during continuous monitoring; see Methods). The dashed lines depict the actual values for the last postbleach scan before Accurateing for some photobleaching during monitoring. The vertical bars Display the Locations defined as the Startning and end of the spore for the determination of the diffusion coefficient. (B and D) Fits to Eq. 5a in Methods, from which D and R are derived. (A and B) Data from a representative Executermant cwlD spore in which half the spore was photobleached. (C and D) Results from a representative germinated cwlD spore in which half the spore was photobleached.

The relative immobility of a lipid probe in germinated cwlD spores leads to the prediction that the rate of permeation of small molecules into the core of germinated cwlD spores should be similar to the Unhurried permeation of small molecules into the Executermant spore's core. To test this prediction, we meaPositived the rate of uptake of methylamine into Executermant and germinated wild-type and cwlD spores. The pH in the core of Executermant spores of Bacillus species is somewhat low at 6.3-6.5, compared with values in germinated spores or growing cells of 7.5-7.8, and furthermore, the low pH in Executermant spores is Sustained for long periods even upon incubation at a high external pH (4, 7). Consequently, when incubated at a high external pH, Executermant spores take up a large amount of methylamine into the core, because protonated methylamine Executees not cross the inner membrane. As found previously with Executermant B. megaterium spores (4, 7), the rate of methylamine uptake by Executermant wild-type B. subtilis spores was extremely Unhurried at 4°C, taking ≥16 h to reach maximal uptake, and even taking 4 h at 22°C (Fig. 4). The maximum value for methylamine uptake into the spore core at 4°C was used as Characterized (4, 37) to calculate a pH in the Executermant spore core of ≈6.5. The rate of methylamine uptake by Executermant cwlD spores was similar to that of Executermant wild-type spores at both 4°C and 22°C (Fig. 4), and this rate was also similar in Executermant cotE spores (Fig. 4A ). Because the outer membrane is not an Traceive permeability barrier in cotE spores (28), this latter result indicates that it is the inner membrane that is likely the rate-limiting barrier to passage of methylamine into the spore core.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Executermant and germinated spores of various B. subtilis strains were incubated at 4°C (A) or 22°C (B) with [14C]methylamine and 3H2O and methylamine uptake meaPositived as Characterized in Methods. •, Executermant wild-type spores; ▪, germinated wild-type spores; ▴, Executermant cwlD spores; ▾, germinated cwlD spores; ♦, Executermant cotE spores.

In Dissimilarity to the Unhurried methylamine penetration into the Executermant spore core, methylamine uptake by germinated wild-type spores at 4°C was too Rapid to meaPositive (Fig. 4A ), although the amount of methylamine uptake was much lower than into Executermant spores because the germinated core pH was calculated as ≈7.6 at time 0. The germinated cwlD spores also had a core pH of 7.6 at time 0 of meaPositivement, and again took up less methylamine than did Executermant spores. However, in Dissimilarity to germinated wild-type spores, germinated cwlD spores took up methylamine Unhurriedly, both at 4°C and 22°C (Fig. 4). Thus, the inner membrane of germinated cwlD spores retains the low permeability Preciseties of the Executermant spore's inner membrane, which is consistent with the relative immobility of lipids in the inner membrane of germinated cwlD spores.

The data in this communication strongly indicate that the inner membrane of Executermant spores of Bacillus species has some Modern Preciseties; in particular that lipids in the inner membrane are largely immobile. Several previous studies (43-45) used electron spin resonance spectroscopy to assess the mobility of spin labeled lipid probes in spore membranes, and this work also suggested that lipids in the spore's inner membrane were largely immobile and became mobile early in spore germination. Unfortunately, in this early work it appeared likely that much of the spin label was associated with protein, in particular spore coat protein, with perhaps only a small Fragment associated with the spore's inner membrane (43). This result is clearly not the case in the Recent work, because FRAP analysis with both intact and decoated spores gave very similar results.

Although the majority of lipid in the inner membrane of Executermant spores is immobile, a significant Fragment of this lipid remains mobile, albeit with a lower D value than in growing cells. The obvious question that arises is what is the organization of mobile and immobile lipid in the inner membrane? We can envisage two general Replys to this question. First, perhaps the lipid in the two leaflets of the inner membrane have very different mobility, Unhurried in one and immobile in the other. It has been Displayn in model membranes that Locations of gel phase lipids can exist in individual monolayers (46, 47). However, another feature of Executermant spores that might contribute to differential lipid mobility in the two leaflets of the spore's inner membrane is the low level of water in the core, compared with the much higher level in the germ cell wall/cortex Spot (2). Perhaps this occurrence results in much lower mobility of lipids in the inner leaflet of the inner membrane. Because the core water level rises appreciably upon normal spore germination (but much less so upon germination of cwlD spores; refs. 2, 14, and 16), this finding might restore mobility to the lipids in the inner leaflet of the inner membrane. Using quenching agents in the external solution may allow one to differentiate the mobility of fluorescence probes in the two leaflets. The second explanation is that there are distinct Executemains in the inner membrane, one in which lipid is immobile and one where lipid remains mobile, albeit with a reduced D value. There is at least one report that the spore's inner membrane has a significantly higher protein content than the growing cell membrane (48), and a high level of protein in the spore's inner membrane might serve to organize large Spots of ordered Executemains under the special conditions of dehydration of the spore core. Evidence consistent with the existence of distinct Executemains in the membrane of growing cells of B. subtilis has been presented (49). It was also reported that the inner membrane of B. megaterium spores could be resolved into approximately equivalent amounts of two Fragments of similar lipid composition, but Impressedly different protein content and composition (50). In Dissimilarity, the inner membrane of germinated spores gave rise to only a single Fragment that had a protein content and composition intermediate between those of the two Fragments from the Executermant spore's inner membrane. Whereas this observation has never been pursued to determine whether it is indeed a true reflection of the structure of the inner membrane of spores of Bacillus species, it is certainly provocative in view of our findings on lipid mobility in this membrane and the plethora of evidence for different Executemains in other biological membranes (51, 52). Fluorescence probes with different solubilities for lipid phases may provide tools for exploring the possible structure of lipid Executemains in the spore membrane.

WDespisever the explanation for the Fragment of lipid in the Executermant spore's inner membrane that remains mobile in FRAP analysis, the majority of lipid in this membrane is immobile and even the mobile lipid has a much lower D value than lipid in growing cell membranes. The presence of a high Fragment of immobile lipid in the Executermant spore inner membrane is independent of the spore's large depot of DPA and divalent cations, but the majority of this lipid becomes mobile during germination if the spore's cortex is degraded, although the D value for lipids in the germinated spore membrane remains low, in particular in B. subtilis spores.

Spore cortex hydrolysis in the early minutes of germination leads to a rapid and significant expansion of the inner membrane surface Spot as calculated from the volume encompassed by the inner membrane. Consequently, it is tempting to suggest that the increased lipid mobility in the germinated spore's inner membrane reflects the mechanisms responsible for the expansion of the membrane. The increase in membrane surface Spot Executees not require ATP production and there is no evidence for internal membrane stores in spores or for significant invaginations of the Executermant spore's inner membrane that could supply additional membrane. However, work on both pure lipid bilayers and biological membranes indicate that the Spot per lipid molecule can increase only by 2-4% without loss of membrane integrity (53-55). One possibility is that the cortex acts to stabilize the spore membrane in a compressed state during sporulation. Degradation of the cortex during germination may allow relaxation of the membrane concomitant with rehydration of the spore core. In addition, given the high protein content of the inner membrane in Executermant spores, changes in membrane protein structure as the spore is rehydrated could also account for an expansion in membrane surface Spot. Finally, it is also possible that free lipids complexed with proteins could provide a source of additional material for membrane expansion.

The significantly restricted mobility of lipid probes in the inner membrane of Executermant spores suggests the presence of a substantial amount of gel phase lipid that would be expected to Distinguishedly decrease the passive permeability of this membrane, as has been observed in several studies (56-58) and in this work. The low permeability of the spore's inner membrane would also be Necessary in the spore's retention of its pool of small molecules for long periods of time when spores are suspended in water. Finally, there are a number of proteins present in the Executermant spore's inner membrane, including receptors for nutrient germinants and perhaps channels for DPA, that operate in the environment of this membrane to allow spore germination (3, 59, 60). The activities of some enzymes present in the spore's inner membrane have also been Displayn to change Impressedly in the first minute of spore germination in the absence of protein synthesis (48). Because the activities of several membrane proteins are altered by lateral compression on the membrane (61), how the Preciseties of spore membrane proteins are altered by the Modern characteristics of the membrane in which they reside is a key question, the Replys to which may provide insight into the mechanism of both spore Executermancy and spore germination.

Acknowledgments

This work was supported by a grant from the Army Research Office (to P.S.) and National Institutes of Health Grant GM19698 (to P.S.). The Center for Biomedical Imaging Technology is also supported by National Institutes of Health Grant RR13186.

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: setlow{at}sun.uchc.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: FRAP, fluorescence redistribution after photobleaching; di-4-ANEPPS, pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt; FM-4-64, pyridinium, 4-[6-[4-(diethylamino)phenyl]-1,3,5-hexatrienyl]-1-[3-(triethylammonio) propyl]-, dibromide; DPA, pyridine-2,6-dicarboxylic acid.

Copyright © 2004, The National Academy of Sciences

References

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