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Journal of Bacteriology, May 2004, p. 3195-3201, Vol. 186, No. 10
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.10.3195-3201.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of Bacillus subtilis by Inclusion in a Model Membrane

Tim Searls,1 Xingyong Chen,2 Stephanie Allen,2 and Michael D. Yudkin1*

Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU,1 School of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom2

Received 3 October 2003/ Accepted 27 January 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor {sigma}F, and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of B. subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Spore formation by the gram-positive soil bacterium Bacillus subtilis has been extensively studied as a simple example of cellular development and differentiation (21, 67). During vegetative growth of B. subtilis, medial division gives rise to two identical daughter cells, but under conditions of nutritional starvation, the cell forms a large mother cell and a smaller prespore, which develops into a tough, dormant spore. Sporulation in prokaryotes is a complex process, involving an ordered multiprotein division apparatus, which initiates an annular invagination of the cell wall and the formation of an asymmetric septal membrane of two bilayers, separated by peptidoglycan. Differentiation in the prespore is initiated by activation of the transcription factor {sigma}F, the first sporulation-specific sigma factor in the developmental cascade (21, 37, 44, 63). The sigma factor {sigma}F and its regulatory proteins SpoIIAA, SpoIIAB, and SpoIIE are synthesized before septation (6, 25, 56), but {sigma}F remains bound to the anti-sigma factor SpoIIAB, and therefore in an inhibited state, prior to closure of the asymmetric septum (3, 19, 51). In the predivisional cell, the anti-anti-sigma factor SpoIIAA is phosphorylated at a single site (16, 18, 51, 52). The resulting SpoIIAA-P cannot bind SpoIIAB (the protein kinase for SpoIIAA) to release {sigma}F from the SpoIIAB-{sigma}F complex (16, 19, 51). The phosphatase domain of the 91.5-kDa enzyme SpoIIE is responsible for the hydrolysis of SpoIIAA-P (4, 17, 24). Expression of SpoIIE during the early stages of sporulation does not lead to discernible concentrations of nonphosphorylated SpoIIAA. Rapid dephosphorylation of SpoIIAA-P occurs after closure of the septal membrane, even though cellular concentrations of SpoIIAB remain equivalent to or higher than that of the phosphatase SpoIIE (45). The resulting SpoIIAA protein activates {sigma}F by displacing it from the complex with SpoIIAB (4, 17, 18, 24, 47). Free SpoIIAB (but not SpoIIAB in complex with {sigma}F) is subject to ATP-dependent proteolysis by ClpCP, and SpoIIAB lost in this way is not immediately replenished since there is a delay before the spoIIAB gene is translocated into the prespore; this mechanism creates a self-reinforcing cycle that effectively locks {sigma}F activity on and helps to commit the cell to sporulation (54, 55). The prespore-specific activation of {sigma}F is observed only after closure of the asymmetric septum (10, 13, 41), suggesting that a regulatory mechanism exists that couples gene expression to the completion of this morphological event (23, 66).

SpoIIE is a multidomain integral membrane protein (5, 8) recruited by the division protein FtsZ to an asymmetrically positioned site of septum formation (42, 43), and it plays a role in septal morphogenesis (32, 58). In spoIIE null mutations, polar FtsZ ring formation and asymmetric division are impaired, suggesting that SpoIIE also contributes to the switch from medial to polar septation (9, 11, 24). Localization of SpoIIE to the asymmetric septum is important for proper temporal, and possibly spatial, activation of {sigma}F (5, 70). Hydrophobic N-terminal domain I of SpoIIE is composed of 10 membrane-spanning helices that localize the protein to the septal membrane in the early stages of sporulation (6, 8). The function of large central domain II is not well understood, but the domain has been found to self-associate and to interact with cell division protein FtsZ (46). Cytoplasmic C-terminal domain III functions as a serine phosphatase (17), with sequence similarity to the PP2C family of eukaryotic Ser/Thr protein phosphatases (1, 14). The phosphatase activity of SpoIIE is at least partially independent of asymmetric septation (nonphosphorylated SpoIIAA can be detected readily in cell division mutants that are unable to form an asymmetric septum) and is necessary but not sufficient to activate {sigma}F (23, 34), suggesting that for development to proceed, regulation of the phosphatase activity might be involved. How the PP2C domain is regulated is unclear, but several mechanisms have been proposed. One possibility is that completion of the asymmetric septum and an increase in the concentration of SpoIIE could trigger a conformational change in the phosphatase domain. Alternatively, a regulatory protein could bind to SpoIIE and control phosphatase activity. Arigoni et al. (5) have suggested that an unstable inhibitor is lost in the prespore. It has also been proposed that a regulatory site on SpoIIE might play a direct role in retaining SpoIIAA at the cytokinetic ring until after the septum has formed (34).

SpoIIE has been purified, and its kinetic properties have been investigated in a variety of micellar environments (45). In a detergent environment, the SpoIIE protein dephosphorylates SpoIIAA-P at a rate approximately 100 times greater than the rate of phosphorylation by SpoIIAB (45). Similarly, after closure of the asymmetric septum in B. subtilis, the previously quiescent SpoIIE protein dephosphorylates SpoIIAA-P at a rate far greater than the rate of phosphorylation by SpoIIAB. The reason that SpoIIE activity is low before septum formation is not well understood, but it may be relevant that Aubry and Firtel (7) have found that the C-terminal PP2C-like phosphatase domain of the Dictyostelium Spalten protein is autoregulated by its membrane-associated N-terminal domain. The ability of domain II to self-associate, and the rapid increase in the SpoIIE concentration after septum closure and invagination, suggested a similar method for self-regulation through conformational change. It has been observed in B. subtilis that replacement of the transmembrane domain of SpoIIE with MalF caused {sigma}F to become active before septation (34); these results reinforce the possibility that regulation lies in the transmembrane domain. In a recent study (22), mutations affecting the beginning of domain II of spoIIE were found to activate {sigma}F independently of septum formation, and the mutant proteins showed the same phosphatase activity as the wild-type enzyme in vitro. The mutant proteins were fully functional in their localization to sites of asymmetric septation and their morphogenic activity in the formation of the asymmetric septum, suggesting a regulatory site in SpoIIE that tightly controls the activity of the phosphatase domain in response to asymmetric septation (22).

To begin to address the question of how the activity of SpoIIE is regulated in vivo, we have developed methods for reconstituting this transmembrane protein into a model lipid bilayer. Liposomes have been used in recent years as analytic biosensors (53, 68); as a tool for delivery of vaccines, therapeutic drugs, and hormones (26, 27); and as an ideal model for biomembranes. We now describe the unidirectional incorporation of SpoIIE into lipid vesicles by the method of Rigaud et al. (57, 62), which involves stepwise partial solubilization of preformed liposomes with low concentrations of detergent, incorporation of the membrane protein, and removal of the detergent with polystyrene beads. The rates of SpoIIAA-P dephosphorylation by vesicle-bound SpoIIE at a range of protein-to-lipid concentrations were directly compared to the rates of dephosphorylation by equimolar concentrations of micelle-engulfed protein in order to test whether phosphatase activity was diminished at low protein-to-lipid concentrations.

We have also examined the arrangement of the SpoIIE protein inserted into unilamellar vesicle (ULV) bilayers by atomic force microscopy (AFM). A range of scanning probe microscopes has been developed in recent years, among which AFM is increasingly popular for biological applications (2, 20). Conformational changes of membrane protein surfaces in solution at subnanometer resolution have been reported by the use of AFM in the tapping mode (29, 64). The arrangement of the SpoIIE proteins in the lipid bilayer could be directly inspected for oligomerization caused by self-association.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning, overexpression, and membrane isolation of SpoIIE. Recombinant plasmid pRB1011 was transformed into Escherichia coli strain C41(DE3) as described previously (45). Overexpression and membrane isolation were carried out by a method modified from that previously described (45). Cells grown overnight at room temperature after induction with isopropyl-ß-D-thiogalactopyranoside (IPTG) were harvested by centrifugation at 4°C and resuspended in 50 mM Tris-HCl (pH 8.0) containing 250 mM NaCl, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) (buffer A) and 10 mM MgCl2, with traces of DNase I, RNase, and lysozyme added as dry powders. The cells were disrupted in a precooled French press, and the homogenate was centrifuged for 20 min at 3,000 x g to remove the cell debris. The membrane fraction was recovered by centrifugation for 2 h at 40,000 x g. The pellet was resuspended in buffer A containing 0.5 mM EDTA and centrifuged for 1 h at 40,000 x g.

Purification. To extract the SpoIIE protein, the pellet was agitated gently in buffer A containing 0.5 mM EDTA and 5% (vol/vol; 85 mM) Triton X-100 for at least 4 h at 4°C, and the resulting homogenate was centrifuged for 2 h at 40,000 x g. The filtered supernatant was dialyzed into buffer A containing 25 mM NaCl, 0.5 mM EDTA, and 1% Triton X-100 and loaded onto a 30-ml DEAE-Sepharose fast protein liquid chromatography (FPLC) column equilibrated in the same buffer. Proteins were eluted with a linear gradient (0.025 to 0.7 M) of NaCl in the same buffer. The fractions containing SpoIIE eluted between 0.25 and 0.3 M NaCl. The pooled fractions were combined; concentrated with the aid of Centriplus concentrators (Amicon); dialyzed against a solution of Tris-HCl (pH 6.8) containing 25 mM NaCl, 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM EDTA, and 1% Triton X-100; and applied to an SP Hi-trap (Pharmacia) FPLC cation-exchange column equilibrated in the same buffer. The column was developed at a flow rate of 1 ml/min with a 30-ml linear gradient (0.025 to 0.8 M) of NaCl. The fractions containing purified SpoIIE eluted at approximately 0.35 M NaCl. They were dialyzed into a solution of 50 mM Tris-HCl (pH 7.5) containing 50 mM NaCl and 0.5 mM DTT (buffer B) and 0.5 mM EDTA, 0.5 mM PMSF, 1% Triton X-100, and 50% glycerol and stored at –70°C. Concentration of the protein was estimated to two significant digits by subjecting an aliquot to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), staining with Coomassie brilliant blue, and comparison of the density of the stained bands against molecular markers of known concentration from scanned jpeg images (Scion Image software, Frederick, Md.). The concentration was also determined by complete amino acid digestion (Applied Biosystems).

Reconstitution of SpoIIE into unilamellar lipid vesicles. Liposomes of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC; Aventi Polar Lipids) were prepared by suspending dry lipids in buffer B to a final lipid concentration of 10 mM (7.86 mg/ml), followed by vigorous vortexing for solubilization. ULVs with a mean pore size of 200 nm were produced by repeated extrusion through syringe-based polycarbonate filters (Whatman) (48). Reconstitution of the SpoIIE protein into 200-nm ULVs was carried out essentially as described by Knol et al. (35), with the following amendments. To remove glycerol, SpoIIE was dialyzed at 4°C in buffer B containing 0.12% (vol/vol; 2.0 mM) Triton X-100 with at least two changes of dialysis solution, while the ULVs were gently agitated with 0.12% Triton X-100 for 60 min. Liposomes and SpoIIE were mixed with purified lipid-to-protein ratios of 5,000:1, 2,500:1, 1,000:1, 500:1, 200:1, 120:1, and 60:1 (mol/mol), producing solutions with protein concentrations of 2, 4, 10, 20, 50, 83, and 167 µM, respectively. The mixtures were very mildly agitated for 90 min at 4°C. To remove the detergent, polystyrene Bio-Beads (SM-2; Bio-Rad) were added to a wet weight of 50 mg/ml and the samples were incubated with mild agitation for 1 h at 4°C. Two subsequent aliquots of fresh Bio-Beads were added over 20 h at 4°C. Proteoliposomes were harvested by three 60-min centrifugations at 22,000 x g and 4°C, resuspended in 100 µl of buffer B, and stored at 4°C. The protein content of the lipid and supernatant fractions was analyzed by sodium dodecyl sulfate-PAGE; the gels were stained with Coomassie brilliant blue, and percent incorporation was determined by densitometry as described above. Final protein concentrations in the absence of Triton X-100 were determined by UV absorption (Bio-Rad). Samples created in this way were used within 5 days of production. Proteoliposomes in buffer B, obtained as described above, were purified by discontinuous ultracentrifugation over 18 h in a Beckman L7-65 bucket-arm ultracentrifuge at 50,000 x g and 4°C with stepwise gradations of 5, 13, 21, 30, 45, and 60% sucrose.

A control sample of SpoIIE dialyzed into buffer B containing 0.12% Triton X-100 was subjected to conditions similar to those required for proteoliposome production so as to evaluate the effects on SpoIIE activity of physical agitation and exposure to increased temperature.

Overproduction and purification of SpoIIAA-P. E. coli BL21(DE3) cells harboring pEAAB were grown and induced by the method of Diederich et al. (16). Cell extracts were subjected to anion-exchange FPLC as described by Min et al. (51). The column fractions eluting at approximately 0.25 M NaCl, enriched in SpoIIAA-P, were subjected to gel filtration chromatography over Sephadex G-75 as described by Diederich et al. (16). Analysis of the purified SpoIIAA-P protein by native PAGE failed to reveal any nonphosphorylated SpoIIAA.

Kinetic assays of reconstituted SpoIIE. To one-half of the SpoIIE-containing DOPC vesicles was added 2% (34 mM) Triton X-100, a concentration sufficient to resolubilize the proteoliposomes to the micellar condition (35), as seen by clearing of the cloudy vesicle solution. The rates of SpoIIAA-P dephosphorylation for lipid- and detergent-engulfed SpoIIE were compared to the rate for SpoIIE withdrawn from –70°C immediately before the assay, and to the rate for the control sample described above, by the method of Lucet et al. (45). The dephosphorylation assay, carried out at 30°C in buffer B containing 10 mM MnCl2 and 30 nmol of SpoIIAA-P, was started by the addition of 200 pmol of SpoIIE. Aliquots were taken at 20, 40, 60, 90, 120, 180, and 240 min. SpoIIAA-P and SpoIIAA were separated on a native polyacrylamide gel and stained with Coomassie brilliant blue, and relative concentrations were evaluated quantitatively by densitometry.

AFM. Proteoliposomes were examined by tapping-mode AFM in a liquid environment with a Dimension 3000 microscope and a Nanoscope IIIa scanning probe microscope controller equipped with a type G scanner (scan range: 90 by 90 by 10 µm) and a liquid cell (all from Veeco Instruments Ltd.). Images with a resolution of 512 by 512 pixels were acquired in buffer consisting of 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 0.5 mM DTT at a scan speed of 2 lines/s. Silicon nitride AFM probes with a spring constant of approximately 0.2 N/m were driven vibrated at approximately 8 kHz close to the surface to achieve the tapping mode. The probe position was maintained vertical to the sample surface by monitoring the oscillation amplitude damping due to probe-sample interactions. AFM measurements of SpoIIE-loaded DOPC vesicles were taken at room temperature on samples that had condensed to single bilayers on 1-cm2 plates of mica after overnight incubation at 4°C (15, 39). Histograms of the surface height of the lipid bilayers were produced by the University of Texas Health Center, San Antonio, Image Tool software package.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The integral membrane protein SpoIIE is sequestered to an asymmetric site in B. subtilis early in sporulation, and on completion of the septum and invagination of the cell walls, it rapidly dephosphorylates SpoIIAA-P. It is known that SpoIIE molecules can interact through the central domain II (46). Given the rapid rise of the local concentration of SpoIIE in the septum during ring formation, the possibility that the enzyme was self-regulated by a conformational change through aggregation of domain II suggested itself. To assess whether self-association of the SpoIIE protein in the septal membrane plays a role in regulating phosphatase activity, it was important to assay the phosphatase activity of high and low concentrations of SpoIIE in a model lipid bilayer. Transmembrane proteins such as SpoIIE have been increasingly a subject of study, particularly since improved techniques have been developed for production of synthetic membrane bilayers, or liposomes. The kinetics of SpoIIE phosphatase activity in proteoliposomes were directly compared to the kinetics of an equimolar concentration of SpoIIE in detergent micelles of Triton X-100, previously found to have the highest observed in vitro activities (45). It can be estimated that approximately 100 molecules of DOPC are required to surround a 10-helix transmembrane protein. Self-association, if it does occur, would be unlikely at protein concentrations lower than 1 molecule per 500 molecules of lipid, owing to screening by a multilipid barrier, although it is not unheard of for membrane proteins to assemble at far greater lipid-to-protein ratios. If activity was found to be severely diminished at low protein concentrations in DOPC, it could be argued that the activity change after the formation of the asymmetric septum was at least partly due to the effects of a high local SpoIIE concentration in the septum.

High-yield reconstitution of the full-length SpoIIE protein into a model lipid bilayer. Our procedures followed those previously described (45, 62), relying on differential centrifugation and purification of the protein in Triton X-100 by column chromatography. Addition of DNase I, RNase, and lysozyme facilitated the removal of cell debris from B. subtilis at low rotor speeds, and elimination of glycerol in the solution improved the preparative purity of SpoIIE. The increased purity allowed the omission of the Affi-gel Blue step, increasing the overall final yield. Calculations of protein concentration by UV absorption methods were impeded by absorption of Triton X-100, and protein concentrations were estimated from densitometric analysis of an overloaded polyacrylamide gel (not shown). The final preparation was more than 95% pure (45), and protein concentrations determined in this manner agreed to two significant digits with the results of complete amino acid digestion. Western blot analysis with antibodies raised against the C-terminal fragment of SpoIIE confirmed the identity of the isolated material as SpoIIE. The activity of detergent-protected SpoIIE diminished with time, despite storage with 50% glycerol at –80°C.

The protein concentrations of the manufactured proteoliposomes were chosen to approximate to the maximum concentration of SpoIIE (about 28 µM) found in the cell some 120 min after the onset of sporulation (45). The efficiency of SpoIIE reconstitution into DOPC ULVs was diminished by the presence of even minute quantities of glycerol in the solution, and SpoIIE was added to Triton X-100 saturated liposomes only after extensive dialysis. As reported for other membrane proteins (35), maximum reconstitution of SpoIIE into liposomes occurred if vesicles of DOPC were incubated with the nonionic detergent Triton X-100 at concentrations between 2.0 and 2.5 mM. Removal of the detergent with SM-2 polystyrene Bio-Beads, a mechanism widely favored for detergent extraction (61), required at least 20 h of incubation with three changes of Bio-Beads (12, 31). Up to 90% of the SpoIIE protein could be incorporated into ULVs of DOPC. The concentration of SpoIIE in the reconstituted vesicles was determined by UV absorption at 280 nm, as the lipid materials did not absorb this radiation. Vesicles composed of lipid-to-protein ratios of 5,000:1, 500:1, and 200:1 (mol/mol) in buffer B migrated through sucrose during discontinuous ultracentrifugation in a way that depended on their protein concentrations, with 200:1 ratio vesicles migrating to 60% sucrose, 500:1 ratio vesicles migrating to 45%, and 5,000:1 ratio vesicles migrating to 13% sucrose.

Phosphatase activity is not diminished at low concentration in a ULV. Purified SpoIIE was incubated with excess purified SpoIIAA-P, and the relative concentrations of SpoIIAA and SpoIIAA-P were evaluated by native gel electrophoresis. The turnover number of micellar SpoIIE in Triton X-100 (moles of SpoIIAA-P dephosphorylated per mole of SpoIIE) was 6.1 x 10–2 s–1, a value similar to that determined previously (45). A control SpoIIE sample subjected to agitation, a diminished Triton X-100 concentration, and an extended time at elevated temperature similar to that required for SpoIIE reconstitution (described in Materials and Methods) was found to dephosphorylate SpoIIAA-P at an approximately threefold slower rate (Table 1). Activity was best conserved if the reconstitution and detergent removal processes were carried out at 4°C and assays were performed immediately after resuspension of the centrifuged proteoliposomes in buffer B. Turnover rates for all protein-to-lipid ratios were diminished by an order of magnitude versus SpoIIE removed directly from –70°C (Table 1). At all protein-to-lipid ratios, the rates of dephosphorylation were effectively identical to those for SpoIIE resolubilized back into detergent micelles. If the increased rate of dephosphorylation observed in the enclosed septum were caused by conformational changes, consequent upon oligomerization, the phosphatase activity at low protein-to-lipid ratios would be expected to have been severely diminished. This effect was not observed in any of the assays performed.


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  		TABLE 1. Rates of SpoIIAA-P dephosphorylation by SpoIIE

 
Evaluation of SpoIIE self-association by AFM. To show directly whether the expected protein-to-lipid ratios had been achieved, and to show by image analysis whether SpoIIE aggregated or was randomly distributed in the lipid bilayers, we used AFM. AFM requires a sharp tip (ca. 10- to 20-nm apex radius), typically made from silicon nitride or silicon, which is raster scanned over a sample with subnanometer precision with piezoceramic scanners. The tip is attached to the underside of a reflective cantilever, which deflects up and down in response to forces acting against the tip during scanning. To produce a three-dimensional image of sample topography, cantilever deflection is monitored, as a function of the lateral position of the probe on the sample, by shining a diode laser, which deflects off the back of the cantilever onto a position-sensitive photodiode. During this process, computer-controlled feedback mechanisms enable piezoelectric scanners to maintain a constant cantilever deflection or force between the tip and the sample surface. The feedback signal, rather than raw cantilever deflection, is then used to produce an image of the sample surface. In our study, tapping-mode imaging was used as it allows high-resolution topographic imaging of delicate sample surfaces by alternately moving the tip into and out of contact with the surface, avoiding the potentially damaging lateral forces associated with other imaging modes in which the tip is in permanent contact with the sample surface during scanning (2, 20, 29). No information concerning protein secondary structure can be derived from the AFM images obtained, but they are instructive in terms of evaluating relative protein concentrations in the different samples and in identifying any nonentropic protein aggregation. ULVs of DOPC in the absence of protein failed to collapse to a bilayer sheet on slightly negatively charged mica even after overnight incubation, and the same was true of samples at lipid-to-protein ratios of 5,000:1. Proteoliposomes at 500:1 and 200:1, however, formed bilayer sheets with a mean thickness of less than 3.5 nm (Fig. 1). Bilayer thickness measurements were verified at the end of our experiments by imaging a 1-µm2 area of the surface with high force. This process effectively sweeps or clears away an area of the sample to reveal the underlying mica, which can be subsequently seen as a hole within the sample layer in larger-scale images (data not shown) obtained as in Fig. 1. AFM images of samples purified by sucrose ultracentrifugation were indistinguishable from images taken of proteoliposomes not purified in this manner. Images of a protein-to-lipid ratio of 200:1 were observed to have a far higher density of protein than images of a protein-to-lipid ratio of 500:1. Evaluation of the ASCII data files indicated that 200:1 ratio samples contained approximately 2.5 times more material (i.e., protein) above the lipid bilayer than did 500:1 ratio samples, with an error of less than 10% (data not shown). The images obtained from AFM were studied by examining the occurrences of all heights above the mica surface per equivalent surface area. Histograms of small or large segments of these images reproducibly showed two overlapping peaks, the DOPC bilayers at 0 to 4 nm and the protein above the bilayers at 4 to 6 nm (Fig. 2). The shapes of the graphs immediately suggest higher concentrations of protein at 200:1 than at 500:1, and quantitative analysis of the histogram peaks produced a ratio of 2.5:1 with an error consistently less than 5%. The reproducible shapes and ratios of these histograms confirmed that the two images contained proteins that were randomly distributed throughout the lipid bilayers. These images gave no evidence of aggregation of SpoIIE protein in a membrane designed to represent the membrane of the sporulation septum.



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  		FIG. 1. Tapping-mode AFM images (with scan areas of 1,000 by 1,000 nm) of SpoIIE reconstituted into vesicles of DOPC at lipid-to-protein ratios of 200:1 (A) and 500:1 (B). Topological manipulations of 200:1 (C) and 500:1 (D) suggest the increased concentration of protein at 200:1 versus 500:1 and display a lack of nonentropic order to the arrangement of SpoIIE in the model membrane. The large peaks above the mica surface observed at 500:1 in panels B and D are due to liposomes that could not settle to a lipid bilayer because of the high concentration of liposomal material in the solution.

 


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  		FIG. 2. Histograms of a segment measuring 500 by 1,000 nm taken from Fig. 1A and B. Lipid bilayers occur at heights of 0 to 4 Å, with proteins above the membrane at heights of approximately 4 to 6 Å. Statistical analysis of protein occurrence above the bilayers reproducibly revealed a 2.5 times greater value for proteins incorporated at 1 molecule of SpoIIE to 200 molecules of DOPC (A) than for the sample with 1 molecule of SpoIIE to 500 molecules of DOPC (B).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Purification of integral membrane protein SpoIIE requires that it be solubilized in detergent to avoid aggregation. Several detergents have been tested as solubilizing agents; maximum activity was found in Triton X-100 (45). After the incorporation of SpoIIE into liposomes in our experiments, the material was exhaustively extracted with Bio-Beads (polystyrene beads) in order to eliminate interference from any remaining Triton X-100. The choice of a diacyl-phosphatidylcholine for forming lipid vesicles was based on the mean thickness of the hydrophobic bilayer, similar to the thickness of bacterial membranes at approximately 3 to 4 nm (69), and on the stability of the liquid crystalline state over a wide range of pHs and at temperatures below 0°C. It has been shown that insertion of membrane proteins is optimal in membranes composed of DOPC (49, 59, 60). Triton X-100 is considered to be ideal for the reconstitution of a membrane protein containing large cytoplasmic domains (28, 35, 36). At low concentrations of this detergent, the lipid bilayer is not sufficiently solubilized to allow large hydrophilic domains such as domains II and III of SpoIIE to penetrate the membrane, ensuring that the phosphatase domain points out of the vesicle sphere. In this way, the activity of the membrane-bound protein could be directly compared to the activity of the protein in a micellar state.

From the cross-sectional areas of SpoIIE and DOPC (approximately 7.5- and 0.3-nm radii, respectively), it can be estimated that 100 lipid molecules are required to surround the enzyme. If self-association through domain II of SpoIIE occurred, it would be expected at lipid-to-protein ratios of up to 500:1. In fact, however, no significant difference in activity was observed in any of the lipid-to-protein ratios assayed. Although in vitro studies have indicated that SpoIIE can self-associate through the central domain, the elevated rate of SpoIIAA-P dephosphorylation by SpoIIE that is the crucial step in initiating differential gene expression is not triggered by a conformational change due to high localized concentrations of SpoIIE in the enclosed septum. The kinetic activity of the liposomal SpoIIE protein was seen to be effectively identical to that for the enzyme reconstituted back into detergent micelles. These results therefore favor models in which an external regulator, so far unidentified, regulates the activity of SpoIIE until the septum has closed.

That the proteoliposomes contained the expected lipid-to-protein ratios was confirmed by AFM. AFM has been increasingly used as a method for evaluating membrane proteins. While there is a potential risk of deformation of the biological sample through the direct interaction of the scanning stylus and the sample, there is no electron-induced damage to the sample as could occur with electron microscopy. Moreover, AFM allows for observation in a liquid environment, more closely replicating the physiological environment of the cell surface. Unexpectedly, control vesicles with DOPC alone, and vesicles containing very small relative concentrations of SpoIIE, failed to collapse to lipid bilayers on mica surfaces, despite overnight incubations at 4°C. It may be that zwitterionic DOPC is overall slightly negative, providing no electrostatic advantage to vesicle collapse. The pI of SpoIIE is 6.14, and membrane loop regions I and IV, which are both directed away from cytoplasmic domains II and III, contain positively charged arginine residues (5), providing the electrostatic forces required to create the observable lipid bilayer along the mica surface at higher relative concentrations of the enzyme.

The in vivo intracellular concentration of SpoIIE has been observed to increase approximately fivefold between 90 and 120 min after the onset of sporulation. However, this increase in protein concentration and the sequestration of the SpoIIE protein to the sporulating septum are not sufficient to account for an increase in phosphatase activity to nearly 100 times greater than the kinase activity of the anti-sigma factor SpoIIAB (45). At a lipid-to-protein ratio of either 200:1 or 500:1, the imaged proteins were not seen to aggregate, suggesting that an increase in SpoIIE at the sporulating septum does not cause conformational changes due to self-association that increase phosphatase activity. We therefore suggest that an external regulator or regulators may be responsible for the activation of the septum-engulfed SpoIIE protein. One possible candidate is the FtsZ protein, responsible for SpoIIE sequestration to the asymmetric septum, required for phosphatase activity, and known to interact with domain II (46), which leaves the enclosed septum at the same time as the phosphatase domain is activated. It is also possible that SpoIIE forms a complex with SpoIIAA and that the release of SpoIIAA coupled to completion of the asymmetric septum initiates the release of {sigma}F (34). The two mutations identified by Feucht et al. and Hilbert and Piggot (22, 30) might be able to release SpoIIAA independently of septum formation and therefore activate {sigma}F prematurely. Alternatively, extensions of various lengths and conformations found in a number of PP2C homologues have been shown to regulate (positively or negatively) the activity of the phosphatase domain in response to a specific signal (5, 7, 23, 33, 38, 40, 50, 65, 71). Elucidation of the means by which SpoIIE is regulated may well rely on membrane-bound assays similar to those performed here, and on the advancing technology of AFM analysis of biological membranes.


   ACKNOWLEDGMENTS
 
We thank Boyan Bonev of the University of Nottingham and Anthony Watts of the Department of Biochemistry at the University of Oxford for invaluable insights. Protein concentrations were established by amino acid analysis in the Oxford University Department of Biophysics with the assistance of Tony Willis. We also thank Helen Prescott for outstanding technical assistance.

This work was supported by the Biotechnology and Biological Sciences Research Council. Pfizer Global Research and Development provided the funding for Stephanie Allen's lectureship.


   FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom. Phone: 44 1865 275302. Fax: 44 1865 275297. E-mail: michael.yudkin{at}bioch.ox.ac.uk. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, May 2004, p. 3195-3201, Vol. 186, No. 10
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.10.3195-3201.2004
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