De Novo Fatty Acid Synthesis Is Required for Establishment of Cell Type-Specific Gene Transcription During Sporulation in Bacill

Molecular Microbiology (1998) 29(5), 1215–1224

De novo fatty acid synthesis is required for establishment of cell type-speciﬁc gene transcription during sporulation in Bacillus subtilis

Gustavo E. Schujman, Roberto Grau, Hugo C. compartment (Lutkenhaus, 1994). The unequal-sized pro- Gramajo, Leonardo Ornella and Diego de Mendoza* geny resulting from the formation of the polar septum Programa Multidisciplinario de Biologıá Experimental have different developmental fates and express different (PROMUBIE) and Departamento de Microbiologıá, sets of genes (for reviews see Errington, 1993; Losick Facultad de Ciencias Bioquı´micas y Farmaceúticas, and Stragier, 1996). The fate of the forespore chamber Universidad Nacional de Rosario, Suipacha 531, 2000- is determined by the transcription factor ␴F, which is pre- Rosario, Argentina. sent before the formation of the polar septum but does not become active in directing gene transcription until completion of asymmetric division, when its activity is con- Summary fined to the smaller compartment of the sporangium (for A hallmark of sporulation of Bacillus subtilis is the for- reviews see Losick and Stragier, 1996) mation of two distinct cells by an asymmetric septum. The activity of ␴F is regulated by a pathway consisting of The developmental programme of these two cells the proteins SpoIIAB, SpoIIAA and SpoIIE, all of which are involves the compartmentalized activities of ␴E in the produced before the formation of the polar septum (Duncan larger mother cell and of ␴F in the smaller prespore. and Losick, 1993; Min et al., 1993; Alper et al., 1994; Die- A potential role of de novo lipid synthesis on develop- derich et al., 1994; Arigoni et al., 1995; Duncan et al., ment was investigated by treating B. subtilis cells with 1995). SpoIIAB is an anti-sigma factor that binds to ␴F cerulenin, a specific inhibitor of fatty acid biosynth- and holds it in an inactive complex (Duncan and Losick, esis. These experiments demonstrated that spore for- 1993; Min et al., 1993; Magnin et al., 1996). SpoIIAA, on mation requires de novo fatty acid synthesis at the the other hand, is an anti-anti-sigma factor that binds to onset of sporulation. The transcription of the sporula- SpoIIAB, thereby causing the release of free and active tion genes that are induced before the formation of ␴F from the SpoIIAB–␴F complex (Alper et al., 1994; Die- two cell types or that are under the exclusive control derich et al., 1994). SpoIIAB is also a protein kinase that of ␴F occurred in the absence of fatty acid synthesis, phosphorylates and thereby inactivates SpoIIAA (Min et as monitored by spo–lacZ fusions. However, expres- al., 1993). Finally, SpoIIE is a serine phosphatase that is sion of lacZ fusions to genes that required activation capable of dephosphorylating SpoIIAA-P, thereby restor- of ␴E for transcription was inhibited in the absence ing the ability of SpoIIAA to bind SpoIIAB (Duncan et al., of fatty acid synthesis. The block in ␴E-directed gene 1995; Arigoni et al., 1996; Feucht et al., 1996). Interest- expression in cerulenin-treated cells was caused by ingly, SpoIIE has an additional function beyond its role in an inability to process pro-␴E to its active form. Elec- ␴F activation (Bara´k and Youngman, 1996; Feucht et tron microscopy revealed that these fatty acid-starved al., 1996). In its absence, but not in mutants specifically cells initiate abnormal polar septation, suggesting that affected in SpoIIE phosphatase activity, the sporulation de novo fatty acid synthesis may be essential to couple septum develops an aberrantly thick layer of peptidogly- the activation of the mother cell transcription factors can and the time of septum formation is delayed (Bara´k with the formation of the differentiating cells. and Youngman, 1996; Feucht et al., 1996); the later effect is attributed to an amino acid sequence at the extreme COOH-terminus of SpoIIE (Feucht et al., 1996). Thus, Introduction combination of these two functions in a single polypeptide A hallmark of the process of sporulation in the Gram- may serve to couple the release of the cell-specific tran- positive bacterium Bacillus subtilis is the formation of an scription factors with the formation of the differentiating asymmetrically positioned (polar) septum that divides the cells. cell into a small forespore compartment and a large cell Establishment of forespore specificity through activation of ␴F is soon followed by induction in the mother cell of Received 26 March, 1998; revised 1 June, 1998; accepted 5 June, 1998. *For correspondence. E-mail [email protected]; a large set of genes under the control of the transcription Tel. (41) 350 661; Fax (41) 390 465. factor ␴E (Losick and Stragier, 1996). ␴E is an unusual

ᮊ 1998 Blackwell Science Ltd 1216 G. E. Schujman et al. sigma factor in that it is synthesized as an inactive large synthesis and septation in B. subtilis (Strauch et al., 1992). precursor, pro-␴E, the primary product of the promoter Moreover, it has recently been demonstrated that branched- distal member (spoIIGB ) of the two cistron spoIIG operon chain fatty acids play an important role in cell–cell com- (LaBell et al., 1987). Pro-␴E is converted to its active form munication and development in Mixococcus xantus (Down- by proteolytic removal of its pro-amino acid sequence, an ward and Toal, 1995; Toal et al., 1995). N-terminal extension of 27 residues. The product of the In this report, we demonstrate that de novo fatty acid first gene of the operon, spoIIGA, is a multispanning mem- synthesis after the end of exponential phase is required brane protein that is sufficient for activation of pro-␴E and for spore formation and that a reduced level of fatty acid that is believed to be the processing enzyme (Stragier et synthesis allows formation of asymmetric septa but pre- al., 1988). As both pro-␴E and SpoIIGA are present before vents activation of ␴E. We speculate that septal membrane septation, and are presumably segregated into each of the lipids play a key role in the establishment of differential cells after asymmetric division, confinement of ␴E activity gene expression during the morphogenetic programme to the mother cell implies that SpoIIGA-mediated pro-␴E of sporulation in B. subtilis. processing is delayed until after septation, with the possibility that it occurs only in the mother cell (Losick and Stragier, 1996). The proteolytic activation of ␴E requires Results the activity of ␴F through the activity of the ␴F-directed The effect of inhibition of the de novo fatty acid gene spoIIR (Karow et al., 1995; Londonõ-Vallejo and synthesis on sporulation Stragier, 1995). It has been suggested by Losick and Stragier (1996) that spoIIR would be transcribed by ␴F in To determine the role of de novo fatty acid synthesis on the prespore and act as a vectorial cell–cell signal that sporulation, we used the antibiotic cerulenin. This antibiotic would activate ␴E only in the mother cell. Consistent with is a potent inhibitor of lipid synthesis known to irreversibly SpoIIR acting as a cell–cell signal, characterization of alkylate an essential thiol group of ␤-ketoacyl-ACP syn- the spoIIR gene indicated that it encoded a protein with thase, the enzyme that catalyses the condensation of a secretion signal, and recent biochemical analysis has malonyl-CoA with acyl-ACP (D’Agnolo et al., 1973). Thus, shown that SpoIIR is secreted (Hoffmeister et al., 1995). cerulenin inhibits the fatty acid chain elongation step and Once secreted, SpoIIR could interact with the membrane- thereby lipid synthesis (Fig. 1). Cerulenin (10 ␮gml¹1)was bound SpoIIGA that cleaves the pro sequence from ␴E added to strain JH642 grown in DS medium at different to activate it. Thus, the main function of SpoIIR would be times: 1.5 h (t¹1.5) and 0.5 h (t¹0.5) before cells enter sta- to tie the transcription programme in the mother cell to tionary phase, at the onset of stationary phase (t0)orat the transcription programme in the forespore by allowing several times after t0 (Fig. 2). Cerulenin was a potent inhi- E F ␴ to become active only after ␴ has itself been activated bitor of spore formation when added to cells at t¹1, t0 or E and thus ensuring that ␴ does not become active in the t0.5; however, this inhibitory effect disappeared when the predivisional sporangium. Although this model is intriguing, antibiotic was added at more advanced stages of sporula- E the notion that SpoIIR must act vectorially to activate pro-␴ tion (t1 to t4) (Fig. 2). Typically, 16 h after reaching station- processing is not without controversy. Zhang et al. (1996) ary phase, cells treated with cerulenin at t0 had a viable observed that ␴E activation was limited to the mother cell count of about 108 ml¹1 and produced about 106 spores compartment, even when SpoIIR was synthesized before ml¹1. In contrast, in the absence of cerulenin, strain septation and therefore presumed to be present in both JH642 had a viable count of about 6 × 108 ml¹1 and pro- compartments. These authors conclude that a ␴E activa- duced about 5 × 108 spores ml¹1. Monitoring the levels of tion factor other than SpoIIR, possibly SpoIIGA in a parti- fatty acid synthesis in the experiments shown in Fig. 2 cular orientation, could be restricted to the mother cell. revealed that de novo incorporation of [14C]-acetate into The identity of the factor(s) responsible for cell compart- phospholipids of cerulenin-treated cells decreased about ment-specific activation of ␴E and the details of how the 90% when compared with non-treated cells (data not directional processing is accomplished remain to be shown). To establish whether the primary action of cerulenin resolved. This intercellular signal transduction pathway on spore formation is due to its inhibitory effect on de novo and the mechanism that prevents activation of ␴E in the fatty acid synthesis, we used strain GS77. The growth of forespore could be connected to the asymmetrical septum this strain is not inhibited by the addition of cerulenin up biosynthesis because this event is the key morphological to 50 ␮gml¹1, whereas the growth of the isogenic strain step in the formation of the two cell types and compart- JH642 is inhibited at a concentration of 5 ␮gml¹1 (data mentalization of gene expression (for a review see Duncan not shown). We found that extracts of strain GS77 con- et al., 1994). In connection with this possibility, it has been tained a fatty acid synthetase (FAS) activity about seven- suggested that specific fatty acids may act as signals link- fold higher than the isogenic strain JH642 (Table 1). ing the initiation of sporulation to the status of membrane Although the FAS activity of strain GS77 is inhibited

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1215–1224 Role of lipid synthesis in B. subtilis sporulation 1217

Fig. 1. Elongation of fatty acid biosynthesis. Each elongation cycle is initiated by the condensation of malonyl-acyl carrier protein (ACP) with acyl-ACP carried out by ␤-ketoacyl-ACP-synthase. This step is blocked by cerulenin (D’Agnolo et al., 1973). The next step is the reduction of ␤-ketoesters by ␤-ketoacyl reductase. The ␤-D-hydroxyacyl-ACP is then dehydrated to the trans-2 unsaturated acyl-ACP, which is reduced by enoyl reductase to generate an acyl-ACP two carbons longer than the original acyl-ACP. Several lines of evidence indicate that this pathway is used in B. subtilis to elongate straight and branched-chain fatty acids (de Mendoza et al., 1993; Morbidoni et al., 1996). R denotes the terminal group of branched-chain fatty acids derived from ketoacids from valine, leucine or isoleucine (de Mendoza et al., 1993).

about 70% by cerulenin (40 ␮gml¹1), its residual activity is [whose expression early in stationary phase depends on still about twofold higher than the basal FAS activity of the the activation of Spo0A by phosphorylation (Hoch, 1993)] wild-type isogenic strain JH642 (Table 1). Therefore, the were similar in cells treated or untreated with cerulenin, resistance to cerulenin belonging to strain GS77 seems at least within the first and second hour after the addition to be due to overproduction of FAS activity rather than a of the antibiotic; at later times, the level of ␤-galactosidase decrease in the enzyme affinity by the antibiotic. In agree- ment with these findings, the frequency of sporulation of GS77 was essentially the same in the presence or in the ¹1 absence of 10 ␮gml cerulenin added at t0 (data not shown). These experiments indicate that, under our assay conditions, the only target of cerulenin is the B. subtilis FAS. Thus, de novo fatty acid synthesis at the onset of stationary phase is essential for Bacillus cell differentiation.

Sporulation gene expression in the absence of fatty acid synthesis

To determine the stage in the developmental gene expression pathway inhibited by the addition of cerulenin, we used a series of strains containing lacZ fusions to promoters known to be under the control of speciﬁc regulatory proteins and to be expressed at characteristic sporulation stages. Fig. 2. Effect of cerulenin on the extent of sporulation. Strain Cerulenin was added at the onset of sporulation to strains JH642 was grown in DS medium at 37ЊC and cerulenin was added at the indicated times relatives to the end of exponential growth carrying lacZ fusions to each of these genes (Fig. 3). The (t0). The spore titre was measured 24 h after t0. Values represent expression levels of the genes spoIIA, spoIIG and spoIIE the average of at least ﬁve independent experiments.

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Table 1. Effect of cerulenin (40 ␮gml¹1)onin vitro synthesis of fatty The gene spoIIQ is under the exclusive control of ␴F acids by cell-free extracts. (Londonõ-Vallejo et al., 1997), whereas the genes spoIID E Specific activitya and spoIIID require ␴ for transcription (Losick and Stragier, (pmol min¹1 mg¹1 protein) 1996). A fusion of lacZ to the promoter of spoIIQ was expressed in cells treated with cerulenin, although at about Strain ¹Cerulenin þCerulenin 50% of the rate observed in non-treated cells (Fig. 3C JH642 4.1 (1.00)b 0.3 (0.07) and D). In contrast to the expression of spoIIQ, the tran- GS77 27.7 (6.80) 8.8 (2.10) scription of the ␴E-directed spoIID–lacZ and spoIIID–lacZ a. Activities were determined in cell extracts as described under was totally curtailed in the presence of cerulenin (Fig. 3E Experimental procedures. and F). These results suggest that, in the absence of b. Overproduction relative to the isogenic wild-type strain grown in lipid synthesis, cells can initiate septation exhibiting acti- parallel without treatment. vation of ␴F but not activation of ␴E. expressed from those fusions was significantly higher after Cells blocked in lipid synthesis do not process Pro-␴E the addition of cerulenin (Fig. 3A and B). These results are significant because they indicate that cerulenin-treated To examine whether the inhibition of lipid synthesis blocks cells were capable of entering sporulation and that the ␴E activation by affecting its synthesis, processing or presence of the antibiotic does not inhibit transcription of another activation step, we performed a Western blot ana- sporulation genes expressed before septation. lysis of protein extracted from cerulenin-treated cells using

Fig. 3. Effect of cerulenin on ␤-galactosidase activity expressed from various spo–lacZ fusions. In A, B, C and E the strains were grown in DS medium at 37ЊC. Cerulenin (10 ␮gml¹1) was added to one half of each culture at the end of exponential growth (t0 ). In D and F strains were induced to sporulate by the resuspention method. Cerulenin (5 ␮gml¹1) was added to one half of each culture at the time of resuspention (t0 ). Samples were collected at the indicated times relative to t0 and assayed for ␤-galactosidase activity. Control without cerulenin, ﬁlled symbols; cerulenin-treated cells, open symbols.

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1215–1224 Role of lipid synthesis in B. subtilis sporulation 1219 (Figs 5 and 6). We have chosen to distinguish between cells on the basis of septum thickness, number and position, according to the key given by Bara´k and Youngman (1996) (Fig. 6). At least 40% of the scored cells displayed complete septal structures, and some of the scored cells contained two polar asymmetric septa or double asymmetric septa (Fig. 6). These results indicate that cells with a limited supply of fatty acids at the onset of sporulation Fig. 4. Effect of cerulenin on pro-␴E processing. Crude protein are still able to form septa. About 70% of the septated extracts were processed and analysed as described in cells (Fig. 6) make thick, morphological abnormal spore Experimental procedures. Lanes 1–3, extracts from JH642 cells septa (Fig. 5A and D) similar to those shown by strains harvested 1.0, 2.2 and 3.5 h after t ; lane 4, extracts from strain 0 ´ MO1190 (⌬spoIIGA) cells harvested 3 h after t0. Lanes 5–7, containing spoIIE null mutations (Barak and Youngman, ¹1 extracts from JH642 cells treated with 10 ␮gml of cerulenin at t0 1996; Feucht et al., 1996). Our results are quantitatively and harvested 1.0, 2.2 and 3.5 h after the addition of the antibiotic. similar to those reported by Bara´k and Youngman (1996) for spoIIE null mutants, except for the presence anti-␴E antibodies. As shown in Fig. 4, cells that do not in our studies of Ϸ30% of cells with thin asymmetric synthesize lipids at the onset of sporulation produced septa (class 5 in Fig. 6) among the cells that produce pro-␴E normally but did not process it, indicating that de septa in the presence of cerulenin. novo lipid synthesis is required for pro-␴E processing. To obtain more precise information about the effect of cerulenin on morphological development, we investigated the effect of the antibiotic inducing sporulation by resus- Morphological analysis pension of exponentially growing cells in a nutrient-limited Examination of more than 150 complete longitudinal sec- medium, essentially by the method of Sterlini and Mandel- tions from randomly selected ﬁelds of cerulenin-treated stam (1969). Cells treated with cerulenin at the time of cells by electron microscopy showed that the septation fre- resuspention (t0) were collected 3 and 16 h later and ana- quency was reduced after 6 h in stationary phase in DS lysed by electron microscopy. This experiment showed medium and revealed septa with different abnormalities basically the same septum morphologies as those obtained

Fig. 5. Representative examples of septum morphologies scored in strain JH642 treated with cerulenin. At the end of exponential growth ¹1 cerulenin was added at a concentration of 10 ␮gml and the incubation continued until t6. Cells were harvested and processed for electron microscopy as described in Experimental procedures. A.Cells with thick septa at one polar position. B. Cells with thin septa at one polar position. C. Cells with thin septa at two polar positions. D. Cells with double asymmetric septum at one pole.

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1215–1224 1220 G. E. Schujman et al. depends on the availability of sufficient metabolic reserves to complete the developmental process. Morphological and physiological studies have pointed to the importance of membrane synthesis during sporulation for asymmetric septation and prespore engulfment (for a review see Piggot et al., 1994). Freese and Oh (1974) reported that growing glycerol auxotrophs without glycerol prevented sporulation and septum formation. However, glycerol starvation also produced membrane collapse, and much of the cellular ATP was released in the medium. In addition, these mani- pulations did not stop lipid synthesis at the end of exponential growth because to observe effects in sporulation efficiency the auxotrophs were deprived of glycerol during the exponential phase of growth (Freese and Oh, 1974). In this work, we show that sporulation was blocked by the addition of the antibiotic cerulenin, which is a specific inhibitor of fatty acid synthesis, at the end of exponential growth. Furthermore, we demonstrated that the inhibition of spore formation is not due to a side-effect of cerulenin because no effect of the antibiotic was observed in a mutant that overproduces FAS activity. The inhibition of fatty acid F Fig. 6. Quantification of morphological classes in populations of synthesis at t0 allowed the expression of ␴ -directed JH642 cells treated with cerulenin. Cells were treated with cerulenin genes but not ␴E-directed genes. Lipid-starved cells pro- and harvested for examination by electron microscopy as duced pro-␴E but were unable to process it, indicating described in the legend of Fig. 5. Class 1, no septa or incomplete septum; Class 2, thick septum at polar position; Class 3, double that de novo lipid synthesis at the onset of sporulation asymmetric thick septum at polar position; Class 4, thick septum at plays a role in pro-␴E processing. In addition, lipid-starved the mid-cell position; Class 5, thin septum at polar position; Class cells form primarily thick septal structures. The thick sep- 6, double asymmetric thin septum. tum phenotype is apparently caused by the incorporation of excess peptidoglycan into the space between the septum in DS medium. However, all the septa formed by cerulenin- membranes (Bara´k and Youngman, 1996). It has been treated cells in the resuspension medium were abnormally proposed that the SpoIIE protein could be involved in thick (data not shown). In addition, as observed in DS determining the extent to which peptidoglycan becomes medium, the forespore sigma ␴F was active, as judged inserted between the lipid bilayers that constitute the sporu- from spoIIQ–lacZ expression (Fig. 3D), whereas the mother lation septum (Bara´k and Youngman, 1996). This proposal cell sigma factor ␴E was completely inactive, as judged by is based on the finding that SpoIIE is a membrane-bound spoIID–lacZ and spoIIID–lacZ expression (Fig. 3F) and protein that accumulates at sites of asymmetric septum Western blot experiments (data not shown) in cells induced synthesis and that spoIIE null mutants form a thick septum to sporulate by the resuspension method in the presence (Arigoni et al., 1995; Bara´k and Youngman, 1996; Bara´k et of cerulenin. al., 1996). Thus, inhibition of phospholipid synthesis could The results of the resuspension experiments suggest interfere with the role of SpoIIE in septum assembly, caus- that some cells in DS medium are able to form a thin ing a thick phenotype. If this is the case, new phospholipid sporulation septum before sporulation is arrested by lipid synthesis at the initiation of sporulation would be essential starvation. The different results obtained by the addition for the morphogenetic activity of SpoIIE, but not for its of cerulenin to cells induced to sporulate with the nutrient phosphatase activity needed to release the first cell-specific or resuspension method could be explained by the fact sigma factor ␴F in the prespore. Alternatively, the rate of that the last procedure provides a fixed point of the initia- phospholipid and murein synthesis could be tightly coupled tion of sporulation from which subsequent events can be during septum formation. The increased peptidoglycan accurately timed (Errington, 1993). deposition between the septum membranes may thus be a consequence of an unbalanced synthesis of membrane and murein. Discussion How might the deprivation of fatty acid synthesis at t0 To form spores, B. subtilis needs to expend energy and prevent the compartment-specific activation of ␴E?Itis synthesize specialized structures in an environment in well established that the mother cell transcription factor which nutrients are scarce. Thus, the ability to sporulate ␴E is derived by proteolytic cleavage from pro-␴E (LaBell,

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1215–1224 Role of lipid synthesis in B. subtilis sporulation 1221

1987; Stragier et al., 1988). Much experimental evidence could affect the ability of SpoIIR to activate SpoIIGA. To suggests that SpoIIGA, a multispanning membrane pro- this end they constructed a strain in which spoIIR expres- tein, is the processing enzyme (for a review see Losick sion was independent of ␴F and that also contained the and Stragier, 1996). Hofmeister et al. (1995) proposed spoIIE20 mutation (Zhang et al., 1996). However, the resul- that SpoIIGA may be a two-domain protein that resides tant strain was unable to form septa and was prevented within the forespore septum with one domain serving as from testing the role of septum morphology in compartmen- the pro-␴E protease and the second serving as a signal talized activation of ␴E (Zhang et al., 1996). We report transduction domain that responds to transmembrane here that cerulenin treatment produces cells that synthe- activation by SpoIIR. Thus, it is possible that lipids might size thick asymmetric septa expressing spoIIR (data not be essential for the catalytic activity, assembly or SpoIIR- shown) but unable to activate ␴E. Thus, our results are mediated activation of SpoIIGA into the septal membrane. also compatible with the hypothesis that the spoIIE-like A large body of evidence attests that lipids have a strong sporulation septum found in cells treated with cerulenin influence on the properties of membrane-bound enzymes, could affect the ability of SpoIIR to act as a cell-to-cell signal and many solubilized or reconstituted enzymes are speci- for ␴E activation. fically stimulated by the addition of particular lipids (for a Although little is understood about septum formation, it review see Gennis, 1989). Moreover, recent experiments is accepted that its assembly requires the synthesis of a established that a phospholipid acts as a chaperone in great deal of new lipid (Piggot et al., 1994; Feucht et al., the assembly of a membrane transport protein (Bogdanov 1996). Our results show that cells with a deficiency in et al., 1996). If the amphipatic environment of the septal fatty acid synthesis can still form an asymmetric septum. membrane is an important determinant in the assembly However, the lipid composition of this asymmetric septum or activity of SpoIIGA, the addition of cerulenin could inhi- must be determined by ‘old lipids’ synthesized in the pre- bit the accumulation of lipids synthesized at t0 necessary divisional cell. Under these conditions, the polar septum for SpoIIGA activation. If this hypothesis is correct, lipid- is not suitable to activate ␴E-directed gene expression. A mediated activation of SpoIIGA could be relevant for the key challenge now will be to directly determine whether compartment-specific activation of ␴E. A topological lipids affect the activity, assembly or localization of the model for SpoIIGA proposes that multiple membrane- SpoIIGA protein, or if they are important for the transmis- spanning segments in the N-terminus of the protein anchor sion of the cell–cell signalling pathway mediated by SpoIIR. it to the membrane and present the C-terminus to the interior of the cell (Stragier et al., 1988). If SpoIIGA is Experimental procedures inserted in this manner into both septum membranes, it might be available to interact with pro-␴E in both the fore- Bacterial strains spore and the mother cell compartments. Localized acti- The B. subtilis strains used in this study are listed in Table 2. vation of ␴E in the mother cell might then occur as a The strain carrying the original resistance to cerulenin (Cer20 ) result of an asymmetric lipid composition in the sporan- was a gift from Donald Zeigler (BGSC). gium septal membranes. The particular phospholipid composition of the mother cell could be essential for the General methods activation of the protease domain to cleave pro-␴E into Difco sporulation medium (DS) was used to induce sporula- E mature ␴ . In this regard, it is important to mention that tion in B. subtilis in liquid media (Shaeffer et al., 1965). The phospholipids in growing and sporulating cells of B. subtilis method used for induction of sporulation by resuspension in can vary qualitatively and quantitatively and are greatly poor medium was essentially that of Sterlini and Mandelstan affected by spo mutations (Rigomier et al., 1974; Rigomier (1969). The amount of sporulated cells was determined by et al., 1978). It has also been demonstrated that mem- heat treatment (20 min at 80ЊC). branes of B. megaterium show extremely fast lipid trans- Competent cells were prepared and transformed as previously described (Mansilla and de Mendoza, 1997). Selection location, presumably mediated by specific translocases for resistance to cerulenin was performed on LB medium (Rothman and Kennedy, 1977). This transmembrane traffic (Sambrook et al., 1989) containing 10 ␮g of cerulenin per ml. activity could also be important determining a differential Cerulenin was stored as a stock solution at 10 mg ml¹1 in phospholipid composition or lipid microdomains in the fore- ethanol at ¹20ЊC. Aliquots of the solution were added to a spore and mother cell septal membranes. Moreover, evi- shaking culture in order to rapidly disperse the antibiotic. dence consistent with the existence of a variety of types Electron microscopy of sporulating cells sampled 6 h after of membrane domains within prokaryotic cells has been the end of exponential phase was performed as described previously (Chatter et al., 1989). obtained using many different techniques (for a review see Norris and Madsen, 1995). ␤-Galactosidase assays It is worth noting that Zhang et al. (1996) explored the possibility that the septum morphology of spoIIE mutants To test the effect of cerulenin on the expression of lacZ

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Table 2. Bacillus subtilis strains used in this study. Strain Relevant characteristics Source or reference

PY79 Prototrophic Youngman et al. (1984) PL85 PY79, spoIID⍀ spoIID–lacZ Levin and Losick (1994) RL905 PY79, SP␤[spoIIAB–lacZ ] Levin and Losick (1994) RL1306 PY79, usd-spoIIID–lacZ R. Losick JH642 trpC2 phe1 J. Hoch JH16840 JH642, amyE ::spoIIE–lacZ J. Hoch JH16182 JH642, amyE ::spoIIG–lacZ J. Hoch MO2051 JH642, amyE ::spoIIQ–lacZ London˜o-Vallejo et al. (1997) BR151 Lys-3 metB10 trpC2 Young et al. (1969) SL6418 BR151, amyE ::spoIIR–lacZ P. Piggot 1A577 trpC2 Cer20 BGSCb GS77a trpC2 phe1 Cer20 This work

a. The cer20 mutation was introduced into JH642 by transformation with DNA of strain 1A577 and selection for CerR. b. BGSC denote strain obtained from Bacillus Genetics Stock Center. fusions to sporulation genes, ␤-galactosidase levels in sporu- deﬁned as pmoles [2-14C]-malonyl-CoA incorporated into lating cultures were determined as described (Mansilla and de hexane-extractable fatty acids per mg of protein per min. Mendoza, 1997).

Enzyme preparation Metabolic labelling To determine de novo fatty acid synthesis in vivo,strainJH642 Strains were grown to mid-exponential phase of growth in LB was grown in SM medium at 37ЊC and labelled at the times indi- media (250 ml of cell culture); the cells were harvested by cen- cated in Fig. 2 with 10 ␮Ci of [14C]-acetate (56.7 mCi mmol¹1) trifugation and washed twice with 20 mM Tris-HCl, pH 7.5, con- for 1 h. Cellular lipids were extracted (Grau and de Mendoza, taining 0.15 M NaCl. Subsequent operations were carried out at 1993), saponified and the amount of [14C]-acetate incorpo- 4ЊC. The washed cell pellets were resuspended in twice their rated into long-chain fatty acids was determined as previously wet weight in 100 mM potassium phosphate buffer pH 7.0 con- described (de Mendoza et al., 1983). taining 1 mM EDTA, 1 mM ␤-mercaptoethanol and 10 mM phenylmethylsulphonyl fluoride and lysed in a French pressure cell at 18 000 psi. The lysate was centrifuged for 1 h at 100 000 × g using a 80 Ti rotor in a Beckman L8-70 ultracen- Immunoblot analysis trifuge. The supernatant fluid was removed, fractionated with Strain JH642 was grown in DS medium grown at 37ЊC. At the ammonium sulphate, and the precipitate formed between end of exponential growth, the culture was split and cerulenin 40% and 70% saturation was collected. The precipitated pro- was added at a concentration of 10 ␮gml¹1 to one half of the tein was removed by centrifugation. The protein pellet was dis- culture. At the times indicated in Fig. 4 cells (20 ml) were har- solved in 1 ml of lysis buffer and dialysed against the same vested, washed with 5 ml of a buffer containing 100 mM Tris- buffer. HCl pH 7.5 and 150 mM NaCl and resuspended in 1 ml of breaking buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 1 mM Fatty acid synthase assays PMSF and 0.1 mM DTT). Cells were then disrupted by pas- sage through a French pressure and the lysates centrifuged Fatty acid synthase was measured in vitro by a modification of for 45 min at 190 000 × g. Each sample (100 ␮g of protein) the assay described by Butterworth and Bloch (1970). Under was subjected to SDS–PAGE in a 12.5% acrylamide gel. Pro- the conditions described, product formation was linear with teins were electroeluted to a nitrocellulose membrane and respect to time and protein concentration. The reaction mix- revealed using anti-␴E monoclonal antibody and a secondary ture consisted of 0.5 mM NADP, 5 mM glucose-6-phosphate, antibody conjugated to alkaline phosphatase. 0.25 units of glucose-6-phosphate dehydrogenase, 15 ␮M of isobutyril-CoA, 20 ␮M malonyl-CoA, 0.4 ␮Ci of [2-14C]- ¹1 malonyl-CoA (56.7 mCi mmol ), 10 ␮M Escherichia coli ACP, Acknowledgements 100 mM sodium phosphate and 1.25 or 0.75 mg ml¹1 protein from free-cell extracts of strains JH642 or GS77 respectively. We gratefully acknowledge James A. Hoch, Richard Losick, Cerulenin was added to the assay tube in ethanol solution, Patrick Stragier, Patrick Piggot and Donald Zeigler for the preincubated at room temperature for 10 min, and the other gift of bacterial strains and to William Haldewang for the assay components were added to the tube. The [2-14C]-malo- anti-␴E antibody. This work was supported by the Commission nyl CoA was added last to start incubation at 37ЊC for 20 min of European Communities Grant 937004 AR, Fundacioń followed by hydrolysis with 0.2 ml of 60% KOH (w/v) at 100ЊC Antorchas, FONCYT and the Consejo Nacional de Investiga- for 30 min. After acidification the products were extracted into ciones Cientı´ficas y Tećnicas (CONICET). G. E. Schujman hexane and counted into vials containing scintillation solution is a fellow from CONICET and D. de Mendoza and H. C. to determine their radioactivity content. Specific activity is Gramajo are Career Investigators from the same institution.

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1215–1224 Role of lipid synthesis in B. subtilis sporulation 1223

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