Journal of Bacteriology, March 2004, p . 1475-1483, Vol . 186, No . 5

Cardiolipin Domains in Bacillus subtilis Marburg Membranes

Fumitaka Kawai, Momoko Shoda, Rie Harashima, Yoshito Sadaie, Hiroshi Hara, and Kouji Matsumoto^*

Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-ohkubo, Sakura, Saitama, Saitama 338-8570, Japan

Received 31 July 2003/ Accepted 24 November 2003

 
Recently, use of the cardiolipin [CL]-specific fluorescent dye10-N-nonyl-acridine orange [NAO] revealed CL-rich domains inthe Escherichia coli membrane [E . Mileykovskaya and W . Dowhan,J . Bacteriol . 182: 1172-1175, 2000] . Staining of Bacillus subtiliscells with NAO showed that there were green fluorescence domainsin the septal regions and at the poles . These fluorescence domainswere scarcely detectable in exponentially growing cells of theclsA-disrupted mutant lacking detectable CL . In sporulatingcells with a wild-type lipid composition, fluorescence domainswere observed in the polar septa and on the engulfment and foresporemembranes . Both in the clsA-disrupted mutant and in a mutantwith disruptions in all three of the paralogous genes [clsA,ywjE, and ywiE] for CL synthase, these domains did not vanishbut appeared later, after sporulation initiation . A red shiftin the fluorescence due to stacking of two dye molecules andthe lipid composition suggested that a small amount of CL waspresent in sporulating cells of the mutants . Mass spectrometryanalyses revealed the presence of CL in these mutant cells.At a later stage during sporulation of the mutants the frequencyof heat-resistant cells that could form colonies after heattreatment was lower . The frequency of sporulation of these cellsat 24 h after sporulation initiation was 30 to 50% of the frequencyof the wild type . These results indicate that CL-rich domainsare present in the polar septal membrane and in the engulfmentand forespore membranes during the sporulation phase even ina B . subtilis mutant with disruptions in all three paralogousgenes, as well as in the membranes of the medial septa and atthe poles during the exponential growth phase of wild-type cells. The results further suggest that the CL-rich domains in the polar septal membrane and engulfment and forespore membranesare involved in sporulation.

 
The bacterial cell membrane is widely recognized as a matrixin which lipid molecules are homogeneously distributed . However,it has been noticed that lipid molecules are heterogeneouslydistributed in bacterial membranes, and the observations increasinglyinclude those obtained by using fluorescent lipophilic probes.Immunoelectron microscopic observations showing the polar localizationof the chemoreceptor complexes in Caulobacter crescentus and Escherichia coli cells provided early indications of membrane heterogeneity [1, 27] . By using the lipophilic fluorescent styryldye FM4-64, laterally uneven distribution of the fluorescence,which could be an indication of heterogeneous distribution ofphospholipids in E . coli membranes, was then discovered [14].Recently, the cardiolipin [CL]-specific fluorescent dye 10-N-nonyl-acridine orange [NAO] was used to demonstrate that there are CL-containing domains in E . coli membranes, which were observed mostly in the septal regions and at the poles of the cells [31, 32] . Thehypothesis that there are CL-containing domains in these regionsof E . coli cells was supported by an analysis of the lipid compositionof minicells, which consist mainly of polar materials of theenvelope [24].

CL in the presence of certain divalent cations, as well as phosphatidylethanolamine,has the potential to form nonbilayer structures, which couldintroduce discontinuity into the bilayer membrane structuresfor dynamic membrane functions, such as membrane fusion duringcell division, formation of adhesion sites between the outerand inner membranes, integration of proteins into the membrane, and stabilization of membrane proteins [12] . Mutants of E . colilacking either CL or phosphatidylethanolamine are viable, butconstruction of a mutant lacking both phospholipids is not possible[8, 34, 41], suggesting that a common structural feature of the lipids, the potential to form nonbilayer structures, is required . Additionally, CL and phosphatidylglycerol, both ofwhich have an anionic nature, play a role in recruitment ofmembrane proteins having positively charged amphitropic -helices onto the anionic surface of the membrane [12, 29] . In spiteof the anticipated roles, the significance of CL in vivo isstill clouded by its dispensability [23, 29, 34].

The Bacillus subtilis membrane undergoes dynamic rearrangements, which include formation of polar septa and engulfment and forespore membranes, during the sporulation process in addition to the rearrangements that occur during cell division during vegetative growth . The membranes contain CL [9, 30], which could play importantroles in the processes, but little is known about the anticipatedroles and the genes responsible for biosynthesis of this compound.B . subtilis has three homologues [ywnE, ywjE, and ywiE] [25] of E . coli cls, the structural gene for CL synthase, but the contribution of these genes to CL synthesis has not been examined previously . Here, we characterized mutants with disruptionsin each or all three of these homologues . We found that ywnE plays a dominant role in CL synthesis; thus, ywnE is renamed clsA . We also found a small but significant amount of CL in the sporulation-phase cells of the mutant with disruptions inall three genes . By using NAO staining, we found that B . subtilis cells contain CL-rich domains in the polar septal membrane andin the engulfment and forespore membranes in the sporulationphase, as well as in the membrane of the medial septa and atthe poles in vegetative growth phase . We suggest that the CL-containingdomains in the former membranes are involved in sporulation.

 
Bacterial strains and plasmids. The B . subtilis Marburg and E . coli K-12 strains and the plasmidsused in this study are listed in Table 1 . The strains with disrupted alleles of clsA, ywjE, and ywiE were constructed as follows.Plasmids pJESPC and pIENEO with the disrupted alleles ywjE1::spcand ywiE2::neo, respectively, were first constructed with pBR322,and wild-type strain 168 was transformed with the plasmid DNAto obtain SDB201 [ywjE1::spc] and SDB202 [ywiE2::neo], respectively.BFS219 [clsA::pMutin4] was then transformed with the chromosomalDNA of SDB202 [ywiE2::neo] to form SDB203 [clsA::pMutin4 ywiE2::neo]. Finally, SDB203 was transformed with the DNA of SDB201 [ywjE1::spc] to construct SDB206 [clsA::pMutin4 ywiE2::neo ywjE1::spc] . Thedisrupted alleles in the mutant stains were confirmed by the increase in size by using PCR.

 
The pJESPC and pIENEO plasmids were constructed as follows.For construction of pJESPC, the fragment containing ywjE wasfirst obtained by PCR amplification from B . subtilis 168 chromosomal DNA with primer ywjEEco [5'-TATTgAATTCCTGCTGTTCG-3'], which introduced an EcoRI recognition sequence [underlined] with a mismatch [lowercase letter] starting 56 nucleotides [nt] upstream from the initiation codon, and primer ywjESphAS [5'-TCATCGCATgcGAAACGAACCG-3'], which introduced an SphI recognition sequence [underlined] with two mismatches [lowercase letters] starting 112 nt downstream from the termination codon . The fragment was digested with EcoRI and SphI and then inserted into EcoRI-SphI-digested pBR322.The resulting plasmid was digested with HindIII at a site located646 nt downstream from the initiation codon, and the HindIIIfragment containing spc from pDG1726 [16] was inserted to constructpJESPC.

To construct pIENEO, fragment ywiE-F-B [1,590 bp] containing ywiE that had a newly introduced XbaI site 819 nt downstream from the initiation codon was obtained by a second PCR amplification of the first two PCR products [ywiE-F and ywiE-B] with primersywiEEco and ywiEHinAS . The ywiE-F product was obtained by PCRamplification from the chromosomal DNA by using primer ywiEEco [5'-ACAGAGGgAATTcAATCAGATTGGAG-3'], which introduced an EcoRI recognition sequence [underlined] with two mismatches [lowercase letters] starting 26 nt upstream from the initiation codon,and primer ywiEXbaAS [5'-TATCTCTagAAAATCCGATGTACG-3'], which introduced an XbaI recognition sequence [underlined] with two mismatches [lowercase letters] starting 829 nt downstream fromthe initiation codon . The ywiE-B product was obtained from the chromosomal DNA by using primer ywiEXba [5'-GATTTTctAGAGATACACACCTGCGGC-3'], which introduced an XbaI recognition sequence [underlined] with two mismatches [lowercase letters] starting 814 nt downstream from the initiation codon, and primer ywiEHinAS [5'-TTCTCAAgctTACTATACACGGGC-3'], which introduced a HindIII recognition sequence [underlined] with three mismatches [lowercase letters] starting 52 nt downstream from the termination codon . The ywiE-F-B fragment was introduced into EcoRI-HindIII-digested pBR322 . The resulting plasmid wasdigested with XbaI, and then the XbaI fragment containing neofrom pBEST502 [21] was inserted to form pIENEO.

Cloning of clsA, ywjE, and ywiE into expression vector pSK6was performed as follows . The clsA fragment was amplified fromB . subtilis wild-type chromosomal DNA by using primer nesNcoI[5'-GGGTTACAccaTGgGTATTTCTTCC-3'], which introduced an NcoIsite [underlined] starting 11 nt upstream of the initiationcodon GTG with four mismatches [lowercase letters], and primerneasXbaI [5'-CGGGGAAGTcTAgATCACTGACG-3'], which introduced anXbaI site [underlined] with two mismatches [lowercase letters]starting 147 nt downstream from the stop codon . The amplifiedfragment was digested with NcoI and XbaI and then inserted intoNcoI-XbaI-digested pSK6 to construct pMMS1 . The ywjE fragmentwas amplified by using primer jesNcoI [5'-GCCGccATGgAGGTATTTATC-3'],which introduced an NcoI site [underlined] starting 6 nt upstreamof the initiation codon with three mismatches [lowercase letters],and primer jeasSphI [5'-GGCCTCAAGCATGCGGTA-3'], which had an SphI site [underlined] starting 435 nt downstream of the termination codon . The amplified fragment was digested with NcoI and SphI and inserted into NcoI-SphI-digested pSK6 to construct pMMS2.The ywiE fragment was amplified by using primer iesNcoI [5'-GAGAGAACCAccATGgTGAAAAGG-3'],which introduced an NcoI site [underlined] starting 12 nt upstreamof the initiation codon with three mismatches [lowercase letters],and primer ieasHindIII [5'-CCATTAAAgCTtCCGCATCG-3'], which introduceda HindIII site [underlined] with two mismatches [lowercase letters]starting 176 nt downstream of the stop codon . The amplifiedfragment was inserted into NcoI-HindIII-digested pSK6 to construct pMMS3 . These plasmids were introduced into E . coli SD9 cells to examine their ability to produce CL . PCR amplification was conducted by using the Expand High Fidelity PCR system [Boehringer Mannheim Biochemicals], and other methods used for DNA manipulations have been described previously [42].

Media and bacterial growth. Luria-Bertani [LB] broth contained 1% tryptone [Difco, Detroit,Mich.], 0.5% yeast extract [Difco], and 1% NaCl . Difco sporulationmedium [DSM], which contained 0.8% nutrient broth [Difco], 0.1%KCl, 0.025% MgSO₄ · 7H₂O, 1.0 mM Ca[NO₃]₂, 10 µMMnCl₂, and 1.0 µM FeSO₄ [43], was used for cultivation of B . subtilis cells . Synthetic media CI and CII were used for competence development [3] . When required, the following supplementswere added to the media [per liter]; 50 mg of ampicillin [Sigma],20 mg of neomycin [Wako Pure Chemicals], 50 mg of spectinomycin[Sigma], and 0.3 mg of erythromycin [Sigma] . Growth of bacteriawas monitored by measuring turbidity with a Klett-Summerson photoelectric colorimeter [no . 54 filter] . For membrane depolarization carbonyl cyanamide 3-chlorophenylhydrazone from Sigma was added at a concentration of 10 µM [17] . For solid media, 1.5% agar [Difco] was included.

Fluorescence microscopy. NAO [catalog no . A-1372; Molecular Probes] was added to a finalconcentration of 100 nM to a cell culture in DSM at 37°Cthat was harvested in the exponential growth phase and in thesporulation phase at 2 and 4 h after the end of log-phase growth[T₂ and T₄, respectively] . After incubation at room temperaturefor 20 min, the cells were fixed on object slides coated witha layer of 1% [wt/vol] agarose gel in water . FM4-64 [catalogno . T-3166; Molecular Probes] staining was performed similarlyby using a concentration of 0.1 to 0.2 µM in DSM . Fluorescenceimages were viewed by using an ECLIPS E600 fluorescence microscope[Nikon] and a cooled charge-coupled device camera [ORCA-ER;Hamamatsu Photonics Co., Hamamatsu, Japan] . Green fluorescence[with excitation at 495 nm and emission at 525 nm] from NAOwas detected by using a standard GFP[R]-BP filter unit [excitationat 460 to 500 nm and emission at 510 to 560 nm] . Red fluorescence[emission at 640 nm] from NAO was detected by using a set offilters [excitation at 450 to 490 nm and emission at 610 nm]. Fluorescence from FM4-64 [excitation at 510 nm and emissionat 626 nm] was detected by using a G-2A filter unit [excitationat 510 to 560 nm and emission at 590 nm] . To minimize the toxicityof high-energy emission light, the focus was set under phase-contrast conditions, and then fluorescence images were captured shortlyafter the shift to emission light conditions . The exposure timesfor green and red fluorescence of NAO were 0.2 to 0.8 and 7.7s, respectively . The exposure time for FM4-64 fluorescence was3.3 to 4.5 s . Captured images were processed by using AdobePhotoshop 6.0 . The relative intensities of fluorescence werequantified by using NIH Image [Scion Image 4.0.2].

Lipid analysis. Membrane lipids were labeled with 0.5 µCi of [1-¹⁴C]aceticacid [57.2 mCi/mmol; Amersham] per ml for more than six generationsduring cultivation of the mutant and wild-type cells in DSMbroth [5 ml] and were harvested at the late exponential phaseand T₂ and T₄ of the sporulation phase . In some cases, includingE . coli, cells were cultivated in LB broth with no radioisotope.Lipids were extracted by the method of Bligh and Dyer [2] orby the method developed for sporulating B . subtilis cells by Lacombe and Lubochinsky [26], with minor modifications . For the latter method we incorporated the following acidic treatment into the former method . The treatment included incubation of the cells in suspension with 0.9 M [final concentration] perchloric acid in 1% NaCl at 0°C for 30 min, followed by additionof 1.88 ml of chloroform-methanol [1:2 [vol/vol]] to 0.5 mlof the cell suspension . One-half of each lipid fraction wasseparated by two-dimensional thin-layer chromatography [TLC]on Silica Gel 60 [Merck, Darmstadt, Germany], first [x dimension]with chloroform-methanol-water [65:25:4 [vol/vol/vol]] and then[y dimension] with chloroform-methanol-acetic acid [65:25:10[vol/vol/vol]] . Spots of ¹⁴C-labeled lipid were visualized andquantified with a BAS 1000 bioimaging analyzer [Fuji Photo Film,Tokyo, Japan] . In some cases phospholipids were visualized byuniformly spraying the gel with Dittmer-Lester reagent [11]and were quantified with a high-speed TLC scanner [model CS-920;Shimadzu, Kyoto, Japan] . The molar percentage of each componentwas calculated.

Mass spectrometry analysis. The CL fractions that eluted from the CL spots on TLC plateswere mixed with 2,5-dihydrobenzoic acid [Aldrich Chemical Co.]to saturation and subjected to mass spectrometry . Matrix-assistedlaser desorption ionization—time of flight mass spectrawere acquired with a KRATOS KOMPACT MALDI 4 [Shimadzu/Kratos,Tokyo, Japan] equipped with a pulsed nitrogen laser [337 nm]in the linear mode by using an acceleration voltage of 20 kV.

 
NAO staining of wild-type B . subtilis cells. A previous study with E . coli cells indicated that NAO at aconcentration of 100 to 200 nM in the growth medium resultedin staining of the cells having the wild-type phospholipid compositionand had no noticeable effect on the growth rate [31] . B . subtilis cells also exhibited no noticeable reduction in the growth rate with the same and much higher concentrations [100, 200, and500 nM] of NAO in LB and DSM media . After staining of wild-type168 cells in the late exponential growth phase with 100 nM NAOfor 20 min at room temperature, the cells were mounted on agarose-coatedobject slides and viewed with an ECLIPS E600 fluorescence microscope[Nikon] equipped with a standard GFP[R]-BP filter unit [excitationat 460 to 500 nm and emission at 510 to 560 nm] . Green fluorescentdomains were clearly observed in the septal regions and at thepoles [Fig. 1A] . A sharp band in the center of the cell was observed in most cells . In some cases two fluorescent dots were located in the center of the cell . The two-dot structures were interpreted to be two-dimensional projections of three-dimensional rings with a small amount of fluorescent material forming anopen ring in immunofluorescence studies of the FtsZ ring [10, 40] . Sharp bands were interpreted as a ring that contained morefluorescent material in a closed-ring structure . Thus, we concludedthat the NAO fluorescent domains were localized in the septalmembrane . Membranes at the nascent poles in separating cells were also stained with NAO [Fig . 1A and B] . The fluorescenceat these poles was therefore considered to be fluorescence fromthe remnant of the material in the septal membrane . In sporulatingcells NAO fluorescent domains were observed in the polar septaand on the engulfment and forespore membranes [Fig. 1B and C].The fluorescence was restricted to these membranes, and themother cell membrane was not stained with NAO . In order to identifythe fluorescence as specific to CL, control experiments withmutants lacking CL were required . However, the gene[s] responsiblefor CL synthesis in B . subtilis has not been determined.

 
NAO staining of mutant cells with a disrupted allele of clsA coding for CL synthase. B . subtilis has three candidates for the gene coding for CLsynthase, ywiE, ywjE, and ywnE [25] . To determine which of theseparalogous genes is involved in CL synthesis, lipid was extractedby the Bligh-Dyer method [2] from mutant cells having a pMutin-disrupted allele of each candidate gene cultivated to the mid-exponential growth phase, and the phospholipid composition was examined.No noticeable reduction in CL content was observed in the extractsfrom the late-log-phase cells of the ywiE and ywjE disruptants, strains BFS1244 and BFS1245, respectively . However, there was no CL in the ywnE disruptant, strain BFS219, suggesting that ywnE is involved in CL synthesis but ywiE and ywjE are not. On the basis of this finding and the results described below ywnE was renamed clsA.

In clsA disruptant cells fluorescence was scarcely detectable [Fig . 2A], as expected because of the lack of CL . In sporulation-phasecells, however, fluorescent domains were observed in the medialand polar septa and engulfment membranes . This might suggestthat the clsA-disrupted cells contained CL in the sporulationphase . Since all three gene products have highly conserved duplicatedHxK[x]₄D[x]₆G[x]₂N motifs [18, 28], we expected that all threemight be active in CL synthesis . The remaining two, ywiE andywjE, were anticipated to be responsible for minor CL synthesisin the clsA-disrupted cells in the sporulation phase.

 
CL contents of mutant cells with disrupted clsA and three [clsA, ywiE, and ywjE] disrupted alleles. To prove that the product of ywiE and ywjE has CL synthase activity, these paralogous genes were examined for CL production in E.coli mutant SD9 [pssA1 cls-1] lacking detectable CL [35] . Eachopen reading frame, from its initiation codon obtained by using the new NcoI site created by PCR on the initiation codon, was placed under control of the arabinose promoter of pSK6, a derivative of pSC101 [46], and was expressed in SD9 cells . The cells harboringclsA on plasmid pMMS1 contained CL [4.6% of the total phospholipid]after induction by addition of L-arabinose . The cells harboringywjE on pMMS2 contained CL that accounted for 2% of the totallipid . The ywjE gene was, therefore, active in CL synthesis.In the cells harboring ywiE on pMMS3, however, we did not detectCL [less than 0.1% of the total phospholipid] . When the cellsharboring each of these paralogues were incubated at 42°C,expression of clsA and ywjE complemented the temperature-sensitivegrowth of SD9 cells, although ywjE did this very weakly; however,expression of ywiE did not . The ywiE gene may therefore be inactive in CL synthesis.

To clarify the possible contribution of ywjE and ywiE to CL synthesis, we constructed multiply disrupted mutant strainsand examined their lipid compositions by ¹⁴C labeling . The drug resistance genes spc and neo were inserted into the unique HindIIIsite in ywjE and the XbaI site in ywiE, respectively, to constructywjE1::spc and ywiE2::neo alleles . The disrupted alleles were successively introduced into BFS219 [clsA::pMutin], and multiply disrupted mutant strains were constructed . The triply disrupted mutant, designated SDB206 [clsA::pMutin ywjE1::spc ywiE2::neo], was examined in the studies described below.

Lipids were extracted by the perchloric acid method that was described previously for efficient extraction of CL from sporulating B . subtilis cells [26] from SDB206 and BFS219 cells cultivatedin DSM containing [1-¹⁴C]acetic acid, and the CL contents ofthe mutants were examined after two-dimensional TLC on silicagel plates . The results of this lipid extraction analysis indicatedthat both of the mutant strains contained CL in the sporulationphase [Table 2] . The clsA-disrupted mutant cells contained CL[0.3% of the total lipid at T₄], although in the exponentialgrowth phase the level was below the detection limit . Note thateven in the triply disrupted mutant cells CL accounted for 0.2and 0.3% of total lipid at T₂ and T₄, respectively.

 
To confirm the presence of CL in sporulating cells of thesemutants, we obtained mass spectra of the fraction that elutedfrom the CL spot on a TLC plate . The region at mass/charge ratiosranging from 1,200 to 1,500 contained peaks of charged CL ionswith gross acyl chain compositions ranging from 56 to 72 carbonatoms; this range of fatty acid distribution is the result ofCL from B . subtilis [5] . In the extracts from SDB206 cells wedetected typical peaks of CL species with gross acyl chain compositionsthat varied from 61 to 69 carbon atoms with zero to three unsaturatedbonds . The extract from BSF219 cells produced peaks at similarmass values . These results indicated that both the clsA-disruptedand triply disrupted mutant cells contained CL in the sporulationphase.

Fluorescence of NAO-stained cells of SDB206 with three disrupted alleles [clsA, ywiE, and ywjE]. In the cells of the triply disrupted mutant, fluorescence wasscarcely detectable during the vegetative growth phase [Fig.3C-1]; this was similar to the results obtained with the cellsof the clsA-disrupted mutant [Fig . 2A and Fig . 3B-1] . Theseresults contrasted with the results obtained for red fluorescenceafter staining with FM4-64, a stain used to label plasma membranesof B . subtilis cells approximately uniformly [14, 38]; the cellmembranes of the mutants were uniformly bright lines and couldnot be distinguished from those of the wild type [Fig . 3D-1, E-1, and F-1].In the wild-type cells stained with the two dyesthe membranes formed uniformly bright red fluorescent lineswith FM4-64 [Fig. 3G-1-F] and showed an uneven distributionof green NAO fluorescence [Fig . 3G-1-N] . Under such conditions the relative intensity of the FM4-64 fluorescence of the septal membranes was nearly double that of other regions of the membrane,as shown previously [38] . The intensities of the NAO fluorescenceof the region of the lateral membranes relative to those ofthe septal membranes were, however, much less [1/8 to 1/10]. Similar relative intensity values were obtained for the cells stained with either one of the two dyes . These results indicatedthat the septal and polar membrane localization of NAO fluorescencewas dependent on CL and that it was not caused by cell lysis.

 
In the sporulation-phase cells of the mutants, however, the fluorescent domains of NAO appeared in the medial septa andat the poles 2 h after the end of exponential growth [T₂] [Fig. 3A-2, B-2, and C-2] and then on the engulfment and forespore membranes [Fig . 3A-3, B-3, and C-3] . The intensity of the fluorescencein these mutants seemed to be weaker than that in the wild type[Fig . 3A-3], which probably reflected the low CL content . Notethat the formation of the polar septa and engulfment membraneswas delayed for ca. 1 h in these mutant cells.

NAO binding to CL results in dimerization of the two dye molecules in a stacking form in which the molecules are in close proximity,and thus the emitted peak of the fluorescence for the monomer[at 525 nm] shifts to red [at 640 nm] due to the metachromaticeffect [36] . Thus, red fluorescence emission indicates labelingof CL domains in the membrane [15, 36] . When the GFP[R]-BP filterunit was replaced with a set of filters for red fluorescence [excitation at 450 to 490 nm and emission at 610 nm], red fluorescence was observed in the same regions [Fig . 4] . The red fluorescencewas weaker than the green fluorescence, which is consistentwith the results of a spectral analysis of the NAO-CL interactionwhich showed that the red fluorescence is approximately 14-foldweaker than the green fluorescence [36] . The brightness of thered fluorescence was thus intensified with Photoshop 6.0 forcomparison with the localization of the green fluorescence.The red fluorescent domains in the septa and at the poles ofthe wild-type cells colocalized with the green fluorescence [Fig . 4A-2 and A-3] . The images in polar septa and on engulfmentmembranes in sporulation phase [T₄] were colocalized as well[Fig . 4B-2 and B-3] . In the mutant cells the red fluorescencedomains were observed in the sporulation phase, and they werecolocalized with the green fluorescence domains [Fig . 4C-2 and C-3], indicating that the green fluorescence of the mutant cellswas from NAO bound to CL in the membranes . These results confirmedthe notion, based on TLC and mass spectrometry analyses, thatboth the clsA-disrupted and triply disrupted mutant cells containedCL in the sporulation phase.

 
In the mutant cells the appearance of the polar septa and engulfment membranes was delayed during the sporulation process [Fig . 3]. We therefore examined the frequency of heat-resistant cells during sporulation . In the case of the mutant strains the frequency of heat-resistant cells that could form colonies after 80°Ctreatment was lower in the later stage of sporulation . The frequenciesof heat-resistant spores of SDB206 and BFS219 at 24 h aftersporulation initiation were 30 and 50%, respectively, of thefrequency of heat-resistant spores of the wild type . These resultssuggest that the CL-rich domains in the membranes are involvedin a process required for sporulation.

 
Examination with the fluorescent dye NAO showed that B . subtilis cells contain CL-specific fluorescence domains in the septaand at the poles in the exponential growth phase and in thepolar septa and on the engulfment and forespore membranes duringthe sporulation phase . The specificity of NAO for CL in theseexperiments was supported by examination of the mutant strainswith defects in putative CL synthase genes [clsA, ywiE, andywjE] . Fluorescence was not observed in the clsA-disrupted mutant cells that lacked detectable CL in the exponential growth phase. During sporulation the CL content increased in both wild-typeand clsA-disrupted mutant cells [Table 2], which is analogous to the findings obtained for E . coli, in which the CL content increased during the stationary phase [7, 34, 44] . The increasein the CL content during the sporulation phase is consistentwith the results of ß-galactosidase transcriptionalfusion experiments which indicated that both clsA and ywjE exhibitedthe maximal activity, although the activity of ywjE was quitelow, in the early stationary phase and that ywiE had almostno activity [unpublished results] . Indeed, the clsA and ywjEmRNA levels exhibited 2.1- and 11.7-fold ^E-dependent increases,respectively [13] . The increase in transcription is analogousto the increase in E . coli CL synthase activity, which increasedin the stationary phase [20] . The product of ywjE is thus anticipatedto produce CL in sporulating cells of the clsA-disrupted mutant,although it was not effective under the conditions which weexamined [Table 2] . In spite of its highly conserved duplicated HxK[x]₄D[x]₆G[x]₂N motifs for bacterial CL synthase [18, 28],the product of ywiE may not be involved in CL synthesis . Thismolecule may have a role in the heat shock response since thelevel of the transcript of ywiE increases after a heat shock[19].

Two-dimensional TLC analysis of the lipid extracted from ¹⁴C-labeled cells indicated that in the triply disrupted mutant CL accounted for 0.2 and 0.3% of total lipid at T₂ and T₄, respectively,in the sporulation phase [Table 2] . Mass spectrometry analysisof the material eluted from the CL spot supported the identification.The red fluorescence in the stationary-phase cells of the triplydisrupted mutant [Fig. 4] confirmed that CL was present in thepolar septa and on the engulfment and forespore membranes, sinceNAO binding to CL results in dimerization of the two dye moleculesin a stacking form in which the molecules are in close proximity,which shifts the emitted peak of green fluorescence for themonomer [at 525 nm] to red [at 640 nm] due to the metachromaticeffect [36] . What enzyme is responsible for production of CLin the triply disrupted mutant cells? This enzyme could be theproduct of pss, phosphatidylserine synthase, since a significantamount of CL was synthesized [0.15% of the total phospholipidas determined by 3 min of pulse-labeling with ³²P] in E . colinull pssA cls double-mutant cells harboring a complementingB . subtilis pss gene on the pBR322 plasmid [41], similar to CL production in E . coli cls mutants by the product of E . coli pssA [34] . The activity of the enzyme or the side reaction maybe activated to produce CL during the early sporulation phase.The increase in the CL content during the sporulation phase may also be explained in part by the inactivity of a putative CL-specific phosphodiesterase [phospholipase D] that dependson ATP, as suggested by examination of stationary-phase cellsof E . coli [6, 44].

CL-containing domains are visualized with NAO in the septal regions and at the poles of E . coli cells [31], indicating thatthe phospholipid distribution in the membrane is heterogeneous.This conclusion was supported by an examination showing thatE . coli minicells, which consist mainly of polar materials ofthe envelope, are rich in CL [24] . In our work, we obtainedevidence of heterogeneous distribution of CL in the gram-positivebacterium B . subtilis. In eukaryotic cells loading and retentionof NAO on membranes are thought to be dependent on the membranepotential of mitochondria, since the intensity and stainingpattern of the cells are affected by depolarization treatment[22] . However, isolated mitochondria that are devoid of membranepotential after fixation in glutaraldehyde are stained brightby NAO [22] . Fixation of B . subtilis cells in 4% paraformaldehydedid not alter the pattern of NAO fluorescence [data not shown]. Depolarization treatment of B . subtilis cells by addition of 10 µM carbonyl cyanamide 3-chlorophenylhydrazone did notaffect the staining pattern, although the intensity of the fluorescencewas slightly reduced [data not shown], as observed for eukaryoticcells [22], indicating that the pattern of NAO fluorescencereflects a spatially heterogeneous distribution of CL-rich domainsin B . subtilis membranes . The CL-rich domains were also foundin the sporulating cells in the polar septa and on the engulfmentand forespore membranes and not only in the cells in the exponentialgrowth phase . The biological significance of the CL-rich domainsin sporulation was illustrated by using mutants with retardedemergence of the polar septal and engulfment membranes thatwas accompanied by a low frequency of heat-resistant cells.

What is the role of the CL-rich domains in these membranes?We can imagine two distinct roles that CL-rich domains playin the membranes . First, CL could contribute to form a nonbilayerstructure, which is thought to be required for the formationof septa and for engulfment, since it facilitates a dynamicphase shift that causes formation of nonbilayer structures inthe presence of certain divalent cations [mainly Ca²⁺, Mg²⁺,or Sr²⁺] under physiological conditions [12] . The polar septal membranes that develop into engulfment membranes may requireCL for dynamic progression, since their appearance was retardedin the sporulating cells of the mutants [Fig . 3] . Development of the polar septa may depend on a level of CL that increases in the early sporulation phase . Second, CL could contributeto recruitment of peripheral membrane proteins to localize tospecific regions of the cell membranes, and it could also contributeto maintenance of the optimal activity of these proteins . Theformer type of contribution has recently been described forMinD, which localizes in a horseshoe structure on the membraneat the poles of E . coli cells [39, 45] . MinD, with its C-terminalamphiphilic -helix bound to liposomes containing anionic phospholipids,has a higher affinity for CL-enriched liposomes [33, 47] . Thesecond type of contribution is also feasible, as essential interactionsof CL with integral membrane proteins [e.g., cytochrome c oxidase]are well known [12] and an interaction with CL of a Rhodobactersphaeroides type II reaction center has been demonstrated byX-ray crystallography [48] . Hence, some of the sporulation-phase-specificproteins involved in the formation of the polar septal and theengulfment membranes [37] in B . subtilis could require CL-dependentlocalization and/or maintenance for optimal activity.

 
We thank Marie-Françoise Hullo and Patrick Stragier formutant strains, Hiroaki Takahashi, Ayako Nishibori, and SatokoFuchizawa for assistance, and Fujio Kawamura, Hideaki Nanamiya[St . Paul's University], and Kei Asai for guidance concerningfluorescence microscopy . We also thank Roy H . Doi for reviewingthe manuscript . Thanks are also due to Isao Shibuya, HiroshiMatsuzaki, Junichi Ohnishi, Shigeki Moriya [Nara Institute],and Junichi Sekiguchi [Shinshu University] for encouragementand helpful discussions.

This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Cultureof Japan.

 
* Corresponding author . Mailing address: Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-ohkubo, Sakura, Saitama, Saitama 338-8570, Japan . Phone: 81-48-858-3406 . Fax: 81-48-858-3698 . E-mail: koumatsu@molbiol.saitama-u.ac.jp .

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