Journal of Bacteriology, September 2004, p . 6230-6238, Vol . 186, No . 18

Bacillus subtilis StoA Is a Thiol-Disulfide Oxidoreductase Important for Spore Cortex Synthesis

Lður S . Erlendsson, Mirja Möller, and Lars Hederstedt^*

Department of Cell and Organism Biology, Lund University, Lund, Sweden

Received 16 March 2004/ Accepted 24 June 2004

 
Bacillus subtilis is an endospore-forming bacterium . There are indications that protein disulfide linkages occur in spores,but the role of thiol-disulfide chemistry in spore synthesisis not understood . Thiol-disulfide oxidoreductases catalyzeformation or breakage of disulfide bonds in proteins . CcdA isthe only B . subtilis thiol-disulfide oxidoreductase that haspreviously been shown to play some role in endospore biogenesis.In this work we show that lack of the StoA [YkvV] protein resultsin spores sensitive to heat, lysozyme, and chloroform . Comparedto CcdA deficiency, StoA deficiency results in a 100-fold-strongernegative effect on sporulation efficiency . StoA is a membrane-boundprotein with a predicted thioredoxin-like domain probably localizedin the intermembrane space of the forespore . Electron microscopyof spores of CcdA- and StoA-deficient strains showed that thespore cortex is absent in both cases . The BdbD protein catalyzesformation of disulfide bonds in proteins on the outer side ofthe cytoplasmic membrane but is not required for sporulation.Inactivation of bdbD was found to suppress the sporulation defectof a strain deficient in StoA . Our results indicate that StoAis a thiol-disulfide oxidoreductase that is involved in breakingdisulfide bonds in cortex components or in proteins importantfor cortex synthesis.

 
Bacteria of the genera Bacillus and Clostridium can differentiate into endospores in response to unfavorable growth conditions. This dormant state is more resistant to heat, desiccation, UV radiation, hydrolytic enzymes, and toxic chemicals than the vegetative cell . The outermost protective layers of Bacillus subtilis endospores are the coat and the cortex [7] . The sporecoat is a proteinaceous barrier against bactericidal enzymes and destructive chemicals . The cortex is composed of a thick peptidoglycan layer that helps to maintain the dehydrated stateof the spore core and is required for the extreme heat resistanceof spores . There are indications that proteins in the coat are cross-linked by disulfide bonds [1, 26] . These bonds may contributeto the overall resistance of the spore . Some proteins, e.g.,YkvU and SpmB, encoded by ^E-dependent genes [8, 11] and possibly involved in cortex assembly are also rich in cysteine residues. However, the importance of disulfides and free thiol groupsfor the function of sporulation proteins is not understood.

Thiol-disulfide oxidoreductases catalyze the formation or breakage of disulfide bonds in other proteins . These enzymes have intheir active site a pair of cysteine residues that participatein the reaction . These cysteine residues are often arrangedin a Cys-X-X-Cys motif . Stable disulfide bonds in proteins inBacteria are normally only found in extracytoplasmic compartmentsand in secreted proteins . Thioredoxin and other reductants breakdisulfide bonds formed in cytoplasmic proteins . In both gram-positiveand -negative bacteria, several thiol-disulfide oxidoreductaseshave been identified that are involved in forming or breakingdisulfide bonds in proteins on the outer side of the cytoplasmicmembrane [for a review, see reference 19].

Six membrane-bound thiol-disulfide oxidoreductases that function on the outer side of the cytoplasmic membrane have so far been identified in B . subtilis . Four of these proteins function as pairs, i.e., BdbA/BdbB and BdbD/BdbC, and are similar to Escherichia coli DsbA/DsbB . These protein pairs catalyze formation of disulfide bonds in proteins [4, 6, 10] . B . subtilis CcdA most likely transfers reducing equivalents from thioredoxin in the cytoplasm acrossthe cytoplasmic membrane to ResA on the outer side of the membrane[32] . ResA has a thioredoxin-like domain [5] and functions to break a disulfide bond in the heme binding site of apo-cytochrome c [9] . CcdA is also required for efficient spore synthesis [31].The exact role of CcdA in sporulation has not been determined,but we have proposed that it transfers reducing equivalentsto one or more not-yet-identified thiol-disulfide oxidoreductasesthat function in spore synthesis . As deduced from the B . subtilisgenome sequence, YkvV and YneN are predicted membrane-boundproteins with a thioredoxin-like domain and are overall similarto ResA . These proteins could therefore possibly interact withCcdA . The ykvV gene is in a dicistronic operon together withykvU located at 123° on the B . subtilis chromosome [Fig.1] . The ykvUV operon is transcribed from a ^E-dependent promoter[8, 11] . YkvU is of unknown function but has sequence similarityto SpoVB, which is important for cortex synthesis . The yneNgene is monocistronic and located at 164° on the chromosomalmap.

 
We have analyzed the role of YkvV, YkvU, and YneN in B . subtilis and show that YkvV is involved in spore formation . The ykvV gene is therefore renamed stoA [sporulation thiol-disulfide oxidoreductase A] . We show that StoA is a membrane-bound thiol-disulfide oxidoreductase important for spore cortex synthesis.

 
Bacterial strains and plasmids. The B . subtilis strains and plasmids used in this work are listedin Table 1 . Oligonucleotides used as primers are listed in Table2. E . coli strain JM109 {recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi [lac-proAB] F' [traD36 proAB⁺ lacI^q lacZM15]} and TOP10[mcrA [mrr-hsdRMS-mcrBC] 80lacZM15 lacX74 deoR recA1 araD139 [ara-leu]7697 galU galK rpsL endA1 nupG] were used for the propagationof plasmids . E . coli strains BL21[DE3] [F^– dcm ompT hsdS[r_B^– m_B^–] gal [DE3]] and TOP10 were used for recombinant proteinproduction.

 
Media and growth conditions. E . coli cells were grown at 37°C in Luria-Bertani [LB] mediumor on LB agar plates [29]. B . subtilis strains were cultivatedat 30 or 37°C in LB medium or nutrient sporulation mediumwith phosphate [NSMP] [12] or on tryptose blood agar base [TBAB]plates [Difco] . Antibiotics were used at the following concentrationswhen appropriate: for B . subtilis, chloramphenicol at 3 mg/liter,erythromycin at 1 mg/liter, kanamycin at 10 mg/liter, neomycinat 5 mg/liter, spectinomycin at 150 mg/liter, and tetracyclineat 15 mg/liter; for E . coli, ampicillin at 50 mg/liter and chloramphenicolat 12.5 mg/liter.

DNA techniques. Standard DNA techniques were used [29] . Plasmid DNA was isolatedby using a Quantum prep plasmid miniprep kit [Bio-Rad] or byCsCl density gradient centrifugation . Chromosomal DNA from B.subtilis was isolated according to the method of Marmur [22].E . coli was transformed by electroporation, and B . subtiliswas grown to natural competence essentially as described byHanahan et al . and by Hoch, respectively [15, 17].

Construction of plasmids. Plasmid pLLE9 was constructed by amplifying a region upstreamof bdbA using primers LE001 and LE002 . The PCR product was clonedinto pDG780 at restriction sites XbaI and BamHI . A region downstreamof bdbB was amplified using primers LE003 and LE004 and clonedinto the plasmid at restriction sites ClaI and KpnI . PlasmidpLLE16 was constructed by amplifying regions up- and downstreamof yneN using primers LE009 and LE010 [fragment I] and LE011and LE012 [fragment II], respectively . The PCR products werecloned into pDG647 at restriction sites EcoRI and SmaI [forfragment I] and XbaI and PstI [for fragment II] . Plasmid pLLE34was constructed using primers LE030 and LE031 . The amplifiedDNA fragment was cut with KpnI and HindIII and cloned into thevector pBAD-HisA that had been digested with the same restrictionenzymes . Plasmid pLLE39 was constructed by amplifying an internalfragment of stoA using primers LE034 and LE035 . The PCR productwas cut with PstI and HindIII and cloned into pHV32 that hadbeen cut with the same restriction enzymes . Plasmid pLLE65 was constructed using primers LE051 and LE052 . The amplified DNA fragment was cut with KpnI and HindIII and cloned into the vector pBAD-HisB that had been digested with the same restriction enzymes. Plasmid pLLE77 was constructed by amplifying a region upstreamof ykvU using primers LE053 and LE054 . The PCR product was cloned into pDG1515 at restriction sites XbaI and BamHI . A region downstream of stoA was amplified using primers LE055 and LE056 and cloned into the plasmid at restriction sites SalI and XhoI . PlasmidpLLE83 was constructed using primers LE047 and LE048 . The amplifiedDNA fragment was cut with HindIII and XbaI and cloned into thevector pDG148 that had been cut with the same restriction enzymes.The template used for all the PCRs described above was chromosomalDNA isolated from B . subtilis strain 1A1 . Plasmid pLLE82 was constructed by moving a HindIII and ScaI fragment containingresA from pRAN1 to pDG148.

Construction of B . subtilis strains. Strain LUL20 was obtained by transforming 1A1 with pLLE39 andselecting transformants on TBAB plates containing chloramphenicol.The disruption of the stoA gene by the integrated plasmid wasconfirmed by PCR amplification of a DNA fragment using primersLE034, which hybridizes just upstream of stoA in the chromosome,and HV32P01, which hybridizes to a sequence in the vector partof pLLE39 . Strain LUL30 was constructed by transforming 1A1with SalI-cut pLLE77 and selecting transformants on plates containingtetracycline . Strain LUL110 was obtained by transforming 1A1with SalI-cut pLLE9 and selection on plates containing kanamycin.Strain LUL121 was isolated after transforming 1A1 with SalI-cutpLLE16 and selection on plates containing erythromycin.

Production and affinity purification of water soluble His-tagged variants of StoA and BdbD. Soluble His-tagged StoA was produced in E . coli TOP10 containingpLLE65 . Cells were grown in 100 ml of LB medium at 37°C.At an optical density at 600 nm of 0.6, the expression of stoAin the plasmid was induced with 0.2% [wt/vol] arabinose . Threehours after induction, the cells were collected by centrifugationand washed in buffer A [50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0]containing 10 mM imidazole . The cells were then broken usinga French press . The lysate was centrifuged at 31,000 x g for30 min, and the supernatant was mixed with 1 ml of Ni-nitrilotriaceticacid resin [QIAGEN] . After mixing for 1 h at 4°C, the resinwas washed with 20 mM imidazole in buffer A and the proteinwas eluted from the resin by 250 mM imidazole in buffer A . SolubleHis-tagged BdbD was produced in E . coli TOP10 containing pLLE34.Expression and purification of the protein was done in the sameway as for soluble His-tagged StoA except that gene expressionwas induced with 0.02% [wt/vol] arabinose . The purity of theisolated soluble His-tagged StoA and BdbD was assessed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis analysis[30].

Analysis of StoA transmembrane topology. DNA corresponding to a truncated variant of the stoA readingframe was amplified by PCR using B . subtilis 1A1 chromosomalDNA as template and the primer pair LE049/LE050 . The PCR productwas cut with BamHI and KpnI and cloned into pPHO1 digested withthe same enzymes, resulting in pLLE64 . Alkaline phosphataseactivity of cell lysates of E . coli BL21[DE3] containing pLLE64or pPHO1 grown in LB medium with 50 mM phosphate was measuredusing p-nitrophenyl phosphate as substrate [36].

Spore assay. Cultures were grown in 25 ml of NSMP at 30°C in 500-ml baffledErlenmeyer flasks for 2 days . Sporulation efficiency of strainswas analyzed by heating 5 ml of culture at 80°C for 15 min or by adding 0.6 ml of chloroform to a 5-ml culture followedby vigorous mixing for 10 s . Lysozyme sensitivity of cells wasanalyzed by diluting the culture 100-fold in minimal salts solution[80.4 mM K₂HPO₄, 44.1 mM KH₂PO₄, 15.1 mM [NH₄]₂SO₄, 3 mM sodium-citrate]followed by incubation with lysozyme [0.5 g/liter] at 30°Cfor 30 min . Serial dilutions of treated and untreated sampleswere spread on TBAB plates . After overnight incubation of theplates at 37°C, colonies were counted.

Electron microscopy. Preparation of B . subtilis cells for analysis by electron microscopywas performed essentially as described by Asai et al . [2] . Cellswere grown in NSMP medium at 37°C for 24 h after entry intostationary phase . After fixation in 3% glutaraldehyde in 50mM phosphate buffer [pH 6.5], the cells were postfixed in 1%osmium tetroxide for 1 h, dehydrated, and embedded in Epon.Sections were stained in 2% uranyl acetate and in lead citrate,according to the method of Reynolds [27] . Sections were examinedusing a JEOL 1230 transmission electron microscope.

Microarray analysis. B . subtilis strains 1A1 and LUL20 were grown in 300 ml of NSMPmedium at 37°C . Samples [20 ml] were harvested at the pointof entry into stationary phase [T₀] and two hours into stationaryphase [T₂] . RNA extraction, cDNA synthesis, hybridization ofcDNA to glass microarrays [Eurogentec], and acquisition andanalysis of data were performed essentially as described byHambraeus et al . [14].

Other methods. Membranes were isolated from strains grown in NSMP at 37°Cessentially as described previously [16] . Protein concentrationswere determined using the bicinchoninic acid protein assay [PierceChemical Co.] with bovine serum albumin as standard . N,N,N',N'-Tetramethyl-p-phenylenediamine [TMPD] oxidation assay of colonies on NSMP plates and cytochrome c oxidation activity measurements were performed as described previously [10, 21] . Insulin reduction assay was performed essentiallyas described by Holmgren [18] . Insulin and E . coli thioredoxinwere purchased from Sigma Chemical Co . ß-Galactosidaseactivity measurements using 4-methylumbelliferyl-ß-D-galactosideas substrate were done as described previously [31].

 
StoA and YneN are predicted membrane-bound thiol-disulfide oxidoreductases. StoA and YneN both have a dicysteine motif [Cys-X-Pro-Cys] thatis characteristic for proteins in the thioredoxin family . Alignmentof StoA and YneN with known thiol-disulfide oxidoreductasesshows sequence similarity, particularly around the dicysteinemotif . Secondary structure prediction [using PSIPRED [23]] basedon comparison to E . coli thioredoxin [TrxA] indicates that StoA and YneN have a thioredoxin-like fold . The program TMHMM [20] predicts that StoA and YneN each have one transmembrane segment, constituted by the N-terminal part of the protein and with the thioredoxin-like domain exposed on the outer side of the cytoplasmic membrane.

StoA is involved in sporulation. To investigate the physiological functions of B . subtilis StoAand YneN, the stoA gene in the chromosome was disrupted by aCampbell-type integration of plasmid pLLE39 and the yneN genewas deleted and replaced by an erythromycin resistance gene.The StoA- and YneN-deficient strains were named LUL20 and LUL121,respectively . Survival after heat treatment of LUL20 cells,grown for sporulation, was only 0.05% compared to that of untreatedcells, indicating that StoA has a role in sporulation [Table3] . In a recent study, Eichenberger et al . [8] found a sporulation efficiency of 0.001% with an independent B . subtilis StoA [YkvV]-deficientstrain . StoA deficiency does not completely block formationof heat-resistant spores, as can be seen by comparing the sporulationefficiency of LUL20 with that of LUA14, a strain blocked insporulation [Table 4] . As presented in Table 4, the survivalof LUL20 cells, grown for sporulation, was also low after lysozymeand chloroform treatment compared to that of wild-type cells.The sporulation defect of LUL20 cells was complemented by stoAexpressed in trans from plasmid pLLE83 which contains stoA undercontrol of the IPTG [isopropyl-ß-D-thiogalactopyranoside]-inducible spac promoter [Table 5 and Fig . 1] . This confirmed that thestoA gene is important for sporulation.

 
Strain LUL121, with the yneN gene deleted, was found to havea normal sporulation phenotype . A double mutant strain, LUL25, deficient in both YneN and StoA, showed the same low sporulation efficiency as LUL20 [Table 3] . These results demonstrate that YneN is not important for sporulation and indicate that StoA and YneN do not functionally overlap, i.e., YneN does not contribute to sporulation in a strain deficient in StoA . Strain LU60A1,which is deficient in CcdA, has 2 to 5% of wild-type sporulationefficiency [31] . Strain LUL21, deficient in both StoA and CcdA,showed approximately 10% sporulation efficiency compared toLUL20 [Table 3] . The additive effect of the two defects suggestthat StoA and CcdA act independently of each other, i.e., probablydo not function in the same pathway in spore formation.

Morphology of strains lacking StoA. The effect of StoA deficiency on the morphology of sporulatingcells was examined by light microscopy . LUL20 cells did notshow the characteristic bright light refraction seen for cellsof the parental strain 1A1 grown under the same conditions.Electron microscopic examination of 1A1 cells showed a clearspore cortex 24 h after the initiation of sporulation [Fig.2A and B] . LUL20 cells formed spores but without a visible cortex[Fig . 2C and D] . The electron-dense outer coat and the lamellarinner coat were seen in spores of both the mutant and the wildtype . The heat sensitivity of LUL20 spores can be explainedby the lack of the cortex layer, which is essential for heatresistance . The spore coat is the major protection barrier againstlysozyme and chloroform [7, 33] . A defect in the spore coatas indicated by the lysozyme and chloroform sensitivity of LUL20spores was not revealed by electron microscopy . The defect mightoriginate from the lack of cortex which serves as a supportbase for the synthesis of the spore coat . Strains defectivein cortex synthesis but with spore coat visible in electronmicroscopy images have been reported to be sensitive to lysozymeand chloroform [2, 3]

 
Electron microscopy showed that CcdA deficiency also affectsspore cortex synthesis [Fig . 3A] . Endospores of strain LUL21, which lacks both CcdA and StoA, contained inner and outer coat layers but appeared deficient in cortex [Fig . 3B] . Thus, both StoA and CcdA deficiency affect cortex synthesis, and endospores of strains LUL20, LU60A1, and LUL21 looked the same as judged from electron microscopy analysis . From the available data wecannot exclude the possibility that the lack of a visible cortexin the mutants is due to hydrolysis of cortex material . However,such hydrolysis seems unlikely since at 12 h after the onsetof sporulation spores of strain LUL20 lacked a visible cortexwhereas the wild-type control contained cortex.

 
The sporulation sigma factor cascade is normal in StoA-deficient strains. In two recent studies it has been shown that transcription of the ykvU-stoA operon is dependent on ^E [8, 11] . Genome-wideanalysis of mRNA extracted from cells harvested from culturesin early stationary phase [i.e., T₀ and T₂] by using DNA microarraysshowed no apparent difference between strains 1A1 and LUL20in upregulation of genes known to be under sigma factor ^F, ^E, or ^G control [array data not shown] . Genes dependent on ^K werenot assayed in the microarray experiment . To determine if StoA deficiency affects ^K-dependent gene transcription, expressionof a cotC-lacZ gene fusion integrated in single copy into theamyE locus was analyzed as before [31] . ß-Galactosidaseactivity measurements with this strain, LUL36, and the parentalstrain LUL35 showed that both the time point for induction duringsporulation and activity levels were the same for both strains[data not shown] . The results showed that the sporulation sigmafactor cascade is normal in StoA-deficient cells.

StoA is a membrane protein. The transmembrane topology of StoA was analyzed by using theN-terminal segment of StoA [residues 1 to 45] fused to E . colialkaline phosphatase [PhoA] lacking its native signal sequence.Alkaline phosphatase requires two disulfide bonds and is thereforeonly active in E . coli if it is transported to the outer sideof the cytoplasmic membrane . Lysates of E . coli BL21[DE3] cellsharboring plasmid pLLE64 [containing the stoA-phoA fusion] showedalkaline phosphatase activity {0.15 µmol/[min x [mg of protein]]} when expression was induced with IPTG, whereas lysatesof E . coli BL21[DE3] cells containing the vector pPHO1 showedno detectable activity {<0.01 µmol/[min x [mg of protein]]}.Furthermore, the alkaline phosphatase activity of BL21[DE3]/pLLE64was found to be associated with the particulate subfractionof cell lysates . From these results, and the predicted topology,we conclude that the N-terminal part of StoA functions as a membrane anchor and the C-terminal thioredoxin-like domain of StoA in B . subtilis is most likely exposed on the outer side of the cytoplasmic membrane.

Known oxidizing thiol-disulfide oxidoreductases are not required for efficient sporulation. BdbD/BdbC and the paralogous BdbA/BdbB system catalyze disulfidebond formation in proteins on the outer side of the membranein B . subtilis [4, 6, 10] . The BdbA/BdbB system seems specificallyinvolved in the maturation of the lanthionine sublancin 168.The BdbC/BdbD system is more generally involved in disulfide bond formation in extracytoplasmic proteins . The BdbD/BdbC and BdbA/BdbB systems are not important for sporulation, i.e., strains LUL3, LUL10, LUL110, and LUL27 showed normal sporulation efficiency [Table 3] . Thus, known oxidizing thiol-disulfide oxidoreductasesystems are not important for spore formation.

Effects of StoA deficiency are suppressed by BdbD deficiency. The sporulation defect caused by StoA deficiency was found tobe suppressed by inactivation of bdbD [strain LUL23; Table 3]. This indicated that StoA is a thiol-disulfide oxidoreductase with a reductive function . Addition of the reducing thiol reagent dithiothreitol [DTT] to the growth medium can overcome cytochrome c deficiency in strains lacking CcdA or ResA [9, 10] . However,inclusion of DTT in the growth medium [15 mM DTT in NSMP mediumadded at different time points during vegetative growth andsporulation] did not complement the sporulation defect of strainLUL20 and only slightly complemented the sporulation defectof strain LU60A1 [data not shown] . One reason for these negativeresults might be that DTT is unable to penetrate the cytoplasmicmembrane and thus will not reach the relevant location, i.e.,the space surrounding the engulfed forespore . Colonies lacking CcdA or StoA lyse on plates after a few days of incubation at room temperature . Microcolonies growing among lysed cells are observed after prolonged incubation at room temperature . Bacteria isolated from such microcolonies behave as wild-type cells onagar plates and show normal sporulation efficiency [31] [data not shown] . Mutations that suppress CcdA deficiency are located in the bdbC or bdbD gene [10] . The StoA deficiency suppressormutations have not been identified but are most likely in bdbCor bdbD.

StoA cannot complement ResA deficiency and StoA is not involved in cytochrome c synthesis. CcdA and ResA function in cytochrome c biogenesis by reducingthe heme binding site of apo-cytochrome c prior to covalentattachment of the heme cofactor [9, 10] . StoA and ResA show51% sequence similarity and 28% identity . Both proteins havethe thioredoxin-like domain anchored to the membrane by onetransmembrane segment constituted by the N-terminal part ofthe polypeptide . To investigate if StoA has any role in cytochromec synthesis, TMPD oxidation of colonies and cytochrome c oxidationactivity assay of isolated membranes were preformed on strains1A1 and LUL20 . Both assays test for the presence of functionalcytochrome caa₃ oxidase [9] . LUL20 showed the same cytochromec oxidase phenotype as 1A1 [data not shown] . Therefore, StoAis not involved in cytochrome c synthesis or assembly of theoxidase.

A strain lacking ResA [LUL9] remained TMPD oxidation negativewhen stoA was expressed in trans from the plasmid pLLE83 . StoA therefore does not complement ResA deficiency . Also, plasmid pLLE82 [containing resA] in LUL20 was unable to rescue the sporulation defect . This indicated that ResA cannot function as a substitute for StoA . Thus, ResA and StoA are either targeted to different subcellular locations and/or they have very different substrate specificity.

StoA has thioredoxin-like activity. The thioredoxin-like domain of StoA and BdbD were produced inE . coli TOP10 containing plasmid pLLE65 and pLLE34, respectively.The water-soluble His-tagged StoA and BdbD proteins were purifiedas described in Materials and Methods . In the in vitro insulinreduction assay where the rate of precipitation of reduced insulinis measured using a spectrophotometer, the water-soluble StoAshowed an activity of 7.5 x 10³ A₆₅₀/[min x mol] . Purified E.coli thioredoxin, used as a reference, showed an activity of30.8 x 10³ A₆₅₀/[min x mol] . Water-soluble BdbD showed no detectableactivity in this assay, as could be expected from the oxidativefunction of the protein . The results demonstrated that the water-solubledomain of StoA has thioredoxin-like activity.

YkvU is not an electron donor to StoA. The stoA gene is transcribed from a ^E-dependent promoter [8],which is only active in the mother cell, and StoA is seeminglyinvolved in cortex synthesis . Therefore, the thioredoxin-likedomain of StoA is probably localized in the intermembrane compartmentin the forespore during spore maturation . At this location theprotein is apparently involved in breaking disulfide bonds [Fig.4] . Reducing equivalents need to be transported to StoA fromthe mother cell or forespore cytoplasm to recycle StoA aftereach of its catalytic steps . In cytochrome c synthesis, ResAseems recycled by CcdA which transfers the reducing equivalentsrequired for disulfide bond breakage from the thioredoxin systemin the cytoplasm . As was mentioned, CcdA is unlikely to be an electron donor to StoA because StoA deficiency results in a 100-fold-stronger negative effect on sporulation efficiencythan CcdA deficiency [Table 3] . The ykvU gene upstream of stoA [Fig . 1] encodes a protein of unknown function but with sequencesimilarity to SpoVB, which is involved in cortex synthesis. YkvU has 12 predicted transmembrane segments and four cysteine residues . The protein seems selectively localized in the forespore outer membrane during spore synthesis as determined by usinga fusion to green fluorescent protein and microscopy of cells[8] . YkvU could be a reductase or a substrate protein for StoA.The sporulation efficiency of strain LUL30, with both ykvU and stoA deleted, was found to be lower than that for LUL20 [Table 3] . This indicated that YkvU might play some role in sporulation.Electron microscopy examination of endospores of strain LUL30showed that they were similar to LUL20 endospores, i.e., containedinner and outer spore coat layers but were deficient in cortex[Fig . 3C] . Strains LUL20 and LUL30 containing pLLE83 [whichcarries the stoA gene] showed approximately 1,000-fold-highersporulation efficiency than LUL20 and LUL30 containing onlythe plasmid vector, pDG148 [Table 5] . Thus, stoA on a plasmidcan complement the sporulation defect of strain LUL30 to thesame relative level as it complements that of LUL20 . YkvU istherefore probably not an electron donor to StoA . However, itcannot be excluded that YkvU is a cortex synthesis protein anda substrate for StoA because some other thiol-disulfide oxidoreductasemight partially complement for StoA deficiency.

 
B . subtilis StoA is a membrane-bound thiol-disulfide oxidoreductase important for spore maturation . Spores of StoA-deficient strains are heat, lysozyme, and chloroform sensitive and seemingly lack the cortex layer . The thioredoxin-like domain of StoA is probably present in the intermembrane space of the forespore and catalyzes disulfide bond breakage in cortex components or in proteinsthat are important for cortex synthesis [Fig . 4] . StoA is not required for efficient spore synthesis if BdbD is absent . BdbD together with BdbC constitute a membrane-bound system that catalyzes disulfide bond formation in proteins on the outer side of the cytoplasmic membrane . The BdbD/BdbC system is required for competence by catalyzing disulfide bond formation in two Com proteins [10, 24] but is not needed for spore synthesis . It therefore remainsuncertain whether disulfide bond formation catalyzed by proteinfactors is important for endospore synthesis . Our findings suggestthat BdbD catalyzes formation of disulfide bonds in some presentlyunknown substrate molecules resulting in spore cortex deficiency.StoA counteracts [or corrects] this effect of BdbD activityby breaking disulfide bonds . Such functional counteraction wouldbe analogous to the situation in cytochrome c synthesis whereBdbD catalyzes the formation of a disulfide bond in apo-cytochromec and ResA specifically breaks this bond [9].

 
We are grateful to Ingrid Stål for technical assistanceand Rita Wallén for the expert help with electron microscopy.We thank Fredrik Johansson for contributions regarding the constructionand analysis of strain LUL30.

This work was supported by a grant from The Swedish Research Council [621-2001-3125] to L.H.

 
* Corresponding author . Mailing address: Department of Cell and Organism Biology, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden . Phone: 046 [46] 2228622 . Fax: 046 [46] 2224113 . E-mail: Lars.Hederstedt@cob.lu.se.

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