Spores formed by wild-type Bacillus subtilis are encased ina multilayered protein structure [called the coat] formed bythe ordered assembly of over 30 polypeptides . One polypeptide[CotB] is a surface-exposed coat component that has been usedas a vehicle for the display of heterologous antigens at thespore surface . The cotB gene was initially identified by reversegenetics as encoding an abundant coat component . cotB is predictedto code for a 43-kDa polypeptide, but the form that prevailsin the spore coat has a molecular mass of about 66 kDa [hereindesignated CotB-66] . Here we show that in good agreement withits predicted size, expression of cotB in Escherichia coli resultsin the accumulation of a 46-kDa protein [CotB-46] . Expressionof cotB in sporulating cells of B . subtilis also results ina 46-kDa polypeptide which appears to be rapidly converted intoCotB-66 . These results suggest that soon after synthesis, CotBundergoes a posttranslational modification . Assembly of CotB-66has been shown to depend on expression of both the cotH andcotG loci . We found that CotB-46 is the predominant form foundin extracts prepared from sporulating cells or in spore coatpreparations of cotH or cotG mutants . Therefore, both cotH andcotG are required for the efficient conversion of CotB-46 intoCotB-66 but are dispensable for the association of CotB-46 withthe spore coat . We also show that CotG does not accumulate insporulating cells of a cotH mutant, suggesting that CotH [ora CotH-controlled factor] stabilizes the otherwise unstableCotG . Thus, the need for CotH for formation of CotB-66 resultsin part from its role in the stabilization of CotG . We alsofound that CotB-46 is present in complexes with CotG at thetime when formation of CotB-66 is detected . Moreover, usinga yeast two-hybrid system, we found evidence that CotB directlyinteracts with CotG and that both CotB and CotG self-interact.We suggest that an interaction between CotG and CotB is requiredfor the formation of CotB-66, which may represent a multimericform of CotB.

 
During the process of sporulation in the gram-positive soilbacterium Bacillus subtilis the developing spore is encasedin a complex protein structure called the coat, which confersresistance to several physicochemical agents and contributesto the response of spores to the presence of germinants [7, 8, 15] . The coat is formed by over 30 polypeptides, rangingin size from about 6 to about 70 kDa, which are assembled intoa lamellar inner coat and a thick electron-dense outer coat[7, 8, 15] . With only one possible exception [38], synthesis of the coat structural components is restricted to the mothercell chamber of the sporulating cell and is temporally governedby a cascade of transcription factors in the order ^E, SpoIIID, ^K, and GerE [7, 8, 15, 24, 35, 40]. ^E and SpoIIID drive synthesisof a class of morphogenetic proteins that [irrespective of theirassociation with the final coat structure] appear to guide theassembly of several structural components into the spore coat[reviewed in references 7, 8, and 15] . For instance, spores produced by a cotE mutant fail to assemble the electron-dense outer coat and the remaining coat structure appears to lack,in addition to CotE, several other abundant components [47]. The results of a recent study indicate that specific regionsin CotE are required for the assembly of different proteinsand suggest that CotE might control the assembly of severalouter-coat components by direct protein-protein interactions[2, 28] . Most of the coat structural components are synthesized [under the control of ^K and GerE] at a later stage in coat assembly,and it is only after ^K is activated that assembly of the coatis unequivocally recognized by electron microscopy of sporulatingcells [7, 8, 15] . Activation of ^K results in the expressionof several genes coding for spore coat proteins and also resultsin transcription of the gerE gene [4], which encodes an ambivalenttranscriptional regulator of coat gene expression . GerE actstogether with ^K to activate a late class of cot genes, but italso represses transcription of other cot genes [18, 19, 45,46] . These regulatory circuits suggest that the time and levelof expression of the genes coding for coat structural componentsare important for the correct assembly of the coat structure[7, 8, 15] . Proper assembly of the coat further relies on mechanismssuch as translational control [34] and posttranslational modifications,including proteolytical processing of larger precursors, proteinsecretion, and protein cross-linking [reviewed in references7, 8, and 15] . These modifications may provide an additionallevel of control over the timing of assembly of specific components.For example, SafA is a morphogenetic protein of about 45 kDaproduced under ^E control from hour 2 of sporulation onwardsbut the main form of SafA detected in the coats is a smaller[approximately 30 kDa] species corresponding to the C-terminalregion of the protein [32, 33] . This smaller species is producedby internal translation initiation [34] . In addition, the full-length and 30-kDa forms of SafA are processed by the YabG protease, which is produced under the control of ^K [42, 43] . The exact contribution of these mechanisms to the ordered assembly ofthe various coat components is poorly understood, and determinationof their nature and contribution will ultimately rely on thefunctional and structural characterization of selected components[11, 29] . To learn more about the mechanisms involved in the morphogenesis of the coat structure, we analyzed the assembly of the outer-coat protein CotB . The cotB gene was initially found [by reverse genetics] to encode an abundant spore coat component [6], later shown to be in the outer coat [47], whichappears to be surface exposed [20] . CotB has been utilized asa vehicle for the presentation of heterologous antigens at thespore surface, suggesting its potential use in vaccine development[9, 20] . Thus, the study of the assembly of CotB may allow amore precise manipulation of CotB as a fusion partner for heterologousantigen presentation . Also, it will expand our knowledge ofthe protein-protein interactions underlying assembly of a complex multiprotein structure and may provide us with tools for nanoengineering applications involving the B . subtilis spore . The cotB gene forms a cluster with two cot genes, cotH and cotG [6, 30, 36].Expression of cotH is under the control of ^K, whereas both cotGand cotB are expressed later under the dual control of ^K andGerE [18, 30, 36, 46, 45] . Assembly of CotB-66 was shown torequire expression of both cotG and cotH [30, 36].

We now show that cotB encodes a 46-kDa polypeptide [CotB-46] which is posttranslationally converted into a form of about66 kDa [herein called CotB-66] . This form of CotB [CotB-66]is equivalent to the 59-kDa protein previously reported by Donovanet al . [6] . We show that formation of CotB-66 requires both cotG and cotH and that CotG does not accumulate in a cotH mutant.This suggests that the requirement for CotH or a CotH-dependentprotein for CotB-66 formation results in part from its stabilizationof CotG . We also found that CotB is present in complexes withCotG at the time when formation of CotB-66 is detected . Moreover,CotB was found to interact with itself and with CotG in a Saccharomycescerevisiae two-hybrid assay . We suggest that formation of CotB-66requires a direct interaction with CotG.

 
Bacterial strains, media, and general techniques. The B . subtilis strains used in this study are listed in Table 1 . Sporulation was induced by nutrient exhaustion in Difco sporulationmedium [31] . Pfu polymerase [Stratagene] was used in all PCRs,and the cloned products were sequenced to ensure that no mutationswere introduced . All other general methods were as describedpreviously [5, 16, 17, 31, 38].

 
Integrational vectors. A fragment encompassing a chloramphenicol resistance [Cm^r] genewas generated by PCR using primers cat-958D and cat-2040R [Table 2] and plasmid pHV33 as the template [41] . The 1,080-bp PCRfragment was digested with EcoRI and PstI and inserted betweenthe same sites of pLITMUS 28 and pLITMUS 38 [New England Biolabs],creating the integrational vectors pMS38 and pMS39, respectively.

 
cotB, cotG, ywrJ, and cotH null mutants. DNA from strain DL067 [obtained from the Bacillus Genetic Stock Center] [6] was used to construct AH141 [cotB::cat; Table 1].The cotH [AH1103] and cotG [AH1497] mutants [Table 1] have beendescribed before [17, 48] . To create an insertion within the5' end of the cotB gene, a 395-bp PCR fragment [generated withprimers cotB-7311D and cotB-7706R] [Table 2] was cloned intopCR 2.1-TOPO [Invitrogen] to create pRZ28 . Next, the insertwas released with EcoRI and SphI and cloned between the samesites of pUS19 [3], yielding pRZ29 . The single-reciprocal-crossoverevent [Campbell-type recombination] of pRZ29 at the cotB locusof strain MB24 [verified by PCR] produced the spectinomycin-resistant[Sp^r] strain AH2055 . To disrupt the ywrJ gene, a 780-bp PCRproduct obtained with primers ywrJ-125D and ywrJ-913R [Table 2] was digested with EcoRI and XhoI and inserted into pET30a[+][Novagen] to yield pRZ47 . A Cm^r cassette was then released frompMS38 with HindIII and SnaBI and introduced between the HindIIIand DraI sites of pRZ47 to produce pRZ48 [Fig . 1] . pRZ48 wascut with NdeI and XbaI and used to transform strain MB24 toCm^r, producing AH2119 [Table 1].

 
Construction of strains carrying N- or C-terminal fusions of cotB to the six-His tag at the nonessential amyE locus. A 1,424-bp PCR product encompassing the cotB promoter and codingregion was generated with primers cotB-13D and cotB-1437R [Table 2] and cloned into pCR 2.1 TOPO to yield pRZ33 . Plasmid pRZ33served as a template to insert a six-His tag just downstream of the cotB initiation codon through the use of primers cotB-184Dhis and cotB-213RHis [QuickChange system; Stratagene] [Table 2].The modified cotB gene was released from the resulting plasmid[pRZ36] with EcoRI and HindIII and inserted between the samesites of the amyE integrational vector pMLK83 [21], therebycreating pRZ38 . A neomycin-resistant [Nm^r] transformant of strainMB24 with PstI- and ScaI-linearized pRZ38 that was AmyE^- [indicatinga marker replacement event at amyE] was identified and namedAH2088 [Table 1] . To create a 3'-end fusion of cotB to the six-Histag, a Cm^r cassette was released from pMS39 with SpeI and NheIand introduced into XbaI-digested pMS16 [see below] . This produced pRZ24, whose Campbell-type integration into the cotB locus of strain MB24 generated AH2036 [Table 1].

Construction of a strain expressing cotB from the cotE P1 promoter. The cotB coding region and translation initiation signals werePCR amplified with primers cotB-150D and cotB-1373R [Table 2].The resulting 1,233-bp fragment was cleaved with EcoRI and XbaIand cloned downstream of the cotE P1 promoter in an amyE integrationalvector [Costa and Henriques, unpublished results] derived frompMLK83 [21] . This produced pRZ37, which [following digestionwith ScaI] was used to transform strain MB24 with selectionfor Nm^r . A AmyE^- transformant was identified and named AH2089[Table 1].

Overproduction and partial purification of CotB. Primers cotB-226D and cotB-1345R [Table 2] were used to PCR amplify the 1,120-bp cotB coding region, and the product was cleaved with NdeI and XhoI and inserted between the NdeI andSalI sites of pET-30a[+] [Novagen] to yield pMS16 . Primers cotB-235Dand cotB-990R [Table 2] were used to PCR amplify a 756-bp fragmentcoding for the first 252 residues of CotB, which was cleavedwith EcoRI and inserted between the EcoRI and EcoRV sites ofpET-30a[+], creating pMS8 . pMS16 or pMS8 was introduced intoBL21[DE3] [pLysS] cells [Novagen], generating AH1834 or AH1816[Table 1], respectively, in which the full-length CotB [CotB-FL]or its N-terminal half [CotBn] could be synthesized as C-terminalfusions to the six-His tag under the control of the T7lac promoter. Overproduction and partial purification of the His-tagged proteins was essentially as described previously [38] . Both CotB-FL- and CotBn-6xHis were used for the production of a rabbit polyclonalantiserum [Eurogentec, Herstal, Belgium] . However, the antibodyraised against CotB-FL was not very specific and the anti-CotBnantibody was used in all experiments herein reported.

Generation of an anti-CotG antibody. A peptide [CDDYKRHDDYDSKKE] corresponding to residues 166 to180 of the CotG primary structure [36] was synthesized and conjugated to keyhole limpet hemocyanin, and the conjugate was used to raise rabbit polyclonal antibodies against CotG [Eurogentec].

Preparation of B . subtilis whole-cell extracts and immunoblotting. B . subtilis whole-cell lysates were prepared, and immunoblottingexperiments were conducted as described previously [39] exceptthat gels for sodium dodecyl sulfate-12.5% or -15% polyacrylamidegel electrophoresis [SDS-12.5% or -15% PAGE] were used [as indicatedin the figure legends] . Antibodies were used at the followingconcentrations: anti-CotBn, 1:2,000; anti-CotG, 1:20,000; andanti-His [Novagen] [monoclonal], 1:50,000 . Secondary anti-rabbitor anti-mouse antibodies [Sigma] were used at concentrationsof 1:10,000 or 1:5,000, respectively.

Spore purification and extraction of spore coat proteins. Spores were harvested 24 h after the onset of sporulation and purified by density gradient centrifugation as described previously[16, 17] . Proteins were extracted from purified spores and fractionatedon SDS-12.5% or -15% PAGE gels . The gels were stained with Coomassieblue and then transferred to nitrocellulose for immunoblottingor to polyvinylidene difluoride membranes for N-terminal sequenceanalysis [16, 17].

Ni²⁺-NTA affinity chromatography purification of complexes containing CotB-6xHis. Samples [100 ml] of Difco sporulation medium cultures of strainMB24 or AH2088 were collected at hour 8 of sporulation, andthe cells were collected by centrifugation and resuspended in1 ml of buffer A [1 mM NaH₂PO₄, 1 mM Na₂HPO₄, 50 mM 0.5% [wt/vol]NaCl, 2 mM phenylmethanesulfonyl fluoride] . Lysates were preparedby passage [at 19,000 lb/in²] through a French press, and celldebris was removed by centrifugation [7,000 x g for 10 min at4°C] . The clarified lysate was applied to a Ni²⁺-NTA [nitrilotriaceticacid] agarose column [Qiagen] preequilibrated with 8 volumesof buffer A . The column was washed with 3 volumes of buffer A, and proteins were eluted with increasing concentrations of imidazole [10, 25, 50, 50, 100, 250, and 500 mM] in buffer A. Proteins in the different elution volumes were resolved on SDS-15% PAGE gels and subjected to immunoblot analysis with anti-CotBnor anti-CotG antibodies.

Yeast two-hybrid analysis. The coding regions of cotB and cotG were PCR amplified withprimers B/5/Nco and B/OMO/3 and primers G/5/Nco and G/Bam/3[Table 2] . The cotB and cotG PCR products were digested with NcoI and BamHI and inserted between the same sites of both pAS2-1 and pACT2 [Clontech] . The resulting plasmids are indicated in Table 3 . The cotH coding sequence was obtained by PCR usingtwo different sets of primers, primers H/5/Nde and 3/H/Bam andprimers H/5/Bam and H/3/Xho [Table 2] . The cotH PCR productswere digested with NdeI and BamHI and inserted between the samesites of pAS2-1 or with BamHI and XhoI and fused to the GAL4activation domain [AD] in pACT2 [see Table 3] . Mating of S. cerevisiae strains and colony lift assays for detection of ß-galactosidase activity were performed as described previously [33].

 
cotB encodes a 46-kDa protein. The cotB gene was previously identified [by reverse genetics]as encoding a spore coat component of about 59 kDa [6] . Sporesfrom a cotB::cat insertional mutant [strain DL067] [Fig . 1] exhibited the wild-type pattern of Coomassie-stained coat polypeptides on SDS-PAGE except for the absence of a 59-kDa polypeptide [6]. Under our electrophoresis conditions, a band of about 66 kDa was absent from the coat of a mutant [AH141] bearing the samecotB::cat allele used by Donovan et al . [6] [Fig . 2, lane 2]or from spores of AH2055 [Fig . 2, lane 3, and Table 1], whichbears an insertion of pRZ29 into the 5' end of cotB [Fig . 1].In addition, the N-terminal sequence of the corresponding bandisolated from wild-type spores [Fig . 2, lane 1] was that deduced for the cotB product [data not shown] . In two recent studies in which mass spectrometry techniques were used to identify spore- or coat-associated polypeptides, the cotB-encoded polypeptide was estimated to be 66 kDa [26] or 62 kDa [27] . We infer thatthe 66-kDa protein [which we refer to as CotB-66] is equivalentto the protein found by Donovan et al . [6] . Under our conditions,the closely migrating CotA protein [6] runs slightly slowerthan CotB, as shown by its absence from spores of a cotA::catinsertional mutant [AH140] [6, 29] [Fig . 2, lane 4].

 
Inspection of the B . subtilis genome sequence reveals, however, that cotB is capable of encoding a polypeptide of 380 residues and about 43 kDa [25] . The disparity between the size of thecotB-dependent polypeptide associated with the coat and thatcalculated from the gene's sequence [43 kDa] could be due to abnormal electrophoretic mobility . Alternatively, CotB couldbe modified during its assembly into the spore coat . We reasonedthat if migration of CotB in the 66-kDa region of the gel weredue to its primary structure, CotB should still migrate at thisposition when produced in E . coli. Conversely, if the migrationof CotB at 66 kDa were due to a modification related to theprocess of coat assembly, then this alteration of CotB couldbe restricted to B . subtilis cells . To investigate this, wefirst cloned the cotB gene with a six-His tag at its 3' endunder the control of the T7lac promoter and introduced the resultingplasmid [pMS16] into cells of the E . coli host BL21[DE3] [Novagen]to produce strain AH1834 [Table 1] . During construction of theCotB-6xHis fusion, a leucine codon and a glutamate codon wereintroduced at the 3' end of cotB [before the six-His-encodingsequence], representing 1.082 kDa . Thus, the CotB-6xHis fusionprotein has a predicted molecular mass of about 44 kDa . Inductionof AH1834 with IPTG [isopropyl-ß-D-thiogalactopyranoside] resulted in the production of polypeptides of about 46 kDa that accumulated as inclusion bodies . The 46-kDa polypeptide was solubilized in a urea-containing buffer and was partially purifiedby affinity chromatography on Ni²⁺ columns [Fig . 2, lane 6].The N-terminal sequence [MSKRRMKY] confirmed that it derived from the cotB gene . When expressed in E . coli, therefore, cotBencodes a protein whose apparent molecular mass [46 kDa] is in good agreement with the size [43 kDa] predicted from the sequence of the gene [25] . To directly compare the size of CotB-6xHismade in E . coli to the size of the cotB-derived polypeptidethat associates with the B . subtilis spore coat, we made useof strain AH2036 [Table 1], which resulted from the Campbell-type integration of pRZ24 and expressed a CotB-6xHis fusion underthe control of the cotB promoter . Addition of the six-His tagat the 3' end of CotB did not prevent its assembly into thecoat structure [Fig . 2, lane 5] and did not significantly changethe size of the protein associated with the coat relative tothat of the wild-type protein [Fig . 2; compare lanes 1 and 5].We also note that an anti-six-His tag antibody reacted witha 66-kDa band in AH2036 spore coat extracts but not in extractsprepared from the wild-type strain MB24 [data not shown], suggestingthat the C-terminal region of CotB is represented in the CotB-66form.

The difference in size between the protein produced in E . coli and the polypeptide that accumulates in the B . subtilis spore coat prompts us to suggest that the form of CotB that prevails in the spore coat [CotB-66] bears a modification that increasesits mass by about 20 kDa.

CotB is synthesized as a 46-kDa species [CotB-46] during sporulation and is converted to a 66-kDa form [CotB-66]. To investigate whether CotB could also accumulate in B . subtilisas a polypeptide of about 43 to 46 kDa before being convertedto the CotB-66 form, we used a polyclonal antiserum to followits accumulation during sporulation . In whole-cell extractsprepared from strain MB24 cells harvested at hour 6 of sporulation[but not in those from the hour-4 sample], the antibody detecteda species of approximately 46 kDa [Fig . 3A] . This species hasabout the same size as the CotB-6xHis protein produced in E. coli [in good agreement with the predicted size of CotB] . In the experiment documented in Fig . 3, this form of CotB [hereinafterdesignated CotB-46] was not detected in extracts prepared athour 8 or 10 of sporulation or in spore coat extracts [Fig.3A] . In addition, the antibody detected a 66-kDa species inhour-6 extracts, presumably CotB-66 [see above] [Fig . 3A] . Incontrast to the results seen with CotB-46, the cellular levelof CotB-66 increased from hour 6 to 8 and was the main formof CotB detected at hour 10 of sporulation or in purified coatmaterial [Fig . 3A] . The exact temporal profiles with which bothCotB-46 and CotB-66 could be detected differed to some extent.In the experiment whose results are presented in Fig . 4A, forexample, CotB-46 could still be detected at later times duringsporulation whereas CotB-66 was only detected from hour 8 onwards.In any case, CotB-66 tended to predominate at later time pointsand was the major form of CotB detected in purified coat material.None of the CotB forms were detected in whole-cell extractsor in spore coat extracts prepared from sporulating cells orspores, respectively, of the AH141 [cotB::cat] or AH2055 [cotB::sp]mutants [data not shown].

 
The temporal pattern of accumulation of both the 46- and 66-kDa polypeptides is consistent with the time of expression of cotB during spore development which is induced around hour 6 underthe joint control of ^K and GerE [18, 46] . Recent results indicate that cotB expression may also be governed by the early mother cell-specific regulator ^E [10] . However, this early expressionof CotB was not detected under our conditions . Our results suggestthat CotB is synthesized around hour 6 of sporulation as a 46-kDapolypeptide [CotB-46] which rapidly undergoes a posttranslationalmodification to yield a 66-kDa form [CotB-66] which accumulatesin the spore coat.

Synthesis and modification of CotB can be uncoupled in sporulating cells. To independently test whether CotB could be synthesized and accumulate as a 46-kDa form in B . subtilis, we placed the cotB coding region under the control of the strong cotE P1 promoter [P_{cotE P1}], which is utilized by the ^E-containing form of RNApolymerase [46] . We reasoned that the putative modificationof CotB could depend on other late-expressed genes and thatthe expression of cotB at sufficient levels prior to the onsetof ^K activity could confirm the size of unmodified CotB in B.subtilis . The P_{cotE P1}-cotB fusion was inserted at the amyElocus in a cotB null mutant, and the accumulation of CotB wasmonitored in AH2089 by immunoblot analysis throughout sporulationand in purified coat material . Control experiments have shownthat the insertion of cotB at the amyE locus resulted in theproduction and assembly of CotB in a manner indistinguishablefrom that seen with the wild-type strain MB24 [data not shown].When cotB was expressed from the cotE P1 promoter, CotB wasdetected as a 46-kDa species from hour 4 of sporulation onwards[Fig . 3B].

Thus, as seen with E . coli cells, cotB seems to encode a proteinof about 46 kDa . Even though cotB was expressed at least 2 hearlier than normally, interestingly, CotB-66 was detected onlyfrom hour 6 of sporulation onwards, as seen with the wild-type strain MB24 [Fig . 3A] . We also found in the coats of AH2089at least two relatively abundant bands of about 40 and 34 kDa, which we interpret as stable degradation forms of CotB [Fig. 3B and 4B] . Donovan et al . [6] also described a coat-associatedpolypeptide of 34 kDa whose N-terminal sequence matched thatof CotB . Inspection of the B . subtilis genome sequence indicatesthat this polypeptide cannot be the product of a separate gene[25] . Since the original insertion into cotB [close to its 3'end] [Fig. 1] did not prevent its accumulation, the 34-kDa form must derive from the N-terminal portion of the CotB proteinas initially hypothesized [6] . Presumably this form of CotB represents a stable degradation product of higher-molecular-mass forms of CotB . Spores of AH2089 [P_{cotE P1}-cotB] show wild-typeresistance to heat and lysozyme, and except for the differencesmentioned above [which were detected by immunoblot analysis],the collection of extractable coat polypeptides does not differfrom that of wild-type spores as judged by Coomassie staining [data not shown] . However, while spores of the cotB::sp mutant AH2055 are not impaired in their ability to germinate in response to L-alanine [as also reported for a strain bearing the cotB::catallele] [6], AH2089 spores are slow to germinate upon exposureto L-alanine [data not shown] . This suggests that the timingof the expression of cotB is important for the spore's responseto the germinant L-alanine.

Transcription of cotB normally requires both ^K and GerE [18,46] . That expression of cotB is not sufficient for formationof CotB-66 suggested that at least one additional late geneis needed for the formation of CotB-66 . We cannot exclude thatformation of CotB-66 involves a factor synthesized early inthe mother cell line of gene expression [under the control of ^E], but in any case CotB-66 is only formed late, coincidentlywith the onset of ^K-directed gene transcription . Thus, irrespectiveof the involvement of early gene products, formation of CotB-66appears to be directly or indirectly dependent on the productsof late, ^K-dependent genes or morphogenetic events . We alsonote that in the P_{cotE P1}-cotB strain, CotB-46 accumulates atlater times during sporulation and undergoes assembly into thespore coat [Fig. 3B] . It appears that in the P_{cotE P1}-cotB strain,CotB-46 cannot be completely converted into the 66-kDa form. Alternatively, the protein at 46 kDa that accumulates from hour 6 onwards may result from degradation of CotB-66 . In any event,we infer that formation of CotB-66 is not a strict requirementfor assembly of CotB [see also below].

Formation of CotB-66 requires expression of both cotG and cotH. Previous work has shown that assembly of CotB [CotB-66] into the spore coat requires expression of both the cotG and cotH loci [30, 36] . The cotG gene is found just upstream and divergentlyoriented with respect to cotH, whereas cotB immediately followscotH [25, 30, 36] [Fig . 1] . Both the cotG and cotH genes areunder the control of ^K, but the expression of cotG further dependson gerE [30, 36] . To determine whether cotH or cotG was requiredfor the modification of CotB, we used our polyclonal antibodyto monitor the synthesis of CotB in cotH and cotG null mutants[AH1103 and AH1497, respectively] [Table 1] . In contrast tothe results seen with the wild type [Fig. 4A], we found CotB-46to be the predominant form of CotB in whole-cell extracts ofa cotG mutant prepared 6, 8, or 10 h after the onset of sporulation[Fig . 4B] . We found CotB-66 [or a species of approximately thesame size] to accumulate transiently at hour 8 in the cotG mutantAH1497, but even in this sample CotB-46 was more abundant [Fig. 4B] . At present we do not know whether this species is the sameas the CotB-66 form seen in the wild type [see also Discussion]. Moreover, CotB-46 was the predominant form of CotB found in coat extracts prepared from cotG spores [Fig . 4B] . Several degradationproducts of CotB were found to accumulate in cells or sporesof the cotG mutant [Fig . 4B] . We then investigated the accumulationof CotB in sporulating cells and in the coats of the cotH mutantAH1103 [Table 1] . The results presented in Fig . 4C show that[together with what appear to be degradation products] only CotB-46 is detected in whole-cell extracts of sporulating cells or in material purified from the coats of cotH mutant spores. Therefore, expression of cotH is also a requirement for the formation of CotB-66 . However, CotB-66 was never detected inthe cotH mutant, suggesting that cotH and cotG might have differentroles in the formation of CotB-66 . In any case, the results implicate both cotH and cotG in the formation of CotB-66 . We cannot ascertain whether cotH and cotG are also involved in the assembly of CotB-66, but we note that the expression of neither loci is required for the association of CotB-46 with the coat structure.

The cotB gene is followed by ywrJ [25] [Fig. 1], and the twogenes appear to be cotranscribed during sporulation [10] . Totest whether ywrJ had any role in spore coat assembly, we createda ywrJ deletion mutant [AH2119] [Table 1] by a marker replacement event involving plasmid pRZ48 [Fig . 1] . We found that disruptionof ywrJ did not cause any detectable effect on the coat polypeptidecomposition, heat or lysozyme resistance of the resulting spores,or accumulation of CotG or CotB-66 [data not shown] . Thus, thefunction of the ywrJ gene remains unknown.

CotH is required for the accumulation of CotG. The need for both cotH and cotG for the formation of CotB-66could indicate that the two loci act together or could reflecta hierarchical control of one locus over the other . To beginto investigate these questions, we first tried to overproduceCotG in E . coli for antibody production . In spite of many attemptsto overproduce CotG as N- or C-terminal fusions to the six-Histag or to partners such as maltose binding protein or glutathioneS-transferase, we never observed accumulation of the fusionproteins in E . coli [data not shown] . We therefore decided toraise a rabbit polyclonal antibody against a peptide derivedfrom residues 166 to 180 of the CotG primary structure [seereference 36 and Materials and Methods] . The cotG gene codesfor a protein with 195 amino acid residues with a predictedmolecular mass of 24 kDa [36] . However, CotG is seen mainlyas a wide, diffuse band of about 36 kDa in spore coat gels [Fig.1; see also below] [17, 30, 36] . The discrepancy between the predicted size and the observed size of CotG has been attributed to its unusual primary structure, which bears a central region of 117 amino acids organized in nine repeats of a 13-amino-acid sequence motif [36] . Using the anti-peptide antibody, we detectedCotG as diffuse bands of about 32 and 36 kDa in whole-cell extractsfrom hour 8 of sporulation onwards [Fig . 5, lane 8 [wild-typestrain results]] . Migration of the 36-kDa species appears tocoincide with that of the 36-kDa cotG-dependent band seen inCoomassie-stained coat gels [Fig . 5, lane SpC [wild-type strainresults]] [17, 30, 36] . The 32-kDa band, which is the band thatis the closest in size to the expected size of the cotG-encoded protein [24 kDa], also coincides with a band seen in the Coomassie-stained gel of the same coat protein extract [Fig . 5, lanes Sp and SpC[wild-type strain results]] . Additional higher-molecular-mass forms of CotG [of about 70 and over 100 kDa] were found in hour-10 whole-cell extracts [Fig . 5] . In purified coat material, fivemain forms [around 32, 36, 50, and 70 and over 100 kDa] of CotG were detected [spore coat extract from wild-type spores] [Fig. 5] . Presumably, some of these species are the same as those detected in hour-10 extracts . The antibody also revealed several less-distinct CotG forms between the 100-and 32-kDa species found in spore coat extracts [Fig . 5], which could have been caused by extensive cross-linking of CotG or proteolysis ora combination of the two processes . None of the bands seen in immunoblotting analysis of wild-type cell lysates or coat material were detected in extracts of a cotG insertional mutant, demonstrating the specificity of the antibody [data not shown] . We then examined the accumulation of CotG in a cotH mutant . Unexpectedly, we did not detect any forms of CotG in whole-cell lysates prepared from sporulating cultures or in material extracted from purified spores of the cotH mutant AH1103 [Fig . 5, lanes 6 to 10 andSp [cotH::cat mutant results]] . Analysis [by SDS-PAGE and Coomassiestaining] of the profiles of proteins extracted from AH1103spores [Fig . 5, lane SpC [cotH::cat mutant results]] revealedthe pattern expected for the cotH mutant [30] . Since the cotH::cat mutation does not affect transcription of the cotG gene [30], these observations strongly suggest that CotG is unstable and that CotH or a protein controlled by CotH is required for the accumulation of CotG . In any case, the need for cotH expression for the formation of CotB-66 may derive in part from its requirement for the accumulation of CotG . The possibility that CotH has,in addition, a more direct role in formation of CotB-66 is notexcluded.

 
CotB and CotG form complexes in vivo. The observation that cotH and cotG promote formation of CotB-66prompted us to investigate possible associations in vivo . Weused a B . subtilis strain expressing a CotB-6xHis fusion proteinat the amyE locus [strain AH2088] to purify [by affinity chromatography]complexes containing CotB . We prepared whole-cell extracts fromAH2088 cells at hour 8 of sporulation and applied the extractto a Ni²⁺-NTA agarose column [Fig. 6] . The column was firstwashed and was then eluted with increasing concentrations [from10 to 500 mM] of imidazole [see Materials and Methods] . Theproteins were electrophoretically resolved, and the presenceof CotB or CotG in the various fractions was assessed by immunoblotanalysis . We do not presently have a good antibody against CotHand could not test whether this protein also copurified withCotB . We found that most of the CotB present at hour 8 of sporulationdid not bind to the Ni²⁺-agarose column and that it was detectedin the column flowthrough [Fig. 6A, lane 1] . Part of this CotBis likely to result from expression of the wild-type cotB genein AH2088 [Table 1], suggesting that untagged CotB is not retained by the column . To verify this, we prepared a whole-cell lysatefrom a sporulating culture of strain MB24 at hour 8 of sporulation.The lysate was applied to a Ni²⁺-NTA column, and the column was eluted with a step gradient of imidazole at concentrationsup to 500 mM . No CotB was found in the elution fractions byimmunoblot analysis . We conclude that under these conditions,untagged CotB is not retained by a Ni²⁺ column [data not shown].In strain AH2088 [CotB-6xHis], some CotB did bind to the columnand was eluted at imidazole concentrations of 50 mM [lane 3]and 100 mM [lane 4] . Most of the fusion protein retained by the affinity column was the 46-kDa form [Fig . 6A, lanes 3 and4] . Very little CotB-66 was found in the 50 or 100 mM imidazole fractions, but this form of the protein appeared to be slightly more abundant in the 50 mM fraction [Fig . 6A, lanes 3 and 4]. No CotB was detected upon elution of the column with buffers containing higher imidazole concentrations [up to 500 mM; datanot shown].

 
We then subjected samples of the same fractions to immunoblot analysis using an anti-CotG antibody [Fig . 6B] . We found that a fraction of CotG present in whole-cell extracts prepared at hour 8 of sporulation [and that included at least two forms[about 36 and 70 kDa] of the protein] was not retained by thecolumn [Fig. 6B, lane 1] . However, CotG [mainly a form of about 36 kDa] also coeluted with CotB-46 at an imidazole concentrationof 100 mM [Fig . 6B, lane 4] . No other column fractions [at imidazoleconcentrations up to 500 mM] contained CotG . Control experimentsusing the wild-type strain MB24 [Table 1] demonstrated thatCotG was not retained by the column when CotB did not carrya six-His tag [Fig . 6B, lane 2, and data not shown] . We do notpresently know why CotB elutes at two different imidazole concentrationsor why CotG only coelutes with CotB at the higher [100 mM] imidazoleconcentration . Presumably, CotB exists in several forms thatinteract differently with the column . In any case these resultsindicate that CotB copurifies with CotG, and we infer that CotB[perhaps mostly as CotB-46] and CotG are present in complexesin vivo.

CotG and CotB directly interact. Since CotG and CotB appeared to form complexes in sporulatingcells, we wanted to investigate whether the two proteins couldinteract directly . In addition, we wanted to test whether CotHcould interact with either CotG or CotB . Interactions amongCotH, CotG, and CotB were tested in vivo using a yeast two-hybridsystem [12, 13, 14] . We fused the entire coding sequence ofcotB, cotH, or cotG to either the AD in pACT-2 or the DNA-bindingdomain [DNA-BD] in pAS2-1 of the yeast transcriptional activatorGAL4, and combinations of the fusion plasmids and/or vectorswere introduced into appropriate yeast reporter strains [33][Table 3] . Interaction of the fusion proteins in the yeast strainsresulted in the expression of a lacZ reporter gene and was detectedby a colony lift assay [see Materials and Methods] . As shownin Table 3, no ß-galactosidase activity was detectedwhen individual fusions were expressed with the correspondingcontrol vector . In addition, background levels [in yeast cellscarrying both vectors] were found to be negligible [Table 3]. Using this system, we detected interactions between CotB anditself and between CotG and itself . Additionally, we detectedan interaction between CotB and CotG [Table 3] . All of the interactions were found when the involved proteins were fused to either the GAL4 AD or the GAL4 BD [Table 3] . In contrast, we did not find evidence for an interaction between CotH and either CotG, CotB, or itself . These results suggest that CotB is capable of associating with itself and also with CotG and that CotG is also capableof self-association.

 
We found that cotB encodes a polypeptide of about 46 kDa [CotB-46] that [soon after synthesis around hour 6 of sporulation] undergoes a posttranslational modification that converts it into a 66-kDa form [CotB-66] . The CotB-66 polypeptide is the form of CotB previously found in the spore coat by Donovan et al . [6] . Underour conditions, CotB-66 is the main form of CotB present in the spore coat . That cotB codes for a 46-kDa protein is supported by two lines of evidence . First, expression of cotB in E . coli results in the accumulation of a 46-kDa polypeptide . Second, under genetic conditions in which synthesis of CotB in B . subtilis was artificially induced from the early ^E-dependent cotE P1promoter [46] a 46-kDa polypeptide accumulated from hour 4 ofsporulation onwards . Conversion of CotB-46 into CotB-66 in theP_{cotE P1}-cotB strain only occurred around hour 6 of sporulation,as seen with the wild type, indicating the involvement of other,late-expressed genes in the modification of CotB-46 . Indeed,our results indicate that the expression of both cotH [whichis driven by ^K] [30] and cotG [which requires ^K and GerE] [36]is needed for the formation of CotB-66 . The data also indicatethat the involvement of cotH and cotG in CotB-66 formation resultsin part from a hierarchical sequence of events . In fact, CotGdoes not accumulate in the absence of CotH, suggesting thatthe latter is an unstable protein and that CotH or a CotH-dependentfactor is somehow needed to stabilize CotG . Even though we didnot find evidence for a direct interaction between the two proteinsin a yeast two-hybrid system, CotH might act as a chaperone[binding directly to CotG] to promote its stabilization . Wenote that CotH is found only in minute amounts in the finalcoat structure [48], whereas CotG accounts for a significantproportion of the protein extractable from the coat of wild-typespores [7, 15] . Chaperones play essential roles in the macromolecularassembly of other biological structures [see, for example, references 1 and 37] but have never been proposed to play such roles inthe context of the assembly of the bacterial endospore coat.An alternative explanation for the role of CotH is that CotHor a CotH-dependent protein acts to inhibit a protease that uses CotG as a substrate.

Previous work has shown that in addition to CotH, CotG and CotB are absent from cotH spores [30] . In contrast, in addition toCotG, only CotB [CotB-66] is missing from cotG spores [36].The cotH-dependent stabilization of CotG helps to explain itsrequirement for the assembly of CotG, whereas the cotG-dependentassembly of CotB is in part explained by the role of CotG inCotB-66 formation [Fig. 7] . We do not know whether CotG is directly recruited by CotH to the coat structure or whether the assemblyof CotB-66 is directly controlled by CotH or CotG [Fig . 7]. However, the observation that CotB-46 accumulates in the coats of cotH or cotG mutants indicates that the assembly of at least this form of the protein is independent of the presence of CotH and CotG . Lastly, we note that CotB-66 is detected transientlyin the cotG mutant but not in cotH cells, suggesting that other than its role in the stabilization of CotG, CotH might serve yet another role in the formation of CotB-66 . However, it isalso possible that this band is a cross-reactive species thatonly accumulates in a cotG mutant.

 
CotG is first detected around hour 8 of sporulation as two main antigenic forms of about 32 and 36 kDa [Fig . 5] . The 32-kDa form could represent the unmodified product of the cotG gene [24 kDa] whose abnormal migration may be attributed to its unusual primary structure [36] . CotG appears to be converted into formsof about 36 and 70 kDa in sporulating cells and then to be subjectedto extensive cross-linking as it is assembled into the sporecoat . That CotG is able to form cross-linked multimers has been previously suggested on the basis of the results of analysisof the coat structure in sodA mutant cells [17] and is supportedby the results of the yeast two-hybrid assay [this work] . A more detailed characterization of the synthesis and assemblyof CotG is currently under way.

Our finding that CotG copurifies with CotB strongly suggeststhat the two proteins are present in complexes at the time incoat assembly during which formation of CotB-66 is detected.In that respect it is noteworthy that CotG [a 36-kDa form ofthe protein] appears to copurify mainly with CotB-46 . On thebasis of the results of the yeast two-hybrid experiments, itis tempting to suggest that CotG may directly interact withCotB-46 during assembly of the spore coat to promote formationof CotB-66 [Fig . 7] . It is unknown whether CotH also copurifiesor interacts with CotB, just as it is unknown whether the presenceof CotG and CotH is sufficient to promote the modification ofCotB . Spores produced by cotB and cotG mutants are not affectedin their resistance to lysozyme or germination properties [6, 36] . Therefore, the significance of the interaction between CotB and CotG is presently unclear and may only be apparent under conditions that have not yet been identified.

The nature of the posttranslational modification of CotB remains unclear . On the basis of the size difference from the unmodifiedform and of the observation that the CotB-66 form presents onlyone N-terminal sequence [6; this work], the easiest explanation is that CotB-66 is a cross-linked homodimer with irregular mobility [the expected size would be of 92 kDa [in contrast to the observed size of 66 kDa]] . It is also possible that CotB-66 is a homodimer cleaved near the C terminus of the protein, but, if so, our observation that a C-terminal six-His sequence is present inthe 66-kDa form indicates that at least one of its componentsis the full-length protein . Our finding that CotB is capableof self-interaction in a yeast two-hybrid system supports thesuggestion that CotB undergoes multimerization . Alternatively,CotB-66 is a heterodimer, perhaps containing CotG [given thatthe two proteins appear to interact]; in that case, however,either the cross-linking involves the N terminus of the secondcomponent or its N-terminal sequence is blocked and refractoryto analysis . Several types of covalent cross-links have beenproposed or detected in the coat . These include disulfide bridges[note, however, that cotB does not code for any cysteine], -glutamyl-lysil isopeptide bonds mediated by a coat-associated transglutaminase [see, for example, references 16, 22, 23, and 44], or o,o-dityrosine cross-links [reference 17 and references therein] . Work in progressaims at determining the nature of the posttranslational modificationof CotB and its structural significance as well as the proteincomponents involved and their roles.

 
We thank T . Barbosa for critically reading the manuscript, J.Pohl [Emory Microchemical Facility] for N-terminal sequence determinations, and A . J . Ozin for help with the yeast two-hybrid experiments.

This work was supported by European Union grant QLK5-CT-2001-01729 to E.R . and A.O.H., by MIUR [Cofin 2002 and FIRB 2002] grantsto E.R., and by grants GM54395 to C.P.M . and PRAXIS/PCNA/C/BIA/129/96to R.Z . M.S . holds a Ph.D . fellowship [PRAXIS XXI/BD 18 251/98]from the F.C.T.

 
* Corresponding author . Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras Codex, Portugal . Phone: 351-21-4469521 . Fax: 351-21-4411277 . E-mail: aoh@itqb.unl.pt.

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