Journal of Bacteriology, March 2004, p . 1462-1474, Vol . 186, No . 5

Assembly of an Oxalate Decarboxylase Produced under ^K Control into the Bacillus subtilis Spore Coat

Teresa Costa,¹ Leif Steil,^2,3,4 Lígia O . Martins,^1,5 Uwe Völker,^2,3,4 and Adriano O . Henriques^1*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras Codex,¹ Universidade Lusófona de Humanidades e Tecnologias, Departamento de Engenharias e Tecnologias, 1749-024 Lisbon, Portugal,⁵ Laboratory for Microbiology, Department of Biology, Philipps University of Marburg, D-35032 Marburg,² Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg,³ Laboratory for Functional Genomics, Medical Faculty, Ernst Moritz Arndt University, D-17487 Greifswald, Germany⁴

Received 18 August 2003/ Accepted 25 November 2003

 
Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat . The coat consists of three main layers,an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-densestriated outer layer . The product of the B . subtilis oxdD genewas previously shown to have oxalate decarboxylase activitywhen it was produced in Escherichia coli and to be a spore constituent.In this study, we found that OxdD specifically associates withthe spore coat structure, and in this paper we describe regulationof its synthesis and assembly . We found that transcription ofoxdD is induced during sporulation as a monocistronic unit underthe control of ^K and is negatively regulated by GerE . We alsofound that localization of a functional OxdD-green fluorescentprotein [GFP] at the surface of the developing spore dependson the SafA morphogenetic protein, which localizes at the interfacebetween the spore cortex and coat layers . OxdD-GFP localizesaround the developing spore in a cotE mutant, which does notassemble the spore outer coat layer, but it does not persistin spores produced by the mutant . Together, the data suggestthat OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy alleleof oxdD resulted in production of spores with increased levelsof OxdD that were able to degrade oxalate but were sensitiveto lysozyme.

 
Bacterial endospores are designed to withstand long periodsof dormancy and to resist physical and chemical conditions thatwould rapidly destroy vegetative cells . This extreme endurancecan be attributed to several factors, including the compositionand structural organization of the layers that surround themature spore core [10, 19, 36] . In all endospore formers, thespore core is surrounded by a thick modified peptidoglycan calledthe cortex, which is a key element in heat resistance [36].The cortex is covered by a multilayer protein coat, which confersresistance to noxious chemicals and to peptidoglycan-breakingenzymes, such as lysozyme [10, 19, 36] . In addition, the coat contributes to the ability of the spore to monitor its environment and to initiate germination upon proper stimulation [10, 19,36] . In Bacillus subtilis, the spore coat is composed of over30 polypeptides, which are organized into three main layers,an amorphous undercoat, a lamellar inner coat, and an electron-densestriated outer coat [2, 10, 13, 19, 28, 29] . Assembly of thecoat is initiated soon after the asymmetric division that partitionsthe sporulating cell into a larger mother cell and a smallerforespore . The early events in coat assembly are controlledby the mother cell-specific RNA polymerase sigma factor ^E [16,26, 42] . Several of the proteins whose synthesis is driven by ^E have morphogenetic roles; i.e., irrespective of their association with the final structure they act by laying down an imprintthat prepares the surface of the developing spore for the orderedassembly of the coat structural components [10, 19].

SpoIVA, CotE, SpoVID, and SafA are morphogenetic proteins whose synthesis is under the control of ^E [5, 39, 44, 48, 50, 57]. SpoIVA localizes along the asymmetric division septum, and afterthe engulfment of the forespore by the mother cell, it encirclesthe forespore protoplast close to its outer membrane [11] . SpoIVA is required for assembly of CotE as a ring-like structure about 75 nm from the spore outer membrane . The space between SpoIVAand CotE is presumably filled with a scaffold or matrix andlater becomes the inner coat region, whereas the CotE ring itselfappears to serve as the nucleation site for outer coat assembly[11] . Accordingly, a cotE mutant forms spores that retain someinner coat but are devoid of an outer coat and are lysozymesensitive [11, 56] . The localization of SpoVID at the surfaceof the developing spore also requires SpoIVA, but it is CotEindependent [11, 40] . SpoVID is not required for formation ofthe CotE ring, but it is needed for maintenance of this ringaround the forespore at later stages of coat assembly [11]. An absence of SpoVID leads to misassembly of the coat as swirls of material dispersed throughout the mother cell cytoplasm andin lysozyme-sensitive spores [5] . SpoVID, but not CotE, is also required for the targeting of SafA to the spore surface [40]. SafA has a cell wall-binding motif at its N terminus and has been shown by immunogold labeling to localize to the cortex-coat interface [39] . A safA mutant forms spores that are deficientin lysozyme resistance and germination [39, 50] . Since SafAand SpoVID directly interact, it has been proposed that SafAmay act as a bridge between the cortex and coat structures [39,40].

It is only at a later developmental stage, after engulfmentof the forespore by the mother cell, that assembly of the coatstructure becomes apparent by electron microscopy [10, 19].Conclusion of the engulfment process triggers activation of the late mother cell regulator ^K, which replaces ^E in the mothercell line of gene expression [16, 26, 42] and drives expression of most of the genes that code for coat structural components[10, 19] . Expression of the coat structural genes is additionally modulated by the action of the transcriptional regulator GerE[3, 17, 22, 23, 45, 49, 54, 55, 57] . Spores of a gerE mutant lack the ultrastructural features normally associated with the inner coat layers [10, 33] . In addition, they are deficientin expression of several genes encoding prominent componentsof the outer coat, such as the CotC and CotG proteins [9, 45,57].

The roles of most individual coat structural proteins in the assembly and function of the spore coat are unclear, as null mutations often do not have a measurable phenotypic effect [10, 19] . This suggests that there is extensive redundancy or thatthe various components make minor contributions to the structureand function of the coat layers [10, 19] . Nevertheless, someof the coat proteins are enzymes or exhibit sequence similarityto enzymes that suggest that they are involved in the assemblyprocess or in the final spore attributes . Ultimately, a descriptionof the assembly process that also accounts for the coat propertieswill require detailed functional and structural characterizationof selected components . For example, the CotA protein [9], acomponent of the spore outer coat layers [56], was recently shown to be a highly thermostable laccase involved in spore resistance to UV light and hydrogen peroxide [21, 32] . The crystalstructure of CotA was determined, in anticipation of the possibilitythat it could serve as a platform for detailed analysis of themechanism underlying the assembly and function of CotA in thespore coat [14].

In this study, we were concerned with the regulation of expression and assembly of the product of the yoaN [oxdD] gene, which, when overproduced in Escherichia coli, was shown to have Mn-dependentoxalate decarboxylase activity [52] . Oxalate decarboxylases[EC 4.1.1.2] convert oxalate to formate and CO₂ [12, 51, 52]. The best-characterized enzymes are enzymes that have a fungal origin, are induced by oxalate, and appear to control excessive concentrations of oxalate [12] . The first oxalate decarboxylaseto be identified in a prokaryote was the acid-inducible OxdCenzyme from B . subtilis, which may have a role in proton consumptionwithin the cytoplasm [51] . OxdC belongs to the cupin superfamily,whose members contain a ß-sandwich domain consistingof one six-strand ß-sheet and one five-strand ß-sheet[1, 12, 51] . OxdC is a homohexameric enzyme in which each monomerhas two cupin ß-barrel domains, and hence it belongsto the bicupin subclass of the cupin superfamily [1, 12, 51].OxdD is very similar to OxdC and to known oxalate decarboxylases[51] . No role has been reported for OxdD . However, both OxdDand OxdC were found in a recent proteomics-based study to bespore-associated proteins [28] . Here, we show that OxdD is specificallyassociated with the spore coat . We found that transcriptionof oxdD is under the control of ^K, is monocistronic, and isnegatively regulated by GerE . An enzymatically active OxdD-greenfluorescent protein [GFP] fusion protein localized to the coatlayers in a safA-dependent manner . Mutations in gerE also interferedwith the assembly of OxdD-GFP . In contrast, cotE was not requiredfor the assembly of OxdD-GFP but determined its stable associationwith the coat . The data suggest that OxdD is targeted to theinner layers of the coat . A multicopy allele of oxdD resultedin spores with increased levels of OxdD and with oxalate decarboxylaseactivity.

 
Bacterial strains, media, and general techniques. The bacterial strains used in this study are listed in Table1 . Difco sporulation medium [DSM] was used to induce sporulationby nutrient exhaustion [37] . Genetic manipulations of E . coli and B . subtilis were performed and spore resistance and germination properties were assessed as previously described [8] . The high-fidelityPfu polymerase [Stratagene, La Jolla, Calif.] was used to generatePCR fragments for cloning . These fragments were sequenced, wheneverrequired, to ensure that no mutations were introduced.

 
Insertional inactivation of the oxdD gene. First, a 510-bp DNA fragment comprising the oxdD promoter regionand the 5' end of its coding region was PCR amplified by usingprimers yoaN-295D and yoaN+215R [Table 2] and doubly digestedwith NcoI and BglII, and the resulting 355-bp fragment was clonedbetween the NcoI and BamHI sites of pAH256 [17] to obtain pTC127.Next, the 3' end of oxdD was isolated from pTC120 [see below]as a 766-bp EcoRI-XhoI fragment and cloned into the same sitesof pTC127, yielding pTC128 [Fig . 1] . Transformation of MB24with ScaI-linearized pTC128 produced the spectinomycin-resistant[Sp^r] oxdD null mutant AH2898 [Table 1] by a double-crossover event at the oxdD locus [verified by PCR].

 
Construction of an oxdD-lacZ fusion. A 510-bp fragment carrying 295 bp of DNA upstream of the oxdDstart codon was PCR amplified with primers yoaN-295D and yoaN+215R[see above], doubly digested with EcoRI and BglII, and cloned between the EcoRI and BamHI sites of pSN32 [a gift from IsabelSá-Nogueira], yielding pTC125 [Fig . 1] . PstI-linearizedpTC125 was used to transfer the oxdD-lacZ fusion to the amyElocus of strains MB24, AH77, and AH2721 to produce the Cm^r AmyE^- strains AH2886, AH2890, and AH2891, respectively [Table 1].

Construction of an oxdD-gfp fusion. pTC119 and pTC147 [Fig . 1] were constructed in two steps . First,the oxdD 3' region [716 bp] was PCR amplified with primers yoaN+461D and yoaN-gfpR [Table 2] . Second, a 719-bp fragment comprisingthe coding region of the gfp gene was PCR amplified by usingpEA18 [a gift from Alan Grossman] as the template and primersgfp-30D and gfpmut2-749R [Table 2] . The resulting fragmentswere mixed and subjected to PCR with primers yoaN+461D and gfpmut2-749R [Table 2] . The resulting 1,435-bp oxdD-gfp fragment was cleavedwith SpeI and XhoI and cloned between the same sites of pAH256[17] to generate pTC119 and between the same sites of pMS38[59] to generate pTC147 . Strains AH2873 [Sp^r] and AH2943 [Cm^r] resulted from the integration of pTC119 and pTC147, respectively, into the oxdD locus of wild-type strain MB24 by a single reciprocal crossover [Campbell-type recombination] [Table 1], as verifiedby PCR . AH2873 was transformed with DNA from 1S105 [Table 1],yielding the Sp^r Cm^r strain AH2883 [cotE oxdD-gfp] [Table 1].The Sp^r strain AH2912 [gerE oxdD-gfp] and the Sp^r Cm^r strainAH2913 [cotE gerE oxdD-gfp] resulted from Campbell integrationof pTC119 into AH94 and AH2884, respectively [Table 1] . Thelatter strain was constructed by transforming AH94 with DNAfrom 1S105 . The absence of congression to Ger⁺ was verifiedas described previously [47] . Finally, AH2943 was transformedwith chromosomal DNA from AOB68 [39] to obtain the Sp^r Cm^r strainAH2944 [safA oxdD-gfp] [Table 1].

Construction of a B . subtilis strain bearing a multicopy allele of oxdD. A PCR fragment [1,795 bp] comprising the entire oxdD codingregion and 295 bp upstream of its transcription initiation sitewas generated with primers yoaN-295D and yoaN-1500R [Table 2],digested with HindIII, and cloned between the HindIII and SmaIsites of replicative plasmid pMK3 [32] to generate pTC149 [Fig. 1] . Competent cells of MB24 were transformed with pTC149 andwith its parental plasmid [pMK3] to obtain the neomycin-resistant [Nm^r] strains AH2953 and AH2954, respectively [Table 1].

Overproduction of OxdD and OxdD-GFP. The entire oxdD coding region [1,207 bp] was PCR amplified withprimers yoaN-pETD and yoaN-1500R [Table 2], digested with NcoI and HindIII, and cloned between the same sites of pET33b[+] [Novagen] to obtain pTC120 [Fig . 1] . pTC148 [Fig. 1], whichcan be used to overproduce OxdD-GFP, was the result of a tripleligation involving NcoI- and BamHI-digested pET28a[+] [Novagen]and the following inserts: [i] a PCR fragment obtained withprimers yoaN-pETD and yoaN-1500R [see above] and digested withNcoI and EcoRI to produce a 438-bp fragment corresponding tothe 5' end of the oxdD coding region; and [ii] the 3' oxdD codingregion fused to gfp [1,458 bp], obtained by digesting with EcoRIand BamHI a 2,190-bp PCR fragment generated with primers yoaN-295Dand gfpR [Table 2] and AH2873 chromosomal DNA . pTC120 and pTC148were introduced into the E . coli host Tuner[DE3][pLacI] [Novagen]to create strains AH2892 and AH2950 [Table 1], in which nativeOxdD and OxdD-GFP, respectively, could be produced under controlof the T7lac promoter . Induction of OxdD production was performedas described previously [52] . Following induction, the cellswere harvested and lysed by passage through a French pressurecell as described previously [52].

Purification of spores and analysis of the spore coat fraction. Spores were harvested by centrifugation of DSM cultures 24 hafter the onset of sporulation . Each spore suspension was washed,and the spores were purified with a 20 to 50% Gastrografin [Schering]step gradient as described previously for Renocal-76 gradients[17, 18, 47] . Coat proteins were extracted from purified sporesat an optical density at 580 nm of about 2 as described previously[17, 18] . Coat proteins were subjected to electrophoretic fractionationon 15% polyacrylamide gels containing sodium dodecyl sulfate[SDS] . The gels were stained with Coomassie brilliant blue R-250.

Fluorescence microscopy. Samples [0.5 ml] of DSM cultures of various strains bearinga translational oxdD-gfp fusion [see above] were collected about8, 10, and 24 h after the initiation of sporulation and resuspendedin 0.2 ml of phosphate-buffered saline . Aliquots were appliedto agarose-coated microscope slides, and images were acquiredwith a Leica fluorescence microscope [DMRA2] by using phase-contrastoptics and a standard filter for visualization of the GFP . Allsamples were observed with a x63 objective lens . Images wereacquired with a Cool Snap HQ camera [Roper Scientific, Tucson,Ariz.], recorded, and processed by using Adobe Photoshop.

Enzyme assays. The activity of ß-galactosidase was determined withthe substrate o-nitrophenyl-ß-D-galactopyranoside, as previously described [18, 47] . The specific activity of oxalatedecarboxylase was determined by spectrophotometry at 37°Cby using a coupled reaction assay based on the method describedby Magro et al . [31, 52] . One unit of enzyme activity was definedas the amount of enzyme required to reduce 1 µmol of NADper min . All specific activities are the mean values of threeassays . The protein concentration was determined with a Bio-Radassay kit [Bio-Rad Laboratories, Hercules, Calif.] used as describedby the manufacturer.

RNA isolation and Northern blot analysis of gerE and oxdD. Samples were collected from sporulating cultures of a wild-type strain [JH642] and a congenic sigK::erm mutant [MO1027] [Table1] . RNA was isolated by mechanical disruption of the liquidnitrogen-frozen cell pellets in a Teflon vessel by using a Micro-Dismembrator[B . Braun Biotec International, Melsungen, Germany] as describedby Petersohn et al . [41] . Total RNA [5 µg per lane] wasseparated in a 1.5% agarose gel containing 6% [vol/vol] formaldehydeand vacuum blotted onto nylon membranes [Biodyne Plus; Pall].Digoxigenin [DIG]-labeled antisense RNA probes were generatedby using T7 polymerase and gene-specific PCR products as templates.Two primer pairs [yoaN-fwd plus yoaN-rev-T7 and gerE-fwd plusgerE-rev-T7] were used in PCR with JH642 chromosomal DNA . Ineach of the PCRs one of the DNA primers carried the sequenceof the T7 promoter . PCR fragments were subsequently used forin vitro RNA synthesis with a MAXIScript kit [Ambion, Inc.,Austin, Tex.] and DIG-labeled UTP [Roche, Basel, Switzerland],which yielded hybridization probes for oxdD [444 nucleotides]and gerE [186 nucleotides] . Hybridization and signal detectionwere performed as previously described [46].

Protein identification by peptide mass fingerprinting. Protein spots were excised from Coomassie brilliant blue R-250-stained gels, destained, and digested with trypsin [Promega Corporation, Madison, Wis.]; peptides were then extracted [38] . Peptide mixtureswere desalted on Poros R2/R3 tips and directly eluted onto asample template of a matrix-assisted laser desorption ionization—timeof flight [MALDI-TOF] mass spectrometer with an elution solutioncontaining 70% [vol/vol] acetonitrile, 0.1% [vol/vol] trifluoroaceticacid, and saturating amounts of -cyano-3-hydroxycinnamic acid.Peptide masses were determined in the positive ion reflector mode with a Voyager Elite MALDI-TOF mass spectrometer [Applied Biosystems, Foster City, Calif.] with internal calibration.The mass accuracy was better than 30 ppm . Peptide mass fingerprintswere compared to databases by using the MASCOT program [http://www.matrixscience.com/cgi/index.pl?page=../home.html]. The searches considered oxidation of methionine and pyroglutamic acid formation at the N-terminal glutamine . Proteins were considered identified when the database search revealed a significant MASCOT score with a probability [P] of <0.05 that the observed match was a random event.

 
Identification of the OxdD protein. To learn more about the polypeptide composition of the sporecoat layers, we used MALDI-TOF mass spectrometry to identifypolypeptides extracted from highly purified spore preparations.Our strategy was to subject spores collected 24 h after theonset of sporulation in DSM to extensive washes with water andthen to further purify the enriched spore preparation by centrifugationthrough 20 to 50% metrizoic acid step gradients [see Materialsand Methods] to eliminate remnants of sporulating cells or celldebris . The purified spores were then subjected to an extractionregimen known to preferentially solubilize a fraction [about70%] of the total spore coat-associated proteins [19], whichwere resolved on one-dimensional SDS—15% polyacrylamide gel electrophoresis [PAGE] gels . As a further criterion for specific association with the coat integuments, bands that were present in the wild type but were present at reduced levelsin the coats of a cotE mutant were processed for MALDI-TOF analysis. By using this approach, the 43-kDa product of the yoaN locus[27] [Fig . 1] was identified as a protein associated with the spore coat layers [Fig . 2] . YoaN was recently found to haveoxalate decarboxylase activity when it was expressed in E . colicells and was accordingly renamed OxdD [52] . In two other recentstudies the workers employed mass spectrometry techniques toidentify proteins associated with the spore or spore coat [28,29] . In one of these studies, OxdD was found to be a spore-associatedprotein [28] . The results reported here suggest that the OxdDprotein specifically associates with the coat layers of B . subtilisspores.

 
The 43-kDa OxdD polypeptide is absent from spores produced by an oxdD insertional mutant. To confirm the association of OxdD with the coat and to examineits role, we constructed an oxdD insertional mutant, AH2898[Table 1] . oxdD is flanked by the yoaO gene upstream and bythe yoaM gene downstream [Fig . 1]; the latter genes encode proteinswith unknown functions . yoaM is convergently oriented relativeto oxdD [27] [Fig . 1] . Moreover, results described below indicatethat oxdD is a monocistronic unit transcribed from a promoterjust upstream of the gene's coding sequence . Thus, the mutationis unlikely to cause a polar effect . We purified spores fromMB24 and AH2898 and analyzed the collection of proteins thatwere extracted from their coat layers on SDS—15% PAGE gels [Fig . 2A] . In spores formed by AH2898 [oxdD::sp], the 43-kDaband identified as the OxdD protein was missing [Fig. 2] . Theset of proteins extracted from AH2898 spores differed furtherfrom the proteins extracted from wild-type spores . First, anadditional band present in the AH2898 coat extract was identifiedby MALDI-TOF analysis as flagellin [Hag], a major componentof the flagellum [20, 30] [Fig . 2] . Several preparations ofAH2898 spores were examined, and in all cases Hag was foundin the coat fraction [data not shown] . Since the hag gene isrequired for motility of vegetative cells [30], it seems plausiblethat residual Hag protein present in the sporulation mediumcan associate with the spore after its release from the mothercell . Hag is tightly associated with spores of the oxdD mutant,as washes with 1 M KCl, a treatment known to release proteinsloosely associated with the coat [47], did not reduce its levelor preclude its presence in the collection of extractable coat polypeptides . Perhaps disruption of oxdD induces a subtle change in the properties of the coat that causes the tight adherence of Hag to the coat . Second, two proteins appeared to be lessabundant in AH2898 coat extracts . One of these proteins is thenormally prominent 36-kDa CotG protein [45], and the other produced a wide diffuse band in the 30-kDa region of the gel, which we were not able to identify by either MALDI-TOF or N-terminalsequence analysis [Fig . 2A] . In contrast, most of the other proteins, including CotA, CotB, and CotC, remained unchangedcompared to the wild-type proteins [Fig . 2A] . Finally, we noted that proteins in the 43-kDa region of the gel, presumably including OxdD, were absent from the coats of cotE [AH2835] [Table 1]or gerE [AH94] [Table 1] mutant spores, or the amounts weregreatly reduced [Fig. 2B] . Together, these results are consistentwith localization of OxdD to the spore coat [see below] . Disruptionof the oxdD locus had no detectable effect on spore resistanceto heat or lysozyme [Table 3] or on the capacity to germinate in response to L-alanine or L-asparagine [data not shown] . Oxalateis known to increase the permeability of the spore coat to certainelectron microscopy dyes [25] . Moreover, spores become sensitiveto lysozyme after incubation for 5 min in the presence of oxalateat 80°C but not when they are incubated for 30 min at 30°C[25] . To determine if OxdD contributed to protection of sporesagainst oxalic acid under these conditions, we exposed wild-typeor oxdD spores to oxalic acid [0.025, 0.25, 2.5, 5, or 25 mM]at 30°C [30 min] or 80°C [5 min] . Treatment with 25mM oxalic acid at 80°C caused similar reductions in sporeviability [from about 10⁸ to 10⁵ spores per ml] or lysozymeresistance [from 10⁸ to 10⁴ spores per ml] for wild-type andmutant spores . Also, no effect on viability or resistance ofwild-type and mutant spores was observed when the other concentrationsof oxalic acid were used, at either 80 or 30°C.

 
Spore-associated OxdD can exhibit oxalate decarboxylase activity. Recently, the spore coat protein CotA was shown to be a laccase, which retains enzymatic activity when it is embedded withinthe endospore coat [21, 32] . To determine whether coat-associatedOxdD also retained enzymatic activity, we assayed purified wild-type[MB24] and oxdD mutant spores [AH2898] for oxalate decarboxylaseactivity [see Materials and Methods] . However, we were unableto detect enzymatic activity in wild-type spores . We repeatedthe assay with various amounts of whole-cell extracts of sporulatingcells or intact spores in the presence of 25 µM MnCl₂at different pH values and substrate concentrations, after treatmentof spore suspensions at 80°C for 10 min to facilitate accessof the substrate [32] and after induction of spore germination.In all cases no enzymatic activity was detected . We then generatedan oxdD multicopy allele [oxdDMC] by inserting the oxdD gene[including its promoter [see Materials and Methods]] into thepMK3 replicative plasmid [32] . Suspensions of spores purifiedfrom the oxdD^MC strain [AH2953] [Table 1] and from a strainbearing the pMK3 vector [AH2954] [Table 1] were tested for theability to degrade oxalate . We found enzymatic activity in sporesof oxdD^MC strain AH2953 [about 15 mU/optical density unit ofa spore suspension] but not in spores produced by strain AH2954harboring the parental pMK3 vector . We also looked for enzymaticactivity in whole-cell extracts prepared from cultures of AH2953and AH2954 after 6 and 8 h of sporulation . Under the assay conditionsused no activity was found in whole-cell extracts of AH2953or AH2954 . Therefore, enzyme activity can be detected even inthe oxdD^MC strain only when the enzyme accumulates at the sporesurface.

SDS-PAGE analysis of the coat polypeptides indicated that increased amounts of OxdD were present in AH2953 spores [Fig . 3, lane2] . However, the representation of several other coat polypeptideswas greatly altered in AH2953 spores compared to that in sporesproduced by AH2954 [Fig . 3], and the resulting spores exhibitedreduced resistance to heat and lysozyme treatments [Table 3],indicating that abnormal [increased] levels of OxdD perturbboth the spore coat composition and spore resistance . Presumably,the coat structure of AH2953 spores is also altered . However,these spores have not been examined by electron microscopy.In any case, the results indicate that spore-associated OxdDhas enzymatic activity.

 
oxdD gene is transcribed during sporulation under ^K control. With one possible exception [47], all the genes shown to beinvolved in spore coat assembly are transcribed in the mothercell compartment of the sporulating cell [10, 19] . In addition,expression of a significant number of the genes known to encodecoat structural components relies upon ^K and is either positivelyregulated or repressed by GerE [3, 7, 17, 45, 49, 54, 57] . We used an oxdD-lacZ fusion inserted at the amyE locus to study the regulation of the oxdD gene . Expression of oxdD-lacZ was monitored throughout growth and sporulation in DSM in an otherwise wild-type strain [AH2886] and congenic sigK [AH2890] and gerE [AH2891] mutants [Table 1] . We found that in AH2886, expressionof oxdD-lacZ was induced around hour 4 of sporulation [Fig.4], a temporal profile shared by several other ^K-controlled genes [10, 15, 19, 57] . No ß-galactosidase productionwas detected in the sigK mutant AH2890 [Fig . 4] . In contrast, expression of oxdD-lacZ increased about threefold in the gerE mutant AH2891 [Fig . 4A] . Consistent with involvement of GerEin the regulation of oxdD expression, three possible GerE-bindingsites were recognized in the oxdD promoter region [Fig . 4B][22] . Identical results were obtained when the oxdD-lacZ fusionwas inserted at the oxdD locus [data not shown] . We inferredthat expression of oxdD during sporulation occurs from a promoter present in the 295 bp upstream of the gene's start codon [see Materials and Methods], which is utilized by ^K and repressedby GerE.

 
oxdD is monocistronic. To ensure that no other upstream promoter contributed significantlyto the expression of oxdD [as implied by the analysis of anoxdD-lacZ fusion at the oxdD locus], we performed a Northernblot analysis . RNA samples were prepared from a wild-type strainand from a sigK mutant at various times during sporulation andwere analyzed with probes specific for oxdD or for gerE . Inwild-type cells, a transcript was detected with the oxdD-specificprobe at hours 4 and 6 of sporulation [Fig . 5A] . The size ofthis transcript [about 1,200 nucleotides] is consistent withthe size of the oxdD coding region [1,176 bp] . No other signalappeared to be present in the wild type at any time tested,and no signal was found in the sigK mutant strain [Fig . 5A]. Similarly, the gerE transcript [300 nucleotides] was detected at hours 4 and 6 during development in the wild type but notin a sigK mutant [Fig . 5B] . The results indicate that transcriptionof oxdD during sporulation occurs mainly if not exclusivelyin a monocistronic mode and that it temporally coincides withthe transcription of a gene [gerE] known to be under the controlof ^K [6, 57] . We tried to map the 5' end of the oxdD transcriptby primer extension, but several attempts in which differentprimers were used were unsuccessful . Of note, however, was thepresence of a possible canonical ^K-dependent promoter [16] justupstream of oxdD [Fig. 4B].

 
OxdD-GFP is a functional oxalate decarboxylase. The genetic requirements for assembly of OxdD were studied byusing a GFP fusion . Since the oxdD mutant did not exhibit anyspore resistance or germination phenotype, we wanted to determinewhether the fusion protein retained oxalate decarboxylase activity.The product of the oxdD gene exhibits oxalate decarboxylaseactivity when it is produced in E . coli [52] . We used the same activity assay to test the functionality of the OxdD-GFP fusion protein after overproduction in E . coli . The oxdD-gfp fusion was cloned under the control of the isopropyl-ß-D-thiogalactopyranoside [IPTG]-inducible T7lac promoter, and the resulting plasmid was introduced into an appropriate E . coli host for overproduction [strain AH2950] [Table 1] . Induction was performed as previouslydescribed [52] [see Materials and Methods] . In parallel, weoverproduced the native [unfused] OxdD protein as a positivecontrol for activity [AH2892] [Table 1] . Oxalate decarboxylaseactivity was then assayed in whole-cell extracts prepared frominduced cultures of AH2950 and AH2892 . Activity was detectedin whole-cell extracts of both induced AH2950 and induced AH2892cultures but not in extracts prepared from uninduced culturesof the same strains [data not shown] . The level of overproductionof OxdD was similar to that of OxdD-GFP [about 10% of the totalprotein] [data not shown], and the levels of enzyme activitywere comparable [2.6 U/mg of protein for OxdD and 1.6 U/mg forOxdD-GFP] . Moreover, the levels of activity are comparable to those reported by Tanner et al . [52] for the activity of OxdDin E . coli crude lysates . We inferred that the overall foldof the fusion protein was not altered by fusion to GFP and that assembly of OxdD-GFP into the coat is likely to reflect assembly of the native OxdD coat protein.

Assembly of OxdD into the coat of wild-type spores. Next, we used OxdD-GFP to investigate the assembly of OxdD intothe coat . Strain AH2873 [OxdD-GFP fusion in MB24] [Table 1] produced spores with wild-type resistance [Table 3] and germinationproperties [data not shown] . The 43-kDa OxdD band was absentfrom the collection of AH2873 spore coat polypeptides, but no additional band corresponding to the size of OxdD-GFP [73 kDa] was detected on the Coomassie brilliant blue-stained gels [Fig. 2A, lane 3] . This failure to detect OxdD-GFP could have been caused by comigration with other proteins, to reduced extractability of the fusion protein, or to both of these factors . In any case, immunoblot analysis with an anti-GFP antibody confirmed the association of OxdD-GFP with the coat of AH2873 spores [datanot shown] . Moreover, the results of fluorescence microscopyexperiments [described below] confirmed the presence of OxdD-GFPin the coat of wild-type spores . No other changes were seenin the pattern of coat proteins, indicating that expressionof OxdD-GFP does not interfere in any significant way with theassembly process [Fig . 2A] . To monitor the assembly of OxdD-GFP,samples were harvested from DSM cultures 8, 10, and 24 h afterthe onset of sporulation, and the live AH2873 cells were mountedon agarose slides for examination by fluorescence microscopy[Fig . 6] . We also used phase-contrast optics to mark the positionof the whole cell and to visualize the developing spore [Fig.6] . The number of partially refractile [phase-grey], fully refractile [phase-bright], or free spores and the pattern of GFP decorationof sporulating cells or spores were recorded for each time point[Table 4] . Control experiments showed that, as expected, no fluorescence was detected at any time tested for a strain having a mutation in the sigK gene [data not shown] . Moreover, under the conditions used, no fluorescence signal was found to be associated with cells or spores of a wild-type strain [MB24]bearing no gfp gene or fusion for any time tested [data notshown] . Fluorescence was detected for AH2873 [OxdD-GFP] at hours4 and 6 of sporulation, when the expression of oxdD commencedand reached a maximum, respectively [Fig . 4 and 5], in lessthan 1% of the specimens observed . The frequency of decoration of the developing spore by OxdD-GFP was greatly enhanced around hour 8 of sporulation [Fig . 6a and a'] . The fusion protein was detected as caps at both poles of the developing spore in about 6% of the specimens examined; these caps were phase grey . However, the majority of the specimens showed fluorescence around theentire developing spore [Fig . 6a and a'; Table 4] . In most specimens[76%], the spore was partially refractile, whereas in some specimens[18%] the spore was phase bright [Table 4] . The polar cap patternhas been observed for other coat proteins [e.g., for CotE byusing either GFP fusions [53] or immunofluorescence [4, 43]] and is also consistent with observations made by using immunoelectron microscopy [11] . The fact that the polar cap pattern preferentiallyassociates with partially refractile spores suggests that itrepresents an early stage in the assembly process . By hour 10 of sporulation, 87% of the sporulating cells had spores fully encircled by OxdD-GFP [Fig . 6b and b'], but the frequency of decoration of phase-bright spores relative to the partially refractile spores had increased to 54% [Table 4] . The overall representation of the polar cap pattern associated with phase-grey spores was maintained [4%], but this pattern was also observed for cells having phase-bright spores [4%] . Finally, 24 h afterthe initiation of the developmental process, OxdD-GFP fullyencircled the spore in 98% of the specimens examined; 84% ofthese specimens were free phase-bright spores, and about 14%were cells containing bright spores [Fig . 6c and c'; Table 4]. We noted that the prespore surface often had a punctuated patternof decoration rather than being uniformly covered [see Discussion].No fluorescence was detected throughout the mother cell cytoplasmin the wild-type strain, but occasionally spots of fluorescencewere observed close to the cell pole opposite where the sporewas formed [Fig . 6a' and b'] . Together, the results suggest that following synthesis most of the fusion protein is targetedto the poles of the developing spore and then, as the developingspore attains full refractility, gains access to the entiresurface.

 
cotE and gerE are required for stable association of OxdD with the coat. Native OxdD absent from the coats of cotE or gerE mutant spores[Fig . 2B] or the amount was greatly reduced, but it was stillpossible that in the mutants OxdD was targeted to the developingspore but was not retained upon spore release . We thereforeinvestigated the localization of OxdD-GFP in cells with a cotEmutation, which failed to assemble the outer coat [56], in cellswith a gerE mutation, which lacked several of the inner andouter coat proteins [2, 3, 7, 10, 19, 33, 34, 45, 49], or incells with both mutations . The cotE gerE double mutant formedspores that were missing both coat layers [10] . In strain AH2883[cotE], at hour 8 of sporulation, we observed decoration ofthe polar cap region of the developing spores in both phase-greyspores [19%] and phase-bright spores [8%] [Fig . 6d and d'; Table 4] . Since representation of the polar cap pattern increased in the mutant [to 27% of the specimens observed, compared to6% in the wild type] and this pattern was also found in associationwith phase-bright spores, it seems that complete encirclingof the spore by OxdD-GFP was slowed down in the mutant and uncoupledfrom spore maturation . Accordingly, the proportions of phase-greyspores [63%] and phase-bright spores [9%] in cells of the cotEmutant at hour 8 which were fully encircled by OxdD-GFP decreasedto a total of about 72% [Table 4] . As in the wild type, the prespore surface often displayed a punctuated pattern of decoration [see above and Discussion] . By hour 10, fluorescence was also observed as spots or patches in the mother cell in 25% of thecotE mutant cells carrying phase-grey or phase-bright spores[Fig. 6e and e'; Table 4], a pattern that persisted at hour24 [Table 4] . Decoration of the entire surface of the developingspore was greatly reduced by hour 24 [2% of the specimens scored][Table 4] . Moreover, the fluorescence signal was found in only4% of the free spores [Fig. 6f and f'] . The results suggestthat OxdD-GFP is initially targeted to the surface of the developingspore following the polar cap pattern, as in the wild type,and then fully encircles the spore, albeit more slowly . However,the accumulation of fluorescence in the mother cell cytoplasmfrom hour 10 onward and the lack of decoration of free sporesat hour 24 suggest that OxdD-GFP does not stably associate withthe forming coat and does not persist in spores released bythe cotE mutant cells.

In contrast to the cotE mutant, decoration of the entire surface of the developing spore was never observed for the gerE or cotE gerE mutants, even at a late time in assembly [Fig . 6g to l;Table 4] . Indeed, sporulating cells of both of these mutantsexhibited only polar cap pattern of spore decoration, and nofluorescence signal was ever found to be associated with freespores [Table 4] . These observations indicate that the gerE36mutation is epistatic over the cotE null allele . Consistentwith the observation that GerE represses expression of oxdD[see above], increased fluorescence was observed in cells bearingthe gerE36 allele . It seems that the initial targeting of OxdD-GFPto the polar regions of the developing spore does not requiregerE but that migration of the fusion protein to completelysurround the spore is a gerE-dependent event . The lack of fluorescenceassociated with free spores produced by AH2883, AH2912, or AH2913is consistent with the observation that native OxdD is absentfrom the coats of purified cotE or gerE spores or the amountis greatly reduced [Fig . 2B].

SafA directs OxdD to the developing spore coat. The cotE-independent targeting of OxdD-GFP to the surface ofthe developing spore suggested that the fusion protein couldreside in the inner layers of the coat and that its assemblycould depend on the action of morphogenetic proteins whose assemblyis itself CotE independent . Localization of SpoIVA, SpoVID,and SafA is CotE independent [11, 40] . We found that targetingof OxdD-GFP to the spore surface was prevented by mutationsin spoIVA or spoVID [data not shown] . Since spoIVA governs theassembly of SpoVID and since SpoVID recruits SafA, we also analyzedthe localization of OxdD-GFP in sporulating cells of a safAmutant [AH2944] . In the safA cells at hour 8 of sporulation,OxdD-GFP fluorescence tended to accumulate at the mother cell-presporeborder as spots or large patches [Fig . 6m and m'], a pattern that became more accentuated as sporulation proceeded [Fig. 6n and n'] [for simplicity this pattern is recorded as mother cell spots in Table 4] . Essentially no fluorescence was associatedwith free safA spores [Fig . 6o and o'] [Table 4] . Importantly,expression of oxdD-gfp did not aggravate the lysozyme sensitivityof safA mutant spores [Table 3] . The results suggest that the initial targeting of OxdD-GFP to the spore surface is safA dependent and support the view that OxdD resides in the inner layers of the coat.

 
Our results indicate that the OxdD protein of B . subtilis isa component of the coat layers . Several lines of evidence supportthis claim . First, the OxdD protein was identified by MALDI-TOFanalysis among the collection of polypeptides that can be extractedfrom the coats of wild-type spores but not from cotE or gerE spores, which have abnormal coats [10, 19, 33, 56] . Second,disruption of oxdD consistently led to the absence of the OxdDprotein from coat extracts prepared from spores of the mutant.Moreover, oxdD was found to be expressed in the mother cellfrom hour 4 of sporulation onward under the control of ^K, whichcoincided temporally and spatially with the expression of most genes encoding coat structural components [10, 19] . Finally,studies in which a functional OxdD-GFP fusion was used revealedassembly of the fusion protein around the developing spore ina manner that was influenced by loci known to play key rolesin coat biogenesis.

OxdD shows a high degree of sequence similarity to another B. subtilis protein, OxdC [formerly YvrK], which was recently characterized as an acid-inducible oxalate decarboxylase, and both proteins are very similar to well-characterized fungal enzymes [12, 51].OxdD is likely to have the overall fold and structural featuresof OxdC and was able to convert oxalate to formate and CO₂ inan Mn-dependent manner when it was overproduced in E . coli cells[52] . Both OxdD and OxdC were identified in a previous studyin which liquid chromatography coupled to tandem mass spectrometrywas used to analyze proteins extracted from whole spores [28]. However, no expression data were reported for either oxdD or oxdC, and none of the products were assigned to a specific spore layer or structure [28] . While our results indicate that OxdDis a component of the inner coat layers, we did not observe expression of an oxdC-gfp fusion during sporulation and did not see decoration of mature spores by the fusion protein [data not shown] . It is possible that in contrast to OxdD [this study], minute levels OxdC are associated with the spore, presumablywith the spore core . No OxdD activity was reported in B . subtilis,nor were the conditions that induced expression of the oxdDgene in B . subtilis reported . Using a combination of Northernblot analysis and a fusion of the oxdD promoter region to the lacZ gene, we were able to show that a single monocistronic transcript was produced during sporulation under the controlof ^K and that expression of oxdD was normally repressed by theGerE ancillary regulator . At this time, ^K and GerE are the onlyknown regulators of oxdD expression in B . subtilis.

The role of OxdD was examined by analyzing the coat polypeptide composition and the resistance and germination properties ofspores produced by an oxdD insertional mutant . As observed for mutations in several other coat structural components, disruptionof oxdD did not interfere significantly with the assembly or organization of the coat layers . Spores of the mutant did notdiffer from the wild-type spores in the ability to resist lysozymeexposure or the ability to germinate in response to L-alanine or L-asparagine . Also, they were not more sensitive to oxalatetreatment or to oxalate treatment followed by lysozyme treatment.However, we did not detect oxalate decarboxylase activity inwild-type spores of B . subtilis, as assayed previously for E.coli cells producing OxdC or OxdD and for OxdC in B . subtilisextracts [51, 52] . We do not think that the lack of activityis caused by poor access of oxalate to the enzyme, since thespore coat is permeable to oxalate up to the inner coat-outercoat junction and even further following treatment at 80°C[25;this study] . The activity assay is based on the reductionof NAD upon conversion of formate to CO₂ by formate dehydrogenase[31] . Again, it seems unlikely that the smaller formate moleculedoes not come in contact with formate dehydrogenase . It maybe that upon assembly, OxdD is in a microenvironment that doesnot support enzyme activity . For example, it is possible thatanother coat protein inhibits the enzyme . The observation thatexpression of a multicopy allele of oxdD results in spores thatcan convert oxalate into formate indicates that the proteinthat is synthesized during spore development and that becomesassociated with the spore coat is a functional oxalate decarboxylase.We suggest that the activity observed results in part from thedisorganization of the coat structure caused by the increasedrepresentation of OxdD [as shown by the altered coat proteinprofile and lysozyme sensitivity], which could free the enzymefrom its putative inhibitory microenvironment, and in part fromincreased levels of enzyme . Some fungi produce oxalic acid,which can chelate manganese and stimulate the activity of anextracellular Mn peroxidase involved in lignin degradation [12]. In this context, oxalate decarboxylases appear to confer protection against excess oxalate [12, 51] . Since spore germination inthe soil can occur in association with the growth and developmentof fungal hyphae [35], it is tempting to speculate that OxdDcould also protect the spore [or the germinating spore] fromthe harmful effects of oxalic acid . It is also possible thatOxdD plays only a structural role in coat assembly . In any case,the biological significance of the association of OxdD withthe B . subtilis spore coat is unclear at present . Database searchessuggest that the genome of at least one other spore-formingmicroorganism, Bacillus cereus, encodes an oxalate decarboxylase[24], but this protein has not been characterized.

A functional OxdD-GFP fusion protein seems to be assembled intwo steps . In the first step, OxdD-GFP localizes as caps atboth poles of the engulfed prespore . In the second step, thefusion protein fully encircles the developing spore . The punctuatedpattern of fluorescence often seen in sporulating cells or sporessuggests that the distribution of OxdD-GFP is not uniform inthe prespore surface but rather is patchy . While this patterncould be an artifact caused by the presence of the GFP moiety,we suspect that it may reflect the possible multimeric natureof OxdD, as suggested by its similarity to the hexameric OxdCmolecule [1] . Our results indicate that SafA is necessary forthe initial targeting of OxdD to the cap regions of the prespore[Fig . 7] . In the absence of SafA, fluorescence from OxdD-GFPis observed as patches or spots in the mother cell cytoplasm[Fig . 7] . As observed for SafA [39], it is possible that OxdD localizes to the inner coat layers . This notion is consistentwith the observation that the amounts of both OxdD and OxdD-GFPare greatly reduced or these molecules are missing from thecoats of cotE or gerE spores, and it is further supported bythe drastic effect that the oxdD^MC allele has on the structure and properties of the coat . We note that a multicopy alleleof cotA results in lysozyme-resistant spores with a normal complement of coat proteins except for CotA [32] . Since CotA is an outercoat protein [56], one interpretation is that the effects ofthe oxdD^MC allele result from the more internal localizationof OxdD, perhaps in conjunction with its larger size [see above].We found that the initial targeting of OxdD-GFP to the presporepolar regions was gerE and cotE independent . However, OxdD-GFPfails to fully encircle the prespore in a gerE mutant, implyingthat at least one GerE-dependent protein is necessary for thesecond stage of OxdD localization [Fig. 7] . In contrast, OxdD-GFPwas capable of encircling the prespore in cotE mutant cellsbut was not retained in the mature released spores [Fig . 7].Presumably, OxdD is lost from mature spores lacking completeinner and outer coat layers . Expression of gerE is requiredfor the development of the morphological features normally associatedwith the inner coat, even though a mutation also interfereswith assembly of the outer coat [10, 19, 33] . Mutations in cotEappear to have a much more specific effect on the assembly ofthe outer coat [11, 56] . OxdD could be assembled at the borderbetween the inner and outer coats, which could explain the factthat the protein is retained in association with the inner coatfound in cotE spores . For example, CotH is synthesized underthe control of ^K, and since its assembly is both cotE and gerEdependent, it has been proposed that CotH is close to CotE,at the inner coat-outer coat border [34, 58] . Another coat protein,CotS, produced under the joint control of ^K and GerE, was foundby immunoelectron microscopy in the inner coat and pericortex,yet it was not detected in cotE spores [49] . We speculate thatlike OxdD, CotS cannot be retained in association with the innercoat in spores lacking the outer coat . This pattern of assemblymay have a broader distribution, suggesting that care should be taken in assignment of a protein to the outer coat on the basis of its absence from cotE spores . OxdD is presently the only protein whose targeting to the developing spore specifically requires expression of safA . It will be interesting to determine whether the targeting of OxdD involves a direct interaction with SafA.

 
We thank C . P . Moran, Jr., for critically reading the manuscript.

This work was supported by grants from the Max Planck Institute for Terrestrial Microbiology [Marburg, Germany] and the Bundesministerium für Bildung und Forschung to U.V . and by internal grantsfrom the Instituto de Tecnologia Química e Biológicato A.O.H . T.C . holds a Ph.D . fellowship [PRAXIS XXI/BD/1167/00]from Fundação para a Ciência e a Tecnologia.

 
* Corresponding author . Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av . 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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