INTRODUCTION

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In response to nutrient depletion, Bacillus subtilis can differentiate to produce a dormant cell type known as the endospore (reviewed in references 7, 17, 21, and 23). The endospore can withstand physical and chemical insults such as dehydration, exposure to organic solvents, lysozyme, and extreme temperatures and pressures. These properties of Bacillus subtilis spores can be attributed to the physical and chemical makeup of the structures that encase the spore (6, 10, 21). There are two conspicuous structures surrounding the spore, as viewed by thin-section electron microscopy: a thick central region of modified peptidoglycan (the cortex) and an exterior, multilayer protein structure (the spore coat). The coat is composed of over two dozen proteins, organized into three layers: an amorphous undercoat, a thin lamellar inner coat, and a thick striated outer coat (as reviewed in references 6 and 10).

Assembly of the spore coat is one of several complex morphological changes that occur during endospore formation. Endospore formation begins with an asymmetric cell division that produces a small cell and a large cell (forespore and mother cell, respectively). After the asymmetric division, the membranes of the mother cell engulf the forespore, producing a small cell within a larger cell (7, 17, 21, 23). The two cells have different developmental fates. The forespore develops into the endospore, while the mother cell provides an environment that nurtures development and provides structural components of the endospore. Assembly of the coat is initiated by the expression in the mother cell of a group of morphogenetic proteins, SpoIVA, CotE, and SpoVID, which control the assembly of several of the coat structural components. As a result, their absence has profound impacts on coat structure and function. The production of the coat morphogenetic proteins occurs in the initial stages of the assembly process and is driven by the early mother cell-specific regulator ^E. Some coat structural components are also produced under the control of ^E, but most of the proteins found in the mature spore coat are produced during later stages of endospore development, when ^K becomes active, replacing ^E in the mother cell (6, 10). Although the synthesis of the different coat polypeptides is temporally regulated, their order of assembly and localization within the coat layers may rely mainly on specific protein-protein interactions, as well as a variety of posttranslational modifications, including proteolytic processing, secretion, cross-linking, and glycosylation (6, 10).

The SafA gene is predicted to encode a 45-kDa protein. This protein is found in the spore coat; however, a 30-kDa protein composed of the C-terminal region of SafA is also found in spore coats (20). Takamatsu et al. determined the N-terminal amino acid sequence of this 30-kDa form of SafA and found that the N-terminal residue is a methionine encoded by codon 164 of the safA gene (26, 27). They hypothesized that the 30-kDa form of SafA is produced from proteolytic cleavage of the full-length SafA protein (26, 27). However, in this work, we provide evidence that the 30-kDa form of SafA is produced primarily by initiation of translation at codon 164 of full-length safA mRNA transcript. This is the first report implicating alternative initiation of translation as the mechanism for the generation of a structural component of the bacterial endospore coat. The internal initiation of translation of the safA message must be subjected to precise control, because the slight overproduction of the 30-kDa form of SafA interferes with the transcriptional activity of the late mother cell regulator ^K and severely compromises sporulation.

	MATERIALS AND METHODS

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Bacterial strains, media, and general techniques. The B. subtilis strains used in this study are listed in Table 1. The Escherichia coli strain DH5 (Bethesda Research Laboratories) was used for transformation and amplification of all plasmid constructs. Luria-Bertani medium was routinely used for growth and maintenance of E. coli and B. subtilis strains, with appropriate antibiotic selection when needed (9). Nutrient exhaustion was used to induce sporulation in liquid culture or on plates of Difco sporulation medium (DSM) (19).

Spore resistance tests and spore purification. Spore resistance testsheat and lysozymewere performed as previously described (8, 9). Mature spores were harvested 48 h after the onset of sporulation and subjected to a step gradient for purification. Purified spores were used for downstream tests of germinability or for analysis of the coat protein profile on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (8-10).

Construction of strains carrying a C-terminal FLAG-tagged version of safA. Primers saf + 41-d and FLAG-R (which encodes the FLAG tag, DYKDDDDK; see Table 2) were used to amplify the safA coding region, excluding the C-terminal stop codon. The resulting 1,223-bp PCR product, in which the FLAG tag is fused in frame to the 3' end of safA, was cloned into the pCR2.1 TOPO vector (Invitrogen) to form pOZ130. A chloramphenicol cassette, released from pMS38 (M. Serrano and A. O. Henriques, unpublished data) by digestion with BglII and BamHI, was inserted into the BamHI site of pOZ130 to form pOZ139 (Table 1). Competent cells of MB24 were transformed with pOZ139, selecting for chloramphenicol resistance. Transformants were expected to arise as the result of a single reciprocal crossover (Campbell-type) event at the safA locus. A transformant, whose chromosomal structure in the vicinity of the safA locus was confirmed by PCR analysis, was chosen and named AOB90. Strain AOB90 carries two copies of safA: a functional upstream copy with a C-terminal FLAG tag and a downstream promoterless allele.

Preparation of B. subtilis whole-cell extracts and immunoblotting. For immunoblotting, samples (10 ml for the French press, 1 ml for lysozyme lysis) of DSM cultures of various strains were collected at intervals after the onset of sporulation. Whole-cell lysates were prepared by French press as described by Ozin et al. (20) or by gentle lysozyme lysis as described by Kodama et al. (14). Proteins were resolved on SDS-PAGE (12 or 7.5% polyacrylamide) gels as indicated in the figure legends. Immunoblotting was performed as in reference 20 with the following antibody concentrations: anti-SafA, 1:15,000 (20); anti--galactosidase, 1:50,000 (Promega); anti-FLAG, 1:30,000 (Sigma-Aldrich).

Construction of strains carrying transcriptional fusions of safA to the gusA gene. The safA gene, including 554 bp upstream from its start point of transcription, was amplified by PCR generated from primers saf-554d and saf + 1234R (Table 2). This 1,788-bp DNA fragment was cloned into the TA cloning site of pCR2.1 TOPO (Invitrogen) to produce pOZ115 (Table 1). The 5' region, from 554, 105, 22, or +83 nucleotides from the +1 start site of transcription, up to codon 387 of safA, was amplified from pOZ115 (Table 1) by using the reverse primer saf + 1234R paired with each of the direct primers saf-554d, saf-105d, saf-22, and saf + 83 (Table 2). The oligonucleotides were engineered with either a BamHI site or a BglII site so that the PCR products could be cloned into the BamHI site of pMLK83 (13). The resulting amyE integrational plasmids are pOZ150, pOZ148, pOZ156, and pOZ176, respectively. The orientation of the safA insert was confirmed by restriction digest and PCR analyses.

Construction of plasmids containing mutations in the SafA coding region. Plasmid pOZ115 (Table 1) served as a template for site-directed mutagenesis. Three independent alanine substitutions (M1A, M164A, and M161A) and a nonsense mutation (TGA) at codon 155 were made following the manufacturer's protocol for the Quick Change system (Stratagene) with the following primer sets: safM1A-d and safM1A-R, safM164A-d and safM164A-R, safM161A-d and safM161A-R, and saf155stop-d and saf155stop-R (Table 2). The mutations in the resulting plasmids (pOZ158, pOZ161, pOZ160, and pOZ138, respectively) were sequenced (Table 1).

Construction of strains containing alanine substitutions in safA at codons 164 and 161. Competent cells of a safA deletion mutant, AOB68 (Table 1), were transformed independently with plasmids pOZ178, pOZ179, pOZ140, and pOZ220 (Table 1). Transformants were isolated with selection for chloramphenicol resistance (Cm^r). The resulting clones, AOB145, AOB146, AOB91, and AOB236 (Table 1), were shown by PCR analysis to arise from a Campbell integration event (single crossover) in the regions of homology upstream from the safA gene. The presence of the correct mutations was confirmed by sequencing of the recombinant chromosomes.

Mapping the 5' terminus of safA mRNA. B. subtilis cells were grown in DSM, and 25 ml was harvested at 2.5 and 4.5 h after the onset of sporulation. RNA for primer extension analysis was prepared by using the RNeasy Maxi kit (Qiagen) according to the manufacturer's directions. Primer extension was performed essentially as previously described (11) with primers S + 107 and S + 187 (Table 2) 5' end labeled with -³²P (Promega). The same 5'-end-labeled primers were used to generate a sequence ladder by the dideoxy chain termination method with pOZ115 (Table 1) as a DNA template and the f-mol Cycle Sequencing kit (Promega) (22). The products of the primer extension were subjected to electrophoresis in a 6% (wt/vol) polyacrylamide slab gel containing 8 M urea and were detected on a PhosphorImager (Molecular Dynamics).

Construction of strains carrying safA::lacZ translational fusions ectopically inserted at the amyE locus. Primer set saf-121d and saf-pAC5R1 (Table 2) was used to PCR amplify the safA promoter region up to codon 167 independently from pOZ115, pOZ138, pOZ158, and pOZ160 (Table 1). Primers saf118d and saf-pAC5R2 (contains safA M164A substitution) or saf-pAC5R3 (contains safA M161A and M164A substitutions) (Table 2) were used to PCR amplify the same 663-bp region from pOZ115. The six resulting PCR products, flanked by engineered BamHI sites, were cloned into the BamHI site of pAC5 (Table 1) to create safA::LacZ translational fusion plasmids pOZ168 (safA::LacZ), pOZ175 (safA 155stop::LacZ), pOZ172 (safA M1A::LacZ), pOZ174 (safA M161A::LacZ), pOZ173 (safA M164A::LacZ), and pOZ189 (safA::M161A, M164A::LacZ), respectively. In all cases, the orientation of the insert was confirmed to be in the same direction as lacZ by PCR and restriction digest analysis.

Construction of a strain overproducing the C-terminal form of SafA or the SafA promoter. We used primers saf-554d (BamHI) and saf + 1234R (BglII) to PCR amplify a 1,788-bp fragment, containing safA M1A plus the promoter region from a plasmid template, pOZ158 (Table 1). The resulting PCR products, flanked by engineered BamHI-BglII sites, were cloned into the BamHI site of plasmid pMK3 (24), which replicates in B. subtilis, thereby creating pOZ194 (Table 1). We also cloned the promoter region of safA plus 30 bp of the coding region into pMK3 by PCR amplifying a 584-bp fragment and cloning it first into pCR 2.1 TOPO, to create pOZ217. The insert was then excised from pOZ217 by digestion with EcoRI, gel purified, and ligated into the EcoRI site of pMK3 to create pOZ218 (Table 1).

Enzyme assays. The activities of -galactosidase and -glucuronidase were determined with the substrates o-nitrophenyl--D-galactopyranoside (ONPG) and p-nitrophenyl--D-glucopyranoside (PNPG), respectively, as previously described (9). In both cases, cells were permeabilized by treatment with toluene. Enzyme-specific activity is expressed in nanomoles of substrate (ONPG or PNPG) hydrolyzed per milligram (dry weight) per minute.

	RESULTS

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A single promoter for safA. Takamatsu et al. (27) used Northern blots and primer extension analysis to identify a ^E-dependent transcript of the safA gene. We refer to the promoter driving expression of this transcript as safA P1. In order to seek other promoters of safA expression, we fused a series of DNA fragments from the safA region to a promoterless gusA reporter gene and looked for the minimal region required for gusA expression. All of the DNA fragments from the safA gene contained the C terminus of the safA gene and extended upstream to various points up to 554 bp upstream from the start point of the safA P1. These fusions were moved into the amyE locus of a wild-type strain, and the -glucuronidase activity of the resulting strains was measured during sporulation (Fig. 1). The safA DNA fragment that extended 105 bp upstream from the start point of transcription directed gusA expression, whereas the fragment that extended only 22 bp upstream failed to promote gusA expression. Therefore, any promoter located upstream or within safA requires sequences located at least 23 bp upstream from the start point of safA P1.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Defining the promoter region(s) in safA. (A) Illustration of regions of safA (solid bars) that were cloned into a gusA transcriptional fusion plasmid construct, pMLK83 (Table 1), to make the indicated B. subtilis strains, AOB147, AOB105, AOB95, and AOB98. Numbers indicate the distance from the P1 +1 start site of transcription. (B) Measurement of transcriptional activity of safA-gusA. Circles, AOB98; triangles, AOB95; squares, AOB105; diamonds, AOB147.

Accumulation of the 30-kDa short form of SafA does not require expression of the full-length form. The N-terminal sequence of a 30-kDa form of SafA (SafA-C₃₀) begins with a methionine encoded by codon 164 of safA (26, 27), and the transcriptional analysis suggests that there is only a full-length transcript driving expression of safA (27; this work). Therefore, accumulation of SafA-C₃₀ may arise from proteolytic processing of the full-length protein or from initiation of translation at codon 164 within the full-length transcript. To distinguish between these two possibilities, we constructed a B. subtilis strain, AOB91, carrying a stop codon just upstream of a predicted ribosome-binding site (RBS) preceding M164, to block formation of the full-length form (SafA-FL) (Fig. 2A). We monitored the accumulation of SafA products by immunoblot analysis with anti-SafA antiserum. As we had shown previously (20), no SafA products were detected in an extract from a SafA null mutant (data not shown). However, we found that even in the absence of full-length SafA (SafA-FL), a 30-kDa form of SafA was detectable by immunoblots of extracts from sporulating cells of AOB91 probed with the anti-SafA antibody (Fig. 2B). If this 30-kDa form of SafA (Fig. 2B, lanes a to c) is the same as the 30-kDa form produced in the wild type (SafA-C_30; Fig. 2B, lane d), we would conclude that production of the 30-kDa form is not dependent on the production of full-length SafA. We confirmed that the 30-kDa species produced in AOB91 was the same as the SafA-C₃₀ in the wild type by using an anti-FLAG antibody to probe immunoblots of extracts from strains containing C-terminally FLAG epitope-tagged versions of SafA, with and without the stop codon mutation (Fig. 2C). Therefore, production of the 30-kDa form probably results from initiation of translation at codon 164. This model predicts that the AUG codon at 164 is required for SafA-C₃₀ production. This prediction was tested as described below. In addition to the 30-kDa band, a band of about 21 kDa in extracts of AOB91 reacted with anti-SafA antiserum. Presumably, this band results from termination of translation at the TGA nonsense codon introduced into the safA coding sequence, which would produce a polypeptide of about 17.7 kDa (Fig. 2A). Interestingly, a band with about the same apparent mobility is detected in extracts prepared from the wild-type strain, as is a band that migrates between the 30- and 21-kDa species (Fig. 2B, lane d). The band that migrated between the 30- and 21-kDa species was not detected when an anti-FLAG monoclonal antibody was used to monitor the expression of C-terminal FLAG-tagged alleles of SafA (Fig. 2C). Therefore, it may be derived by proteolytic removal of the C-terminal region.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Detection of different forms of SafA in a safA-stop codon mutant during sporulation. (A) Diagram of the SafA coding region (amino acid residues 1 to 387) showing the position of the stop codon mutation engineered at codon 155 (star), an internal RBS (GGAGG), and the ATG methionine codon at position 164. (B) Immunoblots of whole-cell extracts (French press method) were prepared at the indicated times (hours) after the onset of sporulation and probed with anti-SafA antibodies. Lanes a to c, AOB91 (safAstop155-FLAG); lane d, wild type (MB24). The asterisk marks the position of the 30-kDa form of SafA (C30), the arrow marks the position of the full-length SafA (FL), and the large arrowhead marks the position of the 21-kDa band described in the text. (C) Whole-cell extracts (lysozyme method) of strains AOB232 (safA with a TGA stop codon at 155 and a C-terminal FLAG tag) (lanes a and b) and AOB90 (safA with a C-terminal FLAG tag) (lanes c and d) were harvested at 4 and 6 h after the onset of sporulation and probed with antibodies against the FLAG epitope.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   Accumulation of SafA in safA M164A and safA M161A mutants. Immunoblots of extracts (lysozyme method) from sporulating cells at the indicated times (hours) after the onset of sporulation, probed with anti-SafA antibodies. (A) Lanes a to e, wild type (MB24); lanes f to j, AOB146 (safA M164A). The arrow marks the position of SafA-C30. This form is missing in lanes f to j. (B) Lanes a to e, wild type (MB24); lanes f to j; AOB145 (safA M161A). The arrow marks the position of a band that is not present in wild-type extracts. In both panels, the asterisk indicates the expected position of SafA-C30. This form accumulates in both the wild type and the AOB145 mutant, but not the AOB146 mutant.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Activity and accumulation of safA::lacZ wild-type and mutant translation fusions. (A) Diagram of the safA coding region and the safA::lacZ fusion construct junction. The positions of the alanine substitutions and the stop codon mutation are symbolized by white dots and labeled M1A (a), 155stop (stop codon at residue 155) (b), M161A (c), M164A (d), and M161A and M164A (e), respectively. (B) Comparison of the translational activity of wild-type safA::LacZ and various mutants in the safA portion of safA::LacZ. The error in measurement of -galactosidase activity is ±5%. (C) Immunoblot of extracts (lysozyme method) taken at 5 h after the onset of sporulation from strains containing the wild-type or mutant versions of safA::LacZ. The blot was probed with monoclonal antibodies against -galactosidase. Lane a, wild-type safA::LacZ (strain AOB125); lane b, wild-type safA M1A::LacZ (AOB135); lane c, safA-stop155::LacZ (AOB148); lane d, safA M161A::LacZ (AOB149); lane e, safA M164A::LacZ (AOB150); lane f, safA M161A, M164A::LacZ (AOB200).

Overexpression of SafA-C₃₀ causes a stage IV sporulation block. We examined the effects of mutations in safA on spore coat function and structure by performing heat and lysozyme tests, SDS-PAGE analysis of proteins extracted from the spore coat, germination assays, and analysis of spore coat ultrastructure by electron microscopy. We determined that expression of just SafA-C₃₀ (AOB91) is not sufficient for lysozyme resistance and has the same phenotype as a safA deletion mutant (not shown). Thus, expression of the full-length protein is required for assembly of the spore coat. In contrast, single alanine substitutions at codons 161 and 164 (AOB145 and AOB146) and double alanine substitutions at codons 161 and 164 (AOB236) had no detectable effect on spore coat structure or function (not shown).

View larger version (41K): [in this window] [in a new window]   FIG. 5.   SafA-C30 is overproduced during sporulation on a multicopy plasmid. Immunoblots of extracts (lysozyme method) from sporulating cells at the indicated times (hours) after the onset of sporulation, probed with anti-SafA antibodies. Lanes a to e, wild-type strain containing pOZ194, which contains the safA promoter and coding region and an alanine substitution at codon 1 (AH2655); lanes f to i, wild-type strain containing the pMK3 vector-only control (AH2654).

View larger version (31K): [in this window] [in a new window]   FIG. 6.   The effect of overproducing SafA-C30 on sporulation-specific gene expression. -Galactosidase production was monitored throughout sporulation in DSM cultures of a collection of congenic strains bearing fusions of E-, G-, and K-dependent promoters to the lacZ gene and containing plasmid pMK3, pOZ194, or pOZ218 (E-dependent spoIID-lacZ fusion in panel A, G-dependent sspE-lacZ fusion in panel B, K-dependent sigK-lacZ fusion in panel C, and K-dependent gerE-lacZ fusion in panel D). Symbols represent strains containing vector-only control, pMK3 (diamonds); safA M1A-pMK3, pOZ194 (circles); or safA promoter region-pMK3, pOZ218 (triangles). Strains without a lacZ fusion are represented by squares.

	DISCUSSION

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SafA accumulates as multiple forms during sporulation. The SafA-C₃₀ form is encoded by the C terminus of the safA gene (20, 27; this work). In this study, we have shown that blocking expression of the full-length SafA, by placing a stop codon just upstream of M164, did not block the accumulation of a 30-kDa SafA species (Fig. 2B). Moreover, an alanine substitution of M164 specifically eliminated the accumulation of SafA-C₃₀ (Fig. 3A). These and the other data presented support a mechanism in which initiation of translation at codon 164 produces SafA-C₃₀. The safA gene appears to be transcribed from a single promoter; therefore, translation is initiated at both codons 1 and 164 of a single primary transcript. It is not known whether translation is initiated from both positions on every transcript or whether posttranscriptional modifications determine which initiation site will be used on each transcript. The kinetics of synthesis of both translation products is also not well established. In Fig. 3, for example, it appears that SafA-FL accumulates before SafA-C₃₀. However, this result may be an artifact, because the SafA antiserum may react more strongly to the N-terminal region of the protein. The FLAG-tagged versions of SafA-FL and SafA-C₃₀ appeared to accumulate simultaneously when probed with anti-FLAG antibody (data not shown). Therefore, SafA-FL and SafA-C₃₀ may be synthesized simultaneously. Moreover, the amount of SafA-FL falls in the later stages of sporulation. If SafA-FL is degraded during sporulation more rapidly than SafA-C₃₀, the relative rates of synthesis of these two proteins cannot easily be determined from the apparent rates of accumulation. Recently, Takamatsu et al. (25) presented evidence that SafA is proteolytically processed by YabG. They showed that the 45-kDa form of SafA and a 31-kDa form of SafA accumulate early during sporulation, but disappear as new forms with sizes of 42 and 30 kDa accumulate, and this appearance of the 42- and 30-kDa forms was dependent on YabG. Our evidence supports the model that SafA-C₃₀ is made primarily from translation initiation at codon 164. However, other SafA products may arise from proteolytic events. For example, the 45-kDa form (SafA-FL) may be processed to the 42-kDa form. We do not know whether or which of the 30- or 31-kDa forms of SafA described by Takamatsu et al. (25) corresponds to the SafA-C₃₀ described by us. However, since our data show that SafA-C₃₀ is made primarily from translation initiation at codon 164, we speculate that SafA-C₃₀ is the form that they call the 31-kDa form. Furthermore, the 31-kDa form may be processed at its carboxy terminus by the protease to produce the form they call the 30-kDa form. In some of our experiments, the addition of an epitope tag or fusion of -galactosidase to the carboxy-terminal end of SafA may have inhibited proteolytic processing.

SafA is the first example of a coat protein that is expressed as two forms as a result of internal initiation of translation. There are examples in other bacterial systems where alternatively translated gene products may be involved in subtle functional adaptations and for regulation of protein activity. For example, CheA, a histidine protein kinase of the chemotaxis signal transduction system, is synthesized as two forms by in-frame initiation sites within the cheA gene (18). The full-length form (CheAL) is essential for signal transduction, but the function of the short form (CheAs) is unclear. CheAs is coexpressed with CheAL in a variety of enteric clinical isolates, and it is proposed to be important for chemotactic responses in specialized environmental niches. In contrast, the holins of lambda phages use internal initiation of translation (dual start motifs) for expression of a long form and a short form, differing by only a few codons. The long form interacts with the short form, an interaction that regulates the activity of the short form by holding it in an inactivated state (2, 3, 28)

The long and short forms of SafA appear to have different roles. The full-length form of SafA is necessary for formation of an intact coat, since the stop codon mutant (AOB91; Fig. 2A) had the same phenotype as a safA deletion mutant, AOB68 (20). The function of the 30-kDa short form is less clear. Blocking accumulation of SafA-C₃₀ with single or double alanine substitutions at methionines 161 and 164 did not have a detectable effect on spore coat function or morphology. However, overexpression of SafA-C₃₀ on a multicopy plasmid drastically blocked sporulation at stage IV. Further work is required to discover the role of SafA-C₃₀.