INTRODUCTION

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Dormant spores of Bacillus subtilis contain a number of proteins which are not present in growing cells, including spore coat proteins, components of the spore germination apparatus, a few unique spore enzymes, and a group of small, acid-soluble spore proteins (SASP) (38, 39). Among the latter proteins are the multiple /-type SASP and the single -type SASP; three of these proteins (SASP , , and ) make up the great majority of all SASP in spores (38, 40). However, B. subtilis spores also contain a number of minor SASP, and similar minor proteins are present in spores of other Bacillus species (16, 37, 38, 46). While one of the minor SASP in B. subtilis is a minor /-type SASP termed SspC (46), the identity of the other minor proteins is not known.

Identification and analysis of these additional minor SASP and study of the regulation of their coding genes may be of interest for a number of reasons. First, since the minor SASP are undoubtedly very small, it is possible that their coding regions were not identified as open reading frames (ORFs) in the recently completed B. subtilis genomic sequence (18). Identification of any new ORFs will thus assist in completion of the analysis of the genomic sequence. Second, if the new minor SASP are indeed spore-specific proteins, then their coding genes should exhibit sporulation-specific expression. Study of the regulation of expression of these new genes, and in particular of their dependence on sporulation-specific sigma factors for RNA polymerase and their promoter sequences, would thus expand our knowledge of regulation of sporulation-specific genes. Finally and most importantly, several SASP, particularly the major /-type SASP, have major functions in the dormant spore in (i) providing resistance to spore DNA against damage caused by heat and oxidizing agents (6, 40); (ii) altering spore DNA photochemistry, thus providing a major element of spore UV resistance (25, 36, 40); and (iii) generating free amino acids for protein synthesis by their degradation early in spore germination (38). It is certainly possible that the new minor SASP has redundant functions in the spore, and thus loss of only one may have no phenotypic effect or at most a minor one, as is the case for the two major /-type SASP (25, 40). However, the essential role of the latter proteins in several of the properties unique to or characteristic of bacterial spores suggests that the new minor SASP might also have some specific function in sporulation, spores, or spore germination. Consequently, mutagenesis of the genes encoding these new minor SASP, alone or in various combinations, might give new insight into mechanisms determining various aspects of sporulation, spore properties, and spore germination. Given these reasons, we have determined the N-terminal amino acid sequences of minor B. subtilis SASP and have identified the genes encoding 11 of these proteins; five of these genes were not identified as ORFs in the B. subtilis genomic sequence. We also report detailed studies on the regulation of expression and function of two of the latter genes, both of which are new sporulation-specific genes.

	MATERIALS AND METHODS

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Bacterial strains and spore preparation. Escherichia coli TG1 (33) and DH5 (11) were used for cloning; the B. subtilis strains used in this study are listed in Table 1. B. subtilis PS482 was used for identification of minor SASP, as this strain carries deletions of the sspA, -B, and -E genes, which code for the three major B. subtilis SASP, , , and , respectively (9). This strain (termed ) was sporulated at 37°C in 2× SG medium (8), and the spores were purified and stored as described previously (29). Growing cells of strain PS482 were prepared in the same medium but harvested in the late log phase of growth (optical density at 600 nm [OD₆₀₀]  1) and washed once with 0.15 M NaCl, and the cell pellet fraction was frozen and lyophilized. B. subtilis strains with the PS832 background were used to study sspG and sspJ expression and for analysis of the phenotypes of the sspG and sspJ mutants; B. subtilis strains with a PY79 genetic background were used for studies of the genetic dependence of sspG and sspJ expression. PS832 and PY79 are very similar wild-type strains of B. subtilis, but PS832 sporulates slightly more efficiently, while a number of mutations in genes for sporulation sigma factors are available in the PY79 background. All transformations of B. subtilis strains were carried out as described previously (1).

Identification and analysis of minor SASP. Lyophilized dormant spores (100 mg [dry weight]) or late-log-phase cells were broken in a dental amalgamator (Wig-L-Bug) with glass beads (100 mg) as the abrasive for either 10 min (spores) or 2 min (growing cells). The dry powder was extracted twice at 4°C for 30 min with 5 ml of 3% acetic acid, and the supernatant fluids were combined and dialyzed against 1 liter of 1% acetic acid in Spectrapor 3 tubing (molecular weight cutoff, 3,500) for 16 h at 6°C with two changes. The pellet from the acetic acid extract was further extracted twice with 5 ml of 0.3 N HCl at 4°C, and the supernatant fluids were pooled and dialyzed as described above. The dialyzed material was lyophilized, and the dry residue was dissolved in 200 µl of 8 M urea plus 100 µl of acid gel diluent (31). Aliquots of the redissolved material were subjected to polyacrylamide gel electrophoresis (PAGE) at low pH (31), proteins were electrophoretically transferred to polyvinylidine difluoride paper (Immobilon) in 10% methanol-0.7% acetic acid for 45 min at 200 mA (24), and the proteins on the paper were stained lightly with Coomassie blue, destained, and air dried. Selected protein bands were cut from the paper with a clean razor blade and subjected to protein sequence analysis as described previously (46).

Construction of B. subtilis strains containing translational sspG- and sspJ-lacZ fusions. Fragments encompassing 220 bp upstream of the sspG ORF as well as 22 bp of the coding region or 201 bp upstream of the sspJ ORF and 20 bp of the coding region were amplified by PCR. The primers used for sspG were prot2TXN5' (5'-GGAATTCGAGATGATAAGCCGTCG-3') and prot2TXN3' (5'-CGGGATCCTTTTCATGACGATTTTCGCTC-3'). The primers used for sspJ were prot3-5' (5'-GGAATTCGCGATGCCTCCCATGATG-3') and prot3-3' (5'-CGGGATCCTCTTTATTAAAGAAACCCATTC-3'). In all cases, the primers had extra residues at the 5' end, including an EcoRI or a BamHI site (underlined). The PCR fragments were cut with EcoRI and BamHI and cloned between the EcoRI and BamHI sites of pJF751, a vector for construction of translational lacZ fusions (7). The resulting plasmids, termed pIB454 (sspG) and pIB452 (sspJ), were integrated into the PS832 chromosome by a single crossover event with selection for Cm^r. Transformants containing a single copy of the translational sspG- or sspJ-lacZ fusion at the sspG or sspJ locus as shown by Southern blot analysis were called strains IB464 and IB465, respectively. Chromosomal DNA was isolated from these strains and used to transform B. subtilis strains containing different spo mutations in the PY79 background or strain 522.2 to Cm^r.

Analysis of -galactosidase activity in sporulating cells, spores, and vegetative cells. Sporulation of B. subtilis was induced at 37°C by the resuspension method (43) or by the nutrient exhaustion method in 2× SG medium (8, 20), and samples (1 ml each) were harvested by centrifugation and stored frozen. Strains carrying genes encoding ^F, ^G, or ^K under the control of the isopropyl--D-thiogalactopyranoside (IPTG)-inducible spac promoter (Pspac) were grown at 37°C in 2× YT medium to an OD₆₀₀ of 0.25. The culture was then divided in half, IPTG was added to 2 mM to one-half, incubation was continued, and samples were taken and stored frozen as described above. -Galactosidase activity was determined with o-nitrophenyl--D-galactopyranoside as the substrate as described previously (29); lysozyme (200 µg/ml) was used for cell permeabilization prior to enzyme assay. To analyze -galactosidase activity in spores, the spores were first decoated and then treated with lysozyme prior to enzyme assays as described previously (29). All -galactosidase specific activities are expressed in Miller units (27).

Determination of the sspG-yurS and sspJ transcription start sites. Total RNA was extracted from cells of strains IB464 and IB465 sporulating in 2× SG medium as described previously (28). The RNA was used in primer extension reactions at 47°C with avian myeloblastosis virus reverse transcriptase (28). The primers used were prot2-55 (5'-CTTTTGCTAATCCGCTGTTTTGG-3'), which anneals to sspG mRNA; yurS-140 (5'-GTTAAGAGGAATGATGTTTTCGTTC-3'), which anneals to yurS mRNA; prot3-50 (5'-TCTTCAAGAGCTCCTTGGATTAC-3'), which anneals to sspJ mRNA; and lacZ-70 (5'-AAGGCGATTAAGTTGGGTAACG-3'), which anneals to the lacZ portion of sspG-lacZ or sspJ-lacZ mRNAs. Size standards for analysis of the primer extension products were produced with the same four primers in DNA sequencing reactions. The prot2-55 and yurS-140 primers were used with plasmid pIB517, which carries a 1,055-bp fragment encompassing the sspG and yurS region (see below); the prot3-50 primer was used with plasmid pIB460, which carries a 1,190-bp fragment encompassing the sspJ region (see below); and the lacZ-70 primer was used with plasmids pIB454 and pIB452, which carry the translational sspG-lacZ and sspJ-lacZ fusions, respectively, in plasmid pJF751.

Cloning of a fragment encompassing the sspG-yurS and sspJ regions. A fragment encompassing 1,055 bp of the sspG-yurS region was amplified by PCR. The primers used were prot2TXN5' (see above) and prot2mut4 (5'-GCTCTAGATCAAGACATGGCACTGG-3'). The PCR product was cloned in the TA-cloning vector pCR2.1 (Invitrogen) according to the manufacturer's instructions, and the resulting plasmid was called pIB517.

Construction of an sspG-yurS null mutant. A 490-bp fragment containing the region directly upstream of the sspG ORF was amplified by PCR. The primers used were prot2mut1 (5'-GGAATTCTTATACGCCCTTTCCTCC-3') and prot2mut2 (5'-AACTGCAGGTATCATCCTTTCTCTATTG-3'), each containing extra residues, including an EcoRI or PstI site at their 5' ends (underlined). The PCR fragment was cut with EcoRI and PstI and cloned between the EcoRI and PstI sites of plasmid pJL74 (19), which contains an Sp^r cassette, giving plasmid pIB458. A 539-bp fragment containing the region starting 149 bp downstream of the sspG ORF and encompassing the second half of the yurS ORF was amplified by PCR. The primers used were prot2mut3 (5'-CGGGATCCGCCGCAAAGCAGATGAC-3') and prot2mut4new (5'-ATAAGAATGCGGCCGCATCAAGACATGGCACTGG-3'), each containing extra residues, including a BamHI or NotI site at their 5' ends (underlined). The PCR fragment was cut with BamHI and NotI and cloned between the BamHI and NotI sites of plasmid pIB458. The resulting plasmid (pIB479) contains the sspG flanking regions, with the Sp^r cassette replacing the sspG ORF and the first half of the yurS ORF. pIB479 was linearized with XhoI and used to transform B. subtilis PS832 to Sp^r (100 µg/ml). In this transformation the Sp^r cassette was integrated into the B. subtilis chromosome by a double crossover event removing the sspG ORF and the first half of the yurS ORF. One Sp^r transformant whose expected chromosomal structure was confirmed by Southern blot analysis (data not shown) was termed IB488. To construct an sspG-yurS null mutant that did not produce SASP , , and , we transformed strain PS482 to Sp^r with chromosomal DNA from strain IB488. The resulting Cm^r Sp^r strain was called IB511.

Construction of the sspJ null mutant. A 521-bp fragment containing the region immediately downstream of the sspJ ORF was amplified by PCR. The primers used were prot3mut1 (5'-GGAATTCGCTCCAAACGGACTCGC-3') and prot3mut2 (5'-AACTGCAGCCACATGCGGATAGGGC-3'), each containing extra residues, including an EcoRI or PstI site at their 5' ends (underlined). The PCR fragment was cut with EcoRI and PstI and cloned between the EcoRI and PstI sites of plasmid pJL74 (19), giving plasmid pIB476. A 513-bp fragment containing the first 7 bp of the sspJ ORF and 506 upstream bp was amplified by PCR. The primers used were prot3mut3 (5'-CGGGATCCAACCCATTCGTATCACCTC-3') and prot3mut4 (5'-TCCCCGCGGTGATTCCGTTCACCGTCC-3'), each containing extra residues, including a BamHI or SacII site at their 5' ends (underlined). The PCR fragment was cut with BamHI and SacII and cloned between the BamHI and SacII sites of plasmid pIB476. The resulting plasmid (pIB482) contains the sspJ flanking regions, with the Sp^r cassette replacing the sspJ ORF. pIB482 was linearized with XhoI and used to transform B. subtilis PS832 to Sp^r. In this transformation the Sp^r cassette was integrated into the B. subtilis chromosome by a double crossover event, removing the sspJ ORF. One Sp^r transformant whose chromosomal structure was confirmed by Southern blot analysis (data not shown) was termed IB490. To construct an sspJ null mutant which does not produce SASP , , and , we transformed strain PS482 to Sp^r with chromosomal DNA from strain IB490. The resulting Cm^r Sp^r strain was called IB515.

Analysis of resistance, germination, and outgrowth of B. subtilis spores. Spores were harvested from cultures grown for 48 h at 37°C in 2× SG medium and purified as described previously (25, 29). Spores in water were heat treated (85°C) or UV irradiated with 254-nm light, and survival was measured as described previously (6, 25). For analyses of spore lysozyme and chloroform resistance, spores were diluted to an OD₆₀₀ of 1 in 10 mM potassium phosphate buffer (pH 7.4) containing 50 mM KCl and 1 mM MgSO₄ and treated with lysozyme at 1.5 mg per ml for 30 min at 37°C or with chloroform as described previously (29).

	RESULTS

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Identification of new SASP and their coding genes. PAGE at low pH of acetic acid extracts of B. subtilis spores (termed ) lacking the three major SASP gave a series of protein bands, none of which were present in acetic acid extracts of growing cells (Fig. 1, lanes a and b). The levels of at least some of these acetic acid-soluble proteins unique to spores were similar in both and wild-type spores (data not shown); however, the presence of SASP , , and  precluded the observation of several of the minor protein bands in extracts of wild-type spores. Consequently, all routine analysis of these proteins was carried out with spores. Subsequent extraction of spores with HCl dissolved more of the proteins that were present in the acetic acid extract as well as a number of additional proteins (Fig. 1, lane c). However, many of these latter additional proteins appeared to be present in the HCl extract of growing cells (Fig. 1, lane d). Consequently, we focused on the acetic acid-soluble proteins from spores and identified 10 distinct protein bands which were present in significant amounts in acetic acid extracts from spores but not in HCl extracts of growing cells (Fig. 1, lane e). Automated protein sequence analysis showed that band 8 was the product of the cotK gene, while band 5 has been shown previously to be the minor /-type SASP SspC (Table 2) (46). Band 4 gave two different protein sequences, one identical to the product of an additional gene, sspD, coding for a minor /-type SASP (SspD), with the other sequence derived from an ORF encoded by a gene of unknown function termed ysfA. All other bands gave single unambiguous amino acid sequences. Two were products of additional ORFs of unknown function which had been identified in the B. subtilis genomic sequencing project, while five were products of small ORFs in intergenic regions which had not previously been identified as coding genes (Table 2). None of these five new proteins exhibits any significant sequence similarity to known proteins (data not shown). Since the new proteins identified are small, acid-soluble proteins that appear unique to spores, with two exceptions we have termed them SASP and their coding genes ssp (Table 2). The exceptions are (i) the gene encoding band 2, which had been termed tlp, based on the encoded protein's homology to thioredoxin; and (ii) the gene encoding band 8, termed cotK, for which there is some evidence that the encoded protein is a spore coat protein (12).

View larger version (66K): [in this window] [in a new window]   FIG. 1.   Low-pH PAGE analysis of minor SASP from B. subtilis. Acetic acid and HCl extracts from dormant spores and growing cells of strain PS482 were prepared and redissolved as described in Materials and Methods, aliquots were run on PAGE at low pH, either the gel was stained with Coomassie blue (lanes a to d) or proteins were transferred to polyvinylidene difluoride paper, and the paper was stained with Coomassie blue (lane e). The samples (and amounts) of redissolved extract run in the various lanes were as follows: a and e, acetic acid extract of spores (20 µl); b, acetic acid extract of growing cells (20 µl); c, HCl extract of spores (20 µl); and d, HCl extract of growing cells (5 µl). The numbers adjacent to lane e denote protein bands present in spores but not in growing cells and are the numbers of the bands analyzed in Table 2.

Properties of sspG and sspJ. While the new SASP described above appeared unique to the spore, we decided to investigate this in more detail by examining the regulation of expression of two of the genes encoding these proteins. The two we chose for this detailed analysis were sspG and sspJ.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Nucleotide sequence of the sspG-yurS (A) and sspJ (B) regions. (A) The sequence shown includes nucleotides 3352955 to 3353300 in the B. subtilis genome. The sspG start and stop codons are in bold and underlined. The start codon for yurR is underlined, and that for yurS is in bold. Sequences corresponding to 10 and 35 promoter elements and the sspG and yurS ribosome binding sites (RBS) as well as those corresponding to the start of transcription of sspG-yurS (+1) are underlined. The consensus 35 and 10 sequences for K-dependent promoters (10, 48) are shown in bold below these elements in the sspG sequence; the abbreviation used in the 35 consensus sequence is H for A or C. The boxed residues denote putative GerE binding sites (32); one is from positions 3353061 to 3353050 on the strand transcribed to give sspG, and the other is from positions 3353013 to 3353024 on the nontranscribed strand. (B) The sequence shown includes nucleotides 3420842 to 3420491 in the B. subtilis genome (note that the direction of transcription of sspJ is counterclockwise). The sspJ start and stop codons are in bold and underlined; the start codon for yvsG is underlined. Sequences corresponding to 10 and 35 promoter elements and the sspJ ribosome binding site (RBS) as well as those corresponding to the start of transcription (+1) are underlined. The consensus 10 and 35 sequences for G-dependent promoters (10) are shown in bold below these elements in the sspJ sequence. The designations in the consensus sequence are H for A or C, R for A or G, and X for A or T. The two underlined G residues upstream of the 10 consensus sequence show the position of the two G residues in promoters recognized primarily by F (44). The apposed arrows denote the putative transcriptional terminator of sspJ.

Expression and regulation of sspG and sspJ during sporulation. To examine sspG and sspJ expression, we integrated translational sspG- and sspJ-lacZ fusions at the sspG and sspJ loci, respectively, and measured -galactosidase activity during vegetative growth and sporulation and in dormant spores. No significant expression of either fusion was observed in vegetatively growing cells (data not shown and see below). However, the sspG-lacZ fusion was expressed beginning ~4 h after the induction of sporulation, with maximum -galactosidase specific activity attained ~6 h after induction of sporulation (Fig. 3A and data not shown); the sspJ-lacZ fusion was expressed beginning at ~2.5 h after induction of sporulation, with maximum specific activity attained ~4 h after induction of sporulation (Fig. 4A). No detectable -galactosidase activity was found in purified spores of the sspG-lacZ fusion, but ~100% of sspJ-driven -galactosidase was found in purified spores (data not shown). These data indicate that both sspG and sspJ are sporulation-specific genes which are likely expressed in the mother cell and the forespore compartment, respectively, of the sporulating cell.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Expression of the translational sspG-lacZ fusion in various spo mutants. Strains with a PY79 background (A) and a CU267 background (B) were sporulated by the resuspension method, and -galactosidase was assayed as described in Materials and Methods. Time 0 is when sporulation was initiated. The symbols used for the various strains are as follows. (A) , IB470 (spo+); , IB469 (spoIVCB), (B) , IB492 (spo+); , IB494 (gerE36); , IB498 (this strain does not contain an sspG-lacZ fusion but rather has a cotA-lacZ fusion in a spo+ background).

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Expression of the translational sspJ-lacZ fusion in various spo mutants. Strains with a PY79 background were sporulated by the resuspension method, and -galactosidase was assayed as described in Materials and Methods. Time 0 is when sporulation was initiated. The symbols used for the various strains are as follows. (A) , IB475 (spo+); , IB474 (spoIVCB), (B) , IB471 (spoIIAC); , IB472 (spoIIGB); , IB473 (spoIIIG).

View larger version (11K): [in this window] [in a new window]   FIG. 5.   Induction of expression in vegetative growing cells of sspG-lacZ in cells expressing K (A) or sspJ-lacZ in cells expressing F (B) or G (C). Cells of strains IB502 (sspG-lacZ Pspac-K) (A), IB480 (sspJ-lacZ Pspac-F) (B), or IB481 (sspJ-lacZ Pspac-G) (C) were grown at 37°C in 2× YT medium. An OD600 of 0.25 (time 0 in the figure), the cultures were divided in half, one-half was made 2 mM in IPTG, incubation was continued, and samples were taken from both cultures for assay of -galactosidase. , without IPTG; , with IPTG. Note the different scales in panels B and C.

Localization of the sspG-yurS and sspJ promoters. To localize the sspG promoter and to determine if yurS is also transcribed from the same promoter, we carried out primer extension analysis with RNA from sporulating cells of strain IB464 containing the translational sspG-lacZ fusion at the sspG locus. Three different primers were used for this analysis; one was annealed to the lacZ portion of sspG-lacZ mRNA, another annealed only to the sspG mRNA, and the third was complementary to RNA transcribed from yurS. All three primers gave the same transcription start site, indicating that sspG and yurS are indeed transcribed from the same promoter, with transcription initiating 23 nt upstream of the sspG AUG codon, at an A residue (Fig. 2 and 6 and data not shown). Additionally, sequences centered approximately 10 and 35 nt upstream of the transcription start site show good similarity to the 10 and 35 consensus sequences recognized by ^K, and upstream of the 35 element are several putative GerE binding sites (see Discussion).

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Primer extension analysis of the start site for transcription of sspG and yurS. RNA from cells of strain IB464 was isolated 7.5 h into sporulation, and the primer extension product was obtained and analyzed as described in Materials and Methods. The primer used is yurS-140, which anneals only to yurS. Lanes a, g, c, and t are DNA sequencing reactions with the same primer and plasmid pIB517; lane 1 is a primer extension reaction with sporulating-cell RNA. The primer extension product is marked with an arrow, and the transcription start site on the sspG upstream sequence to the left of the figure is marked with a square. Note that the sequence shown is the complement of the mRNA sequence.

View larger version (33K): [in this window] [in a new window]   FIG. 7.   Primer extension analysis of the sspJ transcription start site. RNA from cells of strain IB465 was isolated 5 h into sporulation, and primer extension products were obtained and analyzed as described in Materials and Methods. The primer used is prot3-50, which anneals only to sspJ mRNA. Lanes a, g, c, and t are DNA sequencing reactions with the same primer and plasmid pIB460; lane 1 is the primer extension reaction with sporulating-cell RNA. The primer extension product is marked with an arrow, and the transcription start site on the sspJ upstream sequence to the right of the figure is marked with a square. Note that the sequence shown is the complement of the mRNA sequence.

Characterization of sspG-yurS and sspJ null mutants. While it was clear that both SspG and SspJ were spore-specific gene products, it was not clear if these proteins had any function in the spore or any role in sporulation or spore germination. Consequently, we generated and analyzed sspG and sspJ null mutants. In the sspG-yurS mutant, the entire sspG ORF and the first half of the yurS ORF were removed, and a Sp^r cassette was inserted; in the sspJ mutant the Sp^r cassette was substituted for the sspJ ORF. To confirm that the mutations eliminated synthesis of SspG and SspJ, we transferred each of the mutations into a strain lacking the three major SASP, purified spores of the resultant strains, and extracted and analyzed SASP from these spores by PAGE at low pH. As expected, the bands corresponding to SspG and SspJ were absent from the extracts of the sspG-yurS and sspJ mutant spores, respectively (data not shown). In an attempt to localize SspG, and possibly YurS, to spore coats, we also extracted coat proteins from wild-type and sspG yurS spores by boiling and analyzed the extracts by SDS-PAGE as described in Materials and Methods. However, the coat proteins revealed by this analysis had molecular weights that did not even approximate those of the proteins that were absent from the mutant spores (data not shown).

	DISCUSSION

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The identification of 10 new proteins in spores from their amino-terminal sequences confirms that four ORFs identified by sequence analysis of the B. subtilis genome (tlpA, yfjU, ysfA, and cotK) do indeed code for proteins and also identifies five new coding genes which were not identified as such in the analysis of the genome's sequence. All five of the latter code for quite small proteins, which is presumably why the genes were not initially identified as coding regions. However, the translational initiation codon for all five is ATG, the preferred initiation codon in B. subtilis, and these are preceded by sequences with good to reasonable homology to the 3' end of 16s rRNA, which is presumably a ribosome binding site; all coding sequences terminate with TAA, which is also the preferred termination codon in B. subtilis (18). The new proteins, however, appear present in spores at rather low levels. Previous work has shown that the level of SspC, the most prominent minor SASP, is only ~35% of that of SASP-, indicating that SspC is ~0.5% of total spore protein, since SASP- is ~1.5% (38, 46). Comparison of the intensities of the other minor SASP bands with those of SspC indicates that each, including the minor /-type SASP SspD, comprises only 0.05 to 0.2% of total spore protein, assuming that all bands stain equally. Even though these latter values are rather low, given the small size of these proteins, there are clearly a large number of molecules of each of them per spore.

All SASP analyzed previously in B. subtilis have been shown to be located in the spore core, and the presence of the -galactosidase expressed from an sspJ-lacZ fusion in the dormant spore, specifically the spore core, suggests that SspJ is also present in the spore core. The situation with SspG, however, is different, as sspG is clearly expressed in the mother cell and -galactosidase from an sspG-lacZ fusion is not found in the spore. These data are consistent with SspG being a spore coat protein, but we were unable to detect SspG in spore coat extracts. While SspG might be in the spore cortex, it is not obvious why it should move there. Clearly, further work, possibly using direct protein localization techniques such as immunofluorescence or immunoelectron microscopy, will be needed to definitively establish the location of SspG in the spore.

The identification of SspD in spores brings to four the number of /-type SASP identified in B. subtilis spores, which is the number of genes encoding /-type SASP present in the B. subtilis genome (18). Interestingly, one Bacillus species, B. megaterium, has at least seven genes encoding /-type SASP (38, 40), with the additional genes encoding minor proteins. The reason(s) for the loss of multiple genes encoding /-type SASP in B. subtilis (or their gain in B. megaterium) is not clear, but it is known that deletion of genes encoding at least one minor /-type SASP has no obvious phenotypic effect in B. subtilis (38, 40).

Information available to date indicates that all the minor SASP identified in this work are sporulation-specific proteins. This was shown previously for SspC and SspD, whose coding genes are expressed only in the developing forespore during sporulation under the control of ^G (26). The two genes studied in this work, sspG and sspJ, are also sporulation specific, and preliminary work in our laboratory has shown that expression of tlp, sspH, and sspL is also sporulation specific (4). If, as seems likely, given the absence of obvious vegetative cell protein bands comigrating with CotK, SspI, SspK, and SspM, all of these new genes are indeed sporulation specific, a question to ask is what function, if any, is served by these genes or their products. As shown in this work, deletion of sspG (and also yurS) has no obvious phenotypic effect, while deletion of sspJ has only a minor effect on spore outgrowth in a wild-type genetic background. In previous work, we found that loss of another minor small spore-specific protein (but one which is not acid soluble) termed YhcN also causes a slight spore outgrowth defect (3). However, the reason(s) for these outgrowth defects is not clear. The combination of mutations deleting yhcN, sspJ, and other minor SASP may be responsible for a major phenotype. However, it also seems possible that the situation will be similar to that observed with deletions removing many of the spore coat proteins, as these mutations often have no discernible effect beyond the loss of an individual protein from spores (39). Perhaps these new minor SASP have no function individually, have a function that requires special conditions to become obvious, or have functions that are redundant with one or more other minor SASP. Indeed, the latter is the case for the minor /-type SASP SspC and SspF, as loss of these proteins has no noticeable effect on spore resistance (24, 38, 40). However, in a strain lacking the major /-type SASP  and , overexpression of SspC (or SspF) can restore some to much of the resistance to spores (24, 38, 40). Perhaps overexpression of some of the new minor SASP in spores (wild type or ) may assist in elucidating their function.

The detailed analysis of the regulation of the two new ssp genes analyzed in this work indicates that transcription of the sspG operon is directed by E^K and also requires GerE, while transcription of sspJ depends primarily on E^G. Examination of the sequence upstream of the transcription start site of sspG reveals sequences with good matches to the consensus 10 and 35 sequences recognized by E^K (Fig. 2A). Slightly further upstream but still in the intergenic region between yurR and sspG are also two sequences with a reasonable match to the consensus sequence recognized and bound by GerE (Fig. 2A). Preliminary in vitro transcription of sspG templates with partially purified E^K has also given transcripts of the size expected based on the 5' end of sspG mRNA determined in vivo, and this in vitro transcription by E^K was strongly stimulated by GerE (14). While the precise location of the GerE binding site(s) in the sspG promoter has not yet been determined, the analysis of sspG transcription has added another promoter sequence to define the ^K consensus recognition sequence. Upstream of the sspJ transcription start site are also 10 and 35 sequences, which match rather well the consensus 10 and 35 sequences recognized by E^G (Fig. 2B). The sspJ promoter was recognized not only by E^G but also to a slight degree by E^F, both during sporulation and upon induction of ^F synthesis in growing cells. The 10 and 35 promoter sequences recognized by ^G and ^F are quite similar, with a major difference being the presence of G residues at both positions 14 and 15 in good ^F promoters (2, 44). The presence of one G residue in these positions in the sspJ promoter is consistent with the poor but significant transcription of sspJ by E^F compared to that of E^G (44).

The transcription of the sspG yurS operon by E^K plus GerE suggests that SspG and YurS may well be spore coat proteins. However, SspG was not removed from spores by solubilization of much of the spore coat, and neither SspG nor YurS was detected in spore coat extracts. Since certainly SspG should be soluble in the solutions used for preparing spore coat extracts, the precise location of this protein in the spore is presently unclear. The product of the cotK gene was also previously suggested to be a coat protein (12), but again CotK was not removed from spores by an extraction procedure removing most spore coat proteins. Consequently, the identity of CotK as a coat protein is also problematic at present. However, neither CotK nor SspG was lost upon spore germination, in contrast to SspC, -D, -H, -I, -K, -L, and -M and Tlp, which were lost, presumably by their degradation. However, with the exception of SspC and -D, none of these latter proteins have sequences similar to the somewhat-loose recognition sequence of the spore-specific protease that initiates degradation of both /- and -type SASP during spore germination (5, 15, 38). Consequently, the identity of the protease that initiates degradation of Tlp and SspH, -I, -K, -L, and M during spore germination is not evident. Clearly, there is much yet to be learned about the metabolism and function of these minor spore proteins.