JOURNAL OF BACTERIOLOGY, Aug. 1996, p. 4375–4380 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 178, No. 15
Bacillus subtilis Spore Coat Assembly Requires cotH Gene Expression G. NACLERIO,1 L. BACCIGALUPI,1 R. ZILHAO,2 M. DE FELICE,3
AND
E. RICCA3*
Institute of Food Science and Technology, Consiglio Nazionale delle Ricerche, 83100 Avellino,1 and Department of General and Environmental Physiology, University Federico II, 80134 Naples,3 Italy, and Department of Plant Physiology, University of Lisbon, 1700 Lisbon, Portugal2 Received 29 February 1996/Accepted 17 May 1996
Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300& on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat. cotG (7, 18). cotH codes for a 42.8-kDa protein, apparently located in the inner layer of the coat. cotH null mutations show pleiotropic effects on several components of the outer coat and cause a germination-deficient phenotype.
Endospores of the gram-positive bacterium Bacillus subtilis are encased in a thick protein shell known as the coat (2). The coat is composed of 15 or more polypeptides arranged in an electron-dense outer layer and a lamellar inner layer. These layers protect the spore from bactericidal enzymes and chemicals, such as lysozyme and chloroform. So far, the genes for 13 of these coat proteins have been identified. These are located at diverse positions on the chromosome and code for polypeptides of 65 (CotA), 59 (CotB), 10 (CotC), 9 (CotD), 24 (CotE), 19 (CotF), 24 (CotG and CotJ), 41 (CotS), 10 (CotT), 19 (CotX), 26 (CotY), and 18 (CotZ) kDa (1, 3, 6, 7, 10, 18, 22, 24). Certain proteins, such as CotD, are located in the inner coat, and others, such as CotA, CotB, CotC, and CotG, are located in the outer coat (18, 24). The coat is produced at a relatively late stage in the process of sporulation, when the developing spore (or forespore) is present as a free protoplast within the mother cell compartment of the sporangium (14). Coat proteins are organized on the outer surface of the membrane surrounding the forespore by the sporulation protein SpoIVA (8, 17, 21). SpoIVA controls the assembly of a ring of CotE proteins around the forespore (8). The CotE ring is thought to regulate the assembly of the proteins of the outer coat and is separated from the outer surface of the forespore membrane by a small gap, which is believed to be the site at which the inner coat will be assembled (8). The production of coat proteins is governed by a regulatory cascade of four transcription factors acting in the mother cell compartment of the sporangium in the sequence sE, SpoIIID, sK, and GerE (26). sE and sK are RNA polymerase sigma factors, whereas SpoIIID and GerE are DNA-binding proteins that act in conjunction with sE- and sK-containing forms of RNA polymerase, respectively (4, 9, 13, 23, 25). We report the identification of a gene, herein called cotH, located at 3008 on the B. subtilis chromosomal map, where it is clustered with two previously described cot genes, cotB and
MATERIALS AND METHODS Bacterial strains. B. subtilis PY17 (trpC2 SPbS) was used for the purification of spore coat proteins and for the construction of the cotH null mutants (24). Cloning experiments were carried out with the Escherichia coli strain DH5a (19). Gene cloning and sequencing. Plasmid pER110 (18) (Fig. 1) contains a 3.7-kb HindII-HindIII fragment of the B. subtilis chromosome cloned into the integrational vector pER19 (16). Plasmid pER110 carries the entire cotG gene and the 59 half of the cotB gene separated by about 1.3 kb (Fig. 1) (18). DNA sequence analysis of the 1.3-kb region separating the cotG and cotB genes was performed by the dideoxy chain termination method (20). Construction of cotH null mutants. Two different cotH null mutations were constructed. One mutation was created by inserting a chloramphenicol resistance (cat) cassette in the cotH coding region. Plasmid pER110 contains a unique EcoRI site within the cotH coding region into which was inserted an EcoRI cassette containing the cat gene (from plasmid pMI1101 [16]). The resulting plasmid, pER111, was linearized and used to replace cotH on the chromosome of strain PY17 by a double recombination event. The second mutation was created by transforming competent cells of strain PY17 with plasmid pER117 (Fig. 1). This plasmid contains a 435-bp EcoRV-DraI fragment, completely internal to the cotH coding region, cloned into the integrative vector pER19. Chloramphenicol-resistant cells were the result of integration of the plasmid into the chromosome by single reciprocal (Campbell-like) recombination between B. subtilis DNA sequences in pER117 and homologous sequences on the chromosome. Purification of spore coat protein. B. subtilis cells were grown in DS sporulation medium for 48 h at 378C. Spores were harvested by centrifugation and purified by washing and lysozyme treatment as previously described (11). Coat proteins were solubilized by treatment of the spores with 1% (wt/vol) sodium dodecyl sulfate (SDS)–50 mM dithiothreitol at 658C for 30 min (7). After centrifugation to remove the extracted spores, the supernatant was subjected to electrophoretic fractionation in 10 and 15% polyacrylamide gels containing SDS. b-Galactosidase assays. b-Galactosidase activity was determined by using the substrate o-nitrophenol-b-D-galactoside as described previously (15). One unit of enzyme hydrolizes 1 mmol of substrate per min per A595 of initial cell density. Mapping of the 5* terminus of cotH mRNA. Primer extension experiments were performed as previously described (18). Sporulation was induced by resuspension of cells in SM medium (5). RNA was extracted as previously described (18) from wild-type and mutant cells (50 ml) harvested after the onset of sporulation. Two synthetic oligonucleotides, H1 (59-GGATCATCGTCCCATA-39 [see Fig. 2]) and H5 (59-GCTGATAAAGCGGTAA-39 [see Fig. 2]), were used to prime cDNA synthesis with sporulation RNA as the template. The products of primer extension were analyzed by electrophoresis in 6% polyacrylamide gels (20). Germination efficiency and lysozyme resistance. Purified spores were heat
* Corresponding author. Mailing address: Department of General and Environmental Physiology, University Federico II, via Mezzocannone 16, 80134 Naples, Italy. Phone: 39-81-5514299. Fax: 39-815514437. Electronic mail address:
[email protected] or ericca@ds .unina.it. 4375
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FIG. 1. Restriction map of the cotG cotB region. Dashed and solid lines indicate the direction of transcription and subcloned DNA fragments, respectively. The open triangle indicates the site of insertion of the cat gene. Restriction site abbreviations: H, HindIII; Pv, PvuII; Rv, EcoRV; H2, HindII; K, KpnI; Ms, MspI; S, Sau96I; Bg, BglII; D, DraI; R, EcoRI. The indicated MspI and Sau96I sites are not unique in the fragment.
activated as previously described (5) and diluted in 10 mM Tris-HCl (pH 8.0) buffer containing 1 mM glucose, 1 mM fructose, and 10 mM KCl. After 15 min at 378C, germination was induced by adding 10 mM L-alanine or 10 mM L-asparagine and the optical density at 580 nm was measured at 5-min intervals until a constant reading was reached. Qualitative germination assays were performed as previously described (5) on tetrazolium plates. Sensitivity to lysozyme was measured as described by Zheng et al. (24). Spores were prepared as previously described (11), omitting the lysozyme step and eliminating vegetative cells by heat treatment (10 min at 808C). Purified spores were then suspended in 10 mM Tris-HCl (pH 7.0) buffer containing lysozyme (50 mg/ml), and the decrease in optical density was monitored at 595 nm at 1-min intervals for 10 min. Spore viability was measured after 30 min as CFU on TY agar plates. Nucleotide sequence accession number. The sequence has been deposited in GenBank under accession number X98342.
RESULTS AND DISCUSSION Identification of an open reading frame in the cotG-cotB region. It has been previously reported that two cot genes, cotB and cotG, are clustered around 3008 on the B. subtilis chromosomal map. These two genes are similarly regulated, both being under the transcriptional control of sK-driven RNA polymerase and of the transcriptional activator GerE. cotB and cotG are divergently transcribed and are separated by about 1,300 bp (18). We analyzed the DNA region separating cotB and cotG to investigate whether a third gene was clustered in that region. Nucleotide sequence analysis revealed the presence of a single open reading frame extending for 1,086 bp. This open reading frame, hereafter called orfH, is transcribed on the cotB coding strand of DNA, and its 59 regulatory region is adjacent to the cotG promoter region (Fig. 1). Figure 2 shows orfH nucleotide and deduced amino acid sequences. orfH codes for a presumptive polypeptide of 362 amino acids with a molecular mass of 42.8 kDa. A computer-assisted analysis did not reveal homology of orfH with any of the sequences present in the data bank. Construction of orfH null mutations. To study the role of the orfH gene product in the cell, orfH null mutants were constructed in two ways. A chloramphenicol resistance (cat) cassette was inserted into a unique EcoRI site of plasmid pER110 (Fig. 1) and the resulting plasmid, pER111, was linearized and used to transform competent cells of the B. subtilis strain PY17. Chloramphenicol-resistant clones were obtained as a result of a double crossover recombination event between homologous DNA regions present on plasmid pER111 and on the chromosome. One of the clones, ER209, was tested by Southern analysis to verify the site of insertion of the cat gene (data not shown).
An independent orfH null mutation was constructed by transforming competent cells of the wild-type strain, PY17, with plasmid pER117 (Fig. 1), a derivative of the integrational plasmid pER19 (16) carrying a 435-bp EcoRV-DraI DNA fragment, entirely internal to the cotH coding region. Chloramphenicol-resistant clones were the result of a single Campbelllike recombination event between homologous DNA regions present on the plasmid and on the chromosome. One of these clones, ER223, was tested by Southern analysis to verify the site of insertion of the cat gene (data not shown). The orfH gene product is required for spore coat assembly. Strains ER209 and ER223 did not differ from the isogenic wild-type strain, PY17, in cell viability or sporulation efficiency (data not shown). Mutant spores appeared to be normally resistant to chloroform, lysozyme, and heat treatments (data not shown). To test whether the orfH null mutations had an effect on the pattern of coat proteins released after SDS treatment, spores of the isogenic strain PY17 and of the cotH null mutant strains were purified, and Cot proteins were solubilized by SDS treatments as described in Materials and Methods and separated on 10 and 15% acrylamide denaturing gels. The pattern of released proteins for strains ER209 and ER223 appeared to be dramatically different from that of their isogenic wild-type strain (Fig. 3). In particular, bands of about 68, 43, 41, 32, and 16 kDa that were present in the wild-type strain were absent in the orfH null mutants, and the intensity of bands of 27 and 12 kDa appeared to be reduced. Some polypeptide bands were more intense in mutant than in wild-type spores. Although we cannot exclude the possibility that those polypeptides are degradation products of highermolecular-mass proteins, this effect could be due to the solubilization of proteins either no longer protected by more external proteins or misassembled and more exposed than in the wild-type spores. Two polypeptides of about 41 and 43 kDa, which correspond to the predicted size of the orfH gene product (42.8 kDa), were missing from spores of the ER209 strain (Fig. 3A). The absence of several bands in the pattern obtained from mutant spores did not allow us to assign a specific Cot protein as the orfH gene product, and immunological data will be needed for this purpose. Although we have not proven that the orfH gene product is a Cot protein, the pleiotropic effect of orfH null mutations on several Cot proteins suggested that the orfH gene product plays an important role in the formation of the spore coat; we will refer to it as CotH and to its structural gene as cotH.
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FIG. 2. Nucleotide sequences of the nontranscribed strand of the cotH (orfH) gene and adjacent DNA regions. The deduced amino acid sequences of cotH (orfH) and of the 59 end of cotB are reported. The cotH (orfH) proposed ribosome binding site (RBS) and the putative recognition sequences for sK-containing RNA polymerase (210 and 235) are indicated, as are relevant restriction sites. The nucleotide sequence from bp 1 to 349 (PvuII site) has been previously reported (18).
CotH effects on the assembly of CotB, CotG, and CotC. Spores of strains mutated in several cot genes were solubilized by SDS treatment, and the released proteins were compared with those released by cotH spores (Fig. 4). This analysis showed that bands of 68 and 32 kDa, which were missing in the cotH mutants, and the band of 12 kDa, whose intensity is reduced in the mutants, corresponded to the previously characterized outer coat proteins CotB, CotG, and CotC, respectively. Figure 4 (lanes 1 and 2) also shows that CotH does not interfere with the assembly of the inner coat protein CotD. It has previously been reported that CotG plays a morphogenic role in the assembly of CotB (18). The effect of CotH on CotB shown in Fig. 4 (lanes 4 and 5) could reflect either a dual control of CotG and CotH on CotB assembly or a hierarchical control of CotH on CotG, which in turn controls CotB. cotH transcriptional analysis. Primer extension experiments were carried out to map the 59 end of cotH mRNA and to analyze the timing and dependency of cotH expression. The experiment whose results are shown in Fig. 5A showed that cotH mRNA originated from a site located 99 bp upstream of the beginning of the open reading frame. A cotH-specific transcript was most abundant in RNA from cells harvested 6 to 7 h after the start of sporulation, while no extension products were detected in vegetatively growing and in early sporulating cells (data not shown). Primer extension experiments were also performed with total RNA extracted from various isogenic mutant cells altered in
the sigE, sigK, or gerE gene, all harvested 6 h after the onset of sporulation. No extension products from sigE and sigK mutants were detected, while an identical transcript was obtained with RNA extracted from the wild-type and the isogenic gerE mutant (Fig. 5B). The same sigK RNA preparation used in the experiment whose results are shown in Fig. 5B (lane sK) was used to prime cDNA synthesis with Pr1, a synthetic oligonucleotide that specifically anneals 187 bp downstream of the transcriptional start site of the sE-controlled gene spoVM (12). An extension product of the predicted size was obtained (Fig. 5B, lane VM/sK), thus indicating that the absence of a cotHspecific transcript in sigK RNA was actually due to a lack of cotH transcription. Although sequences located upstream of the 59 end (11) of the cotH mRNA (Fig. 2) did not show a high degree of homology with promoters recognized by sK-containing RNA polymerase (13), the results shown in Fig. 5 suggested that cotH is under the transcriptional control of sK-driven RNA polymerase and that the transcriptional activator GerE is not required for expression of the gene. Therefore, cotH is to be considered, together with other coat-related genes, such as gerE, cotA, cotD (26), cotF (6), and cotS (1), part of sK regulon. CotH does not affect the transcriptional efficiency of cotC and cotG. The requirement of CotH for correct assembly of the outer coat could be explained by (i) a morphogenic effect of CotH on several Cot proteins or (ii) a regulatory role of CotH on the expression of cot genes.
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FIG. 3. Pattern of released coat proteins fractionated on 10% (A) and 15% (B) polyacrylamide gels. Lanes: 1, strain ER209 (orfH); 2, strain PY17 (wild type). The gels were stained with Coomassie brilliant blue. The arrows indicate polypeptides of about 41 and 43 kDa appearing only in lanes 2.
To test whether CotH is a transcriptional factor affecting the expression of cot genes, we measured the b-galactosidase activity of strains carrying the E. coli lacZ gene translationally fused to cotC (26) and to cotG (18). The time course experiments whose results are shown in Fig. 6 show that cotC- and cotG-driven b-galactosidase activity in the cotH null mutant
FIG. 4. Pattern of released coat proteins extracted from wild-type and mutant strains. Lanes: 1, ER209 (cotH); 2, BZ109 (cotD); 3, BD071 (cotC); 4, MS33 (cotG); 5, BD067 (cotB); 6, ER223 (cotH); 7, PY17 (wild type). In contrast to the absence of CotB and CotG in cotH mutant spores, the CotC band is only reduced in its intensity. The gel (15% polyacrylamide) was stained with Coomassie brilliant blue.
and the wild-type strain were identical. Similar results were obtained by measuring the b-galactosidase activity of strains carrying lacZ translationally fused to cotA and cotD (data not shown). Since translational fusions of cotB to a reporter gene were not available, primer extension experiments were carried out to verify that the CotH effect on CotB was not at the transcriptional level. The synthetic oligonucleotide Pr2 (26), annealing 109 bp downstream of the cotB transcriptional start site, was used to prime cDNA synthesis with total RNA extracted from wild-type and cotH null mutant cells harvested 8 h after the onset of sporulation. An identical extension product was obtained, indicating that CotH does not affect cotB transcription (data not shown). Taken together, these results suggest that the CotH role in spore coat formation is more likely to be morphogenic than regulatory. CotH is an inner coat protein. The pleiotropic effect of cotH null mutations on several Cot proteins induced us to investigate further the phenotypic properties of cotH spores. The cat cassette of strain ER209 was replaced by a spectinomycin resistance cassette (spc) by transforming ER209 competent cells with a linearized form of plasmid pJL62 (cat::spc) (a gift from A. Grossman). Spectinomycin-resistant clones were obtained as a result of a double crossover recombination event between homologous DNA regions present on plasmid pJL62 and on the chromosome. One of the clones (ER220) was used as the source of chromosomal DNA which, in turn, was used to transform strain BZ213 (cotED::cat) (23), yielding the double cotE cotH null mutant strain ER221. The germination efficiencies and resistance to lysozyme treatment of spores purified from the cotE, cotH, and cotE cotH mutants and from the isogenic wild-type strain were measured. The data in Fig. 7 show that with L-asparagine as a
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FIG. 5. Primer extension analysis. (A) RNA extracted from wild-type cells at the indicated hours after the onset of sporulation. Primer extension and sequencing reactions were primed with the synthetic oligonucleotide H1. Similar results were obtained with oligonucleotide H5 (data not shown). (B) RNA extracted from gerE, sK, sE, and wild-type (wt) cells 6 h after the start of sporulation. Primer extension and sequencing reactions were primed with the synthetic oligonucleotide H1. The primer extension reaction of lane VM/sK was performed with RNA extracted from sK cells and the spoVM-specific oligonucleotide Pr1.
germination inducer, cotH and cotE cotH spores had a strongly reduced germination efficiency compared with the wild-type and the cotE strain, respectively. Similar results were obtained with L-alanine as a germination inducer and in qualitative assays on tetrazolium plates (data not shown).
FIG. 6. Effect of cotH null mutation on cotC (A) and cotG (B) transcription. Shown are results for cotC- and cotG-directed b-galactosidase synthesis in an otherwise-wild-type strain (open symbols) and in a cotH null background (shaded symbols). Samples were collected at various times after the onset of sporulation.
Although cotH spores did not show any difference with respect to the wild type, spores of the double cotE cotH null mutant had an increased sensitivity to lysozyme compared with the single cotE null mutant (Fig. 8), thus indicating that none of the CotH-dependent proteins are required for lysozyme resistance. Similar results were obtained when spore viability was measured as CFU on TY agar plates (data not shown). With the exception of cotE, none of the cot mutants so far characterized have noticeable phenotypic effects on spore resistance and germination. The observed phenotypic effects of cotH spores are probably due to the pleiotropic effect of CotH on several outer coat components. Zheng et al. (24) have previously shown by electron microscopy that cotE spores completely lack the electron-dense outer layer of the coat. The differences we found between cotE and cotE cotH mutants in
FIG. 7. Efficiency of germination of spores purified from wild-type (open squares) and cotH (darkly shaded triangles), cotE (shaded squares), and cotE cotH (lightly shaded triangles) strains. Germination was induced by Asn-GFK as previously reported (5). The data are averages of two independent experiments. The variation between different experiments was less than 10%.
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FIG. 8. Effect of lysozyme on spores of the wild type (solid squares), a cotH null mutant (darkly shaded triangles), a cotE null mutant (lightly shaded squares), and a double cotE cotH null mutant (lightly shaded triangles). The data are the means of three independent experiments. OD, optical density.
germination efficiency and lysozyme resistance indicate that CotH is still present in a cotE mutant and, therefore, is most probably localized in the inner coat (or at the interface between the two layers). From its inner localization, CotH exerts its morphogenic influence on several components of the outer part of the coat, whose correct assembly is thought to be responsible for the resistance to lysozyme (9). The absence of the CotH-controlled coat proteins is not sufficient to cause sensitivity of spores to lysozyme but increases the lysozyme sensitivity of spores depleted of the entire outer coat, suggesting that the inner coat also plays a role in resistance to lysozyme. The germination deficiency of cotH spores and the proposed inner coat localization of CotH are in agreement with previous reports (9, 11) suggesting that the inner coat is important for spore permeability or response to germinants. ACKNOWLEDGMENTS We are grateful to Alan Grossman for the gift of plasmid pJL62 and to Richard Losick for the gift of several strains and for critical reading of the manuscript. We thank Giovanni Manna and Giovanni Varriale for technical assistance. R.Z. was the recipient of a short-term EMBO fellowship. This work was supported by CNR (Progetti Finalizzati “Biotecnologie e Biostrumentazione” and “Ingegneria Genetica”) and by MIRAAF (Piano Nazionale Biotecnologie Vegetali). REFERENCES 1. Abe, A., H. Koide, T. Kohno, and K. Watabe. 1995. A Bacillus subtilis spore coat polypeptide gene, cotS. Microbiology 141:1433–1442. 2. Aronson, A. I., and P. Fitz-James. 1976. Structure and morphogenesis of the
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