Assembly and interactions of cotJ-encoded proteins, constituents of the inner layers of the Bacillus subtilis spore coat

During Bacillus subtilis endospore formation, a complex protein coat is assembled around the maturing spore. The coat is made up of more than two dozen proteins that form an outer layer, which provides chemical resistance, and an inner layer, which may play a role in the activation of germination. A third, amorphous layer of the coat occupies the space between the inner coat and the cortex, and is referred to as the undercoat. Although several coat proteins have been characterized, little is known about their interactions during assembly of the coat. We show here that at least two open reading frames of the cotJ operon ( cotJA and cotJC ) encode spore coat proteins. We suggest that CotJC is a component of the undercoat, since we found that its assembly onto the forespore is not prevented by mutations that block both inner and outer coat assembly, and because CotJC is more accessible to antibody staining in spores lacking both of these coat layers. Assembly of CotJC into the coat is dependent upon expression of cotJA . Conversely, CotJA is not detected in the coats of a cotJC insertional mutant. Co-immunoprecipitation was used to demonstrate the formation of complexes containing CotJA and CotJC 6 h after the onset of sporulation. Experiments with the yeast two-hybrid system indicate that CotJC may interact with itself and with CotJA. We suggest that interaction of CotJA with CotJC is required for the assembly of both CotJA and CotJC into the spore coat.     

Regulatory studies on the promoter for a gene governing synthesis and assembly of the spore coat in Bacillus subtilis

gerE is a regulatory gene of Bacillus subtilis that governs the synthesis and assembly of the spore coat and is required for the production of spores that are lysozyme-resistant and germination-proficient. We report the identification of the promoter for gerE and studies on the regulation of its expression. We show that gerE is switched on at the fourth hour of sporulation (stage-V) and that this expression is restricted to the mother-cell chamber of the sporangium. Dependency studies in which the level of gerE expression was measured in 36 different developmental mutants indicate that efficient expression of gerE requires the products of almost all spo0-IV genes tested as well as certain spoV genes. On the basis of its time of induction, compartmentalization of expression and pattern of dependence on other spo genes, gerE is inferred to be regulated co-ordinately with the previously studied spore coat protein gene cotA. gerE and cotA may be members of a developmental regulon of genes whose products are involved in the assembly of the spore coat.     

Characterization of spores of Bacillus subtilis that lack most coat layers

Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.     

Protein filaments may initiate the assembly of the Bacillus subtilis spore coat.

The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores

The spores of Bacillus subtilis show remarkable resistance to many environmental stresses, due in part to the presence of an outer proteinaceous structure known as the spore coat. GerQ is a spore coat protein essential for the presence of CwlJ, an enzyme involved in the hydrolysis of the cortex during spore germination, in the spore coat. Here we show that GerQ is cross-linked into higher-molecular-mass forms due in large part to a transglutaminase. GerQ is the only substrate for this transglutaminase identified to date. In addition, we show that cross-linking of GerQ into high-molecular-mass forms occurs only very late in sporulation, after mother cell lysis. These findings, as well as studies of GerQ cross-linking in mutant strains where spore coat assembly is perturbed, lead us to suggest that coat proteins must assemble first and that their cross-linking follows as a final step in the spore coat formation pathway.     

Germination properties of a spore coat-defective mutant of Bacillus subtilis.

The presence of the gerE36 mutation in strains of Bacillus subtilis 168 resulted in poor germination of their spores in a range of germinants, as measured by the fall in absorbance of spore suspensions. Although resistant to heat and organic solvents, spores were sensitive to lysozyme; electron microscopy revealed that their coat structure was incomplete. These spores responded to germinants by losing heat resistance and changing from phase bright to phase gray. The release of dipicolinic acid and the fall in absorbance of spore suspensions reached only 75 and 50% of wild-type levels, respectively, but followed the same time course as the loss of heat resistance. Although the germination response was incomplete, the concentration of L-alanine required to elicit it was the same for the mutant as for the wild type. The properties of mutant spores suggest that an intact spore coat is not required for the initial interaction between germinant and spore, but that the coat layers may contain molecules important in later stages of germination. In transduction with phage SPP1, the gerE36 mutation mapped between citF and ilvB and was 90% cotransduced with citF2. The gerE mutation identifies the location of a gene important for the progress of late stages of spore formation.     

spoVIC, a new sporulation locus in Bacillus subtilis affecting spore coats, germination and the rate of sporulation

A mutation near cysB on the Bacillus subtilis chromosome marks a new sporulation locus, spoVIC. It causes spores to germinate more slowly than those of the wild-type under all conditions and, from indirect evidence, it does not appear to alter the affinity for the germinant L-alanine. The mutant spores have some deficiency of coat proteins (particularly the alkalisoluble coat protein, Mr = 12 000) and the spore coat layers are disorganized. The mutant strain grows normally and sporulates normally until stage II, after which its sporulation is delayed by about 2 h compared to that of the wild-type. This delay results in the prolonged synthesis of some coat proteins and the late synthesis of others. The abnormal coat may be the cause of the germination deficiency. A double mutant strain carrying the spoVIC610 mutation together with gerE36 sporulates slowly. Its spores have very little coat protein, are sensitive to heat, lysozyme and organic solvents, but germinate as well as the strain carrying the spoVIC mutation alone. The role of the spore coat in germination is discussed in the light of these findings.     

Dynamics of the assembly of a complex macromolecular structure--the coat of spores of the bacterium Bacillus subtilis

The coat is the outermost layer of spores of many Bacillus species, and plays a key role in these spores' resistance. The Bacillus subtilis spore coat contains > 70 proteins in four distinct layers: the basement layer, inner coat, outer coat and crust. In this issue of Molecular Microbiology, McKenney and Eichenberger study the dynamics of spore coat assembly using GFP-fusions to 41 B. subtilis coat proteins. A key finding in the work is that formation of the spore coat is initiated by the apparently simultaneous assembly of foci of proteins from all four coat layers on the developing spore just as forespore engulfment by the mother cell begins. The expansion of these foci before completion of forespore engulfment then sets up the scaffold to which coat proteins added later in sporulation are added. This study provides new understanding of the mechanism of the assembly of a multi-protein, multi-lamellar structure.     

A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains epsilon-(gamma-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under sigma(K) control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.     

Protease production during sporulation of germination mutants of Bacillus subtilis and the cloning of a functional gerE gene

Early in sporulation, cells of wild-type Bacillus subtilis produce three proteases (b, c and d) with monomeric Mr values of about 65 000, 53 000 and 43 500, and a further protease, e (Mr about 30 000) at the time of coat assembly. An additional protease, f (Mr about 15 000) appears transiently in sporangia at about the time of spore release. Three strains with defective spore coats were examined for alterations in sporulation proteases. A strain carrying the gerE36 mutation produces b, c and d normally, fails to produce e and accumulates f on or in its spores. A strain carrying the spoVIC610 mutation produces normal quantities of proteases b, c and d, but has a reduced amount of proteases e and f. A strain carrying both the gerE36 and the spoVIC610 mutations accumulates neither protease e nor f. The wild-type allele of the gerE gene was cloned in the vector, phage phi 105J9. Complementation tests with the cloned gene showed that the gerE36 mutation is recessive to the wild-type allele.     

Altered arrangement of proteins in the spore coat of a germination mutant of Bacillus subtilis

Spores produced by a mutant of Bacillus subtilis were slow to develop their resistance properties during sporulation, and were slower to germinate than were wild-type spores. The coat protein composition of the mutant spores, as analysed by SDS-PAGE, was similar to that of the wild-type spores. However, one of the proteins (mol. wt 12000) which is normally present in the outer-most layers of mature wild-type spores and which is surface-exposed, was assembled abnormally into the coat of the mutant spores and not surface-exposed. The mutation responsible for this phenotype (spo-520) has been mapped between pheA and leuB on the B. subtilis chromosome, and was 47% cotransformable with leuB16. This mutation, and three others closely linked to it, define a new sporulation locus, spoVIB, which is involved in spore coat assembly. The phenotype of the mutant(s) supports the contention that spore germination and resistance properties may be determined by the assembly of the coat.     

Involvement of the spore coat in germination of Bacillus cereus T spores.

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

Involvement of the spore coat in germination of Bacillus cereus T spores

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.