Assembly of multiple CotC forms into the Bacillus subtilis spore coat

We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.     

CotE Binds to CotC and CotU and Mediates Their Interaction during Spore Coat Formation in Bacillus subtilis

CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of Bacillus subtilis. CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.The spore of Bacillus subtilis is a dormant cell, resistant to harsh conditions and able to survive extreme environmental conditions (25). Spores are produced in a sporangium that consists of an inner cell, the forespore, that will become the mature spore and an outer cell, the mother cell, that will lyse, liberating the mature spore (18, 26). Resistance of the spore to noxious chemicals, lytic enzymes, and predation by soil protozoans is in part due to the coat, a complex, multilayered structure of more than 50 proteins that encases the spore (5, 8, 13). Proteins that constitute the coat are produced in the mother cell and deposited around the outer membrane surface of the forespore in an ordered manner (8).A small subset of coat proteins have a regulatory role on the formation of the coat. Those proteins, referred to as morphogenic factors, do not affect the synthesis of the coat components but drive their correct assembly outside of the outer forespore membrane (8). Within this subset of regulatory coat proteins, SpoIVA and CotE play a crucial role. SpoIVA (6, 20, 23) is assembled into the basement layer of the coat and is anchored to the outer membrane of the forespore through its C terminus that contacts SpoVM, a small, amphipathic peptide embedded in the forespore membrane (16, 21, 22). A spoIVA-null mutation impairs the assembly of the coat around the forming spore, and as a consequence, coat material accumulates in the mother cell cytoplasm (23).CotE (28) assembles into a ring and surrounds the SpoIVA basement structure. The inner layer of the coat is then formed between the SpoIVA basement layer and the CotE ring by coat components produced in the mother cell that infiltrate through the CotE ring, while the outer layer of the coat is formed outside of CotE (6). However, not all CotE molecules are assembled into the ring-like structure, and CotE molecules are also found in the mother cell cytoplasm, at least up to 8 h after the start of sporulation (3). CotE was first identified as a morphogenic factor in a seminal study in which an ultrastructural analysis indicated that a cotE-null mutation prevented formation of the electron-dense outer layer of the coat while it did not affect inner coat formation (28). A subsequent mutagenesis study has revealed that CotE has a modular structure with a C-terminal domain involved in directing the assembly of various coat proteins, an internal domain involved in the targeting of CotE to the forespore, and a N-terminal domain that, together with the internal domain, directs the formation of CotE multimers (17). More recently, formation of CotE multimers has been also confirmed by a yeast two-hybrid approach (14). In a global study of protein interactions in the B. subtilis coat, performed by a fluorescence microscopy analysis of a collection of strains carrying cot-gfp fusions, CotE has been proposed to interact with most outer coat components (12).From those and other studies, the interactions of CotE with coat structural components have been exclusively inferred on the basis of genetic experiment results, i.e., cotE mutants that failed to assemble one or more coat components. Evidence of a direct interaction between CotE and another coat component has never been provided. We addressed this issue by using as a model two coat components, CotC and CotU, known to be controlled by CotE and to form a heterodimer (10, 28).CotC is an abundant, 66-amino-acid protein known to assemble in the outer coat in various forms: a monomer of 12 kDa, a homodimer of 21 kDa, and two less abundant forms of 12.5 and 30 kDa, probably due to posttranslational modifications of CotC (9). CotU is a structural homolog of CotC of 86 amino acids. The two proteins, which share an almost identical N terminus and a less conserved C terminus, interact, originating the formation of a heterodimer of 23 kDa (10). Heterodimer formation most likely requires a B. subtilis-specific factor since it does not occur in Escherichia coli or Saccharomyces cerevisiae (10). CotC and CotU are synthesized in the mother cell compartment of the sporulating cell but do not accumulate there since they are immediately assembled around the forming spore (10). In a strain carrying a cotE-null mutation, CotC and CotU, together with all other outer coat components, do not assemble around the forming spore (10). CotC and CotU are also dependent on CotH, an additional morphogenic factor involved in coat formation (9). A cotH-null mutation prevents CotC and CotU assembly in the coat as well as their accumulation in the mother cell cytoplasm (10). Since a mutation causing cotH overexpression allows CotC and CotU accumulation in the mother cell cytoplasm (1), it has been proposed that CotH acts by stabilizing CotC and CotU in the mother cell cytoplasm (1, 10).Here we provide the first direct evidence that CotE interacts with two other coat components, CotC and CotU, and show that CotE is essential and sufficient to mediate CotC-CotU interaction to form a heterodimer.     

CotL,a new morphogenetic spore coat protein of Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σ^E and σ^K. CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.     

Localization of Proteins to Different Layers and Regions of Bacillus subtilis Spore Coats

Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring- or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.Proper localization and assembly of proteins in cells and subcellular structures are essential features of living organisms. Complex protein assemblies, including ribosomes, flagella, and the cytokinetic machinery, play important roles in bacteria (26, 27, 40). Studying how these complex structures are formed is a fundamental theme in molecular biology. In this work, we developed a method to analyze one of the most complex bacterial protein assemblies: the spore coat of Bacillus subtilis.Sporulation of B. subtilis is initiated in response to nutrient limitation, and it involves a highly ordered program of gene expression and morphological change (33, 42). The first morphological change of sporulation is the appearance of an asymmetrically positioned septum that divides the cell into a larger mother cell and a smaller forespore. Next, the mother cell membrane migrates around the forespore membrane during a phagocytosis-like process called engulfment. The completion of engulfment involves fusion of the mother cell membrane to pinch off the forespore within the mother cell. Compartment-specific gene expression brings about maturation of the spore and its release upon lysis of the mother cell (reviewed in reference 19). Mature spores remain viable during long periods of starvation and are resistant to heat, toxic chemicals, lytic enzymes, and other factors capable of damaging vegetative cells (30). Spores germinate and resume growth when nutrients become available (32).The outer portions of Bacillus spores consist of a cortex, a spore coat layer, and in some cases, an exosporium. The cortex, a thick layer of peptidoglycan, is deposited between the inner and the outer membranes of the forespore, and it is responsible for maintaining the highly dehydrated state of the core, thereby contributing to the extreme dormancy and heat resistance of spores. Spore coat assembly involves the deposition of at least 50 protein species (12, 21, 24) into two major layers: an electron-dense outer layer, called the outer coat, and a less electron-dense inner layer with a lamellar appearance, called the inner coat (50). These layers provide a protective barrier against bactericidal enzymes and chemicals, such as lysozyme and organic solvents (30). Although disruption of any one gene encoding a spore coat protein typically has little or no effect on spore resistance, morphology, or germination, a few proteins, referred to as morphogenetic proteins, play central roles in the assembly of the spore coat (7, 10, 13). One of the morphogenetic proteins, CotE, is located between the inner and outer coats and directs the assembly of most or all of the outer coat proteins and also a few of the inner coat proteins (2, 9, 17, 25, 52). The locations of CotE, CotS, and SpoIVA in the spore coat were determined previously by immunoelectron microscopy (9, 43). CotA, CotB, CotC, and CotG were shown to be externally exposed on the surface of the spore by single-molecule recognition force spectroscopy or antibody accessibility (15, 18, 45, 28). However, the positions of most of the spore coat proteins in the coat have not been determined experimentally, although provisional assignments were made based largely on the control of assembly into the coat by CotE (17). In this study, we developed methods to estimate the positions of proteins in the spore coat layers by using fluorescence microscopy analysis of coat protein-fluorescent protein fusions, with resolution that allowed us to distinguish between the inner and outer coats. In addition, we discovered an asymmetric spatial distribution of four spore coat proteins and a ring- or spiral-like structure of CotB. These observations suggest that spore coat assembly is more intricate than previously appreciated.     

CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis

Bacterial spores of the Bacillus genus are ubiquitous in nature and are commonly isolated from a variety of diverse environments. Such wide distribution mainly reflects the spore resistance properties but some Bacillus species can grow/sporulate in at least some of the environments where they have been originally isolated. Growing and sporulating at different conditions is known to affect the structure and the resistance properties of the produced spore. In B. subtilis the temperature of growth and sporulation has been shown to influence the structure of the spore surface throughout the action of a sporulation-specific and heat-labile kinase CotH. Here we report that CotG, an abundant component of the B. subtilis spore surface and a substrate of the CotH kinase, assembles around the forming spore but also accumulates in the mother cell cytoplasm where it forms aggregates with at least two other coat components. Our data suggest that the thermo-regulator CotH contributes to the switch between the coat of 25°C and that of 42°C spores by controlling the phosphorylation levels of CotG that, in turn, regulates the assembly of at least two other coat components.     

The Direct Interaction between Two Morphogenetic Proteins Is Essential for Spore Coat Formation in Bacillus subtilis

In Bacillus subtilis the protective layers that surround the mature spore are formed by over seventy different proteins. Some of those proteins have a regulatory role on the assembly of other coat proteins and are referred to as morphogenetic factors. CotE is a major morphogenetic factor, known to form a ring around the forming spore and organize the deposition of the outer surface layers. CotH is a CotE-dependent protein known to control the assembly of at least nine other coat proteins. We report that CotH also controls the assembly of CotE and that this mutual dependency is due to a direct interaction between the two proteins. The C-terminal end of CotE is essential for this direct interaction and CotH cannot bind to mutant CotE deleted of six or nine C-terminal amino acids. However, addition of a negatively charged amino acid to those deleted versions of CotE rescues the interaction.     

Role of spore coat proteins in the resistance of Bacillus subtilis spores to Caenorhabditis elegans predation

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.     

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 (H₂O₂) 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% H₂O₂, 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.     

SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins

Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.     

Antagonistic Role of CotG and CotH on Spore Germination and Coat Formation in Bacillus subtilis

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.     

A protein phosphorylation module patterns the Bacillus subtilis spore outer coat

Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self-organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C-terminal moiety of CotB, SKR^B, formed by serine-rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine-repeat region, SKR^G, interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKR^B region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers.     

Surface display of lipolytic enzyme,Lipase A and Lipase B of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spore

For the enhancement of lipase stability in organic solvent containing reaction, live immobilization method, using Bacillus subtilis spore as a display vehicle was attempted. Bacillus subtilis coat protein cotE was used as an anchoring motif for the display of lipA and lipB of Bacillus subtilis. Using this motif, lipolytic enzyme Lipase A and Lipase B were functionally displayed on the surface of Bacillus subtilis spore. Purified spore displaying CotE-LipB fusion protein showed higher lipolytic activity compared to that of CotE-LipA fusion protein. The surface localization of Lipase B was verified with flow cytometry and protease accessibility experiment. Spore displayed lipase retained its activity against acetone and benzene which completely deactivated free soluble lipase in the same reaction condition.     

Study of the interactions between the key spore coat morphogenetic proteins CotE and SpoVID

The capability of Bacillus subtilis spores to withstand extreme environmental conditions is thought to be conferred especially by their outermost proteinaceous protective layer, called the spore coat. Of the over 70 proteins that form the spore coat, only a small subset of them affect its morphogenesis, they are referred to as morphogenetic proteins. In this study we investigated the interaction between two spore coat morphogenetic proteins SpoVID and CotE. SpoVID is involved in the process of spore surface encirclement by individual coat proteins, these include CotE, which controls the assembly of the outer coat layer. Both proteins were proposed to be recruited to a common protein scaffold, but their direct association has not been previously shown. Here we studied the interactions between CotE and SpoVID in vitro for the first time by using molecule recognition force spectroscopy, which allows the detection of piconewton forces between conjugated biological pairs and also facilitates the investigation of dynamic processes. The most probable CotE–CotE unbinding force was 49.4 ± 0.1 pN at a loading rate of 3.16 × 10³ pN/s while that of SpoVID–CotE was 26.5 ± 0.6 pN at a loading rate of 7.8 × 10² pN/s. We further analyzed the interactions with the bacterial two hybrid system and pull-down experiments, which also indicate that SpoVID interacts directly with CotE. In combination with the previously identified direct contacts among SpoIVA, SpoVID and SafA, our data imply that the physical association of key morphogenetic proteins forms a basic skeleton where other coat proteins could be attached.