Bacillus subtilis Spore Coat

In response to starvation, bacilli and clostridia undergo a specialized program of development that results in the production of a highly resistant dormant cell type known as the spore. A proteinacious shell, called the coat, encases the spore and plays a major role in spore survival. The coat is composed of over 25 polypeptide species, organized into several morphologically distinct layers. The mechanisms that guide coat assembly have been largely unknown until recently. We now know that proper formation of the coat relies on the genetic program that guides the synthesis of spore components during development as well as on morphogenetic proteins dedicated to coat assembly. Over 20 structural and morphogenetic genes have been cloned. In this review, we consider the contributions of the known coat and morphogenetic proteins to coat function and assembly. We present a model that describes how morphogenetic proteins direct coat assembly to the specific subcellular site of the nascent spore surface and how they establish the coat layers. We also discuss the importance of posttranslational processing of coat proteins in coat morphogenesis. Finally, we review some of the major outstanding questions in the field.     

Architecture and Assembly of the Bacillus subtilis Spore Coat

Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization pattern in a biological organism.     

The Presence of Keratin-like Substance in Spore Coat of Bacillus subtilis

Unique crystalline structures were found by X-ray diffractometry to be present in spore coats of Bacillus subtilis. By crystallographical and chemical studies of the purified spore coats it was found that these crystalline structures of the spore coats were essentially similar to those of α- and β-keratin, and that the spore coats were composed of keratin-like substance (or keratin). This keratin-like substance was found to be synthesized during sporogenesis from sulfur-containing water-soluble substances in the cells.     

枯草芽孢杆菌芽孢中的芽孢衣

芽孢衣是赋予芽孢对有机溶剂和溶菌酶的抗性,以及对外界出芽诱导物的感应能力的保护性结构。该主要介绍由枯草芽孢杆菌(bacillus subtilis)所形成的芽孢的芽孢衣中已鉴定的主要蛋白质．及其基因表达调控和应用等方面的研究进展。     

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.     

Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments

The germination of spore-forming bacteria in high-salinity environments is of applied interest for food microbiology and soil ecology. It has previously been shown that high salt concentrations detrimentally affect Bacillus subtilis spore germination, rendering this process slower and less efficient. The mechanistic details of these salt effects, however, remained obscure. Since initiation of nutrient germination first requires germinant passage through the spores'' protective integuments, the aim of this study was to elucidate the role of the proteinaceous spore coat in germination in high-salinity environments. Spores lacking major layers of the coat due to chemical decoating or mutation germinated much worse in the presence of NaCl than untreated wild-type spores at comparable salinities. However, the absence of the crust, the absence of some individual nonmorphogenetic proteins, and the absence of either CwlJ or SleB had no or little effect on germination in high-salinity environments. Although the germination of spores lacking GerP (which is assumed to facilitate germinant flow through the coat) was generally less efficient than the germination of wild-type spores, the presence of up to 2.4 M NaCl enhanced the germination of these mutant spores. Interestingly, nutrient-independent germination by high pressure was also inhibited by NaCl. Taken together, these results suggest that (i) the coat has a protective function during germination in high-salinity environments; (ii) germination inhibition by NaCl is probably not exerted at the level of cortex hydrolysis, germinant accessibility, or germinant-receptor binding; and (iii) the most likely germination processes to be inhibited by NaCl are ion, Ca²⁺-dipicolinic acid, and water fluxes.     

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.     

Ultrahigh-Temperature Activation of a Low-Temperature Bacillus subtilis Spore Germination System

Exposure of Bacillus subtilis A spores to 115 to 125 C for several seconds activated a low-temperature germination system that remained dormant after a heat treatment that activated the normal l-alanine- and glucose-stimulated germination systems. The low-temperature germination system was characterized by an optimum temperature lower than that of the l-alanine or glucose germination systems (30 C versus 45 C), germination in the absence of exogenous germination stimulants, and the capacity for heat-induced deactivation and subsequent reactivation. The rates of activation at 115 to 125 C were exponential and were not influenced by a previous heat treatment that activated the l-alanine- or glucose-stimulated germination systems. Although activation of the low-temperature germination system was accompanied by suppression of l-alanine-stimulated germination, it did not appear to be a modification of the l-alanine germination system.     

Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores'' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.     

枯草芽胞杆菌芽胞表面展示研究进展

枯草芽胞杆菌芽胞表面展示技术是把枯草芽胞杆菌作为芽胞表面展示的宿主来展示目的蛋白的一种技术。该技术不仅具备芽胞表面展示技术可展示分子量较大的目的蛋白、目的蛋白无需跨膜及芽胞的极强抗逆性等特点外,同时由于该技术的宿主菌--枯草芽胞杆菌的分子生物学信息研究得比较清楚、安全性高而被广泛应用。介绍了枯草芽胞杆菌表面展示近10年在生产疫苗和固定化酶方面的进展,并对如何提高表面展示目的蛋白的产量做了简要概述。     

Purification of Bacillus subtilis Spore Coat Protein by Electrophoretic Elution Procedure and Determination of NH2-Terminal Amino Acid Sequences

Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure. Solubilized coat protein components were separated on SDS-PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined. Two purified polypeptides with molecular weights of about 40 kDa were obtained; each of them was in very closed size on SDS-PAGE, both retaining antigenic activity against anti-spore coat protein serum on immunoblot analysis. The N-terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.     

Spore Peptidoglycan Structure in a cwlD dacB Double Mutant of Bacillus subtilis

Bacillus subtilis cwlD and dacB mutants produce spore peptidoglycan (PG) with increased cross-linking but with little change in spore core hydration compared to the wild type. A cwlD dacB double mutant produced spores with a two- to fourfold greater increase in PG cross-linking and novel muropeptides containing glycine residues but no significant changes in spore resistance or core hydration.     

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.