Immunological detection of the GerA spore germination proteins in the spore integuments of Bacillus subtilis using scanning electron microscopy

Abstract To clarify the molecular mechanisms that trigger spore germination of Bacillus subtilis , the location of GerA proteins (GerAA, GerAB and GerAC), which were reported to be putative gene products of a receptor for one of the germinants, l-alanine, was investigated by immunological techniques using anti-GerA peptide antibodies. Four antibodies were raised against the corresponding epitopes, two in GerAA, one in GerAB and the other in GerAC molecules. The binding of all four antibodies to the inner surface of the cortex-less spore coat fragments could be seen by scanning immunoelectron microscopy with colloidal gold particles. The result agreed with the fact, previously reported, that the colloidal gold particles were visualized just inside the spore coat layer by transmission immunoelectron microscopy using another anti-GerAB peptide antibody.     

Presence of proteins derived from the vegetative cell membrane in the dormant spore coat of Bacillus subtilis

To confirm the presence of the outer spore membrane in dormant spore coats of Bacillus subtilis, the proteins from vegetative cell membrane and dormant spore coat fractions were compared by immunoblot assay with antibodies prepared against both preparations. The spore coat fraction contained at least 11 proteins antigenically identical to those in the vegetative cell membranes. Further, the cytochemical localization of the proteins derived from vegetative cell membrane in dormant spores was examined by an immunoelectron microscopy method with a colloidal gold-immunoglobulin G complex. The colloidal gold particles were observed in the coat region and around the core region of dormant spore. These results have provided evidence that some proteins from vegetative cell membrane remain in the dormant spore coat region of B. subtilis, although it is not clear whether the outer membrane persists as an intact functional entity or not.     

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.     

Immunological Detection of 22K Protein in Sporulating Cells of Bacillus megaterium ATCC 12872

The synthesis and deposition of 22,000-dalton (22K) spore coat protein were examined immunochemically on the sporulating cells of Bacillus megaterium ATCC 12872 using the antibody to purified 22K spore coat protein. This antibody cross-reacted with 44K and 25K proteins in immunoblot analysis of dormant spore coat proteins. Immunoblot analysis on the sporulating cells showed that 22K protein was detected from t₈ in forespore coat protein fractions. Sandwich enzyme immunoassay revealed that 22K protein in the spore coat protein fraction appeared at t₆ and reached a plateau at t₉, and 22K protein in the mother cell cytoplasmic fraction was detected at only t₇ and t₈ at a very low level.     

Synthesis and deposition of spore coat proteins during sporulation of Bacillus megaterium

Rabbit (anti-spore coat protein) IgG was prepared by immunization with coat proteins extracted with sodium dodecyl sulfate and dithiothreitol from isolated spore coats of Bacillus megaterium ATCC 12872. Coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fraction by sandwich enzyme immunoassay using this antibody. The proteins in the forespore coat protein fraction increased from t3 and reached a plateau at t10. Immunoblot analysis for the coat proteins in sporulating cells revealed the sequential synthesis of various proteins in the mother cell cytoplasmic fraction and simultaneous deposition of the same proteins as in the forespore coat fraction. These results suggest that turnover of precursor proteins of the spore coat is very rapid if precursor proteins are produced and they are proteolytically processed to produce mature proteins. Specific antibody to the 48,000-dalton protein, which is a major protein, did not cross-react with any other major (36,000, 22,000, 19,500, and 17,500-dalton) proteins. Specific antibody to the 22,000-dalton protein did not cross-react with the 48,000, 36,000, 19,500, 17,500, and 16,000-dalton proteins, but did cross-react with the 44,000, 25,000, and 12,000-dalton proteins.     

The Clostridium botulinum GerAB germination protein is located in the inner membrane of spores

Clostridium botulinum dormant spores germinate in presence of l-alanine via a specific receptor composed of GerAA, GerAB and GerAC proteins. In Bacillus subtilis spores, GerAA and GerAC proteins were located in the inner membrane of the spore. We studied the location of the GerAB protein in C. botulinum spore fractions by Western-blot analysis, using an antipeptidic antibody. The protein GerAB was in vitro translated and used to confirm the specificity of the antibodies. GerAB was not present in a coat and spore outer membrane fraction but was present in a fraction of decoated spores containing inner membrane. These results strongly suggest that the protein GerAB is located in the inner membrane of the spore.     

Spore coat proteins of Dictyostelium discoideum are packaged in prespore vesicles

Previous studies have shown that Dictyostelium discoideum spore coat proteins are found in prespore cells, which are localized to the posterior region of migrating slugs, and in the coats of mature spores. Prespore vesicles, identified by morphology and by staining with anti-D. mucoroides spore serum, are also localized in the posterior region of migrating slugs. Using antisera specific to the spore coat proteins, we show that the spore coat proteins are packaged in prespore vesicles. They are present in the vesicles as a complex which can be dissociated by denaturation. The anti-D. mucoroides spore serum reacts with at least five proteins in whole spore extracts including the spore coat proteins SP96 and SP70.     

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.     

Coat and enterotoxin-related proteins in Clostridium perfringens spores

Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.     

Characterization of aBacillus subtilis germination mutant with pleiotropic alterations in spore coat structure

One class of spore germination mutants ofBacillus subtilis produces lysozymesensitive spores with altered surface structure. These mutations were pleiotropic in that the pattern of soluble and insoluble spore coat proteins was extensively changed with the virtual absence of a major 12kd polypeptide. Reversion to the lysozyme-resistant phenotype (and wild-type spore coat profile) at or near the site of the original mutation occurred at a frequency consistent with an initial point mutation.The 12kd protein was also absent from extracts of sporulating cells of the mutant although antigens of 14kd and 32kd protein cross-reacting with antibody to the 12kd polypeptide were detected. The 32kd antigen was also present in extracts of sporulating cells but not in the extracts of the spore coat of the wild type and is probably a precursor. Improper processing of such a precursor could account for the extensive alterations of coat structure.     

Multifunctional inorganic-binding beads self-assembled inside engineered bacteria

Multifunctional shell-core nano/microbeads with a hydrophobic biopolymer core and a designed protein coat for selective binding of an inorganic substance and antibodies were self-assembled inside engineered bacteria. Hybrid genes were constructed to produce tailormade bead-coating proteins in the bacterium Escherichia coli. These fusion proteins contained a binding peptide for an inorganic material, the antibody binding ZZ domain, and a self-assembly promoting as well as biopolymer synthesizing enzyme. Production of these multidomain fusion proteins inside E. coli resulted in self-assembly of beads comprising a biopolyester core and displaying covalently bound binding sites for specific and selective binding of an inorganic substance and any antibody belonging to the immunoglobulin G class. Engineered beads were isolated and purified from the respective E. coli cells by standard cell disruption procedures. Bead morphology and the binding functionalities displayed at the bead surface were assessed by the enzyme-linked immunosorbent assay, transmission electron microscopy, elemental analysis, backscattering electron density, analytical density ultracentrifugation, and atomic force microscopy. These analyses showed that bacteria can be engineered to produce fusion proteins mediating self-assembly of spherical biopolymer beads with binding affinity to gold and/or silica and antibodies. Spherical structures of this type could conceivably serve as nano/microdevices for bioimaging in medical approaches where an antibody mediated targeted delivery of an inorganic contrast agent would be desired.     

东方蜜蜂微孢子虫一新型孢壁蛋白的基因克隆、 表达及亚细胞定位

【目的】微孢子虫(Microsporidia)孢壁在孢子构成及孢子侵染宿主过程中扮演重要的角色。本研究旨在鉴定获得的东方蜜蜂微孢子虫Nosema ceranae新型孢壁蛋白,并进行基因克隆和原核表达,明确其亚细胞定位。【方法】通过在线软件对东方蜜蜂微孢子虫新型孢壁蛋白AAJ76_1400036761序列进行生物信息学分析。利用PCR法获取目的片段并将其克隆至原核表达载体pCold II中,利用IPTG诱导表达重组蛋白并通过镍柱亲和层析法纯化目的蛋白。以获得的重组蛋白为抗原免疫小鼠制备多克隆抗体,通过间接免疫荧光技术和免疫胶体金定位技术对该蛋白进行亚细胞定位分析;利用蛋白质免疫印迹法检测该蛋白与东方蜜蜂微孢子虫几丁质壳的互作。【结果】在MicrosporidiaDB数据库中获得AAJ76_1400036761基因序列,基因全长681 bp,编码226个氨基酸;预测等电点为6.84,分子量为26.19 kD。SDS-PAGE电泳和Western blot结果表明AAJ76_1400036761重组蛋白能够在大肠杆菌Eescherichia coli Rosetta中高量表达。Western blot结果表明,制备的多克隆抗体能够特异地识别东方蜜蜂微孢子虫总蛋白中的AAJ76_1400036761,说明其在成熟东方蜜蜂微孢子虫中有表达。亚细胞定位结果显示,AAJ76_1400036761定位于东方蜜蜂微孢子虫孢壁上。重组蛋白AAJ76_1400036761能够与蜜蜂微孢子虫的几丁质壳结合。【结论】AAJ76_1400036761蛋白在东方蜜蜂微孢子虫成熟孢子中有表达;该蛋白定位于东方蜜蜂微孢子虫孢壁上,为东方蜜蜂微孢子虫新的孢壁蛋白。本研究为深入研究该蛋白的生物学功能奠定了基础。     

Proteomic analysis of a developmentally regulated secretory vesicle

Secretion of spore coat proteins from the prespore secretory vesicles (PSVs) in Dictyostelium discoideum is a signal mediated event that underlies terminal cell differentiation, and represents an important case of developmentally regulated secretion. In order to study the biochemical mechanisms that govern the regulated fusion of the PSVs with the plasma membrane and the subsequent secretion of their cargo, we purified this organelle from prespore cells. Analysis of protein extracts of highly purified PSVs indicated that, in addition to the cargo of structural spore coat proteins, many more proteins are associated with the PSVs. Their identification is paramount to the understanding of the mechanism of regulated secretion in this system. In this study we have taken the first comprehensive proteomic approach to the analysis of an entire, previously uncharacterized, organelle, with the goal of identifying the major proteins associated with the PSVs. We show that in addition to the structural spore coat proteins, the PSVs contain the enzymes needed for proper spore coat assembly (thioredoxin 2 and 3), regulatory proteins which we predict receive and transduce the developmental signal for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding proteins calfumirin-1 and calreticulin) as well as proteins that interact with the cytoskeleton to mediate movement of the PSVs to the plasma membrane (actin binding proteins coactosin and profilin 1). In addition, the results suggest that proteins can play multiple roles in the cell, and that protein function can be dictated in part by subcellular localization. The identification of the PSV proteins is allowing us to develop testable hypotheses about the roles of these proteins within the functional context of developmentally regulated secretion.     

Ultrastructural changes occurring during germination and outgrowth of spores of the thermophile Bacillus acidocaldarius.

Spores of the thermophilic, acidophilic, Bacillus acidocaldarius were covered by a thick outer coat and a laminated inner coat (5.5 nm periodicity). Small membranous vesicles were present in the spore core and they disappeared as germination proceeded. After depolymerization of the cortex, and a 30% increase in spore diameter a localized gap appeared in the laminated inner coat only. This inner coat gap was narrow and could be the whole length of the spore. The germ cell appeared to grow, or to be pushed towards the inner coat gap, at which stage the outer coat disappeared in the same localized area. As the vegetative cell grew out the spore coat fell away, with loose cortical material still attached to it. The young germ cell developed a large spherical electron dense inclusion body in the cytoplasm, at the same time as the ribosomal and nuclear areas became distinct.     

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.     

Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.