Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.     

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.     

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 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.     

Peptide Ligands That Bind Selectively to Spores of Bacillus subtilis and Closely Related Species

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.     

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.     

Heat activation and germination–promotion of Bacillus subtilis spores by infrared irradiation

The influence of infrared (IR) radiation on the viability and heat-activation of Bacillus subtilis spores, suspended in phosphate-buffered saline, was investigated. Two types of IR heaters with different spectral distributions were used. Near-infrared (NIR) and far-infrared (FIR) heaters with main wavelengths of approximately 1 μm and 3–6 μm, respectively, were utilized. Although both irradiation treatments decreased the number of B. subtilis colonies at a bulk temperature of approximately 75 °C, the mode of action was clearly different. In the case of the NIR heater, the number of colony-counts decreased gradually. In contrast, use of the FIR heater resulted in heat activation of the spores during the early stage of irradiation at a low bulk temperature (40–60 °C) over several minutes, followed by a decrease in the number of colonies. Consequently, FIR irradiation inactivated 90% of B. subtilis spores more effectively as compared to NIR irradiation for 20 min with a suspension volume of 20 ml and irradiation energy of 7.57 kW m^?2. Spore exposure to FIR irradiation accelerated their germination rate in nutrient broth; however, this was not true for treatment with the NIR heater. The absorption IR spectrum of B. subtilis spores indicated that FIR radiation was absorbed easily by the spore cell components and might activate the bioactive substances involved in germination. Even at the same irradiation energy, the influence of infrared radiation on spore germination was dependent on the IR spectral distribution. Bacterial spores undergoing germination lose their resistance to stressors, such as heat, chemicals and ultraviolet rays. FIR heating promotes heat activation and germination, thereby producing vegetative cells that are more susceptible to other killing methods, enabling the killing of bacterial spores at lower stress without product damage.     

CotC-CotU heterodimerization during assembly of the Bacillus subtilis spore coat

We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of σ^K and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to form a coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis. Monomeric forms of both CotU and CotC failed to be assembled at the surface of the developing spore and accumulated in the mother cell compartment of cells mutant for cotE. In contrast, neither CotU nor CotC accumulated in the mother cell compartment of cells mutant for cotH. These results suggest that CotH is required to protect both CotU and CotC in the mother cell compartment of the sporangium and that CotE is needed to allow their assembly and subsequent interaction at the spore surface.     

Diverse supramolecular structures formed by self‐assembling proteins of the Bacillus subtilis spore coat

Bacterial spores (endospores), such as those of the pathogens Clostridium difficile and Bacillus anthracis, are uniquely stable cell forms, highly resistant to harsh environmental insults. Bacillus subtilis is the best studied spore‐former and we have used it to address the question of how the spore coat is assembled from multiple components to form a robust, protective superstructure. B. subtilis coat proteins (CotY, CotE, CotV and CotW) expressed in Escherichia coli can arrange intracellularly into highly stable macro‐structures through processes of self‐assembly. Using electron microscopy, we demonstrate the capacity of these proteins to generate ordered one‐dimensional fibres, two‐dimensional sheets and three‐dimensional stacks. In one case (CotY), the high degree of order favours strong, cooperative intracellular disulfide cross‐linking. Assemblies of this kind could form exquisitely adapted building blocks for higher‐order assembly across all spore‐formers. These physically robust arrayed units could also have novel applications in nano‐biotechnology processes.     

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.     

Kinetics of Inactivation of Bacillus Spores Using Low Temperature Argon Plasma at Different Microwave Power Densities

The objective of this study was to determine the remarkable role of the microwave power density of argon plasma in the inactivation of Bacillus subtilis, Bacillus stearothermophilus and Bacillus pumilus spores deposited on polypropylene bio‐indicator carriers. In particular, spore survival by argon plasma was determined as a function of the initial spore density of the bio‐indicators. The microwave induced argon plasmas were generated at 1.47, 2.63 and 4.21 w/cm³ microwave power densities under a low gas pressure of 50 Pa at an ambient temperature of 15 °C to reach low temperature distribution of 31, 35 and 43 °C, respectively. Our results indicate that the different Bacillus spores showed distinct degrees of argon plasma sensitivity, and spore survival was significantly reduced when the microwave power density of the plasma treatments was increased. Among the three Bacillus strains, Bacillus subtilis was the most argon plasma resistant, whereas Bacillus stearothermophilus was the most sensitive. However, spore survival was not affected by the initial spore density of the bio‐indicators. Only a certain degree of the spore inactivation log (No/N) from 1.67 to 1.95 was observed despite the 4‐order differences in the initial spore density of the Bacillus pumilus bio‐indicators.     

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log₁₀ reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.     

Interactions among CotB, CotG, and CotH during assembly of the Bacillus subtilis spore coat

Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides. One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface. The cotB gene was initially identified by reverse genetics as encoding an abundant coat component. cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66). Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46). Expression of cotB in sporulating cells of B. subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66. These results suggest that soon after synthesis, CotB undergoes a posttranslational modification. Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci. We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants. Therefore, both cotH and cotG are required for the efficient conversion of CotB-46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat. We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG. Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG. We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact. We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB.     

The response to a specific germinant by <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores in primary mouse macrophages is modulated by a protein encoded on the pXO1 plasmid

A Bacillus anthracis Sterne pXO1 plasmid-encoded protein designated Cot43 was found in coat extracts of purified spores. Cot43 is a tetratricopeptide repeat domain protein related to those which function as phosphatases in the sporulation phosphorelay and as regulators of competence and pathogenic factors. The synthesis of Cot43 began in the late exponential phase downstream from a sigmaA promoter (as mapped by RACE) and it was present at least until the formation of phase white endospores. There was specificity in the association of Cot43 with B. anthracis spores since Bacillus cereus producing Cot43 from a cloned gene had very little of this protein in spore coat extracts. In addition, Cot43 was synthesized by B. anthracis cells to the same extent in glucose-yeast extract and nutrient sporulation media, but was essentially absent from spores formed in the former. l-histidine is an important germinant for B. anthracis spores in macrophages, Spores produced by a mutant with a disruption of cot43 germinated in response to l-histidine both in vitro and within primary mouse macrophages earlier and more extensively than Sterne strain spores. The germination delay due to the presence of Cot43 would enhance spore survival and thus increase the chances for a successful infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Presence and function of a thick mucous layer rich in polysaccharides around Bacillus subtilis spores

This study was designed to establish the presence and function of the mucous layer surrounding spores of Bacillus subtilis. First, an external layer of variable thickness and regularity was often observed on B. subtilis spores. Further analyses were performed on B. subtilis 98/7 spores surrounded by a thick layer. The mechanical removal of the layer did not affect their resistance to heat or their ability to germinate but rendered the spore less hydrophilic, more adherent to stainless steel, and more resistant to cleaning. This layer was mainly composed of 6-deoxyhexoses, ie rhamnose, 3-O-methyl-rhamnose and quinovose, but also of glucosamine and muramic lactam, known also to be a part of the bacterial peptidoglycan. The specific hydrolysis of the peptidoglycan using lysozyme altered the structure of the required mucous layer and affected the physico-chemical properties of the spores. Such an outermost mucous layer has also been seen on spores of B. licheniformis and B. clausii isolated from food environments.     

Hydrophobic characteristics ofBacillus spores

Spores from severalBacillus species displayed a strong affinity for hexadecane and other hydrophobic solvents. The binding ofBacillus subtilis spore suspensions to octyl-Sepharose was enhanced by ammonium sulfate and other salts, but was inhibited by detergents. Treatment of spore suspensions with strong denaturants promoted their adherence to hexadecane, presumably by exposing hydrophobic residues in coat proteins. The hydrophobic characteristics of spores may be important in the ecological adaptation of certain bacteria.     

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.     

Extreme Spore UV Resistance of <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Isolates Obtained from an Ultraclean Spacecraft Assembly Facility

Recent environmental microbial sampling of the ultraclean Spacecraft Assembly Facility at NASA Jet Propulsion Laboratory (JPL-SAF) identified spores of Bacillus pumilus as major culturable bacterial contaminants found on and around spacecraft. As part of an effort to assess the efficacy of various spacecraft sterilants, purified spores of 10 JPL-SAF B. pumilus isolates were subjected to 254-nm UV and their UV resistance was compared to spores of standard B. subtilis biodosimetry strains. Spores of six of the 10 JPL-SAF isolates were significantly more resistant to UV than the B. subtilis biodosimetry strain, and one of the JPL-SAF isolates, B. pumilus SAFR-032, exhibited the highest degree of spore UV resistance observed by any Bacillus spp. encountered to date.     

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.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.