Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores.
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ABSTRACT:
SUBMITTER: Perez-Valdespino A
PROVIDER: S-EPMC4010985 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.
| S-EPMC100119 | biostudies-literature
Project description:To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.
| S-EPMC3147665 | biostudies-literature
Project description: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.
| S-EPMC4570822 | biostudies-literature
Project description:Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.
| S-EPMC6597383 | biostudies-literature
Project description:Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.
| S-EPMC3994143 | biostudies-other
Project description:Sporulene, a C 35-terpenoid hydrocarbon with an unusual pentacyclic structure, is produced by Bacillus subtilis during sporulation.
| S-EPMC2646877 | biostudies-other
Project description:Large-scale shotgun sequencing (RNA-seq) analysis of mRNAs in dormant Bacillus subtilis spores prepared on plates or in liquid generally found the same ∼46 abundant mRNA species, with >250 mRNAs detected at much lower abundances. Knowledge of the amount of phosphate in a single B. subtilis spore allowed calculation of the amount of mRNA in an individual spore as ∼106 nucleotides (nt). Given the levels of abundant spore mRNAs compared to those of other mRNAs, it was calculated that the great majority of low-abundance mRNAs are present in only small fractions of spores in populations. Almost all of the most abundant spore mRNAs are encoded by genes expressed late in sporulation in the developing spore under the control of the forespore-specific RNA polymerase sigma factor, σG, and most of the encoded proteins are in spores. Levels of the most abundant spore mRNAs were also relatively stable for a week at 4°C after spore harvest. RNA-seq analysis of mRNAs in highly purified and less-well-purified spores made in liquid, as well as from spores that were chemically decoated to remove possible contaminating mRNA, indicated that low-abundance mRNAs in spores were not contaminants in purified spore preparations, and several sources of low-abundance mRNAs in spores are suggested. The function of at least the great majority of spore mRNAs seems most likely to be the generation of ribonucleotides for new RNA synthesis by their degradation early in spore revival.IMPORTANCE Previous work indicates that dormant Bacillus subtilis spores have many hundreds of mRNAs, some of which are suggested to play roles in spores' "return to life" or revival. The present work finds only ∼46 mRNAs at ≥1 molecule spore, with others in only fractions of spores in populations, often very small fractions. Less-abundant spore mRNAs are not contaminants in spore preparations, but how spores accumulate them is not clear. Almost all abundant spore mRNAs are synthesized in the developing spore late in its development, most encode proteins in spores, and abundant mRNAs in spores are relatively stable at 4°C. These findings will have a major impact on thinking about the roles that spore mRNAs may play in spore revival.
| S-EPMC6456856 | biostudies-literature
Project description:The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric beta-glucuronidase enzyme from Escherichia coli.
| S-EPMC2935070 | biostudies-literature
Project description:Dormant bacterial spores are extraordinarily resistant to environmental insults and are vectors of various illnesses. However, spores cannot cause disease unless they germinate and become vegetative cells. The molecular details of initiation of germination are not understood, but proteins essential in early stages of germination, such as nutrient germinant receptors (GRs) and GerD, are located in the spore inner membrane. In this study, we examine how these germination proteins are organized in dormant Bacillus subtilis spores by expressing fluorescent protein fusions that were at least partially functional and observing spores by fluorescence microscopy. We show that GRs and GerD colocalize primarily to a single cluster in dormant spores, reminiscent of the organization of chemoreceptor signalling complexes in Escherichia coli. GRs require all their subunits as well as GerD for clustering, and also require diacylglycerol addition to GerD and GRs' C protein subunits. However, different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. We predict that the clusters represent a functional germination unit or 'germinosome' in the spore inner membrane that is necessary for rapid and cooperative response to nutrients, as conditions known to block nutrient germination also disrupt the protein clusters.
| S-EPMC7959159 | biostudies-literature
Project description:We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ?200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl3 to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability.Spores of Bacillus species cause food spoilage and disease and are extremely resistant to standard decontamination methods. This hardiness is partly due to spores' extremely low permeability to chemicals, including water. We present a method to directly monitor the uptake of molecules into B. subtilis spores by weighing spores in fluid. The results demonstrate the exchange of core water with subsecond resolution and show a correlation between water permeability and the rate at which small molecules can initiate or inhibit germination in coat-damaged spores. The ability to directly measure the uptake of molecules in the context of spores with known structural or genetic deficiencies is expected to provide insight into the determinants of spores' extreme resistance.
| S-EPMC4686191 | biostudies-literature