Bacillus Subtilis Spores Research Articles

Identification and characterization of the Bacillus subtilis spore germination protein GerY.

Pathogens in the orders Bacillales and Clostridiales resist sterilization by differentiating into stress-resistant spores. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients, they rapidly resume growth, causing food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, called germination, is a key target in combating these important pathogens. Here, we report a high-throughput genetic screen using transposon sequencing to identify novel germination factors that ensure the efficient exit from dormancy. We identify several new factors and characterize one in greater detail. This factor, renamed GerY, is part of the proteinaceous coat that encapsulates the dormant spore. Our data suggest that GerY enables efficient transit of nutrients through the coat to trigger germination.

Neutrophil-activating protein in Bacillus spores inhibits casein allergy via TLR2 signaling

BackgroundMilk allergy commonly occurs in children, mainly caused by bovine-derived casein (CAS) protein. Neutrophil-activating protein (NAP) of Helicobacter pylori plays an immunomodulatory role with potential to suppress Th2-type immune responses. Bacillus subtilis (B. subtilis) spores are commonly used as oral vectors for drug delivery.ObjectiveTo investigate whether recombinantly expressed NAP on B. subtilis spores could be an effective treatment for CAS allergy in mouse.MethodsAfter CAS sensitization, mice were orally administered B. subtilis spores expressing recombinant NAP for 6 weeks. Allergic symptoms and parameters were evaluated after CAS challenge oral gavage, including allergic inflammation, splenic cytokines, and serum-specific antibodies. Protein levels of Toll-like receptor 2 (TLR2) and c-JUN in the jejunum tissue were measured by western blot. Bone marrow-derived macrophages (BMDMs) were stimulated with inactivated NAP spores to measure the influence on cytokine profiles in vitro.ResultsNAP recombinant spore treatment significantly reduced allergic symptoms and intestinal inflammation. Interleukin-12 and interferon-gamma levels increased, whereas serum CAS-specific IgG1 and IgE levels decreased. TLR2 and c-JUN expression levels were elevated in the jejunal tissue. Inactivated NAP spores polarized BMDMs to the M1 phenotype and enhanced cytokine expression, which were inhibited by a TLR2 neutralizing antibody.ConclusionNAP offers a new strategy in the treatment of CAS allergy by inhibiting the Th2 response, while eliciting macrophages to promote Th1 immune responses.

Bacterial spore surface nanoenvironment requires a AAA+ ATPase to promote MurG function

Bacillus subtilis spores are produced inside the cytosol of a mother cell. Spore surface assembly requires the SpoVK protein in the mother cell, but its function is unknown. Here, we report that SpoVK is a sporulation-specific, forespore-localized putative chaperone from a distinct higher-order clade of AAA+ ATPases that promotes the peptidoglycan glycosyltransferase activity of MurG during sporulation, even though MurG does not normally require activation during vegetative growth. MurG redeploys to the forespore surface during sporulation, where we show that the local pH is reduced and propose that this change in cytosolic nanoenvironment abrogates MurG function. Further, we show that SpoVK participates in a developmental checkpoint in which improper spore surface assembly mis-localizes SpoVK, which leads to sporulation arrest. The AAA+ ATPase clade containing SpoVK includes specialized chaperones involved in secretion, cell envelope biosynthesis, and carbohydrate metabolism, suggesting that such fine-tuning might be a widespread feature of different subcellular nanoenvironments.

Display of Bacterial Exochitanase on Bacillus subtilis Spores Improved Enzyme Stability and Recyclability.

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry.

Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation

The transcriptional control of sporulation in Bacillus subtilis is reasonably well understood, but its translational control is underexplored. Here, we use RNA-seq, ribosome profiling and fluorescence microscopy to study the translational dynamics of B. subtilis sporulation. We identify two events of translation silencing and describe spatiotemporal changes in subcellular localization of ribosomes during sporulation. We investigate the potential regulatory role of ribosomes during sporulation using a strain lacking zinc-independent paralogs of three zinc-dependent ribosomal proteins (L31, L33 and S14). The mutant strain exhibits delayed sporulation, reduced germination efficiency, dysregulated translation of metabolic and sporulation-related genes, and disruptions in translation silencing, particularly in late sporulation.

Degradable living plastics programmed by engineered spores.

Plastics are widely used materials that pose an ecological challenge because their wastes are difficult to degrade. Embedding enzymes and biomachinery within polymers could enable the biodegradation and disposal of plastics. However, enzymes rarely function under conditions suitable for polymer processing. Here, we report degradable living plastics by harnessing synthetic biology and polymer engineering. We engineered Bacillus subtilis spores harboring the gene circuit for the xylose-inducible secretory expression of Burkholderia cepacia lipase (BC-lipase). The spores that were resilient to stresses during material processing were mixed with poly(caprolactone) to produce living plastics in various formats. Spore incorporation did not compromise the physical properties of the materials. Spore recovery was triggered by eroding the plastic surface, after which the BC-lipase released by the germinated cells caused near-complete depolymerization of the polymer matrix. This study showcases a method for fabricating green plastics that can function when the spores are latent and decay when the spores are activated and sheds light on the development of materials for sustainability.

Isolation and characterization of superdormant Bacillus subtilis spores under high hydrostatic pressure at 200 MPa and 500 MPa

High hydrostatic pressure (HHP) (200 MPa/500 MPa) superdormant (SD) spores were isolated and characterized for germination and resistance properties. Compared with untreated spores, two-hundred MPa-SD spores showed germination defect with L-alanine, AGFK, dodecylamine, 200 MPa or 500 MPa. Meanwhile, they exhibited elevated resistance to 93 °C and UV254 nm with decimal reduction time (D-value) increased by 2.7 and 1.5 fold respectively, reduced resistance to 0.22 M HCl and 3.2 M H2O2 with D-value decreased by 1.4 and 1.3 fold respectively, and no change in resistance to 0.83 M formaldehyde and 0.34 M NaClO. Five-hundred MPa-SD spores showed germination defect with 500 MPa, no change in germination with L-alanine, AGFK or 200 MPa, but increased germination efficiency with dodecylamine. Accordingly, their resistance to 0.34 M NaClO and UV254 nm elevated with D-value increased by 1.8 and 1.3 fold respectively, while resistance to 93 °C, 0.22 M HCl, 0.83 M formaldehyde and 3.2 M H2O2 reduced with D-value decreased by 2, 1.4, 1.4 and 2.3 fold respectively. These properties were noninheritable as re-sporulated spores from SD spores showed identical germination behavior compared to initial untreated spores. Proteomics analysis revealed that the mechanism of SD spores' superdomancy was likely attributed to the variation of coat proteins and SASPs, and inner membrane modifications. Industrial relevanceIt has long been acknowledged that the existence of SD spores greatly limits the successful application of HHP technology in low-acid foods. The data in this work provide valuable knowledge to understand the properties of SD spores under HHP, and thus hopefully can promote developing new strategies to eliminate bacterial spores in low-acid foods during industrial HHP processing.

The effects of Bacillus subtilis spores and yeast cell wall supplementation on growth and health in Holstein dairy calves

The study aimed to assess the impact of Bacillus subtilis spores and yeast cell wall (YCW) on the performance and health of dairy calves during the milk-feeding period. Thirty female Holstein calves (birth body weight [BW] of 36.7 ± 4.81 kg) were randomly assigned to three treatments: Control (CTL, no additives), T1 (Bacillus subtilis spores), and T2 (Bacillus subtilis + YCW). Animals were individually housed with free access to water and commercial pellet starter. Calves received 8 L/d of pasteurized waste milk and were weaned at 63 d of age. The treatments (T1 and T2) were mixed into milk and administered daily from 3 to 63 d of age. Dry matter (DM) intake (from liquid and starter feed), fecal score, and rectal temperature were recorded daily. Additionally, heart girth was measured at 0, 15, 30, 45, and 60 d of age, and BW was measured at 0 and 60 d of age. Blood was sampled from the jugular vein at 0, 20, and 60 d of age for analysis of serum immunoglobulin G (IgG). There was treatment × time interaction (P = 0.04) for fecal consistency scores. Calves supplemented with Bacillus subtilis tended (χ2 test, P = 0.07) to have lower cases of diarrhea than other groups. T1 or T2 supplementations did not affect milk DM intake, starter DM intake, or total DM intake. Growth performance (BW at weaning and average daily gain) was similar between treatments. Dairy calves fed Bacillus subtilis plus YCW tended to have (P = 0.09) higher rectal temperature and had higher frequency (χ2 test, P < 0.01) of rectal temperature >39.1°C compared to the CTL and Bacillus subtilis groups. However, treatments had similar levels of serum IgG (CTL = 18.3, T1 = 16.7, and T2 = 18.6 mg/mL; P = 0.58). Our results suggest that Bacillus subtills and YCW may interact in the digestive tract of young calves as evidenced by the increased rectal temperature in T2. While the cause of this interaction remains uncertain, there was no negative effect on the animal health or growth performance. The use of Bacillus subtilis spores shows promise in improving fecal consistency scoring in dairy calves fed pasteurized waste milk during the pre-weaning phase.

Inactivation effects of combined thermosonication and potassium sorbate treatments on Bacillus subtilis spores.

This study aimed to investigate the inactivation effect of combined TS (thermosonication) and PS (potassium sorbate) treatments on Bacillus subtilis spores. The inactivation effect and potential mechanisms were examined using plate counts, OD600 values, nucleic acid leakage, DPA (dipicolinic acid) leakage, flow cytometry, and FTIR (Fourier transform infrared spectroscopy). The results showed that, after TS + PS treatments, the integrity of the inner membrane was lost, the permeability of the inner membrane to water molecules was increased, and the intraspore substances leaked. Furthermore, the OD600 value was reduced, indicating that the spore core hydration was enhanced. Spores proportion with damaged inner membrane was significantly increased to 66%, the ordered secondary structure of the protein was changed into a disordered structure and nucleic acid was fragmented after TS + PS treatment. The results indicated that the combined TS and PS treatments may be a useful method for inactivating bacterial spores in food processing and sterilization.

A Dual-Mode Fluorescent Nanoprobe for the Detection and Visual Screening of Pathogenic Bacterial Spores.

Bacterial vegetative cells turn into metabolically dormant spores in certain environmental situations. Once suitable conditions trigger the germination of spores belonging to the pathogenic bacterial category, public safety and environmental hygiene will be threatened, and lives will even be endangered when encountering fatal ones. Instant identification of pathogenic bacterial spores remains a challenging task, since most current approaches belonging to complicated biological methods unsuitable for onsite sensing or emerging alternative chemical techniques are still inseparable from professional instruments. Here we developed a polychromatic fluorescent nanoprobe for ratiometric detection and visual inspection of the pathogenic bacterial spore biomarker, dipicolinic acid (DPA), realizing rapidly accurate screening of pathogenic bacterial spores such as Bacillus anthracis spores. The nanoprobe is made of aminoclay-coated silicon nanoparticles and functionalized with europium ions, exhibiting selective and sensitive response toward DPA and Bacillus subtilis spores (simulants for Bacillus anthracis spores) with excellent linearity. The proposed sensing strategy allowing spore determination of as few as 0.3 × 105 CFU/mL within 10 s was further applied to real environmental sample detection with good accuracy and reliability. Visual quantitative determination can be achieved by analyzing the RGB values of the corresponding test solution color via a color recognition APP on a smartphone. Different test samples can be photographed at the same time, hence the efficient accomplishment of examining bulk samples within minutes. Potentially employed in various on-site sensing occasions, this strategy may develop into a powerful means for distinguishing hazardous pathogens to facilitate timely and proper actions of dealing with multifarious security issues.

Impact of sporulation temperature on germination of Bacillus subtilis spores under optimal and adverse environmental conditions

Bacillus subtilis spores are important food spoilage agents and are occasionally involved in food poisoning. In foods that are not processed with intense heat, such bacterial spores are controlled by a combination of different hurdles, such as refrigeration, acidification, and low water activity (aw), which inhibit or delay germination and/or growth. Sporulation temperature has long been regarded as a relevant factor for the assessment of germination in chemically defined media, but little is known about its impact on food preservation environments. In this study, we compared germination dynamics of B. subtilis spores produced at optimal temperature (37 °C) with others incubated at suboptimal (20 °C) and supraoptimal (43 °C) temperatures in a variety of nutrients (rich-growth medium, L-alanine, L-valine, and AGFK) under optimal conditions as well as under food-related stresses (low aw, pH, and temperature). Spores produced at 20 °C had a lower germination rate and efficiency than those incubated at 37 °C in all the nutrients, while those sporulated at 43 °C displayed a higher germination rate and/or efficiency in response to rich-growth medium and mostly to L-alanine and AGFK under optimal environmental conditions. However, differences in germination induced by changes in sporulation temperature decreased when spores were activated by heat, mainly due to the greater benefit of heat for spores produced at 20 °C and 37 °C than at 43 °C, especially in AGFK. Non-heat-activated spores produced at 43 °C still displayed superior germination fitness under certain stresses that had considerably impaired the germination of the other two populations, such as reduced temperature and aw. Moreover, they presented lower temperature and pH boundaries for the inhibition of germination in rich-growth medium, while requiring a higher NaCl concentration threshold compared to spores obtained at optimal and suboptimal temperature. Sporulation temperature is therefore a relevant source of variability in spore germination that should be taken into account for the accurate prediction of spore behaviour under variable food preservation conditions with the aim of improving food safety and stability.

Detoxification of aflatoxin B1 by a Bacillus subtilis spore coat protein through formation of the main metabolites AFQ1 and epi-AFQ1.

A variety of important agricultural crops host fungi from the Aspergillus genus can produce cancerogenic secondary metabolites such as aflatoxins. Consequently, novel strategies for detoxification and their removal from food and feed chains are required. Here, detoxification of Aflatoxin B1 (AFB1) by the Bacillus subtilis multi-copper oxidase CotA (BsCotA) was investigated. This laccase was recombinantly produced in E. coli while codon optimization led to duplication of the amount of active protein obtained. CuCl2 was added to the cultivation medium leading to a 25-fold increase of V max corresponding to improved incorporation of Cu2+ into the enzyme protein which is essential for the catalytic reaction. To avoid potential cytotoxicity of Cu2+, cultivation was performed at microaerobic conditions indeed leading to 100x more functional protein when compared to standard aerobic conditions. This was indicated by an increase of V max from 0.30 ± 0.02 to 33.56 ± 2.02 U/mg. Degradation kinetics of AFB1 using HPLC with fluorescence detection (HPLC-FLD) analysis indicated a theoretical substrate saturation above solubility in water. At a relatively high concentration of 500 μg/L, AFB1 was decomposed at 10.75 μg/Lh (0.17 nmol*min-1*mg-1) at a dosage of 0.2 μM BsCotA. AFQ1 and epi-AFQ1 were identified as the initial oxidation products according to mass spectrometry (i.e., HPLC-MS, HPLC-QTOF). None of these molecules were substrates for laccase but both decomposed in buffer. However, decomposition does not seem to be due to hydration of the vinyl ether in the terminal furan ring. Genotoxicity of the formed AFB1 was assessed in several dilutions based on the de-repression of the bacterial SOS response to DNA damage indicating about 80-times reduction in toxicity when compared to AFQ1. The results of this study indicate that BsCotA has high potential for the biological detoxification of aflatoxin B1.

Effects of spore purity on the wet heat resistance of Clostridium perfringens, Bacillus cereus and Bacillus subtilis spores

Heat resistance of spores of Clostridium perfringens 8238 (Hobbs Serotype 2), Bacillus cereus NCTC 11143 (4810/72), and Bacillus subtilis PS533, an isogenic derivative of strain PS832 (a 168 strain) was determined in ground beef at 95 °C. Spore purification was by centrifugation and washing with sterile distilled water (dH2O), followed by sonication and then Histodenz centrifugation for B. subtilis and C. perfringens, and centrifugation and washing with sterile dH2O followed by Histodenz centrifugation for B. cereus. Bags containing inoculated beef samples were submerged in a temperature-controlled water bath and held at 95 °C for predetermined lengths of time. Surviving spore populations were enumerated by plating on mannitol egg yolk polymyxin agar (MYP) agar plates for B. cereus and B. subtilis, and on tryptose-sulfite-cycloserine agar (TSC) agar plates for C. perfringens. Survivor curves were fitted to linear, linear with tail, and Weibull models using the USDA Integrated Pathogen Modeling Program (IPMP) 2013 software. The Weibull model provided a relatively better fit to the data since the root mean square error (RMSE), mean square error (MSE), sum of squared errors (SSE), and Akaike information criterion (AIC) values were lower than the values obtained using the linear or the linear with tail models. Additionally, the Weibull model accurately predicted the observed D-values at 95 °C for the three spore-formers since the accuracy factor (Af) values ranged from 1.03 to 1.08 and the bias factor (Bf) values were either 1.00 or 1.01. Times at 95 °C to achieve a 3-log reduction decreased from 206 min for C. perfringens spores purified with water washes alone to 191 min with water washes followed by sonication and Histodenz centrifugation, from 7.9 min for B. cereus spores purified with water washes alone to 1.4 min with water washes followed by Histodenz centrifugation, and from 20.6 min for B. subtilis spores purified with water washes alone to 6.7 min for water washes followed by sonication and Histodenz centrifugation. Thermal-death-time values reported in this study will assist food processors to design thermal processes to guard against bacterial spores in cooked foods. In addition, clearly spore purity is an additional factor in spore wet heat resistance, although the cause of this effect is not clear.

Development of fluorescent GO-AgNPs-Eu3+ nanoparticles based paper visual sensor for foodborne spores detection

Foodborne spores are ubiquitous with extremely strong resistance, and pose a serious threat to food safety and human health. Therefore, rapid, sensitive, and selective detection of spores are crucial. In this study, a fluorescent probe was developed based on lanthanide ion (Eu3+)-labeled nano-silver-modified graphene oxide (GO-AgNPs-Eu3+) for the detection of 2,6-dipicolinic acid (DPA), a biomarker unique to spores, to allow quantitative spores detection. The GO-AgNPs-Eu3+ nano-fluorescent probe was loaded onto a polyvinylidene fluoride microfiltration membrane, and a smartphone-assisted portable GO-AgNPs-Eu3+ nanoparticles-based paper visual sensor was designed for rapid on-site quantitative and real-time online detection of spores. The results indicated that the developed probe achieved equilibrium binding with DPA within 5 min, and enhanced fluorescence emission through antenna effect. The fluorescence detection presented a good linear relationship in the DPA concentration range of 0–45 μM, with a DPA detection limit of 4.62 nM and spore detection limit of 104 cfu/mL. The developed sensor showed a change in fluorescence from blue to red with increasing DPA concentration, and this color change was quantitatively detected through smartphone RGB variations, with a detection limit of 13.1 μM for DPA and 6.3 cfu/mL for Bacillus subtilis spores. Subsequently, the sensitivity and selectivity of the developed sensor were verified using actual milk and water samples spiked with B. subtilis spores. The results of this study provided objective technological support for rapid detection of spores, which is important for reducing the occurrence of foodborne diseases and improving food safety.

The novel sterilization device: the prototype testing

Currently, there are numerous methods that can be used to neutralize pathogens (i.e., devices, tools, or protective clothing), but the sterilizing agent must be selected so that it does not damage or change the properties of the material to which it is applied. Dry sterilization with hydrogen peroxide gas (VHP) in combination with UV-C radiation is well described and effective method of sterilization. This paper presents the design, construction, and analysis of a novel model of sterilization device. Verification of the sterilization process was performed, using classical microbiological methods and flow cytometry, on samples containing Geobacillus stearothermophilus spores, Bacillus subtilis spores, Escherichia coli, and Candida albicans. Flow cytometry results were in line with the standardized microbiological tests and confirmed the effectiveness of the sterilization process. It was also determined that mobile sterilization stations represent a valuable solution when dedicated to public institutions and businesses in the tourism sector, sports & fitness industry, or other types of services, e.g., cosmetic services. A key feature of this solution is the ability to adapt the device within specific constraints to the user’s needs.

Assembling the Bacillus subtilis Spore Coat Basement Layer on Spherical Supported Lipid Bilayers.

Micro- and nanoparticles are often designed by mimicking naturally occurring structures. Bacterial spores are dormant cells elaborated by some Gram-positive bacteria during poor growth conditions to protect their genetic material from harsh environmental stresses. In Bacillus subtilis, this protection is, in part, conferred by a proteinaceous shell, the "coat", which is composed of ~80 different proteins. The basement layer of the coat contains two unusual proteins, which we have recently reconstituted around silica beads to generate synthetic spore-like particles termed "SSHELs". Here, we describe the protocol for generating SSHEL particles, and describe the procedure to covalently link molecules of interest (in this case an anti-HER2 affibody) to SSHEL surfaces. SSHELs therefore represent a versatile platform for the display of ligands or antigens for the site-specific delivery of cargo or vaccines.

Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.

Effect of Humidity on the Inactivation of Bacillus Subtilis Spore by Non-thermal Atmospheric Air Plasma

Objective: If a medical device is not properly disinfected, it will pose a potential danger to patients. Spore is an experimental strain to verify the effect of disinfection on medical devices. It has a complex structure and is somewhat resistant to conventional disinfection techniques. Based on the advantages of simple structure and abundant reactive oxygen and nitrogen species (RONS), surface dielectric barrier discharge (SDBD) has been used to deal with inactivate spores, which provided experimental support for the application of sterilization spores in medical devices. Methods: The sterilization efficiency of the plasma was evaluated by calculating the number of viable colonies on Tryptose Soya Agar. RONS including ozone, nitrogen oxides and other reactive species produced in SDBD were measured by ozone analyzer, Fourier infrared spectrometer and chemical probes, respectively. Ultraviolet-visible spectrophotometer was used to determine the concentration of protein and nucleic acid. Results: After 20min of SDBD treatment, the number of inactivated spores could reach more than 4 log at high humidity (&gt;6g/m3), while the spores were hardly inactivated at low humidity (&lt;6g/m3). Ozone production was inhibited with increasing humidity while OH radicals and peroxynitrous acid increased with increasing humidity. The optical density values of protein and nucleic acid released from spores treated with SDBD was detected and a strong correlation was found with the spore inactivation trend. The protein shell and membrane structure of the spores were destroyed by SDBD, and more leakage and destruction of proteins and nucleic acids in the spores were obtained under the high humidity. Conclusion: With the increase of air humidity (from 0.15g/m3 to 70.16g/m3), stable SDBD is obtained showing a typical filamentous discharge pattern. Compared with low humidity, high humidity is more favorable to SDBD inactivation of spores.

Identification of CgeA as a glycoprotein that anchors polysaccharides to the spore surface in Bacillus subtilis.

The Bacillus subtilis spore is composed of a core, containing chromosomal DNA, surrounded by a cortex layer made of peptidoglycan, and a coat composed of concentric proteinaceous layers. A polysaccharide layer is added to the spore surface, and likely anchored to the crust, the coat outermost layer. However, the identity of the coat protein(s) to which the spore polysaccharides (SPS) are attached is uncertain. First, we showed that the crust proteins CotVWXYZ and CgeA were all contained in the peeled SPS layer obtained from a strain missing CotE, the outer coat morphogenetic protein, suggesting that the SPS is indeed bound to at least one of the spore surface proteins. Second, CgeA is known to be located at the most downstream position in the crust assembly pathway. An analysis of truncated variants of CgeA suggested that its N-terminal half is required for localization to the spore surface, while its C-terminal half is necessary for SPS addition. Third, an amino acid substitution strategy revealed that SPS was anchored at threonine 112 (T112), which constitutes a probable O-glycosylation site on CgeA. Our results indicated that CgeA is a glycoprotein required to initiate SPS assembly and serves as an anchor protein linking the crust and SPS layers.

Lethal and mutagenic effects of different LET radiations on Bacillus subtilis spores

New, useful microorganism resources have been generated by ionizing radiation breeding technology. However, the mutagenic effects of ionizing radiation on microorganisms have not been systematically clarified. For a deeper understanding and characterization of ionizing radiation-induced mutations in microorganisms, we investigated the lethal effects of seven different linear energy transfer (LET) radiations based on the survival fraction (SF) and whole-genome sequencing analysis of the mutagenic effects of a dose resulting in an SF of around 1% in Bacillus subtilis spores. Consequently, the lower LET radiations (gamma [surface LET: 0.2 keV/µm] and 4He2+ [24 keV/µm]) showed low lethality and high mutation frequency (MF), resulting in the major induction of single-base substitutions. Whereas higher LET radiations (12C5+ [156 keV/µm] and 12C6+ [179 keV/µm]) showed high lethality and low MF, resulting in the preferential induction of deletion mutations. In addition, 12C6+ (111) ion beams likely possess characteristics of both low- and high-LET radiations simultaneously. A decrease in the relative biological effectiveness and an evaluation of the inactivation cross section indicated that 20Ne8+ (468 keV/µm) and 40Ar13+ (2214 keV/µm) ion beams had overkill effects. In conclusion, in the mutation breeding of microorganisms, it should be possible to regulate the proportions, types, and frequencies of induced mutations by selecting an ionizing radiation of an appropriate LET in accordance with the intended purpose.