Phage SPO1 Protein Gp49 Is a Novel RNA Binding Protein That Is Involved in Host Iron Metabolism.

Bacillus subtilis is a model organism for studying Gram-positive bacteria and serves as a cell factory in the industry for enzyme and chemical production. Additionally, it functions as a probiotic in the gastrointestinal tract, modulating the gut microbiota. Its lytic phage SPO1 is also the most studied phage among the genus Okubovrius, including Bacillus phage SPO1 and Camphawk. One of the notable features of SPO1 is the existence of a "host-takeover module", a cluster of 24 genes which occupies most of the terminal redundancy. Some of the gene products from the module have been characterized, revealing their ability to disrupt host metabolism by inhibiting DNA replication, RNA transcription, cell division, and glycolysis. However, many of the gene products which share limited similarity to known proteins remain under researched. In this study, we highlight the involvement of Gp49, a gene product from the module, in host RNA binding and heme metabolism-no observation has been reported in other phages. Gp49 folds into a structure that does not resemble any protein in the database and has a new putative RNA binding motif. The transcriptome study reveals that Gp49 primarily upregulates host heme synthesis which captures cytosolic iron to facilitate phage development.

Bacillus subtilis KC1 prevents Mycoplasma gallisepticum-induced lung injury by enhancing intestinal Bifidobacterium animalis and regulating indole metabolism in chickens.

It has been reported that dietary administration of Bacillus subtilis KC1 is effective in alleviating lung injury induced by Mycoplasma gallisepticum (MG) infection in chickens. However, the underlying molecular mechanism of B. subtilis KC1 against MG infection is still unclear. The purpose of this study was to determine whether B. subtilis KC1 could alleviate MG infection-induced lung injury in chickens by regulating their gut microbiota. The results of this study indicate that B. subtilis KC1 supplementation has the potential to alleviate MG infection-induced lung injury as reflected by reduced MG colonization, reduced pathologic changes, and decreased production of pro-inflammatory cytokines. In addition, B. subtilis KC1 supplementation was partially effective in alleviating the gut microbiota disorder caused by MG infection. Importantly, B. subtilis KC1 enriched the beneficial Bifidobacterium animalis in gut and thus reversed indole metabolic dysfunction caused by MG infection. B. subtilis KC1 supplementation increased levels of indole, which enhanced aryl hydrocarbon receptor activation, improving barrier function and alleviating lung inflammation caused by MG. Overall, this study indicates that B. subtilis KC1 has a "gut-lung axis" mechanism that can reduce the severity of MG infection by enriching intestinal B. animalis and regulating indole metabolism.

Study on antibacterial properties of heated oyster shell particle against Bacillus subtilis spores in rainwater by response surface methodology based on central composite design.

Taiwan's oyster industry produces shell waste in abundant quantities every year. This study explored the feasibility of applying this resource as a simple and low-cost disinfectant to improve the microbial quality of harvested rainwater. Critical parameters affecting the disinfection efficacy of calcined oyster shell particles, i.e., heating temperature and duration, dosage, and contact time of the calcined shell material against Bacillus subtilis endospores in rainwater, were investigated. A central composite design of response surface methodology was employed to study the relative effects. As estimated from R2 coefficients, a quadratic model was identified to predict the response variable satisfactorily. Results indicated that the heating temperature, dosage, and contact time of the calcined material in the rainwater significantly influenced (p < 0.05) the sporicidal effect, consistent with the prior literature on calcined shells of similar nature. However, heating time had a relatively low influence on the sporicidal impact, suggesting that the rate of shell activation, i.e., conversion of the carbonate compound in the shell material to oxide, occurs rapidly at high calcination temperatures. In addition, the sterilization kinetics for heated oyster shell particles in aqueous media under stagnant storage conditions were investigated and found to be in good agreement with Hom's model.

Spore germination receptors - a new paradigm.

David Rudner and his team (Gao et al.) predict a pentameric structure for the GerA alanine-responsive germination receptor of Bacillus subtilis and demonstrate that it behaves as a nutrient-gated ion channel, finally establishing a function for this novel family of receptors and focussing research on early ion movements in germination.

Microbial Protocols for Spacecraft: 3. Spore Monolayer Preparation Methods for Ultraviolet Irradiation Exposures.

Developing robust microbial survival models for interplanetary and planetary spacecraft requires precise inactivation kinetics for vehicle bioburdens. To generate such data, reliable protocols are required for preparing, testing, and assaying microbial cells or spores on simulated spacecraft materials. New data are presented on the utility of the liquid droplet protocol for applying Bacillus subtilis spores to aluminum coupons. Results indicate that low-density spore monolayers should be created between 2 and 5 × 106 spores per cm2 on individual coupons to prevent the formation of aggregates or multilayers of spores. Such aggregation or multilayers will interfere with the precision of characterizing the effects of UV irradiation on spore survival. Optimum spore monolayers are defined as spore monolayers without overlapping or clustered cells and in which all spores will receive UV photons during assays. The best spore monolayers were created with sterile deionized water (SDIW) on uncoated aluminum coupons, or with SDIW + Triton X-100 (at 0.5 × of the critical micellar concentration) on either uncoated Al-coupons or on Chemfilm Class 1A-coated coupons. The Triton X-100 surfactant improved the uniformity of the monolayers without affecting the sensitivity of the spores to UV irradiation. Furthermore, spore layers created at either 2 × 107 or 2 × 108 spores/cm2 created multi-stacking effects that clearly reduced the precision of the UV irradiation assays. A set of standardized protocols is suggested for spacecraft processing and planetary protection communities to permit directly comparing results from divergent labs.

Complete genome sequencing of Bacillus subtilis (CWTS 5), a siderophore-producing bacterium triggers antagonistic potential against Ralstonia solanacearum.

AIM: The aims of this study were to explore the antagonistic potential of siderophore-producing Bacillus subtilis (CWTS 5) for the suppression of Ralstonia solanacearum and to explore the mechanisms of inhibition by FTIR, LC-MS, and whole genome analysis. METHODS AND RESULTS: A siderophore-producing B. subtilis (CWTS 5) possessing several plant growth-promoting properties such as IAA and ACC deaminase production, phosphate solubilization, and nitrogen fixation was assessed for its inhibitory effect against R. solanacearum, and its mechanisms were explored by in vitro and in vivo analyses. The active secondary metabolites in the siderophore extracts were identified as 2-deoxystreptamine, miserotoxin, fumitremorgin C, pipercide, pipernonaline, gingerone A, and deoxyvasicinone by LC-MS analysis. The Arnow's test and antiSMASH analysis confirmed the presence of catecholate siderophores, and the functional groups determined by FTIR spectroscopy confirmed the presence of secondary metabolites in the siderophore extract possessing antagonistic effect. The complete genome sequence of CWTS 5 revealed the gene clusters responsible for siderophore, antibiotics, secondary metabolite production, and antibacterial and antifungal metabolites. Furthermore, the evaluation of CWTS 5 against R. solanacearum in pot studies demonstrated 40.0% reduced disease severity index (DSI) by CWTS 5, methanolic extract (DSI-26.6%), ethyl acetate extract (DSI-20.0%), and increased plant growth such as root and shoot length, wet weight and dry weight of Solanum lycopersicum L. owing to its antagonistic potential. This genomic insight will support future studies on the application of B. subtilis as a plant growth promoter and biocontrol agent against R. solanacearum for bacterial wilt management. CONCLUSION: The results of this study revealed that B. subtilis (CWTS 5) possesses multiple mechanisms that control R. solanacearum, reduce disease incidence, and improve S. lycopersicum growth.

Acute physiological effects following Bacillus subtilis DE111 oral ingestion - a randomised, double blinded, placebo-controlled study.

Previous studies using ileostomy samples from study participants demonstrated that the spore-forming probiotic Bacillus subtilis DE111® can germinate in the small intestine as early as 4 hours after ingestion. Metabolomics, proteomics and sequencing technologies, enabled further analysis of these samples for the presence of hypoglycaemic, hypolipidemic, antioxidant, anti-inflammatory and antihypertensive molecules. In the DE111 treatment group, the polyphenols trigonelline and 2,5-dihydroxybenzoic acid, orotic acid, the non-essential amino acid cystine and the lipokine 12,13-diHome were increased. DE111 also reduced acetylcholine levels in the ileostomy samples, and increased the expression of leucocyte recruiting proteins, antimicrobial peptides and intestinal alkaline phosphatases of the brush border in the small intestine. The combination of B. subtilis DE111 and the diet administered during the study increased the expression of the proteins phosphodiesterase ENPP7, ceramidase ASAH2 and the adipokine Zn-alpha-2-glycoprotein that are involved in fatty acid and lipid metabolism. Acute B. subtilis DE111 ingestion had limited detectable effect on the microbiome, with the main change being its increased presence. These findings support previous data suggesting a beneficial role of DE111 in digestion, metabolism, and immune health that appears to begin within hours of consumption.

Prediction and validation of novel SigB regulon members in Bacillus subtilis and regulon structure comparison to Bacillales members.

BACKGROUND: Sigma factor B (SigB) is the central regulator of the general stress response in Bacillus subtilis and regulates a group of genes in response to various stressors, known as the SigB regulon members. Genes that are directly regulated by SigB contain a promotor binding motif (PBM) with a previously identified consensus sequence. RESULTS: In this study, refined SigB PBMs were derived and different spacer compositions and lengths (N12-N17) were taken into account. These were used to identify putative SigB-regulated genes in the B. subtilis genome, revealing 255 genes: 99 had been described in the literature and 156 genes were newly identified, increasing the number of SigB putative regulon members (with and without a SigB PBM) to > 500 in B. subtilis. The 255 genes were assigned to five categories (I-V) based on their similarity to the original SigB consensus sequences. The functionalities of selected representatives per category were assessed using promoter-reporter fusions in wt and ΔsigB mutants upon exposure to heat, ethanol, and salt stress. The activity of the PrsbV (I) positive control was induced upon exposure to all three stressors. PytoQ (II) showed SigB-dependent activity only upon exposure to ethanol, whereas PpucI (II) with a N17 spacer and PylaL (III) with a N16 spacer showed mild induction regardless of heat/ethanol/salt stress. PywzA (III) and PyaaI (IV) displayed ethanol-specific SigB-dependent activities despite a lower-level conserved - 10 binding motif. PgtaB (V) was SigB-induced under ethanol and salt stress while lacking a conserved - 10 binding region. The activities of PygaO and PykaA (III) did not show evident changes under the conditions tested despite having a SigB PBM that highly resembled the consensus. The identified extended SigB regulon candidates in B. subtilis are mainly involved in coping with stress but are also engaged in other cellular processes. Orthologs of SigB regulon candidates with SigB PBMs were identified in other Bacillales genomes, but not all showed a SigB PBM. Additionally, genes involved in the integration of stress signals to activate SigB were predicted in these genomes, indicating that SigB signaling and regulon genes are species-specific. CONCLUSION: The entire SigB regulatory network is sophisticated and not yet fully understood even for the well-characterized organism B. subtilis 168. Knowledge and information gained in this study can be used in further SigB studies to uncover a complete picture of the role of SigB in B. subtilis and other species.

Inhibition and disintegration of Bacillus subtilis biofilm with small molecule inhibitors identified through virtual screening for targeting TasA(28-261), the major protein component of ECM.

Microbial biofilms have been recognized for a vital role in antibiotic resistance and chronic microbial infections for 2-3 decades; still, there are no 'anti-biofilm drugs' available for human applications. There is an urgent need to develop novel 'anti-biofilms' therapeutics to manage biofilm-associated infectious diseases. Several reports have suggested that targeting molecules involved in quorum sensing or biofilm-specific transcription may inhibit biofilm formation. However, the possibility of targeting other vital components of microbial biofilms, especially the extracellular matrix (ECM) components, has remained largely unexplored. Here we report targeting TasA(28-261), the major proteinaceous component of Bacillus subtilis ECM with two small molecule inhibitors (lovastatin and simvastatin) identified through virtual screening and drug repurposing, resulted in complete inhibition of biofilm. In molecular docking and dynamics simulation studies, lovastatin was observed to make stable interactions with TasA(28-261), whereas the simvastatin - TasA(28-261) interactions were relatively less stable. However, in subsequent in vitro studies, both lovastatin and simvastatin successfully inhibited B. subtilis biofilm formation at MIC values of < 10 µg/ml. Besides, these potential inhibitors also caused the disintegration of pre-formed biofilms. Results presented here provide 'proof of concept' for the hypothesis that targeting the extracellular matrix's vital component(s) could be one of the most efficient approaches for inhibiting microbial biofilms and disintegrating the pre-formed biofilms. We propose that a similar approach targeting ECM-associated proteins with FDA-approved drugs could be implemented to develop novel anti-biofilm therapeutic strategies against biofilm-forming chronic microbial pathogens.Communicated by Ramaswamy H. Sarma.

Effects of Bacillus subtilis BS-Z15 on Intestinal Microbiota Structure and Body Weight Gain in Mice.

In our previous study, we identified a metabolite of Bacillus subtilis BS-Z15 (a strain with probiotic characteristics) that could improve immunity in mice. In the present study, we examined the effects of B. subtilis BS-Z15 and its metabolites on body weight gain and the intestinal microbiota of mice. Sixty 25-day-old male Kunming white mice were selected and randomly divided into four groups: control group (A), daily saline gavage; B. subtilis-treated group (B), single gavage (1 × 109 CFU/time/animal/day); group D, 14 consecutive gavages (1 × 109 CFU/time/animal/day); and B. subtilis metabolite-treated group (E), 30 consecutive gavages (90 mg kg-1/time/animal/day). High-throughput sequencing technology was used to analyze intergroup differences in the mouse intestinal microbiota. The results showed that the three treated groups had significantly slower body weight gain compared with the control group, which lasted until the 45 days (P < 0.05), and the daily food intake of the treated mice was higher (P < 0.05). The intestinal microbiota structure of the mice in the treated groups was significantly altered compared with that in the control group, suggesting that B. subtilis BS-Z15 may regulate the weight gain of animals by affecting their intestinal bacterial composition. After stopping the gavage of B. subtilis BS-Z15, the abundance of this strain in the small intestine of the mice gradually decreased and its presence was undetectable at 45 days, indicating that B. subtilis BS-Z15 could not colonize the intestine of these mice. These findings suggest that B. subtilis BS-Z15 may regulate intestinal microbiota through its metabolites to reduce weight gain.

Effects of Dietary Supplementation with Lactobacillus acidophilus and Bacillus subtilis on Mucosal Immunity and Intestinal Barrier Are Associated with Its Modulation of Gut Metabolites and Microbiota in Late-Phase Laying Hens.

We investigated the effects of dietary supplementation with Lactobacillus acidophilus and Bacillus subtilis on the intestinal immune response, intestinal barrier function, cecal microbiota profile, and metabolite profile in late-phase laying hens. Hens were divided into three groups and fed with the basal diet (NC group), basal diet supplementation with 250 mg/kg B. subtilis and L. acidophilus mixture powder (LD group), and basal diet supplementation with 500 mg/kg B. subtilis and L. acidophilus mixture powder (HD group), respectively. The results indicated that the dietary supplementation with L. acidophilus and B. subtilis increased the integrity of the intestinal barrier as evidenced by the significant increase in the number of ileal goblet cells and improve the expression of occludin, claudin-1, and ZO-1 genes in the HD group. Moreover, the levels of IL-6, TNF-α, and IFN-γ were significantly decreased in the LD and HD groups. The levels of immunoglobulin G (IgG) increased in the LD and HD group, and the levels of secretory immunoglobulin A (sIgA) increased with the HD treatment. Furthermore, 16 s rRNA sequencing revealed L. acidophilus in combination with B. subtilis increased the diversity of gut microbiota. The metabolomic analysis revealed beneficial changes in the amino acid metabolism and lipid metabolism (decrease in LysoPC and LysoPE levels). In conclusion, dietary supplementation with L. acidophilus and B. subtilis could improve intestinal barrier function and maintain immune homeostasis. These beneficial effects may be associated with the modulation of the intestinal microbiome and metabolites.

SirA inhibits the essential DnaA:DnaD interaction to block helicase recruitment during Bacillus subtilis sporulation.

Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD and DnaA, thereby restricting helicase recruitment from the origin during sporulation to inhibit further initiation events. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.

Host-derived bacillus spp. as probiotic additives for improved growth performance in broilers.

In recent years, the utilization of antibiotics in animal feed has been restricted, probiotics have been increasingly used to replace antibiotics in maintaining animal health. The aim of this study was to screen and evaluate probiotics with excellent probiotic potential from the gut of healthy goslings for clinical application. Thirteen strains of Bacillus (named AH-G201 to AH-G2013), including 2 strains of Bacillus subtilis (B. subtilis), 6 strains of Bacillus licheniformis (B. licheniformis) and 5 strains of Bacillus amyloliquefaciens (B. amyloliquefaciens), were isolated and identified. Then, acid and bile salts tolerance tests were performed to screen probiotics strains that could survive under different environments. The effects of screened probiotics on the growth of pathogenic Escherichia coli (E. coli) and Salmonella were assessed. Furthermore, we performed the drug resistance tests and safety tests in animals. The results showed that B. Subtilis AH-G201, B. licheniformis AH-G202 and AH-G204 exhibited higher gastrointestinal resistance under in vitro conditions, and showed a moderate level of resistance to the tested antibiotics. Importantly, AH-G201 and AH-G202 showed 24 to 60% inhibition rate against pathogenic E. coli and Salmonella. Moreover, the safety analysis of AH-G201 and AH-G202 suggested that the 2 probiotics strains have no adverse effects on body weight gain and feed intake in the broilers, and in addition, they have significantly improved growth performance. Finally, we analyzed effects of B. Subtilis AH-G201and B. licheniformis AH-G202 on growth performance, immune organ index and the feces microbes of broilers. The results showed that broilers fed with high doses (5 × 109 CFU/mL, for single strain) of a mixture of AH-G201 and AH-G202 exhibited good growth performance, and exhibited the greatest gain in spleen weight and the highest lactic acid bacteria counts. These findings indicate that the combined addition of B. Subtilis AH-G201 and B. licheniformis AH-G202 has the potential to replace antibiotics and to improve the growth performance of broilers.

The Biofilm Regulatory Network from Bacillus subtilis: A Structure-Function Analysis.

Bacterial biofilms are notorious for their ability to protect bacteria from environmental challenges, most importantly the action of antibiotics. Bacillus subtilis is an extensively studied model organism used to understand the process of biofilm formation. A complex network of principal regulatory proteins including Spo0A, AbrB, AbbA, Abh, SinR, SinI, SlrR, and RemA, work in concert to transition B. subtilis from the free-swimming planktonic state to the biofilm state. In this review, we explore, connect, and summarize decades worth of structural and biochemical studies that have elucidated this protein signaling network. Since structure dictates function, unraveling aspects of protein molecular mechanisms will allow us to devise ways to exploit critical features of the biofilm regulatory pathway, such as possible therapeutic intervention. This review pools our current knowledge base of B. subtilis biofilm regulatory proteins and highlights potential therapeutic intervention points.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Comparative Genomics and Physiological Characterization of Two Aerobic Spore Formers Isolated from Human Ileal Samples.

Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that niche. In the lower part of the GIT, they re-sporulate and leave the body as spores, therefore passing through their entire life cycle in the animal body. In the intestine, both un-germinated spores and germination-derived cells interact with intestinal and immune cells and have health-beneficial effects, which include the production of useful compounds, protection against pathogenic microorganisms, contribution to the development of an efficient immune system and modulation of the gut microbial composition. We report a genomic and physiological characterization of SF106 and SF174, two aerobic spore former strains previously isolated from ileal biopsies of healthy human volunteers. SF106 and SF174 belong respectively to the B. subtilis and Alkalihalobacillus clausii (formerly Bacillus clausii) species, are unable to produce toxins or other metabolites with cytotoxic activity against cultured human cells, efficiently bind mucin and human epithelial cells in vitro and produce molecules with antimicrobial and antibiofilm activities.

Water-Content-Dependent Morphologies and Mechanical Properties of Bacillus subtilis Spores' Cortex Peptidoglycan.

Peptidoglycan (PG), bacterial spores' major structural component in their cortex layers, was recently found to regulate the spore's water content and deform in response to relative humidity (RH) changes. Here, we report that the cortex PG dominates the Bacillus subtilis spores' water-content-dependent morphological and mechanical properties. When exposed to an environment having RH varied between 10% and 90%, the spores and their cortex PG reversibly expand and contract by 30.7% and 43.2% in volume, which indicates that the cortex PG contributes to 67.3% of a spore's volume change. The spores' and cortex PG's significant volumetric changes also lead to changes in their Young's moduli from 5.7 and 9.0 GPa at 10% RH to 0.62 and 1.2 GPa at 90% RH, respectively. Interestingly, these significant changes in the spores' and cortex PG's morphological and mechanical properties are only caused by a minute amount of the cortex PG's water exchange that occupies 28.0% of the cortex PG's volume. The cortex PG's capability in sensing and responding to environmental RH and effectively changing its structures and properties could provide insight into spores' high desiccation resistance and dormancy mechanisms.

An electric alarm clock for spores.

Inactive spores integrate stimuli over time through stored electrochemical potential.

Electrochemical potential enables dormant spores to integrate environmental signals.

The dormant state of bacterial spores is generally thought to be devoid of biological activity. We show that despite continued dormancy, spores can integrate environmental signals over time through a preexisting electrochemical potential. Specifically, we studied thousands of individual Bacillus subtilis spores that remain dormant when exposed to transient nutrient pulses. Guided by a mathematical model of bacterial electrophysiology, we modulated the decision to exit dormancy by genetically and chemically targeting potassium ion flux. We confirmed that short nutrient pulses result in step-like changes in the electrochemical potential of persistent spores. During dormancy, spores thus gradually release their stored electrochemical potential to integrate extracellular information over time. These findings reveal a decision-making mechanism that operates in physiologically inactive cells.

Dietary supplementation of two indigenous Bacillus spp on the intestinal morphology, intestinal immune barrier and intestinal microbial diversity of Rhynchocypris lagowskii.

This study evaluated the effects of dietary administration of two indigenous Bacillus (A: basal control diet; B: 0.15 g/kg of Bacillus subtilis; C: 0.1 g/kg of Bacillus subtilis and 0.05 g/kg of Bacillus licheniformis; D: 0.05 g/kg of Bacillus subtilis and 0.1 g/kg of Bacillus licheniformis; E: 0.15 g/kg of Bacillus licheniformis) on the digestive enzyme activities, intestinal morphology, intestinal immune and barrier-related genes relative expression levels, and intestinal flora of Rhynchocypris lagowskii. The results showed that the fold height, lamina propria width, and muscle layer thickness of midgut and hindgut in group C were significantly higher than that of group A (P < 0.05). The activities of protease, amylase, and lipase in group C were significantly higher than those of group A (P < 0.05). The relative expression levels of IL-1ß and IL-8 in the intestine of group C were significantly downregulated, and the relative expression levels of IL-10 and TGF-ß were significantly upregulated (P < 0.05). The relative expression levels of Claudin-2 in group A significantly increased and the relative expression levels of Claudin-4 in group A significantly reduced compared with other groups (P < 0.05). The relative expression levels of ZO-1 in groups C and D were significantly higher than those of other groups (P < 0.05). The Bacillus in the intestine of group C has the highest relative abundance among all groups. Overall, it can generally be concluded that dietary supplementation of indigenous Bacillus subtilis and Bacillus licheniformis (group C) can improve the intestinal morphology, digestion, and absorption enzyme activities, enhance intestinal mucosal immunity and barrier function, and maintain the intestinal microbial balance of R. lagowskii.