Overexpressing the cpr1953 Orphan Histidine Kinase Gene in the Absence of cpr1954 Orphan Histidine Kinase Gene Expression, or Vice Versa, Is Sufficient to Obtain Significant Sporulation and Strong Production of Clostridium perfringens Enterotoxin or Spo0A by Clostridium perfringens Type F Strain SM101.

The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan-Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10 min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Biological, physical and morphological factors for the programming of a novel microbial hygromorphic material.

The urgency for energy efficient, responsive architectures has propelled smart material development to the forefront of scientific and architectural research. This paper explores biological, physical, and morphological factors influencing the programming of a novel microbial-based smart hybrid material which is responsive to changes in environmental humidity. Hygromorphs respond passively, without energy input, by expanding in high humidity and contracting in low humidity.Bacillus subtilisdevelops environmentally robust, hygromorphic spores which may be harnessed within a bilayer to generate a deflection response with potential for programmability. The bacterial spore-based hygromorph biocomposites (HBCs) were developed and aggregated to enable them to open and close apertures and demonstrate programmable responses to changes in environmental humidity. This study spans many fields including microbiology, materials science, design, fabrication and architectural technology, working at multiple scales from single cells to 'bench-top' prototype.Exploration of biological factors at cellular and ultracellular levels enabled optimisation of growth and sporulation conditions to biologically preprogramme optimum spore hygromorphic response and yield. Material explorations revealed physical factors influencing biomechanics, preprogramming shape and response complexity through fabrication and inert substrate interactions, to produce a palette of HBCs. Morphological aggregation was designed to harness and scale-up the HBC palette into programmable humidity responsive aperture openings. This culminated in pilot performance testing of a humidity-responsive ventilation panel fabricated with aggregatedBacillusHBCs as a bench-top prototype and suggests potential for this novel biotechnology to be further developed.

Germination and outgrowth of Bacillus mycoides KBAB4 spores are impacted by environmental pH, quantitatively analyzed at single cell level with sporetracker.

Quantifying spore germination and outgrowth heterogeneity is challenging. Single cell level analysis should provide supplementary knowledge regarding the impact of unfavorable conditions on germination and outgrowth dynamics. This work aimed to quantify the impact of pH on spore germination and outgrowth, investigating the behavior of individual spore crops, produced under optimal and suboptimal conditions. Bacillus mycoides (formerly B. weihenstephanensis) KBAB4 spores, produced at pH 7.4 and at pH 5.5 were incubated at different pH values, from pH 5.2 to 7.4. The spores were monitored by microscopy live imaging, in controlled conditions, at 30 °C. The images were analyzed using SporeTracker, to determine the state of single cells. The impact of pH on germination and outgrowth times and rates was estimated and the correlation between these parameters was quantified. The correlation between germination and outgrowth times was significantly higher at low pH. These results suggest that an environmental pressure highlights the heterogeneity of spore germination and outgrowth within a spore population. Results were consistent with previous observations at population level, now confirmed and extended to single cell level. Therefore, single cell level analyses can be used to quantify the heterogeneity of spore populations, which is of interest in order to control the development of spore-forming bacteria, responsible for food safety issues.

Comparing resistance of bacterial spores and fungal conidia to pulsed light and UVC radiation at a wavelength of 254 nm.

Pulsed light (PL) inactivates microorganisms by UV-rich, high-irradiance and short time pulses (250 µs) of white light with wavelengths from 200 nm to 1100 nm. PL is applied for disinfection of food packaging material and food-contact equipment. Spores of seven Bacillus ssp. strains and one Geobacillus stearothermophilus strain and conidia of filamentous fungi (One strain of Aspergillus brasiliensis, A. carbonarius and Penicillium rubens) were submitted to PL (fluence from 0.23 J/cm2 to 4.0 J/cm2) and UVC (at λ = 254 nm; fluence from 0.01 J/cm2 to 3.0 J/cm2). One PL flash at 3 J/cm2 allowed at least 3 log-reduction of all tested microorganisms. The emetic B. cereus strain F4810/72 was the most resistant of the tested spore-forming bacteria. The PL fluence to 3 log-reduction (F3 PL) of its spores suspended in water was 2.9 J/cm2 and F3 UVC was 0.21 J/cm2, higher than F3 PL and F3 UVC of spores of B. pumilus SAFR-032 2.0 J/cm2 and 0.15 J/cm2, respectively), yet reported as a highly UV-resistant spore-forming bacterium. PL and UVC sensitivity of bacterial spores was correlated. Aspergillus spp. conidia suspended in water were poorly sensitive to PL. In contrast, PL inactivated Aspergillus spp. conidia spread on a dry surface more efficiently than UVC. The F2 PL of A. brasiliensis DSM1988 was 0.39 J/cm2 and F2 UVC was 0.83 J/cm2. The resistance of spore-forming bacteria to PL could be reasonably predicted from the knowledge of their UVC resistance. In contrast, the sensitivity of fungal conidia to PL must be specifically explored.

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination.

BACKGROUND: Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. RESULTS: Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. CONCLUSIONS: This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

Phenotypic, genotypic, and resistome of mesophilic spore-forming bacteria isolated from pasteurized liquid whole egg.

The production of whole-liquid eggs is of significant economic and nutritional importance. This study aimed to assess the phenotypic and genotypic diversity of mesophilic aerobic spore-forming bacteria (n = 200) isolated from pasteurized whole liquid egg and liquid egg yolk. The majority of the isolates were identified as belonging to the genera Bacillus (86 %), followed by Brevibacillus (10 %) and Lysinibacillus (4 %). For the phenotypic characterization, isolates were subjected to various heat shocks, with the most significant reductions observed at 80 °C/30 min and 90 °C/10 min for isolates recovered from raw materials. On the other hand, the decrease was similar for isolates recovered from raw material and final product at 100 °C/5 min and 110 °C/5 min. Genotypic genes related to heat resistance (cdnL, spoVAD, dacB, clpC, dnaK, and yitF/Tn1546) were examined for genotypic characterization. The dnaK gene showed a positive correlation with the highest thermal condition tested (110 °C/5 min), while 100 °C/5 min had the highest number of positively correlated genes (clpC, cdnL, yitF/Tn1546, and spoVAD). Whole Genome Sequencing of four strains revealed genes related to sporulation, structure formation, initiation and regulation, stress response, and DNA repair in vegetative cells. The findings of this study indicate that these mesophilic aerobic spore-forming bacteria may adopt several strategies to persist through the process and reach the final product. As the inactivation of these microorganisms during egg processing is challenging, preventing raw materials contamination and their establishment in processing premises must be reinforced.

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.

Counts of mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic sporeforming bacteria and persistence of Bacillus cereus spores throughout cocoa powder processing chain.

Sporeforming bacteria are a concern in some food raw materials, such as cocoa powder. Samples (n = 618) were collected on two farms and at several stages during cocoa powder manufacture in three commercial processing lines to determine the impact of each stage on bacterial spore populations. Mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic, and Bacillus cereus spore populations were enumerated in all the samples. Genetic diversity in B. cereus strains (n = 110) isolated from the samples was examined by M13 sequence-based PCR typing, partial sequencing of the panC gene, and the presence/absence of ces and cspA genes. The counts of different groups of sporeforming bacteria varied amongst farms and processing lines. For example, the counts of mesophilic aerobic spore-forming (MAS) populations of cocoa bean fermentation were lower than 1 log spore/g in Farm 1 but higher than 4 log spore/g in Farm 2. B. cereus isolated from cocoa powder was also recovered from cocoa beans, nibs, and samples after roasting, refining, and pressing, which indicated that B. cereus spores persist throughout cocoa processing. Phylogenetic group IV was the most frequent (73%), along with processing. Strains from phylogenetic group III (14 %) did not show the ces gene's presence.

Effect of novel and conventional food processing technologies on Bacillus cereus spores.

This chapter provides a summary of the effect of thermal and non-thermal processing technologies on Bacillus cereus spores, a well-known pathogenic bacterium associated with foodborne illnesses. B. cereus has been frequently detected in rice, milk products, infant food, liquid eggs products and meat products all over the world. This Gram positive, rod-shaped, facultative anaerobe can produce endospores that can withstand pasteurization, UV radiation, and chemical reagents commonly used for sanitization. B. cereus spores can germinate into vegetative cells that can produce toxins. The conventional regime for eliminating spores from food is retorting which uses the application of high temperature (121 °C). However, at this temperature, there could be a significant amount of loss in the organoleptic and functional qualities of the food components, especially proteins. This leads to the research on the preventive measures against germination and if possible, to reduce the resistance before using a non-thermal technology (temperatures less than retorting-121 °C) for inactivation. This chapter reviews the development and success of several food processing technologies in their ability to inactivate B. cereus spores in food.

Redefining development in Streptomyces venezuelae: integrating exploration into the classical sporulating life cycle.

Two growth modes have been described for the filamentous Streptomyces bacteria. Their classic developmental life cycle culminates in the formation of dormant spores, where movement to new environments is mediated through spore dispersal. In contrast, exploratory growth proceeds as a rapidly expanding vegetative mycelium that leads to extensive surface colonization and is associated with the release of volatile compounds that promote alkalinization (and reduced iron bioavailability) of its surrounding environment. Here, we report that exploratory growth in Streptomyces venezuelae can proceed in tandem with classic sporulating development in response to specific nutritional cues. Sporulating exploration is not accompanied by a rise in environmental pH but has the same iron acquisition requirements as conventional exploration. We found that mutants that were defective in their ability to sporulate were unaffected in exploration, but mutants undergoing precocious sporulation were compromised in their exploratory growth and this appeared to be mediated through premature activation of the developmental regulator WhiI. Cell envelope integrity was also found to be critical for exploration, as mutations in the cell envelope stress-responsive extracytoplasmic function sigma factor SigE led to a failure to explore robustly under all exploration-promoting conditions. Finally, in expanding the known exploration-promoting conditions, we discovered that the model species Streptomyces lividans exhibited exploration capabilities, supporting the proposal that exploration is conserved across diverse streptomycetes. IMPORTANCE: Streptomyces bacteria have evolved diverse developmental and metabolic strategies to thrive in dynamic environmental niches. Here, we report the amalgamation of previously disparate developmental pathways, showing that colony expansion via exploration can proceed in tandem with colony sporulation. This developmental integration extends beyond phenotype to include shared genetic elements, with sporulation-specific repressors being required for successful exploration. Comparing this new exploration mode with previously identified strategies has revealed key differences (e.g., no need for environmental alkalinization), and simultaneously allowed us to define unifying requirements for Streptomyces exploration. The "reproductive exploration" phenomenon reported here represents a unique bet-hedging strategy, with the Streptomyces colony engaging in an aggressive colonization strategy while transporting a protected genetic repository.

The impact of orphan histidine kinases and phosphotransfer proteins on the regulation of clostridial sporulation initiation.

Sporulation is an important feature of the clostridial life cycle, facilitating survival of these bacteria in harsh environments, contributing to disease transmission for pathogenic species, and sharing common early steps that are also involved in regulating industrially important solvent production by some non-pathogenic species. Initial genomics studies suggested that Clostridia lack the classical phosphorelay that phosphorylates Spo0A and initiates sporulation in Bacillus, leading to the hypothesis that sporulation in Clostridia universally begins when Spo0A is phosphorylated by orphan histidine kinases (OHKs). However, components of the classical Bacillus phosphorelay were recently identified in some Clostridia. Similar Bacillus phosphorelay components have not yet been found in the pathogenic Clostridia or the solventogenic Clostridia of industrial importance. For some of those Clostridia lacking a classical phosphorelay, the involvement of OHKs in sporulation initiation has received support from genetic studies demonstrating the involvement of several apparent OHKs in their sporulation. In addition, several clostridial OHKs directly phosphorylate Spo0A in vitro. Interestingly, there is considerable protein domain diversity among the sporulation-associated OHKs in Clostridia. Further adding to the emergent complexity of sporulation initiation in Clostridia, several candidate OHK phosphotransfer proteins that were OHK candidates were shown to function as phosphatases that reduce sporulation in some Clostridia. The mounting evidence indicates that no single pathway explains sporulation initiation in all Clostridia and supports the need for further study to fully understand the unexpected and biologically fascinating mechanistic diversity of this important process among these medically and industrially important bacteria.

The impact of indole and mucin on sporulation, biofilm formation, and enterotoxin production in foodborne Clostridium perfringens.

AIMS: Indole and mucin are compounds found in the host environment as they are produced by the host or by the host-associated microbiota. This study investigated whether indole and mucin impact Clostridium perfringens growth and sporulation, as well as enterotoxin production and biofilm formation. METHODS AND RESULTS: There was no impact on growth of Cl. perfringens for up to 400 µM indole and 240 mg/l mucin, and neither indole nor mucin affected sporulation. Reverse-transcriptase qPCR showed that mucin strongly upregulated the expression of Cl. perfringens enterotoxin (up to 121-fold increase), whereas indole had a much more modest effect (2-fold). This was also reflected in increased Cl. perfringens enterotoxin levels in mucin-treated Cl. perfringens (as assessed by a reversed passive latex agglutination assay). Finally, mucin and indole significantly increased biofilm formation of Cl. perfringens, although the effect size was relatively small (less than 1.5 fold). CONCLUSION: These results indicate that Cl. perfringens can sense its presence in a host environment by responding to mucin, and thereby markedly increased enterotoxin production.

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.

Promising clinical and immunological efficacy of Bacillus clausii spore probiotics for supportive treatment of persistent diarrhea in children.

Persistent diarrhea is a severe gastroenteric disease with relatively high risk of pediatric mortality in developing countries. We conducted a randomized, double-blind, controlled clinical trial to evaluate the efficacy of liquid-form Bacillus clausii spore probiotics (LiveSpo CLAUSY; 2 billion CFU/5 mL ampoule) at high dosages of 4-6 ampoules a day in supporting treatment of children with persistent diarrhea. Our findings showed that B. clausii spores significantly improved treatment outcomes, resulting in a 2-day shorter recovery period (p < 0.05) and a 1.5-1.6 folds greater efficacy in reducing diarrhea symptoms, such as high frequency of bowel movement of ≥ 3 stools a day, presence of fecal mucus, and diapered infant stool scale types 4-5B. LiveSpo CLAUSY supportive treatment achieved 3 days (p < 0.0001) faster recovery from diarrhea disease, with 1.6-fold improved treatment efficacy. At day 5 of treatment, a significant decrease in blood levels of pro-inflammatory cytokines TNF-α, IL-17, and IL-23 by 3.24% (p = 0.0409), 29.76% (p = 0.0001), and 10.87% (p = 0.0036), respectively, was observed in the Clausy group. Simultaneously, there was a significant 37.97% decrease (p = 0.0326) in the excreted IgA in stool at day 5 in the Clausy group. Overall, the clinical study demonstrates the efficacy of B. clausii spores (LiveSpo CLAUSY) as an effective symptomatic treatment and immunomodulatory agent for persistent diarrhea in children.Trial registration: NCT05812820.

Spore-DNA localization and extraction efficiencies of Bacillus subtilis for accurate results in quantitative real-time polymerase chain reaction.

Mechanical bead disruption is an efficient DNA extraction method from spore cells for subsequent quantification of the spore population by quantitative polymerase chain reaction（qPCR）. In this study, to validate spore DNA localization and extraction efficiencies, the fractionated DNA included the total DNA（tDNA）extracted from spore cells and intracellular（iDNA）and extracellular DNA（eDNA）extracted from fractionated spores through chemical decoating and alkaline lysis buffers, each followed by bead disruption. Furthermore, alkaline lysis buffer-treated spore cells were intensively washed three and five times after each centrifugation to determine how the amount of DNA is affected by repeated centrifugation. This process was achieved through fractionated spore pellet and suspension treatments with propidium monoazide xx（PMAxx）before mechanical bead disruption. Three fractionated and extracted DNAs were assessed with qPCR. The amount of eDNA was higher than that of iDNA, and closer to tDNA levels in the qPCR assay. These results indicted the following: 1）amount of eDNA was more than iDNA and responsible for majority of amount of tDNA through the combination method involving alkaline lysis buffer and bead disruption, 2）lysis buffer partially eliminated the eDNA fragments through multiple washing steps, but it was not largely independent of the number of times centrifugation was performed.

Modeling and optimization of sporulation by Bacillus licheniformis BF-002 based on dynamics and recurrent neural networks.

Bacillus licheniformis is widely utilized in disease prevention and environmental remediation. Spore quantity is a critical factor in determining the quality of microbiological agents containing vegetative cells. To improve the understanding of Bacillus licheniformis BF-002 strain culture, a hybrid model integrating traditional dynamic modeling and recurrent neural network was developed. This model enabled the optimization of carbon/nitrogen source feeding rates, pH, temperature and agitation speed using genetic algorithms. Carbon and nitrogen source consumption in the optimal duplicate batches showed no significant difference compared to the control batch. However, the spore quantity in the broth increased by 16.2% and 35.2% in the respective duplicate batches. Overall, the hybrid model outperformed the traditional dynamic model in accurately tracking the cultivation dynamics of Bacillus licheniformis, leading to increased spore production when used for optimizing cultivation conditions.

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis.

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Effect of Bacillus subtilis and Bacillus coagulans spores on induced allergic contact dermatitis in dogs.

BACKGROUND: Probiotic strains have the potential to modulate immune responses, reduce intestinal inflammation, normalize intestinal mucosal function and decrease allergic reactions. OBJECTIVE: This study aimed to investigate the effect of oral probiotic supplements containing Bacillus subtilis and Bacillus coagulans spores on clinical symptoms, haematological factors and immune responses to allergic contact dermatitis in dogs induced by dinitrochlorobenzene (DNCB). METHODS: DNCB was injected subcutaneously into the scapular region of 20 healthy adult dogs of both sexes, divided into four groups, to induce experimental allergic contact dermatitis. Dogs in Group 1 received food without probiotics or medication. Oral prednisolone was administered to Group 2 for 30 days at a dosage of 0.25 mg/kg every other day. The dogs in Group 3 were treated with a combination of oral prednisolone and probiotics. The dogs in Group 4 were fed daily with a mixture of 109 B. subtilis and B. coagulans bacteria for 30 days. The immune system responses and related gene expression were analysed in the treated animals. RESULTS: The administration of probiotics for 30 days resulted in a reduction in clinical symptoms and duration of wound repair. The probiotics treatment also significantly increased the serum bactericidal effects against Staphylococcus aureus and Escherichia coli. It enhanced both the classic and alternative activity of the complement, as well as lysozyme activity. Additionally, the probiotics led to higher total immunoglobulin levels and significant reductions in anti-trypsin and C-reactive protein levels. Furthermore, the expression of IgE, induction of interferon-gamma and IL-4 genes were also reduced. CONCLUSIONS: According to the results, B. subtilis and B. coagulans can be further investigated as a viable alternative to corticosteroids in treating allergic contact dermatitis in dogs.

Assessing CaDPA levels, metabolic activity, and spore detection through deuterium labeling.

Many strains among spore-forming bacteria species are associated with food spoilage, foodborne disease, and hospital-acquired infections. Understanding the impact of environmental conditions and decontamination techniques on the metabolic activity, viability, and biomarkers of these spores is crucial for combatting them. To distinguish and track spores and to understand metabolic mechanisms, spores must be labeled. Staining or genetic modification are current methods for this, however, these methods can be time-consuming, and affect the viability and function of spore samples. In this work, we investigate the use of heavy water for permanent isotope labeling of spores and Raman spectroscopy for tracking sporulation/germination mechanisms. We also discuss the potential of this method in observing decontamination. We find that steady-state deuterium levels in the spore are achieved after only ∼48 h of incubation with 30% D2O-infused broth and sporulation, generating Raman peaks at cell silent region of 2200 and 2300 cm-1. These deuterium levels then decrease rapidly upon spore germination in non-deuterated media. We further find that unlike live spores, spores inactivated using various methods do not lose these Raman peaks upon incubation in growth media, suggesting these peaks may be used to indicate the viability of a spore sample. We further observe several Raman peaks exclusive to deuterated DPA, a spore-specific chemical biomarker, at e.g. 988 and 2300 cm-1, which can be used to track underlying changes in spores involving DPA. In conclusion, permanent spore labeling using deuterium offers a robust and non-invasive way of labeling bacterial spores for marking, viability determination, and characterising spore activity.