Alanine and glutamate catabolism collaborate to ensure the success of Bacillus subtilis sporulation.

Sporulation as a typical bacterial differentiation process has been studied for decades. However, two crucial aspects of sporulation, (i) the energy sources supporting the process, and (ii) the maintenance of spore dormancy throughout sporulation, are scarcely explored. Here, we reported the crucial role of RocG-mediated glutamate catabolism in regulating mother cell lysis, a critical step for sporulation completion of Bacillus subtilis, likely by providing energy metabolite ATP. Notably, rocG overexpression resulted in an excessive ATP accumulation in sporulating cells, leading to adverse effects on future spore properties, e.g. increased germination efficiency, reduced DPA content, and lowered heat resistance. Additionally, we revealed that Ald-mediated alanine metabolism was highly related to the inhibition of premature germination and the maintenance of spore dormancy during sporulation, which might be achieved by decreasing the typical germinant L-alanine concentration in sporulating environment. Our data inferred that sporulation of B. subtilis was a highly orchestrated biological process requiring a delicate balance in diverse metabolic pathways, hence ensuring both the completion of sporulation and production of high-quality spores.

Improvement of Bacillus subtilis PI agarase production, hydrolysate scavenging capability assessment, and saccharification of algal biomass for green ethanol generation.

The goal of the current work was to optimize the growth parameters needed to manufacture agarase enzyme from a non-marine PI strain of Bacillus subtilis on an agar-based medium. Using Plackett-Burman design (PBD), nine process parameters were evaluated, and agar, peptone, and yeast-extract were identified as the most significant independent factors influencing agarase production with confidence levels more than 90%. To evaluate the optimal concentrations of the indicated process parameters on agarase production, the Box-Behnken design (BBD) was applied. After optimization, B. subtilis strain PI produced 119.8 U/ml of agarase, representing a 1.36-fold increase. In addition the agar hydrolysate fermented products contain the liberated oligosaccharide acts as strong antioxidant which has 62.4% scavenging activity. Also, the agarase yields increased (1141.12, 1350.253, 1684.854 and 1921.863 U/ml) after substitution the agar with algal biomass of Carolina officinalis at different concentrations (2, 5, 10 and 15%), respectively. After completing the saccharification process, the resulted hydrolysate was used to produce ethanol through fermentation with Pichia pastoris yeast strain as an economical method giving yields (6.68317, 7.09748, 7.75648 and 8.22332 mg/ml), that are higher than using yeast extract peptone dextrose (YPD) medium (4.461 mg/ml).

Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - Shedding light on spore superdormancy at 550 MPa.

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency. Bacillus subtilis spores were pressurized for 50 s - 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions. The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43-52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions. The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

Bioremediation of petroleum refinery wastewater using Bacillus subtilis IH-1 and assessment of its toxicity.

Environmental contamination from petroleum refinery operations has increased due to the rapid population growth and modernization of society, necessitating urgent repair. Microbial remediation of petroleum wastewater by prominent bacterial cultures holds promise in circumventing the issue of petroleum-related pollution. Herein, the bacterial culture was isolated from petroleum-contaminated sludge samples for the valorization of polyaromatic hydrocarbons and biodegradation of petroleum wastewater samples. The bacterial strain was screened and identified as Bacillus subtilis IH-1. After six days of incubation, the bacteria had degraded 25.9% of phenanthrene and 20.3% of naphthalene. The treatment of wastewater samples was assessed using physico-chemical and Fourier-transform infrared spectroscopy analysis, which revealed that the level of pollutants was elevated and above the allowed limits. Following bacterial degradation, the reduction in pollution parameters viz. EC (82.7%), BOD (87.0%), COD (80.0%), total phenols (96.3%), oil and grease (79.7%), TKN (68.8%), TOC (96.3%) and TPH (52.4%) were observed. The reduction in pH and heavy metals were also observed after bacterial treatment. V. mungo was used in the phytotoxicity test, which revealed at 50% wastewater concentration the reduction in biomass (30.3%), root length (87.7%), shoot length (93.9%), and seed germination (30.0%) was observed in comparison to control. When A. cepa root tips immersed in varying concentrations of wastewater samples, the mitotic index significantly decreased, suggesting the induction of cytotoxicity. However, following the bacterial treatment, there was a noticeable decrease in phytotoxicity and cytotoxicity. The bacterial culture produces lignin peroxidase enzyme and has the potential to degrade the toxic pollutants of petroleum wastewater. Therefore the bacterium may be immobilised or directly used at reactor scale or pilot scale study to benefit the industry and environmental safety.

Assessment of Plant Growth Promotion Potential of Endophytic Bacterium B. subtilis KU21 Isolated from Rosmarinus officinalis.

The current study aimed to evaluate the plant growth-promoting (PGP) potential of endophytic strain Bacillus subtilis KU21 isolated from the roots of Rosmarinus officinalis. The strain exhibited multiple traits of plant growth promotion viz., phosphate (P) solubilization, nitrogen fixation, indole-3-acetic acid (IAA), siderophore, hydrogen cyanide (HCN), lytic enzymes production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. The isolate also exhibited antagonistic activity against phytopathogenic fungi, i.e., Fusarium oxysporum, Fusarium graminiarum, and Rhizoctonia solani. The P-solubilization activity of B. subtilis KU21 was further elucidated via detection of glucose dehydrogenase (gdh) gene involved in the production of gluconic acid which is responsible for P-solubilization. Further, B. subtilis KU21 was evaluated for in vivo growth promotion studies of tomato (test crop) under net house conditions. A remarkable increase in seed germination, plant growth parameters, nutrient acquisition, and soil quality parameters (NPK) was observed in B. subtilis KU21-treated plants over untreated control. Hence, the proposed module could be recommended for sustainable tomato production in the Northwest Himalayan region without compromising soil health and fertility.

Sustainable menaquinone-7 production through continuous fermentation in biofilm bioreactors.

Menaquinone-7 (MK-7), a vital vitamin with numerous health benefits, is synthesized and secreted extracellularly by the formation of biofilm, dominantly in Bacillus strains. Our team developed an innovative biofilm reactor utilizing Bacillus subtilis natto cells to foster biofilm growth on plastic composite supports to produce MK-7. Continuous fermentation in biofilm reactors offers a promising strategy for achieving sustainable and efficient production of Menaquinone-7 (MK-7). Unlike conventional batch fermentation, continuous biofilm reactors maintain a steady state of operation, which reduces resource consumption and waste generation, contributing to sustainability. By optimizing fermentation conditions, MK-7 production was significantly enhanced in this study, demonstrating the potential for sustainable industrial-scale production. To determine the optimal operational parameters, various dilution rates were tested. These rates were selected based on their potential to enhance nutrient supply and biofilm stability, thereby improving MK-7 production. By carefully considering the fermentation conditions and systematically varying the dilution rates, MK-7 production was significantly enhanced during continuous fermentation. The MK-7 productivity was found to increase from 0.12 mg/L/h to 0.33 mg/L/h with a dilution rate increment from 0.007 to 0.042 h-1). This range was chosen to explore the impact of various nutrient supply rates on MK-7 production and to identify the optimal conditions for maximizing productivity. However, a further increase in the dilution rate to 0.084 h-1 led to reduced productivity at approximately 0.16 mg/L/h, likely due to insufficient retention time for effective biofilm formation. Consequently, a dilution rate of 0.042 h-1 exhibited the highest productivity of 0.33 mg/L/h, outperforming all investigated dilution rates and demonstrating the critical balance between nutrient supply and retention time in continuous fermentation. These findings validate the feasibility of operating continuous fermentation at a 0.084 h-1 dilution rate, corresponding to a 48 h retention time, to achieve the highest MK-7 productivity compared to conventional batch fermentation. The significant advancements achieved in enhancing Menaquinone-7 (MK-7) productivity through continuous fermentation at optimal dilution rates in the present work indicate promising prospects for even greater efficiency and sustainability in MK-7 production through future developments.

SpoIIQ-dependent localization of SpoIIE contributes to septal stability and compartmentalization during the engulfment stage of Bacillus subtilis sporulation.

During spore development in bacteria, a polar septum separates two transcriptionally distinct cellular compartments, the mother cell and the forespore. The conserved serine phosphatase SpoIIE is known for its critical role in the formation of this septum and activation of compartment-specific transcription in the forespore. Signaling between the mother cell and forespore then leads to activation of mother cell transcription and a phagocytic-like process called engulfment, which involves dramatic remodeling of the septum and requires a balance between peptidoglycan synthesis and hydrolysis to ensure septal stability and compartmentalization. Using Bacillus subtilis, we identify an additional role for SpoIIE in maintaining septal stability and compartmentalization at the onset of engulfment. This role for SpoIIE is mediated by SpoIIQ, which anchors SpoIIE in the engulfing membrane. A SpoIIQ mutant (SpoIIQ Y28A) that fails to anchor SpoIIE, results in septal instability and miscompartmentalization during septal peptidoglycan hydrolysis, when other septal stabilization factors are absent. Our data support a model whereby SpoIIE and its interactions with the peptidoglycan synthetic machinery contribute to the stabilization of the asymmetric septum early in engulfment, thereby ensuring compartmentalization during spore development.IMPORTANCEBacterial sporulation is a complex process involving a vast array of proteins. Some of these proteins are absolutely critical and regulate key points in the developmental process. Once such protein is SpoIIE, known for its role in the formation of the polar septum, a hallmark of the early stages of sporulation, and activation of the first sporulation-specific sigma factor, σF, in the developing spore. Interestingly, SpoIIE has been shown to interact with SpoIIQ, an important σF-regulated protein that functions during the engulfment stage. However, the significance of this interaction has remained unclear. Here, we unveil the importance of the SpoIIQ-SpoIIE interaction and identify a role for SpoIIE in the stabilization of the polar septum and maintenance of compartmentalization at the onset of engulfment. In this way, we demonstrate that key sporulation proteins, like SpoIIQ and SpoIIE, function in multiple processes during spore development.

Enhanced surface colonisation and competition during bacterial adaptation to a fungus.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

Influence of PepF peptidase and sporulation on microcin J25 production in Bacillus subtilis.

The lasso peptide microcin J25 (MccJ25) possesses strong antibacterial properties and is considered a potential effective component of bacterial disease treatment drugs and safe food preservatives. Although MccJ25 can be heterologously expressed in Bacillus subtilis as we have previously reported, its regulation and accumulation are yet to be understood. Here, we investigated the expression level and stability of MccJ25 in B. subtilis strains with disruption in peptidase genes pepA, pepF, and pepT. Oligoendopeptidase F (PepF) was found to be involved in reduction of the production of MccJ25 by degradation of its precursor peptide. In the pepF mutant, the MccJ25 reached a concentration of 1.68 µM after a cultivation time exceeding 60 hours, while the wild-type strain exhibited a concentration of only 0.14 µM. Moreover, the production of MccJ25 in B. subtilis downregulated the genes associated with sporulation, and this may contribute to its accumulation. Finally, this study provides a strategy to improve the stability and production of MccJ25 in B. subtilis. IMPORTANCE: MccJ25 displays significant antibacterial activity, a well-defined mode of action, exceptional safety, and remarkable stability. Hence, it presents itself as a compelling candidate for an optimal antibacterial or anti-endotoxin medication. The successful establishment of exogenous production of MccJ25 in Bacillus subtilis provides a strategy for reducing its production cost and diversifying its utilization. In this study, we have provided evidence indicating that both peptidase PepF and sporulation are significant factors that limit the expression of MccJ25 in B. subtilis. The ΔpepF and ΔsigF mutants of B. subtilis express MccJ25 with higher production yield and enhanced stability. To sum up, this study developed several better engineered strains of B. subtilis, which greatly reduced the consumption of MccJ25 during the nutrient depletion stage of the host strain, improved its production, and elucidated factors that may be involved in reducing MccJ25 accumulation in B. subtilis.

Novel cereal bran based low-cost liquid medium for enhanced growth, multifunctional traits and shelf life of consortium biofertilizer containing Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.

The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log10 CFU/ml and 12.63 ± 0.04 Log10 CFU/ml for Azotobacter chroococcum and Bacillus subtilis whereas for Pseudomonas sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log10 CFU/ml) after 72 h of incubation. RB51 and WB52liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.

Bacillus subtilis promotes plant phosphorus (P) acquisition through P solubilization and stimulation of root and root hair growth.

Bacteria can be applied as biofertilizers to improve crop growth in phosphorus (P)-limited conditions. However, their mode of action in a soil environment is still elusive. We used the strain ALC_02 as a case study to elucidate how Bacillus subtilis affects dwarf tomato cultivated in soil-filled rhizoboxes over time. ALC_02 improved plant P acquisition by increasing the size and P content of P-limited plants. We assessed three possible mechanisms, namely root growth stimulation, root hair elongation, and solubilization of soil P. ALC_02 produced auxin, and inoculation with ALC_02 promoted root growth. ALC_02 promoted root hair elongation as the earliest observed response and colonized root hairs specifically. Root and root hair growth stimulation was associated with a subsequent increase in plant P content, indicating that a better soil exploration by the root system improved plant P acquisition. Furthermore, ALC_02 affected the plant-available P content in sterilized soil differently over time and released P from native P pools in the soil. Collectively, ALC_02 exhibited all three mechanisms in a soil environment. To our knowledge, bacterial P biofertilizers have not been reported to colonize and elongate root hairs in the soil so far, and we propose that these traits contribute to the overall effect of ALC_02. The knowledge gained in this research can be applied in the future quest for bacterial P biofertilizers, where we recommend assessing all three parameters, not only root growth and P solubilization, but also root hair elongation. This will ultimately support the development of sustainable agricultural practices.

Efficacy of DAP coated with bacterial strains and their metabolites for soil phosphorus availability and maize growth.

Phosphorus (P) use efficiency in alkaline/calcareous soils is only 20% due to precipitation of P2O5 with calcium and magnesium. However, coating Diammonium Phosphate (DAP) with phosphorus solubilizing bacteria (PSB) is more appropriate to increase fertilizer use efficiency. Therefore, with the aim to use inorganic fertilizers more effectively present study was conducted to investigate comparative effect of coated DAP with PSB strains Bacillus subtilis ZE15 (MN003400), Bacillus subtilis ZR3 (MN007185), Bacillus megaterium ZE32 (MN003401) and Bacillus megaterium ZR19 (MN007186) and their extracted metabolites with uncoated DAP under axenic conditions. Gene sequencing was done against various sources of phosphorus to analyze genes responsible for phosphatase activity. Alkaline phosphatase (ALP) gene amplicon of 380bp from all tested strains was showed in 1% w/v gel. Release pattern of P was also improved with coated fertilizer. The results showed that coated phosphatic fertilizer enhanced shoot dry weight by 43 and 46% under bacterial and metabolites coating respectively. Shoot and root length up to 44 and 42% with metabolites coated DAP and 41% with bacterial coated DAP. Physiological attributes also showed significant improvement with coated DAP over conventional. The results supported the application of coated DAP as a useful medium to raise crop yield even at lower application rates i.e., 50 and 75% DAP than non-coated 100% DAP application which advocated this coating technique a promising approach for advancing circular economy and sustainable development in modern agriculture.

From growth inhibition to ultrastructural changes: Toxicological assessment of lambda cyhalothrin and fosetyl aluminium against Bacillus subtilis and Pseudomonas aeruginosa.

In modern agricultural practices, agrochemicals and pesticides play an important role in protecting the crops from pests and elevating agricultural productivity. This strategic utilization is essential to meet global food demand due to the relentless growth of the world's population. However, the indiscriminate application of these substances may result in environmental hazards and directly affect the soil microorganisms and crop production. Considering this, an in vitro study was carried out to evaluate the pesticides' effects i.e. lambda cyhalothrin (insecticide) and fosetyl aluminum (fungicide) at lower, recommended, and higher doses on growth behavior, enzymatic profile, total soluble protein production, and lipid peroxidation of bacterial specimens (Pseudomonas aeruginosa and Bacillus subtilis). The experimental findings demonstrated a concentration-dependent decrease in growth of both tested bacteria, when exposed to fosetyl aluminium concentrations exceeding the recommended dose. This decline was statistically significant (p < 0.000). However, lambda cyhalothrin at three times of recommended dose induces 10% increase in growth of Pseudomonas aeruginosa (P. aeruginosa) and 76.8% decrease in growth of Bacillus subtilis (B. subtilis) respectively as compared to control. These results showed the stimulatory effect of lambda cyhalothrin on P. aeruginosa and inhibitory effect on B. subtilis. Pesticides induced notable alterations in biomarker enzymatic assays and other parameters related to oxidative stress among bacterial strains, resulting in increased oxidative stress and membrane permeability. Generally, the maximum toxicity of both (P. aeruginosa and B. subtilis) was shown by fosetyl aluminium, at three times of recommended dose. Fosetyl aluminium induced morphological changes like cellular cracking, reduced viability, aberrant margins and more damage in both bacterial strains as compared to lambda cyhalothrin when observed under scanning electron microscope (SEM). Conclusively the, present study provide an insights into a mechanistic approach of pyrethroid insecticide and phosphonite fungicide induced cellular toxicity towards bacteria.

Catabolism of germinant amino acids is required to prevent premature spore germination in Bacillus subtilis.

Spores of Bacillus subtilis germinate in response to specific germinant molecules that are recognized by receptors in the spore envelope. Germinants signal to the dormant spore that the environment can support vegetative growth, so many germinants, such as alanine and valine, are also essential metabolites. As such, they are also required to build the spore. Here we show that these germinants cause premature germination if they are still present at the latter stages of spore formation and beyond, but that B. subtilis metabolism is configured to prevent this: alanine and valine are catabolized and cleared from wild-type cultures even when alternative carbon and nitrogen sources are present. Alanine and valine accumulate in the spent media of mutants that are unable to catabolize these amino acids, and premature germination is pervasive. Premature germination does not occur if the germinant receptor that responds to alanine and valine is eliminated, or if wild-type strains that are able to catabolize and clear alanine and valine are also present in coculture. Our findings demonstrate that spore-forming bacteria must fine-tune the concentration of any metabolite that can also function as a germinant to a level that is high enough to allow for spore development to proceed, but not so high as to promote premature germination. These results indicate that germinant selection and metabolism are tightly linked, and suggest that germinant receptors evolve in tandem with the catabolic priorities of the spore-forming bacterium. IMPORTANCE: Many bacterial species produce dormant cells called endospores, which are not killed by antibiotics or common disinfection practices. Endospores pose critical challenges in the food industry, where endospore contaminations cause food spoilage, and in hospitals, where infections by pathogenic endospore formers threaten the life of millions every year. Endospores lose their resistance properties and can be killed easily when they germinate and exit dormancy. We have discovered that the enzymes that break down the amino acids alanine and valine are critical for the production of stable endospores. If these enzymes are absent, endospores germinate as they are formed or shortly thereafter in response to alanine, which can initiate the germination of many different species' endospores, or to valine. By blocking the activity of alanine dehydrogenase, the enzyme that breaks down alanine and is not present in mammals, it may be possible to inactivate endospores by triggering premature and unproductive germination.

Pilot-scale production of Bacillus subtilis MSCL 897 spore biomass and antifungal secondary metabolites in a low-cost medium.

OBJECTIVES: Bacillus subtilis is a plant growth promoting bacterium (PGPB) that acts as a microbial fertilizer and biocontrol agent, providing benefits such as boosting crop productivity and improving nutrient content. It is able to produce secondary metabolites and endospores simultaneously, enhancing its ability to survive in unfavorable conditions and eliminate competing microorganisms. Optimizing cultivation methods to produce B. subtilis MSCL 897 spores on an industrial scale, requires a suitable medium, typically made from food industry by-products, and optimal temperature and pH levels to achieve high vegetative cell and spore densities with maximum productivity. RESULTS: This research demonstrates successful pilot-scale (100 L bioreactor) production of a biocontrol agent B. subtilis with good spore yields (1.5 × 109 spores mL-1) and a high degree of sporulation (>80%) using a low-cost cultivation medium. Culture samples showed excellent antifungal activity (1.6-2.3 cm) against several phytopathogenic fungi. An improved methodology for inoculum preparation was investigated to ensure an optimal seed culture state prior to inoculation, promoting process batch-to-batch repeatability. Increasing the molasses concentration in the medium and operating the process in fed-batch mode with additional molasses feed, did not improve the overall spore yield, hence, process operation in batch mode with 10 g molasses L-1 is preferred. Results also showed that the product quality was not significantly impacted for up to 12 months of storage at room temperature. CONCLUSION: An economically-feasible process for B. subtilis-based biocontrol agent production was successfully developed at the pilot scale.

Enhanced control efficacy of Bacillus subtilis NM4 via integration of chlorothalonil on potato early blight caused by Alternaria solani.

Early blight caused by Alternaria solani is a common foliar disease of potato around the world, and serious infections result in reduced yields and marketability due to infected tubers. The major aim of this study is to figure out the synergistic effect between microorganism and fungicides and to evaluate the effectiveness of Bacillus subtilis NM4 in the control of early blight in potato. Based on its colonial morphology and a 16S rRNA analysis, a bacterial antagonist isolated from kimchi was identified as B. subtilis NM4 and it has strong antifungal and anti-oomycete activity against several phytopathogenic fungi and oomycetes. The culture filtrate of strain NM4 with the fungicide effectively suppressed the mycelial growth of A. solani, with the highest growth inhibition rate of 83.48%. Although exposure to culture filtrate prompted hyphal alterations in A. solani, including bulging, combining it with the fungicide caused more severe hyphal damage with continuous bulging. Surfactins and fengycins, two lipopeptide groups, were isolated and identified as the main compounds in two fractions using LC-ESI-MS. Although the surfactin-containing fraction failed to inhibit growth, the fengycin-containing fraction, alone and in combination with chlorothalonil, restricted mycelial development, producing severe hyphal deformations with formation of chlamydospores. A pot experiment combining strain NM4, applied as a broth culture, with fungicide, at half the recommended concentration, resulted in a significant reduction in potato early blight severity. Our results indicate the feasibility of an integrated approach for the management of early blight in potato that can reduce fungicide application rates, promoting a healthy ecosystem in agriculture.

Multifarious Potential of Biopolymer-Producing Bacillus subtilis NJ14 for Plant Growth Promotion and Stress Tolerance in Solanum lycopercicum L. and Cicer arietinum L: A Way Toward Sustainable Agriculture.

Diverse practices implementing biopolymer-producing bacteria have been examined in various domains lately. PHAs are among the major biopolymers whose relevance of PHA-producing bacteria in the field of crop improvement is one of the radical unexplored aspects in the field of agriculture. Prolonging shelf life is one serious issue hindering the establishment of biofertilizers. Studies support that PHA can help bacteria survive stressed conditions by providing energy. Therefore, PHA-producing bacteria with Plant Growth-Promoting ability can alter the existing problem of short shelf life in biofertilizers. In the present study, Bacillus subtilis NJ14 was isolated from the soil. It was explored to understand the ability of the strain to produce PHA and augment growth in Solanum lycopersicum and Cicer arietinum. NJ14 strain improved the root and shoot length of both plants significantly. The root and shoot length of S. lycopersicum was increased by 3.49 and 0.41 cm, respectively. Similarly, C. arietinum showed a 9.55 and 8.24 cm increase in root and shoot length, respectively. The strain also exhibited halotolerant activity (up to 10%), metal tolerance to lead (up to 1000 µg/mL) and mercury (up to 100 µg/mL), indicating that the NJ14 strain can be an ideal candidate for a potent biofertilizer.

Programming bacteria for multiplexed DNA detection.

DNA is a universal and programmable signal of living organisms. Here we develop cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA-sensing mechanism and simple detection procedure could enable programmable DNA sensing for a wide range of applications.

Genetic analysis of Bacillus subtilis stable L-forms obtained via long-term cultivation.

Various bacteria can change to a spherical cell-wall-deficient state, called L-from, in the presence of antibiotics that inhibit cell wall synthesis. L-forms are classified into two types: unstable and stable L-forms. Unstable L-forms revert to a normal walled state in the absence of antibiotics, while stable L-forms remain in their wall-deficient state. The conversion from unstable to stable L-forms has been often observed during long-term cultivation. However, the genetic cause for this conversion is not yet fully understood. Here, we obtained stable Bacillus subtilis L-form strains from unstable L-form strains via three independent long-term culturing experiments. The whole genome sequencing of the long-cultured strains identified many mutations, and some mutations were commonly found in all three long-cultured strains. The knockout strain of one of the commonly mutated genes, tagF, in the ancestral strain lost the ability to revert to walled state (rod shape), supporting that eliminating the function of tagF gene is one of the possible methods to convert unstable L forms to a stable state.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Isla Arena está localizada na coordenada 20°70’N - 90°45’W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.