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.

De novo engineering riboflavin production Bacillus subtilis by overexpressing the downstream genes in the purine biosynthesis pathway.

BACKGROUND: Bacillus subtilis is widely used in industrial-scale riboflavin production. Previous studies have shown that targeted mutagenesis of the ribulose 5-phosphate 3-epimerase in B. subtilis can significantly enhance riboflavin production. This modification also leads to an increase in purine intermediate concentrations in the medium. Interestingly, B. subtilis exhibits remarkable efficiency in purine nucleoside synthesis, often exceeding riboflavin yields. These observations highlight the importance of the conversion steps from inosine-5'-monophosphate (IMP) to 2,5-diamino-6-ribosylamino-4(3 H)-pyrimidinone-5'-phosphate (DARPP) in riboflavin production by B. subtilis. However, research elucidating the specific impact of these reactions on riboflavin production remains limited. RESULT: We expressed the genes encoding enzymes involved in these reactions (guaB, guaA, gmk, ndk, ribA) using a synthetic operon. Introduction of the plasmid carrying this synthetic operon led to a 3.09-fold increase in riboflavin production compared to the control strain. Exclusion of gmk from the synthetic operon resulted in a 36% decrease in riboflavin production, which was further reduced when guaB and guaA were not co-expressed. By integrating the synthetic operon into the genome and employing additional engineering strategies, we achieved riboflavin production levels of 2702 mg/L. Medium optimization further increased production to 3477 mg/L, with a yield of 0.0869 g riboflavin per g of sucrose. CONCLUSION: The conversion steps from IMP to DARPP play a critical role in riboflavin production by B. subtilis. Our overexpression strategies have demonstrated their effectiveness in overcoming these limiting factors and enhancing riboflavin production.

Effective of different industrial disinfection in subzero cold-chain environment.

Effective disinfection methods are crucial in the cold chain transportation process of food due to the specificity of temperature and the diversity of contaminated flora. The objective of this study was to investigate the sanitizing effect of different disinfectants on various fungi at - 20 °C to achieve accurate disinfection of diverse bacterial populations. Peracetic acid, hydrogen peroxide, and potassium bisulfate were selected as low-temperature disinfectants and were combined with antifreeze. The sanitizing effect of these cryogenic disinfectants on pathogens such as Bacillus subtilis black variant spores (ATCC9372), Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231), Escherichia coli (8099), and poliovirus (PV-1) was sequentially verified by bactericidal and virus inactivation experiments. After a specified time of disinfection, a neutralizing agent was used to halt the sanitizing process. The study demonstrates that different disinfectants exhibit selective effects during the low-temperature disinfection process. Peracetic acid, hydrogen peroxide, and potassium monopersulfate are suitable for the low-temperature environmental disinfection of bacterial propagules, viruses, and fungal contaminants. However, for microorganisms with strong resistance to spores, a low-temperature disinfectant based on peracetic acid should be chosen for effective disinfection treatment. Our results provide a valuable reference for selecting appropriate disinfectants to sanitize various potential pathogens in the future.

Chemical Composition, Anti-bacterial Activity and Molecular Docking Studies of Essential Oil Isolated from Sa Sam Nam (Launaea sarmentosa).

Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.

Activation of immune pathways in common bed bugs, Cimex lectularius, in response to bacterial immune challenges - a transcriptomics analysis.

The common bed bug, Cimex lectularius, is an urban pest of global health significance, severely affecting the physical and mental health of humans. In contrast to most other blood-feeding arthropods, bed bugs are not major vectors of pathogens, but the underlying mechanisms for this phenomenon are largely unexplored. Here, we present the first transcriptomics study of bed bugs in response to immune challenges. To study transcriptional variations in bed bugs following ingestion of bacteria, we extracted and processed mRNA from body tissues of adult male bed bugs after ingestion of sterile blood or blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis or the Gram-negative (Gr-) bacterium Escherichia coli. We analyzed mRNA from the bed bugs' midgut (the primary tissue involved in blood ingestion) and from the rest of their bodies (RoB; body minus head and midgut tissues). We show that the midgut exhibits a stronger immune response to ingestion of bacteria than the RoB, as indicated by the expression of genes encoding antimicrobial peptides (AMPs). Both the Toll and Imd signaling pathways, associated with immune responses, were highly activated by the ingestion of bacteria. Bacterial infection in bed bugs further provides evidence for metabolic reconfiguration and resource allocation in the bed bugs' midgut and RoB to promote production of AMPs. Our data suggest that infection with particular pathogens in bed bugs may be associated with altered metabolic pathways within the midgut and RoB that favors immune responses. We further show that multiple established cellular immune responses are preserved and are activated by the presence of specific pathogens. Our study provides a greater understanding of nuances in the immune responses of bed bugs towards pathogens that ultimately might contribute to novel bed bug control tactics.

Two promising natural lipopeptides from Bacillus subtilis effectively induced membrane permeabilization in Candida glabrata.

Candida glabrata is an important opportunistic human pathogen well known to develop resistance to antifungal drugs. Due to their numerous desirable qualities, antimicrobial lipopeptides have gained significant attention as promising candidates for antifungal drugs. In the present study, two bioactive lipopeptides (AF4 and AF5 m/z 1071.5 and 1085.5, respectively), coproduced and purified from Bacillus subtilis RLID12.1, consist of seven amino acid residues with lipid moieties. In our previous studies, the reversed phased-HPLC purified lipopeptides demonstrated broad-spectrum of antifungal activities against over 110 Candida albicans, Candida non-albicans and mycelial fungi. Two lipopeptides triggered membrane permeabilization of C. glabrata cells, as confirmed by propidium iodide-based flow cytometry, with PI uptake up to 99% demonstrating fungicidal effects. Metabolic inactivation in treated cells was confirmed by FUN-1-based confocal microscopy. Together, the results indicate that these lipopeptides have potentials to be developed into a new set of antifungals for combating fungal infections.

Structural basis and synergism of ATP and Na+ activation in bacterial K+ uptake system KtrAB.

The K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous system.

Preservation of Bacillus subtilis' cellular liquid state at deep sub-zero temperatures in perchlorate brines.

Although a low temperature limit for life has not been established, it is thought that there exists a physical limit imposed by the onset of intracellular vitrification, typically occurring at ~-20 °C for unicellular organisms. Here, we show, through differential scanning calorimetry, that molar concentrations of magnesium perchlorate can depress the intracellular vitrification point of Bacillus subtilis cells to temperatures much lower than those previously reported. At 2.5 M Mg(ClO4)2, the peak vitrification temperature was lowered to -83 °C. Our results show that inorganic eutectic salts can in principle maintain liquid water in cells at much lower temperatures than those previously claimed as a lower limit to life, raising the prospects of active biochemical processes in low temperature natural settings. Our results may have implications for the habitability of Mars, where perchlorate salts are pervasive and potentially other terrestrial and extraterrestrial, cryosphere environments.

Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

Development of a DC-Biased AC-Stimulated Microfluidic Device for the Electrokinetic Separation of Bacterial and Yeast Cells.

Electrokinetic (EK) microsystems, which are capable of performing separations without the need for labeling analytes, are a rapidly growing area in microfluidics. The present work demonstrated three distinct binary microbial separations, computationally modeled and experimentally performed, in an insulator-based EK (iEK) system stimulated by DC-biased AC potentials. The separations had an increasing order of difficulty. First, a separation between cells of two distinct domains (Escherichia coli and Saccharomyces cerevisiae) was demonstrated. The second separation was for cells from the same domain but different species (Bacillus subtilis and Bacillus cereus). The last separation included cells from two closely related microbial strains of the same domain and the same species (two distinct S. cerevisiae strains). For each separation, a novel computational model, employing a continuous spatial and temporal function for predicting the particle velocity, was used to predict the retention time (tR,p) of each cell type, which aided the experimentation. All three cases resulted in separation resolution values Rs>1.5, indicating complete separation between the two cell species, with good reproducibility between the experimental repetitions (deviations < 6%) and good agreement (deviations < 18%) between the predicted tR,p and experimental (tR,e) retention time values. This study demonstrated the potential of DC-biased AC iEK systems for performing challenging microbial separations.

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.

Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion.

Defense-associated sirtuin 2 (DSR2) systems are widely distributed across prokaryotic genomes, providing robust protection against phage infection. DSR2 recognizes phage tail tube proteins and induces abortive infection by depleting intracellular NAD+, a process that is counteracted by another phage-encoded protein, DSR Anti Defense 1 (DSAD1). Here, we present cryo-EM structures of Bacillus subtilis DSR2 in its apo, Tube-bound, and DSAD1-bound states. DSR2 assembles into an elongated tetramer, with four NADase catalytic modules clustered in the center and the regulatory-sensing modules distributed at four distal corners. Interestingly, monomeric Tube protein, rather than its oligomeric states, docks at each corner of the DSR2 tetramer to form a 4:4 DSR2-Tube assembly, which is essential for DSR2 NADase activity. DSAD1 competes with Tube for binding to DSR2 by occupying an overlapping region, thereby inhibiting DSR2 immunity. Thus, our results provide important insights into the assembly, activation and inhibition of the DSR2 anti-phage defense system.

Surface modification of the laser powder bed-fused Ti-Zr-Nb scaffolds by dynamic chemical etching and Ag nanoparticles decoration.

Metallic lattice scaffolds are designed to mimic the architecture and mechanical properties of bone tissue and their surface compatibility is of primary importance. This study presents a novel surface modification protocol for metallic lattice scaffolds printed from a superelastic Ti-Zr-Nb alloy. This protocol consists of dynamic chemical etching (DCE) followed by silver nanoparticles (AgNP) decoration. DCE, using an 1HF + 3HNO3 + 12H2O23% based solution, was used to remove partially-fused particles from the surfaces of different as-built lattice structures (rhombic dodecahedron, sheet gyroid, and Voronoi polyhedra). Subsequently, an antibacterial coating was synthesized on the surface of the scaffolds by a controlled (20 min at a fixed volume flowrate of 500 mL/min) pumping of the functionalization solutions (NaBH4 (2 mg/mL) and AgNO3 (1 mg/mL)) through the porous structures. Following these treatments, the scaffolds' surfaces were found to be densely populated with Ag nanoparticles and their agglomerates, and manifested an excellent antibacterial effect (Ag ion release rate of 4-8 ppm) suppressing the growth of both E. coli and B. subtilis bacteria up to 99 %. The scaffold extracts showed no cytotoxicity and did not affect cell proliferation, indicating their safety for subsequent use as implants. A cytocompatibility assessment using MG-63 spheroids demonstrated good attachment, spreading, and active migration of cells on the scaffold surface (over 96 % of living cells), confirming their biotolerance. These findings suggest the promise of this surface modification approach for developing superelastic Ti-Zr-Nb scaffolds with superior antibacterial properties and biocompatibility, making them highly suitable for bone implant applications.

Expression and characterization of a novel ß-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

Bacillus subtilis: current and future modification strategies as a protein secreting factory.

Bacillus subtilis is regarded as a promising microbial expression system in bioengineering due to its high stress resistance, nontoxic, low codon preference and grow fast. The strain has a relatively efficient expression system, as it has at least three protein secretion pathways and abundant molecular chaperones, which guarantee its expression ability and compatibility. Currently, many proteins are expressed in Bacillus subtilis, and their application prospects are broad. Although Bacillus subtilis has great advantages compared with other prokaryotes related to protein expression and secretion, it still faces deficiencies, such as low wild-type expression, low product activity, and easy gene loss, which limit its large-scale application. Over the years, many researchers have achieved abundant results in the modification of Bacillus subtilis expression systems, especially the optimization of promoters, expression vectors, signal peptides, transport pathways and molecular chaperones. An optimal vector with a suitable promoter strength and other regulatory elements could increase protein synthesis and secretion, increasing industrial profits. This review highlights the research status of optimization strategies related to the expression system of Bacillus subtilis. Moreover, research progress on its application as a food-grade expression system is also presented, along with some future modification and application directions.

A novel glycosyltransferase from Bacillus subtilis achieves zearalenone detoxification by diglycosylation modification.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin produced by Fusarium spp., contaminates cereals and threatens human and animal health by inducing hepatotoxicity, immunotoxicity, and genotoxicity. In this study, a new Bacillus subtilis strain, YQ-1, with a strong ability to detoxify ZEN, was isolated from soil samples and characterized. YQ-1 was confirmed to degrade more than 46.26% of 20 µg mL-1 ZEN in Luria-Bertani broth and 98.36% in fermentation broth within 16 h at 37 °C; one of the two resulting products was ZEN-diglucoside. Under optimal reaction conditions (50 °C and pH 5.0-9.0), the reaction mixture generated by YQ-1 catalyzing ZEN significantly reduced the promoting effect of ZEN on MCF-7 cell proliferation, effectively eliminating the estrogenic toxicity of ZEN. In addition, a new glycosyltransferase gene (yqgt) from B. subtilis YQ-1 was cloned with 98% similarity to Bs-YjiC from B. subtilis 168 and over-expressed in E. coli BL21 (DE3). ZEN glycosylation activity converted 25.63% of ZEN (20 µg mL-1) to ZEN-diG after 48 h of reaction at 37 °C. The characterization of ZEN degradation by B. subtilis YQ-1 and the expression of YQGT provide a theoretical basis for analyzing the mechanism by which Bacillus spp. degrades ZEN.

Biomaterial-based sponge for efficient and environmentally sound removal of bacteria from water.

Designing materials capable of disinfecting water without releasing harmful by-products is an ongoing challenge. Here, we report a novel polycationic sponge material synthesized from chitosan derivatives and cellulose fibers, exhibiting antibacterial properties. The design of such material is based on three key principles. First, the formation of a highly porous structure through cryogelation for an extensive surface area. Second, the incorporation of cationic quaternary ammonium moieties onto chitosan to enhance bacterial adsorption and antibacterial activity. Lastly, the reinforcement of mechanical properties through integration of cellulose fibers. The presented sponge materials exhibit up to a 4-log (99.99%) reduction within 6 h against both gram-positive B. subtilis and gram-negative E. coli. Notably, QCHI90/Cell, with the highest surface charge, exhibits a 2-4.5 log reduction within 1 h of incubation time. The eco-friendly synthesis from water and readily available biomaterials, along with cost-effectiveness and simplicity, underscores its versatility and feasibility of upscaling. Together with its outstanding antibacterial activity, this macroporous biomaterial emerges as a promising candidate for water disinfection applications.

Combined control of plant diseases by Bacillus subtilis SL44 and Enterobacter hormaechei Wu15.

Co-incubation of plant growth promoting rhizobacteria (PGPRs) have been proposed as a potential alternative to pesticides for controlling fungal pathogens in crops, but their synergism mechanisms are not yet fully understood. In this study, combined use of Bacillus subtilis SL44 and Enterobacter hormaechei Wu15 could decrease the density of Colletotrichum gloeosporioides and Rhizoctonia solani and enhance the growth of beneficial bacteria on the mycelial surface, thereby mitigating disease severity. Meanwhile, PGPR application led to a reorganization of the rhizosphere microbial community through modulating its metabolites, such as extracellular polymeric substances and chitinase. These metabolites demonstrated positive effects on attracting and enhancing conventional periphery bacteria, inhibiting fungal pathogens and promoting soil health effectively. The improvement in the microbial community structure altered the trophic mode of soil fungal communities, effectively decreasing the proportion of saprotrophic soil and reducing fungal plant diseases. Certain combinations of PGPR have the potential to serve as precise instruments for managing plant pathogens.

Two distinct regulatory systems control pulcherrimin biosynthesis in Bacillus subtilis.

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MarR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important role for pulcherrimin in B. subtilis physiology.

Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach.

BACKGROUND: Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form. METHODS: Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures. RESULTS: According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures. CONCLUSION: The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.