Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes. KEY POINTS: • A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD • Both SwiKR and GDH are identified with ketosteroid isomerase activity. • Development of catalytic strategy to control by-product and achieve highly selective DHEA production.

Surface display of the COE antigen of porcine epidemic diarrhoea virus on Bacillus subtilis spores.

Porcine epidemic diarrhoea virus (PEDV) infects pigs of all ages by invading small intestine, causing acute diarrhoea, vomiting, and dehydration with high morbidity and mortality among newborn piglets. However, current PEDV vaccines are not effective to protect the pigs from field epidemic strains because of poor mucosal immune response and strain variation. Therefore, it is indispensable to develop a novel oral vaccine based on epidemic strains. Bacillus subtilis spores are attractive delivery vehicles for oral vaccination on account of the safety, high stability, and low cost. In this study, a chimeric gene CotC-Linker-COE (CLE), comprising of the B. subtilis spore coat gene cotC fused to the core neutralizing epitope CO-26 K equivalent (COE) of the epidemic strain PEDV-AJ1102 spike protein gene, was constructed. Then recombinant B. subtilis displaying the CLE on the spore surface was developed by homologous recombination. Mice were immunized by oral route with B. subtilis 168-CLE, B. subtilis 168, or phosphate-buffered saline (PBS) as control. Results showed that the IgG antibodies and cytokine (IL-4, IFN-γ) levels in the B. subtilis 168-CLE group were significantly higher than the control groups. This study demonstrates that B. subtilis 168-CLE can generate specific systemic immune and mucosal immune responses and is a potential vaccine candidate against PEDV infection.

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.

Microbially induced calcium carbonate precipitation through CO2 sequestration via an engineered Bacillus subtilis.

BACKGROUND: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase-an enzyme with the capability to hydrolyse CO2, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO3 biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry. RESULTS: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO2 and convert it to calcium carbonate. A significant decrease in CO2 was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO2 sequestration. CONCLUSION: Recombinantly produced carbonic anhydrase successfully sequestered CO2 and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Surface-associated residues in subtilisins contribute to poly-L-lactic acid depolymerization via enzyme adsorption.

Poly-L-lactic acid (PLLA) is currently the most abundant bioplastic; however, limited environmental biodegradability and few recycling options diminish its value as a biodegradable commodity. Enzymatic recycling is one strategy for ensuring circularity of PLLA, but this approach requires a thorough understanding of enzymatic mechanisms and protein engineering strategies to enhance activity. In this study, we engineer PLLA depolymerizing subtilisin enzymes originating from Bacillus species to elucidate the molecular mechanisms dictating their PLLA depolymerization activity and to improve their function. The surface-associated amino acids of two closely related subtilisin homologues originating from Bacillus subtilis (BsAprE) and Bacillus pumilus (BpAprE) were compared, as they were previously engineered to have nearly identical active sites, but still varied greatly in PLLA depolymerizing activity. Further analysis identified several surface-associated amino acids in BpAprE that lead to enhanced PLLA depolymerization activity when engineered into BsAprE. In silico protein modelling demonstrated increased enzyme surface hydrophobicity in engineered BsAprE variants and revealed a structural motif favoured for PLLA depolymerization. Experimental evidence suggests that increases in activity are associated with enhanced polymer binding as opposed to substrate specificity. These data highlight enzyme adsorption as a key factor in PLLA depolymerization by subtilisins.

Efficient production of guanosine in Escherichia coli by combinatorial metabolic engineering.

BACKGROUND: Guanosine is a purine nucleoside that is widely used as a raw material for food additives and pharmaceutical products. Microbial fermentation is the main production method of guanosine. However, the guanosine-producing strains possess multiple metabolic pathway interactions and complex regulatory mechanisms. The lack of strains with efficiently producing-guanosine greatly limited industrial application. RESULTS: We attempted to efficiently produce guanosine in Escherichia coli using systematic metabolic engineering. First, we overexpressed the purine synthesis pathway from Bacillus subtilis and the prs gene, and deleted three genes involved in guanosine catabolism to increase guanosine accumulation. Subsequently, we attenuated purA expression and eliminated feedback and transcription dual inhibition. Then, we modified the metabolic flux of the glycolysis and Entner-Doudoroff (ED) pathways and performed redox cofactors rebalancing. Finally, transporter engineering and enhancing the guanosine synthesis pathway further increased the guanosine titre to 134.9 mg/L. After 72 h of the fed-batch fermentation in shake-flask, the guanosine titre achieved 289.8 mg/L. CONCLUSIONS: Our results reveal that the guanosine synthesis pathway was successfully optimized by combinatorial metabolic engineering, which could be applicable to the efficient synthesis of other nucleoside products.

Regulation of biofilm gene expression by DNA replication in Bacillus subtilis.

Bacillus subtilis relies on biofilms for survival in harsh environments. Extracellular polymeric substance (EPS) is a crucial component of biofilms, yet the dynamics of EPS production in single cells remain elusive. To unveil the modulation of EPS synthesis, we built a minimal network model comprising the SinI-SinR-SlrR module, Spo0A, and EPS. Stochastic simulations revealed that antagonistic interplay between SinI and SinR enables EPS production in bursts. SlrR widens these bursts and increases their frequency by stabilizing SinR-SlrR complexes and depleting free SinR. DNA replication and chromosomal positioning of key genes dictate pulsatile changes in the slrR:sinR gene dosage ratio (gr) and Spo0A-P levels, each promoting EPS production in distinct phases of the cell cycle. As the cell cycle lengthens with nutrient stress, the duty cycle of gr pulsing decreases, whereas the amplitude of Spo0A-P pulses elevates. This coordinated response facilitates keeping a constant proportion of EPS-secreting cells within colonies across diverse nutrient conditions. Our results suggest that bacteria may 'encode' eps expression through strategic chromosomal organization. This work illuminates how stochastic protein interactions, gene copy number imbalance, and cell-cycle dynamics orchestrate EPS synthesis, offering a deeper understanding of biofilm formation.

Enhanced Thermostability of Nattokinase by Computation-Based Rational Redesign of Flexible Regions.

Nattokinase is a nutrient in healthy food natto that has the function of preventing and treating blood thrombus. However, its low thermostability and fibrinolytic activity limit its application in food and pharmaceuticals. In this study, we used bioinformatics analysis to identify two loops (loop10 and loop12) in the flexible region of nattokinase rAprY. Using this basis, we screened the G131S-S161T variant, which showed a 2.38-fold increase in half-life at 55 °C, and the M3 variant, which showed a 2.01-fold increase in activity, by using a thermostability prediction algorithm. Bioinformatics analysis revealed that the enhanced thermostability of the G131S-S161T variant was due to the increased rigidity and structural shrinkage of the overall structure. Additionally, the increased rigidity of the local region surrounding the active center and its mutated sites helps maintain its normal conformation in high-temperature environments. The increased catalytic activity of the M3 variant may be due to its more efficient substrate binding mechanism. We investigated strategies to improve the thermostability and fibrinolytic activity of nattokinase, and the resulting variants show promise for industrial production and application.

Secretion of the cytoplasmic and high molecular weight ß-galactosidase of Paenibacillus wynnii with Bacillus subtilis.

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

Cell-Free Supernatant of Bacillus subtilis G2B9-Q Improves Intestinal Health and Modulates Immune Response to Promote Mouse Recovery in Clostridium perfringens Infection.

Clostridium perfringens is one of the critical causative agents causing diarrhea in piglets, with significant economic losses to the pig industry. Under normal gut microbiota homeostasis and well-managed barns, diarrhea caused by C. perfringens could be controlled. Some reports show that probiotics, such as Bacillus subtilis, are beneficial in preventing necrotic enteritis (NE) in chickens, but few reports on piglets. Clostridium perfringens was found in the piglets' diarrhea with intestinal microbiota dysbiosis in our survey. Bacillus subtilis G2B9-Q, which was isolated from the feces of healthy pigs, was found to have anti-Clostridium activity after screening. Clostridium perfringens was used to challenge mice by intraperitoneal injection for modeling to evaluate the anti-infective activity of cell-free supernatant (CFS) of B. subtilis G2B9-Q and different concentrations of B. subtilis G2B9-Q by oral administration. The results showed that G2B9-Q can mitigate intestinal lesions caused by C. perfringens infection, reduce inflammatory reactions, and modulate intestinal microbiota. The CFS of G2B9-Q can alleviate the pathological damage of intestinal tissues caused by C. perfringens infection, reduce the concentration of TNF-α and IL-10 in the sera of mice, as well as the relative expression levels of alpha toxin (CPA), perfringolysin O (PFO) toxin, IL-10, IL-22, and TNF-α in the jejunum and colon tissues, and alleviate the changes in gut microbiota structure caused by C. perfringens infection, which showed better therapeutic effects and indicated that the metabolites of G2B9-Q are essential mediators for their beneficial effects. Therefore, the CFS of G2B9-Q could potentially replace antibiotics in treating C. perfringens infection.

The inhibition mechanism of co-cultured probiotics on biofilm formation of Klebsiella pneumoniae.

AIMS: Klebsiella pneumoniae, an important opportunistic pathogen of nosocomial inflection, is known for its ability to form biofilm. The purpose of the current study is to assess how co- or mono-cultured probiotics affect K. pneumoniae's ability to produce biofilms and investigate the potential mechanisms by using a polyester nonwoven chemostat and a Caco-2 cell line. METHODS AND RESULTS: Compared with pure cultures of Lactobacillus rhamnosus and Lactobacillus sake, the formation of K. pneumoniae biofilm was remarkably inhibited by the mixture of L. rhamnosus, L. sake, and Bacillus subtilis at a ratio of 5:5:1 by means of qPCR and FISH assays. In addition, Lactobacillus in combination with B. subtilis could considerably reduce the adherence of K. pneumoniae to Caco-2 cells by using inhibition, competition, and displacement assays. According to the RT-PCR assay, the adsorption of K. pneumoniae to Caco-2 cells was effectively inhibited by the co-cultured probiotics, leading to significant reduction in the expression of proinflammatory cytokines induced by K. pneumoniae. Furthermore, the HPLC and RT-PCR analyses showed that the co-cultured probiotics were able to successfully prevent the expression of the biofilm-related genes of K. pneumoniae by secreting plenty of organic acids as well as the second signal molecule (c-di-GMP), resulting in inhibition on biofilm formation. CONCLUSION: Co-culture of L. sake, L. rhamnosus, and B. subtilis at a ratio of 5:5:1 could exert an antagonistic effect on the colonization of pathogenic K. pneumoniae by down-regulating the expression of biofilm-related genes. At the same time, the co-cultured probiotics could effectively inhibit the adhesion of K. pneumoniae to Caco-2 cells and block the expression of proinflammatory cytokines induced by K. pneumoniae.

A limited concentration range of diaphorin, a polyketide produced by a bacterial symbiont of the Asian citrus psyllid, promotes the in vitro gene expression with bacterial ribosomes.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Physiological concentrations of diaphorin, which D. citri contains at levels as high as 2-20 mM, are inhibitory to various eukaryotes and Bacillus subtilis (Firmicutes: Bacilli) but promote the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales). Our previous study demonstrated that 5-mM diaphorin, which exhibits significant inhibitory and promoting effects on cultured B. subtilis and E. coli, respectively, inhibits in vitro gene expression utilizing purified B. subtilis and E. coli ribosomes. This suggested that the adverse effects of diaphorin on B. subtilis are partly due to its influence on gene expression. However, the result appeared inconsistent with the positive impact on E. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressed in vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 µM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to 5-mM diaphorin, promotes the in vitro translation with the B. subtilis and E. coli ribosomes, suggesting that the positive effects of diaphorin on E. coli are due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications.IMPORTANCEThis study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes and Bacillus subtilis but promotes the growth and metabolic activity of Escherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production by E. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the distinct property to pest management and industrial use.

Mechanistic insight into roles of α/ß-type small acid-soluble proteins, RecA, and inner membrane proteins during bacterial spore inactivation by ohmic heating.

AIM: Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores. METHODS AND RESULTS: We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/ß-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50 V/cm. CONCLUSIONS: In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.

Whole genome sequencing of the poly-γ-glutamic acid-producing novel Bacillus subtilis Tamang strain, isolated from spontaneously fermented kinema.

Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain's Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

Non-consecutive enzyme interactions within TCA cycle supramolecular assembly regulate carbon-nitrogen metabolism.

Enzymes of the central metabolism tend to assemble into transient supramolecular complexes. However, the functional significance of the interactions, particularly between enzymes catalyzing non-consecutive reactions, remains unclear. Here, by co-localizing two non-consecutive enzymes of the TCA cycle from Bacillus subtilis, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD), in phase separated droplets we show that MDH-ICD interaction leads to enzyme agglomeration with a concomitant enhancement of ICD catalytic rate and an apparent sequestration of its reaction product, 2-oxoglutarate. Theory demonstrates that MDH-mediated clustering of ICD molecules explains the observed phenomena. In vivo analyses reveal that MDH overexpression leads to accumulation of 2-oxoglutarate and reduction of fluxes flowing through both the catabolic and anabolic branches of the carbon-nitrogen intersection occupied by 2-oxoglutarate, resulting in impeded ammonium assimilation and reduced biomass production. Our findings suggest that the MDH-ICD interaction is an important coordinator of carbon-nitrogen metabolism.

[The expression and secretion of human lactoferrin in Bacillus subtilis].

Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P21, P43, and Pveg) and three inducible promoters (Pgrac100, PxylA, and Ptet*) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 µg/L, was achieved when regulated by the Ptet* promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SPyycP resulted in increased HLF expression to 1 099.87 µg/L, with 498.68 µg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 µg/L. This study successfully demonstrated the secretory expression of HLF in B. subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B. subtilis cell factories for lactoprotein synthesis.

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

Connection between protein-tyrosine kinase inhibition and coping with oxidative stress in Bacillus subtilis.

In bacteria, attenuation of protein-tyrosine phosphorylation occurs during oxidative stress. The main described mechanism behind this effect is the H2O2-triggered conversion of bacterial phospho-tyrosines to protein-bound 3,4-dihydroxyphenylalanine. This disrupts the bacterial tyrosine phosphorylation-based signaling network, which alters the bacterial polysaccharide biosynthesis. Herein, we report an alternative mechanism, in which oxidative stress leads to a direct inhibition of bacterial protein-tyrosine kinases (BY-kinases). We show that DefA, a minor peptide deformylase, inhibits the activity of BY-kinase PtkA when Bacillus subtilis is exposed to oxidative stress. High levels of PtkA activity are known to destabilize B. subtilis pellicle formation, which leads to higher sensitivity to oxidative stress. Interaction with DefA inhibits both PtkA autophosphorylation and phosphorylation of its substrate Ugd, which is involved in exopolysaccharide formation. Inactivation of defA drastically reduces the capacity of B. subtilis to cope with oxidative stress, but it does not affect the major oxidative stress regulons PerR, OhrR, and Spx, indicating that PtkA inhibition is the main pathway for DefA involvement in this stress response. Structural analysis identified DefA residues Asn95, Tyr150, and Glu152 as essential for interaction with PtkA. Inhibition of PtkA depends also on the presence of a C-terminal α-helix of DefA, which resembles PtkA-interacting motifs from known PtkA activators, TkmA, SalA, and MinD. Loss of either the key interacting residues or the inhibitory helix of DefA abolishes inhibition of PtkA in vitro and impairs postoxidative stress recovery in vivo, confirming the involvement of these structural features in the proposed mechanism.

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.