The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σA-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a ...published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σA-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also report expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.
•Purification of nucleic acid-free, His-tagged σA-RNAP from Bacillus subtilis•Fast and robust single affinity step protocol•Thorough verification of constructed SG7 strain•Robust low temperature expression of B. subtilis σA in E. coli•Fast single affinity step preparation of functional σA
Food waste compost (FWC) is a sustainable recycling approach employed in soil media, offering extensive advantages to urban areas by promoting resource circulation and effectively managing water ...pollution. To improve value, Bacillus subtilis (B. subtilis)-induced FWC-based biomedia (BIBMFWCs) was produced via a secondary treatment involving selective meso-thermophilic stages. During the production of BIBMFWCs, physicochemical properties were found to have favorable characteristics for the efficient removal of metal ions. The produced organic-carbonate complex structure demonstrated the synergistic effect involving simultaneous sorption/precipitation mechanisms for the removal of Pb(II) and Cr(III). Also, the dose of B. subtilis has an impact on the pseudo-second-order (PSO) and intra-particle diffusion (IPD) reaction, leading to distinct removal capacities for Pb(II) and Cr(III) 24.26–24.74 mg g−1 in Pb(II) and 12.7–23.93 mg g−1 in Cr(III). Furthermore, B. subtilis, an inducing mediator for microbial metabolites, exhibits the potential to facilitate the removal of Pb(II) and Cr(III) through biological modification of raw materials, which are transformed, facilitating the presence of hydroxyl groups, immobilizing metal ions, and enabling ion exchange via biogenic carbonate formation processes. Finally, the developed BIBMFWCs could be used as a nature-based solution (NBS) material without in-situ pH control.
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•The composting strategy improves urban soil and water management.•Bacillus subtilis improves the maturity and pore properties of biomedia.•BIBMFWCs effectively remove Pb(II) and Cr(III) metal ions.•BIBMFWCs is a nature-based solution for sustainable Pb(II) and Cr(III) removal.
The aim of this study was to increase the efficacy of probiotic Bacillus subtilis E20 by encapsulating the probiotic in alginate and coating it with chitosan. The protective effect was evaluated by ...firstly ensuring the viability of encapsulated probiotics in simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) conditions and then at different storage temperatures. In addition, the encapsulated probiotic was incorporated into the diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei. B. subtilis E20 has the ability to survive in SGF when encapsulated in 1.5–2% alginate and coated with 0.4% chitosan. Furthermore, viability increased significantly in SIF compared to the probiotic encapsulated in 1% alginate and coated with 0.4% chitosan and the non-encapsulated probiotic. Longer storage time and adverse conditions affected probiotics' survival, which was improved by the encapsulation with significantly higher viability than the non-encapsulated probiotic at different temperatures and storage duration. Encapsulation of B. subtilis E20 and dietary administration at 107 CFU kg−1 decreased shrimp mortality after a Vibrio infection, thereby improving shrimp's disease resistance, while the non-encapsulated probiotic required 109 CFU kg−1 to achieve better resistance. Although the best results of growth performance, immune response, and disease resistance against Vibrio alginolyticus were found in the shrimp fed with the diets supplemented with encapsulated probiotic at >108 CFU kg−1, shrimp's growth performance and health status improved after being fed 107 CFU kg−1 encapsulated probiotic for 56 days. Together, the results of this study prove that encapsulation could improve the viability of probiotic in different gastrointestinal conditions and adverse storage temperatures. Overall, lower concentrations of encapsulated probiotic B. subtilis E20 (107 CFU kg−1) was able to increase the growth performance and health status of shrimp.
•Probiotic efficacy of Bacillus subtilis E20 was improved by encapsulation.•Encapsulated probiotic had better resistance against adverts conditions of gastrointestinal tract and storage.•Encapsulated probiotic in diet at a level of 107 CFU kg−1 were able to improve the growth and health status of shrimp.
MXenes are a family of atomically thin, two-dimensional (2D) transition metal carbides and carbonitrides with many attractive properties. Two-dimensional Ti3C2Tx (MXene) has been recently explored ...for applications in water desalination/purification membranes. A major success indicator for any water treatment membrane is the resistance to biofouling. To validate this and to understand better the health and environmental impacts of the new 2D carbides, we investigated the antibacterial properties of single- and few-layer Ti3C2Tx MXene flakes in colloidal solution. The antibacterial properties of Ti3C2Tx were tested against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) by using bacterial growth curves based on optical densities (OD) and colonies growth on agar nutritive plates. Ti3C2Tx shows a higher antibacterial efficiency toward both Gram-negative E. coli and Gram-positive B. subtilis compared with graphene oxide (GO), which has been widely reported as an antibacterial agent. Concentration dependent antibacterial activity was observed and more than 98% bacterial cell viability loss was found at 200 μg/mL Ti3C2Tx for both bacterial cells within 4 h of exposure, as confirmed by colony forming unit (CFU) and regrowth curve. Antibacterial mechanism investigation by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) coupled with lactate dehydrogenase (LDH) release assay indicated the damage to the cell membrane, which resulted in release of cytoplasmic materials from the bacterial cells. Reactive oxygen species (ROS) dependent and independent stress induction by Ti3C2Tx was investigated in two separate abiotic assays. MXenes are expected to be resistant to biofouling and offer bactericidal properties.
In this study, we have isolated and characterized proteolytic soil bacteria and their alkaline protease. Based on 16S rRNA sequence analysis, 12 isolates with the highest protease activity were ...classified as B. subtilis and B. cereus groups. B. subtilis D9 isolate showing the highest protease activity was selected for in vitro and in silico analysis for its ِِAKD9 protease. The enzyme has a molecular mass of 48 kDa, exhibiting optimal activity at 50 °C pH 9.5, and showed high stability till 65 °C and pH 8–11 for 1 h. Fe3+ stimulated, but Zn2+ and Hg2+ strongly inhibited the protease activity. Also, the maximum inhibition with PMSF indicated serine protease-type of AKD9 protease. AkD9 alkaline serine protease gene showed high sequence similarity and close phylogenetic relationship with AprX serine protease of B. subtilis isolates. Functional prediction of AKD9 resulted in the detection of subtilase domain, peptidase_S8 family, and subtilase active sites. Moreover, prediction of physicochemical properties indicated that AKD9 serine protease is hydrophilic, thermostable, and alkali-halo stable. Secondary structure prediction revealed the dominance of the coils enhances AKD9 activity and stability under saline and alkaline conditions. Based on molecular docking, AKD9 showed very promising binding affinities towards casein substrate with expected intrinsic proteolytic activities matching our obtained in vitro results. In conclusion, AKD9 alkaline serine protease seems to be a significant candidate for industrial applications because of its stability, hydrophilicity, enhanced thermostability, and alkali-halo stability.
Alkaline serine protease gene; Phylogenetic analysis; B. subtilis; B. cereus; Subtilase domain; Catalytic triad.
Lipopeptide biosurfactants produced by
sp. were assessed regarding their antimicrobial activity against foodborne pathogenic and food spoilage microorganisms. Both Gram-positive and Gram-negative ...bacteria were found not to be susceptible to these lipopeptides. However, mycosubtilin and mycosubtilin/surfactin mixtures were very active against the filamentous fungi
and
, with minimum inhibitory concentrations (MICs) of 1-16 mg/L. They were also active against
, MIC = 16-64 mg/L. Moreover it was found that the antifungal activity of these lipopeptides was not affected by differences in isoform composition and/or purity. Furthermore their cytotoxicity tested on two different cell lines mimicking ingestion and detoxification was comparable to those of approved food preservatives such as nisin. Overall, for the first time here mycosubtilin and mycosubtilin/surfactin mixtures were found to have high antifungal activity against food relevant fungi at concentrations lower than their toxicity level hence, suggesting their application for extending the shelf-life of products susceptible to these moulds. In addition combining nisin with mycosubtilin or mycosubtiliin/surfactin mixtures proved to be an effective approach to produce antimicrobials with broader spectrum of action.
Microorganisms can facilitate their survival in stressful environments by entering a state of metabolic inactivity or dormancy 1. However, this state impairs the function of the very sensory systems ...necessary to detect favorable growth conditions. Thus, how can a metabolically quiescent cell accurately monitor environmental conditions in order to best decide when to exit dormancy? One strategy employed by microbes to deal with changing environments is the generation of phenotypes that may be less well adapted to a current condition but might confer an advantage in the future 2, 3. This bet-hedging depends on phenotypic diversity in the population 4, which itself can derive from naturally occurring stochastic differences in gene expression 5, 6. In the case of metabolic dormancy, a bet-hedging strategy that has been proposed is the “scout model” where cells comprising a fraction of the dormant population reinitiate growth stochastically, independent of environmental cues 7, 8. Here, we provide experimental evidence that such a mechanism exists in dormant spores produced by the ubiquitous soil bacterium Bacillus subtilis. We observe that these spores reinitiate growth at a low but measureable frequency even in the absence of an inducing signal. This phenomenon is the result of phenotypic variation in the propensity of individual spores to reinitiate growth spontaneously. Since this bet-hedging mechanism produces individuals that will either grow under favorable conditions or die under unfavorable conditions, a population can properly respond to environmental changes despite the impaired sensory ability of individual cells.
•A sub-population of Bacillus subtilis bacterial spores germinate spontaneously•Phenotypic variability underlies spontaneous germination•The phenotypic variability of spontaneous germination is under genetic control
Sturm and Dworkin demonstrate that a sub-population of dormant bacterial spores exhibit stochastic behavior when re-initiating growth. This strategy, based on phenotypic diversity, allows a population to effectively respond to the environment without requiring that any individual cell be competent to sense environmental conditions.
FeS clusters are prosthetic groups present in all organisms. Proteins with FeS centers are involved in most cellular processes. ISC and SUF are machineries necessary for the formation and insertion ...of FeS in proteins. Recently, a phylogenetic analysis on more than 10,000 genomes of prokaryotes have uncovered two new systems, MIS and SMS, which were proposed to be ancestral to ISC and SUF. SMS is composed of SmsBC, two homologs of SufBC(D), the scaffolding complex of SUF. In this review, we will specifically focus on the current knowledge of the SUF system and on the new perspectives given by the recent discovery of its ancestor, the SMS system.
•SufS and SufU sulfurtransferase for SufS sulfur transfer•SufU as zinc-containing protein•The two cofactors (Flavin and Fe-S) of SufBCD scaffold.•SmsCB is the scaffold of the ancestral system•Unknown sulfur donor for SMS system
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The combination of silver (I) oxide (Ag2O) with zinc oxide (ZnO) evolves the magnificent response of composite for antimicrobial and photocatalytic applications concomitantly. A ...novel, cost-effective and facile wet-chemical recipe for developing ZnO/Ag2O nanocomposites is realized by customizing pseudo-successive ionic layer adsorption and reaction (p-SILAR) process. Quantitatively as well as qualitatively controlled Ag2O was deposited on flower-like ZnO, termed as ZnO NFs, by carrying out 1–4 cycles of customized p-SILAR. It was affirmed that the cationic adsorption of Ag+ from AgNO3 solution instigates the formation of Ag2O over ZnO NFs via a simple heat-treatment process. Followed by the customary material characterizations including SEM, TEM, XPS etc., the ZnO/Ag2O nanocomposites were tested for controlling the toxic organic dye (Rhodamine B) and harmful bacterial species i.e. E. coli and B. subtilis. A significant increase in the photocatalytic performance of ZnO NFs under ultra-violet (UV) as well as visible (vis) irradiation was obtained by incorporating 2 cycles of Ag2O deposition. Consequently, the degradation rate constant of optimal ZnO/Ag2O-2C was increased to 0.1077 and 0.0104 from 0.0235 and 0.0011 min−1, under UV and vis light respectively. Similarly, ZnO/Ag2O evolved a significant increase in the inhibition zones without exhibiting a considerable decrease in the cell viability of HepG2, which further avowed the bio-compatibility of ZnO NFs having Ag2O.
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