Potential antitumor and anti-inflammatory activities of an extracellular polymeric substance (EPS) from Bacillus subtilis isolated from a housefly.

Bacillus subtilis, a probiotic, has been applied in the medical, food, and feed industries among others. However, the mechanisms of its benefits to hosts are not yet fully understood. Here the characterization and bioactivities of an extracellular polymeric substance (EPS) from Bacillus subtilis were investigated to reveal its partial mechanisms and provide the theoretical basics for further development and utilization of Bacillus subtilis. In this study, the novel strain Bacillus subtilis xztubd1 (GenBank: MG458322.1) was isolated from a housefly's body, identified according to phenotypical and genotypical analyses, and found to produce large amounts of an EPS. Through ultraviolet spectroscopy and Fourier transform infrared spectroscopy (FTIR spectroscopy), the EPS was found to contain a variety of chemical functional groups, such as O-H groups, C=C, C=O, CH3, C-O-H and C-O-C bonds, and alpha-type pyranose. Furthermore, the in vitro antioxidant activity of the EPS on DPPH radicals at a concentration of 90 µg/ml was 62%; on the superoxide radical at a concentration of 90 µg/ml, this value was 75%; and on hydroxyl radicals at a concentration of 90 µg/ml, the activity was 54%. EPS also enhanced significantly phagocytosis, lysozyme activity in macrophages, IL-2 content in mice and inhibited dramatically the growth of HeLa cells. These results showed that the EPS with reductive groups have the strong capacity to scavenge reactive oxygen species (ROS), reinforce the immune system and inhibit the growth of cancer cell, which helps theirs hosts defence against many diseases, including inflammation and cancer. The EPS from Bacillus subtilis has the potential to be an anticancer and anti-inflammatory drug candidate in the pharmaceutical industries, which provide scientific evidence for the development and utilization of probiotic-derived medicines.

Spatial disinfection potential of slightly acidic electrolyzed water.

Slightly acidic electrolyzed water (SAEW) was developed by Japanese companies over 20 years ago. SAEW has the advantage of potent sterilizing action while being relatively safe. This study evaluated the potential application of SAEW in spatial disinfection. Prior to experiments involving spatial spraying, the ability of SAEW to remove seven type of microorganisms that cause food poisoning was studied in vitro. Results indicated that free chlorine in SAEW, even at a low concentration (30 mg/L), was able to remove Cladosporium cladosporioides, a typical airborne fungus that degrades food, and spores such as Bacillus subtilis, a hardy bacterium. In an experiment involving spatial spraying, 3.43 log10 CFU/100 L of Staphylococcus epidermidis was sprayed in a room-sized space; the same space was then sprayed with SAEW. The number of settling microbes was measured and the sterilizing ability of SAEW was assessed. Results indicated that the concentration of S. epidermidis in the space was completely removed after 20 minutes of SAEW spraying. The above findings indicate that SAEW may be used to remove airborne microorganisms via spatial spraying.

Genome mining reveals the genes of carboxypeptidase for OTA-detoxification in Bacillus subtilis CW14.

Bacillus subtilis CW14, isolated from fresh elk droppings in Beijing Zoo, is a Gram-positive, conferred Generally Recognized as Safe (GRAS) bacterium with the capacity of ochratoxin A (OTA) detoxification. The genome sequence of the CW14 strain showed a size of 4,287,522 bp with 44.06% GC content. It was predicted many putative enzymes involved in degrading mycotoxin by analyzing the signal peptides and the transmembrane regions. Nine extracellular enzymes were predicted relating to OTA detoxification, including four D-Ala-D-Ala carboxypeptidases, two hydrolases, two amidases, and one lactamase. Indeed, two of the carboxypeptidase genes dacA and dacB, expressed in Escherichia coli, were verified contributing to OTA detoxification. DacA and OTA were mixed incubated for 24 h, and the degradation rate reached 71.3%. After purification, the concentration of recombinant DacA protein was 0.5 mg/mL. Bacillus subtilis CW14 and its carboxypeptidases may be used as OTA detoxification agents in food and feed industry production.

Bioprocess development for enhanced endoglucanase production by newly isolated bacteria, purification, characterization and in-vitro efficacy as anti-biofilm of Pseudomonas aeruginosa.

Endoglucanase producing bacteria were isolated from Egyptian soils and the most active bacterial strain was identified as Bacillus subtilis strain Fatma/1. Plackett-Burman statistical design was carried out to assess the effect of seven process variables on endoglucanase production. Carboxymethyl cellulose (CMC), yeast extract and peptone were the most significant variables that enhanced the endoglucanase production and thus were selected for further optimization using face-centered central composite design. The highest yield of endoglucanase (32.37 U/mL) was obtained in run no. 9, using 18 g/L CMC, 8 g/L peptone, 7 g/L yeast extract and 0.1 g/L FeSO4.7H2O. The optimized medium showed about eightfold increase in endoglucanase production compared to the unoptimized medium. The produced crude enzyme was further purified by ammonium sulfate precipitation, then DEAE-Sepharose CL6B column. The purified enzyme was shown to have a molecular weight of 37 kDa. The enzyme showed maximum activity at pH 8.0, temperature of 50 °C, incubation time of 60 min. The half-life time (T1/2) was 139.53 min at 50 °C, while being 82.67 min at 60 °C. Endoglucanase at concentration of 12 U/mL effectively removed 84.61% of biofilm matrix of Pseudomonas aeruginosa with marked reduction in carbohydrate content of the biofilm from 63.4 to 7.9 µg.

Genome Mining, Heterologous Expression, Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60.

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D-F (1-3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D-F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS+ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.

Whole genome sequence insight of two plant growth-promoting bacteria (B. subtilis BS87 and B. megaterium BM89) isolated and characterized from sugarcane rhizosphere depicting better crop yield potentiality.

Since sugarcane is a ratoon crop, genome analysis of plant growth-promoting bacteria that exist in its soil rhizosphere, can provide opportunity to better understand their characteristics and use of such bacteria in turn, may especially improve perennial crop productivity. In the present study, genome of two bacterial strains, one each of B. megaterium (BM89) and B. subtilis (BS87), isolated and reported earlier (Chandra et al., 2018), were sequenced and characterized. Though both strains have demonstrated plant growth promoting properties and enhanced in-vitro plant growth responses, functional annotation and analysis of genes indicated superiority of BS87 as it possessed more plant growth promotion attributable genes over BM89. Apart from some common genes, trehalose metabolism, glycine betaine production, peroxidases, super oxide dismutase, cold shock proteins and phenazine production associated genes were selectively identified in BS87 genome indicating better plant growth performances and survival potential under harsh environmental conditions. Genes for chitinase, d-cysteine desulfhydrase and γ-aminobutyric acid (GABA), as found in BM89, propose its selective utilization in defense and bio-control measures. Concomitant with better settlings' growth, scanning electron micrographs indicated these isolated and characterized bacteria exhibiting healthy colonization within root of sugarcane crop. Kegg pathways' assignment also revealed added pathways namely carbohydrate and amino acid metabolism attached to B. subtilis strain BS87, a preferable candidate for bio-fertilizer and its utilization to promote growth of both plant and ratoon crops of sugarcane usually experiencing harsh environmental conditions.

Screening and Identification of Sponge-Associated Chitinolytic Bacteria by Forming Chitosan from Manado Bay, Indonesia.

BACKGROUND AND OBJECTIVE: Chitosan can be produced through the enzymatic process catalyzed by chitin deacetylase which can be produced by bacteria. The biotransformation of chitin to chitosan by bacteria is interesting because the process is economical and environmentally friendly. This study described the potential of sponge-associated bacterium capability in degrading chitin and forming chitosan. MATERIALS AND METHODS: The bacteria were isolated from sponge Cribrochalina sp. at Manado Bay, Indonesia. In the screening of the chitinase activity of bacteria, chitin media was used. Meanwhile, the transformation of chitin to chitosan was tested by using Chitinase Degrading Activity media. Molecular identification of bacteria was based on 16S rRNA gene sequences. RESULTS: The results showed that the SS1, SS2, SS3, SS4 and SS5 bacterial isolates could degrade chitin based on chitinolytic indexes. These five bacteria could also form chitosan exhibited through the presence of chitosan in the form of precipitation in the fermented broth of bacteria. SS1 had the highest chitinase activity based on the chitinolytic index identified as Bacillus subtilis (100% identity), hence it is called B. subtilis strain SS1. The partial rRNA gene sequences data were deposited at GenBank under accession number MN999892. CONCLUSION: The bacteria strain isolated from Cribrochalina sp. can be utilized in degrading chitin and form chitosan which could be a promising candidate for an economical and eco-friendly process of chitosan.

Isolation, stability, and characteristics of high-pressure superdormant Bacillus subtilis spores.

Bacterial spores are a major challenge in industrial decontamination processes owing to their extreme resistance. High-pressure (HP) of 150 MPa at 37 °C can trigger the germination of spores, making them lose their extreme resistance. Once their resistance is lost, germinated spores can easily be inactivated by a mild decontamination step. The implementation of this gentle germination-inactivation strategy is hindered by the presence of a subpopulation of so-called high-pressure superdormant (HPSD) spores, which resist germination or germinate only very slowly in response to HP. It is essential to understand the properties of HPSD spores and the underlying causes of superdormancy to tackle superdormant spores and further develop germination-inactivation strategies involving HP. This study investigated factors influencing the prevalence of HPSD spores and successfully isolated them by combining buoyant density centrifugation and fluorescence-activated cell sorting, which allowed further characterisation of HPSD spores for the first time. The prevalence of HPSD spores was shown to be strongly dependent on the HP dwell time, with increasing treatment times reducing their prevalence. Spore mutants lacking major germinant receptors further showed a highly increased prevalence of HPSD spores; 93% of the spores remained dormant even after a prolonged HP dwell time of 40 min. In contrast to nutrient germination, sublethal heat treatment of 75 °C for 30 min prior to pressure treatment did not induce spore activation and increase germination. The isolated HPSD spores did not show visible structural differences compared to the initial dormant spores when investigated with transmission electron microscopy. Re-sporulated HPSD spores showed similar germination capacity compared to the initial dormant spores, indicating that HPSD spores are most likely not genetically different from the rest of the population. Moreover, the majority of HPSD spores germinated when exposed a second time to the same germination treatment; however, the germination capacity was lower than that of the initial population. The fact that the majority of spores lost superdormancy when exposed a second time to the same trigger makes it unlikely that there is one factor that determines whether a spore germinates with a certain HP treatment or not. Instead, it seems possible that there are other reversible or cumulative causes. This study investigated the factors influencing spore HP superdormancy to improve the understanding of HPSD spores with regard to their stability, germination capacity, and potential underlying causes of spore HP superdormancy. This knowledge will contribute to the development of HP-based germination-inactivation strategies for gentle but effective spore control.

A deep-sea pathogenic Bacillus subtilis isolate employs different strategies to escape the killing of teleost and murine complements.

Bacillus subtilis subsp. subtilis G7 was isolated from a deep-sea hydrothermal vent and is pathogenic to pathogenic to fish (Japanese flounder) and mice. G7 is able to survive in host sera and phagocytes. In this study, we investigated the underlying mechanism of G7 serum resistance. We found that (i) the remaining complement activity was very low in G7-incubated flounder serum but high in G7-incubated mouse serum; (ii) cleaved C3 and C5 components were detected on flounder serum-incubated G7 but not on mouse serum-incubated G7; (iii) abundant uncleaved C5 was localized in G7-incubated mouse, but not flounder, serum; (iv) G7-incubated flounder, but not mouse, serum exhibited strong chemotactic activity; (v) pre-treatment with low-dose lysozyme abolished the serum resistance of G7. Hence, G7 activates flounder complement but is protected from complement-mediated destruction by its cell wall structure, while G7 prevents the activation of mouse complement. These results indicate that G7 employs different mechanisms to avoid the complement killing of different hosts.

Experimental elucidation of an antimycobacterial bacteriocin produced by ethnomedicinal plant-derived Bacillus subtilis (MK733983).

A bacteriocin from Bacillus subtilis (MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with Mycobacterium smegmatis as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0-8.0) and preservation (- 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe2+ and Mn2+. Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin's action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum with MICs ranging 0.225 ± 0.02-0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of S. aureus and P. aeruginosa treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects.

Spoilage potential of Bacillus subtilis in a neutral-pH dairy dessert.

The objective of this study was the characterization of the microbiota associated with spoilage of vanilla cream pudding during storage at different temperatures. Commercial cream samples were stored aerobically at 4, 8, 12 and 15 °C for a maximum time period of 40 days. At appropriate time intervals, cream samples were subjected to: (i) microbiological analyses, and (ii) high-performance liquid chromatography (HPLC). Furthermore, the spoilage microbiota was identified through repetitive extragenic palindrome-PCR, while selected isolates were further characterized based on sequencing of the V1-V3 region of the 16S rRNA gene. Microbial growth was observed only during storage of cream samples at 12 and 15 °C, with the applied genotypic analysis demonstrating that Bacillus subtilis subsp. subtilis was the dominant spoilage microorganism of this product. Based on the HPLC analysis results, citric acid and sucrose were the most abundant organic acid and sugar, respectively throughout storage of cream pudding, whereas notable changes mainly included: (i) increase in the concentration of lactic acid and to a lesser extent of formic and acetic acids, and (ii) increase in the concentration of glucose and fructose at the expense of sucrose and lactose. The results of this study should be useful for the dairy industry in detecting and controlling microbiological spoilage in cream pudding and other chilled, neutral-pH dairy desserts.

Optimization of low-cost biosurfactant produced by Bacillus subtilis SASCBT01 and their environmental remediation potential.

The present research aims to enhance the biosurfactant (BS) production using agricultural by-products as a low-cost substrate with the statistical approach. BS production from Bacillus subtilis SASCBT01 was carried out with four different variables such as pH, incubation time, cassava peel waste (CPW) and palmira sprout (PS). The model expected the highest emulsification activity of 65 ± 1·2% after 96-h incubation with 3·0 g l-1 of CPW and PS at pH 7·0. The SASCBT01 strain-based BS was successful at retrieving up to 18% and the highest Pb removal rates were found at 65%. These BS have considered high quality in bioremediation applications.

Safety Evaluation and In vivo Strain-Specific Functionality of Bacillus Strains Isolated from Korean Traditional Fermented Foods.

Unveiling and understanding differences in physiological features below the species level may serve as an essential fast-screening tool for selecting strains that can promote a specific probiotic effect. To study the intra-species diversity of Bacillus, a genus with a wide range of enzyme activities and specificity, 190 Bacillus strains were isolated from traditional Korean fermented food products. Altogether, in the preliminary safety screening, 8 of these strains were found negative for lecithinase and hemolysis activity and were selected for further investigations. On the basis of different levels of enzyme functionalities (high or low proteolytic, amylolytic, and lipolytic (PAL) activities), two Bacillus subtilis strains were selected for an in vivo study. Each of the two strains was separately administered at a level of 1 × 108 CFU per day to C57BL/6 mice that were fed 60% high-fat diet ad libitum for 8 weeks, while Xenical, an anti-obesity drug, was used as a positive control in the experimental setup. B. subtilis M34 and B. subtilis GS40a with low and high amylolytic activities, respectively, induced significantly different and contrasting physiological effects. The production of short-chain fatty acids appeared to be closely associated with a shift in the gut microbiota.

Bacillus subtilis CP4, isolated from native soil in combination with arbuscular mycorrhizal fungi promotes biofortification, yield and metabolite production in wheat under field conditions.

AIMS: The aim of this study was to identify the best combination of plant growth promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) for biofortification and enhancing yield in wheat as well as improve soil health under field conditions. Another aim was to get insights into metabolite dynamics in plants treated with PGPB and AMF. METHODS AND RESULTS: Different combinations of PGPB and AMF that gave good results in greenhouse study were used in a field study. The combined application of Bacillus subtilis CP4 (native PGPB) and AMF gave the best results with a significant increase in biomass, macronutrient and micronutrient content in wheat grains and improvement in yield-related parameters relative to the untreated control. PGPB and AMF treatment increased antioxidant enzymes and compounds and decreased the level of an oxidation marker. Metabolite profiling performed using Gas Chromatography-Mass Spectrometry (GC-MS) showed significant upregulation of specific organic acids, amino acids, sugars and sugar alcohols in plants treated with CP4 and AMF. The altered pathways due to CP4 and AMF inoculation mainly belong to carbohydrate and amino acid metabolism. A positive correlation was observed between some organic acids, sugars and amino acids with wheat growth and yield parameters. The activities of soil enzymes increased significantly with the best results shown by native PGPB and AMF combination. CONCLUSIONS: A native bacterial isolate Bacillus subtilis CP4 in combination with AMF showed exceptional ability for biofortification and yield enhancement under field conditions. The upregulation of a number of metabolites showed correlation plant growth promotion and nutrients. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined application of native B. subtilis CP4 and AMF could offer a more sustainable approach for the development of a biofertilizer to enhance wheat nutrient content and production and soil health thereby advancing agriculture.

Production and characterization of exopolysaccharide from the sponge-associated Bacillus subtilis MKU SERB2 and its in-vitro biological properties.

In this study, the sponge-associated a potential endosymbiotic bacterium, Bacillus subtilis MKU SERB2 was identified and optimized the production of exopolysaccharide (EPS) by using response surface methodology (RSM). The central composite rotatable design (CCRD) exhibited the highest yield of EPS (617.81 µg/mL) obtained from the optimized medium containing 11.5 g/L of sucrose, 3.5 g/L of yeast extract, 3.0 g/L of peptone, and 2.5 g/L of calcium chloride. Fourier transform infrared (FTIR) spectrum of purified EPS indicated that the presence of carboxyl, hydroxyl, and amide as functional groups, and their structural composition was confirmed by 1H and 13C nuclear magnetic resonance (NMR) analysis. Moreover, the fibrous, porous and semi-crystalline nature of EPS was confirmed by SEM and X-ray powder diffraction (XRD) analysis and the EDX inferred demonstrated the presence of C, Na, O, N, S, and Cl respectively. Further, the isolated EPS exhibited potent antioxidant activity and moderate anticoagulant efficacy whereas there was no hemolytic and lymphocytes toxicity. Overall, our result suggests that the functional and biological properties of the EPS imply the potential applications in food and pharmaceutical industries in the future.

The inhibitory effect of volatile organic compounds produced by Bacillus subtilis CL2 on pathogenic fungi of wolfberry.

Bacillus subtilis strain CL2 is antagonistic to wolfberry postharvest pathogenic fungi. In this study, we isolated and screened this strain for in vitro experiments. The result of the two-sealed-base-plates method revealed that volatile organic compounds (VOCs) emitted from the strain CL2 inhibited the hyphal growth of four pathogenic fungi Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. After exposure to VOCs for 5 days, the hyphal growth of the pathogen C. fioriniae LB8 was inhibited by 73%. Scanning electron microscopy revealed that the VOCs produced by B. subtilis CL2 caused the mycelium morphology of the pathogenic fungi to deform, twist, fold, and shrink. In the in vivo experiments, we noticed that VOCs could significantly reduce the weight loss rate of wolfberry fruits caused by the pathogenic fungus M. circinelloides LB1 and that the decay incidence rate were caused by the pathogenic fungi F. arcuatisporum LB5, A. iridiaustralis LB7, and C. fioriniae LB8. On the basis of the headspace-gas chromatography-ion mobility spectrometry analysis, seven VOCs produced by strain CL2 were identified. Among them, 2,3-butanedione and 3-methylbutyric acid are the main antifungal active substances. This study investigated the antifungal properties of VOCs produced by the strain CL2 on postharvest pathogenic fungi isolated from wolfberry fruits both in vivo and in vitro, thereby providing the theoretical basis for its future applications.

Integrated SERS Platform for Reliable Detection and Photothermal Elimination of Bacteria in Whole Blood Samples.

Herein, an interference-free surface-enhanced Raman scattering (SERS) platform with a "sandwich" structure has been developed for reliable detection and photothermal killing of bacteria with whole blood as the real sample. The multifunctional platform comprised a plasmonic gold film (pAu) functionalized with bacteria-capturing units of 4-mercaptophenylboronic acid and internal reference of 4-mercaptobenzonitrile as the SERS substrate and vancomycin-modified core (gold)-shell (Prussian blue) nanoparticles (Au@PB@Van NPs) as the SERS tag. The detected SERS signals were from the Raman-silent region where no background signals occurred from biological sources, eliminating the interference and improving the detection sensitivity and accuracy. As a proof-of-concept, model bacterial strain, Staphylococcus aureus, was captured and detected in the whole blood samples. Furthermore, high antibacterial efficiency of approximately 100% was reached under the synergistic photothermal effect from pAu and Au@PB@Van NPs. This study provides a new avenue for bacteria detection in real samples and their subsequent in situ elimination.

Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix.

Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm2) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.

Rampant loss of social traits during domestication of a Bacillus subtilis natural isolate.

Most model bacteria have been domesticated in laboratory conditions. Yet, the tempo with which a natural isolate diverges from its ancestral phenotype under domestication to a novel laboratory environment is poorly understood. Such knowledge, however is essential to understanding the rate of evolution, the time scale over which a natural isolate can be propagated without loss of its natural adaptive traits, and the reliability of experimental results across labs. Using experimental evolution, phenotypic assays, and whole-genome sequencing, we show that within a week of propagation in a common laboratory environment, a natural isolate of Bacillus subtilis acquires mutations that cause changes in a multitude of traits. A single adaptive mutational step in the gene coding for the transcriptional regulator DegU impairs a DegU-dependent positive autoregulatory loop and leads to loss of robust biofilm architecture, impaired swarming motility, reduced secretion of exoproteases, and to changes in the dynamics of sporulation across environments. Importantly, domestication also resulted in improved survival when the bacteria face pressure from cells of the innate immune system. These results show that degU is a target for mutations during domestication and underscores the importance of performing careful and extremely short-term propagations of natural isolates to conserve the traits encoded in their original genomes.

Molecular analysis of a fibrin-degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean-based fermented food moromi.

The screening of proteolytic and fibrinolytic bacteria from moromi (an Indonesian soybean-based fermented food) yielded a number of isolates. Based on morphological and biochemical analyses and sequencing of the 16S rRNA gene, the isolate that exhibited the highest proteolytic and fibrinolytic activity was identified as Bacillus subtilis K2. The study was performed to analyze molecular characteristic of a fibrin-degrading enzyme from B. subtilis K2. BLASTn analysis of the nucleotide sequence encoding this fibrinolytic protein demonstrated 73.6% homology with the gene encoding the fibrin-degrading enzyme nattokinase of the B. subtilis subsp. natto, which was isolated from fermented soybean in Japan. An analysis of the putative amino-acid sequence of this protein indicated that it is a serine protease enzyme with aspartate, histidine, and serine in the catalytic triad. This enzyme was determined to be a 26-kDa molecule, as confirmed with a zymogram assay. Further bioinformatic analysis using Protparam demonstrated that the enzyme has a pI of 6.02, low instability index, high aliphatic index, and low GRAVY value. Molecular docking analysis using HADDOCK indicated that there are favorable interactions between subtilisin K2 and the fibrin substrate, as demonstrated by a high binding affinity (ΔG: - 19.4 kcal/mol) and low Kd value (6.3E-15 M). Overall, the study concluded that subtilisin K2 belong to serine protease enzyme has strong interactions with its fibrin substrate and fibrin can be rapidly degraded by this enzyme, suggesting its application as a treatment for thrombus diseases.