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The safety of microbially-derived food enzymes must be carefully assessed before market introduction. The production strain's safety is central to the assessment. In this paper, we have determined that DSM's Bacillus subtilis strain lineage can be considered safe for food enzyme production. The mutations introduced into this non-pathogenic and non-toxigenic microorganism do not lead to any safety concerns, as ensured by a thorough characterization of the strain lineage. The safety of both targeted and randomly introduced changes into the production strain's genome is confirmed by validating the absence of vector sequences and antibiotic resistance genes in all relevant production strains, and by demonstrating absence of cytotoxic peptide production. Furthermore, three food enzyme preparations produced by strains within this lineage did not show genotoxic potential. 90-day oral toxicity studies performed with the same enzyme preparations did not reveal toxicologically significant adverse effects. These results demonstrate absence of safety concerns from the introduced genetic modifications. Based on the establishment of this safe strain lineage, we postulate that future enzymes produced by current and new strains derived from the lineage can be safely developed without additional genotoxicity and systemic toxicity studies, allowing for a reduction of animal testing without compromising on product safety., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pan-genome analysis is widely used to study the evolution and genetic diversity of species, particularly in bacteria. However, the impact of strain selection on the outcome of pan-genome analysis is poorly understood. Furthermore, a standard protocol to ensure high-quality pan-genome results is lacking. In this study, we carried out a series of pan-genome analyses of different strain sets of Bacillus subtilis to understand the impact of various strains on the performance and output quality of pan-genome analyses. Consequently, we found that the results obtained by pan-genome analyses of B. subtilis can be influenced by the inclusion of incorrectly classified Bacillus subspecies strains, phylogenetically distinct strains, engineered genome-reduced strains, chimeric strains, strains with a large number of unique genes or a large proportion of pseudogenes, and multiple clonal strains. Since the presence of these confounding strains can seriously affect the quality and true landscape of the pan-genome, we should remove these deviations in the process of pan-genome analyses. Our study provides new insights into the removal of biases from confounding strains in pan-genome analyses at the beginning of data processing, which enables the achievement of a closer representation of a high-quality pan-genome landscape of B. subtilis that better reflects the performance and credibility of the B. subtilis pan-genome. This procedure could be added as an important quality control step in pan-genome analyses for improving the efficiency of analyses, and ultimately contributing to a better understanding of genome function, evolution and genome-reduction strategies for B. subtilis in the future., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

With the growing popularity of probiotics in dietary supplements, foods, and beverages, it is important to substantiate not only the health benefits and efficacy of unique strains but also safety. In the interest of consumer safety and product transparency, strain identification should include whole-genome sequencing and safety assessment should include genotypic and phenotypic studies. Bacillus subtilis MB40, a unique strain marketed for use in dietary supplements, and food and beverage, was assessed for safety and tolerability across in silico, in vitro, and in vivo studies. MB40 was assessed for the absence of undesirable genetic elements encoding toxins and mobile antibiotic resistance. Tolerability was assessed in both rats and healthy human volunteers. In silico and in vitro testing confirmed the absence of enterotoxin and mobile antibiotic resistance genes of safety concern to humans. In rats, the no-observed-adverse-effect level (NOAEL) for MB40 after repeated oral administration for 14 days was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 10 11 CFU/kg bw/day). In a 28 day human tolerability trial, 10 × 10 9 CFU/day of MB40 was well tolerated. Based on genome sequencing, strain characterization, screening for undesirable attributes and evidence of safety by appropriately designed safety evaluation studies in rats and humans, Bacillus subtilis MB40 does not pose any human health concerns under the conditions tested.

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 × 10 8 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.

Although recombination is accepted to be common in bacteria, for many species robust phylogenies with well-resolved branches can be reconstructed from whole genome alignments of strains, and these are generally interpreted to reflect clonal relationships. Using new methods based on the statistics of single-nucleotide polymorphism (SNP) splits, we show that this interpretation is incorrect. For many species, each locus has recombined many times along its line of descent, and instead of many loci supporting a common phylogeny, the phylogeny changes many thousands of times along the genome alignment. Analysis of the patterns of allele sharing among strains shows that bacterial populations cannot be approximated as either clonal or freely recombining but are structured such that recombination rates between lineages vary over several orders of magnitude, with a unique pattern of rates for each lineage. Thus, rather than reflecting clonal ancestry, whole genome phylogenies reflect distributions of recombination rates., Competing Interests: TS, CF, Ev No competing interests declared, (© 2021, Sakoparnig et al.)

The genomes of two historical Bacillus species strains isolated from the roots of oilseed rape and used routinely in PR China as biocontrol agents to suppress Sclerotinia disease were sequenced. Average nucleotide identity (ANI) and digital DNA-DNA hybridization analyses demonstrated that they were originally misclassified as Bacillus subtilis and now belong to the bacterial species Bacillus velezensis . A broader ANI analysis of available Bacillus genomes identified 292 B. velezensis genomes that were then subjected to core gene analysis and phylogenomics. Prediction and dereplication of specialized metabolite biosynthetic gene clusters (BGCs) defined the prevalence of multiple antimicrobial-associated BGCs and highlighted the natural product potential of B. velezensis . By defining the core and accessory antimicrobial biosynthetic capacity of the species, we offer an in-depth understanding of B. velezensis natural product capacity to facilitate the selection and testing of B. velezensis strains for use as biological control agents.

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.

Bacillus based probiotics are becoming relevant as alternatives to antibiotics used in poultry production and in other animal husbandry. This study describes the isolation of 48 Bacillus spp. candidates, from chickens and chicken environments, for use as potential probiotics in poultry production. These isolates, plus a further 18, were tested in a comprehensive in vitro screening regime that was specifically designed to select the best isolates that satisfied multiple modes of action desirable for commercial poultry probiotics. This screening programme involved the evaluation of the ability of the isolates to survive and grow in the limiting conditions of the chicken gastrointestinal tract. Only 11 of the isolates fulfilled these criteria; hence, they were further evaluated for the ability to adhere to epithelial cells, produce extracellular enzymes, and to demonstrate antagonistic activity against selected pathogens of significant importance in poultry production. Of these, a total of 6 isolates were selected, due to their all-round probiotic capability. Identification by 16S RNA sequencing confirmed these isolates as B. subtilis and B. velezensis, identities which are generally regarded as safe. The Bacillus isolates reported in our study exhibit strong all-inclusive probiotic effects and can potentially be formulated as a probiotic preparation for poultry production.

Plant growth-promoting rhizobacteria are a group of free-living bacteria that colonize plant rhizosphere and benefit plant root growth, thereby increasing host plant to cope with salinity induced stress. The aim of this study was to (1) isolate and characterize auxin-producing bacteria showing a high plant growth-promoting (PGP) potential, and (2) evaluate the PGP effects on the growth of Medicago sativa L under salinity stress (130 mM NaCl). Of thirteen isolates, Bacillus megaterium NRCB001 (NRCB001), B. subtilis subsp. subtilis NRCB002 (NRCB002) and B. subtilis NRCB003 (NRCB003) had the ability to produce auxin, which ranged from 47.53 to 154.38 μg ml -1 . The three auxin-producing bacterial strains were shown multiple PGP traits, such as producing siderophore and NH 3 , showing ACC deaminase activity, solubilize phosphate and potassium. Furthermore, NRCB001, NRCB002, and NRCB003 could survive in LB medium containing 1750 mM NaCl. The three auxin-producing with salinity tolerance strains were selected for further analyses. In greenhouse experiments, when inoculated with NRCB001, NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 24.1%, 23.1% and 38.5% respectively, compared with those of non-inoculated control seedlings under normal growth condition. When inoculated with NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 96.9 and 71.6% respectively, compared with those of non-inoculated control seedlings under 130 mM NaCl condition. Our results indicated that NRCB002 and NRCB003 having PGP traits are promising candidate strains to develop biofertilizers, especially used under salinity stress conditions.

Endophytic bacteria, which are common in plant tissues, may help to control plant pathogens and enhance plant growth. Camellia oleifera, an oil-producing plant, is widely grown in warm, subtropical, hilly regions in China. However, C. oleifera is strongly negatively affected by C. oleifera anthracnose, which is caused by Colletetrichum fructicola. To find a suitable biocontrol agent for C. oleifera anthracnose, 41 endophytes were isolated from the stems, leaves, and roots of C. oleifera. Bacterial cultures were identified based on analyses of 16S rDNA sequences; most strains belonged to the genus Bacillus. The antagonistic effects of these strains on C. fructicola were tested in vitro. In total, 16 strains inhibited C. fructicola growth, with B. subtilis strain 1-L-29 being the most efficient. Strain 1-L-29 demonstrated antagonistic activity against C. siamense, C. asianum, Fusarium proliferatum, Agaricodochium camellia, and Pseudomonas syringae. In addition, this strain produced indole acetic acid, solubilized phosphate, grew on N-free media, and produced siderophores. To facilitate further microecological studies of this strain, a rifampicin-resistant, green fluorescent protein (GFP)-labeled strain, 1-L-29gfpr, was created using protoplast transformation. This plasmid had good segregational stability. Strain 1-L-29gfpr was re-introduced into C. oleifera and successfully colonized root, stem, and leaf tissues. This strain remained at a stable concentration in the root more than 20 d after inoculation. Fluorescence microscopic analysis showed that strain 1-L-29gfpr thoroughly colonized the root surfaces of C. fructicola as well as the root vascular tissues of Arabidopsis thaliana., Competing Interests: The authors have declared that no competing interests exist.

Food fermentation can improve food nutritional value and sensory performance, it is considered as an ecofriendly bioprocessing technology. In this work, a fermented natto chestnut food was firstly developed and its active ingredients and functional properties were systematically studied. Through systematic experimental screening, including a single factor experiment and Box-Behnken design, the fermentation parameters of chestnut were optimized and selected. Under the optimal fermentation conditions, fermentation time 56 h, temperature 38 ℃ and 5% inoculum concentration, the fibrinolytic activity of the natto-chestnut reached 6479 IU/g. Meanwhile, higher antioxidant activity of the natto-chestnut was obtained due to the increased contents of total phenolic, total flavonoid and VC. In addition, α-glucosidase inhibition activity was also improved in the natto-chestnut. These results indicated that fermented chestnut could be a new dietary supplement with higher quality and better activities for people's health., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The sheath blight disease of rice caused by Rhizoctonia solani is widely prevalent and one of the most destructive diseases, affecting rice cultivation and loss worldwide. In the present study, a set of twenty Bacillus isolates from saline soil of Uttar Pradesh were tested for their biocontrol activity against R. solani with the aim to obtain a potential strain for the control of sheath blight disease toward ecofriendly and sustainable agriculture. The results of dual-culture assay and scanning electron microscopic studies showed that the strain RH5 exhibited significant antagonistic activity (84.41%) against the fungal pathogen R. solani. On the basis of 16S rDNA sequencing analysis, the potential biocontrol strain RH5 was identified as Bacillus subtilis. Furthermore, the strain RH5 was characterized by different plant growth-promoting (PGP) activities and induction of defense-related enzymes in rice plants against R. solani. The strain RH5 posses various PGP attributes (indole acetic acid, siderophore, hydrogen cyanide production and phosphate, Zn, K solubility), hydrolytic enzymatic (chitinase, protease, cellulase, xylanase) activity, and presence of antimicrobial peptide biosynthetic genes (bacylisin, surfactin, and fengycin), which support the strain for efficient colonization of hyphae and its inhibition. Finally, the results of the greenhouse study confirmed that strain RH5 significantly increased plant growth and triggered resistance in rice plants through the production of defense-related antioxidant enzymes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: Bacillus subtilis, a typical plant growth-promoting rhizobacteria, can benefit plant through promoting growth and reducing disease. The colonization intensity of B. subtilis in rhizosphere is a key factor for improving their effectiveness of field application. In this study, we developed a rapid and sensitive method for detecting B. subtilis in rhizosphere via TaqMan qPCR and droplet digital PCR (ddPCR) methods., Methods and Results: The primers/probe set targeting gyrB gene could successfully distinguish B. subtilis from its close-related species. Both the TaqMan qPCR and ddPCR methods exhibited a good linear relationship in the sensitivity assay, suggesting the developed method was specific, effective and reliable. Finally, the two methods were used to detect the colonization dynamic of B. subtilis within Arabidopsis rhizosphere. Both of them showed a consistent trend compared with the traditional cultivation-based and microscopy-based methods., Conclusions: The TaqMan qPCR and droplet digital PCR (ddPCR) methods we developed could be used to rapidly detect B. subtilis in rhizosphere., Significance and Impact of the Study: The TaqMan qPCR and ddPCR methods developed in this study can be applied to rapid quantitative detection of B. subtilis populations, and will be helpful to evaluate their effectiveness of field application., (© 2019 The Society for Applied Microbiology.)

Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the improvement of the properties of biotechnological strains. B. subtilis strains with improved characteristics maybe obtained by rational design and the directed evolution technologies, or be found among newly described strains. In the course of the long-term microbiome composition studies in the Russian segment of the International Space Station, the B. subtilis 20 strain was isolated, this strain shows the capacity for rapid growth and considerable biomass accumulation, as well as increased resistance to acidification of the environment in comparison to the "terrestrial" B. subtilis 168 strain. What is more, B. subtilis 20 is hyperresistant to the DNA and protein damaging factors that are linked to the overexpression of the genes controlling DNA repair, hydrogen sulfide production, and reactive oxygen species neutralization. The described properties of B. subtilis 20 are indicative of its considerable potential as a promising producer of biologically active compounds.

Bacillus subtilis currently encompasses four subspecies, Bacillus subtilis subsp. subtilis, Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris. Several studies based on genomic comparisons have suggested these subspecies should be promoted to species status. Previously, one of the main reasons for leaving them as subspecies was the lack of distinguishing phenotypes. In this study, we used comparative genomics to determine the genes unique to each subspecies and used these to lead us to the unique phenotypes. The results show that one difference among the subspecies is they produce different bioactive secondary metabolites. B. subtilis subsp. spizizenii is shown conserve the genes to produce mycosubtilin, bacillaene and 3,3'-neotrehalosadiamine. B. subtilis subsp. inaquosorum is shown conserve the genes to produce bacillomycin F, fengycin and an unknown PKS/NRPS cluster. B. subtilis subsp. stercoris is shown conserve the genes to produce fengycin and an unknown PKS/NRPS cluster. While B. subtilis subsp. subtilis is shown to conserve the genes to produce 3,3'-neotrehalosadiamine. In addition, we update the chemotaxonomy and phenotyping to support their promotion to species status.

A total of 104 exopolysaccharide (gum)-producing bacteria were isolated from the juice screen and juice tank in a sugarcane processing factory at times of low- and high dextran concentrations in the produced sugar. Dextran is an indicator of cane deterioration and sucrose loss after harvesting of the cane. The isolates were identified as Bacillus amyloliquefaciens (96 isolates) and Bacillus subtilis (eight isolates) based on restriction enzyme banding patterns of amplified 16S rRNA genes and rpoB gene sequence analysis. Exopolysaccharide production in sugarcane is normally associated with dextran produced by Leuconostoc mesenteroides. B. amyloliquefaciens, and to a lesser extent B. subtilis, could, however, also be responsible for exopolysaccharide (slime or gum) production in cane processing factories.

Microbial degradation plays a major role in the dissipation of pendimethalin, and nitroreduction is an initial and detoxicating step. Previously, a pendimethalin nitroreductase, PNR, was identified in Bacillus subtilis Y3. Here, another pendimethalin nitroreductase from strain Y3, LNR, was identified. LNR shares only 40% identity with PNR and reduces the aromatic ring C-6 nitro group of pendimethalin and both nitro groups of trifluralin, butralin, and oryzalin. The catalytic activities against the four dinitroanilines were much higher for LNR than for PNR. lnr deletion significantly reduced the pendimethalin-reduction activity (60% activity loss), while pnr deletion led to only 30% activity loss, indicating that both LNR and PNR were involved in pendimethalin nitroreduction in strain Y3; however, LNR played the major role. This study facilitates the elucidation of pendimethalin catabolism and provides degrading enzyme resources for the removal of dinitroaniline herbicide residues in environment and agricultural products.

β-Mannanases are crucial enzymes for the breakdown of mannans. As Mannans are being considered as antinutritional factors in poultry production, the search for mannanase-producing probiotic bacteria is now attracting considerable attention as a strategy to enhance nutrients bioavailability. Five soil born Bacilli (US134, US150, US176, US180, and US191) were selected for their ability to produce extracellular β-mannanases that were biochemically characterized. The probiotic properties of these strains were determined to assess their potential as animal feed supplements. Bacillus subtilis US191 was shown to be sensitive to all antibiotics tested, to inhibit growth of the bacterial pathogens tested, and to produce a thermostable β-mannanase. It exhibited a notable acid and bovine bile tolerance and high ability to form biofilm. These features may favor its adherence to the intestinal epithelial cells allowing its survival and persistence in the digestive tract. Furthermore, our study revealed that US191 was among the strains showing the highest ability to digest wheat dry matter in vitro when compared to the commercial feed additive Rovabio ® Max. Altogether, our findings suggest that the β-mannanase producer B.subtilis US191 is a promising probiotic candidate for the feed industry., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Two colonies of Bacillus subtilis of identical strains growing adjacent to each other on an agar plate exhibit two distinct types of interactions: they either merge as they grow or demarcation occurs leading to formation of a line of demarcation at the colony fronts. The nature of this interaction depends on the agar concentration in the growth medium and the initial separation between the colonies. When the agar concentration was 0.67% or lower, the two sibling colonies were found to always merge. At 1% or higher concentrations, the colonies formed a demarcation line only when their initial separation was 20 mm or higher. Interactions of a colony with solid structures and liquid drops have indicated that biochemical factors rather than the presence of physical obstacles are responsible for the demarcation line formation. A reaction diffusion model has been formulated to predict if two sibling colonies will form a demarcation line under given agar concentration and initial separation. The model prediction agrees well with experimental findings and generates a dimensionless phase diagram containing merging and demarcation regimes. The phase diagram is in terms of a dimensionless initial separation, d[combining macron], and a dimensionless diffusion coefficient, D[combining macron], of the colonies. The phase boundary between the two interaction regimes can be described by a power law relation between d[combining macron] and D[combining macron].

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis . In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610 T , G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis .

Several Bacillus strains are used as biocontrol agents, as they frequently have strong antagonistic effects against microbial plant pathogens. Bacillus strain SZMC 6179J, isolated from tomato rhizosphere, was previously shown to have excellent in vitro antagonistic properties against the most important fungal pathogens of tomato (Alternaria solani, Botrytis cinerea, Phytophthora infestans and Sclerotinia sclerotiorum) as well as several Fusarium species. Taxonomic investigations revealed that it is a member of the B. subtilis subsp. subtilis group and very closely related with the reference type strain B. subtilis subsp. subtilis 168. The sequenced genome of strain SZMC 6179J contains the genes responsible for the synthesis of the extracellular antibiotics surfactin, fengycin and bacilysin. Compared to strain 168, a prophage-like region is missing from the genome of SZMC 6179J, while there are 106 single nucleotide polymorphisms and 23 deletion-insertion polymorphisms. The high biocontrol potential of strain SZMC 6179J may results from a single base deletion in the sfp gene encoding the transcription factor of the surfactin and fengycin operons. Hypermutated regions reflecting short-time evolutionary processes could be detected in SZMC 6179J. The deletion-insertion polymorphism in the sfp gene and the detected hypermutations can be suggested as genetic determinants of biocontrol features in B. subtilis.

The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi. The presence of aprN, a gene-encoding nattokinase, was verified with PCR method. The predicted amino acid sequence was aligned with homologous sequences, and a phylogenetic tree was constructed. Nattokinase was sublimated with ammonium sulfate, using a DEAE-Sepharose Fast Flow column, a CM-Sepharose Fast Flow column and a Sephadex G-75 gel filtration column. SDS-PAGE analysis indicated that the molecular weight of the purified nattokinase from Bacillus subtilis MX-6 was about 28 kDa. Fermentation of Bacillus subtilis MX-6 nattokinase showed that nattokinase production was maximized after 72 h; the diameter of clear zone reached 21.60 mm on the plasminogen-free fibrin plate. Nattokinase production by Bacillus subtilis MX-6 increased significantly after supplementation with supernatant I, supernatant II and soy peptone but decreased substantially after the addition of amino acids. This result indicated that the nattokinase production by B. subtilis MX-6 might be induced by soybean polypeptides. The addition of MgSO 4 and CaCl 2 increased B. subtilis MX-6 nattokinase production.

Medicago sativa L. (alfalfa) is an important forage crop throughout the world. Despite the abiotic nutritional components of alfalfa having been extensively studied, there is only limited information on alfalfa endophytes. In this study, thirteen endophytic bacteria were isolated from alfalfa seeds. Bacillus (76·9%) was the most abundant genus, followed by Enterobacter (15·4%), Brevibacterium (7·7%), Geobacillus (7·7%) and Staphylococcus (7·7%). Four of the 13 endophytic bacteria, including Bacillus amyloliquefaciens EnB-alf1, Bacillus subtilis EnB-alf3, EnB-alf5 and EnB-alf13, were capable of significantly extending the lifespan of Caenorhabditis elegans. In addition, B. amyloliquefaciens EnB-alf1 enhanced the resistance of C. elegans to thermal stress whereas B. subtilis EnB-alf3 enhanced the resistance to oxidative stress. Further studies demonstrated that the enhanced lifespan of the worm was depended on the function of DAF-2/DAF-16 and was associated with the colonization of strain in the worms' intestines when strain EnB-alf1 or strain EnB-alf3 was presented to the worms as food sources. Our results suggest that some endophytic Bacillus strains isolated from alfalfa are beneficial on C. elegans health. SIGNIFICANCE AND IMPACT OF THE STUDY: Medicago sativa L. (alfalfa) is an important forage crop throughout the world. Despite the abiotic nutritional components of alfalfa having been extensively studied, there is only limited information available on alfalfa endophytes. Beneficial bacteria residing in the host intestine have been shown to affect host longevity. However, there is limited information available on the functions of alfalfa seed endophytes to nematodes. In this study, four endophytic Bacillus strains isolated from alfalfa seeds were found to significantly extend the lifespan of Caenorhabditis elegans and enhance resistance to thermal and oxidative stress. Our results suggest that some endophytic Bacillus strains isolated from alfalfa seeds can promote good health in C. elegans., (© 2018 The Society for Applied Microbiology.)

Fusarium wilt is a devastating soil-borne disease caused mainly by highly host-specific formae speciales of Fusarium oxysporum. Antagonistic microorganisms play a very important role in Fusarium wilt control, and the isolation of potential biocontrol strains is becoming more and more important. We isolated a bacterial strain (SEM-9) from the high-temperature stage of silkworm excrement composting, which had a marked ability to solubilize phosphorus, promote the growth and increase the yield of the small Chinese cabbage, and which also exhibited considerable antagonistic effect towards Fusarium sambucinum and other fungi. The result of physiological and biochemical analyses, as well as genome sequencing, showed that SEM-9 was a strain of Bacillus subtilis. Through genome annotation and analysis, it was found that SEM-9 contained genes related to the regulation of biofilm formation, which may play an important role in colonization, and gene clusters encoding the biosynthesis of antimicrobials, such as surfactin, bacilysin, fengycin, and subtilosin-A. The production of such antifungal compounds may constitute the basis of the mode-of-action of SEM-9 against Fusarium spp. These data suggested that the SEM-9 strain has potential as both a biofertilizer and a biocontrol agent, with the potential to manage Fusarium wilt disease in crops.

Here, we review the multiple mechanisms that the Gram-positive bacterium Bacillus subtilis uses to allow it to communicate between cells and establish community structures. The modes of action that are used are highly varied and include routes that sense pheromone levels during quorum sensing and control gene regulation, the intimate coupling of cells via nanotubes to share cytoplasmic contents, and long-range electrical signalling to couple metabolic processes both within and between biofilms. We explore the ability of B. subtilis to detect 'kin' (and 'cheater cells') by looking at the mechanisms used to potentially ensure beneficial sharing (or limit exploitation) of extracellular 'public goods'. Finally, reflecting on the array of methods that a single bacterium has at its disposal to ensure maximal benefit for its progeny, we highlight that a large future challenge will be integrating how these systems interact in mixed-species communities., (© 2018 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

The emergence of extensive antibiotics resistant bacteria increased the demands for finding out new sources of antimicrobial agents. Marine niches were reported to be rich in many competent producers of significant bioactive compounds. On the course of screening program for new antimicrobials, a Bacillus strain was isolated from Alexandria sea shores, Egypt. According to the morphological, cultural and biochemical characteristics, 16S rRNA sequence analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the strain was identified as Bacillus subtilis and designated as B. subtilis AD35. One phthalate derivative namely Di-(2-ethylhexyl) phthalate (DEHP) was purified from the crude extract of B. subtilis AD35 by high performance liquid chromatography (HPLC) and the structural elucidation of this compound was confirmed on the basis of gas chromatography-mass spectroscopy (GC-MS), Fourier infrared spectroscopy (FT-IR) and UV spectrum. The results of MIC of the purified DEHP were as follow: 16 μg/ml (Salmonella typhimurium), 32 μg/ml (Methicillin resistant Staphylococcus aureus, MRSA), 0.25 μg/ml (Listeria monocytogenes), 0.5 μg/ml (Aeromonas hydrophila), 8 μg/ml (Staphylococcus aureus), 4 μg/ml (Staphylococcus epidermidis), 4 μg/ml (Escherichia coli), and 8 μg/ml (Pseudomonas aeruginosa). DEHP produced by B. subtilis AD35 up to a concentration of 2500 μg/ml exhibited no cytotoxic effect against normal Vero cells. In addition, it did not show an antiviral activity against HAV or a significant growth inhibitory effect toward human colorectal adenocarcinoma and human mammary gland adenocarcinoma cell-lines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A strain of bacteria in the Bacillus subtilis species complex was isolated from a building's air vent in the Washington DC area, USA, and produced strong antifungal activity with in vitro assays. This strain, designated (HU Biol-II), showed pronounced inhibitory effects on mycelial growth of a wide spectrum of fungi. The objectives of this study were to use genome sequencing to confirm the taxonomy of HU Biol-II, evaluate its antifungal activity and implement genome mining and HPLC-MS/MS to characterize the bioactive secondary metabolites. The strain, as determined by multilocus sequence alignment analysis, was identified as a member of Bacillus subtilis subsp. inaquosorum clade. Core genome phylogeny showed that the isolate is most closely related to B. subtilis subsp. inaquosorum strain DE111, a commercially produced human probiotic. The investigation identified eight bioactive metabolite clusters in the genome. HPLC MS/MS was able to confirm the production of seven of the metabolites. This study is the first to report the production of two antifungal cyclic lipopeptides (bacillomycin F and fengycin) from a member of B. subtilis subsp. inaquosorum. The strain also produced the antibacterial aurantinin B, which confirms the biosynthetic cluster responsible for its production. Comparative genomics and metabolomics demonstrated the commercial probiotic strain DE111 produced the same metabolites, with the exception of aurantinin B. These findings are the first description of the secondary metabolites produced by a strain of B. subtilis subsp. inaquosorum., (Published by Elsevier GmbH.)

The type strain Bacillus subtilis subsp. subtilis KCTC 3135 T was deeply sequenced and annotated, replacing a previous draft genome in this study. The tar and tag genes were involved in synthesizing wall teichoic acids (WTAs), and these genes and their products were previously regarded as the distinguishing difference between B. s. subtilis and B. s. spizizenii . However, a comparative genomic analysis of B. subtilis spp. revealed that both B. s. subtilis and B. s. spizizenii had various types of cell walls. These tar and tag operons were mutually exclusive and the tar genes from B. s. spizizenii were very similar to the genes from non- Bacillus bacteria, unlike the tag genes from B. s. subtilis . The results and previous studies suggest that the tar genes and the tag genes are not inherited after subspecies speciation. The phylogenetic tree based on whole genome sequences showed that each subspecies clearly formed a monophyletic group, while the tree based on tar genes showed that monophyletic groups were formed according to the cell wall type rather than the subspecies. These findings indicate that the tar genes and the presence of ribitol as a cell-wall constituent were not the distinguishing difference between the subspecies of B. subtilis and that the description of subspecies B. s. spizizenii should be updated.

This paper describes a novel technique to quantify and identify bacterial cultures of Bacillus Subtilis (2.10-1.30 × 10 9  CFU mL -1 ) and Escherichia Coli (1.60-1.00 × 10 9  CFU mL -1 ), in corn oil using dielectric spectroscopy at elevated frequencies of 0.0100-20.0 GHz. This technique is using the electrophysiological/biophysical differences (e.g. gram positive and gram negative) between various bacteria types, as a basis to distinguish between bacteria concentrations and bacteria types. A close-ended, coaxial probe of 20.0 mm long sample-holder was developed and used to calculate the dielectric constant from the measured S parameters of the bacterial cultures, using the Nicolson-Ross-Weir method. This technique shows a linear relationship (r 2  ≥ 0.999) between the dielectric constant and the cell concentration, at 16.0 GHz. The sensitivity of the technique is 0.177 × 10 9 (CFU mL -1 ) -1 for B. Subtilis (with a size of 10.0 × 1.00 μm), 0.322 × 10 9 (CFU mL -1 ) -1 for E. Coli (with a size of 2.00 × 0.500 μm) and 0.913 × 10 9 (CFU mL -1 ) -1 for their 1:1 mixture, while the response time is 60.0s. The dependency of dielectric constant on the bacterial cell concentration at a given frequency can be potentially exploited for measuring bacterial concentrations and biophysical differences., (Copyright © 2018 Elsevier B.V. All rights reserved.)

One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra recorded from single bacterial colonies picked from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis strains in <30 min solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin, caused by a single frameshift mutation. Next, we used IDBac to detect subtle intraspecies differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we used IDBac to simultaneously extract protein and specialized metabolite MS profiles from unidentified Lake Michigan sponge-associated bacteria isolated from an agar plate. In just 3 h, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 spectra) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on interspecies and intraspecies differences in specialized metabolite production. IDBac is an attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow for facile discrimination of closely related bacterial colonies., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Rhizobacteria are an active part of microbial population in the rhizosphere of plants. In this study, twenty rhizobacteria were isolated from the rhizosphere of a perennial grass, Haloxylon salicornicum, found in Cholistan desert, an arid landmass near Bahawalpur Pakistan, in one set of experimental conditions. Colony characteristics, biochemical and molecular analyses of these isolates were performed. All isolates were bacilli, gram positive with off-white colonies and exhibited typical bacilli colony morphology. None of the isolates was gelatinase, urease, indole, H2S and catalase producer. Eleven isolates were amylase producers and 8 isolates were acid producers. All isolates fermented glucose, 3 fermented lactose and 19 fermented fructose. Molecular data revealed that out of twenty isolates, 14 isolates showed 91-99% identity with Brevibacillus borstelensis, 4 with Bacillus subtilis (97-98%) and 2 with Bacillus licheniformis (94-99%) through BLAST analysis. All identified bacterial isolates cladded with their respective groups in the phylogenetic tree. Many (11-15 out of 20) of the isolates were more effective in inhibiting growth of the tested bacterial strains as compared to the positive control (Ampicillin 50 μg/disc). We conclude that bacilli are the predominant form populating rhizosphere of this desert grass. Among the isolated bacteria Brevibacillus borstelensis, Bacillus subtilis and Bacillus licheniformis are the most predominant species.

The objective of this study was to investigate the effects of Bacillus subtilis-based probiotic supplementation in broiler chicken diets on growth performance, feed efficiency, intestinal cytokine, and tight junction (TJ) protein mRNA expression. Zero-day-old broiler chicks (n = 140) were randomly assigned to one of five dietary treatments: basal diet (CON); basal diet supplemented with either antibiotic bacitracin methylene disalicylate (BMD); or probiotics, namely, B. subtilis strain 1781 (PB1), a combination of B. subtilis strain 1104 + strain 747 (PB2), or B. subtilis strain 1781 + strain 747 (PB3). Body weight and feed intake were measured at 14 days of age, and the feed conversion ratio (FCR) was calculated. At 14 days of age, ileal samples were collected and used for intestinal cytokine, TJ protein, and mucin gene expression analysis using qRT-PCR. The chickens supplemented with antibiotic (BMD) and B. subtilis strain 1781 alone (PB1) had significantly higher body weights compared to controls of the same age. Dietary supplementation with antibiotic (BMD) or probiotics (PB1, PB2, PB3) significantly improved the feed efficiency as evidenced by decreased FCR compared to controls. No differences were observed in the expression of IL1β, IL17F, IFNγ, and MUC2 gene among the different treatment groups. However, elevated expression of IL6 (BMD, PB1, PB2), IL8 (PB2), and TNFSF15 (PB1, PB2, PB3) compared to controls was observed in the ileum. IL2 and IL10 expression was upregulated in chicks in the PB2 and PB3 groups, and IL4 was elevated in the PB1 group. IL13 was elevated in all probiotic-fed groups (PB1, PB2, PB3). Probiotic supplementation was also shown to significantly increase the expression of TJ proteins JAM2, ZO1 (PB2, PB3), and occludin (PB1, PB2). Taken together, B. subtilis supplementation altered intestinal immune activity and influenced gut barrier integrity through increased tight junction gene expression.

Bacillus subtilis is a Gram-positive bacterium widely used in medicine and agriculture. So far, little is known about its pathogenicity in animals. In this study, a strain of Bacillus subtilis, HFBF-B11 isolated from brain tissue of ducklings co-infected with Riemerella anatipestifer was characterized. The strain demonstrated consistent characteristics of B. subtilis in staining and morphological, biochemical and physiological analyses. Moreover, its DNA sequence, which was obtained via PCR sequencing of 16S rRNA, exhibited 99% homology with the B. subtilis reference strain. In in vitro cultures HFBF-B11 exhibited β-hemolysis. The results of experiments showed that a single infection of HFBF-B11 in 9-day-old ducklings did not result in clear clinical symptoms. However, following co-infection with HFBF-B11 and R. anatipestifer, the animals demonstrated liver injury and blood-brain barrier disruption leading to infection and brain damage with a mortality rate of 100%. These results suggest that the HFBF-B11 strain of B. subtilis is an opportunistic pathogen of ducklings. This is the first report about the isolation of a B. subtilis strain with pathogenicity in ducklings., (Copyright© by the Polish Academy of Sciences.)

Pectinase is an important kind of enzyme with many industrial applications, among which pectinases produced by bacteria were scarce compared with fungal sources. In this study, a novel bacterium which produced extracellular pectinase was firstly isolated from flue-cured tobacco leaves and identified as Bacillus subtilis PB1 according to its 16S rRNA gene. The pectinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography, after which molecular weight was determined as 43.1 ± 0.5 kDa by SDS-PAGE. Peptide mass fingerprinting of the pectinase by MALDI-TOF MS showed that the purified enzyme shared homology with pectate lyase and was designated as BsPel-PB1. The optimal temperature for BsPel-PB1 was 50 °C. The optimal pH was pH 9.5 for BsPel-PB1 while it had a broad pH stability from 5 to 11. The values of K m and V max were 0.312 mg/mL and 1248 U/mL, respectively. Accordingly, the BsPel-PB1 was a novel alkaline pectate lyase which could find potential application as a commercial candidate in the pectinolytic related industries.

Alkaline pectinase has important applications in the pretreatment of waste water from food processing and in both the fabric and paper industries. In this study, a 2-level factorial design was used to screen significant factors that affect the activity of alkaline pectinase, and the response surface methodology (RSM) with a Box-Behnken design (BBD) was used to optimize their concentrations. Starch, peptone, KH 2 PO 4 and K 2 HPO 4 ·3H 2 O were found to significantly affect the activity of alkaline pectinase. Their optimal concentrations were as follows: 4.68% starch, 1.6% peptone, 0.26% KH 2 PO 4 and 0.68% K 2 HPO 4 ·3H 2 O. Under the above conditions, the activity of alkaline pectinase was significantly improved to 734.11 U/mL. Alkaline pectinase was purified to homogeneity with a recovery rate of 9.6% and a specific activity of 52372.52 U/mg. Its optimal temperature and pH were 50°C and 8.6, respectively. The purified enzyme showed strong thermo-stability and good alkali resistance. In addition, the activity of alkaline pectinase was improved in the presence of Mg 2+ . Cu 2+ , Mn 2+ , and Co 2+ significantly inhibited its activity. This study provides an important basis for the future development and use of a heat-tolerant alkaline pectinase from B. subtilis ZGL14.

The study reports the effects on broiler performance of a newly isolated Bacillus subtilis strain, which is phylogenetically not closely related to already well-described strains of B. subtilis. In the first experiment, birds were reared in battery cages and exposed to C. perfringens. An increase in growth performance was observed with the strain when compared to the challenged animals. Three additional growth trials were conducted to 35 d of age, in different rearing conditions (genetic breeds, corn-soybean meal-based diet with or without animal proteins, in presence or absence of phytase, on fresh or used litter) to investigate the efficacy and the specificity of this new B. subtilis strain on the improvement of BWG and FCR of broilers in comparison with a B. subtilis-based DFM already used in the field. Whatever the rearing conditions tested, the new B. subtilis strain led to an average 3.2% improvement in feed conversion ratio or bodyweight. Comparatively, the commercial Bacillus strain significantly improved broiler performance in only one trial out of 3 with an average improvement reaching 2%. All these results indicate that this new B. subtilis strain consistently improves broiler performances., (© 2017 Poultry Science Association Inc.)

The draft genome here presents the sequence of Bacillus subtilis UMX-103. The bacterial strain was isolated from hydrocarbon-contaminated soil from Terengganu, Malaysia. The whole genome of the bacterium was sequenced using Illumina HiSeq 2000 sequencing platform. The genome was assembled using de novo approach. The genome size of UMX-103 is 4,234,627 bp with 4399 genes comprising 4301 protein-coding genes and 98 RNA genes. The analysis of assembled genes revealed the presence of 25 genes involved in biosurfactant production, where 14 of the genes are related to biosynthesis and 11 of the genes are in the regulation of biosurfactant productions. This draft genome will provide insights into the genetic bases of its biosurfactant-producing capabilities.

Given the close genetic relationship between Bacillus amyloliquefaciens and B. subtilis, distinguishing the two solely based on their physiological and biochemical characteristics and 16S rRNA sequences is difficult. Molecular identification was used to discover suitable genes for distinguishing the two bacteria, and to identify the bio-controlling strain B29, due to molecular identification has been paid more and more attention. The similarity of four genes, cheA, gyrB, groEL and phoR, of the two species was compared by the software BLASTN and MAGA, and phylogenetic tree was constructed. The B29 strain was re-identified by using the screened genes. The similarities of the four genes, gyrB, groEL, cheA and phoR, of the two species were 93-95%, 82-84%, 76-78% and 76-77%, respectively. The homologies of the four genes of the strain B29 and the strains of B. amyloliquefaciens strains were more than 95%. We determined how well the phoR and cheA genes could be used to differentiate B. amyloliquefacien and B. subtilis. The previously isolated biological control strain B29, initially classified as B. subtilis, was re-classified as B. amyloliquefaciens. Our data indicate that other than the phoR gene, the cheA gene might be a useful phylogenetic marker for differentiating B. subtilis and B. amyloliquefaciens.

Bacteria within communities can interact to organize their behavior. It has been unclear whether such interactions can extend beyond a single community to coordinate the behavior of distant populations. We discovered that two Bacillus subtilis biofilm communities undergoing metabolic oscillations can become coupled through electrical signaling and synchronize their growth dynamics. Coupling increases competition by also synchronizing demand for limited nutrients. As predicted by mathematical modeling, we confirm that biofilms resolve this conflict by switching from in-phase to antiphase oscillations. This results in time-sharing behavior, where each community takes turns consuming nutrients. Time-sharing enables biofilms to counterintuitively increase growth under reduced nutrient supply. Distant biofilms can thus coordinate their behavior to resolve nutrient competition through time-sharing, a strategy used in engineered systems to allocate limited resources., (Copyright © 2017, American Association for the Advancement of Science.)

Plant growth-promoting bacteria (PGPB) may trigger tolerance against biotic/abiotic stresses and growth enhancement in plants. In this study, an endophytic bacterial strain from rapeseed was isolated to assess its role in enhancing plant growth and tolerance to abiotic stresses, as well as banded leaf and sheath blight disease in maize. Based on 16S rDNA and BIOLOG test analysis, the 330-2 strain was identified as Bacillus subtilis. The strain produced indole-3-acetic acid, siderophores, lytic enzymes and solubilized different sources of organic/inorganic phosphates and zinc. Furthermore, the strain strongly suppressed the in vitro growth of Rhizoctonia solani AG1-IA, Botrytis cinerea, Fusarium oxysporum, Alternaria alternata, Cochliobolus heterostrophus, and Nigrospora oryzae. The strain also significantly increased the seedling growth (ranging 14-37%) of rice and maize. Removing PCR analysis indicated that 114 genes were differentially expressed, among which 10%, 32% and 10% were involved in antibiotic production (e.g., srfAA, bae, fen, mln, and dfnI), metabolism (e.g., gltA, pabA, and ggt) and transportation of nutrients (e.g., fhu, glpT, and gltT), respectively. In summary, these results clearly indicate the effectiveness and mechanisms of B. subtilis strain 330-2 in enhancing plant growth, as well as tolerance to biotic/abiotic stresses, which suggests that the strain has great potential for commercialization as a vital biological control agent.

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg -1  protein ml -1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

We previously discovered that lagging strand genes evolve faster in Bacillus subtilis (and potentially other bacteria). Lagging strand genes are transcribed in the head-on orientation with respect to DNA replication, leading to collisions between the two machineries that stall replication and can destabilize genomes. Our previous work indicated that the increased mutagenesis of head-on genes depends on transcription-coupled repair and the activity of an error prone polymerase which is likely activated in response to these collisions. Recently, it was proposed that sequence context is a major contributor to the increased mutagenesis and evolution of head-on genes. These models are based on laboratory-based evolution experiments performed in B. subtilis. However, critical evolutionary analyses of naturally occurring single nucleotide polymorphisms (SNPs) in wild strains were not performed. Using the genomic sequences from nine closely related wild B. subtilis strains, we analyzed over 200,000 naturally occurring SNPs as a proxy for natural mutation patterns for all genes and in particular, head-on genes. Our analysis suggests that (frame-independent) triplet sequence context can impact mutation rates: certain triplet sequences (TAG, CCC, CTA, and ACC) accumulate SNPs at a higher rate and are depleted from the genome. However, the triplet sequences previously identified as mutagenic in laboratory experiments (CCG, GCG, and CAC) do not have an elevated rate of SNP accumulation and are not depleted from the genome. Importantly, dN/dS analyses indicate that the accelerated evolution of head-on genes is not dependent on any particular triplet sequence. Thus, in agreement with our previous results, mutagenic transcription-coupled repair, rather than sequence context, is sufficient to explain the accelerated evolution of head-on genes., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Starch-degrading amylase enzyme is important in biotechnological applications as food, fermentation, textile, paper and pharmaceutical purposes. The aim of current study to isolate alkaline thermostable α-amylase bacteria and then study the composition of medium and culture conditions to optimize cells growth and a-amylase production., Materials and Methods: Thermophilic amylase producing bacterium was isolated from local hot water-springs in Gazan city Saudi Arabia., Results: Phylogenetic analysis of 16 S rRNA sequence for the strain revealed that the strain have the same sequence of Bacillus subtilis . Maximum amylase production was observed, when B. subtilis cultured in medium containing starch at concentration 0.5%, and 10 g/L peptones as nitrogen source at pH 8.5 in when it was incubated for 48 h at 45°C., Conclusion: An amylase-producing bacterium were isolated from hot-spring water and was identified as B. subtilis . Amylase produced from B.subtilis had optimum temperature 45°C and pH 8.5 in shaking media.

The possible application of a bacterial strain - Bacillus subtilis R1, isolated from an oil contaminated desert site in India, as biocontrol agent and its biosurfactant in microbial enhanced oil recovery are discussed. The biosurfactant production in minimal medium was carried out at different temperatures and salt concentrations, where it produced an efficient biosurfactant at 30-45°C and in presence of up to 7% salt. It significantly reduced the surface tension from 66±1.25mN/m to 29±0.85mN/m within 24h. In order to enhance the biosurfactant production, random mutagenesis of B. subtilis R1 was performed using chemical mutagen - ethyl methanesulfonate. Majority of the isolated 42 mutants showed biosurfactant production, but the difference was statistically insignificant as compared with parent strain R1. Therefore none of the mutants were selected for further study, and only parent strain R1 was studied. The biosurfactant was quite stable under harsh conditions for up to 10 days. The biosurfactant was extracted and characterized as similar to the lipopeptide group - surfactins and fengycin. The crude oil displacement experiments using biosurfactant broth in sand pack glass columns showed 33±1.25% additional oil recovery. The strain also showed inhibition of various plant pathogenic fungi on potato dextrose agar medium., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Urinary tract infections are a common disease in humans. Therefore, new methods are needed to destroy biofilms that are formed by uropathogens. Iturin A lipopeptides (LPs) C14 and C15 are potent biosurfactants synthetized by the Bacillus subtilis I'1a strain. The biological activity of extracted LPs was confirmed by examining extracts from I'1a cultures against uropathogenic bacteria that had been isolated from biofilms on urinary catheters. Compared with cultures of DSM 3257, which produce surfactin at a relatively low level, the extract obtained from strain I'1a exhibited a greater inhibitory effect against both planktonic and sessile forms of Escherichia coli, Serratia marcescens, Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii and Enterococcus faecalis. Moreover, cyclic LP biosurfactants may disturb the integrity of cytoplasmic membranes; therefore, we investigated the effects of synthetized LPs on fatty acids and phospholipids of B. subtilis. LPs and lipids were analyzed using GC-MS, LC-MS/MS and MALDI-TOF/TOF techniques. Compared with B. subtilis DSM 3257, membranes of the I'1a strain were characterized by an increased amount of anteiso fatty acids and a ten-fold higher ratio of phosphatidylglycerol (PG)-to-phosphatidylethanolamine (PE). Interestingly, in cultures of B. subtilis DSM 3257 supplemented with LP extracts of the I'1a strain, the PG-to-PE ratio was fourfold higher, and the amount of anteiso fatty acids was also increased., Competing Interests: The authors declare no conflict of interest.

Regulation of translation is critical for maintaining cellular protein levels, and thus protein homeostasis. The conserved RNA-binding protein CsrA (also called RsmA; for carbon storage regulator and regulator of secondary metabolism, respectively; hereafter called CsrA) represents a well-characterized example of regulation at the level of translation initiation in bacteria. Binding of a CsrA homodimer to the 5'UTR of an mRNA occludes the Shine-Dalgarno sequence, blocking ribosome access for translation. Small noncoding RNAs (sRNAs) can competitively antagonize CsrA activity by a well-understood mechanism. However, the regulation of CsrA by the protein FliW is just emerging. FliW antagonizes the CsrA-dependent repression of translation of the flagellar filament protein, flagellin. Crystal structures of the FliW monomer reveal a novel, minimal β-barrel-like fold. Structural analysis of the CsrA/FliW heterotetramer shows that FliW interacts with a C-terminal extension of CsrA. In contrast to the competitive regulation of CsrA by sRNAs, FliW allosterically antagonizes CsrA in a noncompetitive manner by excluding the 5'UTR from the CsrA-RNA binding site. Our phylogenetic analysis shows that the FliW-mediated regulation of CsrA regulation is the ancestral state in flagellated bacteria. We thus demonstrate fundamental mechanistic differences in the regulation of CsrA by sRNA in comparison with an ancient regulatory protein., Competing Interests: The authors declare no conflict of interest.