Sample records for bacillus subtilis cells

  1. Cosegregation of cell wall and DNA in Bacillus subtilis.

    Schlaeppi, J M; Karamata, D


    Cosegregation of cell wall and DNA of a lysis-negative mutant of Bacillus subtilis was examined by continuously labeling (i) cell wall, (ii) DNA, and (iii) both cell wall and DNA. After four to five generations of chase in liquid media it was found by light microscope autoradiography that the numbers of wall segregation units per cell are 29 and 9 in rich and minimal medium, respectively. Under the same conditions the numbers of segregation units of DNA were almost 50% lower: 15 and 5, respec...

  2. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells.

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David


    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent. PMID:26858704

  3. Taxonomy Icon Data: Bacillus subtilis [Taxonomy Icon

    Full Text Available Bacillus subtilis Bacillus subtilis Bacillus_subtilis_L.png Bacillus_subtilis_NL.png Bacillus..._subtilis_S.png Bacillus_subtilis_NS.png http://biosciencedbc.j...p/taxonomy_icon/icon.cgi?i=Bacillus+subtilis&t=S

  4. Metabolic protein interactions in Bacillus subtilis studied at the single cell level

    Detert Oude Weme, Ruud Gerardus Johannes


    We have investigated protein-protein interactions in live Bacillus subtilis cells (a bacterium). B. subtilis’ natural habitat is the soil and the roots of plants, but also the human microbiota. B. subtilis is used worldwide as a model organism. Unlike eukaryotic cells, bacteria do not have organelle

  5. [Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells].

    Iakorski, P; Kuntsova, M M; Loseva, E F; Khasanov, F K


    The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions. PMID:1944323

  6. Essential Bacillus subtilis genes

    Kobayashi, K.; Ehrlich, S.D.; Albertini, A.;


    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were...... predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to...... cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from...

  7. Absence of correlation between rates of cell wall turnover and autolysis shown by Bacillus subtilis mutants.

    Vitković, L; Cheung, H. Y.; Freese, E


    Bacillus subtilis mutants with reduced rates of cell wall autolysis reached a constant rate of wall turnover after a longer lag than the standard strain but eventually showed the same turnover rate. In reverse, a turnover-deficient mutant autolysed at a slightly higher rate than the standard strain. Consequently, there is no correlation between the rates of cell wall turnover and autolysis.

  8. Regulation of Growth of the Mother Cell and Chromosome Replication during Sporulation of Bacillus subtilis

    Xenopoulos, Panagiotis; Piggot, Patrick J.


    During spore formation, Bacillus subtilis divides asymmetrically, resulting in two cells with different fates. Immediately after division, the transcription factor σF becomes active in the smaller prespore, followed by activation of σE in the larger mother cell. We recently showed that a delay in σE activation resulted in the novel phenotype of two spores (twins) forming within the same mother cell. Mother cells bearing twins are substantially longer than mother cells with single spores. Here...

  9. Biocalcifying Bacillus subtilis cells effectively consolidate deteriorated Globigerina limestone.

    Micallef, Roderick; Vella, Daniel; Sinagra, Emmanuel; Zammit, Gabrielle


    Microbially induced calcite precipitation occurs naturally on ancient limestone surfaces in Maltese hypogea. We exploited this phenomenon and treated deteriorated limestone with biocalcifying bacteria. The limestone was subjected to various mechanical and physical tests to present a statistically robust data set to prove that treatment was indeed effective. Bacillus subtilis conferred uniform bioconsolidation to a depth of 30 mm. Drilling resistance values were similar to those obtained for freshly quarried limestone (9 N) and increased up to 15 N. Treatment resulted in a high resistance to salt deterioration and a slow rate of water absorption. The overall percentage porosity of treated limestone varied by ±6 %, thus the pore network was preserved. We report an eco-friendly treatment that closely resembles the mineral composition of limestone and that penetrates into the porous structure without affecting the limestones' natural properties. The treatment is of industrial relevance since it compares well with stone consolidants available commercially. PMID:27072564

  10. Sigma factors, asymmetry, and the determination of cell fate in Bacillus subtilis.

    Lewis, P J; Partridge, S R; Errington, J


    Soon after the initiation of sporulation, Bacillus subtilis divides asymmetrically to produce sister cells that have very different developmental fates. Recently, it has been proposed that the differential gene expression which begins soon after this division is due to cell-specific activation of the transcription factors sigma F and sigma E in the prespore and the mother cell, respectively. We describe the use of a method for the localization of gene expression in individual sporulating cell...

  11. Differential Actions of Chlorhexidine on the Cell Wall of Bacillus subtilis and Escherichia coli

    Cheung, Hon-Yeung; Wong, Matthew Man-Kin; Cheung, Sau-Ha; Liang, Longman Yimin; Lam, Yun-Wah; Chiu, Sung-Kay


    Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of Gram-positive and Gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Esc...

  12. Cell wall synthesis and initiation of deoxyribonucleic acid replication in Bacillus subtilis.

    Sandler, N.; Keynan, A


    We have observed a connection between cell wall synthesis and the initiation of chromosome replication in Bacillus subtilis. Initiation of chromosome replication was prevented in synchronous cultures in the presence of the cell wall synthesis inhibitor vancomycin. When vancomycin was added to the cultures after initiation of chromosome replication, one round of replication was completed but no reinitiation occurred. Similar results were obtained when cell wall synthesis was inhibited by risto...

  13. Cell division of cycle of Bacillus subtilis: evidence of variability in period D.

    Holmes, M.; Rickert, M; Pierucci, O


    In Bacillus subtilis the deoxyribonucleic acid content and the extent of cell division during inhibition of chromosome replication increased as a function of the average cell mass, independent of the growth rate. At each growth rate, mass, deoxyribonucleic acid, and residual division varied in different cultures. The variation is consistent with a large variability in the D period. At growth rates higher than 1.5 doublings per h at 37 degrees C, the change in D accounts for the growth rate de...

  14. Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells.

    O'Hara, M B; Hageman, J H


    Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent....

  15. Free and attached cells of Bacillus subtilis as starters for production of a soup flavouring (“ogiri egusi”)

    Peter-Ikechukwu, A. I.; Ahaotu, I.; Owuamanam, C. I.; Ogueke, C. C.


    Aims: This Bacillus subtilis has been identified to be the main fermenting bacterium during indigenous production of “ogiri egusi”; a traditional soup flavouring rich in protein. Evaluation of the use of starter and broth cultures of this bacterium in the production of ‘ogiri egusi’ was therefore undertaken with the view to improve the fermentation process and quality of product. Methodology and Results: Cowpea granules in association with Bacillus subtilis cells were developed as starter cul...

  16. Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilis

    Levdikov, Vladimir M; Blagova, Elena V.; Rawlings, Andrea E.; Jameson, Katie; Tunaley, James; Hart, Darren J.; Barak, Imrich; Wilkinson, Anthony J.


    Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA pol...

  17. Analysis of Peptidoglycan Structure from Vegetative Cells of Bacillus subtilis 168 and Role of PBP 5 in Peptidoglycan Maturation

    Atrih, Abdelmadjid; Bacher, Gerold; Allmaier, Günter; Williamson, Michael P; Foster, Simon J.


    The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4...

  18. From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate.

    Jordi van Gestel


    Full Text Available The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles" of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

  19. Cell wall mechanical properties as measured with bacterial thread made from Bacillus subtilis.

    Mendelson, N H; Thwaites, J J


    Engineering approaches used in the study of textile fibers have been applied to the measurement of mechanical properties of bacterial cell walls by using the Bacillus subtilis bacterial thread system. Improved methods have been developed for the production of thread and for measuring its mechanical properties. The best specimens of thread produced from cultures of strain FJ7 grown in TB medium at 20 degrees C varied in diameter by a factor of 1.09 over a 30-mm thread length. The stress-strain...

  20. Bacillus subtilis Deoxyribonucleic Acid Gyrase

    Sugino, A; Bott, K F


    Bacillus subtilis 168 was shown to contain a deoxyribonucleic acid (DNA) gyrase activity which closely resembled those of the enzymes isolated from Escherichia coli and Micrococcus luteus in its enzymatic requirements, substrate specificity, and sensitivity to several antibiotics. The enzyme was purified from the wild type and nalidixic acid-resistant and novobiocin-resistant mutants of B. subtilis and was functionally characterized in vitro. The genetic loci nalA and novA but not novB were s...

  1. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

    Soufi, Boumediene; Kumar, C.; Gnad, F.;


    We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic for...... most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

  2. Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells

    The authors have shown, with an optimized [14C]leucine-labeling and chasing procedure, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (≤ 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, the authors found that chloramphenicol strongly inhibited proteolysis even when added 6 h into the sporulation process. Restricting the calcium ion concentration in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation, and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells. Restricting the Ca2+ concentration in the medium reduced by threefold of the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2+-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for period of 8 h

  3. Dynamic expression of the translational machinery during Bacillus subtilis life cycle at a single cell level.

    Alex Rosenberg

    Full Text Available The ability of bacteria to responsively regulate the expression of translation components is crucial for rapid adaptation to fluctuating environments. Utilizing Bacillus subtilis (B. subtilis as a model organism, we followed the dynamics of the translational machinery at a single cell resolution during growth and differentiation. By comprehensive monitoring the activity of the major rrn promoters and ribosomal protein production, we revealed diverse dynamics between cells grown in rich and poor medium, with the most prominent dissimilarities exhibited during deep stationary phase. Further, the variability pattern of translational activity varied among the cells, being affected by nutrient availability. We have monitored for the first time translational dynamics during the developmental process of sporulation within the two distinct cellular compartments of forespore and mother-cell. Our study uncovers a transient forespore specific increase in expression of translational components. Finally, the contribution of each rrn promoter throughout the bacterium life cycle was found to be relatively constant, implying that differential expression is not the main purpose for the existence of multiple rrn genes. Instead, we propose that coordination of the rrn operons serves as a strategy to rapidly fine tune translational activities in a synchronized fashion to achieve an optimal translation level for a given condition.

  4. Incorporation of glycine and serine into sporulating cells of Bacillus subtilis

    The changes during growth and sporulation in activities of cells of Bacillus subtilis to incorporate various amino acids were investigated with wild-type strain and its asporogenous mutant. In the case of wild type strain the uptake of valine, phenylalanine, and proline was largest during the logarithmic growth period. The uptake of these amino acids decreased rapidly during the early stationary phase. The uptake of valine and cysteine increased again to some extent just prior to the forespore stage. The uptake of glycine and serine, however, was largest at the forespore stage at which the formation of spore coat took place. From these observed phenomena it was assumed that the remarkable incorporation of glycine and serine into the wild type strain during sporulation was closely related to the formation of spore coat. (auth.)

  5. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

    Marciniak, Bogumila C.; Trip, Hein; van-der Veek, Patricia J.; Kuipers, Oscar P.; Marciniak, Bogumiła C.


    Background: Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentatio

  6. The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis

    Bisicchia, Paola; Noone, David; Lioliou, Efthimia;


    Adaptation of bacteria to the prevailing environmental and nutritional conditions is often mediated by two-component signal transduction systems (TCS). The Bacillus subtilis YycFG TCS has attracted special attention as it is essential for viability and its regulon is poorly defined. Here we show...

  7. Abnormal morphology of Bacillus subtilis ugtP mutant cells lacking glucolipids.

    Matsuoka, Satoshi; Chiba, Minako; Tanimura, Yu; Hashimoto, Michihiro; Hara, Hiroshi; Matsumoto, Kouji


    Bacillus subtilis Marburg 168 cells with disrupted ugtP, which encodes UDP-glucosyltransferase involved in glucolipid synthesis, were bent and distended. In the ugtP mutant cells, the extracytoplasmic function sigmas SigM, SigV and SigX, were found to be activated. Introduction of a disrupted allele of sigM into the ugtP strain caused even more abnormal morphology, with cells taking on a balloon-like shape; growth of these cells in LB medium was hampered by addition of 1.5% NaCl. Addition of MgSO4 or MnCl2 suppressed the abnormal morphology. In ugtP mutant cells the transcription of the mreB operon from an upstream promoter in maf (designated Pupstream mreB) and PmreBH was 4.3- and 2.3-fold higher, respectively, and localization of GFP-MreB was not in discrete dots (in an apparently helical pattern), but faint and in irregular clusters. GFP-MreB protein was reduced in the ugtP mutant cells. We suggest that glucolipids are important for MreB isoforms to take on the configuration that appears as discrete dots and plays a role in shaping cells into straight rods. PMID:22362028

  8. The membrane-induced proton motive force influences the metal binding ability of Bacillus subtilis cell walls.

    Urrutia Mera, M; Kemper, M; Doyle, R.; Beveridge, T. J.


    Bacillus subtilis 168 is a gram-positive bacterium whose cell wall contains the highly electronegative polymers peptidoglycan (chemotype A1 gamma) and glycerol-based teichoic acid to produce a surface with a net negative charge with high metal binding capacity. During metabolism, a membrane-induced proton motive force continuously pumps protons into the wall fabric. As a result, a competition between protons and metal ions for anionic wall sites occurs, and less metal is bound in living cells...

  9. Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

    Anderson, Iain; Sorokin, Alexei; Kapatral, Vinayak; Reznik, Gary; Bhattacharya, Anamitra; Mikhailova, Natalia; Burd, Henry; Joukov, Victor; Kaznadzey, Denis; Walunas, Theresa; D'Souza, Mark; Larsen, Niels; Pusch, Gordon; Liolios, Konstantinos; Grechkin, Yuri


    Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1,381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-...

  10. [Sporulation or competence development? A genetic regulatory network model of cell-fate determination in Bacillus subtilis].

    Lu, Zhenghui; Zhou, Yuling; Zhang, Xiaozhou; Zhang, Guimin


    Bacillus subtilis is a generally recognized as safe (GRAS) strain that has been widely used in industries including fodder, food, and biological control. In addition, B. subtilis expression system also plays a significant role in the production of industrial enzymes. However, its application is limited by its low sporulation frequency and transformation efficiency. Immense studies have been done on interpreting the molecular mechanisms of sporulation and competence development, whereas only few of them were focused on improving sporulation frequency and transformation efficiency of B. subtilis by genetic modification. The main challenge is that sporulation and competence development, as the two major developmental events in the stationary phase of B. subtilis, are regulated by the complicated intracellular genetic regulatory systems. In addition, mutual regulatory mechanisms also exist in these two developmental events. With the development of genetic and metabolic engineering, constructing genetic regulatory networks is currently one of the most attractive research fields, together with the genetic information of cell growth, metabolism, and development, to guide the industrial application. In this review, the mechanisms of sporulation and competence development of B. subtilis, their interactions, and the genetic regulation of cell growth were interpreted. In addition, the roles of these regulatory networks in guiding basic and applied research of B. subtilis and its related species were discussed. PMID:26939438

  11. Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant Bacillus anthracis Protective Antigen from B. subtilis

    Thwaite, Joanne E.; Baillie, Les W. J.; Carter, Noel M; Stephenson, Keith; Rees, Mark; Harwood, Colin R.; Emmerson, Peter T.


    The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process general...

  12. Induction of extracytoplasmic function sigma factors in Bacillus subtilis cells with membranes of reduced phosphatidylglycerol content.

    Hashimoto, Michihiro; Takahashi, Hiroaki; Hara, Yoshinori; Hara, Hiroshi; Asai, Kei; Sadaie, Yoshito; Matsumoto, Kouji


    The Bacillus subtilis gene pgsA, which codes for the phosphatidylglycerophosphate synthase that catalyzes the committed step for the synthesis of phosphatidylglycerol (PG), is essential since Pspac-pgsA cells require IPTG for growth. Removal of the inducer caused a dramatic decrease of PG content in the membranes of cells and retarded growth. At 60 min and 120 min after removal, it was reduced to 14.1% and 8.9% of total lipid, respectively, from an initial content of 28.1%. We conjectured that the activity of some extracytoplasmic function (ECF) sigma factors, most of which are caught and regulated directly by cognate transmembrane anti-sigma factors, are affected by altered lipid composition of the membranes. Induction of the activities of ECF sigma factors (sigma(M) and sigma(V)) was observed after removal of IPTG, though that of sigma(V) was small. But other ECF sigma factors (sigma(W), sigma(X), sigma(Y), sigma(YlaC) and sigma(Z)) and the general stress sigmas sigma(B) and sigma(I) were not induced. Especially sigma(M) was activated strongly with the reduction of PG content and sustained a high level of activity, in contrast to the transient activation in PG normal cells after exposure to high salinity. This study demonstrates a new relationship between the alterations of lipid composition in the membranes and the activation of ECF sigma factors. PMID:19745567

  13. Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall.

    Nikola Ojkic


    Full Text Available To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in [Formula: see text] 60 [Formula: see text] of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

  14. Influence of phosphate supply on teichoic acid and teichuronic acid content of Bacillus subtilis cell walls.

    Lang, W K; Glassey, K; Archibald, A R


    Bacillus subtilis 168 was grown in chemostat culture in fully defined media containing a constant concentration of magnesium and concentrations of phosphate that varied from those giving phosphate-limited growth to those in which phosphate was present in excess and magnesium was limiting. Phosphate-limited bacteria were deficient in wall teichoic acid and contained less than half as much cellular phosphate as did bacteria grown in excess of phosphate. Approximately 70% of the additional phosp...

  15. On the effect of N-methyl-bis (3-mesyloxypropyl) amine hydroxychloride on Bacillus subtilis cells.

    Shimi, I R; Shoukry, S


    N-Methyl-bis (3-mesyloxypropyl)amine hydrochloride is now in use as an antitumer drug. In view of its activity against some bacteria the present work was conducted to study its mode of action of Bacillus subtilis. The compound was found to induce irreversible damage to bacterial DNA whereas its effect on RNA was temporary and depending on maintenance of effective concentrations of the compound. PMID:168172

  16. Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

    Heath Murray


    Full Text Available In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

  17. Transformation of Bacillus subtilis by single-stranded plasmid DNA.

    Rudolph, C F; Schmidt, B J; Saunders, C W


    The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.

  18. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

    Marciniak Bogumiła C


    Full Text Available Abstract Background Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 β-lactamase of Escherichia coli, membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus and lipoproteins (MntA and YcdH of B. subtilis. Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes. Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 β-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue specifically under membrane proteins overproduction. Conclusions The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.

  19. The sigma E subunit of Bacillus subtilis RNA polymerase is present in both forespore and mother cell compartments.

    Carlson, H. C.; Haldenwang, W G


    Bacillus subtilis cells harvested 3.5 h after the onset of sporulation (t3.5) were fractionated into extracts enriched in either mother cell or forespore components and were analyzed immunologically for sigma E and its precursor protein, P31. We determined by Western blot (immunoblot) analysis that equivalent amounts of P31 and sigma E were present in both mother cell and forespore extracts. This result implies that, although sigma E is not synthesized until a stage in development when the ce...

  20. Characterization of the involvement of two compensatory autolysins in mother cell lysis during sporulation of Bacillus subtilis 168.

    Smith, T J; Foster, S. J.


    The 30-kDa sporulation-specific peptidoglycan hydrolase CwlC of Bacillus subtilis 168 was purified and characterized. It is an N-acetylmuramoyl-L-alanine amidase (amidase) that is associated with the mother cell wall of sporulating cells, and although it is secreted, it undergoes no N-terminal processing except removal of the initial methionine. It was found that mother cells of a strain insertionally inactivated in cwlC and lytC (the major vegetative amidase gene) did not lyse at the end of ...

  1. Cannibalism stress response in Bacillus subtilis.

    Höfler, Carolin; Heckmann, Judith; Fritsch, Anne; Popp, Philipp; Gebhard, Susanne; Fritz, Georg; Mascher, Thorsten


    When faced with carbon source limitation, the Gram-positive soil organism Bacillus subtilis initiates a survival strategy called sporulation, which leads to the formation of highly resistant endospores that allow B. subtilis to survive even long periods of starvation. In order to avoid commitment to this energy-demanding and irreversible process, B. subtilis employs another strategy called 'cannibalism' to delay sporulation as long as possible. Cannibalism involves the production and secretion of two cannibalism toxins, sporulation delaying protein (SDP) and sporulation killing factor (SKF), which are able to lyse sensitive siblings. The lysed cells are thought to then provide nutrients for the cannibals to slow down or even prevent them from entering sporulation. In this study, we uncovered the role of the cell envelope stress response (CESR), especially the Bce-like antimicrobial peptide detoxification modules, in the cannibalism stress response during the stationary phase. SDP and SKF specifically induce Bce-like systems and some extracytoplasmic function σ factors in stationary-phase cultures, but only the latter provide some degree of protection. A full Bce response is only triggered by mature toxins, and not by toxin precursors. Our study provides insights into the close relationship between stationary-phase survival and the CESR of B. subtilis. PMID:26364265

  2. Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering.

    Juhas, Mario; Reuß, Daniel R; Zhu, Bingyao; Commichau, Fabian M


    Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell 'chassis'. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications. PMID:25092907

  3. The Regularities of Mutagenic Action of gamma-Radiation on Vegetative Bacillus subtilis Cells with Different Repair Genotype

    Boreyko, A V; Krasavin, E A


    The regularities of induction of his^-\\to his^+ mutations in vegetative Bacillus subtilis cells with different repair capacity after gamma-irradiation have been studied. The wild type cells, polA1, recE4, recA, recP, add5, recH were used in experiments. It was shown that radiation-induced mutagenesis is determined by a repair genotype of cells. The blocking of different reparation genes is reflected on mutagenesis ratio by the various ways. A frequency of induction mutations in polA strain is higher than in wild type cells and it is characterized by the linearly-quadratic dose curve. The different rec^- strains that belong to various epistatic groups reveal an unequal mutation induction. The add5 and recP strains are characterized by the high-level induction mutations in contrast with the wild type cells. The mutagenesis in recE and recH strains, on the contrary, sharply reduces. The different influence of rec genes inhering to various epistatic groups on mutagenesis in Bacillus subtilis cells probably reflec...

  4. Triple fixation of Bacillus subtilis dormant spores.

    Kozuka, S; Tochikubo, K


    A triple-fixation method with a sequential application of 5% glutaraldehyde, 1% osmium tetroxide, and 2% potassium permanganate gave superior preservation of the ultrastructure of Bacillus subtilis dormant spores with a thick spore coat.

  5. Bacillus subtilis regulatory protein GerE

    Ducros, V M A; Brannigan, J.A.; Lewis, R J; Wilkinson, A.J.


    GerE is the latest-acting of a series of factors which regulate gene expression in the mother cell during sporulation in Bacillus. The gene encoding GerE has been cloned from B. subtilis and overexpressed in Escherichia coli. Purified GerE has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The small plate-like crystals belong to the monoclinic space group C2 and diffract beyond 2.2 Angstrom resolution with a synchrotron radiation X-ra...

  6. Immobilization of CotA, an extremophilic laccase from Bacillus subtilis, on glassy carbon electrodes for biofuel cell applications

    Beneyton, T.; El Harrak, A.; Griffiths, A.D.; Taly, V. [Institut de Science et d' Ingenierie Supramoleculaire, CNRS UMR, Strasbourg (France); Hellwig, P. [Institut de Chimie, Universite de Strasbourg, CNRS UMR, Strasbourg (France)


    Thanks to their high stability over a wide range of experimental conditions, extremophilic enzymes represent an interesting alternative to mesophilic enzymes as catalysts for biofuel cell applications. In the present work, we report for the first time the immobilization of a thermophilic laccase (CotA from Bacillus subtilis endospore coat) on glassy carbon electrodes functionalized via electrochemical reduction of in situ generated aminophenyl monodiazonium salts. We compare the performance of CotA-modified electrodes for the reduction of O{sub 2} to mutant variants and demonstrate that the measured electrical current is directly correlated to the catalytic efficiencies (k{sub cat}/K{sub m}) of the immobilized enzyme. CotA-modified electrodes showed an optimal operation temperature of 45-50 C and stable catalytic activity for at least 7 weeks. (author)

  7. Bacillus subtilis Spore Inner Membrane Proteome.

    Zheng, Linli; Abhyankar, Wishwas; Ouwerling, Natasja; Dekker, Henk L; van Veen, Henk; van der Wel, Nicole N; Roseboom, Winfried; de Koning, Leo J; Brul, Stanley; de Koster, Chris G


    The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation, these spore formers can survive very harsh physical and chemical conditions. Yet, they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics-based membrane protein predictions, ca. one-third could be predicted to be membrane-localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were identified. In addition to previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology, unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets. PMID:26731423

  8. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.; Deutscher, J.; Jensen, Peter Ruhdal


    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge on...

  9. Complete Genome of Bacillus subtilis Myophage Grass

    Miller, Stanton Y.; Colquhoun, Jennifer M.; Perl, Abbey L.; Chamakura, Karthik R.; Kuty Everett, Gabriel F.


    Bacillus subtilis is a ubiquitous Gram-positive model organism. Here, we describe the complete genome of B. subtilus myophage Grass. Aside from genes encoding core proteins pertinent to the life cycle of the phage, Grass has several interesting features, including an FtsK/SpoIIIE protein.

  10. Characterization of a new cell-bound alpha-amylase in Bacillus subtilis 168 Marburg that is only immunologically related to the exocellular alpha-amylase.

    Haddaoui, E; Petit-Glatron, M F; Chambert, R


    Immunoblot analysis of Bacillus subtilis cell extracts with polyclonal antibodies, raised against purified exocellular alpha-amylase, revealed one protein species of 82,000 Da. This protein was found even in cells in which the amyE gene, encoding exocellular alpha-amylase, was disrupted. Isolated from the membrane fraction, the 82,000-M(r) protein displayed an alpha-amylase activity in vitro.

  11. Insights into the Defense-Related Events Occuring in Plant Cells Following Perception of Surfactin-Type Lipopeptide from Bacillus subtilis

    Jourdan, Emmanuel; Henry, Guillaume; Duby, F.; Dommes, Jacques; Barthelemy, Jean-Paul; Thonart, Philippe; Ongena, Marc


    Multiple strains of Bacillus subtilis were demonstrated to stimulate plant defense responses, and cyclic lipopeptides may be involved in the elicitation of this induced systemic resistance phenomenon. Here, we further investigated molecular events underlying the interaction between wuch lipopeptides and plant cells. Addition of surfactin but not fengycin or iturin in the micromolar range to tobacco cell suspensions induced defense-related early events such as extracellular medium alkalinizati...

  12. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.;


    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge on...... protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  13. Isolation and characterization of protease from Bacillus subtilis 1012M15



    Full Text Available A local strain of Bacillus sp. BAC4, is known to produce penicillin G acylase (PGA enzyme with relatively high activity. This strain secretes the PGA into the culture medium. However, it has been reported that PGA activity fall and rise during culture, and the activity plummets during storege at –200C, which probably due to usage protease activity of Bacillus sp. BAC4. To study the possible use of Bacillus subtilis 1012M15 as a host cell for cloning the pga gene from Bacillus sp. BAC4, the protease activity of Bacillus subtilis 1012M15 were studied. Protease activity was determined by Horikoshi method. In this experiment, maximum protease activity in Bacillus subtilis 1012M15 culture was obsereved after 8 hours. At this optimum condition, protease activity of Bacillus sp. BAC4 is five time higher than that of Bacillus subtilis 1012M15. This situation promised the possible usage of Bacillus subtilis 1012M15 as a host cell for pga expression. For protease characterization, the bacterial culture had been separated from the cell debris by centrifugation. The filtrate was concentrated by freeze drying, fractionated by ammonium sulphate, dialyzed in selovan tube, and then fractionated by ion exchance chromatography employing DEAE-cellulose. The five peaks resulted indicated the presence of five protease. Based on inhibitor and activator influence analysis, it could be concluded that proteases from Bacillus subtilis 1012M15 contained of serin protease as well as metalloprotease and serin protease mixture.

  14. Simple, fast and high‐efficiency transformation system for directed evolution of cellulase in Bacillus subtilis

    Zhang, Xiao‐Zhou; Zhang, Y.‐H. Percival


    Summary Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme‐, phosphatase‐ and ligas...

  15. Changes of lipid domains in Bacillus subtilis cells with disrupted cell wall peptidoglycan

    Muchová, Katarína; Wilkinson, Anthony J.; Barák, Imrich


    The cell wall is responsible for cell integrity and the maintenance of cell shape in bacteria. The Gram-positive bacterial cell wall consists of a thick peptidoglycan layer located on the outside of the cytoplasmic membrane. Bacterial cell membranes, like eukaryotic cell membranes, are known to contain domains of specific lipid and protein composition. Recently, using the membrane-binding fluorescent dye FM4-64, helix-like lipid structures extending along the long axis of the cell and consist...

  16. From cell differentiation to cell collectives : Bacillus subtilis uses division of labor to migrate

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto


    The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In thi

  17. A cell factory of Bacillus subtilis engineered for the simple bioconversion of myo-inositol to scyllo-inositol, a potential therapeutic agent for Alzheimer's disease

    Takenaka Shinji


    Full Text Available Abstract Background A stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimer's disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound is relatively rare in nature, whereas another stereoisomer of inositol, myo-inositol, is abundantly available. Results Bacillus subtilis possesses a unique inositol metabolism involving both stereoisomers. We manipulated the inositol metabolism in B. subtilis to permit the possible bioconversion from myo-inositol to scyllo-inositol. Within 48 h of cultivation, the engineered strain was able to convert almost half of 10 g/L myo-inositol to scyllo-inositol that accumulated in the culture medium. Conclusions The engineered B. subtilis serves as a prototype of cell factory enabling a novel and inexpensive supply of scyllo-inositol.

  18. Free and attached cells of Bacillus subtilis as starters for production of a soup flavouring (“ogiri egusi”

    Peter-Ikechukwu, A. I.


    Full Text Available Aims: This Bacillus subtilis has been identified to be the main fermenting bacterium during indigenous production of “ogiri egusi”; a traditional soup flavouring rich in protein. Evaluation of the use of starter and broth cultures of this bacterium in the production of ‘ogiri egusi’ was therefore undertaken with the view to improve the fermentation process and quality of product. Methodology and Results: Cowpea granules in association with Bacillus subtilis cells were developed as starter cultures for the fermentation. Results obtained showed that the starter cultures resulted in an increase in the aminonitrogen from 1.67±0.02 to 19.96±0.05 mg N/100 g dry matter in 48 h while the broth cultures increased the aminonitrogen from 1.63±0.03 to 16.54±0.05 mg N/100 g dry matter in 72 h. There was also a corresponding increase in the protease activity of the fermentation conducted with the starter cultures from 2.69±0.03 to 54.98±0.04 mg N/min in 48 h. The broth cultures produced an increase from 2.65±0.02 to 47.61±0.06 mg N/min in 72 h. Changes in these parameters for the natural process were gradual and reached their peaks at 120 h with values of 9.89±0.13 mg N/100g dry matter and 31.92±0.03 mg N/min respectively. Peroxide values for the fermentation processes increased throughout the period; however the starter cultures produced the lowest value (10.20±0.10 meq/kg showing that rancidity may not occur in the product fermented by the starter culture. Conclusion, significance and impact of study: The starter cultures significantly reduced fermentation time from 96 – 120 h in the natural process to 48 h. Thus use of starter cultures optimized the process of fermentation and will eliminate chances of contamination of product with pathogens and spoilage organisms. This ultimately will improve product quality.

  19. Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

    Serrano, Mónica; Gao, JinXin; Bota, João; Bate, Ashley R; Meisner, Jeffrey; Eichenberger, Patrick; Moran, Charles P; Henriques, Adriano O


    Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue. PMID:25835496

  20. Comparative genome analysis of Bacillus cereus group genomes withBacillus subtilis

    Anderson, Iain; Sorokin, Alexei; Kapatral, Vinayak; Reznik, Gary; Bhattacharya, Anamitra; Mikhailova, Natalia; Burd, Henry; Joukov, Victor; Kaznadzey, Denis; Walunas, Theresa; D' Souza, Mark; Larsen, Niels; Pusch,Gordon; Liolios, Konstantinos; Grechkin, Yuri; Lapidus, Alla; Goltsman,Eugene; Chu, Lien; Fonstein, Michael; Ehrlich, S. Dusko; Overbeek, Ross; Kyrpides, Nikos; Ivanova, Natalia


    Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1,381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.

  1. Cytoplasmic pH Response to Acid Stress in Individual Cells of Escherichia coli and Bacillus subtilis Observed by Fluorescence Ratio Imaging Microscopy

    Martinez, Keith A.; Ryan D Kitko; Mershon, J. Patrick; Adcox, Haley E.; Malek, Kotiba A.; Berkmen, Melanie B.; Slonczewski, Joan L.


    The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switch...


    Supartono Supartono


    Full Text Available PRODUCTION OF ANTIBIOTICS BY Bacillus subtilis M10 IN UREA-SORBITOL MEDIUM. Infection diseases still become the main health problems that suffered by people in Indonesia. Besides, there were many pathogen bacteria found to be resistant to the some antibiotics. Therefore, the efforts to get a new antibiotic require to be done continuously. A new local strain of Bacillus subtilis BAC4 has been known producing an antibiotic that inhibit Serratia marcescens ATCC 27117 growth. To make efficient the local strain, mutation on Bacillus subtilis BAC4 was done by using acridine orange and a mutant cell of Bacillus subtilis M10 that overproduction for producing antibiotic was obtained. Nevertheless, the production kinetics of antibiotic by this mutant has not been reported. The objective of this research was to study the production kinetics of antibiotic by Bacillus subtilis M10 mutant. The production of antibiotic was conducted using batch fermentation and antibiotic assay was performed with agar absorption method using Serratia marcescens ATCC 27117 as bacteria assay. Research result provided that Bacillus subtilis M10 mutant with overproduction of antibiotic produced an antibiotic since 8th hour’s fermentation and optimum of it production was at 14th hours after inoculation.  Penyakit infeksi masih menjadi masalah yang utama diderita oleh masyarakat Indonesia. Di samping itu, banyak bakteri patogen yang ditemukan resisten terhadap beberapa antibiotika. Oleh karena itu, upaya-upaya untuk mendapatkan antibiotika baru perlu dilakukan secara terus-menerus. Suatu galur lokal baru Bacillus subtilis BAC4 teridentifikasi memproduksi senyawa antibiotika yang menghambat pertumbuhan Serratia marcescens ATCC27117. Untuk memberdayakan galur tersebut, terhadap Bacillus subtilis BAC4 dilakukan mutasi dengan larutan akridin oranye dan diperoleh mutan Bacillus subtilis M10 yang memproduksi antibiotika berlebihan. Namun, kinetika produksi antibiotika oleh Bacillus

  3. Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

    Wood, Joseph P.; Meyer, Kathryn M.; Kelly, Thomas J.; Choi, Young W.; Rogers, James V.; Riggs, Karen B.; Willenberg, Zachary J.


    There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log10 reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially. PMID:26372011

  4. Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

    Perkins, J B; Dean, D H


    DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.

  5. Comparison of different Bacillus subtilis expression systems.

    Vavrová, Ludmila; Muchová, Katarína; Barák, Imrich


    Bacillus subtilis is considered to have great potential as a host for the production and secretion of recombinant proteins. Many different expression systems have been developed for B. subtilis. Here we compare two widely used expression systems, the IPTG-inducible derivative of spac system (hyper-spank) and the xylose-inducible (xyl) to the SURE (subtilin-regulated gene expression) system. Western blot analysis of the membrane protein SpoIISA together with its protein partner SpoIISB showed that the highest expression level of this complex is obtained using the SURE system. Measurement of β-galactosidase activities of the promoter-lacZ fusions in individual expression systems confirmed that the P(spaS) promoter of the SURE system is the strongest of those compared, although the induction/repression ratio reached only 1.84. Based on these results, we conclude that the SURE system is the most efficient of these three B. subtilis expression systems in terms of the amount of expressed product. Remarkably, the yield of the SpoIISA-SpoIISB complex obtained from B. subtilis was comparable to that normally obtained from the Escherichia coli arabinose-inducible expression system. PMID:20863884

  6. Enhanced hydrocarbon biodegradation by a newly isolated bacillus subtilis strain

    The relation between hydrocarbon degradation and biosurfactant (rhamnolipid) production by a new bacillus subtilis 22BN strain was investigated. The strain was isolated for its capacity to utilize n-hexadecane and naphthalene and at the same time to produce surface-active compound at high concentrations (1.5 - 2.0 g l-1). Biosurfactant production was detected by surface tension lowering and emulsifying activity. The strain is a good degrader of both hydrocarbons used with degradability of 98.3 ± 1% and 75 ± 2% for n-hexadecane and naphthalene, respectively. Measurement of cell hydrophobicity showed that the combination of slightly soluble substrate and rhamnolipid developed higher hydrophobicity correlated with increased utilization of both hydrocarbon substrates. To our knowledge, this is the first report of bacillus subtilis strain that degrades hydrophobic compounds and at the same time produces rhamnolipid biosurfactant. (orig.)

  7. A non-invasive method for studying viral DNA delivery to bacteria reveals key requirements for phage SPP1 DNA entry in Bacillus subtilis cells.

    Fernandes, Sofia; Labarde, Audrey; Baptista, Catarina; Jakutytè, Lina; Tavares, Paulo; São-José, Carlos


    Bacteriophages use most frequently a tail apparatus to create a channel across the entire bacterial cell envelope to transfer the viral genome to the host cell cytoplasm, initiating infection. Characterization of this critical step remains a major challenge due to the difficulty to monitor DNA entry in the bacterium and its requirements. In this work we developed a new method to study phage DNA entry that has the potential to be extended to many tailed phages. Its application to study genome delivery of bacteriophage SPP1 into Bacillus subtilis disclosed a key role of the host cell membrane potential in the DNA entry process. An energized B. subtilis membrane and a millimolar concentration of calcium ions are shown to be major requirements for SPP1 DNA entry following the irreversible binding of phage particles to the receptor YueB. PMID:27179995

  8. Hyaluronic Acid Production in Bacillus subtilis

    Widner, Bill; Behr, Régine; Von Dollen, Steve; Tang, Maria; Heu, Tia; Sloma, Alan; Sternberg, Dave; DeAngelis, Paul L; Paul H. Weigel; Brown, Steve


    The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in...

  9. Bacillus subtilis pur operon expression and regulation.

    Ebbole, D J; Zalkin, H


    The Bacillus subtilis pur operon is a 12-gene cluster, purEKB-purC(orf)QLF-purMNH(J)-purD, organized in groups of overlapping coding units separated by intercistronic gaps. Translational fusions of Escherichia coli lacZ were constructed to purE, purC, and purM, the first gene of each group. Analyses of gene fusions integrated into the chromosomal pur operon exclude the possibility of internal promoters in intercistronic regions and support the view that transcription is from the single sigma ...

  10. Investigation of biosurfactant production by Bacillus pumilus 1529 and Bacillus subtilis WPI

    shila khajavi shojaei


    Full Text Available Introduction: Biosurfactants are unique amphipathic molecules with extensive application in removing organic and metal contaminants. The purpose of this study was to investigate production of biosurfactant and determine optimal conditions to produce biosurfactant by Bacillus pumilus 1529 and Bacillus subtilis WPI. Materials and methods: In this study, effect of carbon source, temperature and incubation time on biosurfactant production was evaluated. Hemolytic activity, emulsification activity, oil spreading, drop collapse, cell hydrophobicity and measurement of surface tension were used to detect biosurfactant production. Then, according to the results, the optimal conditions for biosurfactant production by and Bacillus subtilis WPI was determined. Results: In this study, both bacteria were able to produce biosurfactant at an acceptable level. Glucose, kerosene, sugarcane molasses and phenanthrene used as a sole carbon source and energy for the mentioned bacteria. Bacillus subtilis WPI produced maximum biosurfactant in the medium containing kerosene and reduced surface tension of the medium to 33.1 mN/m after 156 hours of the cultivation at 37°C. Also, the highest surface tension reduction by Bacillus pumilus 1529 occurred in the medium containing sugarcane molasses and reduce the surface tension of culture medium after 156 hours at 37°C from 50.4 to 28.83 mN/m. Discussion and conclusion: Bacillus pumilus 1529 and Bacillus subtilis WPI had high potential in production of biosurfactant and degradation of petroleum hydrocarbons and Phenanthrene. Therefore, it could be said that these bacteria had a great potential for applications in bioremediation and other environmental process.

  11. Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis

    van Dijl, J M; de Jong, A; Smith, H; Bron, S; Venema, G


    The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half t

  12. Fast Neutron Radiation Effects on Bacillus Subtili

    CHEN Xiaoming; REN Zhenglong; ZHANG Jianguo; ZHENG Chun; TAN Bisheng; YANG Chengde; CHU Shijin


    To examine the sterilizing effect and mechanism of neutron radiation, Bacillus sub-tilis vat. niger, strain (ATCC 9372) spores were irradiated with the fast neutron from the Chinese fast burst reactor Ⅱ(CFBR-Ⅱ). The plate-count results indicated that the D10 value was 384.6 Gy with a neutron radiation dose rate of 7.4 Gy/min. The rudimental catalase activity of the spores declined obviously with the increase in the radiation dose. Meanwhile, under the scanning electron microscope, no visible influence of the neutron radiation on the spore configuration was detected even if the dose was increased to 4 kGy. The content and distribution of DNA double-strand breaks induced by neutron radiation at different doses were measured and quantified by pulsed-field gel electrophoresis (PFGE). Further analysis of the DNA release percentage (PR), the DNA breakage level (L), and the average molecular weight, indicated that DNA fragments were obvi-ously distributed around the 5 kb regions at different radiation doses, which suggests that some points in the DNA molecule were sensitive to neutron radiation. Both PR and L varied regularly to some extent with the increase in radiation dose. Thus neutron radiation has a high sterilization power, and can induce falling enzyme activity and DNA breakage in Bacillus subtilis spores

  13. Enhancement of natural killer cell activity by supplementation of extract of metabolic products of Bacillus subtilis AK%纳豆菌(Bacillus subtilis)提取物提高NK细胞活性

    韩德权; 曾伟民; 田中道子


    研究了从纳豆菌(Bacillus subtilis)中筛选得到的AK株的代谢产物经口服后对NK细胞活性的影响.其结果为:白鼠服用群的50% NK细胞活性增强;用于人群试验的合成品AKH通过人群7天服用后,被试者中末梢血中NK细胞活性增强者数量增加.纳豆菌提取物可作为保健食品.

  14. Bacillus subtilis as potential producer for polyhydroxyalkanoates

    Patel Sanjay KS


    Full Text Available Abstract Polyhydroxyalkanoates (PHAs are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB, the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.

  15. Pseudosecretion of Escherichia coli chloramphenicol acetyltransferase by Bacillus subtilis.

    Le Grice, S F; Gentz, R; Bannwarth, W; Kocher, H. P.


    Bacillus subtilis harboring the vector 25RBSII secrets an Escherichia coli-derived chloramphenicol acetyltransferase into culture supernatants. The secreted enzyme lacks 18 amino acids; these are removed externally rather than during secretion.

  16. Two Genes Encoding Uracil Phosphoribosyltransferase Are Present in Bacillus subtilis

    Martinussen, Jan; Glaser, Philippe; Andersen, Paal S.;


    Uracil phosphoribosyltransferase (UPRTase) catalyzes the key reaction in the salvage of uracil in many microorganisms. Surprisingly, two genes encoding UPRTase activity were cloned from Bacillus subtilis by complementation of an Escherichia coli mutant. The genes were sequenced, and the putative...

  17. Selection of Bacillus subtilis mutants impaired in ammonia assimilation.

    Dean, D R; Aronson, A I


    The selection of Bacillus subtilis mutants capable of using D-histidine to fulfill a requirement for L-histidine resulted in mutants with either no glutamate synthase activity or increased amounts of an altered glutamine synthetase.

  18. Proteins that interact with GTP during sporulation of Bacillus subtilis

    During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link [α-32P]GTP to proteins in cell extracts at different stages of growth. After electrophoresis, 11 bands of radioactivity were found in vegetative cells, 4 more appeared during sporulation, and only 9 remained in mature spores. Based on the labeling pattern with or without UV light to cross-link either [α-32P]GTP or [γ-32P]GTP, 11 bands of radioactivity were apparent guanine nucleotide-binding proteins, and 5 bands appeared to be phosphorylated and/or guanylated. Similar results were found with Bacillus megaterium. Assuming the GTP might be a type of signal for sporulation, it could interact with and regulate proteins by at least three mechanisms

  19. Water surface tension modulates the swarming mechanics of Bacillus subtilis

    Ke, Wan-Ju; Hsueh, Yi-Huang; Cheng, Yu-Chieh; Wu, Chih-Ching; Liu, Shih-Tung


    Many Bacillus subtilis strains swarm, often forming colonies with tendrils on agar medium. It is known that B. subtilis swarming requires flagella and a biosurfactant, surfactin. In this study, we find that water surface tension plays a role in swarming dynamics. B. subtilis colonies were found to contain water, and when a low amount of surfactin is produced, the water surface tension of the colony restricts expansion, causing bacterial density to rise. The increased density induces a quorum ...

  20. Expression of UGA-Containing Mycoplasma Genes in Bacillus subtilis

    Kannan, T. R.; Baseman, Joel B.


    We used Bacillus subtilis to express UGA-containing Mycoplasma genes encoding the P30 adhesin (one UGA) of Mycoplasma pneumoniae and methionine sulfoxide reductase (two UGAs) of Mycoplasma genitalium. Due to natural UGA suppression, these Mycoplasma genes were expressed as full-length protein products, but at relatively low efficiency, in recombinant wild-type Bacillus. The B. subtilis-expressed Mycoplasma proteins appeared as single bands and not as multiple bands compared to expression in r...

  1. Uracil incorporation in the forespore and the mother cell during spore development in Bacillus subtilis

    The transcriptional activity of the two genomes of the sporangium during spore formation was determined by pulse-labeling bacteria with 3H-uracil at different times of sporulation and preparing them for high resolution autoradiography. The quantitative analysis of autoradiographs shows that uracile incorporation in the whole sporangium decreases considerably between stages II and IV. However, the variations of the transpcriptional activity are not identical in the mother cell and in the forespore. The one of the mother cell decreases rapidly between stages II and III and then remains stable until the end of stage IV, whereas that of the forespore which is low at stage II increases as the forespore grows ovoid and then quickly diminishes. It is very weak at the beginning of stage IV and negligible at the end of this stage. (orig.)

  2. Draft Genome Sequence of Bacillus subtilis strain KATMIRA1933

    Karlyshev, Andrey V.; Melnikov, Vyacheslav G.; Chikindas, Michael L.


    In this report, we present a draft sequence of Bacillus subtilis KATMIRA1933. Previous studies demonstrated probiotic properties of this strain partially attributed to production of an antibacterial compound, subtilosin. Comparative analysis of this strain’s genome with that of a commercial probiotic strain, B. subtilis Natto, is presented.

  3. Peptidoglycan metabolism is controlled by the WalRK (YycFG) and PhoPR two-component systems in phosphate-limited Bacillus subtilis cells.

    Bisicchia, Paola; Lioliou, Efthimia; Noone, David; Salzberg, Letal I; Botella, Eric; Hübner, Sebastian; Devine, Kevin M


    In Bacillus subtilis, the WalRK (YycFG) two-component system controls peptidoglycan metabolism in exponentially growing cells while PhoPR controls the response to phosphate limitation. Here we examine the roles of WalRK and PhoPR in peptidoglycan metabolism in phosphate-limited cells. We show that B. subtilis cells remain viable in a phosphate-limited state for an extended period and resume growth rapidly upon phosphate addition, even in the absence of a PhoPR-mediated response. Peptidoglycan synthesis occurs in phosphate-limited wild-type cells at approximately 27% the rate of exponentially growing cells, and at approximately 18% the rate of exponentially growing cells in the absence of PhoPR. In phosphate-limited cells, the WalRK regulon genes yocH, cwlO(yvcE), lytE and ydjM are expressed in a manner that is dependent on the WalR recognition sequence and deleting these genes individually reduces the rate of peptidoglycan synthesis. We show that ydjM expression can be activated by PhoP approximately P in vitro and that PhoP occupies its promoter in phosphate-limited cells. However, iseA(yoeB) expression cannot be repressed by PhoP approximately P in vitro, but can be repressed by non-phosphorylated WalR in vitro. Therefore, we conclude that peptidoglycan metabolism is controlled by both WalRK and PhoPR in phosphate-limited B. subtilis cells. PMID:20487291

  4. Biocontrol of Rhizoctonia solani Damping-Off of Tomato with Bacillus subtilis RB14

    Asaka, O.; Shoda, M


    Bacillus subtilis RB14, which showed antibiotic activities against several phytopathogens in vitro by producing the antibiotics iturin A and surfactin, was subjected to a pot test to investigate its ability to suppress damping-off of tomato seedlings caused by Rhizoctonia solani. To facilitate recovery from soil, B. subtilis RB14-C, a spontaneous streptomycin-resistant mutant of RB14, was used. Damping-off was suppressed when the culture broth, cell suspension, or cell-free culture broth of R...

  5. Live cell imaging of germination and outgrowth of individual bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker.

    Rachna Pandey

    Full Text Available Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program "SporeTracker" allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less and fewer grew out (48.4% less after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased and the distribution and average of the duration of germination itself (increased. However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.

  6. Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

    Ruud G J Detert Oude Weme

    Full Text Available Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET. Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM. For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

  7. Inactivation of Bacillus Subtilis by Atomic Oxygen Radical Anion

    LI Longchun; WANG Lian; YU Zhou; LV Xuanzhong; LI Quanxin


    UAtomic oxygen radical anion (O- ) is one of the most active oxygen species, and has extremely high oxidation ability toward small-molecules of hydrocarbons. However, to our knowledge, little is known about the effects of O- on cells of micro-organisms. This work showed that O- could quickly react with the Bacillus subtilis cells and seriously damage the cell walls a s well as their other contents, leading to a fast and irreversible inactivation. SEM micrographs revealed that the cell structures were dramatically destroyed by their exposure to O-. The inactivation efficiencies of B. subtilis depend on the O-- intensity, the initial population of cells and the treatment temperature, but not on the pH in the range of our investigation. For a cell concentration of 106 cfu/ml, the number of survived cells dropped from 106 cfu/ml to 103 cfu/ml after about five-minute irradiation by an O- flux in an intensity of 233 nA/cm2 under a dry argon environment (30 ℃, 1 atm, exposed size: 1.8 cm2). The inactivation mechanism of micro-organisms induced by O- is also discussed.

  8. Genetic transformation of Bacillus strains close to bacillus subtilis and isolated from the soil

    Chromosomal and plasmid transformation was found in five out of 118 Bacillus strains, close or identical to Bacillus subtilis, and isolated from soil in Moscow or in the Moscow district. The efficiency of transformation in these strains was lower than that in derivatives of Bac. subtilis strain 168. In these strains the ability to undergo transformation was dependent on the rate of sporulation and the presence of restrictases. As in the case of Bac. subtilis 168 the strains isolated may be used as models in genetic transformation studies on Bac. subtilis

  9. Amino acid efflux in response to chemotactic and osmotic signals in Bacillus subtilis.

    Wong, L S; Johnson, M. S.; Sandberg, L. B.; Taylor, B L


    We observed a large efflux of nonvolatile radioactivity from Bacillus subtilis in response to the addition of 31 mM butyrate or the withdrawal of 0.1 M aspartate in a flow assay. The major nonvolatile components effluxed were methionine, proline, histidine, and lysine. In studies of the release of volatile radioactivity in chemotaxis by B. subtilis cells that had been labeled with [3H]methionine, the breakdown of methionine to methanethiol can contribute substantially to the volatile radioact...

  10. Effect of Riboflavin Operon Dosage on Riboflavin Productivity in Bacillus Subtilis

    CHEN Tao; CHEN Xun; WANG Jingyu; ZHAO Xueming


    After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7-8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.

  11. 40 CFR 180.1128 - Bacillus subtilis MBI 600; exemption from the requirement of a tolerance.


    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis MBI 600; exemption... FOOD Exemptions From Tolerances § 180.1128 Bacillus subtilis MBI 600; exemption from the requirement of... biofungicide Bacillus subtilis MBI 600 in or on all food commodities, including residues resulting from...

  12. Fitness Trade-Offs in Competence Differentiation of Bacillus subtilis

    Yüksel, Melih; Power, Jeffrey J.; Ribbe, Jan; Volkmann, Thorsten; Maier, Berenike


    In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state). The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relative fitness decreased with increasing differentiation probability both during the stationary phase and during outgrowth. When exposed to antibiotics inhibiting cell wall synthesis, transcription, and translation, cells that differentiated into the K-state showed a selective advantage compared to differentiation-deficient bacteria; this benefit did not require transformation. Although beneficial, the K-state was not induced by sub-MIC concentrations of antibiotics. Increasing the differentiation probability beyond the wt level did not significantly affect the competition dynamics with transient antibiotic exposure. We conclude that the competition dynamics are very sensitive to the fraction of competent cells under benign conditions but less sensitive during antibiotic exposure, supporting the picture of stochastic differentiation as a fitness trade-off. PMID:27375604

  13. Fitness Trade-Offs in Competence Differentiation of Bacillus subtilis.

    Yüksel, Melih; Power, Jeffrey J; Ribbe, Jan; Volkmann, Thorsten; Maier, Berenike


    In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state). The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relative fitness decreased with increasing differentiation probability both during the stationary phase and during outgrowth. When exposed to antibiotics inhibiting cell wall synthesis, transcription, and translation, cells that differentiated into the K-state showed a selective advantage compared to differentiation-deficient bacteria; this benefit did not require transformation. Although beneficial, the K-state was not induced by sub-MIC concentrations of antibiotics. Increasing the differentiation probability beyond the wt level did not significantly affect the competition dynamics with transient antibiotic exposure. We conclude that the competition dynamics are very sensitive to the fraction of competent cells under benign conditions but less sensitive during antibiotic exposure, supporting the picture of stochastic differentiation as a fitness trade-off. PMID:27375604

  14. Fitness trade-offs in competence differentiation of Bacillus subtilis

    Melih Yüksel


    Full Text Available In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state. The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relative fitness decreased with increasing differentiation probability both during the stationary phase and during outgrowth. When exposed to antibiotics inhibiting cell wall synthesis, transcription, and translation, cells that differentiated into the K-state showed a selective advantage compared to differentiation-deficient bacteria; this benefit did not require transformation. Although beneficial, the K-state was not induced by sub-MIC concentrations of antibiotics. Increasing the differentiation probability beyond the wt level did not significantly affect the competition dynamics with transient antibiotic exposure. We conclude that the competition dynamics are very sensitive to the fraction of competent cells under benign conditions but less sensitive during antibiotic exposure, supporting the picture of stochastic differentiation as a fitness trade-off.

  15. Characterization of an L-arabinose isomerase from Bacillus subtilis

    Kim, Jin-Ha; Prabhu, Ponnandy; Jeya, Marimuthu;


    An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypep......An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding...

  16. Enhancement of Cellulase Production by Cellulomonas Fimi and Bacillus Subtilis

    Two bacterial strains identified as Cellulomonas fimi and Baciliius subtilus are cosidered as highly active cellulytic bacteria. Trials for maximizing the cellulolytic activites of the two strains were conducted. A maximum cellulase production was achieved at 1 and 1.5%carboxy methyl cellulose as carbon source, sodium nitrate and yeast as nitrogen source for Cellulomonas fimi and Bacillus subtilis, respectively. Incubation temprature at 30 and 45 degree C, ph at 6 and 7 achieved the highest activity of cellulase for Cellulomonas fimi and bacillus subtilis, respectively

  17. N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

    Kuhn, H; Fietzek, P P; Lampen, J. O.


    The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.

  18. Menaquinone and iron are essential for complex colony development in Bacillus subtilis.

    Gidi Pelchovich

    Full Text Available Cells of undomesticated species of Bacillus subtilis frequently form complex colonies during spreading on agar surfaces. Given that menaquinone is involved in another form of coordinated behavior, namely, sporulation, we looked for a possible role for menaquinone in complex colony development (CCD in the B. subtilis strain NCIB 3610. Here we show that inhibition of menaquinone biosynthesis in B. subtilis indeed abolished its ability to develop complex colonies. Additionally some mutations of B. subtilis which confer defective CCD could be suppressed by menaquinone derivatives. Several such mutants mapped to the dhb operon encoding the genes responsible for the biosynthesis of the iron siderophore, bacillibactin. Our results demonstrate that both menaquinone and iron are essential for CCD in B. subtilis.

  19. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    Rubin, Edward M.


    Bacillus subtilis carries two genes, thyA and thyB, each encoding different protein products, with thymidylate synthetase (TSase) activity. Either of these genes alone is sufficient for thymidine independence in B. subtilis. In addition there exist two B. subtilis temperate bacteriophages which upon infection of thymine requiring auxotrophs results in conversion of the organism to thymine independence. Chimeric plasmids selected for Thy/sup +/ transforming activity in E. coli were constructed and then used as a source of defined highly enriched DNA with which to transform competent B. subtilis. These plasmids were studied for their: (1) abiility to transform B. subtilis to thymine independence; (2) site of integration within the B. subtilis chromosome upon transformation; (3) phenotype of Thy/sup +/ plasmid generated transformants; and (4) nucleotide sequence homology among the cloned DNA fragments conferring thymine independence. Plasmids containing the two bacteriophage thy genes displayed the phenotype associated with thyA, whereas the plasmids containing the cloned B. subtilis chromosomal genes displayed the phenotype associated with thyB. Utilizing similar technology, the ability of an entirely foreign hybred bacterial plasmiid to transform B. subtilis was examined. In this case the gene from E. coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid was effective in transforming both E. coli and B. subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine requiring strains of B. subtilis to thymine independence. Although the Thy/sup +/ transformants of E. coli contained plasmid DNA, the Thy/sup +/ transformants derived from the transformation of B. subtilis did not contain detectable extrachromosomal DNA. Instead the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis. (ERB)

  20. Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism

    Buescher, Joerg Martin; Liebermeister, Wolfram; Jules, Matthieu;


    known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control......Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and...... model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the...

  1. Protective effects of SOD on Bacillus subtilis irradiated by γ-rays

    In order to investigate the protective effect of SOD, Bacillus subtilis vegetative cells treated with SOD of different concentrations were irradiated to different doses by 60Co γ-rays. Cell survival rate was examined with the standard plate-count method. Intercellular SOD activity was measured by SOD kit with xanthine oxidase method, and DNA double-strand breaks was analyzed by pulsed-field gel electrophoresis (PFGE). The results showed that the cell survival rate increased obviously with the SOD-added Bacillus subtilis, though we did not find a clear relation among the intercellular SOD activity, concentration of added SOD and irradiation dose. The DNA release percentage(PR) value decreased evidently with the SOD-treated Bacillus subtilis. Basically,the trend of cell survival rate with concentration of the added SOD and irradiation dose is consistent with the PR value. It can be concluded that exogenous SOD could protect Bacillus subtilis vegetative cell from damage induced by γ-rays, and the protective efficiency of added SOD is related to the concentration. (authors)

  2. Sigma A recognition sites in the Bacillus subtilis genome

    Jarmer, Hanne Østergaard; Larsen, Thomas Schou; Krogh, Anders Stærmose;


    A hidden Markov model of sigma (A) RNA polymerase cofactor recognition sites in Bacillus subtilis, containing either the common or the extended -10 motifs, has been constructed based on experimentally verified sigma (A) recognition sites. This work suggests that more information exists at the ini...

  3. Molecular physiology of weak organic acid stress in Bacillus subtilis

    Brul, S.; Beilen, van, J.W.A.


    The mechanism by which weak organic acid (WOA) preservatives inhibit growth of microorganisms may differ between different WOAs and these differences are not well understood. The aim of this thesis has been to obtain a better understanding of the mode of action of these preservatives by which they inhibit the growth of spore-forming bacteria (more specifically Bacillus subtilis).

  4. A New Saponin Transformed from Ginsenoside Rhl by Bacillus subtilis

    Guo Hong LI; Yue Mao SHEN; Ke Qin ZHANG


    A novel saponin was isolated from the transformed products of ginsenoside Rh1 by Bacillus subtilis. It's structure was determined to be 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20 (S)-protopanaxatriol on the basis of the spectral data.

  5. The transcriptionally active regions in the genome of Bacillus subtilis

    Rasmussen, Simon; Nielsen, Henrik Bjørn; Jarmer, Hanne Østergaard


    The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome...

  6. Sigma A recognition sites in the Bacillus subtilis genome

    Jarmer, Hanne Østergaard; Larsen, Thomas Schou; Krogh, Anders Stærmose; Saxild, Hans Henrik; Brunak, Søren; Knudsen, Steen


    A hidden Markov model of sigma (A) RNA polymerase cofactor recognition sites in Bacillus subtilis, containing either the common or the extended -10 motifs, has been constructed based on experimentally verified sigma (A) recognition sites. This work suggests that more information exists at the...

  7. Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution

    Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium

  8. Post-translational control of vegetative cell separation enzymes through a direct interaction with specific inhibitor IseA in Bacillus subtilis.

    Yamamoto, Hiroki; Hashimoto, Masayuki; Higashitsuji, Yuhei; Harada, Hiroyuki; Hariyama, Nozomi; Takahashi, Lisa; Iwashita, Tomoaki; Ooiwa, Seika; Sekiguchi, Junichi


    Three D,L-endopeptidases, LytE, LytF and CwlS, are involved in the vegetative cell separation in Bacillus subtilis. A novel cell surface protein, IseA, inhibits the cell wall lytic activities of these d,l-endopeptidases in vitro, and IseA negatively regulates the cell separation enzymes at the post-translational level. Immunofluorescence microscopy indicated that the IseA-3xFLAG fusion protein was specifically localized at cell separation sites and poles on the vegetative cell surface in a similar manner of the d,l-endopeptidases. Furthermore, pull-down assay showed that IseA binds to the catalytic domain of LytF, indicating that IseA is localized on the cell surface through the catalytic domain of LytF. Overexpression of IseA caused a long-chained cell morphology in the exponential growth phase, indicating that IseA inhibits the cell separation D,L-endopeptidases in vivo. Besides, overexpression of IseA in a cwlO disruptant affected cell growth, implying that IseA is also involved in the cell elongation event. However, although IseA inhibits the activities of LytE, LytF, CwlS and CwlO in vitro, it is unlikely to inhibit CwlS and CwlO in vivo. This is the first demonstration that the cell separation event is post-translationally controlled through a direct interaction between cell separation enzymes and a specific novel inhibitor in bacteria. PMID:18761694

  9. Influence of physical, chemical and inducer treatments on menaquinone-7 biosynthesis by Bacillus subtilis MTCC 2756

    Alka Puri


    Full Text Available Effects of physical and chemical treatment on nutrient mobility, their utilization for menaquinone-7 (MK-7 biosynthesis; growth of microbial cells has been investigated in the present research. Bacillus subtilis MTCC 2756 fermented medium was supplied with 1-naphthol and hypoxanthine resulted in a significant increase in MK-7 production. Ultrasonication, electric shock, heat shock, and tween 80 were used for inducer uptake by Bacillus subtilis and menaquinone-7 production. Induction of Bacillus subtilis (at 16 hours of fermentation using 1-naphthol (2 mg/ml, along with tween 80 (0.1% was found to increase the MK-7 production by 3 fold i.e. 14.4 µg/ml as compared to the untreated fermentation medium. The ultrasonicated (ultrasonic power 33 W, treatment time 4 min and frequency 36 KHz microbial cells yielded higher biomass and 2.5 fold increase in the MK-7 production i.e.10.3 µg/ml than control. 1-naphthol along with physical or chemical treatment is required for maximum MK-7 production by Bacillus subtilis.

  10. Phylogeny and Molecular Taxonomy of the Bacillus subtilis species Complex and the Description of Bacillus subtilis subsp. inaquosorum subsp. nov

    The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S ribosomal RNA gen...

  11. Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase

    Jensen, Kaj Frank


    Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared to be a...

  12. Study of levan productivity from Bacillus subtilis Natto by surface response methodology and its antitumor activity against HepG2 cells using metabolomic approach.

    Cabral de Melo, Fernando Cesar Bazani; Borsato, Dionísio; de Macedo Júnior, Fernando César; Mantovani, Mario Sérgio; Luiz, Rodrigo Cabral; Colabone-Celligoi, Maria Antonia-Pedrine


    Levan productivity of Bacillus subtilis Natto was evaluated in submerged culture varying the pH, temperature and culture time, using factorial design and response surface methodology. The characterization of levan molecular weight was performed by HPSEC and its antitumor activity against HepG2 cells using metabolomic approach was also evaluated. At first, the variables investigated, as well as their interactions, demonstrated significant effect. Further, a second design using the same variables at different levels was developed. Thus, according to the model, an optimized value corresponding to 5.82 g.L⁻¹.h⁻¹ was achieved at pH 8, 39.5°C in 21 hours, the highest value reported so far. After analysis by HPSEC, two molecular weights were obtained corresponding to 72.37 and 4146 kDa. The levan promoted an increase of acetate, alanine, lactate and phosphocreatine in HepG2 cells suggesting an alteration in the bioenergetics pathways and cellular homeostasis by intracellular accumulation of lactate, justifying its antitumor activity. PMID:26639487

  13. Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

    Neyfakh, A A; Bidnenko, V E; L. B. CHEN


    Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycop...

  14. A microbial transformation using Bacillus subtilis B7-S to produce natural vanillin from ferulic acid.

    Chen, Peng; Yan, Lei; Wu, Zhengrong; Li, Suyue; Bai, Zhongtian; Yan, Xiaojuan; Wang, Ningbo; Liang, Ning; Li, Hongyu


    Bacillus subtilis strain B7-S screened from18 strains is an aerobic, endospore-forming, model organism of Gram-positive bacteria which is capable to form vanillin during ferulic acid bioconversion. The bioconversion of ferulic acid to vanillin by Bacillus subtilis B7-S (B. subtilis B7-S) was investigated. Based on our results, the optimum bioconversion conditions for the production of vanillin by B. subtilis B7-S can be summarized as follows: temperature 35 °C; initial pH 9.0; inoculum volume 5%; ferulic acid concentration 0.6 g/L; volume of culture medium 20%; and shaking speed 200 r/min. Under these conditions, several repeated small-scale batch experiments showed that the maximum conversion efficiency was 63.30% after 3 h of bioconversion. The vanillin products were confirmed by spectral data achieved from UV-vis, inductively coupled plasma atomic emission spectroscope (ICP-AES) and Fourier transform infrared spectrometer (FT-IR) spectra. Scanning electron microscopy (SEM) and transmission electron spectroscopy (TEM) results confirmed that the cell surface of B. subtilis plays a role in the induction of ferulic acid tolerance. These results demonstrate that B. subtilis B7-S has the potential for use in vanillin production through bioconversion of ferulic acid. PMID:26841717

  15. Probing phenotypic growth in expanding Bacillus subtilis biofilms.

    Wang, Xiaoling; Koehler, Stephan A; Wilking, James N; Sinha, Naveen N; Cabeen, Matthew T; Srinivasan, Siddarth; Seminara, Agnese; Rubinstein, Shmuel; Sun, Qingping; Brenner, Michael P; Weitz, David A


    We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert's law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation. PMID:27003268

  16. Decolorization of Distillery Effluent by Thermotolerant Bacillus subtilis

    Soni Tiwari


    Full Text Available Problem statement: Ethanol production from sugarcane molasses generate large volume of effluent containing high Biological Oxygen Demand (BOD and Chemical Oxygen Demand (COD along with melanoidin, a color compound generally produced by Millard reaction. Melanodin is a recalcitrant compound degraded by specific microorganisms having ability to produce mono and di-oxygenases peroxidases, phenoxidases and laccases, are mainly responsible for degradation of complex aromatic hydrocarbons like color compound. These compounds causes several toxic effects on living system, therefore may be treated before disposal. Approach: The purpose of this study was to isolate a potential thermotolerant melanoidin decolorizing bacterium from natural resources for treatment of distillery effluent at industrial level. Results: Total 10 isolates were screened on solid medium containing molasses pigments. Three potential melanoidin decolorizing thermotolerant bacterial isolates identified as Bacillus subtilis, Bacillus cereus and Pseudomonas sp. were further optimized for decolorization at different physico-chemical and nutritional level. Out of these three, Bacillus subtilis showed maximum decolorization (85% at 45°C using (w/v 0.1%, glucose; 0.1%, peptone; 0.05%, MgSO4; 0.01%, KH2PO4; pH-6.0 within 24h of incubation under static condition. Conclusion/Recommendations: The strain of Bacillus subtilis can tolerate higher temperature and required very less carbon (0.1%, w/v and nitrogen sources (0.1%, w/v in submerged fermentation. It can be utilized for melanoidin decolorization of distillery effluent at industrial scale.

  17. CotC-CotU Heterodimerization during Assembly of the Bacillus subtilis Spore Coat▿

    Isticato, Rachele; Pelosi, Assunta; Zilhão, Rita, 1959-; Baccigalupi, Loredana; Henriques, Adriano O.; De Felice, Maurilio; Ricca, Ezio


    We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of σK and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to form a coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis. Monomeric forms of both CotU a...

  18. Nutrient depletion in Bacillus subtilis biofilms triggers matrix production

    Many types of bacteria form colonies that grow into physically robust and strongly adhesive aggregates known as biofilms. A distinguishing characteristic of bacterial biofilms is an extracellular polymeric substance (EPS) matrix that encases the cells and provides physical integrity to the colony. The EPS matrix consists of a large amount of polysaccharide, as well as protein filaments, DNA and degraded cellular materials. The genetic pathways that control the transformation of a colony into a biofilm have been widely studied, and yield a spatiotemporal heterogeneity in EPS production. Spatial gradients in metabolites parallel this heterogeneity in EPS, but nutrient concentration as an underlying physiological initiator of EPS production has not been explored. Here, we study the role of nutrient depletion in EPS production in Bacillus subtilis biofilms. By monitoring simultaneously biofilm size and matrix production, we find that EPS production increases at a critical colony thickness that depends on the initial amount of carbon sources in the medium. Through studies of individual cells in liquid culture we find that EPS production can be triggered at the single-cell level by reducing nutrient concentration. To connect the single-cell assays with conditions in the biofilm, we calculate carbon concentration with a model for the reaction and diffusion of nutrients in the biofilm. This model predicts the relationship between the initial concentration of carbon and the thickness of the colony at the point of internal nutrient deprivation. (paper)

  19. Bacillus subtilis Hfq: A role in chemotaxis and motility



    Hfq is a global post-transcriptional regulator that modulates the translation and stability of target mRNAs and therebyregulates pleiotropic functions, such as growth, stress, virulence and motility, in many Gram-negative bacteria.However, comparatively little is known about the regulation and function(s) of Hfq in Gram-positive bacteria.Recently, in Bacillus subtilis, a role for Hfq in stationary phase survival has been suggested, although the possibilityof Hfq having an additional role(s) cannot be ruled out. In this study we show that an ortholog of Hfq in B. subtilis isregulated by the stress sigma factor, σB, in addition to the stationary phase sigma factor, σH. We further demonstratethat Hfq positively regulates the expression of flagellum and chemotaxis genes (fla/che) that control chemotaxis andmotility, thus assigning a new function for Hfq in B. subtilis.

  20. Structure-function correlation in glycine oxidase from Bacillus subtilis

    Mörtl, Mario; Diederichs, Kay; Welte, Wolfram; Molla, Gianluca; Motteran, Laura; Andriolo, Gabriella; Pilone, Mirella S.; Pollegioni, Loredano


    Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies. The structure of the GO-glycolate complex was determined at 1.8 Å, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted. GO can be considered a member of the amine oxidase class ...

  1. Maltose- und Maltodextrin-Verwertung in Bacillus subtilis

    Schönert, Stefan


    In seinem natürlichen Habitat findet das Gram - positive Bodenbakterium Bacillus subtilis hauptsächlich polymere Zuckerformen als Kohlenstoffquelle vor, die aus den von anderen Organismen synthetisierten Speicherstoffen, z.B. Stärke und Glykogen, stammen. Jedoch müssen diese Polysaccharide zuerst extrazellulär zu Maltose und Maltodextrinen hydrolysiert werden, bevor sie aufgenommen werden können.Im Gegensatz zu der sehr gut untersuchten Maltose - und Maltodextrin - Aufnahme in Escherichia col...

  2. Use of bacillus subtilis strains to inhibit postharvest pathogenic fungi

    An isolate (87) of the bacillus subtilis strains isolated from cold stored citrus fruit 13 proved to inhibit the growth in vitro of the penicillium italicum used in the experiment (from 50.6% to 92.2%) and to inhibit botrytis cinerea (from 65.3% to 95.9%). A further test, superimposing on plates containing PDA strains Nos. 13, 173, and 160, totally inhibited the fungi. Tested in vivo on artificially bruised oranges, they significantly inhibited two fungi

  3. Extracellular and membrane-bound proteases from Bacillus subtilis.

    Mäntsälä, P; Zalkin, H


    Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The e...

  4. MinCD Proteins Control the Septation Process during Sporulation of Bacillus subtilis

    Barák, Imrich; Prepiak, Peter; Schmeisser, Falko


    Mutation of the divIVB locus in Bacillus subtilis causes misplacement of the septum during cell division and allows the formation of anucleate minicells. The divIVB locus contains five open reading frames (ORFs). The last two ORFs (minCD) are homologous to minC and minD of Escherichia coli but a minE homolog is lacking in B. subtilis. There is some similarity between minicell formation and the asymmetric septation that normally occurs during sporulation in terms of polar septum localization. ...

  5. Selectivity in protein degradation during sporulation of Bacillus subtilis

    The breakdown of cellular protein was investigated in Bacillus subtilis ATCC 6051 labeled with glycine-2-3H or L-phenylalanine-U-14C at the different stages of vegetative growth and sporulation. The growth of the culture was determined by measuring optical density at 660 nm. The heat-resistant spores were scored by plating after heating at 80 deg C for 10 minutes. A question whether the turnover of glycine-labeled protein is similar to that of phenylalanine-labeled protein was experimentally studied. The patterns obtained with the glycine-labeled protein were different from those of phenylalanine-labeled protein. This was not multiple turnover. The cellular protein which was labeled with glycine at an early stage of sporulation showed rapid degradation, but the degradation of the protein labeled with glycine at later stages did not occur at all. Another question whether the labeled glycine incorporated into cells at the different stages of growth and sporulation was present in the spore coat fraction of matured spores was studied. Experiment demonstrated that the glycine incorporated into cells at the late sporulation stage was mainly utilized for the biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amount of glycine is rarely subject to further degradation. (Iwakiri, K.)

  6. Transcriptional regulation of a Bacillus subtilis dipeptide transport operon.

    Slack, F J; Mueller, J P; Strauch, M A; Mathiopoulos, C; Sonenshein, A L


    The Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation. The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase. Post-exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant. This implicated AbrB, another known regulator, as a repressor of dciA. In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion. Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB. PMID:1766371

  7. Identification of Bacillus subtilis genes expressed early during sporulation.

    Mathiopoulos, C; Sonenshein, A L


    Labelled cDNA transcribed in vitro from early-sporulation RNA was enriched for sporulation-specific sequences by subtractive hybridization to an excess of vegetative RNA and used to probe libraries of Bacillus subtilis chromosomal DNA. From the initial collection of clones that coded for RNAs transcribed preferentially during sporulation, several were subcloned and studied in more detail. It was found that two clones contained sequences (dciA and dciB) that had an undetectable level of transcription during vegetative growth but had transcripts that started to appear no later than eight minutes after induction of sporulation. A third DNA segment (dciC) was expressed at a low level in vegetative cells and increased within four minutes after induction of sporulation. The effects of spoO mutations, i.e. mutations that prevent cells from reaching stage I of the sporulation process, were tested. Induction of the dciA and dciB transcripts was significantly reduced in strains carrying mutations in the spoOA and spoOH genes but not in a spoOB mutant strain. In addition, a product of the abrB locus, a locus in which mutations are known to partially overcome the pleiotropic effect of spoOA and spoOB mutations, seemed to be required for dciA and dciB expression. PMID:2481799

  8. Novel methyl transfer during chemotaxis in Bacillus subtilis

    If Bacillus subtilis is incubated in radioactive methionine in the absence of protein synthesis, the methyl-accepting chemotaxis proteins (MCPs) become radioactively methylated. If the bacteria are further incubated in excess nonradioactive methionine (cold-chased) and then given the attractant aspartate, the MCPs lose about half of their radioactivity due to turnover, in which lower specific activity methyl groups from S-adenosylmethionine (AdoMet) replace higher specific activity ones. Due to the cold-chase, the specific activity of the AdoMet pool is reduced at least 2-fold. If, later, the attractant is removed, higher specific activity methyl groups return to the MCPs. Thus, there must exist an unidentified methyl carrier than can reversibly receive methyl groups from the MCPs. In a similar experiment, labeled cells were transferred to a flow cell and exposed to addition and removal of attractant and of repellent. All four kinds of stimuli were found to cause methanol production. Bacterial with maximally labeled MCPs were exposed to many cycles of addition and removal of attractant; the maximum amount of radioactive methanol was evolved on the third, not the first, cycle. This result suggests that there is a precursor-product relationship between methyl groups on the MCPs and on the unidentified carrier, which might be the direct source of methanol. However, since no methanol was produced when a methyltransferase mutant, whose MCPs were unmethylated, was exposed to addition and removal of attractant or repellent, the methanol must ultimately derive from methylated MCPs

  9. Inhibitory effect of novobiocin on ribonucleic acid synthesis during germination of Bacillus subtilis spores.

    Matsuda, M; Kameyama, T


    Novobiocin inhibited ribonculeic acid synthesis during germination of Bacillus subtilis spores. Transcription of certain kinds of genes probably required a preceding conformational change in deoxyribonucleic acid.

  10. Production of Bioactive Compounds by Bacillus subtilis against Sclerotium rolfsii

    Nalisha, I.


    Full Text Available This study aims to investigate the characteristic of bioactive compound produced by Bacillus subtilis against Sclerotium rolfsii and the influence of additive supplements on the antagonistic activity of B. subtilis. The fact that B. subtilis produced an antifungal substance which has inhibitory effect on wide range of fungi, including S. rolfsii, is well known. To learn the effect of pH, temperature and light condition on the production of antifungal compound, B. subtilis was inoculated in Potato Dextrose Broth at various initial pH, temperatures and light conditions, respectively. This antagonist was found to produce antifungal compound that stable at 80C with 58.3 % inhibition on S. rolfsii. The activity was constant within a wide range of pH (3–11. However, treatment with pH11 lead to higher antifungal activity (31.57 % inhibition and it was also found to produce substance that can endure dark condition (46.24 % inhibition with fungicidal effect on S. rolfsii. A series of experiments also been carried out to enhance the antifungal production by supplementing different carbon source preparation into bacterial liquid culture. B. subtilis were grown in minimal medium containing 1 % of oil palm root, Ganoderma lucidum or chitin, respectively prior to bioassay. Crude culture from oil palm root supplemented culture shown significantly reduction in S. rolfsii growth compared to other carbon source crude culture or the antagonism alone, suggesting that this approach may provide improved biocontrol efficiency.

  11. Regulation of Polyglutamic Acid Synthesis by Glutamate in Bacillus licheniformis and Bacillus subtilis

    Kambourova, Margarita; Tangney, Martin; Priest, Fergus G.


    The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular γ-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreo...

  12. Joint toxicity assessment of heavy metals based on CellSense biosensor of Bacillus Subtilis%基于枯草芽孢杆菌的CellSense生物传感器的重金属联合毒性分析

    赵红宁; 董晓静; 王学江; 夏四清


    采用聚碳酸醑膜直接固定法制备了基于枯草芽孢杆菌(Bacillus Subtilis)的CellSense生物传感器,分别测定了Cd2+、Cu2+、Zn2+和Cr(Ⅵ)对Bacillus Subtilis的单一毒性,以及等毒性配比和等浓度配比下二元混合体系的联合毒性,并用相加指数法对其联合毒性效应进行了评价.结果表明,基于对数生长后期和稳定期的Bacillus Subtilis的CellSense生物传感器具有良好的毒性分析性能;2种联合毒性评价方法下4种重金属离子二元混合体系的联合作用结果一致,均表现出不同程度的拮抗作用.%A CellSense biosensor for pollutants acute toxicity test was developed using Bacillus Subtilis immobilized on thin-film carbon electrodes by polycarbonate membrane. The single toxicity of Cd2+ , Cu2+ , Zn2+ and Cr( Ⅵ ) and joint toxicity of binary mixtures in equiconcentration and equitoxicity ratios to Bacillus Subtilis were determined, respectively, additive index method was adopted to evaluate the joint toxicity effect. The results showed that when Bacillus Subtilis was in logarithmic and stable growth periods, the CellSense biosensor with Bacillus Subtilis showed good performance in heavy metal ions toxicity analysis. The consistent conclusion could be drawn from both equiconcentration mixtures and equitoxicity ones that the joint effect of the binary mixtures of Cd2+ , Cu2+ , Zn2+ and Cr( Ⅵ ) were antagonistic.

  13. Study on the Effect of Penicillin on the Broken Cell Wall of Bacillus subtilis%青霉素对枯草芽孢杆菌破壁效果研究

    周靖波; 周娅; 黄宗泳; 姚正辉; 张新武; 黄继红


    [ Objective ] The effect of the penicillin on the Bacillus subtilis cell wall-breaking was researched. [ Method ] The technique of the different amounts of penicillin for the Bacillus subtilis cell wall-breaking was experimented so that the effective activity-material inside cell could be released. [ Result ] The testing result of the variation value of light density before and after cell well-breaking indicated that the penicillin eould obvionsly affect the variation of light density during the period of Bacillus subtilis growth. When the adding amount of penicillin was 25 mg/kg, the action time was 4 hours and the temperature was 30 ℃, the variation value of light density(ΔOD) was the largest(0.695), which was the best one. [ Conclusion ] The penicillin had a certain role in the Bacillus subtilis cell wall-breaking and the internal material of its cell could be released after the reasonable amount of penicillin was used.%[目的]研究枯草芽孢杆菌生长期添加青霉素时的破壁效果.[方法]为使细菌释放出细胞内的有效活性物质,采用青霉素干扰微生物细胞壁形成的方法,研究不同的青霉素添加量对细胞的破壁效果.[结果]通过检测破壁前后菌体光密度变化值得出,在枯草芽孢杆菌的生长期,青霉素添加量对菌体光密度的变化有明显影响,青霉素添加量为25 mg/Kg,作用时间4 h,温度30℃时,光密度变化值(AOD)最高,达0.695,说明此时破壁效果最佳.[结论]青霉素对细胞具有一定的破壁效果,推测根据需要可以适量添加青霉素,使细菌释放出细胞内的物质.

  14. Small Regulatory RNA-Induced Growth Rate Heterogeneity of Bacillus subtilis

    Mars, Ruben A. T.; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Maeder, Ulrike; Voelker, Uwe; van Dijl, Jan Maarten; Denham, Emma L.


    Author Summary Bacterial cells that share the same genetic information can display very different phenotypes, even if they grow under identical conditions. Despite the relevance of this population heterogeneity for processes like drug resistance and development, the molecular players that induce heterogenic phenotypes are often not known. Here we report that in the Gram-positive model bacterium Bacillus subtilis a small regulatory RNA (sRNA) can induce heterogeneity in growth rates by increas...

  15. Undecaprenyl Pyrophosphate Involvement in Susceptibility of Bacillus subtilis to Rare Earth Elements

    Inaoka, Takashi; Ochi, Kozo


    The rare earth element scandium has weak antibacterial potency. We identified a mutation responsible for a scandium-resistant phenotype in Bacillus subtilis. This mutation was found within the uppS gene, which encodes undecaprenyl pyrophosphate synthase, and designated uppS86 (for the Thr-to-Ile amino acid substitution at residue 86 of undecaprenyl pyrophosphate synthase). The uppS86 mutation also gave rise to increased resistance to bacitracin, which prevents cell wall synthesis by inhibitin...

  16. Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms

    Cairns, Lynne S; Hobley, Laura; Stanley-Wall, Nicola R.


    Biofilm formation is a social behaviour that generates favourable conditions for sustained survival in the natural environment. For the Gram-positive bacterium Bacillus subtilis the process involves the differentiation of cell fate within an isogenic population and the production of communal goods that form the biofilm matrix. Here we review recent progress in understanding the regulatory pathways that control biofilm formation and highlight developments in understanding the composition, func...

  17. Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms.

    Cairns, Lynne S; Hobley, Laura; Stanley-Wall, Nicola R


    Biofilm formation is a social behaviour that generates favourable conditions for sustained survival in the natural environment. For the Gram-positive bacterium Bacillus subtilis the process involves the differentiation of cell fate within an isogenic population and the production of communal goods that form the biofilm matrix. Here we review recent progress in understanding the regulatory pathways that control biofilm formation and highlight developments in understanding the composition, function and structure of the biofilm matrix. PMID:24988880

  18. Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis.

    Chang, S.; Chang, S Y; Gray, O


    The Bacillus plasmid pLS11 partitions faithfully during cell division. Using a partition-deficient plasmid vector, we randomly cloned DNA fragments of plasmid pLS11 and identified the locus that regulates plasmid partition (par) by cis complementation in Bacillus subtilis. The cloned par gene conferred upon the vector plasmid a high degree of segregational stability. The par locus was mapped to a 167-base-pair segment on pLS11, and its nucleotide sequence was determined. The cloned par fragme...

  19. Homolactic fermentation from glucose and cellobiose using Bacillus subtilis

    Martinez Alfredo


    Full Text Available Abstract Backgroung Biodegradable plastics can be made from polylactate, which is a polymer made from lactic acid. This compound can be produced from renewable resources as substrates using microorganisms. Bacillus subtilis is a Gram-positive bacterium recognized as a GRAS microorganism (generally regarded as safe by the FDA. B. subtilis produces and secretes different kind of enzymes, such as proteases, cellulases, xylanases and amylases to utilize carbon sources more complex than the monosaccharides present in the environment. Thus, B. subtilis could be potentially used to hydrolyze carbohydrate polymers contained in lignocellulosic biomass to produce chemical commodities. Enzymatic hydrolysis of the cellulosic fraction of agroindustrial wastes produces cellobiose and a lower amount of glucose. Under aerobic conditions, B. subtilis grows using cellobiose as substrate. Results In this study, we proved that under non-aerated conditions, B. subtilis ferments cellobiose to produce L-lactate with 82% of the theoretical yield, and with a specific rate of L-lactate production similar to that one obtained fermenting glucose. Under fermentative conditions in a complex media supplemented with glucose, B. subtilis produces L-lactate and a low amount of 2,3-butanediol. To increase the L-lactate production of this organism, we generated the B subtilis CH1 alsS- strain that lacks the ability to synthesize 2,3-butanediol. Inactivation of this pathway, that competed for pyruvate availability, let a 15% increase in L-lactate yield from glucose compared with the parental strain. CH1 alsS- fermented 5 and 10% of glucose to completion in mineral medium supplemented with yeast extract in four and nine days, respectively. CH1 alsS- produced 105 g/L of L-lactate in this last medium supplemented with 10% of glucose. The L-lactate yield was up to 95% using mineral media, and the optical purity of L-lactate was of 99.5% since B. subtilis has only one gene (lctE that

  20. An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

    Ogawa, Takafumi; Iwata, Tetsuo; Kaneko, Shinya; Itaya, Mitsuhiro; Hirota, Junji


    Background The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. ...

  1. Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

    Wu, Sau-Ching; Wong, Sui-Lam


    Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. Th...

  2. Sporicidal characteristics of heated dolomite powder against Bacillus subtilis spores.

    Yasue, Syogo; Sawai, Jun; Kikuchi, Mikio; Nakakuki, Takahito; Sano, Kazuo; Kikuchi, Takahide


    Dolomite is a double salt composed of calcium carbonate (CaCO3) and magnesium carbonate (MgCO3). The heat treatment of CaCO3 and MgCO3 respectively generates calcium oxide (CaO) and magnesium oxide (MgO), which have antimicrobial activity. In this study, heated dolomite powder (HDP) slurry was investigated for its sporicidal activity against Bacillus subtilis ATCC 6633 spores. The B. subtilis spores used in this study were not affected by acidic (pH 1) or alkaline (pH 13) conditions, indicating that they were highly resistant. However, dolomite powder heated to 1000℃ for 1 h could kill B. subtilis spores, even at pH 12.7. Sporicidal activity was only apparent when the dolomite powder was heated to 800℃ or higher, and sporicidal activity increased with increases in the heating temperature. This temperature corresponded to that of the generation of CaO. We determined that MgO did not contribute to the sporicidal activity of HDP. To elucidate the sporicidal mechanism of the HDP against B. subtilis spores, the generation of active oxygen from HDP slurry was examined by chemiluminescence analysis. The generation of active oxygen increased when the HDP slurry concentration rose. The results suggested that, in addition to its alkalinity, the active oxygen species generated from HDP were associated with sporicidal activity. PMID:25252642

  3. Complexity in regulation of tryptophan biosynthesis in Bacillus subtilis.

    Gollnick, Paul; Babitzke, Paul; Antson, Alfred; Yanofsky, Charles


    Bacillus subtilis uses novel regulatory mechanisms in controlling expression of its genes of tryptophan synthesis and transport. These mechanisms respond to changes in the intracellular concentrations of free tryptophan and uncharged tRNA(Trp). The major B. subtilis protein that regulates tryptophan biosynthesis is the tryptophan-activated RNA-binding attenuation protein, TRAP. TRAP is a ring-shaped molecule composed of 11 identical subunits. Active TRAP binds to unique RNA segments containing multiple trinucleotide (NAG) repeats. Binding regulates both transcription termination and translation in the trp operon, and translation of other coding regions relevant to tryptophan metabolism. When there is a deficiency of charged tRNA(Trp), B. subtilis forms an anti-TRAP protein, AT. AT antagonizes TRAP function, thereby increasing expression of all the genes regulated by TRAP. Thus B. subtilis and Escherichia coli respond to identical regulatory signals, tryptophan and uncharged tRNA(Trp), yet they employ different mechanisms in regulating trp gene expression. PMID:16285852

  4. A part toolbox to tune genetic expression in Bacillus subtilis.

    Guiziou, Sarah; Sauveplane, Vincent; Chang, Hung-Ju; Clerté, Caroline; Declerck, Nathalie; Jules, Matthieu; Bonnet, Jerome


    Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis. PMID:27402159

  5. Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions

    Jarmer, Hanne Østergaard; Berka, R.; Knudsen, Steen;


    DNA microarrays were used to analyze the changes in gene expression in Bacillus subtilis strain 168 when nitrogen limiting (glutamate) and nitrogen excess (ammonium plus glutamate) growth conditions were compared. Among more than 100 genes that were significantly induced during nitrogen starvation...... we detected the comG, comF, comE, nin-nucA and comK transcription units together with recA. DNA was added to B. subtilis grown in minimal medium with glutamate as the sole nitrogen source and it was demonstrated that the cells were competent. Based on these observations we propose a simplification of...... previously designed one-step transformation procedures for B. subtilis strain 168....

  6. Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure.

    Nguyen Thi Minh, Hue; Durand, Alain; Loison, Pauline; Perrier-Cornet, Jean-Marie; Gervais, Patrick


    Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry. PMID:21380515

  7. Complete Genome Sequence of Bacillus subtilis Strain CU1050, Which Is Sensitive to Phage SPβ

    Johnson, Christopher M.; Grossman, Alan D.


    The Gram-positive bacterium Bacillus subtilis is used as a model organism to study cellular and molecular processes. Here, we announce the complete genomic sequence of B. subtilis strain CU1050, derived from B. subtilis strain 168. CU1050 has historically been used to study suppressor mutations and phage biology, especially the lysogenic phage SPβ.

  8. A Bacillus subtilis dipeptide transport system expressed early during sporulation.

    Mathiopoulos, C; Mueller, J P; Slack, F J; Murphy, C G; Patankar, S; Bukusoglu, G; Sonenshein, A L


    Two previously identified Bacillus subtilis DNA segments, dciA and dciB, whose transcripts accumulate very rapidly after induction of sporulation, were found in the same 6.2 kb transcription unit, now known as the dciA operon. Analysis of the sequence of the dciA operon showed that its putative products are homologous to bacterial peptide transport systems. The product of the fifth gene, DciAE, is similar to peptide-binding proteins from Escherichia coli and Salmonella typhimurium (DppA and OppA) and B. subtilis (OppA). A null mutation in dciAE abolished the ability of a proline auxotroph to grow in a medium containing the dipeptide Pro-Gly as sole proline source, suggesting that the dciA operon encodes a dipeptide transport system. PMID:1766370

  9. Regulation of the anaerobic metabolism in Bacillus subtilis.

    Härtig, Elisabeth; Jahn, Dieter


    The Gram-positive soil bacterium Bacillus subtilis encounters changing environmental conditions in its habitat. The access to oxygen determines the mode of energy generation. A complex regulatory network is employed to switch from oxygen respiration to nitrate respiration and various fermentative processes. During adaptation, oxygen depletion is sensed by the [4Fe-4S](2+) cluster containing Fnr and the two-component regulatory system ResDE consisting of the membrane-bound histidine kinase ResE and the cytoplasmic ResD regulator. Nitric oxide is the signal recognized by NsrR. Acetate formation and decreasing pH are measured via AlsR. Finally, Rex is responding to changes in the cellular NAD(+)/NADH ration. The fine-tuned interplay of these regulators at approximately 400 target gene promoters ensures efficient adaptation of the B. subtilis physiology. PMID:23046954

  10. Stoichiometric growth model for riboflavin-producing Bacillus subtilis.

    Dauner, M; Sauer, U


    Rate equations for measured extracellular rates and macromolecular composition data were combined with a stoichiometric model to describe riboflavin production with an industrial Bacillus subtilis strain using errors in variables regression analysis. On the basis of this combined stoichiometric growth model, we explored the topological features of the B. subtilis metabolic reaction network that was assembled from a large amount of literature. More specifically, we simulated maximum theoretical yields of biomass and riboflavin, including the associated flux regimes. Based on the developed model, the importance of experimental data on building block requirements for maximum yield and flux calculations were investigated. These analyses clearly show that verification of macromolecular composition data is important for optimum flux calculations. PMID:11505383

  11. Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum.

    Yueju Zhao

    Full Text Available Fusarium graminearum causes Fusarium head blight (FHB, a devastating disease that leads to extensive yield and quality loss of wheat and barley. Bacteria isolated from wheat kernels and plant anthers were screened for antagonistic activity against F. graminearum. Based on its in vitro effectiveness, strain SG6 was selected for characterization and identified as Bacillus subtilis. B. subtilis SG6 exhibited a high antifungal effect on the mycelium growth, sporulation and DON production of F. graminearum with the inhibition rate of 87.9%, 95.6% and 100%, respectively. In order to gain insight into biological control effect in situ, we applied B. subtilis SG6 at anthesis through the soft dough stage of kernel development in field test. It was revealed that B. subtilis SG6 significantly reduced disease incidence (DI, FHB index and DON (P ≤ 0.05. Further, ultrastructural examination shows that B. subtilis SG6 strain induced stripping of F. graminearum hyphal surface by destroying the cellular structure. When hypha cell wall was damaged, the organelles and cytoplasm inside cell would exude, leading to cell death. The antifungal activity of SG6 could be associated with the coproduction of chitinase, fengycins and surfactins.

  12. Heat Resistance and Population Stability of Lyophilized Bacillus subtilis Spores

    Odlaug, Theron E.; Caputo, Ross A.; Graham, Gary S.


    Bacillus subtilis 5230 spores were lyophilized in 0.067 M phosphate buffer and stored at 2 to 8°C for 9 to 27 months. The lyophilized spores were reconstituted with buffer or 0.9% saline, and the heat resistance was determined in a thermoresistometer. Lyophilization had no effect on the heat resistance of the spores but did result in a slight decrease in population (≤0.3-logarithm reduction). The lyophilized spores maintained heat resistance and population levels over the test periods. The D-...

  13. Unhairing animal hides using probiotic Bacteria bacillus subtilis

    Данилкович, Анатолій Григорович; Гвоздяк, Петро Ілліч; Романюк, Оксана Олександрівна; Ковтуненко, Ольга Василівна


    The most efficient technology of processing natural raw materials into skin and fur is the use of enzyme products for soaking and liming processes. Therefore, the use of bacterial products, which produce enzymes of various functional effects, is considered to be very promising for the above mentioned processes.Soaking and liming of flint-dried rabbit hides were carried out using probiotic bacreria Bacillus subtilis on 4 samples in a laboratory centrifuge at soaking temperature 36-38°С and wor...

  14. Hyper production of alkaline protease by mutagenized bacillus subtilis

    The purpose of this work was to augment the alkaline protease production from Bacillus subtilis by using chemical mutagen (MMS) and UV mutagenesis. A number of mutants were isolated which produce high levels of extra cellular proteases. Analysis of culture supernatants of these mutants had shown that the total amounts of proteolysis activity were increased from 1 to 2 fold over the wild strain. Clones showing promote response were further characterized by analyzing different parameters; like of Temperature, pH substrate concentration and incubation period, to study the activity of protease enzyme. (author)

  15. Characterization of Bacillus subtilis Colony Biofilms via Mass Spectrometry and Fluorescence Imaging.

    Si, Tong; Li, Bin; Zhang, Ke; Xu, Yiran; Zhao, Huimin; Sweedler, Jonathan V


    Colony biofilms of Bacillus subtilis are a widely used model for studying cellular differentiation. Here, we applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to examine cellular and molecular heterogeneity in B. subtilis colony biofilms. From B. subtilis cells cultivated on a biofilm-promoting medium, we detected two cannibalistic factors not found in previous MALDI MSI studies of the same strain under different culturing conditions. Given the importance of cannibalism in matrix formation of B. subtilis biofilms, we employed a transcriptional reporter to monitor matrix-producing cell subpopulations using fluorescence imaging. These two complementary imaging approaches were used to characterize three B. subtilis strains, the wild type isolate NCIB3610, and two mutants, Δspo0A and ΔabrB, with defective and enhanced biofilm phenotypes, respectively. Upon deletion of key transcriptional factors, correlated changes were observed in biofilm morphology, signaling, cannibalistic factor distribution, and matrix-related gene expression, providing new insights on cannibalism in biofilm development. This work underscores the advantages of using multimodal imaging to compare spatial patterns of selected molecules with the associated protein expression patterns, obtaining information on cellular heterogeneity and function not obtainable when using a single method to characterize biofilm formation. PMID:27136705

  16. Bacillus subtilis spores as vaccine adjuvants: further insights into the mechanisms of action.

    Renata Damásio de Souza

    Full Text Available Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.

  17. A Bacitracin-Resistant Bacillus subtilis Gene Encodes a Homologue of the Membrane-Spanning Subunit of the Bacillus licheniformis ABC Transporter

    Ohki, Reiko; Tateno, Kozue; Okada, Youji; Okajima, Haruo; Asai, Kei; Sadaie, Yoshito; Murata, Makiko; Aiso, Toshiko


    Bacitracin is a peptide antibiotic nonribosomally produced by Bacillus licheniformis. The bcrABC genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells. Bacillus subtilis 168, which has no bacitracin synthesizing operon, has several genes homologous to bcrABC. It was found that the disruption of ywoA, a gene homologous to bcrC, resulted in hypersensitivity to bacit...

  18. Decolourization of 4-chloro-2-nitrophenol by a soil bacterium, Bacillus subtilis RKJ 700.

    Pankaj Kumar Arora

    Full Text Available A 4-Chloro-2-nitrophenol (4C2NP decolourizing strain RKJ 700 was isolated from soil collected from a pesticide contaminated site of India and identified as Bacillus subtilis on the basis of the 16S rRNA gene sequence analysis. Bacillus subtilis RKJ 700 decolourized 4C2NP up to concentration of 1.5 mM in the presence of additional carbon source. The degradation pathway of 4C2NP was studied and 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole (5C2MBZ were identified as metabolites by high performance liquid chromatography and gas chromatography-mass spectrometry. Resting cell studies showed that Bacillus subtilis RKJ 700 depleted 4C2NP completely with stoichiometric formation of 5C2MBZ. This is the first report of (i the degradation of 4C2NP at high concentration (1.5 mM and, (ii the formation of 5C2MBZ by a soil bacterium.

  19. The localization of key Bacillus subtilis penicillin binding proteins during cell growth is determined by substrate availability

    Lages, Marta Carolina Afonso; Beilharz, Katrin; Angeles, Danae Morales; Veening, Jan-Willem; Scheffers, Dirk-Jan


    The shape of bacteria is maintained by the cell wall. The main component of the cell wall is peptidoglycan (PG) that is synthesized by penicillin binding proteins (PBPs). The correct positioning of PBPs is essential for the maintenance of cell shape. In the literature, two different models for local

  20. Isolation of a New Mexican Strain of Bacillus subtilis with Antifungal and Antibacterial Activities

    M. G. L. Basurto-Cadena; M. Vázquez-Arista; García-Jiménez, J.; Salcedo-Hernández, R.; Bideshi, D. K.; Barboza-Corona, J. E.


    Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 2...

  1. Monovalent cations enable cell wall turnover of the turnover-deficient lyt-15 mutant of Bacillus subtilis.

    Cheung, H. Y.; Freese, E


    A lyt-15 mutant reported to be unable to turn over the cell wall exhibited the same rate of wall turnover as the standard strain if the medium contained 0.2 M NaCl, which did not affect growth. Cell wall autolysis was also optimal at 0.2 M NaCl.

  2. Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P.

    Rajkovic, Andrei; Hummels, Katherine R; Witzky, Anne; Erickson, Sarah; Gafken, Philip R; Whitelegge, Julian P; Faull, Kym F; Kearns, Daniel B; Ibba, Michael


    Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys(32) of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility. PMID:27002156

  3. Initiation of decay of Bacillus subtilis trp leader RNA.

    Deikus, Gintaras; Bechhofer, David H


    Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated by binding of the 11-mer TRAP complex to nascent trp RNA, which results in formation of a terminator structure. Rapid decay of trp leader RNA, which is required to release the TRAP complex and maintain a sufficient supply of free TRAP, is mediated by polynucleotide phosphorylase (PNPase). Using purified B. subtilis PNPase, we showed that, when TRAP was present, PNPase binding to the 3' end of trp leader RNA and PNPase digestion of trp leader RNA from the 3' end were inefficient. These results suggested that initiation of trp leader RNA may begin with an endonuclease cleavage upstream of the transcription terminator structure. Such cleavage was observed in vivo. Mutagenesis of nucleotides at the cleavage site abolished processing and resulted in a 4-fold increase in trp leader RNA half-life. This is the first mapping of a decay-initiating endonuclease cleavage site on a native B. subtilis RNA. PMID:17507374

  4. Application of gaseous ozone for inactivation of Bacillus subtilis spores.

    Aydogan, Ahmet; Gurol, Mirat D


    The effectiveness of gaseous ozone (O3) as a disinfectant was tested on Bacillus subtilis spores, which share the same physiological characteristics as Bacillus anthracis spores that cause the anthrax disease. Spores dried on surfaces of different carrier material were exposed to O3 gas in the range of 500-5000 ppm and at relative humidity (RH) of 70-95%. Gaseous O3 was found to be very effective against the B. subtilis spores, and at O3 concentrations as low as 3 mg/L (1500 ppm), approximately 3-log inactivation was obtained within 4 hr of exposure. The inactivation curves consisted of a short lag phase followed by an exponential decrease in the number of surviving spores. Prehydration of the bacterial spores has eliminated the initial lag phase. The inactivation rate increased with increasing O3 concentration but not >3 mg/L. The inactivation rate also increased with increase in RH. Different survival curves were obtained for various surfaces used to carry spores. Inactivation rates of spores on glass, a vinyl floor tile, and office paper were nearly the same. Whereas cut pile carpet and hardwood flooring surfaces resulted in much lower inactivation rates, another type of carpet (loop pile) showed significant enhancement in the inactivation of the spores. PMID:16568801

  5. Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

    Overkamp, Wout; Beilharz, Katrin; Weme, Ruud Detert Oude; Solopova, Ana; Karsens, Harma; Kovacs, Akos T.; Kok, Jan; Kuipers, Oscar P.; Veening, Jan-Willem


    Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental c

  6. The Antimicrobial Properties of Silver Nanoparticles in Bacillus subtilis Are Mediated by Released Ag+ Ions.

    Yi-Huang Hsueh

    Full Text Available The superior antimicrobial properties of silver nanoparticles (Ag NPs are well-documented, but the exact mechanisms underlying Ag-NP microbial toxicity remain the subject of intense debate. Here, we show that Ag-NP concentrations as low as 10 ppm exert significant toxicity against Bacillus subtilis, a beneficial bacterium ubiquitous in the soil. Growth arrest and chromosomal DNA degradation were observed, and flow cytometric quantification of propidium iodide (PI staining also revealed that Ag-NP concentrations of 25 ppm and above increased membrane permeability. RedoxSensor content analysis and Phag-GFP expression analysis further indicated that reductase activity and cytosolic protein expression decreased in B. subtilis cells treated with 10-50 ppm of Ag NPs. We conducted X-ray absorption near-edge structure (XANES and extended X-ray absorption fine structure (EXAFS analyses to directly clarify the valence and fine structure of Ag atoms in B. subtilis cells placed in contact with Ag NPs. The results confirmed the Ag species in Ag NP-treated B. subtilis cells as Ag2O, indicating that Ag-NP toxicity is likely mediated by released Ag+ ions from Ag NPs, which penetrate bacterial cells and are subsequently oxidized intracellularly to Ag2O. These findings provide conclusive evidence for the role of Ag+ ions in Ag-NP microbial toxicity, and suggest that the impact of inappropriately disposed Ag NPs to soil and water ecosystems may warrant further investigation.

  7. Microbial desalination cell for enhanced biodegradation of waste engine oil using a novel bacterial strain Bacillus subtilis moh3.

    Sabina, K; Fayidh, Mohammed A; Archana, G; Sivarajan, M; Babuskin, S; Babu, P Azhagu Saravana; Radha, K Krishnan; Sukumar, M


    Microbial desalination cell (MDC) is a bioelectrochemical system developed recently from microbial fuel cells (MFCs), for producing green energy from organic wastes along with desalination of saltwater. MDC is proved to be a better performer than MFC in terms of power output and chemical oxygen demand removal, with desalination as an additional feature. This study investigates the application potential of MDC for integrated biodegradation of waste engine oil. This study showed, for the first time, that waste engine oil could be used as an organic substrate in MDC, achieving biodegradation of engine oil along with considerable desalination and power production. Utilization of these wastes in MDC can protect the environment from waste engine oil contamination. Indigenous oil-degrading bacteria were isolated and identified from engine oil contaminated sludge. Degradation of waste engine oil by these novel isolates was studied in batch cultures and optimized the growth conditions. The same cultures when used in MDC, gave enhanced biodegradation (70.1 +/- 0.5%) along with desalination (68.3 +/- 0.6%) and power production (3.1 +/- 0.3 mW/m2). Fourier transform-infrared spectroscopy and gas chromatography-mass spectrometry analyses were performed to characterize the degradation metabolites in the anolyte of MDC which clearly indicated the biodegradation of long chain, branched and cyclic hydrocarbons present in waste engine oil. PMID:25145172

  8. Isolation and Identification of the Antimicrobial Substance Produced by Bacillus subtilis fmbR%Bacillus subtilis fmbR抗菌物质的分离和鉴定

    别小妹; 陆兆新; 吕凤霞; 赵海珍; 杨胜远; 孙力军


    [目的]对Bacillus subtilis fmbR产生的抗菌物质进行分离和鉴定研究,以确定抗菌物质的组成和结构.[方法]采用HPLC和TLC层析对Bacillus subtilis fmbR抗菌物质进行分离纯化,通过ESI-MS和MALDI-MS分析对抗菌物质的组成和结构进行初步鉴定.[结果]HPLC层析表明了Bacillus subtilis fmbR抗菌物质含有保留时间与surfactin相似的成分.TLC层析和原位酸解证明了Bacillus subtilis fmbR抗菌物质含有闭合肽键类的物质,其中之一为相对迁移率Rf与标样surfactin相近的组分.采用ESI-MS分析检测到Bacillus subtilis fmbR抗菌物质含有分子量与surfactinA相同的m/z1009.1、m/z1023.2 和m/z1037.0等3种同系物;通过MALDI-MS分析获得[M+H]+为m/z 3403.95抗菌物质,该物质分子量与Bacillus subtilis 168产生的细菌素subtilosin的m/z3403.3 相同.[结论]Bacillus subtilis fmbR抗菌物质由C13~C15的3种surfactinA同系物和一种羊毛硫抗生素subtilosin组成.

  9. A microbial transformation using Bacillus subtilis B7-S to produce natural vanillin from ferulic acid

    Peng Chen; Lei Yan; Zhengrong Wu; Suyue Li; Zhongtian Bai; Xiaojuan Yan; Ningbo Wang; Ning Liang; Hongyu Li


    Bacillus subtilis strain B7-S screened from18 strains is an aerobic, endospore-forming, model organism of Gram-positive bacteria which is capable to form vanillin during ferulic acid bioconversion. The bioconversion of ferulic acid to vanillin by Bacillus subtilis B7-S (B. subtilis B7-S) was investigated. Based on our results, the optimum bioconversion conditions for the production of vanillin by B. subtilis B7-S can be summarized as follows: temperature 35 °C; initial pH 9.0; inoculum volume...

  10. Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916

    Luo, Chuping; Liu, Xuehui; Zhou, Xian; Guo, Junyao; Truong, John; Wang, Xiaoyu; Zhou, Huafei


    Three families of Bacillus cyclic lipopeptides—surfactins, iturins, and fengycins—have well-recognized potential uses in biotechnology and biopharmaceutical applications. This study outlines the isolation and characterization of locillomycins, a novel family of cyclic lipopeptides produced by Bacillus subtilis 916. Elucidation of the locillomycin structure revealed several molecular features not observed in other Bacillus lipopeptides, including a unique nonapeptide sequence and macrocyclization. Locillomycins are active against bacteria and viruses. Biochemical analysis and gene deletion studies have supported the assignment of a 38-kb gene cluster as the locillomycin biosynthetic gene cluster. Interestingly, this gene cluster encodes 4 proteins (LocA, LocB, LocC, and LocD) that form a hexamodular nonribosomal peptide synthetase to biosynthesize cyclic nonapeptides. Genome analysis and the chemical structures of the end products indicated that the biosynthetic pathway exhibits two distinct features: (i) a nonlinear hexamodular assembly line, with three modules in the middle utilized twice and the first and last two modules used only once and (ii) several domains that are skipped or optionally selected. PMID:26162886

  11. Mutations that relieve nutritional repression of the Bacillus subtilis dipeptide permease operon.

    Slack, F J; Mueller, J P; Sonenshein, A L


    The Bacillus subtilis dciA operon encodes a dipeptide transport complex that is induced rapidly as cells enter stationary phase and initiate sporulation. Expression of this operon in growing cells is repressed by glucose, by a mixture of amino acids, and by the AbrB protein. A genetic screen was devised to identify mutations that allow inappropriate expression from the dciA promoter during growth. These mutations resulted in increased dciA transcription during growth in nutrient broth, in min...

  12. Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis

    Smith, Bradley T.; Grossman, Alan D.; Walker, Graham C.


    We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA2B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This recon...

  13. Isolation and characterization of topological specificity mutants of minD in Bacillus subtilis

    Karoui, M E; Errington, J


    In rod-shaped bacteria such as Bacillus subtilis, division site selection is mediated by MinC and MinD, which together function as a division inhibitor. Topological specificity is imposed by DivIVA, which ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at mid-cell. MinD plays a central role in this process, as it positions and activates MinC and is dependent on DivIVA for its own positioning at the poles. To investigate MinD activities furthe...

  14. Detection of Anthrax Simulants with Microcalorimetric Spectroscopy: Bacillus subtilis and Bacillus cereus Spores

    Arakawa, Edward T.; Lavrik, Nickolay V.; Datskos, Panos G.


    Recent advances in the development of ultrasensitive micromechanical thermal detectors have led to the advent of novel subfemtojoule microcalorimetric spectroscopy (CalSpec). On the basis of principles of photothermal IR spectroscopy combined with efficient thermomechanical transduction, CalSpec provides acquisition of vibrational spectra of microscopic samples and absorbates. We use CalSpec as a method of identifying nanogram quantities of biological micro-organisms. Our studies focus on Bacillus subtilis and Bacillus cereus spores as simulants for Bacillus anthracis spores. Using CalSpec, we measured IR spectra of B. subtilis and B. cereus spores present on surfaces in nanogram quantities (approximately 100 -1000 spores). The spectra acquired in the wavelength range of 690 -4000 cm-1 (2.5 -14.5 μm) contain information-rich vibrational signatures that reflect the different ratios of biochemical makeup of the micro-organisms. The distinctive features in the spectra obtained for the two types of micro-organism can be used to distinguish between the spores of the Bacillus family. As compared with conventional IR and Fourier-transform IR microscopic spectroscopy techniques, the advantages of the present technique include significantly improved sensitivity (at least a full order of magnitude), absence of expensive IR detectors, and excellent potential for miniaturization.

  15. Regulation of proteolysis in Bacillus subtilis: effects of calcium ions and energy poisons

    Bacillus subtilis cells carry out extensive intracellular proteolysis (k = 0.15-0.23/h) during sporulation. Protein degradation was measured in cells growing in chemically defined sporulation medium, by following the release of [14C]-leucine from the cells during spore formation. Sodium arsenate, carbonyl cyanide 3-chlorophenyl hydrazone, and sodium azide strongly inhibited proteolysis without altering cell viability greatly, which suggested that bulk proteolysis in B. subtilis is energy dependent. The authors have tested the hypothesis that the energy requirement may be for pumping in Ca2+. When [Ca2+] was -6, rates of proteolysis in sporulating cells were reduced 4-8 times that in cells in calcium ion- sufficient medium. Further, omission of Ca2+ from the medium prevented the increase in the activity of the major intracellular serine protease. However, the presence of energy poisons in the media at levels which inhibited proteolysis, had no detectable effect on the uptake of by cells [45Ca]. The authors concluded that B. subtilis cells required both metabolic energy and calcium ions for normal proteolysis

  16. Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta.

    Kumari, Lata; Kanwar, Shamsher S


    A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105 kDa), multimeric (pentamer of ∼21 kDa protein) nature, organic solvent compatibility, and a half-life of ∼2 h at 37 °C. The bacterial COX exhibited ∼22 % higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05 % (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25 mM and 2.17 μmol min ml(-1), respectively. The purified COX showed very little cytotoxicity associated with it. PMID:26453032

  17. Use of a Novel Report Protein to Study the Secretion Signal of Flagellin in Bacillus subtilis.

    Wang, Guangqiang; Xia, Yongjun; Xiong, Zhiqiang; Zhang, Hui; Ai, Lianzhong


    Flagellin (also called Hag) is the main component of bacterial flagellum and is transported across the cytoplasmic membrane by flagellar secretion apparatus. Because flagella play an essential role in the pathogenesis of numerous pathogens, the flagellins of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Campylobacter jejuni, and Vibrio cholerae have been intensively studied; however, very few studies have focused on the flagellin of Bacillus subtilis, which is considered to be a model organism with which to study the secretion of bacteria and is used on an industrial scale for the secretion of proteins. The signal of B. subtilis flagellin is still debated. This study was performed to seek the export signals of flagellin from B. subtilis. The naturally nonsecretory, intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was used as the reporter protein. Our results demonstrate that the export signal is contained within the first 50 amino acids of B. subtilis flagellin. Nsp is easily degraded inside the cell and can be exported into culture medium with the aid of the signal of flagellin. This method provides a new potential strategy for the expression of proteins with high proteolytic susceptibility via fusion to export signals. PMID:27154466

  18. Preliminary X-ray crystallographic studies of Bacillus subtilis SpeA protein

    In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme the speA gene was amplified from B. subtilis genomic DNA and cloned. The enzyme was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 298 K. The speA gene in Bacillus subtilis encodes arginine decarboxylase, which catalyzes the conversion of arginine to agmatine. Arginine decarboxylase is an important enzyme in polyamine metabolism in B. subtilis. In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme, the speA gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET-28a(+). SpeA was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 289 K. The best crystal diffracted to 2.0 Å resolution and belonged to space group P21, with unit-cell parameters a = 86.4, b = 63.3 c = 103.3 Å, β = 113.9°

  19. D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis.

    Leiman, Sara A; May, Janine M; Lebar, Matthew D; Kahne, Daniel; Kolter, Roberto; Losick, Richard


    The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. PMID:24097941

  20. Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of Bacillus subtilis

    Seydlová, G.; Fišer, R.; Čabala, R.; Kozlík, P.; Svobodová, J.; Pátek, Miroslav


    Roč. 1828, č. 11 (2013), s. 2370-2378. ISSN 0005-2736 Institutional support: RVO:61388971 Keywords : Surfactin * Bacillus subtilis * Membrane Subject RIV: EE - Microbiology, Virology Impact factor: 3.431, year: 2013

  1. Regiospecific Addition of Uracil to Acrylates Catalyzed by Alkaline Protease from Bacillus subtilis

    Ying CAI; Jian Yi WU; Na WANG; Xiao Feng SUN; Xian Fu LIN


    Michael addition reactions of uracil to acrylates were catalyzed by an alkaline protease from Bacillus subtilis in dimethyl sulfoxide at 55 ℃ for 72 h. The adducts were determined by TLC, IR and 1H NMR.

  2. Growth and sporulation of Bacillus subtilis under microgravity (7-IML-1)

    Mennigmann, Horst-Dieter


    The experiment was aimed at measuring the growth and sporulation of Bacillus subtilis under microgravity. The hardware for the experiment consists of a culture chamber (15 ml) made from titanium and closed by a membrane permeable for gases but not for water. Two variants of this basic structure were built which fit into the standard Biorack container types 1 and 2 respectively. Growth of the bacteria will be monitored by continuously measuring the optical density with a built-in miniaturized photometer. Other parameters (viability, sporulation, fine structure, size distribution of cells and spores, growth kinetics, etc.) will be measured on the fixed samples and on those where metabolism was temporarily halted, respectively.

  3. d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

    Leiman, Sara A.; May, Janine M.; Lebar, Matthew D.; Kahne, Daniel; Kolter, Roberto; Losick, Richard


    The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were ...

  4. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    Mars, Ruben A. T.; Pierre Nicolas; Mariano Ciccolini; Ewoud Reilman; Alexander Reder; Marc Schaffer; Ulrike Mäder; Uwe Völker; Jan Maarten van Dijl; Denham, Emma L.


    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 inter...

  5. Septation, dephosphorylation, and the activation of σF during sporulation in Bacillus subtilis

    King, Nicole; Dreesen, Oliver; Stragier, Patrick; Pogliano, Kit; Losick, Richard


    Cell-specific activation of transcription factor σF during sporulation in Bacillus subtilis requires the formation of the polar septum and the activity of a serine phosphatase (SpoIIE) located in the septum. The SpoIIE phosphatase indirectly activates σF by dephosphorylating a protein (SpoIIAA-P) in the pathway that controls the activity of the transcription factor. By use of a SpoIIE–GFP fusion protein in time-course and time-lapse experiments and by direct visualization of septa in living c...

  6. Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.

    Nicholson, W L; Chambliss, G H


    Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strai...

  7. Production, purification, and characterization of a-amylase by Bacillus subtilis and its mutant derivates

    DEMİRKAN, Elif


    The effects of various carbon and nitrogen sources on production of a-amylase by Bacillus subtilis and its mutant derivates were investigated. The maximum production of a-amylase by all strains was obtained in the presence of mesoinositol as the carbon source. There was no more significant increase in enzyme yield in the case of the supplementation of nitrogen sources, whereas malt extract and tryptone were preferred nitrogen sources for amylase production by Bacillus subtilis and mutant U 2-...

  8. Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production.

    Posada-Uribe, Luisa F; Romero-Tabarez, Magally; Villegas-Escobar, Valeska


    Bacillus subtilis spores have important biotechnological applications; however, achieving both, high spore cell densities and sporulation efficiencies in fermentation, is poorly reported. In this study, medium components and culture conditions were optimized with different statistical methods to increase spore production of the plant growth promoting rhizobacteria B. subtilis EA-CB0575. Key medium components were determined with Plackett-Burman (PB) design, and the optimum concentration levels of two components (glucose, MgSO4·7H2O) were optimized with a full factorial and central composite design, achieving 1.37 × 10(9) CFU/mL of spore cell density and 93.5 % of sporulation efficiency in shake flask. The optimized medium was used to determine the effect of culture conditions on spore production at bioreactor level, finding that maintaining pH control did not affect significantly spore production, while the interaction of agitation and aeration rates had a significant effect on spore cell density. The overall optimization generated a 17.2-fold increase in spore cell density (8.78 × 10(9) CFU/mL) and 1.9-fold increase in sporulation efficiency (94.2 %) compared to that of PB design. These results indicate the potential of B. subtilis EA-CB0575 to produce both, high spore cell densities and sporulation efficiencies, with very low nutrient requirements and short incubation period which can represent savings of process production. PMID:26135004

  9. Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid.

    Moran, C P; Losick, R; Sonenshein, A L


    A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation. This is the first demonstration of a spo locus in cloned B. subtilis deoxyribonucleic acid.

  10. Induced sensitivity of Bacillus subtilis colony morphology to mechanical media compression

    Jessica K. Polka


    Full Text Available Bacteria from several taxa, including Kurthia zopfii, Myxococcus xanthus, and Bacillus mycoides, have been reported to align growth of their colonies to small features on the surface of solid media, including anisotropies created by compression. While the function of this phenomenon is unclear, it may help organisms navigate on solid phases, such as soil. The origin of this behavior is also unknown: it may be biological (that is, dependent on components that sense the environment and regulate growth accordingly or merely physical. Here we show that B. subtilis, an organism that typically does not respond to media compression, can be induced to do so with two simple and synergistic perturbations: a mutation that maintains cells in the swarming (chained state, and the addition of EDTA to the growth media, which further increases chain length. EDTA apparently increases chain length by inducing defects in cell separation, as the treatment has only marginal effects on the length of individual cells. These results lead us to three conclusions. First, the wealth of genetic tools available to B. subtilis will provide a new, tractable chassis for engineering compression sensitive organisms. Second, the sensitivity of colony morphology to media compression in Bacillus can be modulated by altering a simple physical property of rod-shaped cells. And third, colony morphology under compression holds promise as a rapid, simple, and low-cost way to screen for changes in the length of rod-shaped cells or chains thereof.

  11. Isolation and Characterization of Phages Infecting Bacillus subtilis

    Anna Krasowska


    Full Text Available Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages or noncontractile (ARπ phage tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0 and alkaline (9.0 and 10.0 pH.

  12. The studies on radiation mutation breeding of Bacillus subtilis with high-yield of amylase

    The mutagenesis effects on the yield of amylase have been investigated with Bacillus subtilis irradiated by γ-rays and fast neutrons in once or twice irradiation at various dose rates and total irradiation doses. Several parameters such as flat transparent circle, colony diameter, transparent circle diameter and the ratio of flat transparent circle to colony diameter (HC) are used to estimate the radiation mutation of Bacillus subtilis. A series of results has been obtained as (1) Irradiation both with neutrons and γ-rays could make Bacillus subtilis mutationed to produce high-yield amylase effectively. (2) The average colony diameter of Bacillus subtilis irradiated by γ-rays or fast neutrons is smaller than that of control group at various total doses and dose rates. And their colony diameter becomes smaller slightly with the increment of γ-rays irradiation dose. (3) After the second neutrons irradiation, the values of average colony diameter, the biggest colony diameter, average transparent circle diameter and the biggest transparent circle diameter of all mutationed Bacillus subtilis exceed that of original strains greatly. (4) Three kinds of mutationed Bacillus subtilis strains with high-yield amylase have been screened out, in which two strains can produce high-yield amylase steadily after 15 times breeding. Their biggest colony diameter, the biggest transparent circle diameter and the biggest HC value are up to 8.32 mm, 22.38 mm and 5.39 respectively. (authors)

  13. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    Mars, Ruben A T; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Mäder, Ulrike; Völker, Uwe; van Dijl, Jan Maarten; Denham, Emma L


    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions. PMID:25790031

  14. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    Ruben A T Mars


    Full Text Available Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

  15. Complete Genome Sequences of Bacillus subtilis subsp. subtilis Laboratory Strains JH642 (AG174) and AG1839

    Smith, Janet L.; Goldberg, Jonathan M.; Grossman, Alan D.


    The Gram-positive bacterium Bacillus subtilis is widely used for studies of cellular and molecular processes. We announce the complete genomic sequences of strain AG174, our stock of the commonly used strain JH642, and strain AG1839, a derivative that contains a mutation in the replication initiation gene dnaB and a linked Tn917.

  16. Efficient whole-cell biocatalyst for acetoin production with NAD+ regeneration system through homologous co-expression of 2,3-butanediol dehydrogenase and NADH oxidase in engineered Bacillus subtilis.

    Teng Bao

    Full Text Available Acetoin (3-hydroxy-2-butanone, an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold and NADH/NAD+ ratio (2.2 fold. By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h, which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products.

  17. Crude glycerol from biodiesel industry as substrate for biosurfactant production by Bacillus subtilis ATCC 6633

    Marylane de Sousa


    Full Text Available Glycerol, a co-product of the biodiesel industry, may be a suitable raw material for the production of high added-value compounds by the microorganisms. This study aimed to use the glycerol obtained from the biodiesel production process as the main carbon source for biosurfactant production by Bacillus subtilis ATCC 6633. Results indicated that the strain lowered the surface tension of the cell-free fermented broth to 31.5 ± 1.6 mN/m, indicating the production of biosurfactant. The critical micelle concentration (CMC = 33.6 mN/m obtained was similar to the previously reported for biossurfactants isolated from other Bacillus. The produced biosurfactant was able to emulsify n-hexadecane and soybean oil.

  18. Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis


    The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 ℃ .

  19. Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis


    The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.

  20. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca

    Romero, Diego; de Vicente, Antonio; Rakotoaly, Rivo H.; Dufour, Samuel E.; Veening, Jan-Willem; Arrebola, Eva; Cazorla, Francisco M.; Kuipers, Oscar P; Paquot, Michel; Perez-Garcia, Alejandro; Stacey, Gary


    Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lip...

  1. Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product from Bacillus subtilis

    Inaoka, Takashi; MATSUMURA, Yoshinobu; TSUCHIDO, Tetsuaki


    Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 ...

  2. Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis

    Meima, R; Haijema, BJ; Haan, GJ; Venema, G; Bron, S


    The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the

  3. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S


    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  4. Bacillus subtilis subsp. subtilis CBMDC3f with antimicrobial activity against Gram-positive foodborne pathogenic bacteria: UV-MALDI-TOF MS analysis of its bioactive compounds.

    Torres, M J; Petroselli, G; Daz, M; Erra-Balsells, R; Audisio, M C


    In this work a new Bacillus sp. strain, isolated from honey, was characterized phylogenetically. Its antibacterial activity against three relevant foodborne pathogenic bacteria was studied; the main bioactive metabolites were analyzed using ultraviolet matrix assisted laser desorption-ionization mass spectrometry (UV-MALDI MS). Bacillus CBMDC3f was phylogenetically characterized as Bacillus subtilis subsp. subtilis after rRNA analysis of the 16S subunit and the gyrA gene (access codes Genbank JX120508 and JX120516, respectively). Its antibacterial potential was evaluated against Listeria monocytogenes (9 strains), B. cereus (3 strains) and Staphylococcus aureus ATCC29213. Its cell suspension and cell-free supernatant (CFS) exerted significant anti-Listeria and anti-S. aureus activities, while the lipopeptides fraction (LF) also showed anti-B. cereus effect. The UV-MALDI-MS analysis revealed surfactin, iturin and fengycin in the CFS, whereas surfactin predominated in the LF. The CFS from CBMDC3f contained surfactin, iturin and fengycin with four, two and four homologues per family, respectively, whereas four surfactin, one iturin and one fengycin homologues were identified in the LF. For some surfactin homologues, their UV-MALDI-TOF/TOF (MS/MS; Laser Induced Decomposition method, LID) spectra were also obtained. Mass spectrometry analysis contributed with relevant information about the type of lipopeptides that Bacillus strains can synthesize. From our results, surfactin would be the main metabolite responsible for the antibacterial effect. PMID:25820813

  5. Bacillus subtilis FZB24® Affects Flower Quantity and Quality of Saffron (Crocus sativus)

    Sharaf-Eldin, Mahmoud; Elkholy, Shereen; Fernández, José-Antonio; Junge, Helmut; Cheetham, Ronald; Guardiola, José; Weathers, Pamela


    The effect of Bacillus subtilis FZB24® on saffron (Crocus sativus L.) was studied using saffron corms from Spain and the powdered form of B. subtilis FZB24®. Corms were soaked in water or in B. subtilis FZB24 spore solution for 15min before sowing. Some corms were further soil drenched with the spore solution 6, 10 or 14 weeks after sowing. Growth and saffron stigma chemical composition were measured. Compared to untreated controls, application of B. subtilis FZB24 significantly increased lea...

  6. Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of Bacillus subtilis by Inclusion in a Model Membrane

    Searls, Tim; Chen, Xingyong; Allen, Stephanie; Yudkin, Michael D.


    Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor σF, and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of B. subtilis precedes dephosphorylation of the anti-anti-sigma fac...

  7. Second Messenger Signaling in Bacillus subtilis: Accumulation of Cyclic di-AMP Inhibits Biofilm Formation.

    Gundlach, Jan; Rath, Hermann; Herzberg, Christina; Mäder, Ulrike; Stülke, Jörg


    The Gram-positive model organism Bacillus subtilis produces the essential second messenger signaling nucleotide cyclic di-AMP. In B. subtilis and other bacteria, c-di-AMP has been implicated in diverse functions such as control of metabolism, cell division and cell wall synthesis, and potassium transport. To enhance our understanding of the multiple functions of this second messenger, we have studied the consequences of c-di-AMP accumulation at a global level by a transcriptome analysis. C-di-AMP accumulation affected the expression of about 700 genes, among them the two major operons required for biofilm formation. The expression of both operons was severely reduced both in the laboratory and a non-domesticated strain upon accumulation of c-di-AMP. In excellent agreement, the corresponding strain was unable to form complex colonies. In B. subtilis, the transcription factor SinR controls the expression of biofilm genes by binding to their promoter regions resulting in transcription repression. Inactivation of the sinR gene restored biofilm formation even at high intracellular c-di-AMP concentrations suggesting that the second messenger acts upstream of SinR in the signal transduction pathway. As c-di-AMP accumulation did not affect the intracellular levels of SinR, we conclude that the nucleotide affects the activity of SinR. PMID:27252699

  8. Second Messenger Signaling in Bacillus subtilis: Accumulation of Cyclic di-AMP Inhibits Biofilm Formation

    Gundlach, Jan; Rath, Hermann; Herzberg, Christina; Mäder, Ulrike; Stülke, Jörg


    The Gram-positive model organism Bacillus subtilis produces the essential second messenger signaling nucleotide cyclic di-AMP. In B. subtilis and other bacteria, c-di-AMP has been implicated in diverse functions such as control of metabolism, cell division and cell wall synthesis, and potassium transport. To enhance our understanding of the multiple functions of this second messenger, we have studied the consequences of c-di-AMP accumulation at a global level by a transcriptome analysis. C-di-AMP accumulation affected the expression of about 700 genes, among them the two major operons required for biofilm formation. The expression of both operons was severely reduced both in the laboratory and a non-domesticated strain upon accumulation of c-di-AMP. In excellent agreement, the corresponding strain was unable to form complex colonies. In B. subtilis, the transcription factor SinR controls the expression of biofilm genes by binding to their promoter regions resulting in transcription repression. Inactivation of the sinR gene restored biofilm formation even at high intracellular c-di-AMP concentrations suggesting that the second messenger acts upstream of SinR in the signal transduction pathway. As c-di-AMP accumulation did not affect the intracellular levels of SinR, we conclude that the nucleotide affects the activity of SinR. PMID:27252699

  9. Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6

    Bashir, Shaista; Sadaf, Saima; Ahmad, Sajjad; Akhtar, Muhammad Waheed


    This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer. PMID:26881203

  10. Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source.

    Saori Kosono

    Full Text Available Lysine residues can be post-translationally modified by various acyl modifications in bacteria and eukarya. Here, we showed that two major acyl modifications, acetylation and succinylation, were changed in response to the carbon source in the Gram-positive model bacterium Bacillus subtilis. Acetylation was more common when the cells were grown on glucose, glycerol, or pyruvate, whereas succinylation was upregulated when the cells were grown on citrate, reflecting the metabolic states that preferentially produce acetyl-CoA and succinyl-CoA, respectively. To identify and quantify changes in acetylation and succinylation in response to the carbon source, we performed a stable isotope labeling by amino acids in cell culture (SILAC-based quantitative proteomic analysis of cells grown on glucose or citrate. We identified 629 acetylated proteins with 1355 unique acetylation sites and 204 succinylated proteins with 327 unique succinylation sites. Acetylation targeted different metabolic pathways under the two growth conditions: branched-chain amino acid biosynthesis and purine metabolism in glucose and the citrate cycle in citrate. Succinylation preferentially targeted the citrate cycle in citrate. Acetylation and succinylation mostly targeted different lysine residues and showed a preference for different residues surrounding the modification sites, suggesting that the two modifications may depend on different factors such as characteristics of acyl-group donors, molecular environment of the lysine substrate, and/or the modifying enzymes. Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome. Mutations that modulate protein acylation affected B. subtilis growth. A mutation in acetate kinase (ackA increased the global acetylation level, suggesting that acetyl phosphate-dependent acetylation is

  11. Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

    Luo, Chuping; Liu, Xuehui; Zhou, Huafei; Wang, Xiaoyu; Chen, Zhiyi


    Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called loc...

  12. Regulation of the tryptophan biosynthetic genes in Bacillus halodurans: common elements but different strategies than those used by Bacillus subtilis.

    Szigeti, Reka; Milescu, Mirela; Gollnick, Paul


    In Bacillus subtilis, an RNA binding protein called TRAP regulates both transcription and translation of the tryptophan biosynthetic genes. Bacillus halodurans is an alkaliphilic Bacillus species that grows at high pHs. Previous studies of this bacterium have focused on mechanisms of adaptation for growth in alkaline environments. We have characterized the regulation of the tryptophan biosynthetic genes in B. halodurans and compared it to that in B. subtilis. B. halodurans encodes a TRAP protein with 71% sequence identity to the B. subtilis protein. Expression of anthranilate synthetase, the first enzyme in the pathway to tryptophan, is regulated significantly less in B. halodurans than in B. subtilis. Examination of the control of the B. halodurans trpEDCFBA operon both in vivo and in vitro shows that only transcription is regulated, whereas in B. subtilis both transcription of the operon and translation of trpE are controlled. The attenuation mechanism that controls transcription in B. halodurans is similar to that in B. subtilis, but there are some differences in the predicted RNA secondary structures in the B. halodurans trp leader region, including the presence of a potential anti-antiterminator structure. Translation of trpG, which is within the folate operon in both bacilli, is regulated similarly in the two species. PMID:14729709

  13. Activity of essential oils against Bacillus subtilis spores.

    Lawrence, Hayley A; Palombo, Enzo A


    Alternative methods for controlling bacterial endospore contamination are desired in a range of industries and applications. Attention has recently turned to natural products, such as essential oils, which have sporicidal activity. In this study, a selection of essential oils was investigated to identify those with activity against Bacillus subtilis spores. Spores were exposed to thirteen essential oils, and surviving spores were enumerated. Cardamom, tea tree, and juniper leaf oils were the most effective, reducing the number of viable spores by 3 logs at concentrations above 1%. Sporicidal activity was enhanced at high temperatures (60 degrees C) or longer exposure times (up to one week). Gas chromatography-mass spectrometry analysis identified the components of the active essential oils. However, none of the major oil components exhibited equivalent activity to the whole oils. The fact that oil components, either alone or in combination, did not show the same level of sporicidal activity as the complete oils suggested that minor components may be involved, or that these act synergistically with major components. Scanning electron microscopy was used to examine spores after exposure to essential oils and suggested that leakage of spore contents was the likely mode of sporicidal action. Our data have shown that essential oils exert sporicidal activity and may be useful in applications where bacterial spore reduction is desired. PMID:20075624

  14. Dynamics of Aerial Tower Formation in Bacillus subtilis Biofilms

    Sinha, Naveen; Seminara, Agnese; Wilking, James; Brenner, Michael; Weitz, Dave


    Biofilms are highly-organized colonies of bacteria that form on surfaces. These colonies form sophisticated structures which make them robust and difficult to remove from environments such as catheters, where they pose serious infection problems. Previous work has shown that sub-mm sized aerial towers form on the surface of Bacillus subtilis colony biofilms. Spore-formation is located preferentially at the tops of these towers, known as fruiting bodies, which aid in the dispersal and propagation of the colony to new sites. The formation of towers is strongly affected by the quorum-sensing molecule surfactin and the cannibalism pathway of the bacteria. In the present work, we use confocal fluorescence microscopy to study the development of individual fruiting bodies, allowing us to visualize the time-dependent spatial distribution of matrix-forming and sporulating bacteria within the towers. With this information, we investigate the physical mechanisms, such as surface tension and polymer concentration gradients, that drive the formation of these structures.

  15. Tip-enhanced Raman scattering of bacillus subtilis spores

    Rusciano, G.; Zito, G.; Pesce, G.; Sasso, A.; Isticato, R.; Ricca, E.


    Understanding of the complex interactions of molecules at biological interfaces is a fundamental issue in biochemistry, biotechnology as well as biomedicine. A plethora of biological processes are ruled by the molecular texture of cellular membrane: cellular communications, drug transportations and cellular recognition are just a few examples of such chemically-mediated processes. Tip-Enhanced Raman Scattering (TERS) is a novel, Raman-based technique which is ideally suited for this purpose. TERS relies on the combination of scanning probe microscopy and Raman spectroscopy. The basic idea is the use of a metalled tip as a sort of optical nano-antenna, which gives place to SERS effect close to the tip end. Herein, we present the application of TERS to analyze the surface of Bacillus subtilis spores. The choice of this biological systems is related to the fact that a number of reasons support the use of spores as a mucosal delivery system. The remarkable and well-documented resistance of spores to various environmental and toxic effects make them clear potentials as a novel, surface-display system. Our experimental outcomes demonstrate that TERS is able to provide a nano-scale chemical imaging of spore surface. Moreover, we demonstrate that TERS allows differentiation between wilde-type spore and genetically modified strains. These results hold promise for the characterization and optimization of spore surface for drug-delivery applications.

  16. A Low Dimensional Approximation For Competence In Bacillus Subtilis.

    Nguyen, An; Prugel-Bennett, Adam; Dasmahapatra, Srinandan


    The behaviour of a high dimensional stochastic system described by a chemical master equation (CME) depends on many parameters, rendering explicit simulation an inefficient method for exploring the properties of such models. Capturing their behaviour by low-dimensional models makes analysis of system behaviour tractable. In this paper, we present low dimensional models for the noise-induced excitable dynamics in Bacillus subtilis, whereby a key protein ComK, which drives a complex chain of reactions leading to bacterial competence, gets expressed rapidly in large quantities (competent state) before subsiding to low levels of expression (vegetative state). These rapid reactions suggest the application of an adiabatic approximation of the dynamics of the regulatory model that, however, lead to competence durations that are incorrect by a factor of 2. We apply a modified version of an iterative functional procedure that faithfully approximates the time-course of the trajectories in terms of a two-dimensional model involving proteins ComK and ComS. Furthermore, in order to describe the bimodal bivariate marginal probability distribution obtained from the Gillespie simulations of the CME, we introduce a tunable multiplicative noise term in a two-dimensional Langevin model whose stationary state is described by the time-independent solution of the corresponding Fokker-Planck equation. PMID:27045827

  17. Mutagenesis of Bacillus subtilis spores exposed to simulated space environment

    Munakata, N.; Natsume, T.; Takahashi, K.; Hieda, K.; Panitz, C.; Horneck, G.

    Bacterial spores can endure in a variety of extreme earthly environments. However, some conditions encountered during the space flight could be detrimental to DNA in the spore, delimiting the possibility of transpermia. We investigate the genetic consequences of the exposure to space environments in a series of preflight simulation project of EXPOSE. Using Bacillus subtilis spores of repair-proficient HA101 and repair-deficient TKJ6312 strains, the mutations conferring resistance to rifampicin were detected, isolated and sequenced. Most of the mutations were located in a N-terminal region of the rpoB gene encoding RNA polymerase beta-subunit. Among several potentially mutagenic factors, high vacuum, UV radiation, heat, and accelerated heavy ions induced mutations with varying efficiencies. A majority of mutations induced by vacuum exposure carried a tandem double-base change (CA to TT) at a unique sequence context of TCAGC. Results indicate that the vacuum and high temperature may act synergistically for the induction of mutations.

  18. Screening of Bacillus subtilis transposon mutants with altered riboflavin production.

    Tännler, Simon; Zamboni, Nicola; Kiraly, Csilla; Aymerich, Stéphane; Sauer, Uwe


    To identify novel targets for metabolic engineering of riboflavin production, we generated about 10,000 random, transposon-tagged mutants of an industrial, riboflavin-producing strain of Bacillus subtilis. Process-relevant screening conditions were established by developing a 96-deep-well plate method with raffinose as the carbon source, which mimics, to some extent, carbon limitation in fed batch cultures. Screening in raffinose and complex LB medium identified more efficiently riboflavin overproducing and underproducing mutants, respectively. As expected for a "loss of function" analysis, most identified mutants were underproducers. Insertion mutants in two genes with yet unknown function, however, were found to attain significantly improved riboflavin titers and yields. These genes and possibly further ones that are related to them are promising candidates for metabolic engineering. While causal links to riboflavin production were not obvious for most underproducers, we demonstrated for the gluconeogenic glyceraldehyde-3-phosphate dehydrogenase GapB how a novel, non-obvious metabolic engineering strategy can be derived from such underproduction mutations. Specifically, we improved riboflavin production on various substrates significantly by deregulating expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor CcpN. This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture. PMID:18582593

  19. Loop grafting of Bacillus subtilis lipase A: inversion of enantioselectivity.

    Boersma, Ykelien L; Pijning, Tjaard; Bosma, Margriet S; van der Sloot, Almer M; Godinho, Luís F; Dröge, Melloney J; Winter, Remko T; van Pouderoyen, Gertie; Dijkstra, Bauke W; Quax, Wim J


    Lipases are successfully applied in enantioselective biocatalysis. Most lipases contain a lid domain controlling access to the active site, but Bacillus subtilis Lipase A (LipA) is a notable exception: its active site is solvent exposed. To improve the enantioselectivity of LipA in the kinetic resolution of 1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the active-site entrance by longer loops originating from Fusarium solani cutinase and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the interaction surface for the substrate. The resulting loop hybrids showed enantioselectivities inverted toward the desired enantiomer of IPG. The acetylxylan esterase-derived variant showed an inversion in enantiomeric excess (ee) from -12.9% to +6.0%, whereas the cutinase-derived variant was improved to an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was further improved by directed evolution to an ee of +57.4%. PMID:18721749

  20. Ectopic integration vectors for generating fluorescent promoter fusions in Bacillus subtilis with minimal dark noise.

    Stephanie Trauth

    Full Text Available Fluorescent protein promoter reporters are important tools that are widely used for diverse purposes in microbiology, systems biology and synthetic biology and considerable engineering efforts are still geared at improving the sensitivity of the reporter systems. Here we focus on dark noise, i.e. the signal that is generated by the empty vector control. We quantitatively characterize the dark noise of a few common bacterial reporter systems by single cell microscopy. All benchmarked reporter systems generated significant amounts of dark noise that exceed the cellular autofluorescence to different extents. We then reengineered a multicolor set of fluorescent ectopic integration vectors for Bacillus subtilis by introducing a terminator immediately upstream of the promoter insertion site, resulting in an up to 2.7-fold reduction of noise levels. The sensitivity and dynamic range of the new high-performance pXFP_Star reporter system is only limited by cellular autofluorescence. Moreover, based on studies of the rapE promoter of B. subtilis we show that the new pXFP_Star reporter system reliably reports on the weak activity of the rapE promoter whereas the original reporter system fails because of transcriptional interference. Since the pXFP_Star reporter system properly isolates the promoter from spurious transcripts, it is a particularly suitable tool for quantitative characterization of weak promoters in B. subtilis.

  1. Protein-tyrosine phosphorylation in Bacillus subtilis: a 10-year retrospective

    Josef eDeutscher


    Full Text Available The discovery of tyrosine-phosphorylated proteins in Bacillus subtilis in the year 2003 was followed by a decade of intensive research activity. Here we provide an overview of the lessons learned in that period. While the number of characterized kinases and phosphatases involved in reversible protein-tyrosine phosphorylation in B. subtilis has remained essentially unchanged, the number of proteins known to be targeted by this post-translational modification has increased dramatically. This is mainly due to phosphoproteomics and interactomics studies, which were instrumental in identifying new tyrosine-phosphorylated proteins. Despite their structural similarity, the two B. subtilis protein-tyrosine kinases (BY-kinases, PtkA and PtkB (EpsB, seem to accomplish different functions in the cell. The PtkB is encoded by a large operon involved in exopolysaccharide production, and its main role appears to be the control of this process. The PtkA seems to have a more complex role; it phosphorylates and regulates a large number of proteins involved in the DNA, fatty acid and carbon metabolism and engages in physical interaction with other types of kinases (Ser/Thr kinases, leading to mutual phosphorylation. PtkA also seems to respond to several activator proteins, which direct its activity towards different substrates. In that respect PtkA seems to function as a highly connected signal integration device.

  2. Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida.

    Troeschel, Sonja Christina; Thies, Stephan; Link, Olga; Real, Catherine Isabell; Knops, Katja; Wilhelm, Susanne; Rosenau, Frank; Jaeger, Karl-Erich


    Novel shuttle vectors named pEBP were constructed to allow the gene expression in different bacterial hosts including Escherichia coli, Bacillus subtilis and Pseudomonas putida. These vectors share the inducible promoters P(T7) and P(Xyl) and a cos site to enable packaging of plasmid DNA into phage, and carry different multiple cloning sites and antibiotic resistance genes. Vector pEBP41 generally replicates episomally while pEBP18 replicates episomally in Gram-negative bacteria only, but integrates into the chromosome of B. subtilis. Plasmid copy numbers determined for E. coli and P. putida were in the range of 5-50 per cell. The functionality of pEBP18 and pEBP41 was confirmed by expression of two lipolytic enzymes, namely lipase A from B. subtilis and cutinase from the eukaryotic fungus Fusarium solani pisi in three different host strains. Additionally, we report here the construction of a T7 RNA polymerase-based expression strain of P. putida. PMID:22440389

  3. Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores.

    Loison, Pauline; Gervais, Patrick; Perrier-Cornet, Jean-Marie; Kuimova, Marina K


    In this work, we investigated how a combination of ethanol and high temperature (70°C), affect the properties of the inner membrane of Bacillus subtilis spores. We observed membrane permeabilization for ethanol concentrations ≥50%, as indicated by the staining of the spores' DNA by the cell impermeable dye Propidium Iodide. The loss of membrane integrity was also confirmed by a decrease in the peak corresponding to dipicolinic acid using infrared spectroscopy. Finally, the spore refractivity (as measured by phase contrast microscopy) was decreased after the ethanol-heat treatment, suggesting a partial rehydration of the protoplast. Previously we have used fluorescent lifetime imaging microscopy (FLIM) combined with the fluorescent molecular rotor Bodipy-C12 to study the microscopic viscosity in the inner membrane of B. subtilis spores, and showed that at normal conditions it is characterized by a very high viscosity. Here we demonstrate that the ethanol/high temperature treatment led to a decrease of the viscosity of the inner membrane, from 1000cP to 860cP for wild type spores at 50% of ethanol. Altogether, our present work confirms the deleterious effect of ethanol on the structure of B. subtilis spores, as well as demonstrates the ability of FLIM - Bodipy-C12 to measure changes in the microviscosity of the spores upon perturbation. PMID:27267704

  4. Tryptophan provision by dietary supplementation of a Bacillus subtilis mutant strain in piglets

    Torres-Pitarch, A; Nielsen, B.; Canibe, Nuria;


    Supplementing Bacillus (B.) subtilis mutants selected to overproduce a specific amino acid (AA) may be an alternative method to provide essential AA in pig diets. Two experiments on a B. subtilis strain selected to overproduce Trp were conducted using 8-kg pigs fed Trp-deficient diets for 20 d. B....... subtilis were supplied in a low or high dose in Experiments 1 and 2, respectively. The Trp-deficient diet (0.15 SID Trp:Lys) reduced (p < .05) both gain and feed intake of piglets compared to the positive control diet (0.17 SID Trp:Lys). Supplementation of the B. subtilis strain was not able to...... counterbalance the Trp deficiency in any of the two experiments. No effect of B. subtilis supplementation to piglet diets was observed on the plasma AA profile. In conclusion, this mutant strain of B. subtilis was not able to compensate a Trp deficiency in the tested doses....

  5. Simulation of Bacillus subtilis biofilm growth on agar plate by diffusion–reaction based continuum model

    Zhang, Xianlong; Wang, Xiaoling; Nie, Kai; Li, Mingpeng; Sun, Qingping


    Various species of bacteria form highly organized spatially-structured aggregates known as biofilms. To understand how microenvironments impact biofilm growth dynamics, we propose a diffusion–reaction continuum model to simulate the formation of Bacillus subtilis biofilm on an agar plate. The extended finite element method combined with level set method are employed to perform the simulation, numerical results show the quantitative relationship between colony morphologies and nutrient depletion over time. Considering that the production of polysaccharide in wild-type cells may enhance biofilm spreading on the agar plate, we inoculate mutant colony incapable of producing polysaccharide to verify our results. Predictions of the glutamate source biofilm’s shape parameters agree with the experimental mutant colony better than that of glycerol source biofilm, suggesting that glutamate is rate limiting nutrient for Bacillus subtilis biofilm growth on agar plate, and the diffusion-limited is a better description to the experiment. In addition, we find that the diffusion time scale is of the same magnitude as growth process, and the common-employed quasi-steady approximation is not applicable here.

  6. Phosphorylation on histidine is accompanied by localized structural changes in the phosphocarrier protein, HPr from Bacillus subtilis.

    Jones, B. E.; Rajagopal, P.; Klevit, R. E.


    The histidine-containing protein (HPr) of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) serves a central role in a series of phosphotransfer reactions used for the translocation of sugars across cell membranes. These studies report the high-definition solution structures of both the unphosphorylated and histidine phosphorylated (P-His) forms of HPr from Bacillus subtilis. Consistent with previous NMR studies, local conformational adjustments occur upon phosphorylation of...

  7. Purification of polypeptide P6 with a molecular weight of 6,000 from the vegetative chromosomes of Bacillus subtilis.

    Nakayama, T.; Kurogi, Y; Irikura, M


    Folded chromosomes were prepared as membrane-associated complexes from vegetative cells of Bacillus subtilis by stepwise sucrose gradient centrifugation. From nucleoids, a deoxyribonucleic acid-bound polypeptide with a molecular weight of 6,000 (P6) was purified by KCl-(NH4)2SO4 salting out, diethylaminoethyl cellulose column chromatography, and deoxyribonucleic acid cellulose column chromatography. The amino acid composition of polypeptide P6 was determined.

  8. Suppression of Magnaporthe oryzae and interaction between Bacillus subtilis and rice plants in the control of rice blast.

    Sha, Yuexia; Wang, Qi; Li, Yan


    Magnaporthe oryzae, the causative pathogen of rice blast, has caused extensive losses to rice cultivation worldwide. Strains of the bacterium Bacillus subtilis have been used as biocontrol agents against rice blast. However, little has been reported about the interaction between B. subtilis and the rice plant and its mechanism of action. Here, the colonization process and induced disease resistance by B. subtilis SYX04 and SYX20 in rice plants was examined. Strains of B. subtilis labeled with green fluorescent protein reached population of more than 5 × 10(6) CFU/g after 20 days on mature rice leaves and were detected after 3 days on newly grown leaves. Results showed that SYX04 and SYX20 not only inhibited spore germination, germ tube length, and appressorial formation but also caused a series of alterations in the structures of hyphae and conidia. The cell walls and membrane structures of the fungus showed ultrastructural abnormalities, which became severely degraded as observed through scanning electron microscopy and transmission electron microscopy. The mixture of both B. subtilis and M. oryzae resulted in enhanced activity of peroxidase, and polyphenol oxidase while there was significantly more superoxide dismutase activity in plants that had been sprayed with B. subtilis alone. The present study suggests that colonized SYX04 and SYX20 strains protected rice plants and exhibited antifungal activity and induced systemic resistance, thus indicating their potential biological control agents. PMID:27536521

  9. Identification of surfactins and iturins produced by potent fungal antagonist, Bacillus subtilis K1 isolated from aerial roots of banyan (Ficus benghalensis) tree using mass spectrometry

    Pathak, Khyati V.; Keharia, Hareshkumar


    The banyan endophyte, Bacillus subtilis K1, produces a complex mixture of lipopeptides exhibiting potent antifungal activity. These lipopeptides were purified by high-performance liquid chromatography and analyzed using MALDI-TOF-MS as well as liquid chromatography coupled with ESI-MS. A heterogenous mixture of lipopeptides belonging to three different families of cyclic lipopeptides, viz., fengycins, iturins and surfactins, was detected in the cell-free extracellular extract of B. subtilis K...

  10. Chitinase production by Bacillus subtilis ATCC 11774 and its effect on biocontrol of Rhizoctonia diseases of potato.

    Saber, Wesam I A; Ghoneem, Khalid M; Al-Askar, Abdulaziz A; Rashad, Younes M; Ali, Abeer A; Rashad, Ehsan M


    Stem canker and black scurf of potato, caused by Rhizoctonia solani, can be serious diseases causing an economically significant damage. Biocontrol activity of Bacillus subtilis ATCC 11774 against the Rhizoctonia diseases of potato was investigated in this study. Chitinase enzyme was optimally produced by B. subtilis under batch fermentation conditions similar to those of the potato-growing soil. The maximum chitinase was obtained at initial pH 8 and 30 °C. In vitro, the lytic action of the B. subtilis chitinase was detected releasing 355 μg GlcNAc ml⁻¹ from the cell wall extract of R. solani and suggesting the presence of various chitinase enzymes in the bacterial filtrate. In dual culture test, the antagonistic behavior of B. subtilis resulted in the inhibition of the radial growth of R. solani by 48.1% after 4 days. Moreover, the extracted B. subtilis chitinase reduced the growth of R. solani by 42.3% when incorporated with the PDA plates. Under greenhouse conditions, application of a bacterial suspension of B. subtilis at 109 cell mL⁻¹ significantly reduced the disease incidence of stem canker and black scurf to 22.3 and 30%, respectively. In addition, it significantly improved some biochemical parameters, growth and tubers yield. Our findings indicate two points; firstly, B. subtilis possesses a good biocontrol activity against Rhizoctonia diseases of potato, secondly, the harmonization and suitability of the soil conditions to the growth and activity of B. subtilis guaranteed a high controlling capacity against the target pathogen. PMID:26616375

  11. ABILITY OF BACTERIAL CONSORTIUM: Bacillus coagulans, Bacilus licheniformis, Bacillus pumilus, Bacillus subtilis, Nitrosomonas sp. and Pseudomonas putida IN BIOREMEDIATION OF WASTE WATER IN CISIRUNG WASTE WATER TREATMENT PLANT

    Ratu SAFITRI; Bambang PRIADIE; Mia MIRANTI; Arum Widi ASTUTI


    This study was conducted in order to determine the ability of bacterial consortium: Bacillus coagulans, Bacilus licheniformis, Bacillus pumilus, Bacillus subtilis, Nitrosomonas sp., and Pseudomonas putida in bioremediation of wastewater origin Cisirung WWTP. This study uses an experimental method completely randomized design (CRD), which consists of two treatment factors (8x8 factorial design). The first factor is a consortium of bacteria (K), consisting of 8 level factors (k1, k2, k3, k4, k5...

  12. Estimation of P-to-O ratio in Bacillus subtilis and its influence on maximum riboflavin yield.

    Sauer, U; Bailey, J E


    Simultaneous growth and riboflavin overproduction were investigated using a previously developed stoichiometric model of Bacillus subtilis metabolism. A fit of model predictions to experimental data was used to obtain estimates of fundamental energetic parameters of B. subtilis. Although multiple solutions describe the experimental data, evidence for a P-to-O ratio of about 1(1/3) mole of ATP produced per atom of oxygen consumed in oxidative phosphorylation was provided by genomic analysis of electron transport components, because no homologue of the proton-translocating NADH dehydrogenase I was found in the B. subtilis genome database. These results allow us to devise a rational metabolic engineering strategy to improve riboflavin production. The potential influence of increased energy coupling in oxidative phosphorylation on riboflavin yield is discussed. Higher coupling is most significant under carbon-limiting conditions in slow-growing cells, that is, in fed-batch processes of industrial interest. PMID:10417225

  13. Bacillus subtilis is a Potential Degrader of Pyrene and Benzo[a]pyrene

    Lynette Ekunwe


    Full Text Available Polycyclic Aromatic Hydrocarbons (PAHs are a group of compounds that pose many health threats to human and animal life. They occur in nature as a result of incomplete combustion of organic matter, as well as from many anthropogenic sources including cigarette smoke and automobile exhaust. PAHs have been reported to cause liver damage, red blood cell damage and a variety of cancers. Because of this, methods to reduce the amount of PAHs in the environment are continuously being sought. The purpose of this study was to find soil bacteria capable of degrading high molecular weight PAHs, such as pyrene (Pyr and benzo[a]pyrene (BaP, which contain more than three benzene rings and so persist in the environment. Bacillus subtilis, identified by fatty acid methyl ester (FAME analysis, was isolated from PAH contaminated soil. Because it grew in the presence of 33μg/ml each of pyrene, 1-AP and 1-HP, its biodegradation capabilities were assessed. It was found that after a four-day incubation period at 30oC in 20μg/ml pyrene or benzo[a]pyrene, B. subtilis was able to transform approximately 40% and 50% pyrene and benzo[a]pyrene, respectively. This is the first report implicating B. subtilis in PAH degradation. Whether or not the intermediates resulting from the transformation are more toxic than their parent compounds, and whether B. subtilis is capable of mineralizing pyrene or benzo[a]pyrene to carbon dioxide and water, remains to be evaluated.




    Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 degrees on the B. subtilis chromosome. It

  15. Enhancement of extracellular expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis: Effects of promoter and host.

    Song, Wan; Nie, Yao; Mu, Xiao Qing; Xu, Yan


    Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed pullulanase was 3.9 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular pullulanase activity of 24.5 U ml(-1). Therefore, promoter and host had an impact on pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis. PMID:27109467

  16. Assessment of the antidiabetic and antilipidemic properties of Bacillus subtilis SPB1 biosurfactant in alloxan-induced diabetic rats.

    Zouari, Raida; Ben Abdallah-Kolsi, Rihab; Hamden, Khaled; Feki, Abdelfattah El; Chaabouni, Khansa; Makni-Ayadi, Fatma; Sallemi, Fahima; Ellouze-Chaabouni, Semia; Ghribi-Aydi, Dhouha


    The present study aimed to scrutinize the potential of Bacillus subtilis SPB1biosurfactant, orally administered, for preventing diabetic complications in rats. The findings revealed that, Bacillus subtilis biosurfactant was an effective reducer of α-amylase activity in the plasma. Moreover, this supplement helped protect the β-cells from death and damage. Both the inhibitory action of SPB1 biosurfactant on α-amylase and the protection of the pancreas' β-cells lead to a decrease of the blood glucose levels, consequently antihyperglycemic effect. Interestingly, this lipopeptide biosurfactant modulated key enzyme related to hyperlipidemia as lipase; which leads to the regulation of the lipid profile in serum by the delay in the absorption of LDL-cholesterol and triglycerides, and a significant increase in HDL-cholesterol. Histological analyses also showed that it exerted a protective action on the pancreases and efficiently preserved the liver-kidney functions of diabetic rats, evidenced by significant decreases in aspartate transaminase, alanine transaminase, gamma-glytamyl transpeptidase and lactate deshydrogenase activities in the plasma, as well as in the creatinine and urea contents. Overall, the present study demonstrated that the hypoglycemic and antilipidemic activities exhibited by Bacillus subtilis biosurfactant were effective enough to alleviate induced diabetes in experimental rats. Therefore, SPB1biosurfactant could be considered as a potential strong candidate for the treatment and prevention of diabetes. PMID:26228442

  17. [Adhesion of Bacillus subtilis on the surface of pectin-calcium gel].

    Gunter, E A; Melekhin, A K


    Pectin-calcium gels obtained based on pectins of callus cultures are able to adhere to the surface of cells of Gram-positive bacteria Bacillus subtilis to various degrees and this is thanks to the structural features of pectin. Rapid adhesion of the cells to gels obtained from the pectin of Tanacetum vulgare (TVC) callus cultures is associated with a high content of the linear region in the carbohydrate chain of pectin, a high molecular weight, and a low degree of methyl etherification of pectin. The number of adherent cells on the surface of gels obtained from pectins of Silene vulgaris callus cultures (SVC), TVC, and Lemna minor (LMC) after 8 h of incubation was close, whereas the number of cells was minimal on a gel produced using the pectin of Silene tatarica (STC) callus culture. This was due to the higher degree of methyl etherification of STC pectin (45%) compared to other pectins (4-12%). The adhesion rate constant (k) of B. subtilis for TCV gel during the first 120 min was the highest in comparison with other gels; the k value for SVC, STC and LMC gels was similar. The lowest level of k was characteristic for the gel from commercial apple pectin. The obtained data can beused for the production of gels with adhesive and antiadhesive properties. PMID:25842905

  18. An unstable donor-recipient DNA complex in transformation of Bacillus subtilis

    In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 51, 8-trimethylpsoralen in conjuction with long-wave-ultra violet light irradiation. This indicates that basepairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation. (orig.)

  19. Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

    Panga Jaipal Reddy

    Full Text Available Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division.

  20. Biological control of Colletotrichum panacicola on Panax ginseng by Bacillus subtilis HK-CSM-1

    Ryu, Hojin; Park, Hoon; Suh, Dong-Sang; Jung, Gun Ho; Park, Kyungseok; Lee, Byung Dae


    Background Biological control of plant pathogens using benign or beneficial microorganisms as antagonistic agents is currently considered to be an important component of integrated pest management in agricultural crops. In this study, we evaluated the potential of Bacillus subtilis strain HK-CSM-1 as a biological control agent against Colletotrichum panacicola. Methods The potential of B. subtilis HK-CSM-1 as a biological control agent for ginseng anthracnose was assessed. C. panacicola was i...

  1. Levan from Bacillus subtilis Natto: its effects in normal and in streptozotocin-diabetic rats

    Fernando Cesar Bazani Cabral de Melo; Cássia Thaïs Bussamra Viera Zaia; Maria Antonia Pedrine Colabone Celligoi


    Levan is an exopolysaccharide of fructose primarily linked by β-(2→6) glycosidic bonds with some β-(2→1) branched chains. Due to its chemical properties, levan has possible applications in both the food and pharmaceutical industries. Bacillus subtilis is a promising industrial levan producer, as it ferments sucrose and has a high levan-formation capacity. A new strain of B. subtilis was recently isolated from Japanese food natto, and it has produced levan in large quanti...

  2. Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB)

    Méndez-Lorenzo, Luz; Jaime R Porras-Domínguez; Raga-Carbajal, Enrique; Olvera, Clarita; Rodríguez-Alegría, Maria Elena; Carrillo-Nava, Ernesto; Costas, Miguel; López Munguía, Agustín


    Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particul...

  3. Methodological approaches to help unravel the intracellular metabolome of Bacillus subtilis

    Meyer, Hanna; Weidmann, Hendrikje; Lalk, Michael


    Background Bacillus subtilis (B. subtilis) has become widely accepted as a model organism for studies on Gram-positive bacteria. A deeper insight into the physiology of this prokaryote requires advanced studies of its metabolism. To provide a reliable basis for metabolome investigations, a validated experimental protocol is needed since the quality of the analytical sample and the final data are strongly affected by the sampling steps. To ensure that the sample analyzed precisely reflects the...

  4. Vacuum-induced Mutations In Bacillus Subtilis Spores

    Munakata, N.; Maeda, M.; Hieda, K.

    During irradiation experiments with vacuum-UV radiation using synchrotron sources, we made unexpected observation that Bacillus subtilis spores of several recombination-deficient strains lost colony-forming ability by the exposure to high vacuum alone. Since this suggested the possible injury in spore DNA, we looked for mutation induction using the spores of strains HA101 (wild-type repair capability) and TKJ6312 (excision and spore repair deficient) that did not lose survivability. It was found that the frequency of nalidixic-acid resistant mutation increased several times in both of these strains by the exposure to high vacuum (10e-4 Pa after 24 hours). The analysis of sequence changes in gyrA gene showed that the majority of mutations carried a unique allele (gyrA12) of tandem double-base substitutions from CA to TT. The observation has been extended to rifampicin resistant mutations, the majority of that carried substitutions from CA to TT or AT in rpoB gene. On the other hand, when the spores of strains PS578 and PS2319 (obtained from P. Setlow) that are defective in a group of small acidic proteins (alpha/beta-type SASP) were similarly treated, none of the mutants analyzed carried such changes. This suggests that the unique mutations might be induced by the interaction of small acidic proteins with spore DNA under forced dehydration. The results indicate that extreme vacuum causes severe damage in spore DNA, and provide additional constraint to the long-term survival of bacterial spores in the space environment.

  5. Effects of Bacillus subtilis natto and Different Components in Culture on Rumen Fermentation and Rumen Functional Bacteria In Vitro.

    Sun, Peng; Li, Jinan; Bu, Dengpan; Nan, Xuemei; Du, Hong


    This study was to investigate the effects of live or autoclaved Bacillus subtilis natto, their fermented products and media on rumen fermentation and rumen functional bacteria in vitro. Rumen fluid from three multiparous lactating Holstein cows was combined and transferred into serum bottles after diluted. Fifteen serum bottles were divided into five treatments, which were designed as following: CTR (the fermentation of 0.5 g TMR and ruminal fluids from dairy cows), LBS (CTR plus a minimum of 10(11) cfu live Bacillus subtilis natto), ABS (CTR plus a minimum of 10(11) cfu autoclaved Bacillus subtilis natto), BSC (CTR plus 1 ml Bacillus subtilis natto fermentation products without bacteria), and BSM (CTR plus 1 ml liquid fermentation medium). When separated from the culture, live Bacillus subtilis natto individually increased the concentrations of ammonia-N (P production (P probiotic in dairy ration. PMID:26821238

  6. Noise Expands the Response Range of the Bacillus subtilis Competence Circuit.

    Mugler, Andrew; Kittisopikul, Mark; Hayden, Luke; Liu, Jintao; Wiggins, Chris H; Süel, Gürol M; Walczak, Aleksandra M


    Gene regulatory circuits must contend with intrinsic noise that arises due to finite numbers of proteins. While some circuits act to reduce this noise, others appear to exploit it. A striking example is the competence circuit in Bacillus subtilis, which exhibits much larger noise in the duration of its competence events than a synthetically constructed analog that performs the same function. Here, using stochastic modeling and fluorescence microscopy, we show that this larger noise allows cells to exit terminal phenotypic states, which expands the range of stress levels to which cells are responsive and leads to phenotypic heterogeneity at the population level. This is an important example of how noise confers a functional benefit in a genetic decision-making circuit. PMID:27003682

  7. Identification of Bacillus subtilis adaptive response genes by subtractive differential hybridization.

    Mueller, J P; Mathiopoulos, C; Slack, F J; Sonenshein, A L


    Subtractive differential hybridization was used to identify genes in Bacillus subtilis that are induced by nutrient limitation. Several transcription units were identified. They exhibited increased transcription when cells were deprived of certain nutrients, such as glucose, ammonium, or phosphate, or when cells were treated with decoyinine. The genes have been designated dci (for decoyinine-inducible) and gsi (for glucose-starvation-inducible). Using lacZ transcriptional fusions, the dependence of dci and gsi expression on gene products of the sensor and activator classes of bacterial two-component regulatory systems was examined. Transcription of dciA was impaired by a mutation in spoOA, while expression of gsiA was dependent on the early competence genes comP and comA. The implications of these findings are discussed, and a provisional scheme for information flow during the transition phase from growth to sporulation is proposed. PMID:1784820

  8. Noise Expands the Response Range of the Bacillus subtilis Competence Circuit

    Hayden, Luke; Liu, Jintao; Wiggins, Chris H.; Süel, Gürol M.; Walczak, Aleksandra M.


    Gene regulatory circuits must contend with intrinsic noise that arises due to finite numbers of proteins. While some circuits act to reduce this noise, others appear to exploit it. A striking example is the competence circuit in Bacillus subtilis, which exhibits much larger noise in the duration of its competence events than a synthetically constructed analog that performs the same function. Here, using stochastic modeling and fluorescence microscopy, we show that this larger noise allows cells to exit terminal phenotypic states, which expands the range of stress levels to which cells are responsive and leads to phenotypic heterogeneity at the population level. This is an important example of how noise confers a functional benefit in a genetic decision-making circuit. PMID:27003682

  9. Effects of the electrolytic treatment on Bacillus subtilis Efeito do tratamento eletrolítico em Bacillus subtis

    Rodolfo Tolentino-Bisneto


    Full Text Available Conventional processes of water disinfection can generate toxic composites. It is the case of the trihalomethanes (carcinogenic formed in the contact of chlorine with organic substances present in the water. The electrolytic treatment can be a substitute for the chlorination process without the need for addition of chemical substances to the process. The effect of the electrolytic treatment using carbon cathode on the viability of the microorganism Bacillus subtilis was tested to determine the death process. By means of electronic microscopy, it was observed that the main cause of the microorganism's death was the cellular lysis due to the electroporation in the cell membrane.Processos convencionais de desinfecção de águas podem gerar compostos tóxicos. Esse é o caso dos trialometanos formados na reação do cloro com compostos orgânicos presentes na água. O tratamento eletrolítico pode ser um substituto à cloração com vantagem de não requer a adição de nenhum composto na água. O efeito do tratamento eletrolítico, utilizando eletrodos de carbono, na viabilidade de Bacillus subtilis foi testado para se determinar o mecanismo de morte. Através de microscopia eletrônica, foi possível evidenciar que a morte do microrganismo se deu pela lise celular, provavelmente provocada pela eletroporação irreversível da membrana celular.

  10. Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

    Juhas, Mario; Ajioka, James W


    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis. PMID:27033694