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Sample records for bacillus subtilis

  1. Transduction in Bacillus subtilis.

    PubMed

    THORNE, C B

    1962-01-01

    Thorne, Curtis B. (Fort Detrick, Frederick, Md.). Transduction in Bacillus subtilis. J. Bacteriol. 83:106-111. 1962.-A bacteriophage, SP-10, isolated from soil carries out general transduction in Bacillus subtilis. Phage propagated on a streptomycin-resistant mutant of the wild-type strain W-23 was capable of transducing to prototrophy strain 168 (indole(-)), as well as all of the auxotrophic mutants of W-23-S(r) tested, which included mutants requiring arginine, histidine, adenine, guanine, thiamine, leucine, or methionine. Although strain 168 was transduced by phage SP-10, lytic activity on this strain could not be detected and attempts to propagate the phage on it failed. Transductions occurred at frequencies in the range of 10(-6) to 10(-5) per plaque-forming unit. Homologous phage was ineffective, deoxyribonuclease had no effect on the frequency of transduction, and transduction was prevented by the addition of phage antiserum. Phage SP-10 was capable of lysogenizing strain W-23-S(r), and this condition was maintained through repeated growth and sporulation cycles in potato-extract medium. Although heating at 65 C for 60 min inactivated free phage particles, spores retained their lysogenic condition after such heat treatment. When heat-treated spores of the lysogenic cultures were used as inocula for growth in a nutrient broth-yeast extract-glucose medium, filtrates contained 10(9), or more, phage particles per ml.

  2. Characterization of Bacillus subtilis Bacteriophages

    PubMed Central

    Brodetsky, Anna M.; Romig, W. R.

    1965-01-01

    Brodetsky, Anna M. (University of California, Los Angeles), and W. R. Romig. Characterization of Bacillus subtilis bacteriophages. J. Bacteriol. 90:1655–1663. 1965.—A group of six phages, SP5, SP6, SP7, SP8, SP9, and SP13, which use the Marburg strain of Bacillus subtilis as host was characterized. These phages, referred to as group 1, were examined for the following properties: host range, plaque morphology, stability, adsorption kinetics, one-step growth characteristics, calcium requirements, serum neutralization, thermal inactivation, and inactivation by ultraviolet irradiation. Five unrelated B. subtilis phages, SP3, SP10, PBS1, SP alpha, and SP beta, were included in the studies. When first isolated, none of the group 1 phages was able to replicate efficiently on B. subtilis SB19, a mutant of the “transforming” B. subtilis 168. Host range mutants capable of growth in SB19 were isolated for all of the group 1 phages except SP13, and are designated the “star” phages (SP5* through SP9*). For characterization, SB19 was used as host for the star phages, and another B. subtilis mutant, 168B, was host for SP13. PMID:4955056

  3. Experimental evolution of Bacillus subtilis.

    PubMed

    Zeigler, Daniel R; Nicholson, Wayne L

    2017-09-01

    The endospore-forming bacteria have persisted on earth perhaps 3Ga, leveraging the flexibility of their distinctive lifestyle to adapt to a remarkably wide range of environments. This process of adaptation can be investigated through the simple but powerful technique of laboratory evolution. Evolved strains can be analyzed by whole genome sequencing and an array of omics technologies. The intensively studied, genetically tractable endospore-former, Bacillus subtilis, is an ideal subject for laboratory evolution experiments. Here, we describe the use of the B. subtilis model system to study the adaptation of these bacteria to reduced and stringent selection for endospore formation, as well as to novel environmental challenges of low atmospheric pressure, high ultraviolet radiation, and unfavourable growth temperatures. In combination with other approaches, including comparative genomics and environmental field work, laboratory evolution may help elucidate how these bacteria have so successfully adapted to life on earth, and perhaps beyond. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Tryptophanless Death in Bacillus subtilis

    PubMed Central

    Majerfeld, Irene; Barlati, Sergio; Ciferri, Orio

    1970-01-01

    A decline in colony-forming ability is observed in actively growing cultures of a tryptophan arginine auxotroph of Bacillus subtilis after removal of tryptophan (tryptophanless death). This phenomenon can be prevented by simultaneous starvation of the other required amino acid or by chloramphenicol administered in bacteriostatic concentration but not by actinomycin. Addition of tryptophan analogues not only prevents the death but also allows recovery of the cells that have lost the ability to form colonies on solid media. The term tryptophanless death is therefore inappropriate. Chloramphenicol but not actinomycin inhibits the recovery brought about by tryptophan analogues. PMID:4189906

  5. Reclassification of bioindicator strains Bacillus subtilis DSM 675 and Bacillus subtilis DSM 2277 as Bacillus atrophaeus.

    PubMed

    Fritze, D; Pukall, R

    2001-01-01

    On the basis of high DNA-DNA reassociation values and confirmatory automated RiboPrint analysis, two aerobic spore-forming strains hitherto allocated to Bacillus subtilis and used as bioindicators (DSM 675, hot-air sterilization control; DSM 2277, ethylene oxide sterilization control) are reclassified as Bacillus atrophaeus.

  6. Whole-genome sequences of Bacillus subtilis and close relatives.

    PubMed

    Earl, Ashlee M; Eppinger, Mark; Fricke, W Florian; Rosovitz, M J; Rasko, David A; Daugherty, Sean; Losick, Richard; Kolter, Roberto; Ravel, Jacques

    2012-05-01

    We sequenced four strains of Bacillus subtilis and the type strains for two closely related species, Bacillus vallismortis and Bacillus mojavensis. We report the high-quality Sanger genome sequences of B. subtilis subspecies subtilis RO-NN-1 and AUSI98, B. subtilis subspecies spizizenii TU-B-10(T) and DV1-B-1, Bacillus mojavensis RO-H-1(T), and Bacillus vallismortis DV1-F-3(T).

  7. Genetic competence in Bacillus subtilis.

    PubMed Central

    Dubnau, D

    1991-01-01

    Genetic competence may be defined as a physiological state enabling a bacterial culture to bind and take up high-molecular-weight exogenous DNA (transformation). In Bacillus subtilis, competence develops postexponentially and only in certain media. In addition, only a minority of the cells in a competent culture become competent, and these are physiologically distinct. Thus, competence is subject to three regulatory modalities: growth stage specific, nutritionally responsive, and cell type specific. This review summarizes the present state of knowledge concerning competence in B. subtilis. The study of genes required for transformability has permitted their classification into two broad categories. Late competence genes are expressed under competence control and specify products required for the binding, uptake, and processing of transforming DNA. Regulatory genes specify products that are needed for the expression of the late genes. Several of the late competence gene products have been shown to be membrane localized, and others are predicted to be membrane associated on the basis of amino acid sequence data. Several of these predicted protein sequences show a striking resemblance to gene products that are involved in the export and/or assembly of extracellular proteins and structures in gram-negative organisms. This observation is consistent with the idea that the late products are directly involved in transport of DNA and is equally consistent with the notion that they play a morphogenetic role in the assembly of a transport apparatus. The competence regulatory apparatus constitutes an elaborate signal transduction system that senses and interprets environmental information and passes this information to the competence-specific transcriptional machinery. Many of the regulatory gene products have been identified and partially characterized, and their interactions have been studied genetically and in some cases biochemically as well. These include several

  8. Characterisation of Potential Antimicrobial Targets in Bacillus spp. I. Aminotransferases and Methionine Regeneration in Bacillus subtilis

    DTIC Science & Technology

    2002-07-01

    targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J. Berger and Marvin H. Knodel Defence R&D...Characterisation of potential antimicrobial targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J...examined in the gram-positive bacterium Bacillus subtilis. Homogenates of this bacterium were able to convert ketomethiobutyrate to methionine, utilising

  9. Microbial genotyping and differentiating between Bacillus mojavensis and Bacillus subtilis

    USDA-ARS?s Scientific Manuscript database

    Bacillus mojavensis, a specie recently distinguished from its previous Bacillus subtilis classification, was discovered in corn kernels and later determined to possess endophytic character. The bacterium was also determined to have biocontrol potential due to its growth inhibition of the maize mycot...

  10. EFFECTS OF COPPER ON BACILLUS SUBTILIS1

    PubMed Central

    Weed, Lawrence L.

    1963-01-01

    Weed, Lawrence L. (Western Reserve University, Cleveland, Ohio). Effects of copper on Bacillus subtilis. J. Bacteriol. 85:1003–1010. 1963.—Variants have been isolated from liquid cultures of Bacillus subtilis 168 after exposure to copper. The variations manifested are in terms of loss of capacity to be transformed from tryptophan auxotrophy to prototrophy as shown by variant NTCu, and in terms of colony size and altered base composition of the deoxyribonucleic acid as shown by variant SC-22. In addition to the data on altered morphology and chemical composition of the variants, detailed studies on the reversion of one of the variants to B. subtilis are presented. Images PMID:14043986

  11. Bacillus subtilis biofilm induction by plant polysaccharides

    PubMed Central

    Beauregard, Pascale B.; Chai, Yunrong; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2013-01-01

    Bacillus subtilis is a plant-beneficial Gram-positive bacterium widely used as a biofertilizer. However, relatively little is known regarding the molecular processes underlying this bacterium's ability to colonize roots. In contrast, much is known about how this bacterium forms matrix-enclosed multicellular communities (biofilms) in vitro. Here, we show that, when B. subtilis colonizes Arabidopsis thaliana roots it forms biofilms that depend on the same matrix genes required in vitro. B. subtilis biofilm formation was triggered by certain plant polysaccharides. These polysaccharides served as a signal for biofilm formation transduced via the kinases controlling the phosphorylation state of the master regulator Spo0A. In addition, plant polysaccharides are used as a source of sugars for the synthesis of the matrix exopolysaccharide. The bacterium's response to plant polysaccharides was observed across several different strains of the species, some of which are known to have beneficial effects on plants. These observations provide evidence that biofilm genes are crucial for Arabidopsis root colonization by B. subtilis and provide insights into how matrix synthesis may be triggered by this plant. PMID:23569226

  12. Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

    PubMed Central

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

    2015-01-01

    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

  13. Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores.

    PubMed

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

    2015-01-01

    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.

  14. Biodegradation of naphthalene and phenanthren by Bacillus subtilis 3KP

    NASA Astrophysics Data System (ADS)

    Ni'matuzahroh, Trikurniadewi, N.; Pramadita, A. R. A.; Pratiwi, I. A.; Salamun, Fatimah, Sumarsih, Sri

    2017-06-01

    The purposes of this research were to know growth response, degradation ability, and uptake mechanism of naphthalene and phenanthrene by Bacillus subtilis 3KP. Bacillus subtilis 3KP was grown on Mineral Synthetic (MS) medium with addition of 1% yeast extract and naphthalene and phenanthrene respectively 200 ppm in different cultures. Bacillus subtilis 3KP growth response was monitored by Total Plate Count (TPC) method, the degradation ability was monitored by UV-Vis spectrophotometer, and the uptake mechanism of hydrocarbon was monitored by emulsification activity, decrease of surface tension, and activity of Bacterial Adherence to Hydrocarbon (BATH). Bacillus subtilis 3KP was able to grow and show biphasic growth pattern on both of substrates. Naphthalene and phenanthrene were used as a carbon source for Bacillus subtilis 3KP growth that indicated by the reduction of substrate concomitant with the growth. At room temperature conditions (± 30°C) and 90 rpm of agitation for 7 days, Bacillus subtilis 3KP could degrade naphthalene in the amount of 70.5% and phenanthrene in the amount of 24.8%. Based on the analysis of UV-Vis spectrophotometer, three metabolites, 1-hydroxy-2-naphthoic acid, salicylic acid, and pyrocatechol were found in both cultures. The metabolite identification became basis of propose degradation pathway of naphthalene and phenanthrene by Bacillus subtilis 3KP. The results of hydrocarbon uptake mechanism test show that Bacillus subtilis 3KP used all of the mechanism to degrade naphthalene and phenanthrene.

  15. Genes controlling xylan utilization by Bacillus subtilis.

    PubMed Central

    Roncero, M I

    1983-01-01

    Eight mutants of Bacillus subtilis deficient in xylan utilization were isolated and characterized genetically and biochemically. Each mutant was obtained independently after nitrosoguanidine mutagenesis. All of the analyzed mutations were shown to be linked. Reciprocal transformation crosses revealed the existence of two genes controlling xylan utilization which have been designated xynA and xynB. Available data have indicated that these two genes code for two xylan-degrading enzymes existing in the wild-type strains, an extracellular beta-xylanase (xynA) and a cell-associated beta-xylosidase (xynB). PMID:6413490

  16. A Love Affair with Bacillus subtilis

    PubMed Central

    Losick, Richard

    2015-01-01

    My career in science was launched when I was an undergraduate at Princeton University and reinforced by graduate training at the Massachusetts Institute of Technology. However, it was only after I moved to Harvard University as a junior fellow that my affections were captured by a seemingly mundane soil bacterium. What Bacillus subtilis offered was endless fascinating biological problems (alternative sigma factors, sporulation, swarming, biofilm formation, stochastic cell fate switching) embedded in a uniquely powerful genetic system. Along the way, my career in science became inseparably interwoven with teaching and mentoring, which proved to be as rewarding as the thrill of discovery. PMID:25533458

  17. Synthesis of Pulcherriminic Acid by Bacillus subtilis

    PubMed Central

    Uffen, Robert L.; Canale-Parola, E.

    1972-01-01

    The pathway of pulcherriminic acid synthesis in Bacillus subtilis strains AM and AM-L11 (a leucine-requiring auxotroph) was investigated. Determinations of radioactivity in pulcherriminic acid synthesized by cells growing in media containing 14C-labeled amino acids indicated that B. subtilis produced pulcherriminic acid from l-leucine. The organism utilized the carbon skeletons of two l-leucine molecules to synthesize one molecule of pulcherriminic acid. Similar results were obtained with starved cell suspensions. Growing cells formed significant amounts of pulcherriminic acid only in media including a carbohydrate such as starch. However, carbohydrate carbon was not required for the synthesis of pulcherriminic acid molecules. Data obtained with cell suspensions supported the hypothesis that cyclo-l-leucyl-l-leucyl is an intermediate in pulcherriminic acid biosynthesis and indicated that molecular oxygen is required for the conversion of cyclo-l-leucyl-l-leucyl to pulcherriminic acid. A pathway for the synthesis of pulcherrimin from l-leucine in B. subtilis is proposed. PMID:4204912

  18. FORMALDEHYDE GAS INACTIVATION OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACE MATERIALS.

    EPA Science Inventory

    Research evaluated the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface material using formaldehyde gas. Spores were dried on seven types of indoor surfaces and exposed to 1100 ppm formaldehyde gas for 10 hr. Fo...

  19. Suppressor System in Bacillus subtilis 168

    PubMed Central

    Georgopoulos, C. P.

    1969-01-01

    Multiple auxotrophic strains of Bacillus subtilis 168 were tested for joint one-step reversion of two or more auxotrophic markers to the wild-type phenotype. Mu8u5u5, a strain requiring leucine, methionine, and threonine, yielded revertants that grew without added methionine or threonine and proved to have a suppressor gene. When transferred by transformation with deoxyribonucleic acid, this suppressor gene also suppressed the adenine mutation in another strain, Mu8u5u6. The one-step double revertants fell into two distinct classes: strains of class su+I grow well in broth; strains of class su+II grow poorly. Strains su+II tend to revert frequently to the su+I or su− state. Conditional lethal mutants of phage φe were isolated which can grow on the su+ and not on the su− strains. PMID:4975748

  20. Bacillus subtilis as potential producer for polyhydroxyalkanoates

    PubMed Central

    Singh, Mamtesh; Patel, Sanjay KS; Kalia, Vipin C

    2009-01-01

    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. PMID:19619289

  1. Protein Targeting during Bacillus subtilis Sporulation.

    PubMed

    Dworkin, Jonathan

    2014-02-01

    The Gram-positive bacterium Bacillus subtilis initiates the formation of an endospore in response to conditions of nutrient limitation. The morphological differentiation that spores undergo initiates with the formation of an asymmetric septum near to one pole of the cell, forming a smaller compartment, the forespore, and a larger compartment, the mother cell. This process continues with the complex morphogenesis of the spore as governed by an intricate series of interactions between forespore and mother cell proteins across the inner and outer forespore membranes. Given that these interactions occur at a particular place in the cell, a critical question is how the proteins involved in these processes get properly targeted, and we discuss recent progress in identifying mechanisms responsible for this targeting.

  2. A love affair with Bacillus subtilis.

    PubMed

    Losick, Richard

    2015-01-30

    My career in science was launched when I was an undergraduate at Princeton University and reinforced by graduate training at the Massachusetts Institute of Technology. However, it was only after I moved to Harvard University as a junior fellow that my affections were captured by a seemingly mundane soil bacterium. What Bacillus subtilis offered was endless fascinating biological problems (alternative sigma factors, sporulation, swarming, biofilm formation, stochastic cell fate switching) embedded in a uniquely powerful genetic system. Along the way, my career in science became inseparably interwoven with teaching and mentoring, which proved to be as rewarding as the thrill of discovery. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. EPS forces in Bacillus subtilis biofilms

    NASA Astrophysics Data System (ADS)

    Zhang, Wenbo; Angelini, Thomas; Tsai, Shih-Ming; Nixon, Ryan

    2014-03-01

    Bacteria have evolved to congregate in complex communities known as biofilms. The structure that holds a biofilm together is a matrix called extracellular polymeric substance (EPS). It has been observed in previous studies that EPS up-regulation occurs when the nutrient levels fall below a threshold concentration; this increase in EPS concentration produces an osmotic pressure that forces the colony to spread outward. This osmotic pressure may drive nutrient uptake, but the stresses generated by the EPS matrix has never been measured. Here we present measurements of the forces exerted by a biofilm on its supporting substrate and on its fluid nutrients. In our experiments, we use a technique analogous to traction force microscopy to measure strain in agar nutrient substrates imposed by Bacillus subtilis biofilms. By running additional test to measure the permeability and elastic modulus of the agar, we can estimate the pressure generated by the biofilm.

  4. Amino acid chemoreceptors of Bacillus subtilis.

    PubMed Central

    Ordal, G W; Villani, D P; Gibson, K J

    1977-01-01

    Specificities of chemoreceptors for the 20 common amino acids, toward which Bacillus subtilis shows chemotaxis, were assessed by competition ("jamming") experiments using a modification of the traditional capillary assay, called the "sensitivity capillary assay." Many amino acids were sensed by at least two chemoreceptors. All the highest affinity chemoreceptors for the amino acids were distinct, except glutamate and aspartate, which may share one chemoreceptor, and tyrosine, for which the data could not be collected due to low solubility. The data suggest the hypothesis that each amino acid-chemoreceptor complex binds to a different signaler (from each amino acid-chemoreceptor complex binds to a different signaler (from which signals travel to the flagella to modify behavior appropriately), and that many of the signalers can also bind other attractant-chemoreceptor complexes as antagonists (no signals to flagella). PMID:401491

  5. Peptidoglycan transformations during Bacillus subtilis sporulation

    PubMed Central

    Tocheva, Elitza I.; López-Garrido, Javier; Hughes, H. Velocity; Fredlund, Jennifer; Kuru, Erkin; VanNieuwenhze, Michael S.; Brun, Yves V.; Pogliano, Kit; Jensen, Grant J.

    2014-01-01

    Summary While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross-linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high-resolution electron cryotomographic imaging. Three-dimensional reconstructions of whole cells in near-native states revealed a thin PG-like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram-negative-like PG layers as well as its thick, archetypal Gram-positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled. PMID:23531131

  6. Characterization of Bacillus subtilis recombinational pathways.

    PubMed Central

    Alonso, J C; Lüder, G; Tailor, R H

    1991-01-01

    Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination. PMID:1905712

  7. Isolation of a novel mutant from Bacillus subtilis natto.

    PubMed

    Yoshida, Kazuo

    2006-01-01

    For the construction of strains with full probiotics function in intestines, deoxycholate resistant mutants were isolated from Bacillus subtilis natto. The partial characterization of the mutants was carried out and described.

  8. Draft Genome Sequence of Bacillus subtilis strain KATMIRA1933

    PubMed Central

    Melnikov, Vyacheslav G.; Chikindas, Michael L.

    2014-01-01

    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. PMID:24948771

  9. DECONTAMINATION ASSESSMENT OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS, AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACTS USING A HYDROGEN PERIOXIDE GAS GENERATOR

    EPA Science Inventory

    Aims: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Methods and Results: B. anthracis, B. subtilis, and G. Stearothermophilus spores were dried on seven...

  10. Alanylated lipoteichoic acid primer in Bacillus subtilis

    PubMed Central

    Luo, Yu

    2016-01-01

    Lipoteichoic acid is a major lipid-anchored polymer in Gram-positive bacteria such as Bacillus subtilis. This polymer typically consists of repeating phosphate-containing units and therefore has a predominant negative charge. The repeating units are attached to a glycolipid anchor which has a diacylglycerol (DAG) moiety attached to a dihexopyranose head group. D-alanylation is known as the major modification of type I and type IV lipoteichoic acids, which partially neutralizes the polymer and plays important roles in bacterial survival and resistance to the host immune system. The biosynthesis pathways of the glycolipid anchor and lipoteichoic acid have been fully characterized. However, the exact mechanism of D-alanyl transfer from the cytosol to cell surface lipoteichoic acid remains unclear. Here I report the use of mass spectrometry in the identification of possible intermediate species in the biosynthesis and D-alanylation of lipoteichoic acid: the glycolipid anchor, nascent lipoteichoic acid primer with one phosphoglycerol unit, as well as mono- and di-alanylated forms of the lipoteichoic acid primer. Monitoring these species as well as the recently reported D-alanyl-phosphatidyl glycerol should aid in shedding light on the mechanism of the D-alanylation pathway of lipoteichoic acid. PMID:27134729

  11. Kin discrimination between sympatric Bacillus subtilis isolates.

    PubMed

    Stefanic, Polonca; Kraigher, Barbara; Lyons, Nicholas Anthony; Kolter, Roberto; Mandic-Mulec, Ines

    2015-11-10

    Kin discrimination, broadly defined as differential treatment of conspecifics according to their relatedness, could help biological systems direct cooperative behavior toward their relatives. Here we investigated the ability of the soil bacterium Bacillus subtilis to discriminate kin from nonkin in the context of swarming, a cooperative multicellular behavior. We tested a collection of sympatric conspecifics from soil in pairwise combinations and found that despite their history of coexistence, the vast majority formed distinct boundaries when the swarms met. Some swarms did merge, and most interestingly, this behavior was only seen in the most highly related strain pairs. Overall the swarm interaction phenotype strongly correlated with phylogenetic relatedness, indicative of kin discrimination. Using a subset of strains, we examined cocolonization patterns on plant roots. Pairs of kin strains were able to cocolonize roots and formed a mixed-strain biofilm. In contrast, inoculating roots with pairs of nonkin strains resulted in biofilms consisting primarily of one strain, suggestive of an antagonistic interaction among nonkin strains. This study firmly establishes kin discrimination in a bacterial multicellular setting and suggests its potential effect on ecological interactions.

  12. Kin discrimination between sympatric Bacillus subtilis isolates

    PubMed Central

    Stefanic, Polonca; Kraigher, Barbara; Lyons, Nicholas Anthony; Kolter, Roberto; Mandic-Mulec, Ines

    2015-01-01

    Kin discrimination, broadly defined as differential treatment of conspecifics according to their relatedness, could help biological systems direct cooperative behavior toward their relatives. Here we investigated the ability of the soil bacterium Bacillus subtilis to discriminate kin from nonkin in the context of swarming, a cooperative multicellular behavior. We tested a collection of sympatric conspecifics from soil in pairwise combinations and found that despite their history of coexistence, the vast majority formed distinct boundaries when the swarms met. Some swarms did merge, and most interestingly, this behavior was only seen in the most highly related strain pairs. Overall the swarm interaction phenotype strongly correlated with phylogenetic relatedness, indicative of kin discrimination. Using a subset of strains, we examined cocolonization patterns on plant roots. Pairs of kin strains were able to cocolonize roots and formed a mixed-strain biofilm. In contrast, inoculating roots with pairs of nonkin strains resulted in biofilms consisting primarily of one strain, suggestive of an antagonistic interaction among nonkin strains. This study firmly establishes kin discrimination in a bacterial multicellular setting and suggests its potential effect on ecological interactions. PMID:26438858

  13. The Cortical Peptidoglycan from Spores of Bacillus Megaterium and Bacillus Subtilis Is Not Highly Cross-Linked

    DTIC Science & Technology

    1993-05-01

    Bacilius megaterium and Bacillus subtilis Is Not Highly Cross-Linked 6. AUTHOR(S) David L. Popham and Peter Setlow 7. PERFORMING ORGANIZATION NAME(S...Determination by amino acid analyses of the percentage of diaminopimelic acid in the spore cortex of Bacillus megaterium and Bacillus subtilis which is...Peptidoglycan from Spores of Bacillus megaterium and Bacillus subtilis Is Not Highly Cross-Linked DAVID L. POPHAM ANDl PETER SETLOW* Department of Biochemistry

  14. Bacillus subtilis Improves Immunity and Disease Resistance in Rabbits

    PubMed Central

    Guo, Mengjiao; Wu, Fahao; Hao, Guangen; Qi, Qin; Li, Rong; Li, Ning; Wei, Liangmeng; Chai, Tongjie

    2017-01-01

    Probiotics such as Lactobacillus and Bifidobacterium have been successfully used to promote growth and prevent diseases. Previous reports have demonstrated that Bacillus subtilis (B. subtilis) was a potential probiotic for animals. In this research, 180 B. subtilis were isolated from the soil, identified, and investigated in vitro. Furthermore, five B. subtilis were selected and mixed to investigate their effect on growth performance, immune response, intestine microbiota, and disease resistance in rabbits. Rabbits with a diet of 106 CFU g−1 mixed B. subtilis exhibited the best growth performance and higher serum IgG and IgA than controls (P < 0.05). Moreover, dairy with B. subtilis can promote the balance of intestinal flora. The major proinflammatory factor and β-defensin were upregulated compared with the controls. After 7 weeks of feeding, the survival rate of the rabbits fed with B. subtilis was significantly higher than those in the control groups postinfected with Escherichia coli. At the same time, this study detected higher expression of β-defensin and reduced bacteria contents of the heart and cecal contents from the diet mixed with B. subtilis compared with the control groups. In conclusion, dietary supplementation with B. subtilis for rabbits could improve growth performance, intestinal homeostasis, and immune organ index and enhance innate immune response as well as disease resistance. These findings showed that the induction of β-defensin by B. subtilis might be an interesting new therapeutic strategy to strengthen innate defense mechanisms. PMID:28424690

  15. [Overview of study on Bacillus subtilis spores].

    PubMed

    Watabe, Kazuhito

    2013-01-01

    This review documents my research for the past 29 years in the work of bacterial sporulation. The Gram-positive bacterium Bacillus subtilis forms spores when conditions are unsuitable for growth. The mature spores remain for long periods of starvation and are resistant to harsh environment. This property is attributed mainly to the unique figures of spore's outer layers, spore coat. The protein composition of the spores was comprehensively analyzed by a combination of SDS-PAGE and LC-MS/MS. The total of 154 proteins were identified and 69 of them were novel. The expression of the genes encoding them was dependent on sporulation-specific sigma factors, σF, σE, σG and σK. The expression of a coat protein gene, cotS, was dependent on σK and GerE. CotE is essential for the assembly of CotS in the coat layer. Many coat genes were identified by reverse genetics and the regulation of the gene expression was studied in detail. Some cot genes are functioned in the resistance to heat and lysozyme, and some of the coat proteins are involved in the specificity of germinants. The yrbA is essential in spore development, yrbA deficient cells revealed abnormal figures of spore coat structure and changed the response to germinants. The location of 16 coat proteins was determined by the observation of fluorescence microscopy using fluorescence-labelled proteins. One protein was assigned to the cortex, nine to the inner coat, and four to the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat.

  16. Chromosomal-DNA amplification in Bacillus subtilis.

    PubMed Central

    Wilson, C R; Morgan, A E

    1985-01-01

    Tetracycline-resistant (Tetr) mutants RAD1, RAD2, RAD6, and RAD7 were isolated from Bacillus subtilis BC92 after protoplasting, polyethylene glycol treatment, and regeneration on a medium containing tetracycline. The Tetr phenotype in RAD1, RAD2, and RAD6 was very stable with less than 5% loss of resistance after 30 generations of growth in the absence of selection. Of the four isolates, three contained amplified chromosomal DNA closely associated with the Tetr phenotype. The intensity of restriction fragments present in HindIII and EcoRI digests of chromosomal DNA from RAD1, RAD6, and RAD7 indicated the presence of tandemly duplicated DNA. Disparity in the size and number of amplified fragments suggested that the tandemly duplicated DNA is different in all three isolates. The sizes of the duplicated DNA present in RAD1, RAD6, and RAD7 were estimated to be 10, 19, and 20 kilobases, respectively. No amplified DNA was detected in RAD2. Results of transductional-mapping studies with PBS1 showed that the tetracycline resistance (tet) loci of RAD1, RAD2, and RAD6 all mapped near the origin of chromosomal replication and close to the guaA locus. Amplified DNA characteristic of RAD1 and RAD6 was cotransduced with the tet locus. Cotransfer of amplified DNA with the guaA locus or other nearby loci in the absence of tet was not observed. In every case, loss of Tetr was accompanied by loss of amplified DNA. A possible explanation for the occurrence of the amplified DNA is presented. Images PMID:2991188

  17. 40 CFR 180.1111 - Bacillus subtilis GB03; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus subtilis GB03; exemption from... FOOD Exemptions From Tolerances § 180.1111 Bacillus subtilis GB03; exemption from the requirement of a tolerance. The biofungicide Bacillus subtilis GB03 is exempted from the requirement of a tolerance in or...

  18. 40 CFR 180.1128 - Bacillus subtilis MBI 600; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 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...

  19. 40 CFR 180.1111 - Bacillus subtilis GB03; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus subtilis GB03; exemption from... FOOD Exemptions From Tolerances § 180.1111 Bacillus subtilis GB03; exemption from the requirement of a tolerance. The biofungicide Bacillus subtilis GB03 is exempted from the requirement of a tolerance in or...

  20. 40 CFR 180.1111 - Bacillus subtilis GB03; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus subtilis GB03; exemption from... FOOD Exemptions From Tolerances § 180.1111 Bacillus subtilis GB03; exemption from the requirement of a tolerance. The biofungicide Bacillus subtilis GB03 is exempted from the requirement of a tolerance in or...

  1. 40 CFR 180.1111 - Bacillus subtilis GB03; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus subtilis GB03; exemption from... FOOD Exemptions From Tolerances § 180.1111 Bacillus subtilis GB03; exemption from the requirement of a tolerance. The biofungicide Bacillus subtilis GB03 is exempted from the requirement of a tolerance in or...

  2. 40 CFR 180.1128 - Bacillus subtilis MBI 600; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 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...

  3. 40 CFR 180.1128 - Bacillus subtilis MBI 600; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 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...

  4. 40 CFR 180.1128 - Bacillus subtilis MBI 600; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 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...

  5. 40 CFR 180.1111 - Bacillus subtilis GB03; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis GB03; exemption from... FOOD Exemptions From Tolerances § 180.1111 Bacillus subtilis GB03; exemption from the requirement of a tolerance. The biofungicide Bacillus subtilis GB03 is exempted from the requirement of a tolerance in or...

  6. 40 CFR 180.1128 - Bacillus subtilis MBI 600; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 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...

  7. [Dependence of Bacillus subtilis cell respiration on monovalent cations].

    PubMed

    Samuilov, V D; Khakimov, S A

    1991-07-01

    Nigericin, monensin, valinomycin + carbonyl-cyanide-m-chlorophenylhydrazone and gramicidin inhibit the respiration of Bacillus subtilis cells incubated with NAD-dependent substrates or succinate, but not with ascorbate + N,N,N',N'-tetramethyl-p- phenylene-diamine. The level of inhibition was decreased by potassium ions and, in a lower degree, by sodium or ammonium ions. The results obtained suggest that the respiration of Bacillus subtilis depends on the presence of monovalent cations whose effects seem to be directed at complexes I, III and probably complex II of the respiratory chain.

  8. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    SciTech Connect

    Rubin, Edward M.

    1980-01-01

    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)

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

    PubMed Central

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

    1982-01-01

    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. PMID:6172418

  10. Combined Bacillus licheniformis and Bacillus subtilis infection in a patient with oesophageal perforation.

    PubMed

    Jeon, You La; Yang, John Jeongseok; Kim, Min Jin; Lim, Gayoung; Cho, Sun Young; Park, Tae Sung; Suh, Jin-Tae; Park, Yong Ho; Lee, Mi Suk; Kim, Soo Cheol; Lee, Hee Joo

    2012-12-01

    Species of the genus Bacillus are a common laboratory contaminant, therefore, isolation of these organisms from blood cultures does not always indicate infection. In fact, except for Bacillus anthracis and Bacillus cereus, most species of the genus Bacillus are not considered human pathogens, especially in immunocompetent individuals. Here, we report an unusual presentation of bacteraemia and mediastinitis due to co-infection with Bacillus subtilis and Bacillus licheniformis, which were identified by 16S RNA gene sequencing, in a patient with an oesophageal perforation.

  11. bmr3, a third multidrug transporter gene of Bacillus subtilis.

    PubMed Central

    Ohki, R; Murata, M

    1997-01-01

    A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase. PMID:9023234

  12. The structure and regulation of flagella in Bacillus subtilis.

    PubMed

    Mukherjee, Sampriti; Kearns, Daniel B

    2014-01-01

    Bacterial flagellar motility is among the most extensively studied physiological systems in biology, but most research has been restricted to using the highly similar Gram-negative species Escherichia coli and Salmonella enterica. Here, we review the recent advances in the study of flagellar structure and regulation of the distantly related and genetically tractable Gram-positive bacterium Bacillus subtilis. B. subtilis has a thicker layer of peptidoglycan and lacks the outer membrane of the Gram-negative bacteria; thus, not only phylogenetic separation but also differences in fundamental cell architecture contribute to deviations in flagellar structure and regulation. We speculate that a large number of flagella and the absence of a periplasm make B. subtilis a premier organism for the study of the earliest events in flagellar morphogenesis and the type III secretion system. Furthermore, B. subtilis has been instrumental in the study of heterogeneous gene transcription in subpopulations and of flagellar regulation at the translational and functional level.

  13. [Phosphatase activity of Bacillus subtilis IMV B-7023].

    PubMed

    Bulavenko, L V; Kurdysh, I K

    2005-01-01

    Phosphatase activity of two strains of bacteria - Bacillus subtilis IMV B-7023 and B. megaterium 12 is investigated. The phosphatase activity is found to reach 260 mkmol/g x hour for B. subtilis IMV B-7023 and 12-100 mkmol/g x hour for B. megaterium 12 at optimal temperature (55 degrees C) and pH (9.5-10.0). Synthesis of alkaline phosphatase is shown to reach its maximum values at the end of logarithmic phase of the culture growth. It is revealed that Mg2+, Ca2+ cations increase phosphotase activity of B. subtilis IMV B-7023, at the same time Cu2+, Mn2+, Zn2+ cations and inorganic phosphate decrease it. Dependence of the rate of phosphatase reaction of B. subtilis IMV B-7023 on substrate concentration is determined.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  15. Metabolic engineering of Bacillus subtilis for growth on overflow metabolites

    PubMed Central

    2013-01-01

    Background The genome of the important industrial host Bacillus subtilis does not encode the glyoxylate shunt, which is necessary to utilize overflow metabolites, like acetate or acetoin, as carbon source. In this study, the operon encoding the isocitrate lyase (aceB) and malate synthase (aceA) from Bacillus licheniformis was transferred into the chromosome of B. subtilis. The resulting strain was examined in respect to growth characteristics and qualities as an expression host. Results Our results show that the modified B. subtilis strain is able to grow on the C2 compound acetate. A combined transcript, protein and metabolite analysis indicated a functional expression of the native glyoxylate shunt of B. lichenifomis in B. subtilis. This metabolically engineered strain revealed better growth behavior and an improved activity of an acetoin-controlled expression system. Conclusions The glyoxylate shunt of B. licheniformis can be functionally transferred to B. subtilis. This novel strain offers improved properties for industrial applications, such as growth on additional carbon sources and a greater robustness towards excess glucose feeding. PMID:23886069

  16. Bacillus subtilis isolated from the human gastrointestinal tract.

    PubMed

    Hong, Huynh A; Khaneja, Reena; Tam, Nguyen M K; Cazzato, Alessia; Tan, Sisareuth; Urdaci, Maria; Brisson, Alain; Gasbarrini, Antonio; Barnes, Ian; Cutting, Simon M

    2009-03-01

    As part of an ongoing study to determine the true habitat of Bacillus species, we report here the isolation and characterisation of Bacillus subtilis from the human gastrointestinal tract (GIT). Strains were obtained from ileum biopsies as well as from faecal samples and their biotypes defined. 16S rRNA analysis revealed that most isolates of B. subtilis were highly conserved, in contrast to RAPD-PCR fingerprinting that showed greater diversity with 23 distinct RAPD types. The majority of B. subtilis strains examined possessed features that could be advantageous to survival within the GIT. This included the ability to form biofilms, to sporulate anaerobically and secretion of antimicrobials. At least one isolate was shown to form spores that carried an exosporium, a loosely attached outer layer to the mature endospore, this being the first report of B. subtilis spores carrying an exosporium. This study reinforces a growing view that B. subtilis and probably other species have adapted to life within the GIT and should be considered gut commensals rather than solely soil microorganisms.

  17. Chromosomal locations of three Bacillus subtilis din genes

    SciTech Connect

    Gillespie, K.; Yasbin, R.E.

    1987-07-01

    Previously isolated DNA damage-inducible (din) genes of Bacillus subtilis have been mapped on the bacterial chromosome by bacteriophage PBS1-mediated transduction. The din genes have been localized to three positions on the B. subtilis map. dinA cotransduction with the hisA locus was 80%, while dinC cotransduction with this marker was about 56%. dinB is unlinked to hisA, but its cotransduction with the dal-1 and purB loci was 84 and 22%, respectively.

  18. Characterization of recombination-deficient mutants of Bacillus subtilis.

    PubMed Central

    Alonso, J C; Tailor, R H; Lüder, G

    1988-01-01

    An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis. PMID:3133357

  19. Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain.

    PubMed

    Geetha, I; Manonmani, A M; Paily, K P

    2010-05-01

    The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC(50) dose of 1 microg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13-C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.

  20. Biodegradation of furfural by Bacillus subtilis strain DS3.

    PubMed

    Zheng, Dan; Bao, Jianguo; Lu, Jueming; Lv, Quanxi

    2015-07-01

    An aerobic bacterial strain DS3, capable of growing on furfural as sole carbon source, was isolated from actived sludge of wastewater treatment plant in a diosgenin factory after enrichment. Based on morphological physiological tests as well as 16SrDNA sequence and Biolog analyses it was identified as Bacillus subtilis. The study revealed that strain DS3 utilized furfural, as analyzed by high-performance liquid chromatography (HPLC). Under following conditions: pH 8.0, temperature 35 degrees C, 150 rpm and 10% inoculum, strain DS3 showed 31.2% furfural degradation. Furthermore, DS3 strain was found to tolerate furfural concentration as high as 6000 mg(-1). The ability of Bacillus subtilis strain DS3 to degrade furfural has been demonstrated for the first time in the present study.

  1. A novel cold-inducible expression system for Bacillus subtilis.

    PubMed

    Thuy Le, Ai Thi; Schumann, Wolfgang

    2007-06-01

    Production of recombinant proteins at low temperatures is one strategy to prevent formation of protein aggregates and the use of an expensive inducer such as IPTG. We report on the construction of two expression vectors both containing the cold-inducible des promoter of Bacillus subtilis, where one allows intra- and the other extracellular synthesis of recombinant proteins. Production of recombinant proteins started within the first 30min after temperature downshock to 25 degrees C and continued for about 5h.

  2. [Bacillus subtilis metabolites as a novel promising probiotic preparations].

    PubMed

    Volkov, M Iu; Tkachenko, E I; Vorobeĭchikov, E V; Sinitsa, A V

    2007-01-01

    Characteristics of Bacillus subtilis metabolites contained in supernatants of its broth cultures are described. Metabolites contained bacterium-produced biologically active components ensuring cells growth and propagation. These components had bacteriostatic and bactericidal effect on gram-positive and gram-negative pathogenic and conditionally pathogenic microflora of gastrointestinal tract of human and animals. Enzymes produced by the bacterium (amylase, protease, cellulose-decomposing enzyme, lipase, pectinase) increased antagonistic properties of preparation and promote its probiotic effect.

  3. Genome engineering using a synthetic gene circuit in Bacillus subtilis

    PubMed Central

    Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

    2015-01-01

    Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac–chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. PMID:25552415

  4. Genome engineering using a synthetic gene circuit in Bacillus subtilis.

    PubMed

    Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

    2015-03-31

    Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.

  5. Removing Bacillus subtilis from fermentation broth using alumina nanoparticles.

    PubMed

    Mu, Dashuai; Mu, Xin; Xu, Zhenxing; Du, Zongjun; Chen, Guanjun

    2015-12-01

    In this study, an efficient separation technology using Al2O3 nanoparticles (NPs) was developed for removing Bacillus subtilis from fermentation broth. The dosage of alumina nanoparticles used for separating B. subtilis increased during the culture process and remained stable in the stationary phase of the culture process. The pH of the culture-broth was also investigated for its effects on flocculation efficiency, and showed an acidic pH could enhance the flocculation efficiency. The attachment mechanisms of Al2O3 NPs to the B. subtilis surface were investigated, and the zeta potential analysis showed that Al2O3 NPs could attach to B. subtilis via electrostatic attachment. Finally, the metabolite content and the antibacterial effect of the fermentation supernatants were detected and did not significantly differ between alumina nanoparticle separation and centrifugation separation. Together, these results indicate a great potential for a highly efficient and economical method for removing B. subtilis from fermentation broth using alumina nanoparticles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Anaerobic growth of a "strict aerobe" (Bacillus subtilis).

    PubMed

    Nakano, M M; Zuber, P

    1998-01-01

    There was a long-held belief that the gram-positive soil bacterium Bacillus subtilis is a strict aerobe. But recent studies have shown that B. subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron acceptor, or by fermentation. How B. subtilis alters its metabolic activity according to the availability of oxygen and alternative electron acceptors is but one focus of study. A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, occupies an early stage in the regulatory pathway governing anaerobic respiration. One of the essential roles of ResD and ResE in anaerobic gene regulation is induction of fnr transcription upon oxygen limitation. FNR is a transcriptional activator for anaerobically induced genes, including those for respiratory nitrate reductase, narGHJI.B. subtilis has two distinct nitrate reductases, one for the assimilation of nitrate nitrogen and the other for nitrate respiration. In contrast, one nitrite reductase functions both in nitrite nitrogen assimilation and nitrite respiration. Unlike many anaerobes, which use pyruvate formate lyase, B. subtilis can carry out fermentation in the absence of external electron acceptors wherein pyruvate dehydrogenase is utilized to metabolize pyruvate.

  7. Metabolic engineering of Bacillus subtilis for terpenoid production.

    PubMed

    Guan, Zheng; Xue, Dan; Abdallah, Ingy I; Dijkshoorn, Linda; Setroikromo, Rita; Lv, Guiyuan; Quax, Wim J

    2015-11-01

    Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in this bacterium have aroused much interest in the scientific community. This review discusses metabolic engineering of B. subtilis for terpenoid production, and encountered challenges will be discussed. We will summarize some major advances and outline future directions for exploiting the potential of B. subtilis as a desired "cell factory" to produce terpenoids.

  8. Expression of low endotoxin 3-O-sulfotransferase in Bacillus subtilis and Bacillus megaterium.

    PubMed

    Wang, Wenya; Englaender, Jacob A; Xu, Peng; Mehta, Krunal K; Suwan, Jiraporn; Dordick, Jonathan S; Zhang, Fuming; Yuan, Qipeng; Linhardt, Robert J; Koffas, Mattheos

    2013-10-01

    A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10(4)-10(5)-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.

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

    SciTech Connect

    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

    2005-09-14

    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.

  10. Characterisation and profiling of Bacillus subtilis, Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting.

    PubMed

    Fernández-No, I C; Böhme, K; Díaz-Bao, M; Cepeda, A; Barros-Velázquez, J; Calo-Mata, P

    2013-04-01

    The Bacillus genus includes species such as Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, some of which may be pathogenic or causative agents in the spoilage of food products. The main goal of this work was to apply matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass fingerprinting to the classification of these Bacillus species. Genetic analyses were also compared to phyloproteomic analyses. A collection of 57 Bacillus strains isolated from fresh and processed food and from culture collections were studied and their mass spectra compiled. The resulting mass fingerprints were compared and characteristic peaks at the strain and species levels were assigned. The results showed that MALDI-TOF was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate classification of Bacillus species, especially for differentiating B. subtilis and B. cereus from Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. MALDI-TOF was also found to provide valuable information at both intra- and interspecies levels in the Bacillus species studied. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Homolactic fermentation from glucose and cellobiose using Bacillus subtilis

    PubMed Central

    Romero-Garcia, Susana; Hernández-Bustos, Claudia; Merino, Enrique; Gosset, Guillermo; Martinez, Alfredo

    2009-01-01

    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 exclusively encodes a L

  12. Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

    PubMed Central

    Lampel, K A; Uratani, B; Chaudhry, G R; Ramaley, R F; Rudikoff, S

    1986-01-01

    The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase. Images PMID:3082854

  13. Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis.

    PubMed

    Chen, H; Wang, L; Su, C X; Gong, G H; Wang, P; Yu, Z L

    2008-09-01

    Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics. The antagonistic activity of JA was tested in vitro; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l(-1) HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C18 column (5 microm, 250 x 4.6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization-mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments. Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens. This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis.

  14. Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis.

    PubMed

    Bleich, Rachel; Watrous, Jeramie D; Dorrestein, Pieter C; Bowers, Albert A; Shank, Elizabeth A

    2015-03-10

    Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called "secondary" metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin's antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects--acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes.

  15. Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis

    PubMed Central

    Bleich, Rachel; Watrous, Jeramie D.; Dorrestein, Pieter C.; Bowers, Albert A.; Shank, Elizabeth A.

    2015-01-01

    Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called “secondary” metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin’s antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects—acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes. PMID:25713360

  16. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells

    PubMed Central

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

    2016-01-01

    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

  17. Analysis of the gluconate (gnt) operon of Bacillus subtilis.

    PubMed

    Reizer, A; Deutscher, J; Saier, M H; Reizer, J

    1991-05-01

    The gluconate (gnt) operon of Bacillus subtilis includes the gntR, gntK, gntP, and gntZ genes, respectively encoding the transcriptional repressor of the operon, gluconate kinase, the gluconate permease, and an unidentified open reading frame (Fujita and Fujita, 1987). We have compared the proteins encoded by the gnt operon of B.subtilis with published sequences and showed that (i) the gluconate repressor is homologous to several putative regulatory proteins in Escherichia coli, (ii) the gluconate kinase of B. subtilis is homologous to xylulose kinase, glycerol kinase and fucose kinase in E. coli (20-26% identity; 12-59 S.D.), (iii) the gluconate permease exhibits a C-terminal domain which is homologous to a hydrophobic protein encoded by an unidentified open reading frame (dsdAp) which precedes the dsdA gene of E. coli (39% identity; 19 S.D.), and (iv) the gntZ gene product is homologous to 6-phosphogluconate dehydrogenases of other bacteria and of animals (48-56%; 82-178 S.D.), thereby suggesting that the B. subtilis gntZ encodes 6-phosphogluconate dehydrogenase. Several conserved regions of the sequenced 6-phosphogluconate dehydrogenases can serve as signature patterns of this protein. Computer analyses have indicated that the previously reported sequences of the porcine and ovine 6-phosphogluconate dehydrogenases, as well as the hypothetical DsdAp protein, are probably erroneous. The probable reasons for the errors are reported along with the proposed revised sequences.

  18. Effects of a dried Bacillus subtilis culture on egg quality.

    PubMed

    Li, L; Xu, C L; Ji, C; Ma, Q; Hao, K; Jin, Z Y; Li, K

    2006-02-01

    The effects of a dried Bacillus subtilis culture on the egg qualities of layers were studied. Nine hundred and sixty 25-wk-old Lohmann Brown layers were randomly divided into 5 groups with 192 layers in each group. Layers in group 1 were fed a control diet. The remaining groups received the control diet that contained either 20 mg of zinc bacitracin/kg and 4 mg of colistin sulfate/kg or 500, 1,000, or 1,500 mg of B. subtilis culture/ kg, respectively. The results showed improvements in egg production, feed consumption, and feed conversion (P < 0.05) of layers when 500 mg of B. subtilis culture/kg was added to the diets. The results also showed some special improvements in this group, including increases in eggshell thickness, yolk color, and Haugh unit, and decreases in yolk cholesterol concentration (P < 0.05). However, excessive doses of B. subtilis culture did not improve the performance of layers.

  19. Engineering a metabolic pathway for isobutanol biosynthesis in Bacillus subtilis.

    PubMed

    Jia, Xiaoqiang; Li, Shanshan; Xie, Sha; Wen, Jianping

    2012-09-01

    Isobutanol can be biosynthesized via α-ketoisovalerate catalyzed by heterologous keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH). In this work, isobutanol biosynthesis pathway was designed in Bacillus subtilis, a notable solvent-tolerant host. In order to do that, a plasmid pPKA expressing KDC and ADH under the control of a B. subtilis strong promoter P(43) was constructed. Isobutanol was detected in the products of the recombinant B. subtilis harboring pPKA plasmid, whereas none was detected by the wild-type strain. Effects of the medium ingredients such as glucose concentration and valine addition, and operating parameters such as initial pH, inoculation volume, and medium work volume on isobutanol production were also investigated. Isobutanol production reached to the maximum of 0.607 g/L after 35-h cultivation under the conditions: glucose concentration of 3%, valine addition of 2%, initial pH of 7.0, inoculum of 1%, and work volume of 50 mL/250 mL. Though the isobutanol production by the recombinant was low, it was the first successful attempt to produce isobutanol in engineered B. subtilis, and the results showed its great potential as an isobutanol-producing cell factory.

  20. Bacillus subtilis antibiotics: structures, syntheses and specific functions.

    PubMed

    Stein, Torsten

    2005-05-01

    The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

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

    2014-01-01

    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.

  3. Effects of Bacillus subtilis KD1 on broiler intestinal flora.

    PubMed

    Wu, B Q; Zhang, T; Guo, L Q; Lin, J F

    2011-11-01

    A novel Bacillus subtilis KD1 strain was isolated and identified from healthy broilers, and its phylogenetic classification was subsequently analyzed. To evaluate its probiotic availability, its growth characteristics and tolerance for the gut environment were evaluated in vitro. The results suggest that B. subtilis KD1 is superior in secreting neutral protease and is highly tolerant of gastric acid and bile salt. In the logarithmic growth phase, the neutral protease reached a maximum of 1,369.3 U/mL. When all live bacteria had become spores in the broth, B. subtilis KD1 was freeze dried and fed to broilers at 10(9), 5 × 10(9), and 10(10) bacilli/kg of feed. The animal trial results suggest that the addition of the new strain significantly improved intestinal flora by increasing lactobacilli and reducing Escherichia coli (P < 0.05) as compared with the control; hence, B. subtilis KD1 is a promising probiotic organism in broilers.

  4. Natural Genetic Competence in Bacillus subtilis Natto OK2

    PubMed Central

    Ashikaga, Sayaka; Nanamiya, Hideaki; Ohashi, Yoshiaki; Kawamura, Fujio

    2000-01-01

    We isolated a Bacillus subtilis natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using a comG-lacZ fusion at the amyE locus. Although transcription of the late competence genes was not detected in the B. subtilis natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the mecA or the clpC mutation derived from B. subtilis 168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for B. subtilis 168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the mecA and clpC mutants of strain OK2 (ComKOK2) could activate the transcription of the whole set of late competence genes and suggested that ComKOK2 was not activated in strain OK2 during competence development. We therefore sequenced the comS gene of OK2 and compared it with that of 168. The comSOK2 had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11. PMID:10762239

  5. Natural genetic competence in Bacillus subtilis natto OK2.

    PubMed

    Ashikaga, S; Nanamiya, H; Ohashi, Y; Kawamura, F

    2000-05-01

    We isolated a Bacillus subtilis natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using a comG-lacZ fusion at the amyE locus. Although transcription of the late competence genes was not detected in the B. subtilis natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the mecA or the clpC mutation derived from B. subtilis 168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for B. subtilis 168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the mecA and clpC mutants of strain OK2 (ComK(OK2)) could activate the transcription of the whole set of late competence genes and suggested that ComK(OK2) was not activated in strain OK2 during competence development. We therefore sequenced the comS gene of OK2 and compared it with that of 168. The comS(OK2) had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.

  6. Growing Bacillus subtilis tendrils sense and avoid each other.

    PubMed

    James, Barry L; Kret, Jennifer; Patrick, Joyce E; Kearns, Daniel B; Fall, Ray

    2009-09-01

    Growing tendrils of aflagellate hag mutants of Bacillus subtilis were found to show an avoidance response when colonizing a semi-solid medium, suggesting a tip-to-tip communication mechanism between colonies. There may be a second sensing mechanism involved in shaping the morphology of tendrils. Tendril growth in B. subtilis was dependent on and possibly shaped by the release of surfactin, a biosurfactant. Transposon mutagenesis yielded two mutants with 'touching' tendrils, and each had a disrupted gspA gene that encodes a putative glycosyltransferase. Tendrils of gspA mutants, unlike the parental strain, were unresponsive to tendril tip growth by surfactin, suggesting disruption of intercellular signaling. Tendril sensing and avoidance could be physiologically relevant in habitats, such as plant roots, where some limiting nutrient might induce this type of multicellular behavior, promoting avoidance of previously explored areas by sibling colonies.

  7. [Molecular cloning and expression of Nattokinase gene in Bacillus subtilis].

    PubMed

    Liu, B Y; Song, H Y

    2002-05-01

    In order to characterize biochemically the nattokinase,the nucleotide sequence of the nattokinase gene was amplified from the chromosomal DNA of B.subtilis (natto) by PCR. The expression plasmid pBL NK was constructed and was used to transform Bacillus subtilis containing a chromosomal deletion in its subtilisin gene. The supernatant of the culture was collected after 15 h culture. The target proteins were identified by SDS-PAGE. Nattokinase was purified by a method including ultrafiltration, Sephacryl S-100 gel filtration and S-Sepharose ion-exchange chromatography, and 100 mg of purified nattokinase was obtained from one liter of culture. The purity of the protein and the specific activity were 95% and 12 000 u/mg (compared to tPA), respectively.

  8. An improved protocol for harvesting Bacillus subtilis colony biofilms.

    PubMed

    Fuchs, Felix Matthias; Driks, Adam; Setlow, Peter; Moeller, Ralf

    2017-03-01

    Bacterial biofilms cause severe problems in medicine and industry due to the high resistance to disinfectants and environmental stress of organisms within biofilms. Addressing challenges caused by biofilms requires full understanding of the underlying mechanisms for bacterial resistance and survival in biofilms. However, such work is hampered by a relative lack of systems for biofilm cultivation that are practical and reproducible. To address this problem, we developed a readily applicable method to culture Bacillus subtilis biofilms on a membrane filter. The method results in biofilms with highly reproducible characteristics, and which can be readily analyzed by a variety of methods with little further manipulation. This biofilm preparation method simplifies routine generation of B. subtilis biofilms for molecular and cellular analysis, and could be applicable to other microbial systems.

  9. Enhanced secretion of natto phytase by Bacillus subtilis.

    PubMed

    Tsuji, Shogo; Tanaka, Kosei; Takenaka, Shinji; Yoshida, Ken-ichi

    2015-01-01

    Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.

  10. Evidence of isoprenoid precursor toxicity in Bacillus subtilis.

    PubMed

    Sivy, Tami L; Fall, Ray; Rosenstiel, Todd N

    2011-01-01

    The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.

  11. Phosphate Starvation Induces the Sporulation Killing Factor of Bacillus subtilis

    PubMed Central

    Allenby, Nicholas E. E.; Watts, Carys A.; Homuth, Georg; Prágai, Zoltán; Wipat, Anil; Ward, Alan C.; Harwood, Colin R.

    2006-01-01

    Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript. PMID:16816204

  12. Phosphate starvation induces the sporulation killing factor of Bacillus subtilis.

    PubMed

    Allenby, Nicholas E E; Watts, Carys A; Homuth, Georg; Prágai, Zoltán; Wipat, Anil; Ward, Alan C; Harwood, Colin R

    2006-07-01

    Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript.

  13. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis.

    PubMed

    Gryczan, T; Shivakumar, A G; Dubnau, D

    1980-01-01

    Restriction endonuclease cleavage maps of seven chimeric plasmids that may be used for molecular cloning in Bacillus subtilis are presented. These plasmids all carry multiple antibiotic resistance markers and were constructed by in vitro molecular cloning techniques. Several of the antibiotic resistance markers were shown to undergo insertional inactivation at specific restriction endonuclease sites. Kanamycin inactivation occurred at the BglII site of pUB110 derivatives, erythromycin inactivation occurred at the HpaI and BclI sites of pE194 derivatives, and streptomycin inactivation occurred at the HindIII site of pSA0501 derivatives. A stable mini-derivative of pBD12 was isolated and characterized. By using these plasmids, we identified proteins involved in plasmid-coded kanamycin and erythromycin resistance. The properties and uses of these chimeric plasmids in the further development of recombinant deoxyribonucleic acid technology in B. subtilis are discussed.

  14. Characterization of an inducible oxidative stress system in Bacillus subtilis.

    PubMed

    Bol, D K; Yasbin, R E

    1990-06-01

    Exponentially growing cells of Bacillus subtilis demonstrated inducible protection against killing by hydrogen peroxide when prechallenged with a nonlethal dose of this oxidative agent. Cells deficient in a functional recE+ gene product were as much as 100 times more sensitive to the H2O2 but still exhibited an inducible protective response. Exposure to hydrogen peroxide also induced the recE(+)-dependent DNA damage-inducible (din) genes, the resident prophage, and the product of the recE+ gene itself. Thus hydrogen peroxide is capable of inducing the SOS-like or SOB system of B. subtilis. However, the induction of this DNA repair system by other DNA-damaging agents is not sufficient to activate the protective response to hydrogen peroxide. Therefore, at least one more regulatory network (besides the SOB system) that responds to oxidative stress must exist. Furthermore, the data presented indicate that a functional catalase gene is necessary for this protective response.

  15. 40 CFR 180.1309 - Bacillus subtilis strain CX-9060; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus subtilis strain CX-9060... RESIDUES IN FOOD Exemptions From Tolerances § 180.1309 Bacillus subtilis strain CX-9060; exemption from the... the microbial pesticide Bacillus subtilis strain CX-9060, in or on all food commodities, when applied...

  16. 40 CFR 180.1309 - Bacillus subtilis strain CX-9060; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus subtilis strain CX-9060... RESIDUES IN FOOD Exemptions From Tolerances § 180.1309 Bacillus subtilis strain CX-9060; exemption from the... the microbial pesticide Bacillus subtilis strain CX-9060, in or on all food commodities, when applied...

  17. 40 CFR 180.1309 - Bacillus subtilis strain CX-9060; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus subtilis strain CX-9060... RESIDUES IN FOOD Exemptions From Tolerances § 180.1309 Bacillus subtilis strain CX-9060; exemption from the... the microbial pesticide Bacillus subtilis strain CX-9060, in or on all food commodities, when applied...

  18. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  19. 40 CFR 180.1209 - Bacillus subtilis strain QST 713; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus subtilis strain QST 713... RESIDUES IN FOOD Exemptions From Tolerances § 180.1209 Bacillus subtilis strain QST 713; exemption from the... the microbial pesticide Bacillus subtilis strain QST 713 when used in or on all food commodities....

  20. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  1. 40 CFR 180.1209 - Bacillus subtilis strain QST 713; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus subtilis strain QST 713... RESIDUES IN FOOD Exemptions From Tolerances § 180.1209 Bacillus subtilis strain QST 713; exemption from the... the microbial pesticide Bacillus subtilis strain QST 713 when used in or on all food commodities....

  2. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  3. 40 CFR 180.1209 - Bacillus subtilis strain QST 713; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis strain QST 713... RESIDUES IN FOOD Exemptions From Tolerances § 180.1209 Bacillus subtilis strain QST 713; exemption from the... the microbial pesticide Bacillus subtilis strain QST 713 when used in or on all food commodities....

  4. Development of an intermolecular transposition assay system in Bacillus subtilis 168 using IS4Bsu1 from Bacillus subtilis (natto).

    PubMed

    Takahashi, Kiwamu; Sekine, Yasuhiko; Chibazakura, Taku; Yoshikawa, Hirofumi

    2007-08-01

    Most of the spontaneous poly-gamma-glutamate (gamma-PGA)-deficient mutants of Bacillus subtilis (natto) appear to have resulted from the insertion of IS4Bsu1 exclusively into the comP gene. However, complete genomic analysis of B. subtilis 168, a close relative of B. subtilis (natto), revealed no IS4Bsu1 insertion. Preliminary experiments using a transformable 'natto' strain indicated that the frequency of transposition of IS4Bsu1 was exceptionally high under competence-developing conditions. On the other hand, such high-frequency transposition was not observed when cells were grown in a rich medium, such as LB medium, suggesting that there must be suitable environmental conditions that give rise to the transposition of IS4Bsu1. To assess the behaviour of IS4Bsu1 and explore any host factors playing roles in IS transposition, an intermolecular transposition assay system was constructed using a modified IS4Bsu1 element in B. subtilis 168. Here, the details of the intermolecular transposition assay system are given, and the increase in transposition frequency observed under high-temperature and competence-inducing conditions is described.

  5. Bacterial determinants of the social behavior of Bacillus subtilis.

    PubMed

    Romero, Diego

    2013-09-01

    Bacteria utilize sophisticated cellular machinery to sense environmental changes and coordinate the most appropriate response. Fine sensors located on cell surfaces recognize a myriad of triggers and initiate genetic cascades leading to activation or repression of certain groups of genes. Structural elements such as pilli, exopolysaccharides and flagella are also exposed at the cell surface and contribute to modulating the intimate interaction with surfaces and host cells. This review will cover the latest advances in our understanding of the biology and functionality of these bacterial determinants within the context of biofilm formation of Bacillus subtilis.

  6. VIABILITY OF BACILLUS SUBTILIS SPORES IN ROCKET PROPELLANTS.

    PubMed

    GODDING, R M; LYNCH, V H

    1965-01-01

    The sporicidal activity of components used in liquid and solid rocket propellants was tested by use of spores of Bacillus subtilis dried on powdered glass. Liquid propellant ingredients tested were N(2)O(4), monomethylhydrazine and 1,1-dimethylhydrazine. N(2)O(4) was immediately sporicidal; the hydrazines were effective within several days. Solid propellants consisted of ammonium perchlorate in combination with epoxy resin (EPON 828), tris-1-(2-methyl) aziridinyl phosphine oxide, bis-1-(2-methyl) aziridinyl phenylphosphine oxide, and three modified polybutadiene polymers. There was no indication of appreciable sporicidal activity of these components.

  7. Viability of Bacillus subtilis Spores in Rocket Propellants

    PubMed Central

    Godding, Rogene M.; Lynch, Victoria H.

    1965-01-01

    The sporicidal activity of components used in liquid and solid rocket propellants was tested by use of spores of Bacillus subtilis dried on powdered glass. Liquid propellant ingredients tested were N2O4, monomethylhydrazine and 1,1-dimethylhydrazine. N2O4 was immediately sporicidal; the hydrazines were effective within several days. Solid propellants consisted of ammonium perchlorate in combination with epoxy resin (EPON 828), tris-1-(2-methyl) aziridinyl phosphine oxide, bis-1-(2-methyl) aziridinyl phenylphosphine oxide, and three modified polybutadiene polymers. There was no indication of appreciable sporicidal activity of these components. PMID:14264838

  8. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis

    PubMed Central

    Elsholz, Alexander K. W.; Birk, Marlene S.; Charpentier, Emmanuelle; Turgay, Kürşad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics. PMID:28748186

  9. Properties of Thermosensitive Extracellular α-Amylases of Bacillus subtilis

    PubMed Central

    Yamane, Kunio; Maruo, Bunji

    1974-01-01

    Enzymological properties of four thermosensitive α-amylases (M3, M9, M18, and M20) brought by different mutation sites in α-amylase structural gene of Bacillus subtilis were compared with those of the parental α-amylase NA64. Two thermosensitive α-amylases (M9 and M20) were altered not only in their thermosensitivity but also in their immunological properties, catalytic properties, molecular weights determined by the gel filtration on a Bio-Gel P-100 column, and others. The other two thermosensitive α-amylases (M3 and M18) were altered only in their thermosensitivity. Images PMID:4218234

  10. Onset of bioconvection in suspensions of Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Jánosi, Imre M.; Kessler, John O.; Horváth, Viktor K.

    1998-10-01

    Bioconvection occurs when upward swimming micro-organisms generate gravitational energy that initiates and maintains dissipative movement of the water in which they swim. Advection, and motion of the organisms relative to the fluid, generate patchiness in concentration that drives and shapes the geometry and rate of convection. This paper presents a method for quantitatively analyzing the development of self-organization, and numerical estimates that connect and interpret theory and experiment. While the oxygen consuming, oxgen-gradient-guided bacteria Bacillus subtilis are the sole subject here, the methods developed will find application to the analysis and modeling of other complex dynamic systems that ineluctably combine physics and biology.

  11. Modelling Fractal Growth of Bacillus subtilis on Agar Plates

    NASA Astrophysics Data System (ADS)

    Fogedby, Hans C.

    1991-02-01

    The observed fractal growth of a bacterial colony of Bacillus subtilis on agar plates is simulated by a simple computer model in two dimensions. Growth morphologies are shown and the fractal dimension is computed. The concentration of nutrients and the time scale ratio of bacterial multiplication and nutrient diffusion are the variable parameters in the model. Fractal growth is observed in the simulations for moderate concentrations and time scale ratios. The simulated morphologies are similar to the ones grown in the biological experiment. The phenomenon is analogous to the fractal morphologies of lipid layers grown on a water surface.

  12. Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

    PubMed

    Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

    2016-08-11

    Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

  13. Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis

    PubMed Central

    Bashir, Abdallah; Hoffmann, Tamara; Smits, Sander H. J.

    2014-01-01

    Glycine betaine is a potent osmotic and thermal stress protectant of many microorganisms. Its synthesis from glycine results in the formation of the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG), and these compounds are also produced when it is catabolized. Bacillus subtilis does not produce sarcosine or DMG, and it cannot metabolize these compounds. Here we have studied the potential of sarcosine and DMG to protect B. subtilis against osmotic, heat, and cold stress. Sarcosine, a compatible solute that possesses considerable protein-stabilizing properties, did not serve as a stress protectant of B. subtilis. DMG, on the other hand, proved to be only moderately effective as an osmotic stress protectant, but it exhibited good heat stress-relieving and excellent cold stress-relieving properties. DMG is imported into B. subtilis cells primarily under osmotic and temperature stress conditions via OpuA, a member of the ABC family of transporters. Ligand-binding studies with the extracellular solute receptor (OpuAC) of the OpuA system showed that OpuAC possesses a moderate affinity for DMG, with a Kd value of approximate 172 μM; its Kd for glycine betaine is about 26 μM. Docking studies using the crystal structures of the OpuAC protein with the sulfur analog of DMG, dimethylsulfonioacetate, as a template suggest a model of how the DMG molecule can be stably accommodated within the aromatic cage of the OpuAC ligand-binding pocket. Collectively, our data show that the ability to acquire DMG from exogenous sources under stressful environmental conditions helps the B. subtilis cell to cope with growth-restricting osmotic and temperature challenges. PMID:24561588

  14. Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

    PubMed

    Shao, Huanhuan; Cao, Qinghua; Zhao, Hongyan; Tan, Xuemei; Feng, Hong

    2015-01-01

    A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus.

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

    SciTech Connect

    Mitchell, C.; Vary, J.C. )

    1989-06-01

    During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link ({alpha}-{sup 32}P)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 ({alpha}-{sup 32}P)GTP or ({gamma}-{sup 32}P)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.

  16. Crystal Structure of the Bacillus subtilis Superoxide Dismutase

    SciTech Connect

    Liu, Ping; Ewis, H.E.; Huang, Y.-J; Lu, C.-D.; Tai, P.C.; Weber, Irene T.

    2008-06-01

    The sodA gene of Bacillus subtilis was expressed in Escherichia coli, purified and crystallized. The crystal structure of MnSOD was solved by molecular replacement with four dimers per asymmetric unit and refined to an R factor of 21.1% at 1.8 {angstrom} resolution. The dimer structure is very similar to that of the related enzyme from B. anthracis. Larger structural differences were observed with the human MnSOD, which has one less helix in the helical domain and a longer loop between two -strands and also showed differences in three amino acids at the intersubunit interface in the dimer compared with the two bacterial MnSODs. These structural differences can be exploited in the design of drugs that selectively target the Bacillus enzymes.

  17. [Dual promoters enhance heterologous enzyme production from bacterial phage based recombinant Bacillus subtilis].

    PubMed

    Liu, Gang; Zhang, Yan; Xing, Miao

    2006-03-01

    The effect of dual promoters on recombinant protein production from bacterial phage based Bacillus subtilis expression system was investigated. Alpha amylase (from Bacillus amyloliquefaciens) and penicillin acylase (from Bacillus megaterium) were selected as the indicating enzymes. Both the promoterless genes and the promoter-bearing genes were isolated through PCR amplification with properly designed primers, and were inserted into plasmid pSG703 that contains the lacZ-cat expression cartridge. The lysogenic B. subtilis (phi105 MU331) was transformed with the resultant recombinant plasmids, and the heterologous genes were thereby integrated into the chromosommal DNA of B. subtilis via homologous recombination. The transformants were designated as B. subtilis AMY1, B. subtilis AMY2, B. subtilis PA1, and B. subtilis PA2, respectively. In the recombinant B. subtilis strains, the inserted sequences were located down stream of a strong phage promoter that could be activated by thermal induction. In B. subtilis AMY1 and B. subtilis PA1, transcription of the heterologous genes was only initiated by the phage promoter after heat shock, whereas in B. subtilis AMY2 and B. subtilis PA2, transcription of the heterologous genes was initiated by dual promoters, the phage promoter and the native promoter. The application of dual promoters increased the productivity of both enzymes, with 133% enhancement for alpha-amylase production and 113% enhancement for penicillin acylase production.

  18. Secretion of heterologous thermostable cellulases in Bacillus subtilis.

    PubMed

    Lan Thanh Bien, Thi; Tsuji, Shogo; Tanaka, Kosei; Takenaka, Shinji; Yoshida, Ken-ichi

    2014-01-01

    Bacillus subtilis is used industrially for the production of secreted enzymes. The most characteristic feature of the secreted enzymes is variation in the N-terminal signal peptides that is recognized by secretion machinery, which is one of the determinants of efficiency and must be customized in each case. Culturing cellulolytic B. subtilis to secrete heterologous cellulases combined with customized signal peptides would be beneficial for producing biocommodities from cellulosic biomass. Four Clostridium thermocellum genes, encoding endoglucanases (celA and celB) and exoglucanases (celK and celS) were cloned to construct random libraries of combinations with 173 different signal peptides originating from the B. subtilis genome. The libraries were successfully screened to identify the signal peptides most efficient in secretion of each of the four cellulases, which were theoretically unpredictable. The secreted cellulases were assayed on carboxymethyl cellulose, phosphoric acid swollen cellulose, and microcrystalline cellulose to determine the possible effects of the signal peptides on substrate specificity. The customized signal peptides for CelA, CelB, and CelS did not affect enzyme performance but those for CelK might influence its substrate specificity.

  19. The cell biology of peritrichous flagella in Bacillus subtilis.

    PubMed

    Guttenplan, Sarah B; Shaw, Sidney; Kearns, Daniel B

    2013-01-01

    Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid-like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.

  20. Genomic Reconstruction of the Transcriptional Regulatory Network in Bacillus subtilis

    PubMed Central

    Leyn, Semen A.; Kazanov, Marat D.; Sernova, Natalia V.; Ermakova, Ekaterina O.; Novichkov, Pavel S.

    2013-01-01

    The adaptation of microorganisms to their environment is controlled by complex transcriptional regulatory networks (TRNs), which are still only partially understood even for model species. Genome scale annotation of regulatory features of genes and TRN reconstruction are challenging tasks of microbial genomics. We used the knowledge-driven comparative-genomics approach implemented in the RegPredict Web server to infer TRN in the model Gram-positive bacterium Bacillus subtilis and 10 related Bacillales species. For transcription factor (TF) regulons, we combined the available information from the DBTBS database and the literature with bioinformatics tools, allowing inference of TF binding sites (TFBSs), comparative analysis of the genomic context of predicted TFBSs, functional assignment of target genes, and effector prediction. For RNA regulons, we used known RNA regulatory motifs collected in the Rfam database to scan genomes and analyze the genomic context of new RNA sites. The inferred TRN in B. subtilis comprises regulons for 129 TFs and 24 regulatory RNA families. First, we analyzed 66 TF regulons with previously known TFBSs in B. subtilis and projected them to other Bacillales genomes, resulting in refinement of TFBS motifs and identification of novel regulon members. Second, we inferred motifs and described regulons for 28 experimentally studied TFs with previously unknown TFBSs. Third, we discovered novel motifs and reconstructed regulons for 36 previously uncharacterized TFs. The inferred collection of regulons is available in the RegPrecise database (http://regprecise.lbl.gov/) and can be used in genetic experiments, metabolic modeling, and evolutionary analysis. PMID:23504016

  1. Assessment of the Requirements for Magnesium Transporters in Bacillus subtilis

    PubMed Central

    Wakeman, Catherine A.; Goodson, Jonathan R.; Zacharia, Vineetha M.

    2014-01-01

    Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions. PMID:24415722

  2. Foam Separation of Pseudomonas fluorescens and Bacillus subtilis var. niger

    PubMed Central

    Grieves, R. B.; Wang, S. L.

    1967-01-01

    An experimental investigation established the effect of the presence of inorganic salts on the foam separation of Pseudomonas fluorescens and of Bacillus subtilis var. niger (B. globigii) from aqueous suspension by use of a cationic surfactant. For P. fluorescens, 5.0 μeq/ml of NaCl, KCl, Na2SO4, K2SO4, CaCl2, CaSO4, MgCl2, or MgSO4 produced increases in the cell concentration in the residual suspension (not carried into the foam) from 2.9 × 105 up to 1.6 × 106 to 2.8 × 107 cells per milliliter (initial suspensions contain from 3.3 × 107 to 4.8 × 107 cells per milliliter). The exceptional influence of magnesium was overcome by bringing the cells into contact first with the surfactant and then the salt. For B. subtilis, the presence of 5.0 μeq/ml of any of the eight salts increased the residual cell concentration by one order of magnitude from 1.2 × 104 to about 4.0 × 105 cells per milliliter. This occurred regardless of the sequence of contact as long as the surfactant contact period was sufficient. The presence of salts increased collapsed foam volumes with P. fluorescens and decreased collapsed foam volumes with B. subtilis. PMID:4961933

  3. Bacillus subtilis spores as adjuvants for DNA vaccines.

    PubMed

    Aps, Luana R M M; Diniz, Mariana O; Porchia, Bruna F M M; Sales, Natiely S; Moreno, Ana Carolina R; Ferreira, Luís C S

    2015-05-11

    Recently, Bacillus subtilis spores were shown to be endowed with strong adjuvant capacity when co-administered with purified antigenic proteins. In the present study we assessed whether spores possess adjuvant properties when combined with DNA vaccines. We showed that B. subtilis spores promoted the activation of dendritic cells in vitro and induced migration of pro-inflammatory cells after parenteral administration to mice. Likewise, co-administration of spores with a DNA vaccine encoding the human papillomavirus type 16 (HPV-16) E7 protein enhanced the activation of antigen-specific CD8(+) T cell responses in vivo. Mice immunized with the DNA vaccine admixed with spores presented a protective immunity increase to previously implanted tumor cells, capable of expressing HPV-16 oncoproteins. Finally, we observed that the adjuvant effect can vary accordingly to the number of co-administered spores which may be ascribed with the ability to induce. Collectively, the present results demonstrate for the first time that B. subtilis spores can also confer adjuvant effects to DNA vaccines.

  4. Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics

    PubMed Central

    Tiyanont, Kittichoat; Doan, Thierry; Lazarus, Michael B.; Fang, Xiao; Rudner, David Z.; Walker, Suzanne

    2006-01-01

    The peptidoglycan (PG) layers surrounding bacterial cells play an important role in determining cell shape. The machinery controlling when and where new PG is made is not understood, but is proposed to involve interactions between bacterial actin homologs such as Mbl, which forms helical cables within cells, and extracellular multiprotein complexes that include penicillin-binding proteins. It has been suggested that labeled antibiotics that bind to PG precursors may be useful for imaging PG to help determine the genes that control the biosynthesis of this polymer. Here, we compare the staining patterns observed in Bacillus subtilis using fluorescent derivatives of two PG-binding antibiotics, vancomycin and ramoplanin. The staining patterns for both probes exhibit a strong dependence on probe concentration, suggesting antibiotic-induced perturbations in PG synthesis. Ramoplanin probes may be better imaging agents than vancomycin probes because they yield clear staining patterns at concentrations well below their minimum inhibitory concentrations. Under some conditions, both ramoplanin and vancomycin probes produce helicoid staining patterns along the cylindrical walls of B. subtilis cells. This sidewall staining is observed in the absence of the cytoskeletal protein Mbl. Although Mbl plays an important role in cell shape determination, our data indicate that other proteins control the spatial localization of the biosynthetic complexes responsible for new PG synthesis along the walls of B. subtilis cells. PMID:16832063

  5. Selected metal ions protect Bacillus subtilis biofilms from erosion.

    PubMed

    Grumbein, S; Opitz, M; Lieleg, O

    2014-08-01

    Many problems caused by bacterial biofilms can be traced back to their high resilience towards chemical perturbations and their extraordinary sturdiness towards mechanical forces. However, the molecular mechanisms that link the mechanical properties of a biofilm with the ability of bacteria to survive in different chemical environments remain enigmatic. Here, we study the erosion stability of Bacillus subtilis (B. subtilis) biofilms in the presence of different chemical environments. We find that these biofilms can utilize the absorption of certain metal ions such as Cu(2+), Zn(2+), Fe(2+), Fe(3+) and Al(3+) into the biofilm matrix to avoid erosion by shear forces. Interestingly, many of these metal ions are toxic for planktonic B. subtilis bacteria. However, their toxic activity is suppressed when the ions are absorbed into the biofilm matrix. Our experiments clearly demonstrate that the biofilm matrix has to fulfill a dual function, i.e. regulating both the mechanical properties of the biofilm and providing a selective barrier towards toxic chemicals.

  6. Computational design of glutamate dehydrogenase in Bacillus subtilis natto.

    PubMed

    Chen, Li-Li; Wang, Jia-Le; Hu, Yu; Qian, Bing-Jun; Yao, Xiao-Min; Wang, Jing-Fang; Zhang, Jian-Hua

    2013-04-01

    Bacillus subtilis natto is widely used in industry to produce natto, a traditional and popular Japanese soybean food. However, during its secondary fermentation, high amounts of ammonia are released to give a negative influence on the flavor of natto. Glutamate dehydrogenase (GDH) is a key enzyme for the ammonia produced and released, because it catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD(+) or NADP(+) as co-factor during carbon and nitrogen metabolism processes. To solve this problem, we employed multiple computational methods model and re-design GDH from Bacillus subtilis natto. Firstly, a structure model of GDH with cofactor NADP(+) was constructed by threading and ab initio modeling. Then the substrate glutamate were flexibly docked into the structure model to form the substrate-binding mode. According to the structural analysis of the substrate-binding mode, Lys80, Lys116, Arg196, Thr200, and Ser351 in the active site were found could form a significant hydrogen bonding network with the substrate, which was thought to play a crucial role in the substrate recognition and position. Thus, these residues were then mutated into other amino acids, and the substrate binding affinities for each mutant were calculated. Finally, three single mutants (K80A, K116Q, and S351A) were found to have significant decrease in the substrate binding affinities, which was further supported by our biochemical experiments.

  7. Proline Utilization by Bacillus subtilis: Uptake and Catabolism

    PubMed Central

    Moses, Susanne; Sinner, Tatjana; Zaprasis, Adrienne; Stöveken, Nadine; Hoffmann, Tamara; Belitsky, Boris R.; Sonenshein, Abraham L.

    2012-01-01

    l-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced l-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the Δ1-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize l-proline to l-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an l-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of l-proline in the growth medium. However, the very large quantities of l-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external l-proline was not dependent on l-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward l-proline. Our findings imply that B. subtilis can distinguish externally supplied l-proline from internal l-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo l-proline synthesis and consumption by not triggering the expression of the putBCP l-proline catabolic genes in response to the osmoadaptive production of the compatible solute l-proline. PMID:22139509

  8. Inactivation of Bacillus subtilis spores with ozone and monochloramine.

    PubMed

    Larson, Matthew A; Mariñas, Benito J

    2003-02-01

    The inactivation kinetics of Bacillus subtilis spores with ozone and monochloramine was characterized by a lag phase followed by a pseudo-first-order rate of inactivation. The lag phase decreased and the post-lag phase rate constant increased with increasing temperature within the range investigated (1-30 degrees C for ozone, 1-20 degrees C for monochloramine). The corresponding activation energies were 46820 J/mol for ozone and 79640 J/mol for monochloramine. The CT concept was found to be valid within the concentration range investigated of 0.44-4.8 mg/l for ozone, and 3.8-7.7 mg/l as Cl(2) for monochloramine. The inactivation kinetics of B. subtilis spores with both ozone and monochloramine varied with pH within the range of pH 6-10 investigated. The fastest ozone and monochloramine inactivation rates were observed at pH 10 and 6, respectively. Different stocks of the same strain of B. subtilis spores had different resistance to ozone and monochloramine mainly because of discrepancies in the extent of the lag phase. B. subtilis spores might not be conservative surrogates for C. parvum oocysts for ozone disinfection at relatively low temperature mainly due to the spores having a lower activation energy compared to that for the oocysts. In contrast, the activation energy for monochloramine was comparable for both microorganisms but differences in the extent of the lag phase might result in the spores being overly conservative surrogates for the oocysts at relatively low temperature.

  9. MutS2 Promotes Homologous Recombination in Bacillus subtilis.

    PubMed

    Burby, Peter E; Simmons, Lyle A

    2017-01-15

    Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Gram-positive bacterium Bacillus subtilis In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2.

  10. Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis.

    PubMed

    Makroczyová, Jana; Jamroškovič, Ján; Krascsenitsová, Eva; Labajová, Nad'a; Barák, Imrich

    2016-06-01

    In rod-shaped bacteria, the proper placement of the division septum at the midcell relies, at least partially, on the proteins of the Min system as an inhibitor of cell division. The main principle of Min system function involves the formation of an inhibitor gradient along the cell axis; however, the establishment of this gradient differs between two well-studied gram-negative and gram-positive bacteria. While in gram-negative Escherichia coli, the Min system undergoes pole-to-pole oscillation, in gram-positive Bacillus subtilis, proper spatial inhibition is achieved by the preferential attraction of the Min proteins to the cell poles. Nevertheless, when E.coli Min proteins are inserted into B.subtilis cells, they still oscillate, which negatively affects asymmetric septation during sporulation in this organism. Interestingly, homologs of both Min systems were found to be present in various combinations in the genomes of anaerobic and endospore-forming Clostridia, including the pathogenic Clostridium difficile. Here, we have investigated the localization and behavior of C.difficile Min protein homologs and showed that MinDE proteins of C.difficile can oscillate when expressed together in B.subtilis cells. We have also investigated the effects of this oscillation on B.subtilis sporulation, and observed decreased sporulation efficiency in strains harboring the MinDE genes. Additionally, we have evaluated the effects of C.difficile Min protein expression on vegetative division in this heterologous host. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

    PubMed Central

    Black, Katherine A.

    2015-01-01

    ABSTRACT The 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s2U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s2U biosynthesis. A conditional B. subtilis knockout strain showed that s2U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis. In vitro experiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli ΔiscS and ΔmnmA strains associated with s2U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA. IMPORTANCE The 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U in Escherichia coli requires

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

    PubMed

    Luo, Chuping; Liu, Xuehui; Zhou, Xian; Guo, Junyao; Truong, John; Wang, Xiaoyu; Zhou, Huafei; Li, Xiangqian; Chen, Zhiyi

    2015-10-01

    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.

  13. [Features of Bacillus subtilis IMB B-7023 and its streptomycin-resistant strain].

    PubMed

    Roĭ, A A; Iatsenko, I P; Gordienko, A S; Kurdish, I K

    2011-01-01

    Features of phosphate-mobilizing bacteria Bacillus subtilis IMB B-7023 and its streptomycin-resistant strain were investigated. While cultivated in medium with glucose and glycerophosphate, the growth rate of the antibiotic-marked strain was approximately similar to this parameter for Bacillus subtilis IMB B-7023 but cell sizes were 1.3-fold less. Both strains significantly stimulated the germinating of plant seeds, attached to their roots, and insignificantly differed in antagonistic activity toward phytopathogens and quantitative content of cell fatty acids and phosphatase activity. Streptomycin-resistant strain may be used for monitoring of Bacillus subtilis introduced to agroecosystem.

  14. [Lactulose plus live binary Bacillus subtilis in the treatment of elders with functional constipation].

    PubMed

    Liu, Ya-Ping; Liu, Xin; Dong, Lei

    2012-11-13

    To explore the therapeutic efficacy and safety of lactulose plus live binary Bacillus subtilis in elders with functional constipation. A total of 97 elder outpatients or inpatients with functional constipation at our department from September 2011 to April 2012 were divided into 3 groups: lactulose treatment (n = 32), live binary Bacillus subtilis treatment (n = 31) and combined treatment (n = 34). Among them, there were 57 males and 40 females with an average age of (74 ± 6) years old. The course of treatment was 4 weeks in all groups. Then we observed the effective time, therapeutic efficacy, adverse reaction and change of life quality. (1) There were 62.5% (20/32) effective patients in the lactulose treatment group, 41.9% (13/31) in live binary Bacillus subtilis treatment group and 82.4% (28/34) in combined treatment group during 48 hours. Combined treatment group was significantly higher than lactulose treatment group (P < 0.05) and live binary Bacillus subtilis treatment group (P < 0.01). And lactulose treatment group was higher than live binary Bacillus subtilis treatment group (P < 0.05). (2) After 4 weeks, the total effective rates were 68.7% (22/32), 41.9% (13/31)and 94.2% (32/34)respectively. Combined treatment group was significantly higher than lactulose treatment group (P < 0.05) and live binary Bacillus subtilis treatment group (P < 0.01). Meanwhile, lactulose treatment group was superior to live binary Bacillus subtilis treatment group (P < 0.05). (3) After 4 weeks, there was no statistic difference in total adverse reaction rates among 3 groups (P > 0.05). (4) The life quality score of combined treatment group was significantly higher than lactulose treatment group (P < 0.05) and live binary Bacillus subtilis treatment group (P < 0.01) and lactulose treatment group was higher than live binary Bacillus subtilis treatment group (P < 0.05) after a 4-week treatment. The combined regimen of lactulose and live binary Bacillus subtilis is more effective

  15. Genome Sequencing of Bacillus subtilis SC-8, Antagonistic to the Bacillus cereus Group, Isolated from Traditional Korean Fermented-Soybean Food

    PubMed Central

    Yeo, In-Cheol; Lee, Nam Keun

    2012-01-01

    Bacillus subtilis SC-8 is a Gram-positive bacterium displaying narrow antagonistic activity for the Bacillus cereus group. B. subtilis SC-8 was isolated from Korean traditional fermented-soybean food. Here we report the draft genome sequence of B. subtilis SC-8, including biosynthetic genes for antibiotics that may have beneficial effects for control of food-borne pathogens. PMID:22207744

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

    NASA Astrophysics Data System (ADS)

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

    2003-04-01

    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.

  17. Metabolic engineering of Bacillus subtilis for enhanced production of acetoin.

    PubMed

    Wang, Meng; Fu, Jing; Zhang, Xueyu; Chen, Tao

    2012-10-01

    Acetoin is widely used in food and other industries. A bdhA and acoA double-knockout strain of Bacillus subtilis produced acetoin at 0.72 mol/mol, a 16.4 % increased compared to the wild type. Subsequent overexpression of the alsSD operon enhanced the acetolactate synthase activity by 52 and 66 % in growth and stationary phases, respectively. However, deletion of pta gene caused little increase of acetoin production. For acetoin production by the final engineered strain, BSUW06, acetoin productivity was improved from 0.087 g/l h, using M9 medium plus 30 g glucose/l under micro-aerobic conditions, to 0.273 g/h l using LB medium plus 50 g glucose/l under aerobic conditions. In fermentor culture, BSUW06 produced acetoin up to 20 g/l.

  18. Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity.

    PubMed

    Harrold, Zoë R; Hertel, Mikaela R; Gorman-Lewis, Drew

    2011-12-01

    Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88±11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.

  19. Safety evaluation of a xylanase expressed in Bacillus subtilis.

    PubMed

    Harbak, L; Thygesen, H V

    2002-01-01

    A programme of studies was conducted to establish the safety of a xylanase expressed in a self-cloned strain of Bacillus subtilis to be used as a processing aid in the baking industry. To assess acute and subchronic oral toxicity, rat feeding studies were conducted. In addition, the potential of the enzyme to cause mutagenicity and chromosomal aberrations was assessed in microbial and tissue culture in vitro studies. Acute and subchronic oral toxicity was not detected at the highest dose recommended by OECD guidelines. There was no evidence of mutagenic potential or chromosomal aberrations. Furthermore, the organism used for production of the xylanase is already accepted as safe by several major national regulatory agencies.

  20. Bioproperties of potent nattokinase from Bacillus subtilis YJ1.

    PubMed

    Yin, Li-Jung; Lin, Hsin-Hung; Jiang, Shann-Tzong

    2010-05-12

    Fibrinolytic enzyme activity was observed during cultivation of Bacillus subtilis YJ1 in a medium containing 1% skim milk, 1% rice husk, 0.5% NaCl, and 0.25% glucose. It was purified to electrophoretical homogeneity after CM-sepharose FF chromatography. The specific activity and yield were 1791.9 FU/mg and 9.5%, respectively. This purified fibrinolytic enzyme had M of 27.5 kDa, optimal temperature and pH at 50 degrees C and 8.5, respectively. It was stable at pH 6.0-10.0 and 10-40 degrees C and inhibited by Fe(3+), Hg(2+), Cu(2+), Zn(2+), and PMSF. Compared the N terminal of amino acids and full DNA sequence with those in NCBI, it was considered to be a nattokinase.

  1. Fractal Growth of Bacillus subtilis on Agar Plates

    NASA Astrophysics Data System (ADS)

    Fujikawa, Hiroshi; Matsushita, Mitsugu

    1989-11-01

    Bacteria have been shown to grow with various morphologies under different conditions on agar plates. A Bacillus subtilis strain was inoculated on the plate surface and incubated at 35°C. Colonies grew two-dimensionally with random branches, similar to clusters of the diffusion-limited aggregation (DLA) model. The colony patterns were analyzed and found to be self-similar with a fractal dimension of 1.716± 0.008, in excellent agreement with the expected value of the DLA model. Interior branches were observed to stop growing in spite of their open neighborhood during the incubation period, implying the existence of a screening effect. These results clearly suggest that the colony pattern of the organism was formed through the DLA process. Moreover, the colonies were found to grow radially with almost regular branches on agar plates with moist surfaces, reminiscent of “dense radial” morphology.

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

    PubMed

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

    2016-07-01

    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.

  3. Effect of polyelectrolytes on serine proteinase secretion by Bacillus subtilis.

    PubMed

    Artemov, A V; Samuilov, V D

    1990-03-12

    Addition of polycations with molecular masses of 5-40 kDa as well as Na+, stimulated serine proteinase secretion by Bacillus subtilis cells. Polyanions and higher-molecular-mass polycations (100-200 kDa) were inefficient. The enzyme yields in the presence of polycations or Na+ were equal in magnitude. The results indicate that the cations, apparently counteracting the negative surface charge of the bacterial plasma membrane, cause the desorption of the serine (alkaline) proteinase. The synthesis of the proteinase is inferred to be stopped as the enzyme is bound to the outer surface of the plasma membrane. The desorption of the enzyme thus induces the synthesis of the new portions of proteinase.

  4. Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755.

    PubMed

    Kerovuo, J; Tynkkynen, S

    2000-04-01

    Phytase enzymes can increase the nutritional value of food and feed by liberating inorganic phosphate from phytate, the major storage form of phosphorus in plants. The phytase (phyC) from Bacillus subtilis VTT E-68013 was expressed in Lactobacillus plantarum strain 755 using Lact. amylovorus alpha-amylase secretion signals. In an overnight cultivation in MRS medium containing cellobiose for induction of the alpha-amylase promoter, catalytically active phytase was secreted as a predominant extracellular protein. However, Western blot analysis revealed unprocessed and processed phytase in the cell fraction. Pulse chase experiments showed that the recombinant phytase was secreted at a slower rate in comparison to the native proteins of Lact. plantarum 755.

  5. Genetic control of the glp system in Bacillus subtilis.

    PubMed Central

    Lindgren, V; Rutberg, L

    1976-01-01

    In pleiotropic negative glycerol utilization mutants (GlpPI mutants) of Bacillus subitilis, glycerol kinase and sn-glycerol 3-phosphate (G3P) dehydrogenase are noninducible. GlpPI mutants also fail to take up exogenous [14C]G3P. To study the regulation of the glp system in B. subtilis phenotypically, Glp+ revertants were isolated from GlpPI mutants. Four classes of revertants were identified: phenotypically, wild type; R1 type, which contains an informational suppressor, R2 type, which produced G3P dehydrogenase constitutively; and R3 type, with a temperature-sensitive Glp phenotype producing G3P dehydrogenase constitutively at permissive temperature (32 degrees C). The properties of the revertants indicate that the glpPI locus codes for a protein with a positive regulatory function. PMID:182672

  6. Periodic growth of Bacillus subtilis colonies on agar plates

    NASA Astrophysics Data System (ADS)

    Fujikawa, Hiroshi

    1992-10-01

    Bacillus subtilis colonies show periodic growth on agar plates. The organism has been observed to show several colony morphologies including diffusion-limited aggregation (DLA) type, dense branching morphology (DBM), Eden type, and spreading without producing openings. The agar concentration for the periodic growth is higher than that of DBM and lower than that of DLA or Eden type. The nutrient (peptone) concentration for the periodic growth is higher than that of DLA and DBM and lower than that of Eden type. The colony grows towards a place with higher peptone concentration. These findings suggest that the diffusion of nutrient particles, i.e. the concentration gradient of peptone particles at the growing perimeter of a colony, would be essentially involved in the periodic growth. The distance between concentric rings of a colony is constant and intervention between two colonies is not observed, unlike the Liesegang ring.

  7. Response of Bacillus subtilis to Cerulenin and Acquisition of Resistance

    PubMed Central

    Schujman, Gustavo E.; Choi, Keum-Hwa; Altabe, Silvia; Rock, Charles O.; de Mendoza, Diego

    2001-01-01

    Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of β-ketoacyl-acyl carrier protein (ACP) synthases. The Bacillus subtilis fabF (yjaY) gene (fabFb) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. There were two mechanisms by which B. subtilis adapted to exposure to this antibiotic. First, reporter gene analysis demonstrated that transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge. This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in fabF reporter gene expression while nalidixic acid did not. Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin. The mutation mapped at the B. subtilis fabF locus, and sequence analysis of the mutant fabF allele showed that a single base change resulted in the synthesis of FabFb[I108F]. The purified FabFb and FabFb[I108F] proteins had similar specific activities with myristoyl-ACP as the substrate. FabFb exhibited a 50% inhibitory concentration (IC50) of cerulenin of 0.1 μM, whereas the IC50 for FabFb[I108] was 50-fold higher (5 μM). These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain. PMID:11325930

  8. Optimization of Microbial Flocculant-Producing Medium for Bacillus subtilis.

    PubMed

    Zhao, Changqing; Yang, Qinhuan; Zhang, Hao

    2017-03-01

    This study aimed to improve microbial flocculant production by optimizing the components of a Bacillus subtilis CZ1003 culture medium. Using the flocculation rate as the dependent variable, single-factor experiments were performed and beef extract at a concentration of 9 g/L was found to be the optimal nitrogen source, while glucose at a concentration of 20 g/L was the optimal carbon source. KCl, MgCl2, NaCl, and CaCl2 at concentrations of 0.75, 2.5, 0.5, and 5.0 g/L, respectively, were the optimum inorganic salts, in order of flocculant production activity. Orthogonal experimental demonstrated that KCl played a dominant role for Bacillus subtilis production of bioflocculants, followed by NaCl and CaCl2. Optimization experiments demonstrated that the optimal combination of the two salts was 0.75 g/L KCl and 0.5 g/L NaCl, resulting in a flocculation rate of 36.2% when included together at these concentrations. The final optimized medium consisted of 20 g/L glucose, 9 g/L beef extract, 0.75 g/L KCl, and 0.5 g/L NaCl. Compared with the initial medium, the optimized medium enhanced the flocculation activity from 12.1 to 36.2%, which equates to an increase of 199.17%. Meanwhile, the flocculant yield was increased from 0.058 g/L to 0.134/L, an increase of 131.03%. The optimized medium could be used to improve microbial flocculant production and provides a basis for further exploration.

  9. Analysis of Spo0M function in Bacillus subtilis

    PubMed Central

    Vega-Cabrera, Luz Adriana; Guerrero, Adán; Rodríguez-Mejía, José Luis; Tabche, María Luisa; Wood, Christopher D.; Gutiérrez-Rios, Rosa-María; Merino, Enrique

    2017-01-01

    Spo0M has been previously reported as a regulator of sporulation in Bacillus subtilis; however, little is known about the mechanisms through which it participates in sporulation, and there is no information to date that relates this protein to other processes in the bacterium. In this work we present evidence from proteomic, protein-protein interaction, morphological, subcellular localization microscopy and bioinformatics studies which indicate that Spo0M function is not necessarily restricted to sporulation, and point towards its involvement in other stages of the vegetative life cycle. In the current study, we provide evidence that Spo0M interacts with cytoskeletal proteins involved in cell division, which suggest a function additional to that previously described in sporulation. Spo0M expression is not restricted to the transition phase or sporulation; rather, its expression begins during the early stages of growth and Spo0M localization in B. subtilis depends on the bacterial life cycle and could be related to an additional proposed function. This is supported by our discovery of homologs in a broad distribution of bacterial genera, even in non-sporulating species. Our work paves the way for re-evaluation of the role of Spo0M in bacterial cell. PMID:28234965

  10. A Combinatorial Kin Discrimination System in Bacillus subtilis

    PubMed Central

    Lyons, Nicholas A.; Kraigher, Barbara; Stefanic, Polonca; Mandic-Mulec, Ines; Kolter, Roberto

    2016-01-01

    SUMMARY Multicellularity inherently involves a number of cooperative behaviors that are potentially susceptible to exploitation but can be protected by mechanisms such as kin discrimination. Discrimination of kin from non-kin has been observed in swarms of the bacterium Bacillus subtilis, but the underlying molecular mechanism has been unknown. We used genetic, transcriptomic, and bioinformatic analyses to uncover kin recognition factors in this organism. Our results identified many molecules involved in cell surface modification and antimicrobial production and response. These genes varied significantly in expression level and mutation phenotype among B. subtilis strains, suggesting interstrain variation in the exact kin discrimination mechanism used. Genome analyses revealed a substantial diversity of antimicrobial genes present in unique combinations in different strains, with many likely acquired by horizontal gene transfer. The dynamic combinatorial effect derived from this plethora of kin discrimination genes creates a tight relatedness cutoff for cooperation that has likely led to rapid diversification within the species. Our data suggest that genes likely originally selected for competitive purposes also generate preferential interactions among kin, thus stabilizing multicellular lifestyles. PMID:26923784

  11. The minimal Bacillus subtilis nonhomologous end joining repair machinery.

    PubMed

    de Vega, Miguel

    2013-01-01

    It is widely accepted that repair of double-strand breaks in bacteria that either sporulate or that undergo extended periods of stationary phase relies not only on homologous recombination but also on a minimal nonhomologous end joining (NHEJ) system consisting of a dedicated multifunctional ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku. Bacillus subtilis is one of the bacterial members with a NHEJ system that contributes to genome stability during the stationary phase and germination of spores, having been characterized exclusively in vivo. Here, the in vitro analysis of the functional properties of the purified B. subtilis LigD (BsuLigD) and Ku (BsuKu) proteins is presented. The results show that the essential biochemical signatures exhibited by BsuLigD agree with its proposed function in NHEJ: i) inherent polymerization activity showing preferential insertion of NMPs, ii) specific recognition of the phosphate group at the downstream 5' end, iii) intrinsic ligase activity, iv) ability to promote realignments of the template and primer strands during elongation of mispaired 3' ends, and v) it is recruited to DNA by BsuKu that stimulates the inherent polymerization and ligase activities of the enzyme allowing it to deal with and to hold different and unstable DNA realignments.

  12. Sporicidal activity of ceragenin CSA-13 against Bacillus subtilis.

    PubMed

    Piktel, Ewelina; Pogoda, Katarzyna; Roman, Maciej; Niemirowicz, Katarzyna; Tokajuk, Grażyna; Wróblewska, Marta; Szynaka, Beata; Kwiatek, Wojciech M; Savage, Paul B; Bucki, Robert

    2017-03-15

    Spore-forming bacteria are a class of microorganisms that possess the ability to survive in extreme environmental conditions. Morphological features of spores assure their resistance to stress factors such as high temperature, radiation, disinfectants, and drying. Consequently, spore elimination in industrial and medical environments is very challenging. Ceragenins are a new class of cationic lipids characterized by a broad spectrum of bactericidal activity resulting from amphipathic nature and membrane-permeabilizing properties. To assess the impact of ceragenin CSA-13 on spores formed by Bacillus subtilis (ATCC 6051), we performed the series of experiments confirming that amphipathic and membrane-permeabilizing properties of CSA-13 are sufficient to disrupt the structure of B. subtilis spores resulting in decreased viability. Raman spectroscopy analysis provided evidence that upon CSA-13 treatment the number of CaDPA-positive spores was clearly diminished. As a consequence, a loss of impermeability of the inner membranes of spores, accompanied by a decrease in spore resistance and killing take place. In addition to their broad antimicrobial spectrum, ceragenins possess great potential for development as new sporicidal agents.

  13. Sporicidal Activities of Various Surfactant Components against Bacillus subtilis Spores.

    PubMed

    Cho, Won-Il; Cheigh, Chan-Ick; Hwang, Hee-Jeong; Chung, Myong-Soo

    2015-06-01

    The sporicidal activities against Bacillus subtilis spores of surfactant components with hydrophilic and hydrophobic properties that can lead to the denaturation of various proteins comprising the spore structure were investigated. The reduction in spore numbers by each of the surfactant components bornyl acetate, geranyl acetate, pinene, p-cymene, camphene, citral, 2,3-dihydrobenzofuran, polylysine, and thiamine dilaurylsulfate at 1% was estimated at 1 to 2 log CFU/ml. The average hydrophilelipophile balance value of surfactants with sporicidal activity causing a reduction of 1 to 2 log CFU/ml was 9.3, with a range from 6.7 to 15.8, which is similar to the values of various chemical surfactants of 9.6 to 16.7. The results also showed that the surfactants that were hydrophobic were more effective than those that were hydrophilic in killing B. subtilis spores. Furthermore, the sporicidal effect of surfactants like geranyl acetate and γ-terpinene was significantly enhanced in the presence of a germinant, because L-alanine and synergistic cofactors (e.g., K(+) ions) trigger cortex hydrolysis in spores.

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

    PubMed Central

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

    2016-01-01

    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

  15. DNA Repair and Genome Maintenance in Bacillus subtilis

    PubMed Central

    Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

    2012-01-01

    Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

  16. A Combinatorial Kin Discrimination System in Bacillus subtilis.

    PubMed

    Lyons, Nicholas A; Kraigher, Barbara; Stefanic, Polonca; Mandic-Mulec, Ines; Kolter, Roberto

    2016-03-21

    Multicellularity inherently involves a number of cooperative behaviors that are potentially susceptible to exploitation but can be protected by mechanisms such as kin discrimination. Discrimination of kin from non-kin has been observed in swarms of the bacterium Bacillus subtilis, but the underlying molecular mechanism has been unknown. We used genetic, transcriptomic, and bioinformatic analyses to uncover kin recognition factors in this organism. Our results identified many molecules involved in cell-surface modification and antimicrobial production and response. These genes varied significantly in expression level and mutation phenotype among B. subtilis strains, suggesting interstrain variation in the exact kin discrimination mechanism used. Genome analyses revealed a substantial diversity of antimicrobial genes present in unique combinations in different strains, with many likely acquired by horizontal gene transfer. The dynamic combinatorial effect derived from this plethora of kin discrimination genes creates a tight relatedness cutoff for cooperation that has likely led to rapid diversification within the species. Our data suggest that genes likely originally selected for competitive purposes also generate preferential interactions among kin, thus stabilizing multicellular lifestyles.

  17. Transient heterogeneity in extracellular protease production by Bacillus subtilis.

    PubMed

    Veening, Jan-Willem; Igoshin, Oleg A; Eijlander, Robyn T; Nijland, Reindert; Hamoen, Leendert W; Kuipers, Oscar P

    2008-01-01

    The most sophisticated survival strategy Bacillus subtilis employs is the differentiation of a subpopulation of cells into highly resistant endospores. To examine the expression patterns of non-sporulating cells within heterogeneous populations, we used buoyant density centrifugation to separate vegetative cells from endospore-containing cells and compared the transcriptome profiles of both subpopulations. This demonstrated the differential expression of various regulons. Subsequent single-cell analyses using promoter-gfp fusions confirmed our microarray results. Surprisingly, only part of the vegetative subpopulation highly and transiently expresses genes encoding the extracellular proteases Bpr (bacillopeptidase) and AprE (subtilisin), both of which are under the control of the DegU transcriptional regulator. As these proteases and their degradation products freely diffuse within the liquid growth medium, all cells within the clonal population are expected to benefit from their activities, suggesting that B. subtilis employs cooperative or even altruistic behavior. To unravel the mechanisms by which protease production heterogeneity within the non-sporulating subpopulation is established, we performed a series of genetic experiments combined with mathematical modeling. Simulations with our model yield valuable insights into how population heterogeneity may arise by the relatively long and variable response times within the DegU autoactivating pathway.

  18. Transient heterogeneity in extracellular protease production by Bacillus subtilis

    PubMed Central

    Veening, Jan-Willem; Igoshin, Oleg A; Eijlander, Robyn T; Nijland, Reindert; Hamoen, Leendert W; Kuipers, Oscar P

    2008-01-01

    The most sophisticated survival strategy Bacillus subtilis employs is the differentiation of a subpopulation of cells into highly resistant endospores. To examine the expression patterns of non-sporulating cells within heterogeneous populations, we used buoyant density centrifugation to separate vegetative cells from endospore-containing cells and compared the transcriptome profiles of both subpopulations. This demonstrated the differential expression of various regulons. Subsequent single-cell analyses using promoter-gfp fusions confirmed our microarray results. Surprisingly, only part of the vegetative subpopulation highly and transiently expresses genes encoding the extracellular proteases Bpr (bacillopeptidase) and AprE (subtilisin), both of which are under the control of the DegU transcriptional regulator. As these proteases and their degradation products freely diffuse within the liquid growth medium, all cells within the clonal population are expected to benefit from their activities, suggesting that B. subtilis employs cooperative or even altruistic behavior. To unravel the mechanisms by which protease production heterogeneity within the non-sporulating subpopulation is established, we performed a series of genetic experiments combined with mathematical modeling. Simulations with our model yield valuable insights into how population heterogeneity may arise by the relatively long and variable response times within the DegU autoactivating pathway. PMID:18414485

  19. Analysis of Spo0M function in Bacillus subtilis.

    PubMed

    Vega-Cabrera, Luz Adriana; Guerrero, Adán; Rodríguez-Mejía, José Luis; Tabche, María Luisa; Wood, Christopher D; Gutiérrez-Rios, Rosa-María; Merino, Enrique; Pardo-López, Liliana

    2017-01-01

    Spo0M has been previously reported as a regulator of sporulation in Bacillus subtilis; however, little is known about the mechanisms through which it participates in sporulation, and there is no information to date that relates this protein to other processes in the bacterium. In this work we present evidence from proteomic, protein-protein interaction, morphological, subcellular localization microscopy and bioinformatics studies which indicate that Spo0M function is not necessarily restricted to sporulation, and point towards its involvement in other stages of the vegetative life cycle. In the current study, we provide evidence that Spo0M interacts with cytoskeletal proteins involved in cell division, which suggest a function additional to that previously described in sporulation. Spo0M expression is not restricted to the transition phase or sporulation; rather, its expression begins during the early stages of growth and Spo0M localization in B. subtilis depends on the bacterial life cycle and could be related to an additional proposed function. This is supported by our discovery of homologs in a broad distribution of bacterial genera, even in non-sporulating species. Our work paves the way for re-evaluation of the role of Spo0M in bacterial cell.

  20. Identification of two distinct Bacillus subtilis citrate synthase genes.

    PubMed

    Jin, S; Sonenshein, A L

    1994-08-01

    Two distinct Bacillus subtilis genes (citA and citZ) were found to encode citrate synthase isozymes that catalyze the first step of the Krebs cycle. The citA gene was cloned by genetic complementation of an Escherichia coli citrate synthase mutant strain (W620) and was in a monocistronic transcriptional unit. A divergently transcribed gene, citR, could encode a protein with strong similarity to the bacterial LysR family of regulatory proteins. A null mutation in citA had little effect on citrate synthase enzyme activity or sporulation. The residual citrate synthase activity was purified from a citA null mutant strain, and the partial amino acid sequence for the purified protein (CitZ) was determined. The citZ gene was cloned from B. subtilis chromosomal DNA by using a PCR-generated probe synthesized with oligonucleotide primers derived from the partial amino acid sequence of purified CitZ. The citZ gene proved to be the first gene in a tricistronic cluster that also included citC (coding for isocitrate dehydrogenase) and citH (coding for malate dehydrogenase). A mutation in citZ caused a substantial loss of citrate synthase enzyme activity, glutamate auxotrophy, and a defect in sporulation.

  1. Sporicidal activity of ceragenin CSA-13 against Bacillus subtilis

    PubMed Central

    Piktel, Ewelina; Pogoda, Katarzyna; Roman, Maciej; Niemirowicz, Katarzyna; Tokajuk, Grażyna; Wróblewska, Marta; Szynaka, Beata; Kwiatek, Wojciech M.; Savage, Paul B.; Bucki, Robert

    2017-01-01

    Spore-forming bacteria are a class of microorganisms that possess the ability to survive in extreme environmental conditions. Morphological features of spores assure their resistance to stress factors such as high temperature, radiation, disinfectants, and drying. Consequently, spore elimination in industrial and medical environments is very challenging. Ceragenins are a new class of cationic lipids characterized by a broad spectrum of bactericidal activity resulting from amphipathic nature and membrane-permeabilizing properties. To assess the impact of ceragenin CSA-13 on spores formed by Bacillus subtilis (ATCC 6051), we performed the series of experiments confirming that amphipathic and membrane-permeabilizing properties of CSA-13 are sufficient to disrupt the structure of B. subtilis spores resulting in decreased viability. Raman spectroscopy analysis provided evidence that upon CSA-13 treatment the number of CaDPA-positive spores was clearly diminished. As a consequence, a loss of impermeability of the inner membranes of spores, accompanied by a decrease in spore resistance and killing take place. In addition to their broad antimicrobial spectrum, ceragenins possess great potential for development as new sporicidal agents. PMID:28294162

  2. Preparation of AN Electrode Modified with a Thermostable Enzyme BACILLUS Subtilis COTA by Electrodeposition

    NASA Astrophysics Data System (ADS)

    Watanabe, Toshio; Yamada, Yohei; Motonaka, Junko; Yabutani, Tomoki; Sakuraba, Haruhiko; Yasuzawa, Mikito

    In this study, electrodeposition of thermostable enzyme Bacillus subtilis CotA, which is a laccase and has a bilirubin oxidase (BOD) activity, was investigated. The electrodeposition was operated in a mixture of Bacillus subtilis CotA in the PBS (pH 8.0) and TritonX-100 under applying potential (1100 mV vs. Ag/AgCl for 5 min.). The current response was measured by linear sweep voltammetry technique (LSV). The thermostable enzyme Bacillus subtilis CotA electrodeposited electrode was compared with a mesophile BOD electrodeposited electrode. As a result, the Bacillus subtilis CotA modified electrode showed better sensitivity and long-term stability than the mesophile BOD modified electrode.

  3. Enhanced production of mosquitocidal cyclic lipopeptide from Bacillus subtilis subsp. subtilis

    PubMed Central

    Manonmani, A.M.; Geetha, I.; Bhuvaneswari, S.

    2011-01-01

    Background & objectives: A cyclic lipopeptide, surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit activity against both the larval and pupal stages of mosquitoes. The present study was aimed at increasing the production of the mosquitocidal metabolite by modifying the conventional medium. Methods: Enhancement of mosquitocidal metabolite production was attempted by replacing the existing micronutrients of the conventional NYSM and supplementing the medium with additional amounts of glucose. The LC50 value of culture supernatant (CS) against the larval and pupal stages of Anopheles stephensi was determined. Crude mosquitocidal metabolite (CMM) was separated from the CS, identified by MALDI-TOF analysis and its LC50 dosage requirement for the pupal stage of the above mosquito species determined. Results: The medium containing a new composition of micronutrients and glucose up to 1 per cent resulted in increased metabolite production. The LC50 value of the CS obtained in the improved medium against larvae and pupae of An. stephensi was 5.57 and 0.71 μl/ml, respectively. The yield of CMM was doubled in the improved medium. MALDI-TOF analysis revealed that the CMM was surfactin. Interpretation & conclusions: The new improved medium enhanced the production of mosquitocidal metabolite as the dosage required for inciting 50 per cent mortality among the pupal stages of mosquitoes was only half of that required when the metabolite was produced in the conventional medium. The mosquitocidal metabolite was identified as surfactin, a cyclic lipopeptide and biosurfactant. PMID:22089610

  4. Enhanced production of mosquitocidal cyclic lipopeptide from Bacillus subtilis subsp. subtilis.

    PubMed

    Manonmani, A M; Geetha, I; Bhuvaneswari, S

    2011-10-01

    A cyclic lipopeptide, surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit activity against both the larval and pupal stages of mosquitoes. The present study was aimed at increasing the production of the mosquitocidal metabolite by modifying the conventional medium. Enhancement of mosquitocidal metabolite production was attempted by replacing the existing micronutrients of the conventional NYSM and supplementing the medium with additional amounts of glucose. The LC₅₀ value of culture supernatant (CS) against the larval and pupal stages of Anopheles stephensi was determined. Crude mosquitocidal metabolite (CMM) was separated from the CS, identified by MALDI-TOF analysis and its LC₅₀ dosage requirement for the pupal stage of the above mosquito species determined. The medium containing a new composition of micronutrients and glucose up to 1 per cent resulted in increased metabolite production. The LC₅₀ value of the CS obtained in the improved medium against larvae and pupae of An. stephensi was 5.57 and 0.71 μl/ml, respectively. The yield of CMM was doubled in the improved medium. MALDI-TOF analysis revealed that the CMM was surfactin. The new improved medium enhanced the production of mosquitocidal metabolite as the dosage required for inciting 50 per cent mortality among the pupal stages of mosquitoes was only half of that required when the metabolite was produced in the conventional medium. The mosquitocidal metabolite was identified as surfactin, a cyclic lipopeptide and biosurfactant.

  5. Genomic comparisons of two Bacillus subtilis biocontrol strains with different modes of actions

    USDA-ARS?s Scientific Manuscript database

    Bacillus subtilis strains AS 43.3 and OH131.1 were isolated from wheat anthers and shown to be efficacious in managing Fusarium head blight in greenhouse and some field trials. Chemical analysis of the cell-free culture supernatant identified B. subtilis strain AS 43.3 to be a potent producer of the...

  6. High-Quality Draft Genome Sequence of Bacillus subtilis Strain WAUSV36.

    PubMed

    Town, Jennifer; Audy, Patrice; Boyetchko, Susan M; Dumonceaux, Tim J

    2016-06-23

    Bacillus subtilis strain WAUSV36 inhibits the growth of and decreases disease symptoms caused by the potato pathogen Phytophthora infestans We determined the sequence of the 4.7-Mbp genome of this strain. WAUSV36 shared very high nucleotide sequence identity with previously sequenced strains of B. subtilis. Copyright © 2016 Town et al.

  7. Engineering the Xylan Utilization System in Bacillus subtilis for Production of Acidic Xylooligosaccharides

    Treesearch

    Mun Su Rhee; Lusha Wei; Neha Sawhney; John D. Rice; Franz J. St. John; Jason C. Hurlbert; James F. Preston

    2014-01-01

    Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and...

  8. Isolation and Characterization of Phages Infecting Bacillus subtilis

    PubMed Central

    Krasowska, Anna; Biegalska, Anna; Augustyniak, Daria; Łoś, Marcin; Richert, Malwina; Łukaszewicz, Marcin

    2015-01-01

    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. PMID:26273592

  9. In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases.

    PubMed Central

    Sogo, J M; Lozano, M; Salas, M

    1984-01-01

    The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase. The transcription of phage phi 29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs. Images PMID:6322128

  10. A Safety and Environmental Assessment of the Biological Simulants Bacillus subtilis and Newcastle Disease Virus. Volume 1: Discussion

    DTIC Science & Technology

    1993-01-01

    1680-82 Enzyme Bio- Systems (1988) GRAS Petition 7G0328 proposing that alpha- amylase from a strain of Bacillus subtilis containing a Bacillus...Image Cover Sheet CLASSIFICATION SYSTEM NUMBER 129413 UNCLASSIFIED 1111111111111111111111111111111111111111 TITLE A SAFETY AND ENVIRONMENTAL...ASSESSMENT OF THE BIOLOGICAL SIMULANTS BACILLUS SUBTILIS AND NEWCASTLE DISEASE VIRUS. VOLUME I: DISCUSSION System Number: Patron Number: Requester

  11. Bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus entering intestinal epithelial cells.

    PubMed

    Wang, Xiaoqing; Hu, Weiwei; Zhu, Liqi; Yang, Qian

    2017-03-07

    Intestinal epithelial cells are the targets for transmissible gastroenteritis virus (TGEV) infection. It is urgently to develop a novel candidate against TGEV entry. Bacillus subtilis is a probiotics with excellent anti-microorganism properties, and one of its secretions, surfactin, has been regarded as the versatile weapons for most plant pathogens, especially for the enveloped virus. We demonstrate for the first time that Bacillus subtilis OKB105 and its surfactin can effectively inhibit one animal coronavirus, TGEV, entering the intestinal porcine epithelial cell line (IPEC-J2). Then, several different experiments were performed to seek for the might mechanisms. The plaque assays showed that surfactant could reduce the plaque generation of TGEV in a dose dependent manner. Meanwhile, the after incubated with TGEV for 1.5 h, Bacillus subtilis could attach TGEV particles to their surface so that the number of virus to bind to the host cells was declined. Furthermore, our data showed that the inhibition of Bacillus subtilis was closely related to the competition with TGEV for the viral-entry receptors, including epidermal growth factor receptor (EGFR) and aminopopeptidase N (APN) protein. In addition, Western blotting and apoptosis analysis indicated that Bacillus subtilis could enhance the resistance of IPEC-J2 cells by up regulating the expression of TLR-6 and reducing the percentage of apoptotic cells. Taken together, our results suggest that Bacillus subtilis OKB105 and its surfactin can antagonize TGEV entry in vitro and may serve as promising new candidates for TGEV prevention.

  12. Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus.

    PubMed

    Orhan, Elif; Omay, Didem; Güvenilir, Yüksel

    2005-01-01

    The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.

  13. Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

    PubMed Central

    Sutyak, K.E.; Wirawan, R.E.; Aroutcheva, A.A.; Chikindas, M.L.

    2008-01-01

    Aims To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product-derived Bacillus amyloliquefaciens. Methods and Results An unknown bacterial species cultured from the Yogu Farm™ probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell-free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. Conclusion The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. Significance and Impact of the Study This is the first report of intra-species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis-associated pathogens. PMID:17976171

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

    PubMed Central

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

    2012-01-01

    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 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants. PMID:22593682

  15. Isolation of a new Mexican strain of Bacillus subtilis with antifungal and antibacterial activities.

    PubMed

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

    2012-01-01

    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 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.

  16. Structural differentiation of the Bacillus subtilis 168 cell wall.

    PubMed Central

    Graham, L L; Beveridge, T J

    1994-01-01

    Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes

  17. Acid and base stress and transcriptomic responses in Bacillus subtilis.

    PubMed

    Wilks, Jessica C; Kitko, Ryan D; Cleeton, Sarah H; Lee, Grace E; Ugwu, Chinagozi S; Jones, Brian D; BonDurant, Sandra S; Slonczewski, Joan L

    2009-02-01

    Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived <15%. Cells grown at pH 9.0 survived 40 to 100% at pH 10, whereas cells grown at pH 7.0 survived <5%. Thus, growth in a moderate acid or base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K(+)/H(+) antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids.

  18. Formulations of Bacillus subtilis BY-2 suppress Sclerotinia sclerotiorum on oilseed rape in the field

    USDA-ARS?s Scientific Manuscript database

    We are developing a collection of Bacillus strains, isolated from different environments, for use in controlling Sclerotinia sclerotiorum on oilseed rape in China and elsewhere. Strain BY-2, isolated from internal tissues of an oilseed rape root, was demonstrated to be Bacillus subtilis based on bi...

  19. The comparative ability of four isolates of Bacillus subtilis to ferment soybeans into dawadawa.

    PubMed

    Terlabie, Nora Narkie; Sakyi-Dawson, Esther; Amoa-Awua, Wisdom Kofi

    2006-02-01

    In an attempt to develop starter cultures for fermenting soybeans into the traditional West African condiment dawadawa, four isolates of Bacillus subtilis: 24BP(2), 72RP(17), 72BP(30), and FpdBP(2), which had been selected from 42 Bacillus cultures in a previous study by the current authors, were used separately to produce soy-dawadawa. The accompanying microbiological and biochemical changes, including enzymatic activities, as well as the organoleptic quality of the products were evaluated including that of a control sample which was fermented spontaneously. Significant differences existed in the ability of the four isolates to hydrolyse the soybean proteins, starch, and fat to produce dawadawa. Bacillus subtilis 24BP(2) recorded the highest protease and amylolytic activities, 101 U/ml and 26.68 mg/ml, respectively, and liberated the most amino acids, 117.64 mg/g dry wt., during fermentation. Bacillus subtilis 24BP(2) also grew to the highest population of cells in the final product. Taste panelists found soybean dawadawa produced by each of the four isolates acceptable and rated soup flavoured with soy-dawadawa produced by Bacillus subtilis FpdBP(2) as the best sample. Panelists scored it higher than the control sample and soy-dawadawa produced by Bacillus subtilis 24BP(2) in that order.

  20. Effects of Bacillus subtilis natto on performance and immune function of preweaning calves.

    PubMed

    Sun, P; Wang, J Q; Zhang, H T

    2010-12-01

    The effects of Bacillus subtilis natto on performance and immune function of dairy calves during the preweaning phase were investigated in this study. Twelve Holstein male calves 7 ± 1 d of age were randomly allotted to 2 treatments of 6 calves. The Bacillus subtilis natto was mixed with milk and fed directly to the calves. The calves were weaned when their starter intake reached 2% of their weight. Blood was collected and IgA, IgE, IgG, IgM, and cytokine levels in the serum of all the calves were determined. The results showed that Bacillus subtilis natto increased general performance by improving the average daily gain and feed efficiency and advanced the weaning age of the calves. No difference was observed in serum IgE, IgA, and IgM, whereas serum IgG was higher in the Bacillus subtilis natto-supplemented calves than in the control calves. Furthermore, calves fed with Bacillus subtilis natto were found to secrete more IFN-γ, but tended to produce less IL-4 than did the control calves, although serum IL-6 and IL-10 were not affected. This study demonstrated that Bacillus subtilis natto did not stimulate IgE-mediated allergic reactions, but increased serum IgG and IFN-γ levels in the probiotic-fed calves. We propose that the viable probiotic characteristics of Bacillus subtilis natto benefit calf immune function. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. [Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

    PubMed

    Kirillova, Iu M; Mikhaĭlova, E O; Balaban, N P; Mardanova, A M; Kaiumov, A R; Rudenskaia, G N; Kostrov, S V; Sharipova, M R

    2006-01-01

    The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

  2. Studies on Sporulation Optimization and Chracterization of Bacillus subtilis Spore Quality

    DTIC Science & Technology

    2011-12-01

    OPTIMIZATION AND CHARACTERIZATION OF BACILLUS SUBTILIS SPORE QUALITY INTRODUCTION Sporulation differentiation, unique to two bacterial species ... Clostridium and Bacillus, is a process induced by reduced levels of nutrients in the environment or in culture (Driks, 2002). Spores can survive for long...days in the 2xSG media; therefore, further work should be performed on other Bacillus species , including B. anthracis. As no growth was achieved on

  3. Dynamics of Aerial Tower Formation in Bacillus subtilis Biofilms

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

    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.

  4. Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation

    PubMed Central

    Ojkic, Nikola; López-Garrido, Javier; Pogliano, Kit; Endres, Robert G

    2016-01-01

    When starved, the Gram-positive bacterium Bacillus subtilis forms durable spores for survival. Sporulation initiates with an asymmetric cell division, creating a large mother cell and a small forespore. Subsequently, the mother cell membrane engulfs the forespore in a phagocytosis-like process. However, the force generation mechanism for forward membrane movement remains unknown. Here, we show that membrane migration is driven by cell wall remodeling at the leading edge of the engulfing membrane, with peptidoglycan synthesis and degradation mediated by penicillin binding proteins in the forespore and a cell wall degradation protein complex in the mother cell. We propose a simple model for engulfment in which the junction between the septum and the lateral cell wall moves around the forespore by a mechanism resembling the ‘template model’. Hence, we establish a biophysical mechanism for the creation of a force for engulfment based on the coordination between cell wall synthesis and degradation. DOI: http://dx.doi.org/10.7554/eLife.18657.001 PMID:27852437

  5. Nutrient depletion in Bacillus subtilis biofilms triggers matrix production

    NASA Astrophysics Data System (ADS)

    Zhang, Wenbo; Seminara, Agnese; Suaris, Melanie; Brenner, Michael P.; Weitz, David A.; Angelini, Thomas E.

    2014-01-01

    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.

  6. Oligonucleotide bias in Bacillus subtilis: general trends and taxonomic comparisons.

    PubMed Central

    Rocha, E P; Viari, A; Danchin, A

    1998-01-01

    We present a general analysis of oligonucleotide usage in the complete genome of Bacillus subtilis . Several datasets were built in order to assign various biological contexts to the biased use of words and to reveal local asymmetries in word usage that may be coupled with replication, the control of gene expression and the restriction/modification system. This analysis was complemented by cross-comparisons with the complete genomes of Escherichia coli , Haemophilus influenzae and Methanococcus jannaschii . We have observed a large number of biased oligonucleotides for words of size up to 8, throughout the datasets and species, indicating that such long strict words play an important role as biological signals. We speculate that some of them are involved in interactions with DNA and/or RNA polymerases. An extensive analysis of palindrome abundances and distributions provides the surprising result that prophage-like elements embedded in the genome exhibit a smaller avoidance of restriction sites. This may reinforce a recently proposed hypothesis of a selfish gene phenomena in the transfer of restriction/modification systems in bacteria. PMID:9611243

  7. Liquid transport facilitated by channels in Bacillus subtilis biofilms.

    PubMed

    Wilking, James N; Zaburdaev, Vasily; De Volder, Michael; Losick, Richard; Brenner, Michael P; Weitz, David A

    2013-01-15

    Many bacteria on earth exist in surface-attached communities known as biofilms. These films are responsible for manifold problems, including hospital-acquired infections and biofouling, but they can also be beneficial. Biofilm growth depends on the transport of nutrients and waste, for which diffusion is thought to be the main source of transport. However, diffusion is ineffective for transport over large distances and thus should limit growth. Nevertheless, biofilms can grow to be very large. Here we report the presence of a remarkable network of well-defined channels that form in wild-type Bacillus subtilis biofilms and provide a system for enhanced transport. We observe that these channels have high permeability to liquid flow and facilitate the transport of liquid through the biofilm. In addition, we find that spatial variations in evaporative flux from the surface of these biofilms provide a driving force for the flow of liquid in the channels. These channels offer a remarkably simple system for liquid transport, and their discovery provides insight into the physiology and growth of biofilms.

  8. Probing phenotypic growth in expanding Bacillus subtilis biofilms.

    PubMed

    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

    2016-05-01

    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.

  9. Tip-enhanced Raman scattering of bacillus subtilis spores

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

    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.

  10. Tetracycline tolerance mediated by gene amplification in Bacillus subtilis.

    PubMed

    Wannarat, Wannasiri; Motoyama, Shiori; Masuda, Kenta; Kawamura, Fujio; Inaoka, Takashi

    2014-11-01

    Bacillus subtilis can acquire a higher tolerance to tetracycline by increasing the gene dosage of its resistance gene tetB. In this study, we estimated the multiplication effect of tetB on tetracycline tolerance. Cells harbouring multiple copies of tetB were found to comprise approximately 30 % of the total tetracycline-resistant cell population when selected on medium containing 10 µg tetracycline ml(-1). Disruption of recA resulted in a significant decrease in the frequency of tetB amplification. Although four direct repeats exist around tetB, the majority of tetB amplicons were found to be flanked by non-homologous sequences, indicating that the initial duplication of tetB can occur largely through RecA-independent recombination. The correlation between the tetB copy number and the MIC values for tetracycline indicated that more than three copies of tetB were required for tolerance to 10 µg tetracycline ml(-1). Thus, the RecA-dependent expansion step appears to be necessary for developing significant tetracycline tolerance mediated by tetB amplification. © 2014 The Authors.

  11. Bacteria-shaped Gymnoplasts (Protoplasts) of Bacillus subtilis

    PubMed Central

    van Iterson, W.; den Kamp, J. A. F. Op

    1969-01-01

    Addition of glucose to the medium in which Bacillus subtilis was grown lowered the pH and increased the amount of lysylphosphatidylglycerol relative to the phosphatidylglycerol content of the membrane fraction. This change in phospholipid composition was accompanied by changes in the shape and behavior of the gymnoplasts obtained by cell wall removal with lysozyme. These gymnoplasts appeared to retain most of their original cell shape and internal organization, often with preservation of the mesosomes. Cells harvested from neutral growth medium gave the usual spherical gymnoplasts. In a hypotonic medium, the spherical gymnoplasts lysed rapidly, whereas the rod-like gymnoplasts lost only part of their cell content while showing a tendency to preserve the original shape. This type of gymnoplast could not be produced from cells grown in neutral medium by simply raising the magnesium concentration. When this was done the gymnoplasts assumed bizarre shapes; they became compact and susceptible to the tonicity of the medium. Gymnoplasts or protoplasts, produced from bacilli exposed to low pH values, were found not to conform to the formulations on “protoplasts” given in 1958 by 13 authors. Cells exposed to a low environmental pH during growth seemed to possess a more rigid membrane structure than cells grown at neutral pH. Images PMID:4979444

  12. An updated metabolic view of the Bacillus subtilis 168 genome.

    PubMed

    Belda, Eugeni; Sekowska, Agnieszka; Le Fèvre, François; Morgat, Anne; Mornico, Damien; Ouzounis, Christos; Vallenet, David; Médigue, Claudine; Danchin, Antoine

    2013-04-01

    Continuous updating of the genome sequence of Bacillus subtilis, the model of the Firmicutes, is a basic requirement needed by the biology community. In this work new genomic objects have been included (toxin/antitoxin genes and small RNA genes) and the metabolic network has been entirely updated. The curated view of the validated metabolic pathways present in the organism as of 2012 shows several significant differences from pathways present in the other bacterial reference, Escherichia coli: variants in synthesis of cofactors (thiamine, biotin, bacillithiol), amino acids (lysine, methionine), branched-chain fatty acids, tRNA modification and RNA degradation. In this new version, gene products that are enzymes or transporters are explicitly linked to the biochemical reactions of the RHEA reaction resource (http://www.ebi.ac.uk/rhea/), while novel compound entries have been created in the database Chemical Entities of Biological Interest (http://www.ebi.ac.uk/chebi/). The newly annotated sequence is deposited at the International Nucleotide Sequence Data Collaboration with accession number AL009126.4.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Recombinant Bacillus subtilis that grows on untreated plant biomass.

    PubMed

    Anderson, Timothy D; Miller, J Izaak; Fierobe, Henri-Pierre; Clubb, Robert T

    2013-02-01

    Lignocellulosic biomass is a promising feedstock to produce biofuels and other valuable biocommodities. A major obstacle to its commercialization is the high cost of degrading biomass into fermentable sugars, which is typically achieved using cellulolytic enzymes from Trichoderma reesei. Here, we explore the use of microbes to break down biomass. Bacillus subtilis was engineered to display a multicellulase-containing minicellulosome. The complex contains a miniscaffoldin protein that is covalently attached to the cell wall and three noncovalently associated cellulase enzymes derived from Clostridium cellulolyticum (Cel48F, Cel9E, and Cel5A). The minicellulosome spontaneously assembles, thus increasing the practicality of the cells. The recombinant bacteria are highly cellulolytic and grew in minimal medium containing industrially relevant forms of biomass as the primary nutrient source (corn stover, hatched straw, and switch grass). Notably, growth did not require dilute acid pretreatment of the biomass and the cells achieved densities approaching those of cells cultured with glucose. An analysis of the sugars released from acid-pretreated corn stover indicates that the cells have stable cellulolytic activity that enables them to break down 62.3% ± 2.6% of the biomass. When supplemented with beta-glucosidase, the cells liberated 21% and 33% of the total available glucose and xylose in the biomass, respectively. As the cells display only three types of enzymes, increasing the number of displayed enzymes should lead to even more potent cellulolytic microbes. This work has important implications for the efficient conversion of lignocellulose to value-added biocommodities.

  15. Detailed structure-function correlations of Bacillus subtilis acetolactate synthase.

    PubMed

    Sommer, Bettina; von Moeller, Holger; Haack, Martina; Qoura, Farah; Langner, Clemens; Bourenkov, Gleb; Garbe, Daniel; Loll, Bernhard; Brück, Thomas

    2015-01-02

    Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Elasticity and wrinkled morphology of Bacillus subtilis pellicles

    PubMed Central

    Trejo, Miguel; Douarche, Carine; Bailleux, Virginie; Poulard, Christophe; Mariot, Sandrine; Regeard, Christophe; Raspaud, Eric

    2013-01-01

    Wrinkled morphology is a distinctive phenotype observed in mature biofilms produced by a great number of bacteria. Here we study the formation of macroscopic structures (wrinkles and folds) observed during the maturation of Bacillus subtilis pellicles in relation to their mechanical response. We show how the mechanical buckling instability can explain their formation. By performing simple tests, we highlight the role of confining geometry and growth in determining the symmetry of wrinkles. We also experimentally demonstrate that the pellicles are soft elastic materials for small deformations induced by a tensile device. The wrinkled structures are then described by using the equations of elastic plates, which include the growth process as a simple parameter representing biomass production. This growth controls buckling instability, which triggers the formation of wrinkles. We also describe how the structure of ripples is modified when capillary effects are dominant. Finally, the experiments performed on a mutant strain indicate that the presence of an extracellular matrix is required to maintain a connective and elastic pellicle. PMID:23341623

  17. Rhizobacteria Bacillus subtilis restricts foliar pathogen entry through stomata.

    PubMed

    Kumar, Amutha Sampath; Lakshmanan, Venkatachalam; Caplan, Jeffrey L; Powell, Deborah; Czymmek, Kirk J; Levia, Delphis F; Bais, Harsh P

    2012-11-01

    Plants exist in a complex multitrophic environment, where they interact with and compete for resources with other plants, microbes and animals. Plants have a complex array of defense mechanisms, such as the cell wall being covered with a waxy cuticle serving as a potent physical barrier. Although some pathogenic fungi infect plants by penetrating through the cell wall, many bacterial pathogens invade plants primarily through stomata on the leaf surface. Entry of the foliar pathogen, Pseudomonas syringae pathovar tomato DC3000 (hereafter PstDC3000), into the plant corpus occurs through stomatal openings, and consequently a key plant innate immune response is the transient closure of stomata, which delays disease progression. Here, we present evidence that the root colonization of the rhizobacteria Bacillus subtilis FB17 (hereafter FB17) restricts the stomata-mediated pathogen entry of PstDC3000 in Arabidopsis thaliana. Root binding of FB17 invokes abscisic acid (ABA) and salicylic acid (SA) signaling pathways to close light-adapted stomata. These results emphasize the importance of rhizospheric processes and environmental conditions as an integral part of the plant innate immune system against foliar bacterial infections.

  18. The sodium effect of Bacillus subtilis growth on aspartate.

    PubMed

    Whiteman, P; Marks, C; Freese, E

    1980-08-01

    aspH mutants of Bacillus subtilis have a constitutive aspartase activity and grow well on aspartate as sole carbon source. aspH aspT mutants, which are deficient in high affinity aspartate transport as a result of the aspT mutation, grow as well as aspH mutants in medium containing high concentrations of aspartate and Na+. This Na+ effect is not due to an enhancement of aspartate transport but is the result of increased cellular metabolism. The ability to grow rapidly in sodium aspartate is induced by prior growth in the presence of Na+. In potassium aspartate, the addition of arginine, citrulline, ornithine, delta 1-pyrroline-5-carboxylase or proline instead of Na+ also allows rapid growth; but in a mutant deficient in ornithine--oxo-acid aminotransferase, only pyrroline-carboxylate or proline can replace Na+. The amino acid pool of cells growing slowly in potassium aspartate contains proline at a low concentration which increases upon addition of proline (but not Na+) to the medium. Thus, Na+ addition does not increase the synthesis of proline, but proline or pyrroline-carboxylate acts similarly to Na+ either in preventing some inhibitory effect (by aspartate or the accumulating NH4+) or in overcoming some deficiency (e.g. in further proline metabolism.

  19. Synthesis of teichoic acid by Bacillus subtilis protoplasts.

    PubMed Central

    Bertram, K C; Hancock, I C; Baddiley, J

    1981-01-01

    Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium. With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit. Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane. During synthesis, which was also achieved readily by whole cells after a brief period of wall lysis, the cytidine phosphate portion of the nucleotide precursors did not pass through the membrane. No evidence could be obtained for a transphosphorylation mechanism for the translocation process. It is suggested that reaction with exogenous substrates was due to temporary exposure of a protein component of the enzyme complex at the outer surface of the membrane during the normal biosynthetic cycle. PMID:6271728

  20. Bioremediation of methylene blue dye using Bacillus subtilis MTCC 441.

    PubMed

    Upendar, Ganta; Dutta, Susmita; Bhattacharya, Pinaki; Dutta, Abhishek

    2017-04-01

    Methylene blue (MB) commonly found in the textile industry effluent has been chosen as a model dye to investigate bioremediation using Bacillus subtilis MTCC 441. Both free cells and calcium alginate immobilized cells have been used to remove MB from the effluent. The operating variables of initial concentration of dye (20-60 mg/L), inoculum size (4-8%) and temperature (25-35 °C) have been varied judiciously during the kinetic study in a batch contactor. A maximum removal of 91.68% is obtained when 20 mg/L MB solution was inoculated with 8% inoculum and cultured for 6 h at 30 °C. Continuous removal of MB has been studied in a fixed bed contactor using immobilized cells as packing materials. Influent concentration (10-30 mg/L) was varied and breakthrough parameters have been determined. With increase in influent concentration from 10 mg/L to 30 mg/L, percentage removal of dye decreases from 72.44% to 49.62%.

  1. Biodegradation of pendimethalin by Bacillus subtilis Y3.

    PubMed

    Ni, Haiyan; Yao, Li; Li, Na; Cao, Qin; Dai, Chen; Zhang, Jun; He, Qin; He, Jian

    2016-03-01

    A bacterium strain Y3, capable of efficiently degrading pendimethalin, was isolated from activated sludge and identified as Bacillus subtilis according to its phenotypic features and 16S rRNA phylogenetic analysis. This strain could grow on pendimethalin as a sole carbon source and degrade 99.5% of 100mg/L pendimethalin within 2.5days in batch liquid culture, demonstrating a greater efficiency than any other reported strains. Three metabolic products, 6-aminopendimethalin, 5-amino-2-methyl-3-nitroso-4-(pentan-3-ylamino) benzoic acid, and 8-amino-2-ethyl-5-(hydroxymethyl)-1,2-dihydroquinoxaline-6-carboxylic acid, were identified by HPLC-MS/MS, and a new microbial degradation pathway was proposed. A nitroreductase catalyzing nitroreduction of pendimethalin to 6-aminopendimethalin was detected in the cell lysate of strain Y3. The cofactor was nicotinamide adenine dinucleotide phosphate (NADPH) or more preferably nicotinamide adenine dinucleotide (NADH). The optimal temperature and pH for the nitroreductase were 30°C and 7.5, respectively. Hg(2+), Ni(2+), Pb(2+), Co(2+), Mn(2+) Cu(2+), Ag(+), and EDTA severely inhibited the nitroreductase activity, whereas Fe(2+), Mg(2+), and Ca(2+) enhanced it. This study provides an efficient pendimethalin-degrading microorganism and broadens the knowledge of the microbial degradation pathway of pendimethalin.

  2. Novel methyl transfer during chemotaxis in Bacillus subtilis

    SciTech Connect

    Thoelke, M.S.; Kirby, J.R.; Ordal, G.W. )

    1989-06-27

    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.

  3. Activity of essential oils against Bacillus subtilis spores.

    PubMed

    Lawrence, Hayley A; Palombo, Enzo A

    2009-12-01

    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.

  4. Liquid transport facilitated by channels in Bacillus subtilis biofilms

    PubMed Central

    Wilking, James N.; Zaburdaev, Vasily; De Volder, Michael; Losick, Richard; Brenner, Michael P.; Weitz, David A.

    2013-01-01

    Many bacteria on earth exist in surface-attached communities known as biofilms. These films are responsible for manifold problems, including hospital-acquired infections and biofouling, but they can also be beneficial. Biofilm growth depends on the transport of nutrients and waste, for which diffusion is thought to be the main source of transport. However, diffusion is ineffective for transport over large distances and thus should limit growth. Nevertheless, biofilms can grow to be very large. Here we report the presence of a remarkable network of well-defined channels that form in wild-type Bacillus subtilis biofilms and provide a system for enhanced transport. We observe that these channels have high permeability to liquid flow and facilitate the transport of liquid through the biofilm. In addition, we find that spatial variations in evaporative flux from the surface of these biofilms provide a driving force for the flow of liquid in the channels. These channels offer a remarkably simple system for liquid transport, and their discovery provides insight into the physiology and growth of biofilms. PMID:23271809

  5. Transcriptional regulation of Bacillus subtilis citrate synthase genes.

    PubMed

    Jin, S; Sonenshein, A L

    1994-08-01

    The Bacillus subtilis citrate synthase genes citA and citZ were repressed during early exponential growth phase in nutrient broth medium and were induced as cells reached the end of exponential phase. Both genes were also induced by treatment of cells with the drug decoyinine. After induction, the steady-state level of citZ mRNA was about five times higher than that of citA mRNA. At least some of the citZ transcripts read through into the isocitrate dehydrogenase (citC) gene. Transcription from an apparent promoter site located near the 3' end of the citZ gene also contributed to expression of citC. In minimal medium, citA transcription was about 6-fold lower when glucose was the sole carbon source than it was when succinate was the carbon source. Expression of the citZ gene was repressed 2-fold by glucose and 10-fold when glucose and glutamate were present simultaneously. This latter synergistic repression is similar to the effect of glucose and glutamate on steady-state citrate synthase enzyme activity. CitR, a protein of the LysR family, appeared to be a repressor of citA but not of citZ.

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

    PubMed Central

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

    2009-01-01

    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-wide expression during mid-exponential growth on rich (LB) and minimal (M9) medium. The identified TARs account for 77.3% of the genes as they are currently annotated and additionally we find 84 putative non-coding RNAs (ncRNAs) and 127 antisense transcripts. One ncRNA, ncr22, is predicted to act as a translational control on cstA and an antisense transcript was observed opposite the housekeeping sigma factor sigA. Through this work we have discovered a long conserved 3′ untranslated region (UTR) in a group of membrane-associated genes that is predicted to fold into a large and highly stable secondary structure. One of the genes having this tail is efeN, which encodes a target of the twin-arginine translocase (Tat) protein translocation system. PMID:19682248

  7. Hydrolysis of black soybean isoflavone glycosides by Bacillus subtilis natto.

    PubMed

    Kuo, Lun-Cheng; Cheng, Wei-Yi; Wu, Ren-Yu; Huang, Ching-Jang; Lee, Kung-Ta

    2006-11-01

    Hydrolysis of isoflavone glycosides by Bacillus subtilis natto NTU-18 in black soymilk is reported. At the concentration of 3-5% (w/v), black soymilk in flask cultures, the isoflavones, daidzin, and genistin were highly deglycosylated within 24 h. Deglycosylation of isoflavones was further carried out in a 7-l fermenter with 5% black soymilk. During the fermentation, viable cells increased from 10(3) to 10(9) CFU ml(-1) in 15 h, and the activity of beta-glucosidase appeared at 8 h after inoculation and reached a maximum (3.3 U/ml) at 12 h, then decreased rapidly. Deglycosylation of isoflavone glycosides was observed at the same period, the deglycosylation rate of daidzin and genistin at 24 h was 100 and 75%, respectively. It is significantly higher than the previous reports of fermentation with lactic acid bacteria. In accordance with the deglycosylation of isoflavone glycosides, the estrogenic activity of the 24 h fermented black soymilk for ERbeta estrogen receptor increased to threefold; meanwhile, the fermented broth activated ERalpha estrogen receptor to a less extent than ERbeta. These results suggest that this fermentation effectively hydrolyzed the glycosides from isoflavone in black soymilk and the fermented black soymilk has the potential to be applied to selective estrogen receptor modulator products.

  8. Periodic Colony Formation by Bacterial Species Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Wakita, Jun-ichi; Shimada, Hirotoshi; Itoh, Hiroto; Matsuyama, Tohey; Matsushita, Mitsugu

    2001-03-01

    We have investigated the periodic colony growth of bacterial species Bacillus subtilis. A colony grows cyclically with the interface repeating an advance (migration phase) and a rest (consolidation phase) alternately on a surface of semi-solid agar plate under appropriate environmental conditions, resulting in a concentric ring-like colony. It was found from macroscopic observations that the characteristic quantities for the periodic growth such as the migration time, the consolidation time and the terrace spacing do not depend so much on nutrient concentration Cn, but do on agar concentration Ca. The consolidation time was a weakly increasing function of Ca, while the migration time and the terrace spacing were, respectively, weakly and strongly decreasing function of Ca. Overall, the cycle (migration-plus-consolidation) time seems to be constant, and does not depend so much on both Cn and Ca. Microscopically, bacterial cells inside the growing front of a colony keep increasing their population during both migration and consolidation phases. It was also confirmed that their secreting surfactant called surfactin does not affect their periodic growth qualitatively, i.e., mutant cells which cannot secrete surfactin produce a concentric ring-like colony. All these results suggest that the diffusion of the nutrient and the surfactin are irrelevant to their periodic growth.

  9. Transformation of Bacillus subtilis in alpha-amylase productivity by deoxyribonucleic acid from B. subtilis var. amylosacchariticus.

    PubMed

    Yoneda, Y; Yamane, K; Yamaguchi, K; Nagata, Y; Maruo, B

    1974-12-01

    Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.

  10. Plant methyl salicylate induces defense responses in the rhizobacterium Bacillus subtilis.

    PubMed

    Kobayashi, Kazuo

    2015-04-01

    Bacillus subtilis is a rhizobacterium that promotes plant growth and health. Cultivation of B. subtilis with an uprooted weed on solid medium produced pleat-like architectures on colonies near the plant. To test whether plants emit signals that affect B. subtilis colony morphology, we examined the effect of plant-related compounds on colony morphology. Bacillus subtilis formed mucoid colonies specifically in response to methyl salicylate, which is a plant-defense signal released in response to pathogen infection. Methyl salicylate induced mucoid colony formation by stimulating poly-γ-glutamic acid biosynthesis, which formed enclosing capsules that protected the cells from exposure to antimicrobial compounds. Poly-γ-glutamic acid synthesis depended on the DegS-DegU two-component regulatory system, which activated DegSU-dependent gene transcription in response to methyl salicylate. Bacillus subtilis did not induce plant methyl salicylate production, indicating that the most probable source of methyl salicylate in the rhizosphere is pathogen-infected plants. Methyl salicylate induced B. subtilis biosynthesis of the antibiotics bacilysin and fengycin, the latter of which exhibited inhibitory activity against the plant pathogenic fungus Fusarium oxysporum. We propose that B. subtilis may sense plants under pathogen attack via methyl salicylate, and express defense responses that protect both B. subtilis and host plants in the rhizosphere.

  11. Inhibitory activity of probiotic Bacillus subtilis UTM 126 against vibrio species confers protection against vibriosis in juvenile shrimp (Litopenaeus vannamei).

    PubMed

    Balcázar, José Luis; Rojas-Luna, Tyrone

    2007-11-01

    The bacterial strain Bacillus subtilis UTM 126 produced antimicrobial activity against pathogenic Vibrio species, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. The probiotic effect of B. subtilis was tested by feeding juvenile shrimp (Litopenaeus vannamei) food supplemented with B. subtilis (10(5 )CFU/g) for 28 days before an immersion challenge with V. harveyi at 10(5 )CFU/mL for 24 h. The treatment with B. subtilis UTM 126 decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp aquaculture.

  12. Spectral and potentiometric analysis of cytochromes from Bacillus subtilis.

    PubMed

    de Vrij, W; van den Burg, B; Konings, W N

    1987-08-03

    Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.

  13. Recombinant Bacillus subtilis That Grows on Untreated Plant Biomass

    PubMed Central

    Anderson, Timothy D.; Miller, J. Izaak; Fierobe, Henri-Pierre

    2013-01-01

    Lignocellulosic biomass is a promising feedstock to produce biofuels and other valuable biocommodities. A major obstacle to its commercialization is the high cost of degrading biomass into fermentable sugars, which is typically achieved using cellulolytic enzymes from Trichoderma reesei. Here, we explore the use of microbes to break down biomass. Bacillus subtilis was engineered to display a multicellulase-containing minicellulosome. The complex contains a miniscaffoldin protein that is covalently attached to the cell wall and three noncovalently associated cellulase enzymes derived from Clostridium cellulolyticum (Cel48F, Cel9E, and Cel5A). The minicellulosome spontaneously assembles, thus increasing the practicality of the cells. The recombinant bacteria are highly cellulolytic and grew in minimal medium containing industrially relevant forms of biomass as the primary nutrient source (corn stover, hatched straw, and switch grass). Notably, growth did not require dilute acid pretreatment of the biomass and the cells achieved densities approaching those of cells cultured with glucose. An analysis of the sugars released from acid-pretreated corn stover indicates that the cells have stable cellulolytic activity that enables them to break down 62.3% ± 2.6% of the biomass. When supplemented with beta-glucosidase, the cells liberated 21% and 33% of the total available glucose and xylose in the biomass, respectively. As the cells display only three types of enzymes, increasing the number of displayed enzymes should lead to even more potent cellulolytic microbes. This work has important implications for the efficient conversion of lignocellulose to value-added biocommodities. PMID:23183968

  14. Completed Chromosomes in Thymine-Requiring Bacillus subtilis Spores

    PubMed Central

    Callister, Heather; Wake, R. G.

    1974-01-01

    Origin:terminus genetic marker ratios (both purA: metB and purA:ilvA) were measured in extracts of spores of Bacillus subtilis strains W23 thy his and 168 thy. For strain W23 thy his, normalized to W23 spore deoxyribonucleic acid, both ratios were equal to unity and were consistent with the presence of only completed chromosomes in the spores. The same ratios in extracts of spores of 168 thy, normalized to strain 168 or the prototroph SB19, were abnormal, i.e., 2.26 ± 0.10 and 0.71 ± 0.06 for purA:metB and purA:ilvA, respectively. These values were unaffected by the extent of extraction of the spore deoxyribonucleic acid, the richness of the medium on which they are formed, and the thymine phenotype. The high ratio for purA:metB is in agreement with the results of earlier workers but, because of the low purA:ilvA ratio, cannot be explained simply by the presence of partially replicated chromosomes in spores of strain 168 thy. Furthermore, purA:leuA in such extracts is 1.01 ± 0.06, consistent with the presence of only completed chromosomes. It is concluded that the abnormal origin:terminus marker ratios are only apparent and result from non-isogenicity between strains 168 thy and 168 in the metB thyB ilvA chromosome region introduced during construction of 168 thy by transformation of strain 168 with W23 thy deoxyribonucleic acid. It is concluded further that the chromosomes of strain 168 thy spores are in a completed form. PMID:4218227

  15. Complete Genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, Two Phylogenetically Distinct Probiotics

    PubMed Central

    Ramya, T. N. C.; Subramanian, Srikrishna

    2016-01-01

    Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions. PMID:27258038

  16. Complete Genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, Two Phylogenetically Distinct Probiotics.

    PubMed

    Khatri, Indu; Sharma, Shailza; Ramya, T N C; Subramanian, Srikrishna

    2016-01-01

    Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions.

  17. UGA can be decoded as tryptophan at low efficiency in Bacillus subtilis.

    PubMed Central

    Lovett, P S; Ambulos, N P; Mulbry, W; Noguchi, N; Rogers, E J

    1991-01-01

    Replacement of cat-86 codon 7 or 144 with the UGA codon permitted the gene to confer chloramphenicol resistance in wild-type Bacillus subtilis. UAA replacements of the same codons resulted in a chloramphenicol-sensitive phenotype in wild-type B. subtilis and a chloramphenicol-resistant phenotype in suppressor-positive strains. N-terminal sequencing showed that UGA at codon 7 was decoded as tryptophan in wild-type cells, at an efficiency of about 6%. Images PMID:1900283

  18. Primary Adsorption Site of Phage PBS1: the Flagellum of Bacillus subtilis

    PubMed Central

    Raimondo, Linda M.; Lundh, Nancy P.; Martinez, Rafael J.

    1968-01-01

    The adsorption of Bacillus subtilis phage PBS1 was studied, and it was demonstrated that the primary adsorption site for this phage is the flagellum of B. subtilis. The capacity of flagella to function for motility may be lost without the loss of their capacity to adsorb the phage and permit infection. Deoxyribonucleic acid injection by the phage is inhibited by cyanide, suggesting the requirement for cellular energy in the infection process. Images PMID:4986906

  19. [Destruction of hexamethylenediamine by a Bacillus subtilis culture in a medium with clay minerals].

    PubMed

    Garbara, S V; Rotmistrov, M N

    1982-01-01

    The object of this work was to study the effect of clay minerals of the montmorillonite group on the oxidative and destructive activity of Bacillus subtilis 21/3 utilizing hexamethylene diamine (HMD) in metabolic processes. HMD destruction by the culture accelerated when clay minerals were added to the chemically defined medium. The respiration activity of Bac. subtilis 21/3 was studied, and the rate of HMD oxidation was found to increase in the presence of montmorillonite.

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

    PubMed

    Juhas, Mario; Ajioka, James W

    2016-06-01

    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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway

    PubMed Central

    Donato, Verónica; Ayala, Facundo Rodríguez; Cogliati, Sebastián; Bauman, Carlos; Costa, Juan Gabriel; Leñini, Cecilia; Grau, Roberto

    2017-01-01

    Beneficial bacteria have been shown to affect host longevity, but the molecular mechanisms mediating such effects remain largely unclear. Here we show that formation of Bacillus subtilis biofilms increases Caenorhabditis elegans lifespan. Biofilm-proficient B. subtilis colonizes the C. elegans gut and extends worm lifespan more than biofilm-deficient isogenic strains. Two molecules produced by B. subtilis — the quorum-sensing pentapeptide CSF and nitric oxide (NO) — are sufficient to extend C. elegans longevity. When B. subtilis is cultured under biofilm-supporting conditions, the synthesis of NO and CSF is increased in comparison with their production under planktonic growth conditions. We further show that the prolongevity effect of B. subtilis biofilms depends on the DAF-2/DAF-16/HSF-1 signalling axis and the downregulation of the insulin-like signalling (ILS) pathway. PMID:28134244

  2. Endophytic colonisation of Bacillus subtilis in the roots of Robinia pseudoacacia L.

    PubMed

    Huang, B; Lv, C; Zhuang, P; Zhang, H; Fan, L

    2011-11-01

    The endophytic colonisation of Bacillus subtilis strain GXJM08, isolated from roots of Podocarpus imbricatus B1. Enum. P1. Jav., in roots of the leguminous plant Robinia pseudoacacia L. was investigated. Ultrastructure observations showed that B. subtilis caused morphological changes in the root hair and colonised the plant through infected root hairs. The structure of the infection thread was similar to that of rhizobia, but the structure of infected cells was different. B. subtilis is also different from rhizobia and plant pathogens in terms of the formation of a peribacteroid membrane and the mode of penetration through the host cell wall. Our results provide a basis for studying development of the mutualistic symbiotic relationship between B. subtilis and plants, and a basis for studying the mechanism of the B. subtilis-plant interaction. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

  3. Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis.

    PubMed Central

    Ohashi, H; Katsuta, Y; Nagashima, M; Kamei, T; Yano, M

    1989-01-01

    The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene. Images PMID:2504107

  4. Menaquinone and Iron Are Essential for Complex Colony Development in Bacillus subtilis

    PubMed Central

    Pelchovich, Gidi; Omer-Bendori, Shira; Gophna, Uri

    2013-01-01

    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. PMID:24223955

  5. The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

    PubMed Central

    Altabe, Silvia G.; Aguilar, Pablo; Caballero, Gerardo M.; de Mendoza, Diego

    2003-01-01

    Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids. PMID:12730185

  6. Deoxyribonucleic acid repair in Bacillus subtilis: development of competent cells into a tester for carcinogens

    SciTech Connect

    Yasbin, R.E.; Miehl, R.

    1980-04-01

    The development of competent transformed Bacillus subtilis into a tester system for carcinogens is described. Precocious or noninduced activation of SOS functions occurs in competent cells. Thus, lower doses or concentrations of SOS inducing agents are needed to cause cell death due to indigenous prophage activation and lysis of bacteria. The two known defective prophages in B. subtilis enhance the sensitivity of competent cells to the carcinogens ultraviolet light, mitomycin C, and methyl methanesulfonate. However, these same cells have no enhanced sensitivity for the non-carcinogenic ethyl methanesulfonate or for nalidixic acid. Therefore, competent B. subtilis appears to be a sensitive tester for carcinogens.

  7. [Factor of salinity and adaptive capacity of recombinant strains of Escherichia coli and Bacillus subtilis].

    PubMed

    Boiandin, A N; Lobova, T I; Krylova, T Iu; Kargatova, T V; Popova, L Iu; Pechurkin, N S

    2000-01-01

    Effect of different concentrations of salts on natural and recombinant strains of Bacillus subtilis and Escherichia coli was studied. The recombinant strain of B. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains of E. coli. Some salts exerted a bacteriostatic effect on E. coli and B. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.

  8. Evaluation of in situ valine production by Bacillus subtilis in young pigs.

    PubMed

    Nørgaard, J V; Canibe, N; Soumeh, E A; Jensen, B B; Nielsen, B; Derkx, P; Cantor, M D; Blaabjerg, K; Poulsen, H D

    2016-11-01

    Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val : Lys, or 0.63 SID Val : Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val : Lys, or 0.63 SID Val : Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.

  9. Novel fluorescent risedronates: synthesis, photodynamic inactivation and imaging of Bacillus subtilis.

    PubMed

    Zhou, Li-Sheng; Yang, Ke-Wu; Feng, Lei; Xiao, Jian-Min; Liu, Cheng-Cheng; Zhang, Yi-Lin; Crowder, Michael W

    2013-02-15

    Novel fluorescently-labeled conjugates of risedronate were synthesized using an epoxide linker, enabling conjugation of risedronate via its pyridyl nitrogen with the aromatic succinimidyl esters. The compounds were characterized by using (1)H NMR, (13)C NMR, (31)P NMR, UV-vis and fluorescence emission spectroscopies. Biological activity assays showed that the conjugates 14 and 15 exhibited photodynamic inactivation of Bacillus subtilis (ATCC 6633) with 91% and 47% bacterial lethality at 10 μM upon visible light irradiation, respectively. Both 14 and 15 could be also used for fluorescence imaging of Bacillus subtilis.

  10. Production of l-Arginine by Arginine Hydroxamate-Resistant Mutants of Bacillus subtilis

    PubMed Central

    Kisumi, Masahiko; Kato, Jyoji; Sugiura, Masaki; Chibata, Ichiro

    1971-01-01

    l-Arginine hydroxamate inhibited the growth of various bacteria, and the inhibition was readily reversed by arginine. l-Arginine hydroxamate (10−3m) completely inhibited the growth of Bacillus subtilis. This inhibitory effect was prevented by 2.5 × 10−4ml-arginine, which was the most effective of all the natural amino acids in reversing the inhibition. l-Arginine hydroxamate-resistant mutants of Bacillus subtilis were isolated and found to excrete l-arginine in relatively high yields. One of the mutants, strain AHr-5, produced 4.5 mg of l-arginine per ml in shaken culture in 3 days. PMID:5002904

  11. Death of Bacillus subtilis Auxotrophs Due to Deprivation of Thymine, Tryptophan, or Uracil

    PubMed Central

    Pritikin, William B.; Romig, W. R.

    1966-01-01

    Pritikin, William B. (University of California, Los Angeles), and W. R. Romig. Death of Bacillus subtilis auxotrophs due to deprivation of thymine, tryptophan, or uracil, J. Bacteriol. 92:291–296. 1966.—Auxotrophic mutants of Bacillus subtilis 168 that require either tryptophan, uracil, or thymine died rapidly when deprived of any of these compounds. Phage PBS1 was produced by infected B. subtilis 168 (thy try-2) deprived of thymine. Phage PBS1 was not produced by infected B. subtilis 168 (try-2) deprived of tryptophan or infected B. subtilis 168-15 (try-2 ura) deprived of uracil. B. subtilis 168 thy try-2 and 168-15 could be transduced by phage PBS1 after prolonged deprivation of tryptophan or uracil, respectively. When B. subtilis 168-15 was transduced to uracil independence by phage PBS1, the uracil-independent transductants became immune to uracil-less death within 10 min of exposure to phage, and began to multiply within 2 hr after exposure to phage at an incubation temperature of 46 C. PMID:16562109

  12. Death of Bacillus subtilis Auxotrophs Due to Deprivation of Thymine, Tryptophan, or Uracil.

    PubMed

    Pritikin, W B; Romig, W R

    1966-08-01

    Pritikin, William B. (University of California, Los Angeles), and W. R. Romig. Death of Bacillus subtilis auxotrophs due to deprivation of thymine, tryptophan, or uracil, J. Bacteriol. 92:291-296. 1966.-Auxotrophic mutants of Bacillus subtilis 168 that require either tryptophan, uracil, or thymine died rapidly when deprived of any of these compounds. Phage PBS1 was produced by infected B. subtilis 168 (thy try-2) deprived of thymine. Phage PBS1 was not produced by infected B. subtilis 168 (try-2) deprived of tryptophan or infected B. subtilis 168-15 (try-2 ura) deprived of uracil. B. subtilis 168 thy try-2 and 168-15 could be transduced by phage PBS1 after prolonged deprivation of tryptophan or uracil, respectively. When B. subtilis 168-15 was transduced to uracil independence by phage PBS1, the uracil-independent transductants became immune to uracil-less death within 10 min of exposure to phage, and began to multiply within 2 hr after exposure to phage at an incubation temperature of 46 C.

  13. Study of the radiation effect of 99Mo/99mTc generator on Bacillus subtilis and Bacillus pumilus species.

    PubMed

    Fukumori, Neuza T O; Endo, Erica M M; Felgueiras, Carlos F; Matsuda, Margareth M N; Osso Junior, João A

    2016-01-01

    In this work, molybdenum-99 loaded columns were challenged with Bacillus subtilis vegetative cells and Bacillus pumilus spores inside and outside the alumina column, and microbial recovery and radiation effect were assessed. Alumina was a barrier for the passage of microorganisms regardless the species, whilst spores were more retained than vegetative cells with a lower microbial recovery, without significant differences between 9.25 and 74 GBq generators. Bacillus pumilus biological indicator showed lower recoveries, suggesting a radiation inactivating effect on microorganisms. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Comparative structural bioinformatics analysis of Bacillus amyloliquefaciens chemotaxis proteins within Bacillus subtilis group.

    PubMed

    Yssel, Anna; Reva, Oleg; Tastan Bishop, Ozlem

    2011-12-01

    Chemotaxis is a process in which bacteria sense their chemical environment and move towards more favorable conditions. Since plant colonization by bacteria is a multifaceted process which requires a response to the complex chemical environment, a finely tuned and sensitive chemotaxis system is needed. Members of the Bacillus subtilis group including Bacillus amyloliquefaciens are industrially important, for example, as bio-pesticides. The group exhibits plant growth-promoting characteristics, with different specificity towards certain host plants. Therefore, we hypothesize that while the principal molecular mechanisms of bacterial chemotaxis may be conserved, the bacterial chemotaxis system may need an evolutionary tweaking to adapt it to specific requirements, particularly in the process of evolution of free-living soil organisms, towards plant colonization behaviour. To date, almost nothing is known about what parts of the chemotaxis proteins are subjected to positive amino acid substitutions, involved in adjusting the chemotaxis system of bacteria during speciation. In this novel study, positively selected and purified sites of chemotaxis proteins were calculated, and these residues were mapped onto homology models that were built for the chemotaxis proteins, in an attempt to understand the spatial evolution of the chemotaxis proteins. Various positively selected amino acids were identified in semi-conserved regions of the proteins away from the known active sites.

  15. Bacillus subtilis HJ18-4 from traditional fermented soybean food inhibits Bacillus cereus growth and toxin-related genes.

    PubMed

    Eom, Jeong Seon; Lee, Sun Young; Choi, Hye Sun

    2014-11-01

    Bacillus subtilis HJ18-4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad-spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18-4. Expression of B. cereus toxin-related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18-4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18-4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18-4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus. © 2014 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists®

  16. Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

    PubMed

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

    2015-01-01

    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 locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

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

    2016-02-04

    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.

  19. Bacillus subtilis Protects Public Goods by Extending Kin Discrimination to Closely Related Species

    PubMed Central

    2017-01-01

    ABSTRACT Kin discrimination systems are found in numerous communal contexts like multicellularity and are theorized to prevent exploitation of cooperative behaviors. The kin discrimination system in Bacillus subtilis differs from most other such systems because it excludes nonkin cells rather than including kin cells. Because nonkin are the target of the system, B. subtilis can potentially distinguish degrees of nonkin relatedness, not just kin versus nonkin. We examined this by testing a large strain collection of diverse Bacillus species against B. subtilis in different multicellular contexts. The effects of kin discrimination extend to nearby species, as the other subtilis clade species were treated with the same antagonism as nonkin. Species in the less-related pumilus clade started to display varied phenotypes but were mostly still discriminated against, while cereus clade members and beyond were no longer subject to kin discrimination. Seeking a reason why other species are perceived as antagonistic nonkin, we tested the ability of B. subtilis to steal communally produced surfactant from these species. We found that the species treated as nonkin were the only ones that made a surfactant that B. subtilis could utilize and that nonkin antagonism prevented such stealing when the two strains were mixed. The nonkin exclusion kin discrimination method thus allows effective protection of the cooperative behaviors prevalent in multicellularity while still permitting interactions with more distant species that are not a threat. PMID:28679746

  20. Three Distinct Phases of Isoprene Formation during Growth and Sporulation of Bacillus subtilis

    PubMed Central

    Wagner, William P.; Nemecek-Marshall, Michele; Fall, Ray

    1999-01-01

    During growth on a glucose-tryptone medium, Bacillus subtilis 6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1,3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation. PMID:10419976

  1. Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in "Bacillus Subtilis"

    ERIC Educational Resources Information Center

    Bredeston, L. M.; Marciano, D.; Albanesi, D.; De Mendoza, D.; Delfino, J. M.

    2011-01-01

    This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of "Bacillus subtilis", composed of a [delta]5-desaturase (encoded by the "des" gene) and the canonical…

  2. Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production

    USDA-ARS?s Scientific Manuscript database

    Spore-forming Bacillus strains that produce extracellular poly-'-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 365 strains, including B. subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting n...

  3. Enhanced production of recombinant nattokinase in Bacillus subtilis by the elimination of limiting factors.

    PubMed

    Chen, Po Ting; Chao, Yun-Peng

    2006-10-01

    By systematic investigation, glutamate and a mixture of metal ions were identified as factors limiting the production of nattokinase in Bacillus subtilis. Consequently, in medium supplemented with these materials, the recombinant strain secreted 4 times more nattokinase (260 mg l(-1)) than when grown in the unsupplemented medium.

  4. Creating new genes by plasmid recombination in Escherichia coli and Bacillus subtilis.

    PubMed

    Gomez, Ana; Galic, Tatjana; Mariet, Jean-François; Matic, Ivan; Radman, Miroslav; Petit, Marie-Agnès

    2005-11-01

    Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence.

  5. Creating New Genes by Plasmid Recombination in Escherichia coli and Bacillus subtilis

    PubMed Central

    Gomez, Ana; Galic, Tatjana; Mariet, Jean-François; Matic, Ivan; Radman, Miroslav; Petit, Marie-Agnès

    2005-01-01

    Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence. PMID:16269814

  6. Transcriptional Profiling in Cotton Associated with Bacillus Subtilis (UFLA285) Induced Biotic-stress Tolerance

    USDA-ARS?s Scientific Manuscript database

    Plant growth promoting rhizobacteria (PGPR) confer disease resistance in many agricultural crops. In the case of Bacillus subtilis (UFLA285) isolated from the cotton producing state of Mato Grosso (Brazil), in addition to inducing foliar and root growth, disease resistance against damping-off cause...

  7. Antagonistic activity of Bacillus subtilis SB1 and its biocontrol effect on tomato bacterial wilt

    USDA-ARS?s Scientific Manuscript database

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, isolated from tomato roots, showed a broad-spectrum of antimicrobial activity in in vitro experiments. It inhibited the growth of many plant pathogens, including Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, Fusarium ox...

  8. Antagonistic activity and mechanisms of Bacillus subtilis SB1 against Ralstonia solanacearum

    USDA-ARS?s Scientific Manuscript database

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, showed a broad-spectrum of antimicrobial activity in vitro experiments. In addition to Ralstonia solanacearum, strain SB1 inhibited the growth of many other plant pathogens, including Fusarium oxysporum, Botrytis cinerea, Phytoph...

  9. The effect of Bacillus subtilis mouth rinsing in patients with periodontitis.

    PubMed

    Tsubura, S; Mizunuma, H; Ishikawa, S; Oyake, I; Okabayashi, M; Katoh, K; Shibata, M; Iizuka, T; Toda, T; Iizuka, T

    2009-11-01

    Bacillus subtilis is an effective probiotic product for prevention of enteric infections both in humans and animals. We hypothesized that a mouth rinse containing Bacillus subtilis should adhere to and colonize part of the oral bacteria on periodontal tissue. The rinsing ability of Extraction 300E (containing Bacillus subtilis: E-300) was compared with that of a mouth wash liquid , Neosteline Green (benzethonium chloride; NG) that is commonly used in Japan. Compared with NG rinsing, E-300 rinsing resulted in a marked change in the BANA-score. The mean BANA values (score +/- SD) over the course of the study from 0 to 30 days were 1.52 +/- 0.51 (p < or = 0.1) and 0.30 +/- 0.47 (p < or = 0.01) for E-300, and 1.56 +/- 0.51 and 0.93 +/- 0.68 for NG, respectively. Gingival Index also had improvement, while probing pocket depth and bleeding on probing showed small improvements. Mouth rinsing with E-300 significantly reduced periodontal pathogens compared with NG. These results suggest that Bacillus subtilis is an appropriate mouth rinse for patients with periodontitis.

  10. Mucosal immune response in broilers following vaccination with inactivated influenza and recombinant Bacillus subtilis

    USDA-ARS?s Scientific Manuscript database

    Mucosal and systemic immunity were observed in broilers vaccinated with mannosylated chitosan adjuvated (MCA) inactivated A/Turkey/Virginia/158512/2002 (H7N2) and administered with and without recombinant Bacillus subtilis to elicit heterologous influenza strain protection. Previously, mucosal immu...

  11. [Synthesis of amino acids of Bacillus subtilis IMV V-7023 in the medium with glycerophosphates].

    PubMed

    Tserkovniak, L S; Roĭ, A O; Kurdysh, I K

    2009-01-01

    It was shown that under cultivation of Bacillus subtilis IMVV-7023 in the nutrient medium with glycerophosphate biologically active substances are accumulated in the culture liquid. They influence positively the seeds growth and formation of plant germs. The bacteria synthesize amino acids in this medium, their quantitative structure differs from the type of carbon nutrition and cultivation time of the cells.

  12. Synchronous Cultures of Bacillus subtilis Obtained by Filtration with Glass Fiber Filters

    PubMed Central

    Sargent, Michael G.

    1973-01-01

    A simple method of potentially wide applicability for obtaining synchronous cultures of Bacillus subtilis based on size selection is described. Using glass fiber filters, a population (about 1 to 2% of the parent population) can be obtained substantially enriched for small cells which grow synchronously. A method for rapidly concentrating dilute suspensions of cells is described. PMID:4200855

  13. Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens

    USDA-ARS?s Scientific Manuscript database

    The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs1...

  14. Cucumber rhizosphere microbial community response to biocontrol agent Bacillus subtilis B068150

    USDA-ARS?s Scientific Manuscript database

    Gram-positive bacteria Bacillus subtilis B068150 has been used as a biocontrol agent against the pathogen Fusarium oxysporum f. sp. Cucumerinum. However, their survival ability in cucumber rhizosphere and non-rhizosphere as well as their influence on native microbial communities has not been fully i...

  15. Complete genome sequence of Bacillus subtilis BSD-2, a microbial germicide isolated from cultivated cotton.

    PubMed

    Liu, Hongwei; Yin, Shuli; An, Likang; Zhang, Genwei; Cheng, Huicai; Xi, Yanhua; Cui, Guanhui; Zhang, Feiyan; Zhang, Liping

    2016-07-20

    Bacillus subtilis BSD-2, isolated from cotton (Gossypium spp.), had strong antagonistic activity to Verticillium dahlia Kleb and Botrytis cinerea. We sequenced and annotated the BSD-2 complete genome to help us the better use of this strain, which has surfactin, bacilysin, bacillibactin, subtilosin A, Tas A and a potential class IV lanthipeptide biosynthetic pathways.

  16. YrxA is the transcriptional regulator that represses de novo NAD biosynthesis in Bacillus subtilis.

    PubMed

    Rossolillo, Paola; Marinoni, Ilaria; Galli, Elisa; Colosimo, Anna; Albertini, Alessandra M

    2005-10-01

    The first genetic, in vivo, and in vitro evidences that YrxA is the regulator of NAD de novo biosynthesis in Bacillus subtilis are hereby reported. The protein is essential to the transcription repression of the divergent operons nadBCA and nifS-yrxA in the presence of nicotinic acid and binds to their shared operator-promoter region.

  17. 77 FR 1633 - Bacillus Subtilis Strain CX-9060; Exemption From the Requirement of a Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-11

    ... decomposing plant residues (Ref. 1). The bacterium produces an endospore that allows it to endure extreme... pathogenic to humans, animals, or plants (Ref. 2). Several strains of Bacillus subtilis are used... agricultural commodities resulting from its use in the treatment of seeds used for growing agricultural crops...

  18. Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in "Bacillus Subtilis"

    ERIC Educational Resources Information Center

    Bredeston, L. M.; Marciano, D.; Albanesi, D.; De Mendoza, D.; Delfino, J. M.

    2011-01-01

    This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of "Bacillus subtilis", composed of a [delta]5-desaturase (encoded by the "des" gene) and the canonical…

  19. Bacillus subtilis as a tool for screening soil metagenomic libraries for antimicrobial activities.

    PubMed

    Biver, Sophie; Steels, Sébastien; Portetelle, Daniel; Vandenbol, Micheline

    2013-06-28

    Finding new antimicrobial activities by functional metagenomics has been shown to depend on the heterologous host used to express the foreign DNA. Therefore, efforts are devoted to developing new tools for constructing metagenomic libraries in shuttle vectors replicatable in phylogenetically distinct hosts. Here we evaluated the use of the Escherichia coli-Bacillus subtilis shuttle vector pHT01 to construct a forest-soil metagenomic library. This library was screened in both hosts for antimicrobial activities against four opportunistic bacteria: Proteus vulgaris, Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus. A new antibacterial activity against B. cereus was found upon screening in B. subtilis. The new antimicrobial agent, sensitive to proteinase K, was not active when the corresponding DNA fragment was expressed in E. coli. Our results validate the use of pHT01 as a shuttle vector and B. subtilis as a host to isolate new activities by functional metagenomics.

  20. Thermoinducible transcription system for Bacillus subtilis that utilizes control elements from temperate phage phi 105.

    PubMed

    Osburne, M S; Craig, R J; Rothstein, D M

    1985-09-01

    We describe a thermoinducible-expression system for Bacillus subtilis which utilized an early promoter-operator sequence from temperate phage phi 105 and the thermolabile prophage repressor from the phage variant phi 105 cts23. The system operated at the transcriptional level to control expression in B. subtilis of the cat-86 gene derived from Bacillus pumilis. Details of the strategies used to isolate the early phage promoter are described. This promoter lay in close proximity to the prophage repressor gene on the phi 105 genome. The sequence of the early promoter differed from that of the vegetative B. subtilis consensus promoter by 1 base pair in both the -10 and -35 regions. We also present evidence that our phage-derived expression system could function in Escherichia coli to effect thermoinducible expression of the galK gene.

  1. Recent progress in Bacillus subtilis spore-surface display: concept, progress, and future.

    PubMed

    Wang, He; Wang, Yunxiang; Yang, Ruijin

    2017-02-01

    With the increased knowledge on spore structure and advances in biotechnology engineering, the newly developed spore-surface display system confers several inherent advantages over other microbial cell-surface display systems including enhanced stability and high safety. Bacillus subtilis is the most commonly used Bacillus species for spore-surface display. The expression of heterologous antigen or protein on the surface of B. subtilis spores has now been practiced for over a decade with noteworthy success. As an update and supplement to other previous reviews, we comprehensively summarize recent studies in the B. subtilis spore-surface display technique. We focus on its benefits as well as the critical factors affecting its display efficiency and offer suggestions for the future success of this field.

  2. Suitability of different β-galactosidases as reporter enzymes in Bacillus subtilis.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2012-01-01

    The suitability of three β-galactosidases as reporter enzymes for promoter expression analyses was investigated in Bacillus subtilis with respect to various temperature conditions during cultivation and assay procedures. Starting from the hypothesis that proteins derived from diverse habitats have different advantages as reporters at different growth temperatures, the beta-galactosidases from the thermophilic organism Bacillus stearothermophilus, from the mesophilic bacterium Escherichia coli and from the psychrophilic organism Pseudoalteromonas haloplanktis TAE79 were analysed under control of the constitutive B. subtilis lepA promoter. Subsequent expression of the β-galactosidase genes and determination of specific activities was performed at different cultivation and assay temperatures using B. subtilis as host. Surprisingly, the obtained results demonstrated that the highest activities over a broad cultivation temperature range were obtained using the β-galactosidase from the mesophilic bacterium E. coli whereas the enzymes from the thermophilic and psychrophilic bacteria revealed a more restricted usability in terms of cultivation temperature.

  3. Characterization of Bacillus subtilis strains in Thua nao, a traditional fermented soybean food in northern Thailand.

    PubMed

    Inatsu, Y; Nakamura, N; Yuriko, Y; Fushimi, T; Watanasiritum, L; Kawamoto, S

    2006-09-01

    To clarify the diversity of Bacillus subtilis strains in Thua nao that produce high concentrations of products useful in food manufacturing and in health-promoting compounds. Production of amylase, protease, subtilisin NAT (nattokinase), and gamma-polyglutamic acid (PGA) by the Bacillus subtilis strains in Thua nao was measured. Productivity of protease NAT by these strains tended to be higher than by Japanese commercial natto-producing strains. Molecular diversity of isolated strains was analysed via randomly amplified polymorphic DNA-PCR fingerprinting. The strains were divided into 19 types, including a type with the same pattern as a Japanese natto-producing strain. B. subtilis strains that could be a resource for effective production of protease, amylase, subtilisin NAT, or PGA were evident in Thua nao produced in various regions in northern Thailand. This study clearly demonstrated the value of Thua nao as a potential resource of food-processing enzymes and health-promoting compounds.

  4. Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production ▿

    PubMed Central

    Kubo, Yuji; Rooney, Alejandro P.; Tsukakoshi, Yoshiki; Nakagawa, Rikio; Hasegawa, Hiromasa; Kimura, Keitarou

    2011-01-01

    Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group. PMID:21764950

  5. Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

    PubMed

    Kubo, Yuji; Rooney, Alejandro P; Tsukakoshi, Yoshiki; Nakagawa, Rikio; Hasegawa, Hiromasa; Kimura, Keitarou

    2011-09-01

    Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.

  6. Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data.

    PubMed

    Nishito, Yukari; Osana, Yasunori; Hachiya, Tsuyoshi; Popendorf, Kris; Toyoda, Atsushi; Fujiyama, Asao; Itaya, Mitsuhiro; Sakakibara, Yasubumi

    2010-04-16

    Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length. We applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1. These are specific for gamma-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases. The determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B. subtilis 168 lacks

  7. Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data

    PubMed Central

    2010-01-01

    Background Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length. Results We applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1. These are specific for γ-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases. Conclusions The determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B

  8. Fed-Batch Biomolecule Production by Bacillus subtilis: A State of the Art Review.

    PubMed

    Öztürk, Sibel; Çalık, Pınar; Özdamar, Tunçer H

    2016-04-01

    Bacillus subtilis is a highly promising production system for various biomolecules. This review begins with the algorithm of fed-batch operations (FBOs) and then illustrates the approaches to design the initial production medium and/or feed stream. Additionally, the feeding strategies developed with or without feedback control for fed-batch B. subtilis fermentations were compiled with a special emphasis on recombinant protein (r-protein) production. For biomolecule production by wild-type B. subtilis, due to the different intracellular production patterns, no consensus exists on the FBO strategy that gives the maximum productivity, whereas for r-protein production appropriate feeding strategies vary depending on the promoter used. Thus, we conclude that the B. subtilis community is still seeking an approved strong promoter and generalized FBO strategies.

  9. Involvement of SpoVG in hemolysis caused by Bacillus subtilis.

    PubMed

    Pan, Xingliang; Chen, Xiuzhen; Su, Xiaoyun; Feng, Yuan; Tao, Yong; Dong, Zhiyang

    2014-01-17

    Bacillus subtilis is a facultative anaerobic Gram-positive non-pathogenic bacterium that includes members displaying hemolytic activity. To identify the genes responsible for hemolysis, a random mariner-based transposon insertion mutant library of B. subtilis 168 was constructed. More than 20,000 colonies were screened for the hypohemolytic phenotype on blood agar plates. One mutant showed significantly less pronounced hemolytic phenotype than the wild type. DNA sequencing and Southern blot analysis showed this mutant has a single transposable element inserted into the open reading frame (ORF) of the spoVG gene; complementation of the spoVG-disrupted mutant with a wild-type copy restored its hemolytic phenotype. It was therefore concluded that the spoVG gene, which plays a role in regulating asymmetric septation during sporulation in B. subtilis, is involved in hemolysis by B. subtilis.

  10. A simple method to isolate biofilm-forming Bacillus subtilis and related species from plant roots.

    PubMed

    Fall, Ray; Kinsinger, Rebecca F; Wheeler, Kevin A

    2004-05-01

    A novel method was developed to isolate pure cultures of wild-type Bacillus subtilis and related species from plant roots, even roots washed free of adhering soil. The method uses casein digest-mannitol agarose (CM) media that promote rapid dendritic growth (low K+ ion) or profuse surface film formation (high K+ ion) of Bacillus species at 40 degrees C. Inoculation from the tips of surface growth on agarose leads to self-purification and streaking on CM agar plates (hard agar and high K+) leads to characteristic colony morphology. Phenotypic and 16S rDNA analysis revealed that most root isolates obtained by this method are spore-forming Bacillus species, with enrichment for B. subtilis and its close relatives. Of particular interest is the finding that the majority of these Bacillus isolates and the B. subtilis Marburg strain also form adhering biofilms on inert surfaces. Thus the methods presented may be useful in isolation of biofilm-forming Bacillus and investigation of their role on plant roots.

  11. Effect of Feeding Bacillus subtilis natto on Hindgut Fermentation and Microbiota of Holstein Dairy Cows.

    PubMed

    Song, D J; Kang, H Y; Wang, J Q; Peng, H; Bu, D P

    2014-04-01

    The effect of Bacillus subtilis natto on hindgut fermentation and microbiota of early lactation Holstein dairy cows was investigated in this study. Thirty-six Holstein dairy cows in early lactation were randomly allocated to three groups: no B. subtilis natto as the control group, B. subtilis natto with 0.5×10(11) cfu as DMF1 group and B. subtilis natto with 1.0×10(11) cfu as DMF2 group. After 14 days of adaptation period, the formal experiment was started and lasted for 63 days. Fecal samples were collected directly from the rectum of each animal on the morning at the end of eighth week and placed into sterile plastic bags. The pH, NH3-N and VFA concentration were determined and fecal bacteria DNA was extracted and analyzed by DGGE. The results showed that the addition of B. subtilus natto at either treatment level resulted in a decrease in fecal NH3-N concentration but had no effect on fecal pH and VFA. The DGGE profile revealed that B. subtilis natto affected the population of fecal bacteria. The diversity index of Shannon-Wiener in DFM1 decreased significantly compared to the control. Fecal Alistipes sp., Clostridium sp., Roseospira sp., beta proteobacterium were decreased and Bifidobacterium was increased after supplementing with B. subtilis natto. This study demonstrated that B. subtilis natto had a tendency to change fecal microbiota balance.

  12. Effect of Bacillus subtilis Natto on Meat Quality and Skatole Content in TOPIGS Pigs.

    PubMed

    Sheng, Q K; Zhou, K F; Hu, H M; Zhao, H B; Zhang, Y; Ying, W

    2016-05-01

    This study investigated the effect of Bacillus subtilis (B. subtilis) natto on meat quality and skatole in TOPIGS pigs. Sixty TOPIGS pigs were randomly assigned to 3 groups (including 5 pens per group, with 4 pigs in each pen) and fed with basic diet (control group), basic diet plus 0.1% B. subtilis natto (B group), and basic diet plus 0.1% B. subtilis natto plus 0.1% B. coagulans (BB group), respectively. All pigs were sacrificed at 100 kg. Growth performance, meat quality, serum parameters and oxidation status in the three groups were assessed and compared. Most parameters regarding growth performance and meat quality were not significantly different among the three groups. However, compared with the control group, meat pH24, fat and feces skatole and the content of Escherichia coli (E. Coli), Clostridium, NH3-N were significantly reduced in the B and BB groups, while serum total cholesterol, high density lipoprotein, the levels of liver P450, CYP2A6, and CYP2E1, total antioxidant capability (T-AOC) and glutathione peroxidase and Lactobacilli in feces were significantly increased in the B and BB groups. Further, the combined supplementation of B. subtilis natto and B. coagulans showed more significant effects on the parameters above compared with B. subtilis, and Clostridium, and NH3-N. Our results indicate that the supplementation of pig feed with B. subtilis natto significantly improves meat quality and flavor, while its combination with B. coagulans enhanced these effects.

  13. Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin.

    PubMed

    Hosoi, T; Ametani, A; Kiuchi, K; Kaminogawa, S

    2000-10-01

    In an effort to demonstrate the potential usefulness of Bacillus subtilis (natto) as a probiotic, we examined the effect of this organism on the growth of three strains of lactobacilli co-cultured aerobically in vitro. Addition of B. subtilis (natto) to the culture medium resulted in an increase in the number of viable cells of all lactobacilli tested. Since B. subtilis (natto) can produce catalase, which has been reported to exhibit a similar growth-promoting effect on lactobacilli, we also examined the effect of bovine catalase on the growth of Lactobacillus reuteri JCM 1112 and L. acidophilus JCM 1132. Both catalase and B. subtilis (natto) enhanced the growth of L. reuteri JCM 1112, whereas B. subtilis (natto) but not catalase enhanced the growth of L. acidophilus JCM 1132. In a medium containing 0.1 mM hydrogen peroxide, its toxic effect on L. reuteri JCM 1112 was abolished by catalase or B. subtilis (natto). In addition, a serine protease from B. licheniformis, subtilisin, improved the growth and viability of L. reuteri JCM 1112 and L. acidophilus JCM 1132 in the absence of hydrogen peroxide. These results indicate that B. subtilis (natto) enhances the growth and (or) viability of lactobacilli, possibly through production of catalase and subtilisin.

  14. Investigating the efficacy of Bacillus subtilis SM21 on controlling Rhizopus rot in peach fruit.

    PubMed

    Wang, Xiaoli; Wang, Jing; Jin, Peng; Zheng, Yonghua

    2013-06-17

    The efficacy of Bacillus subtilis SM21 on controlling Rhizopus rot caused by Rhizopus stolonifer in postharvest peach fruit and the possible mechanisms were investigated. The results indicated B. subtilis SM21 treatment reduced lesion diameter and disease incidence by 37.2% and 26.7% on the 2nd day of inoculation compared with the control. The in vitro test showed significant inhibitory effect of B. subtilis SM21 on mycelial growth of R. stolonifer with an inhibition rate of 48.9%. B. subtilis SM21 treatment significantly enhanced activities of chitinase and β-1,3-glucanase, and promoted accumulation of H2O2. Total phenolic content and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity were also increased by this treatment. Transcription of seven defense related genes was much stronger in fruit treated with B. subtilis SM21 or those both treated with B. subtilis SM21 and inoculated with R. stolonifer compared with fruit inoculated with R. stolonifer alone. These results suggest that B. subtilis SM21 can effectively inhibit Rhizopus rot caused by R. stolonifer in postharvest peach fruit, possibly by directly inhibiting growth of the pathogen, and indirectly inducing disease resistance in the fruit.

  15. Bacillus subtilis Early Colonization of Arabidopsis thaliana Roots Involves Multiple Chemotaxis Receptors

    PubMed Central

    Allard-Massicotte, Rosalie; Tessier, Laurence; Lécuyer, Frédéric; Lakshmanan, Venkatachalam; Lucier, Jean-François; Garneau, Daniel; Caudwell, Larissa; Vlamakis, Hera; Bais, Harsh P.

    2016-01-01

    ABSTRACT Colonization of plant roots by Bacillus subtilis is mutually beneficial to plants and bacteria. Plants can secrete up to 30% of their fixed carbon via root exudates, thereby feeding the bacteria, and in return the associated B. subtilis bacteria provide the plant with many growth-promoting traits. Formation of a biofilm on the root by matrix-producing B. subtilis is a well-established requirement for long-term colonization. However, we observed that cells start forming a biofilm only several hours after motile cells first settle on the plant. We also found that intact chemotaxis machinery is required for early root colonization by B. subtilis and for plant protection. Arabidopsis thaliana root exudates attract B. subtilis in vitro, an activity mediated by the two characterized chemoreceptors, McpB and McpC, as well as by the orphan receptor TlpC. Nonetheless, bacteria lacking these chemoreceptors are still able to colonize the root, suggesting that other chemoreceptors might also play a role in this process. These observations suggest that A. thaliana actively recruits B. subtilis through root-secreted molecules, and our results stress the important roles of B. subtilis chemoreceptors for efficient colonization of plants in natural environments. These results demonstrate a remarkable strategy adapted by beneficial rhizobacteria to utilize carbon-rich root exudates, which may facilitate rhizobacterial colonization and a mutualistic association with the host. PMID:27899502

  16. Effect of Feeding Bacillus subtilis natto on Hindgut Fermentation and Microbiota of Holstein Dairy Cows

    PubMed Central

    Song, D. J.; Kang, H. Y.; Wang, J. Q.; Peng, H.; Bu, D. P.

    2014-01-01

    The effect of Bacillus subtilis natto on hindgut fermentation and microbiota of early lactation Holstein dairy cows was investigated in this study. Thirty-six Holstein dairy cows in early lactation were randomly allocated to three groups: no B. subtilis natto as the control group, B. subtilis natto with 0.5×1011 cfu as DMF1 group and B. subtilis natto with 1.0×1011 cfu as DMF2 group. After 14 days of adaptation period, the formal experiment was started and lasted for 63 days. Fecal samples were collected directly from the rectum of each animal on the morning at the end of eighth week and placed into sterile plastic bags. The pH, NH3-N and VFA concentration were determined and fecal bacteria DNA was extracted and analyzed by DGGE. The results showed that the addition of B. subtilus natto at either treatment level resulted in a decrease in fecal NH3-N concentration but had no effect on fecal pH and VFA. The DGGE profile revealed that B. subtilis natto affected the population of fecal bacteria. The diversity index of Shannon-Wiener in DFM1 decreased significantly compared to the control. Fecal Alistipes sp., Clostridium sp., Roseospira sp., beta proteobacterium were decreased and Bifidobacterium was increased after supplementing with B. subtilis natto. This study demonstrated that B. subtilis natto had a tendency to change fecal microbiota balance. PMID:25049979

  17. Effect of Bacillus subtilis Natto on Meat Quality and Skatole Content in TOPIGS Pigs

    PubMed Central

    Sheng, Q. K.; Zhou, K. F.; Hu, H. M.; Zhao, H. B.; Zhang, Y.; Ying, W.

    2016-01-01

    This study investigated the effect of Bacillus subtilis (B. subtilis) natto on meat quality and skatole in TOPIGS pigs. Sixty TOPIGS pigs were randomly assigned to 3 groups (including 5 pens per group, with 4 pigs in each pen) and fed with basic diet (control group), basic diet plus 0.1% B. subtilis natto (B group), and basic diet plus 0.1% B. subtilis natto plus 0.1% B. coagulans (BB group), respectively. All pigs were sacrificed at 100 kg. Growth performance, meat quality, serum parameters and oxidation status in the three groups were assessed and compared. Most parameters regarding growth performance and meat quality were not significantly different among the three groups. However, compared with the control group, meat pH24, fat and feces skatole and the content of Escherichia coli (E. Coli), Clostridium, NH3-N were significantly reduced in the B and BB groups, while serum total cholesterol, high density lipoprotein, the levels of liver P450, CYP2A6, and CYP2E1, total antioxidant capability (T-AOC) and glutathione peroxidase and Lactobacilli in feces were significantly increased in the B and BB groups. Further, the combined supplementation of B. subtilis natto and B. coagulans showed more significant effects on the parameters above compared with B. subtilis, and Clostridium, and NH3-N. Our results indicate that the supplementation of pig feed with B. subtilis natto significantly improves meat quality and flavor, while its combination with B. coagulans enhanced these effects. PMID:26954164

  18. Production of Enzymes from Agroindustrial Wastes by Biosurfactant-Producing Strains of Bacillus subtilis

    PubMed Central

    Barros, Francisco Fábio Cavalcante; Simiqueli, Ana Paula Resende; de Andrade, Cristiano José; Pastore, Gláucia Maria

    2013-01-01

    Bacteria in the genus Bacillus are the source of several enzymes of current industrial interest. Hydrolases, such as amylases, proteases, and lipases, are the main enzymes consumed worldwide and have applications in a wide range of products and industrial processes. Fermentation processes by Bacillus subtilis using cassava wastewater as a substrate are reported in the technical literature; however, the same combination of microorganisms and this culture medium is limited or nonexistent. In this paper, the amylase, protease, and lipase production of ten Bacillus subtilis strains previously identified as biosurfactant producers in cassava wastewater was evaluated. The LB1a and LB5a strains were selected for analysis using a synthetic medium and cassava wastewater and were identified as good enzyme producers, especially of amylases and proteases. In addition, the enzymatic activity results indicate that cassava wastewater was better than the synthetic medium for the induction of these enzymes. PMID:23533780

  19. Effects of Electrolyzed Oxidizing Water on Inactivation of Bacillus subtilis and Bacillus cereus Spores in Suspension and on Carriers.

    PubMed

    Zhang, Chunling; Li, Baoming; Jadeja, Ravirajsinh; Hung, Yen-Con

    2016-01-01

    Spores of some Bacillus species are responsible for food spoilage and foodborne disease. These spores are highly resistant to various interventions and cooking processes. In this study, the sporicidal efficacy of acidic electrolyzed oxidizing (EO) water (AEW) and slightly acidic EO water (SAEW) with available chlorine concentration (ACC) of 40, 60, 80, 100, and 120 mg/L and treatment time for 1, 2, 3, 4, 5, and 6 min were tested on Bacillus subtilis and Bacillus cereus spores in suspension and on carrier with or without organics. The reduction of spore significantly increased with increasing ACC and treatment time (P < 0.05). Nondetectable level of B. cereus spore in suspension occurred within 2 min after exposure to both EO waters containing 120 mg/L ACC, while only SAEW at 120 mg/L and 2 min treatment achieved >6 log reductions of B. subtilis spore. Both types of EO water with ACC of 60 mg/L and 6 min treatment achieved a reduction of B. subtilis and B. cereus spores to nondetectable level. EO water with ACC of 80 mg/L and treatment time of 3 min on carrier test without organics addition resulted in reductions of B. subtilis spore to nondetectable level. But, addition of 0.3% organics on carrier decreased the inactivation effect of EO water. This study indicated that EO water was highly effective in inactivation of B. subtilis and B. cereus spores in suspension or on carrier, and therefore, rendered it as a promising disinfectant to be applied in food industry.

  20. Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis.

    PubMed Central

    O'Kane, C; Stephens, M A; McConnell, D

    1986-01-01

    An integrable plasmid, pOK4, which replicated independently in Escherichia coli was constructed for generating transcriptional fusions in vivo in Bacillus DNA. It did not replicate independently in Bacillus subtilis, but it could be made to integrate into the chromosome of B. subtilis if sequences homologous to chromosomal sequences were inserted into it. It had a selectable marker for chloramphenicol resistance and carried unique sites for EcoRI and SmaI just to the 5' side of a promoterless alpha-amylase gene from Bacillus licheniformis. When B. subtilis DNA fragments were ligated into one of these sites and the ligation mixture was used to transform an alpha-amylase-negative B. subtilis strain, chloramphenicol-resistant transformants could be isolated conveniently. Many of these were alpha-amylase positive, owing to the fusion of the plasmid amylase gene to chromosomal operons. In principle, because integration need not be mutagenic, it is possible to obtain fusions to any chromosomal operon. The site of each integration can be mapped, and the flanking sequences can be cloned into E. coli. The alpha-amylase gene can be used to detect regulated genes. We used it as an indicator to detect operons which are DNA-damage-inducible (din), and we identified insertions in both SP beta and PBSX prophages. Images PMID:3096966

  1. Bacillus subtilis based-formulation for the control of postbloom fruit drop of citrus.

    PubMed

    Klein, Mariana Nadjara; da Silva, Aline Caroline; Kupper, Katia Cristina

    2016-12-01

    Postbloom fruit drop (PFD) caused by Colletotrichum acutatum affects flowers and causes early fruit drop in all commercial varieties of citrus. Biological control with the isolate ACB-69 of Bacillus subtilis has been considered as a potential method for controlling this disease. This study aimed to develop and optimize a B. subtilis based-formulation with a potential for large-scale applications and evaluate its effect on C. acutatum in vitro and in vivo. Bacillus subtilis based-formulations were developed using different carrier materials, and their ability to control PFD was evaluated. The results of the assays led to the selection of the B. subtilis based-formulation with talc + urea (0.02 %) and talc + ammonium molybdate (1 mM), which inhibited mycelial growth and germination of C. acutatum. Studies with detached citrus flowers showed that the formulations were effective in controlling the pathogen. In field conditions, talc + urea (0.02 %) provided 73 % asymptomatic citrus flowers and 56 % of the average number of effective fruit (ANEF), equating with fungicide treatment. On the contrary, non-treated trees had 8.8 % of asymptomatic citrus flowers and 0.83 % ANEF. The results suggest that B. subtilis based-formulations with talc as the carrier supplemented with a nitrogen source had a high potential for PFD control.

  2. Self-cloning significantly enhances the production of catalase in Bacillus subtilis WSHDZ-01.

    PubMed

    Xu, Sha; Guo, Yaqiong; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-08-01

    The katA gene that encodes catalase (CAT) in Bacillus subtilis WSHDZ-01 was overexpressed in B. subtilis WB600 and B. subtilis WSHDZ-01. The CAT yield in both transformed strains was significantly improved compared to that in the wild-type WSHDZ-01 in shake flask culture. When cultured in a 3-L stirred tank reactor (STR), the recombinant CAT activity in B. subtilis WSHDZ-01 could be improved by 419 %, reaching up to 39,117 U/mL and was 8,149.4 U/mg dry cell weight, which is the highest activity reported in Bacillus sp. However, the recombinant CAT in B. subtilis WB600 cultured in a 3-L STR was not significantly improved by any of the common means for process optimization, and the highest CAT activity was 3,673.5 U/mg dry cell weight. The results suggest that self-cloning of the complete expression cassette in the original strain is a reasonable strategy to improve the yield of wild-type enzymes.

  3. Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection.

    PubMed

    Thirabunyanon, Mongkol; Thongwittaya, Narin

    2012-08-01

    The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, including Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Vibrio cholerae. Beneficial probiotic criteria from these isolates - which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells - exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate was Bacillus subtilis. This B. subtilis NC11 was sensitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction of S. Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium from B. subtilis NC11 was observed by scanning electron microscopy. B. subtilis NC11 cells, as well as the bacterial cultured medium or the cultured medium adjusted to pH 7, significantly inhibited S. Enteritidis invasion (P<0.01) of intestinal epithelial cells. This study indicates that B. subtilis NC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity against S. Enteritidis infection to intestinal epithelial cells.

  4. Bacillus subtilis Early Colonization of Arabidopsis thaliana Roots Involves Multiple Chemotaxis Receptors.

    PubMed

    Allard-Massicotte, Rosalie; Tessier, Laurence; Lécuyer, Frédéric; Lakshmanan, Venkatachalam; Lucier, Jean-François; Garneau, Daniel; Caudwell, Larissa; Vlamakis, Hera; Bais, Harsh P; Beauregard, Pascale B

    2016-11-29

    Colonization of plant roots by Bacillus subtilis is mutually beneficial to plants and bacteria. Plants can secrete up to 30% of their fixed carbon via root exudates, thereby feeding the bacteria, and in return the associated B. subtilis bacteria provide the plant with many growth-promoting traits. Formation of a biofilm on the root by matrix-producing B. subtilis is a well-established requirement for long-term colonization. However, we observed that cells start forming a biofilm only several hours after motile cells first settle on the plant. We also found that intact chemotaxis machinery is required for early root colonization by B. subtilis and for plant protection. Arabidopsis thaliana root exudates attract B. subtilis in vitro, an activity mediated by the two characterized chemoreceptors, McpB and McpC, as well as by the orphan receptor TlpC. Nonetheless, bacteria lacking these chemoreceptors are still able to colonize the root, suggesting that other chemoreceptors might also play a role in this process. These observations suggest that A. thaliana actively recruits B. subtilis through root-secreted molecules, and our results stress the important roles of B. subtilis chemoreceptors for efficient colonization of plants in natural environments. These results demonstrate a remarkable strategy adapted by beneficial rhizobacteria to utilize carbon-rich root exudates, which may facilitate rhizobacterial colonization and a mutualistic association with the host. Bacillus subtilis is a plant growth-promoting rhizobacterium that establishes robust interactions with roots. Many studies have now demonstrated that biofilm formation is required for long-term colonization. However, we observed that motile B. subtilis mediates the first contact with the roots. These cells differentiate into biofilm-producing cells only several hours after the bacteria first contact the root. Our study reveals that intact chemotaxis machinery is required for the bacteria to reach the

  5. Effects of Bacillus subtilis on Epithelial Tight Junctions of Mice with Inflammatory Bowel Disease.

    PubMed

    Gong, Yi; Li, Hui; Li, Yan

    2016-02-01

    Intestinal mucosal barrier dysfunction associated with inflammatory bowel disease (IBD). Effects of Bacillus subtilis on epithelial tight junctions (TJs) and intrinsic regulatory mechanisms of the intestine were studied in pursuit of better treatments for IBD. Fifty Balb/c mice given 5% dextran sulfate sodium (DSS) in tap water ad libitum over a 7-day period (to induce colitis) were randomly assigned to 4 test groups [DSS, DSS+B. subtilis, DSS+5 amino salicylic acid (5ASA), and DSS+B. subtilis+5ASA] to compare with normal controls. In the test groups DSS was administered daily by oral gavage in normal saline (0.2 mL), adding B. subtilis (1 × 10(8) CFU), 5ASA (6 mg), or both for respective test groups. Defecation, body weight, colitis score, pathological features, epithelial TJs proteins [claudin-1, occludin, junctional adhesion molecule (JAM)-A, and zona occludens (ZO)-1], and various cytokines [interleukin (IL)-6, IL-17, IL-23, and tissue necrosis factor (TNF)-α] were evaluated. Relative to the DSS group, disease activity index scores, and graded histologic damage were all significantly reduced by B. subtilis intake. All parameters declined even further when B. subtilis and 5ASA were combined. Analytic testing (immunohistochemical, western blot, and PCR) revealed progressive increase in TJ protein (claudin-1, occludin, JAM-A, and ZO-1) expression in DSS, DSS+B. subtilis, DSS+5ASA, DSS+B. subtilis+5ASA, and normal control groups (P < 0.05), whereas cytokine (IL-6, IL-17, IL-23, and TNF-α) expression similarly declined (P < 0.05). B. subtilis intake upregulated expression of TJ proteins (claudin-1, occludin, JAM-A, and ZO-1), for improved barrier function, and downregulated cytokine expression (IL-6, IL-17, IL-23, and TNF-α) to reduce intestinal epithelial damage.

  6. Comparison of plant growth-promotion with Pseudomonas aeruginosa and Bacillus subtilis in three vegetables

    PubMed Central

    Adesemoye, A.O.; Obini, M.; Ugoji, E.O.

    2008-01-01

    Our objective was to compare some plant growth promoting rhizobacteria (PGPR) properties of Bacillus subtilis and Pseudomonas aeruginosa as representatives of their two genera. Solanum lycopersicum L. (tomato), Abelmoschus esculentus (okra), and Amaranthus sp. (African spinach) were inoculated with the bacterial cultures. At 60 days after planting, dry biomass for plants treated with B. subtilis and P. aeruginosa increased 31% for tomato, 36% and 29% for okra, and 83% and 40% for African spinach respectively over the non-bacterized control. Considering all the parameters tested, there were similarities but no significant difference at P < 0.05 between the overall performances of the two organisms. PMID:24031240

  7. Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

    PubMed Central

    Hoffmann, Tamara; Frankenberg, Nicole; Marino, Marco; Jahn, Dieter

    1998-01-01

    During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation. PMID:9422613

  8. The purification of a novel amylase from Bacillus subtilis and its inhibition by wheat proteins.

    PubMed Central

    Orlando, A R; Ade, P; Di Maggio, D; Fanelli, C; Vittozzi, L

    1983-01-01

    A new alpha-amylase (EC 3.2.1.1) from Bacillus subtilis was purified by affinity chromatography. The molecular weight of the purified enzyme, estimated from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 93000, which is very different from the molecular weights of two well-characterized amylases from B. subtilis. Electrofocusing showed an isoelectric point of 5. Amylase shows a broad maximum of activity between pH 6 and 7; maximal inhibition of enzyme by wheat-protein alpha-amylase inhibitors is displayed at pH 7. Images Fig. 1. PMID:6189482

  9. Mapping of a genetic locus that affects glycerol 3-phosphate transport in Bacillus subtilis.

    PubMed Central

    Lindgren, V

    1978-01-01

    Two types of fosfomycin-resistant mutants of Bacillus subtilis were isolated. Mutants of the first type (GlpT mutants) were resistant to at least 200 microgram of fosfomycin per ml and failed to take up exogenous glycerol 3-phosphate. Mutants of the second type were resistant to lower concentrations of fosfomycin and transported glycerol-3-phosphate as efficiently as wild-type bacteria. The glpT mutations, but not the mutations in the second type of fosfomycin-resistant mutants, map in the cysA-aroI region of the B. subtilis chromosome. PMID:415047

  10. Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity

    NASA Technical Reports Server (NTRS)

    Kacena, M. A.; Smith, E. E.; Todd, P.

    1999-01-01

    The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

  11. Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity

    NASA Technical Reports Server (NTRS)

    Kacena, M. A.; Smith, E. E.; Todd, P.

    1999-01-01

    The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

  12. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... FZB24 in or on all agricultural commodities when applied/used in accordance with label directions....

  13. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... FZB24 in or on all agricultural commodities when applied/used in accordance with label directions. ...

  14. Draft Genome Sequences of 10 Bacillus subtilis Strains That Form Spores with High or Low Heat Resistance

    PubMed Central

    Berendsen, Erwin M.; Wells-Bennik, Marjon H. J.; Krawczyk, Antonina O.; de Jong, Anne; van Heel, Auke; Eijlander, Robyn T.

    2016-01-01

    Here, we report the draft genome sequences of 10 isolates of Bacillus subtilis, a spore forming Gram-positive bacterium. The strains were selected from food products and produced spores with either high or low heat resistance. PMID:26988043

  15. Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans.

    PubMed

    Lefevre, Marie; Racedo, Silvia M; Denayrolles, Muriel; Ripert, Gabrielle; Desfougères, Thomas; Lobach, Alexandra R; Simon, Ryan; Pélerin, Fanny; Jüsten, Peter; Urdaci, Maria C

    2017-02-01

    Bacillus subtilis CU1 is a recently described probiotic strain with beneficial effects on immune health in elderly subjects. The following work describes a series of studies supporting the safety of the strain for use as an ingredient in food and supplement preparations. Using a combination of 16S rDNA and gyrB nucleotide analyses, the species was identified as a member of the Bacillus subtilis complex (B. subtilis subsp. spizizenii). Further characterization of the organism at the strain level was achieved using random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) and pulsed field gel electrophoresis (PFGE) analyses. B. subtilis CU1 did not demonstrate antibiotic resistance greater than existing regulatory cutoffs against clinically important antibiotics, did not induce hemolysis or produce surfactant factors, and was absent of toxigenic activity in vitro. Use of B. subtilis CU1 as a probiotic has recently been evaluated in a 16-week randomized, double-blind, placebo-controlled, parallel-arm study, in which 2 × 10(9) spores per day of B. subtilis CU1 were administered for a total 40 days to healthy elderly subjects (4 consumption periods of 10 days separated by 18-day washouts). This work describes safety related endpoints not previously reported. B. subtilis CU1 was safe and well-tolerated in the clinical subjects without undesirable physiological effects on markers of liver and kidney function, complete blood counts, hemodynamic parameters, and vital signs.

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

    PubMed

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

    2016-05-01

    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 < 0.01), MCP production (P < 0.01), and tended to elevate total VFA (P = 0.07), but decreased the ratio of acetate and propionate (P < 0.01). Autoclaved Bacillus subtilis natto has the similar function with the live bacteria except for the ratio of acetate and propionate. Except B. fibrisolvens, live or autoclaved Bacillus subtilis natto did not influence or decreased the 16S rRNA gene quantification of the detected bacteria. BSC and BSM altered the relative expression of certain functional bacteria in the rumen. These results indicated that it was Bacillus subtilis natto thalli that played the important role in promoting rumen fermentation when applied as a probiotic in dairy ration.

  17. Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages.

    PubMed

    Xu, Xin; Huang, Qin; Mao, Yulong; Cui, Zhiwen; Li, Yali; Huang, Yi; Rajput, Imran Rashid; Yu, Dongyou; Li, Weifen

    2012-12-01

    To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin [IL]-1 beta, IL-6, IL-12, IL-10 and macrophage inflammatory protein-2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up-regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages. © 2012 The Societies and Wiley Publishing Asia Pty Ltd.

  18. Viral mutation affecting bacteriophage phi 1 development in Bacillus subtilis 168.

    PubMed Central

    Rettenmier, C W; Hemphill, H E

    1975-01-01

    Bacillus subtilis bacteriophage phi 1m, a host-range variant, was isolated after mutagenesis of virulent bacteriophage phi 1. Unlike its wild-type antecedent, phi 1m could not form plaques on lawns of B subtilis 168 at 37 C, although it adsorbed to, penetrated, and killed this bacterium. Experiments conducted in liquid medium at 37 C showed that B. subtilis 168 cells allowed reduced levels of phi 1m development at low multiplicities of infection, whereas high multiplicity infections of this strain by the phage were abortive. Certain mutants, derived originally from B. subtilis 168, were observed to be permissive for phi 1m at 37 C; moreover, their permissive phenotype could be duplicated by growing wild-type B. subtilis 168 cells at temperatures above 47 C. Studies on phi 1m and host nucleic acid synthesis under nonpermissive conditions demonstrated that transciption and DNA synthesis proceeded up to 20 min after infection, after which time there was a cessation of all nucleic acid production. These observations are discussed with respect to other abortive bacteriophage infections in B. subtilis. PMID:804042

  19. Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes.

    PubMed Central

    Morbidoni, H R; de Mendoza, D; Cronan, J E

    1996-01-01

    A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome. PMID:8759840

  20. Microbial flora, probiotics, Bacillus subtilis and the search for a long and healthy human longevity

    PubMed Central

    Ayala, Facundo Rodriguez; Bauman, Carlos; Cogliati, Sebastián; Leñini, Cecilia; Bartolini, Marco; Grau, Roberto

    2017-01-01

    Probiotics are live microorganisms that have beneficial effects on host health, including extended lifespan, when they are administered or present in adequate quantities. However, the mechanisms by which probiotics stimulate host longevity remain unclear and very poorly understood. In a recent study (Nat. Commun. 8, 14332 (2017) doi: 10.1038/ncomms14332), we used the spore-forming probiotic bacterium Bacillus subtilis and the model organism Caenorhabditis elegans to study the mechanism by which a probiotic bacterium affects host longevity. We found that biofilm-proficient B. subtilis colonized the C. elegans gut and extended the worm lifespan significantly longer than did biofilm-deficient isogenic strains. In addition to biofilm proficiency, the quorum-sensing pentapeptide CSF and nitric oxide (NO) represent the entire B. subtilis repertoire responsible for the extended longevity of C. elegans. B. subtilis grown under biofilm-supporting conditions synthesized higher levels of NO and CSF than under planktonic growth conditions, emphasizing the key role of the biofilm in slowing host aging. Significantly, the prolongevity effect of B. subtilis was primarily due to a downregulation of the insulin-like signaling system that precisely is a key partaker in the healthy longevity of human centenarians. These findings open the possibility to test if the regular consumption of B. subtilis incorporated in foods and beverages could significantly extend human life expectancy and contribute to stop the development of age-related diseases. PMID:28435840

  1. Genotypic and phenotypic differentiation of an antifungal biocontrol strain belonging to Bacillus subtilis.

    PubMed

    Marten, P; Smalla, K; Berg, G

    2000-09-01

    Physiological and molecular fingerprints of biotechnologically relevant rhizobacteria are necessary for registration, patenting, recognition and quality checking of the strains. To characterize the biological control agent, Bacillus subtilis B2g, the strain was compared with other plant-associated B. subtilis isolates. Phenotypic characterization included biochemical and nutritional properties, in vitro activity and analysis of potential antagonistic mechanisms towards several plant pathogenic fungi. According to the phenotypic characteristics, it was not possible to differentiate the biocontrol agent from the other strains, although the enzymatic fingerprint was unique. Genotypic diversity among the isolates was characterized by molecular fingerprinting methods using REP-PCR (repetitive extragenomic palindromic PCR), and macrorestriction of genomic DNA and electrophoretic separation of DNA fragments by pulsed-field gel electrophoresis (PFGE). A protocol for PFGE analysis using restriction enzyme SfiI for B. subtilis was developed. PFGE typing of B. subtilis B2g resulted in a unique fingerprint. Therefore, it was possible to differentiate B. subtilis B2g, the biocontrol agent of Phytovit, from other antifungal B. subtilis isolates.

  2. Draft Genome Sequence of Bacillus subtilis subsp. natto Strain CGMCC 2108, a High Producer of Poly-γ-Glutamic Acid

    PubMed Central

    Tan, Siyuan; Su, Anping; Zhang, Chen; Ren, Yuanyuan

    2016-01-01

    Here, we report the 4.1-Mb draft genome sequence of Bacillus subtilis subsp. natto strain CGMCC 2108, a high producer of poly-γ-glutamic acid (γ-PGA). This sequence will provide further help for the biosynthesis of γ-PGA and will greatly facilitate research efforts in metabolic engineering of B. subtilis subsp. natto strain CGMCC 2108. PMID:27231363

  3. Genomic analysis of Bacillus subtilis OH 131.1 and coculturing with Cryptococcus flavescens for control of fusarium head blight

    USDA-ARS?s Scientific Manuscript database

    Bacillus subtilis OH131.1 is a bacterial antagonist of Fusarium graminearum, a plant pathogen which causes Fusarium head blight in wheat. The genome of B. subtilis OH131.1 was sequenced, annotated and analyzed to understand its potential to produce bioactive metabolites. The analysis identified 6 sy...

  4. Architecture and Assembly of the Bacillus subtilis Spore Coat

    DTIC Science & Technology

    2014-09-26

    stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose...arrows). EM of ruthenium red stained B. subtilis spores demonstrated the presence of an outermost glycoprotein layer, and it was suggested that this layer...for the adherence and assembly of the coat, and while the peptidoglycan cortex forms relatively normally in spoVID spores, the coat largely assembles

  5. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis

    PubMed Central

    2013-01-01

    Background Standardized and well-characterized genetic building blocks are a prerequisite for the convenient and reproducible assembly of novel genetic modules and devices. While numerous standardized parts exist for Escherichia coli, such tools are still missing for the Gram-positive model organism Bacillus subtilis. The goal of this study was to develop and thoroughly evaluate such a genetic toolbox. Results We developed five BioBrick-compatible integrative B. subtilis vectors by deleting unnecessary parts and removing forbidden restriction sites to allow cloning in BioBrick (RFC10) standard. Three empty backbone vectors with compatible resistance markers and integration sites were generated, allowing the stable chromosomal integration and combination of up to three different devices in one strain. In addition, two integrative reporter vectors, based on the lacZ and luxABCDE cassettes, were BioBrick-adjusted, to enable β-galactosidase and luciferase reporter assays, respectively. Four constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of more than three orders of magnitude in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. Finally, the Bacillus BioBrick Box also provides five widely used epitope tags (FLAG, His10, cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice. Conclusion Our genetic toolbox contains three compatible empty integration vectors, two reporter vectors and a set of six promoters, two of them inducible. Furthermore, five different epitope tags offer convenient protein handling and detection. All parts adhere to the BioBrick standard and hence enable standardized work with B. subtilis. We believe that our well-documented and carefully evaluated Bacillus BioBrick Box represents a very useful genetic tool kit, not only for the iGEM competition but any other

  6. Synthesis of sn-glycerol 3-phosphate, a key precursor of membrane lipids, in Bacillus subtilis.

    PubMed Central

    Morbidoni, H R; de Mendoza, D; Cronan, J E

    1995-01-01

    The Bacillus subtilis gpsA gene was cloned by complementation of an Escherichia coli gpsA strain auxotrophic for sn-glycerol 3-phosphate. The gene was sequenced and found to encode an NAD(P)H-dependent dihydroxyacetone phosphate reductase with a deduced molecular mass of 39.5 kDa. The deduced amino acid sequence showed strong conservation with that of the E. coli homolog and to other procaryotic and eucaryotic dihydroxyacetone phosphate reductases. The physical location of gpsA on the B. subtilis chromosome was at about 200 degrees. Disruption of the chromosomal gpsA gene yielded B. subtilis strains auxotrophic for glycerol, indicating that the gpsA gene product is responsible for synthesis of the sn-glycerol 3-phosphate required for phospholipid synthesis. We also found that transformation of the classical B. subtilis glycerol auxotrophs with a gpsA-containing genomic fragment yielded transformants that grew in the absence of glycerol. In agreement with prior work, our attempts to determine the reductase activity in B. subtilis extracts were unsuccessful. However, expression of the B. subtilis gpsA gene in E. coli gave reductase activity that was only slightly inhibited by sn-glycerol 3-phosphate. Since the E. coli GpsA dihydroxyacetone phosphate reductase is very sensitive to allosteric inhibition by sn-glycerol 3-phosphate, these results indicate that the B. subtilis gpsA-encoded reductase differs from that of E. coli. It seems that B. subtilis regulates sn-glycerol 3-phosphate synthesis at the level of gene expression rather than through the E. coli mechanism of strong allosteric inhibition of an enzyme produced in excess. PMID:7592341

  7. Surface adhesion and confinement variation of Bacillus subtilis on SAM surfaces

    NASA Astrophysics Data System (ADS)

    Swiger, Lauren; Pasquale, Rose; Calabrese, Joseph; Senevirathne, Indrajith

    2012-02-01

    Controlled surface adhesion of non - pathogenic gram positive strain, Bacillus subtilis is interesting as a model system due to possible development of respective biosensors for prevention and detection of the pathogenic variants B. anthracis and B. cereus. Further as a study for bio-machine interfacing systems. Self Assembled Monolayers (SAM) with engineered surfaces of linear thiols on Au(111) were used as the substrate. Sub cultured B. subtilis were used for the analysis. The SAM layered surfaces were dipped in 2 -- 5 Log/ml B. subtilis solution. Subsequent surface adhesion at different bacterial dilutions on surfaces will be discussed, and correlated with quantitative and qualitative adhesion properties of bacteria on the engineered SAM surfaces. The bacteria adhered SAM surfaces were investigated using intermittent contact, noncontact, lateral force and contact modes of Atomic Force Microscopy (AFM).

  8. Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis

    PubMed Central

    Otto, Andreas; Bernhardt, Jörg; Meyer, Hanna; Schaffer, Marc; Herbst, Florian-A.; Siebourg, Juliane; Mäder, Ulrike; Lalk, Michael; Hecker, Michael; Becher, Dörte

    2010-01-01

    Functional genomics of the Gram-positive model organism Bacillus subtilis reveals valuable insights into basic concepts of cell physiology. In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation. For proteomic profiling, a combination of in vivo metabolic labelling and shotgun mass spectrometric analysis was carried out for five different proteomic subfractions (cytosolic, integral membrane, membrane, surface and extracellular proteome fraction), leading to the identification of ∼52% of the predicted proteome of B. subtilis. Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase. The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation. PMID:21266987

  9. Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats.

    PubMed

    Pinchuk, Irina V; Bressollier, Philippe; Sorokulova, Irina B; Verneuil, Bernard; Urdaci, Maria C

    2002-06-01

    One of the most interesting groups of phenolic compounds is comprised of the low molecular weight phenylpropanol derivative substances named isocoumarins, which possess important biological activities. In this study, the isocoumarin production and genetic diversity of 51 Bacillus strains isolated from different geographical and ecological niches were studied. Using molecular identification techniques, 47 strains were identified as B. subtilis, three as B. licheniformis and one as B. pumilus. When these strains were screened for isocumarin production, 11 belonging to the species B. subtilis produced amicoumacins, antibiotics of the isocoumarin group. RAPD analysis demonstrated that these strains fell into two groups which contained only these amicoumacin producers. No association was detected between RAPD profiles and the geographic origin or habitat of the strains tested. In conclusion, production of amicoumacin antibiotics by B. subtilis is a common characteristic of individual strains that presented genetic and physiological homogeneity.

  10. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion.

    PubMed

    Ling Lin Fu; Zi Rong Xu; Wei Fen Li; Jiang Bing Shuai; Ping Lu; Chun Xia Hu

    2007-01-01

    The absence of an outer membrane in Bacillus subtilis can simplify the protein secretion pathways and allow the organism to secrete high levels of extracellular proteins. Of the three known secretory routes, Sec-SRP pathway can direct the majority of secretory proteins into the growth medium. Alternatively, a small number of exoproteins with specific functions are secreted via Tat pathway or ABC transporters in B. subtilis. The discriminating function of precursor proteins among these pathways is largely attributed to the distinct structure of their cleavable signal peptides. Individual secretion machinery components with their special functions are involved in the total flow of proteins from the cytoplasm to the medium. Notably, multiple regulators with signal transduction functions can affect expression of secretion machinery as well as their post-transcriptional actions for protein secretion, resulting in the complicated networks in B. subtilis. Ultimately, according to the available knowledge of secretion machinery, several approaches aimed at optimizing protein secretion are discussed.

  11. Thymine auxotrophy is associated with increased UV sensitivity in Escherichia coli and Bacillus subtilis.

    PubMed

    Lojo, M M

    1995-06-01

    Thymine auxotrophy was shown to be associated with an increase in UV sensitivity both in Bacillus subtilis and in Escherichia coli. This UV sensitization became clearly evident in polA5 mutants of Bacillus subtilis: at UV doses of 16 J/m2, a reduction of more than 10-fold in the survivor population is observed in thymine requiring spontaneous mutants (polA5 thyA thyB) compared to the parental strains (polA5). Reversion of either thyA or thyB mutation led to a partial recovery in the UV resistance. This result suggests that DNA repair polymerization might be improved by the biosynthesis of thymidylate or some effect associated with such activity.

  12. Production, optimization and characterization of fibrinolytic enzyme by Bacillus subtilis RJAS19.

    PubMed

    Kumar, D J Mukesh; Rakshitha, R; Vidhya, M Annu; Jennifer, P Sharon; Prasad, Sandip; Kumar, M Ravi; Kalaichelvan, P T

    2014-04-01

    The present study aimed at the production, purification and characterization of fibrinolytic nattokinase enzyme from the bacteria isolated from natto food. For the purpose, a fibrinolytic bacterium was isolated and identified as Bacillus subtilis based on 16S rDNA sequence analysis. The strain was employed for the production and optimization of fibrinolytic enzyme. The strain showed better enzyme production during 72nd h of incubation time with 50 degrees C at the pH 9. The lactose and peptone were found to be increasing the enzyme production rate. The enzyme produced was purified and also characterized with the help of SDS-PAGE analysis. The activity and stability profile of the purified enzyme was tested against different temperature and pH. The observations suggesting that the potential of fibrinolytic enzyme produced by Bacillus subtilis RJAS 19 for its applications in preventive medicines.

  13. Biosynthesis of rhizocticins, antifungal phosphonate oligopeptides produced by Bacillus subtilis ATCC6633

    PubMed Central

    Borisova, Svetlana A.; Circello, Benjamin T.; Zhang, Jun Kai; van der Donk, Wilfred A.; Metcalf, William W.

    2010-01-01

    Summary Rhizocticins are phosphonate oligopeptide antibiotics containing the C-terminal non-proteinogenic amino acid (Z)-l-2-amino-5-phosphono-3-pentenoic acid (APPA). Here we report the identification and characterization of the rhizocticin biosynthetic gene cluster (rhi) in Bacillus subtilis ATCC6633. Rhizocticin B was heterologously produced in the non-producer strain Bacillus subtilis 168. A biosynthetic pathway is proposed based on bioinformatics analysis of the rhi genes. One of the steps during the biosynthesis of APPA is an unusual aldol reaction between phosphonoacetaldehyde and oxaloacetate catalyzed by an aldolase homolog RhiG. Recombinant RhiG was prepared and the product of an in vitro enzymatic conversion was characterized. Access to this intermediate allows for biochemical characterization of subsequent steps in the pathway. PMID:20142038

  14. Expression, purification and preliminary crystallographic characterization of FlhF from Bacillus subtilis

    SciTech Connect

    Bange, Gert; Petzold, Georg; Wild, Klemens; Sinning, Irmgard

    2007-05-01

    Preliminary crystallographic data are reported for the third SRP GTPase FlhF from Bacillus subtilis. The Gram-positive bacterium Bacillus subtilis contains three proteins belonging to the signal recognition particle (SRP) type GTPase family. The well characterized signal sequence-binding protein SRP54 and the SRP receptor protein FtsY are universally conserved components of the SRP system of protein transport. The third member, FlhF, has been implicated in the placement and assembly of polar flagella. This article describes the overexpression and preliminary X-ray crystallographic analysis of an FlhF fragment that corresponds to the well characterized GTPase domains in SRP54 and FtsY. Three crystal forms are reported with either GDP or GMPPNP and diffract to a resolution of about 3 Å.

  15. Cellular Site in Bacillus subtilis of a Nuclease Which Preferentially Degrades Single-Stranded Nucleic Acids

    PubMed Central

    Birnboim, H. C.

    1966-01-01

    Birnboim, H. C. (Albert Einstein College of Medicine, New York, N.Y.). Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids. J. Bacteriol. 91:1004–1011. 1966.—A nuclease, identified by a marked preference for single-stranded nucleic acids, has been demonstrated in extracts of Bacillus subtilis. The enzyme was associated with the cell wall-membrane fraction of mechanically disrupted cells and was released from cells which had been converted to protoplasts by lysozyme. The nuclease activity prepared by the latter procedure was found to be activated and solubilized by treatment with trypsin. The enzyme had about 2% activity on native deoxyribonucleic acid (DNA) as compared with denatured DNA. By use of CsCl analytical density gradient ultracentrifugation, this preparation was shown to degrade denatured DNA selectively in mixtures of native and denatured DNA. PMID:4956329

  16. Bacillus subtilis as a bioindicator for estimating pentachlorophenol toxicity and concentration.

    PubMed

    Ayude, M A; Okada, E; González, J F; Haure, P M; Murialdo, S E

    2009-05-01

    Pentachlorophenol (PCP) and its sodium salt (Na-PCP) are extremely toxic chemicals responsible for important soil and groundwater pollution, mainly caused by wastes from wood-treatment plants, because chlorinated phenols are widely used as wood preservatives. The methods most commonly used for routine analysis of pesticides such as PCP and Na-PCP are high-performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS). A variety of rapid biological screening tests using marine organisms, bioluminescent bacteria, and enzymes have also been reported. In this study, rapid biological screening analysis using Bacillus subtilis was developed, to assess the biodegradation of PCP and its by-products in liquid samples. An empirical model is proposed for spectrophotometric analysis of Na-PCP concentration after growth of Bacillus subtilis.

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

    PubMed

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

    2014-08-01

    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.

  18. Insulation of the σF Regulatory System in Bacillus subtilis

    PubMed Central

    Carniol, Karen; Kim, Tae-Jong; Price, Chester W.; Losick, Richard

    2004-01-01

    The transcription factors σF and σB are related RNA polymerase sigma factors that govern dissimilar networks of adaptation to stress conditions in Bacillus subtilis. The two factors are controlled by closely related regulatory pathways, involving protein kinases and phosphatases. We report that insulation of the σF pathway from the σB pathway involves the integrated action of both the cognate kinase and the cognate phosphatase. PMID:15205443

  19. Effects of space environment on T-7 bacteriophage and spores of Bacillus subtilis 168

    NASA Technical Reports Server (NTRS)

    Spizizen, J.; Isherwood, J. E.

    1973-01-01

    Two strains of Bacillus subtilis were exposed to components of the ultraviolet spectrum in space. Both strains possess multiple genetic markers, and one of the strains is defective in the ability to repair ultraviolet damage. The T-7 bacteriophage of Escherichia coli was also exposed to selected wavelengths and energy levels of ultraviolet light in space. Preliminary findings do not reveal anomalies in survival rates. Data are not yet available on detailed genetic analyses.

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

    PubMed Central

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

    2014-01-01

    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

  1. Survey of Workers Exposed to Dusts Containing Derivatives of Bacillus Subtilis

    PubMed Central

    Greenberg, M.; Milne, Judith F.; Watt, A.

    1970-01-01

    In a survey of 121 workers exposed to dusts containing derivatives of Bacillus subtilis, mainly proteolytic enzymes, skin tests showed evidence of sentiztation was higher among “atopic” subjects—16 out of tization was higher among “atopic” subjects—16 out of 25 (64%)—than among “non-atopic” subjects—32 out of 96 (33%). Reduced ventilatory capacity was found in 44% of sensitized workers compared with 14% of those not sensitized. PMID:4987928

  2. Structure of the Bacillus subtilis quorum-sensing peptide pheromone ComX.

    PubMed

    Okada, Masahiro; Sato, Isao; Cho, Soo Jeong; Iwata, Hidehisa; Nishio, Toshihiko; Dubnau, David; Sakagami, Youji

    2005-06-01

    The ComX pheromone is an extracellular signaling molecule that stimulates natural competence in response to crowding in the gram-positive bacterium Bacillus subtilis. The pheromone is formed by isoprenylation of an inactive precursor peptide, but its precise structure is not known. Here we report the structure of the ComX pheromone, showing that addition of a geranyl group to a tryptophan residue results in the formation of an unusual ring structure.

  3. Structure of RizA, an L-amino-acid ligase from Bacillus subtilis.

    PubMed

    Kagawa, Wataru; Arai, Toshinobu; Ishikura, Shun; Kino, Kuniki; Kurumizaka, Hitoshi

    2015-09-01

    RizA is an L-amino-acid ligase from Bacillus subtilis that participates in the biosynthesis of rhizocticin, an oligopeptide antibiotic. The substrate-free form of RizA has been crystallized and the structure was solved at 2.8 Å resolution. The amino-acid-binding site appears to be capable of accommodating multiple amino acids, consistent with previous biochemical studies.

  4. Expression and purification of the Bacillus subtilis thioredoxin superfamily protein YkvV.

    PubMed

    Tanaka, Ryoichi; Araki, Yoko; Mizukami, Makoto; Miyauchi, Akira; Ishibashi, Matsujiro; Tokunaga, Hiroko; Tokunaga, Masao

    2004-08-01

    Bacillus subtilis YkvV protein, an extracellular thioredoxin superfamily protein, was successfully expressed both in Brevibacillus choshinensis culture medium using an efficient promoter and the secretion signal of its surface layer protein, and in Escherichia coli cytoplasm with the amino-terminal His-tag (His-YkvV). His-YkvV was purified to homogeneity by Ni-NTA column. Both secreted YkvV and purified His-YkvV exhibited thiol-disulfide oxidoreductase activity.

  5. Effects of space environment on T-7 bacteriophage and spores of Bacillus subtilis 168

    NASA Technical Reports Server (NTRS)

    Spizizen, J.; Isherwood, J. E.

    1973-01-01

    Two strains of Bacillus subtilis were exposed to components of the ultraviolet spectrum in space. Both strains possess multiple genetic markers, and one of the strains is defective in the ability to repair ultraviolet damage. The T-7 bacteriophage of Escherichia coli was also exposed to selected wavelengths and energy levels of ultraviolet light in space. Preliminary findings do not reveal anomalies in survival rates. Data are not yet available on detailed genetic analyses.

  6. YrxA Is the Transcriptional Regulator That Represses De Novo NAD Biosynthesis in Bacillus subtilis

    PubMed Central

    Rossolillo, Paola; Marinoni, Ilaria; Galli, Elisa; Colosimo, Anna; Albertini, Alessandra M.

    2005-01-01

    The first genetic, in vivo, and in vitro evidences that YrxA is the regulator of NAD de novo biosynthesis in Bacillus subtilis are hereby reported. The protein is essential to the transcription repression of the divergent operons nadBCA and nifS-yrxA in the presence of nicotinic acid and binds to their shared operator-promoter region. PMID:16199587

  7. Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

    PubMed Central

    Westers, Helga; Braun, Peter G.; Westers, Lidia; Antelmann, Haike; Hecker, Michael; Jongbloed, Jan D. H.; Yoshikawa, Hirofumi; Tanaka, Teruo; van Dijl, Jan Maarten; Quax, Wim J.

    2005-01-01

    Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor. PMID:15812018

  8. Functional characterization of a Penicillium chrysogenum mutanase gene induced upon co-cultivation with Bacillus subtilis

    PubMed Central

    2014-01-01

    Background Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis. Results Steady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative α-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants

  9. Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis.

    PubMed

    Shirakawa, Márcia Aiko; Cincotto, Maria Alba; Atencio, Daniel; Gaylarde, Christine C; John, Vanderley M

    2011-04-01

    The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials.

  10. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

    2015-03-01

    Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

  11. Thrombolytic effects of Douchi Fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo

    PubMed Central

    2012-01-01

    Background Today, thrombosis is one of the most widely occurring diseases in modern life. Drugs with thrombolytic functions are the most effective methods in the treatment of thrombosis. Among them, Douchi fibrinolytic enzyme (DFE) is a promising agent. DFE was isolated from Douchi, a typical and popular soybean-fermented food in China, and it can dissolve fibrin directly and efficiently. A strain, Bacillus subtilis LD-8547 produced DFE with high fibrinolytic activity has been isolated in our lab previously. Results In the study, thrombolytic effect of DFE from Bacillus subtilis LD-8547 was studied in vitro and in vivo systematically. The results showed that DFE played a significant role in thrombolysis and anticoagulation in vitro. And the thrombolytic effects correlated with DFE in a dose-dependent manner. In vivo, the acute toxicity assay showed that DFE had no obvious acute toxicity to mice. Test of carrageenan-induced thrombosis in mice indicated that the DFE significantly prevented tail thrombosis, and arterial thrombosis model test indicated that Douchi fibrinolytic enzyme DFE had thrombolytic effect on carotid thrombosis of rabbits in vivo. Other results in vivo indicated that DFE could increase bleeding and clotting time obviously. Conclusions The DFE isolated from Bacillus subtilis LD-8547 has obvious thrombolytic effects in vitro and in vivo. This function demonstrates that this enzyme can be a useful tool for preventing and treating clinical thrombus. PMID:22748219

  12. Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis.

    PubMed

    Stickel, Felix; Droz, Sara; Patsenker, Eleonora; Bögli-Stuber, Katja; Aebi, Beat; Leib, Stephen L

    2009-01-01

    Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

  13. Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

    PubMed Central

    Shirakawa, Márcia Aiko; Cincotto, Maria Alba; Atencio, Daniel; Gaylarde, Christine C.; John, Vanderley M.

    2011-01-01

    The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials. PMID:24031661

  14. Decolourization of 4-Chloro-2-Nitrophenol by a Soil Bacterium, Bacillus subtilis RKJ 700

    PubMed Central

    Arora, Pankaj Kumar

    2012-01-01

    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. PMID:23251673

  15. An Exogenous Surfactant-Producing Bacillus subtilis Facilitates Indigenous Microbial Enhanced Oil Recovery.

    PubMed

    Gao, Peike; Li, Guoqiang; Li, Yanshu; Li, Yan; Tian, Huimei; Wang, Yansen; Zhou, Jiefang; Ma, Ting

    2016-01-01

    This study used an exogenous lipopeptide-producing Bacillus subtilis to strengthen the indigenous microbial enhanced oil recovery (IMEOR) process in a water-flooded reservoir in the laboratory. The microbial processes and driving mechanisms were investigated in terms of the changes in oil properties and the interplay between the exogenous B. subtilis and indigenous microbial populations. The exogenous B. subtilis is a lipopeptide producer, with a short growth cycle and no oil-degrading ability. The B. subtilis facilitates the IMEOR process through improving oil emulsification and accelerating microbial growth with oil as the carbon source. Microbial community studies using quantitative PCR and high-throughput sequencing revealed that the exogenous B. subtilis could live together with reservoir microbial populations, and did not exert an observable inhibitory effect on the indigenous microbial populations during nutrient stimulation. Core-flooding tests showed that the combined exogenous and indigenous microbial flooding increased oil displacement efficiency by 16.71%, compared with 7.59% in the control where only nutrients were added, demonstrating the application potential in enhanced oil recovery in water-flooded reservoirs, in particular, for reservoirs where IMEOR treatment cannot effectively improve oil recovery.

  16. Antagonistic Action of Bacillus subtilis Strain SG6 on Fusarium graminearum

    PubMed Central

    Zhao, Yueju; Selvaraj, Jonathan Nimal; Xing, Fuguo; Zhou, Lu; Wang, Yan; Song, Huimin; Tan, Xinxin; Sun, Lichao; Sangare, Lancine; Folly, Yawa Minnie Elodie; Liu, Yang

    2014-01-01

    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. PMID:24651513

  17. Effects of salinomycin and Bacillus subtilis on growth performance and immune responses in broiler chickens.

    PubMed

    Lee, Kyung-Woo; Lillehoj, Hyun S; Jang, Seung I; Lee, Sung-Hyen

    2014-10-01

    The present study was undertaken to compare the effect of salinomycin and Bacillus subtilis on growth performance, serum antibody levels against Clostridium spp. and Eimeria spp., and cytokine mRNA expression levels in broiler chickens raised in the used litter. Broiler chickens fed a diet containing salinomycin showed lower (P < 0.05) body weights compared with the control diet-fed counterparts. Serum nitric oxide levels were significantly (P < 0.05) elevated in chickens fed the B. subtilis-enriched diet compared with those on either the salinomycin-fed or control diet-fed chickens. None of the dietary treatments affected (P > 0.05) serum antibody levels against Clostridium perfringens toxins. Both salinomycin and B.subtilis significantly lowered (P < 0.05) the serum levels of Eimeria-specific antibodies compared with the control group. Salinomycin, but not B. subtilis, significantly modulated (P < 0.05) the expression of cytokines encoding interferon-γ (IFN-γ), interleukin10 (IL-10) and tumor necrosis factor superfamily 15 (TNFSF15) compared with the control group. In conclusion, dietary salinomycin and B. subtilis affected serum anticoccidial antibody and intestinal cytokine expression, but failed to improve growth performance in broiler chickens. Further study is warranted to investigate the mode of action of salinomycin on host immune response and growth performance in broiler chickens.

  18. Effects of water chemistry and surface contact on the toxicity of silver nanoparticles to Bacillus subtilis.

    PubMed

    Yi, Jun; Cheng, Jinping

    2017-07-01

    The growing use of silver nanoparticles (AgNPs) has created concerns about its potential impacts on natural microbial communities. In this study, the physicochemical properties of AgNPs and its toxicity on natural bacteria Bacillus subtilis (B. subtilis) were investigated in aqueous conditions. The characterization data showed that AgNPs highly aggregated in aqueous conditions, and the hydrodynamic diameter of AgNPs in aqueous conditions was larger than its primary size. The studied AgNPs was less toxic to B. subtilis in estuarine water as compared to that in Milli-Q water and artificial seawater, which might be due to the observed enhanced aggregation of AgNPs in estuarine water. The toxicity of AgNPs to B. subtilis was greatly reduced when their surface contact was blocked by a dialysis membrane. Scanning electron microscope images showed that exposure contact to AgNPs resulted in damage of the microbial cell wall and enhanced formation of fibrillar structures. These results suggest that particle-cell contact is largely responsible for the observed toxicity of AgNPs in B. subtilis. This study can help to understand the potential impacts of AgNPs to natural microbes, especially in the complex aquatic environments.

  19. Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto.

    PubMed

    Samanya, Mongkol; Yamauchi, Koh-en

    2002-09-01

    Two experiments were conducted. In experiment 1, chickens were fed dried Bacillus subtilis var. natto for 3 or 28 days. Growth performance and internal organs were not different from controls, but feed efficiency tended to be improved in the 28-day feeding. In these birds, blood ammonia concentration was decreased (P<0.05). Blood glucose concentration, and amylase and lipase activity in the intestinal content were not significantly different among dietary groups. These results suggest that the B. subtilis natto depressed ammonia concentration. In experiment 2, chickens were fed dietary B. subtilis natto for 28 days. These birds had a tendency to display greater growth performance and intestinal histologies, such as villus height, cell area and cell mitosis, than the controls. Flat cell outline on the duodenal villus surface in controls developed large, protruded cell clusters and cell protuberances after feeding of dietary B. subtilis natto. These results indicate that intestinal function was activated by the depressed blood ammonia concentration in the body of the chicken. The present results may suggest that the B. subtilis natto has the potential to be a beneficial microorganism in chickens.

  20. Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.

    PubMed

    Kelwick, Richard; Webb, Alexander J; MacDonald, James T; Freemont, Paul S

    2016-11-01

    Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Predation by Myxococcus xanthus Induces Bacillus subtilis To Form Spore-Filled Megastructures

    PubMed Central

    Müller, Susanne; Strack, Sarah N.; Ryan, Sarah E.; Kearns, Daniel B.

    2014-01-01

    Biofilm formation is a common mechanism for surviving environmental stress and can be triggered by both intraspecies and interspecies interactions. Prolonged predator-prey interactions between the soil bacterium Myxococcus xanthus and Bacillus subtilis were found to induce the formation of a new type of B. subtilis biofilm, termed megastructures. Megastructures are tree-like brachiations that are as large as 500 μm in diameter, are raised above the surface between 150 and 200 μm, and are filled with viable endospores embedded within a dense matrix. Megastructure formation did not depend on TasA, EpsE, SinI, RemA, or surfactin production and thus is genetically distinguishable from colony biofilm formation on MSgg medium. As B. subtilis endospores are not susceptible to predation by M. xanthus, megastructures appear to provide an alternative mechanism for survival. In addition, M. xanthus fruiting bodies were found immediately adjacent to the megastructures in nearly all instances, suggesting that M. xanthus is unable to acquire sufficient nutrients from cells housed within the megastructures. Lastly, a B. subtilis mutant lacking the ability to defend itself via bacillaene production formed megastructures more rapidly than the parent. Together, the results indicate that production of the megastructure facilitates B. subtilis escape into dormancy via sporulation. PMID:25326308

  2. Preparation and biosorption evaluation of Bacillus subtilis/alginate–chitosan microcapsule

    PubMed Central

    Tong, Ke

    2017-01-01

    The aim of this study was to assess the effect of alginate–chitosan microcapsule on viability characteristics of Bacillus subtilis and the ability of B. subtilis/alginate–chitosan microcapsule to remove uranium ion from aqueous solution. The effects of particle size, chitosan molecular weight and inoculum density on viability characteristics were studied using alginate–chitosan microcapsule-immobilized B. subtilis experiments. In addition, the effects of pH, immobilized spherule dosage, temperature, initial uranium ion concentration and contact time on removal of uranium ion were studied using batch adsorption experiments. The results showed that alginate–chitosan microcapsule significantly improved the viability characteristics of B. subtilis and that B. subtilis/alginate–chitosan microcapsule strongly promoted uranium ion absorption. Moreover, the optimum values of pH was 6; immobilized spherule dosage was 3.5; temperature was 20°C; initial uranium ion concentration was 150 mg/L; contact time was 3 h of uranium ion absorption and the maximum adsorption capacity of uranium ion was 376.64 mg/g. PMID:28223783

  3. An Exogenous Surfactant-Producing Bacillus subtilis Facilitates Indigenous Microbial Enhanced Oil Recovery

    PubMed Central

    Gao, Peike; Li, Guoqiang; Li, Yanshu; Li, Yan; Tian, Huimei; Wang, Yansen; Zhou, Jiefang; Ma, Ting

    2016-01-01

    This study used an exogenous lipopeptide-producing Bacillus subtilis to strengthen the indigenous microbial enhanced oil recovery (IMEOR) process in a water-flooded reservoir in the laboratory. The microbial processes and driving mechanisms were investigated in terms of the changes in oil properties and the interplay between the exogenous B. subtilis and indigenous microbial populations. The exogenous B. subtilis is a lipopeptide producer, with a short growth cycle and no oil-degrading ability. The B. subtilis facilitates the IMEOR process through improving oil emulsification and accelerating microbial growth with oil as the carbon source. Microbial community studies using quantitative PCR and high-throughput sequencing revealed that the exogenous B. subtilis could live together with reservoir microbial populations, and did not exert an observable inhibitory effect on the indigenous microbial populations during nutrient stimulation. Core-flooding tests showed that the combined exogenous and indigenous microbial flooding increased oil displacement efficiency by 16.71%, compared with 7.59% in the control where only nutrients were added, demonstrating the application potential in enhanced oil recovery in water-flooded reservoirs, in particular, for reservoirs where IMEOR treatment cannot effectively improve oil recovery. PMID:26925051

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

    PubMed Central

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

    2014-01-01

    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 leaf length, flowers per corm, weight of the first flower stigma, total stigma biomass; microbe addition also significantly decreased the time required for corms to sprout and the number of shoot sprouts. Compared to the controls, picrocrocin, crocetin and safranal compounds were significantly increased when the plants were soil drenched with the spore solution 14 weeks after sowing; in contrast crocin was highest in untreated controls. Results of this study suggest that application of B. subtilis FZB24® may provide some benefit to saffron growers by speeding corm growth (earlier shoot emergence) and increasing stigma biomass yield by 12%. While some treatment conditions also increased saffron chemical composition, these were generally not the same treatments that simultaneously improved growth yields and thus, more study is required. PMID:18622904

  5. Different toxic and hormetic responses of Bombus impatiens to Beauveria bassiana, Bacillus subtilis and spirotetramat.

    PubMed

    Ramanaidu, Krilen; Cutler, G Christopher

    2013-08-01

    Pollinator exposure to pesticides is a concern in agricultural systems that depend on pollinators for crop production. However, not all pesticides elicit toxic effects, and response to a pesticide will vary depending on dose and exposure route. The effects of biopesticide formulations of Bacillus subtilis and Beauveria bassiana and of the tetramic acid insecticide spirotetramat on the common eastern bumblebee, Bombus impatiens, were evaluated. Microcolonies of bees were exposed to field-rate or lower concentrations, and data were collected over 60 days. When ingested, field rates of spirotetramat caused high mortality after 10 days, and B. subtilis significantly reduced drone production, number of days to oviposition and number of days to drone emergence. Converse to effects observed following ingestion, topical applications of B. subtilis at concentrations less than the recommended field rate resulted in a hormetic response, with significantly increased drone production. Topical application of spirotetramat and oral or topical application of B. bassiana had no effects on bees. Spirotetramat and B. subtilis can induce adverse effects on B. impatiens, but hormetic effects following B. subtilis treatment can also occur, depending on exposure route. Additional experiments are required to determine whether similar toxic or hormetic effects occur under more realistic field conditions. © 2012 Society of Chemical Industry.

  6. Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis.

    PubMed

    Zhou, Zheng; Zhou, Li-Ping; Chen, Mei-Juan; Zhang, Yan-Liang; Li, Ren-Bao; Yang, Sheng; Yuan, Zhong-Yi

    2003-05-01

    The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin G acylase (PGA) was cloned and successfully expressed in Escherichia coli and Bacillus subtilis respectively. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cell secreted the recombinant enzyme into the medium. Contrary to limited expression yield of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA) and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This yield increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was fractionated with (NH(4))(2)SO(4) and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as the pH and temperature optimum, the stability against organic solvent and the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing A. faecalis PGA in B. subtilis and purifying secreted enzyme from culture medium one could readily obtain a large amount of an alternative source of PGA.

  7. 2,3-Butanediol production from cellobiose using exogenous beta-glucosidase-expressing Bacillus subtilis.

    PubMed

    Tanimura, Kosuke; Takashima, Shingo; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

    2016-07-01

    We engineered efficient 2,3-butanediol (23BD) production from cellobiose using Bacillus subtilis. First, we found that B. subtilis harboring an empty vector could produce 23BD from cellobiose. However, productivity using cellobiose as a carbon source was lower than that when using glucose. This lower productivity was improved by adding purified beta-glucosidase from Thermobifida fusca YX (Tfu_0937) in the fermentation. Encouraged by these findings, we found that hydrolysis of cellobiose to glucose was an important reaction of 23BD biosynthesis in B. subtilis using cellobiose. Hence, we created efficient 23BD production from cellobiose using exogenous Tfu_0937-expressing B. subtilis. Using the engineered strain, 21.2 g L(-1) of 23BD was produced after 72 h of cultivation. The productivity and yield were 0.294 g L(-1) h(-1) and 0.35 g 23BD/g cellobiose, respectively. We successfully demonstrated efficient 23BD production from cellobiose by using BGL-expressing B. subtilis.

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

    PubMed

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

    2016-05-20

    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.

  9. Effects of sublethal doses of silver nanoparticles on Bacillus subtilis planktonic and sessile cells.

    PubMed

    Gambino, M; Marzano, V; Villa, F; Vitali, A; Vannini, C; Landini, P; Cappitelli, F

    2015-05-01

    Due to their antimicrobial activity, silver nanoparticles (Ag-NPs) are being increasingly used in a number of industrial products. The accumulation of Ag-NPs in the soil might affect plant growth-promoting rhizobacteria and, in turn, the plants. We describe the effects of Ag-NPs on the soil bacteria Azotobacter vinelandii and Bacillus subtilis. In growth-inhibition studies, A. vinelandii showed extreme sensitivity to Ag-NPs, compared to B. subtilis. We investigated the effects of Ag-NPs at subinhibitory concentrations, both on planktonic and sessile B. subtilis cells. As determined by 2,7-dichlorofluorescein-diacetate assays, Ag-NPs increase the formation of reactive oxygen species in planktonic cells, but not in sessile cells, suggesting the activation of scavenging systems in biofilms. Consistently, proteomic analysis in B. subtilis Ag-NPs-treated biofilms showed increased production of proteins related to quorum sensing and involved in stress responses and redox sensing. Extracellular polysaccharides production and inorganic phosphate solubilization were also increased, possibly as part of a coordinated response to stress. At low concentrations, Ag-NPs killed A. vinelandii and affected cellular processes in planktonic and sessile B. subtilis cells. Re-direction of gene expression, linked to selective toxicity, suggests a strong impact of Ag-NPs on soil bacterial communities. © 2015 The Society for Applied Microbiology.

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

    PubMed Central

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

    2012-01-01

    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 Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli. PMID:22606280

  11. Bacillus subtilis FZB24 affects flower quantity and quality of saffron (Crocus sativus).

    PubMed

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

    2008-08-01

    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(R). Corms were soaked in water or in B. subtilis FZB24 spore solution for 15 min 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 leaf length, flowers per corm, weight of the first flower stigma, total stigma biomass; microbe addition also significantly decreased the time required for corms to sprout and the number of shoot sprouts. Compared to the controls, picrocrocin, crocetin and safranal compounds were significantly increased when the plants were soil drenched with the spore solution 14 weeks after sowing; in contrast crocin was highest in untreated controls. Results of this study suggest that application of B. subtilis FZB24 may provide some benefit to saffron growers by speeding corm growth (earlier shoot emergence) and increasing stigma biomass yield by 12 %. While some treatment conditions also increased saffron chemical composition, these were generally not the same treatments that simultaneously improved growth yields and thus, more study is required.

  12. Decrease in spermidine content during logarithmic phase of cell growth delays spore formation of Bacillus subtilis.

    PubMed

    Ishii, I; Takada, H; Terao, K; Kakegawa, T; Igarashi, K; Hirose, S

    1994-11-01

    Bacillus subtilis 168M contained a large amount of spermidine during the logarithmic phase of growth, but the amount decreased drastically during the stationary phase. The extracts, prepared from B. subtilis cells harvested in the logarithmic phase, contained activity of arginine decarboxylase (ADC) rather than the activity of ornithine decarboxylase. In the presence of alpha-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of ADC, the amount of spermidine in B. subtilis during the logarithmic phase decreased to about 25% of the control cells. Under these conditions, spore formation of B. subtilis 168M delayed greatly without significant inhibition of cell growth. The decrease in spermidine content in the logarithmic phase rather than in the stationary phase was involved in the delay of sporulation. Electron microscopy of cells at 24 hrs. of culture confirmed the delay of spore formation by the decrease of spermidine content. Furthermore, the delay of sporulation was negated by the addition of spermidine. These data suggest that a large amount of spermidine existing during the logarithmic phase plays an important role in the sporulation of B. subtilis.

  13. High-salinity growth conditions promote Tat-independent secretion of Tat substrates in Bacillus subtilis.

    PubMed

    van der Ploeg, René; Monteferrante, Carmine G; Piersma, Sjouke; Barnett, James P; Kouwen, Thijs R H M; Robinson, Colin; van Dijl, Jan Maarten

    2012-11-01

    The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.

  14. Spore germination and germinant receptor genes in wild strains of Bacillus subtilis.

    PubMed

    Alzahrani, O M; Moir, A

    2014-09-01

    To compare the germination of laboratory and wild strains of Bacillus subtilis. The spore germination of B. subtilis 168 (subsp. subtilis) was compared with that of the laboratory strain W23 (subsp. spizizenii) and desert-sourced isolates, including one member of subsp. subtilis (RO-NN-1), strains TU-B-10, RO-E-2, N10 and DV1-B-1, (all subsp. spizizenii), the B. mojavensis strain RO-H-1 and a B. subtilis natto strain. All germinated in L-alanine, although some were slower, and some 10-fold less sensitive to germinant. All germinated in calcium dipicolinate (CaDPA). Germination in asparagine, glucose, fructose + KCl was slow and incomplete in many of the strains, and decoating RO-NN-1 and W23 spores did not restore germination rates. Comparing the sequences of B. subtilis strains 168, RO-NN-1, W23, TU-B-10 and DV1-B-1, the operons encoding GerA, B and K germinant receptors were intact, although the two additional operons yndDEF and yfkQRST had suffered deletions or were absent in several spizizenii strains. Wild strains possess an efficient germination machinery for L-alanine germination. AGFK germination is often less efficient, the gerB genes more diverged, and the two germinant receptor operons of unknown function have been lost from the genome in many subsp. spizizenii strains. The two major subspecies of B. subtilis have conserved GerA receptor function, confirming its importance, at least in the natural environments of these strains. © 2014 The Society for Applied Microbiology.

  15. ZnO Nanoparticles Affect Bacillus subtilis Cell Growth and Biofilm Formation

    PubMed Central

    Hsueh, Yi-Huang; Ke, Wan-Ju; Hsieh, Chien-Te; Lin, Kuen-Song; Tzou, Dong-Ying; Chiang, Chao-Lung

    2015-01-01

    Zinc oxide nanoparticles (ZnO NPs) are an important antimicrobial additive in many industrial applications. However, mass-produced ZnO NPs are ultimately disposed of in the environment, which can threaten soil-dwelling microorganisms that play important roles in biodegradation, nutrient recycling, plant protection, and ecological balance. This study sought to understand how ZnO NPs affect Bacillus subtilis, a plant-beneficial bacterium ubiquitously found in soil. The impact of ZnO NPs on B. subtilis growth, FtsZ ring formation, cytosolic protein activity, and biofilm formation were assessed, and our results show that B. subtilis growth is inhibited by high concentrations of ZnO NPs (≥ 50 ppm), with cells exhibiting a prolonged lag phase and delayed medial FtsZ ring formation. RedoxSensor and Phag-GFP fluorescence data further show that at ZnO-NP concentrations above 50 ppm, B. subtilis reductase activity, membrane stability, and protein expression all decrease. SDS-PAGE Stains-All staining results and FT-IR data further demonstrate that ZnO NPs negatively affect exopolysaccharide production. Moreover, it was found that B. subtilis biofilm surface structures became smooth under ZnO-NP concentrations of only 5–10 ppm, with concentrations ≤ 25 ppm significantly reducing biofilm formation activity. XANES and EXAFS spectra analysis further confirmed the presence of ZnO in co-cultured B. subtilis cells, which suggests penetration of cell membranes by either ZnO NPs or toxic Zn+ ions from ionized ZnO NPs, the latter of which may be deionized to ZnO within bacterial cells. Together, these results demonstrate that ZnO NPs can affect B. subtilis viability through the inhibition of cell growth, cytosolic protein expression, and biofilm formation, and suggest that future ZnO-NP waste management strategies would do well to mitigate the potential environmental impact engendered by the disposal of these nanoparticles. PMID:26039692

  16. Inactivation of aprE Gene in Bacillus subtilis 168 by Homologus Recombination

    PubMed Central

    Rabbani, Mohammed; Soleymani, Safoura; Sadeghi, Hamid Mir Mohammad; Soleimani, Narjes; Moazen, Fatemeh

    2014-01-01

    Background One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis (B. Subtilis). One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. Methods The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate (FITC)–labeled casein-substrate. Results The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. Conclusion It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase. PMID:25215183

  17. Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

    PubMed Central

    Zuberi, A R; Bischoff, D S; Ordal, G W

    1991-01-01

    The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes. Images PMID:1898932

  18. ZnO Nanoparticles Affect Bacillus subtilis Cell Growth and Biofilm Formation.

    PubMed

    Hsueh, Yi-Huang; Ke, Wan-Ju; Hsieh, Chien-Te; Lin, Kuen-Song; Tzou, Dong-Ying; Chiang, Chao-Lung

    2015-01-01

    Zinc oxide nanoparticles (ZnO NPs) are an important antimicrobial additive in many industrial applications. However, mass-produced ZnO NPs are ultimately disposed of in the environment, which can threaten soil-dwelling microorganisms that play important roles in biodegradation, nutrient recycling, plant protection, and ecological balance. This study sought to understand how ZnO NPs affect Bacillus subtilis, a plant-beneficial bacterium ubiquitously found in soil. The impact of ZnO NPs on B. subtilis growth, FtsZ ring formation, cytosolic protein activity, and biofilm formation were assessed, and our results show that B. subtilis growth is inhibited by high concentrations of ZnO NPs (≥ 50 ppm), with cells exhibiting a prolonged lag phase and delayed medial FtsZ ring formation. RedoxSensor and Phag-GFP fluorescence data further show that at ZnO-NP concentrations above 50 ppm, B. subtilis reductase activity, membrane stability, and protein expression all decrease. SDS-PAGE Stains-All staining results and FT-IR data further demonstrate that ZnO NPs negatively affect exopolysaccharide production. Moreover, it was found that B. subtilis biofilm surface structures became smooth under ZnO-NP concentrations of only 5-10 ppm, with concentrations ≤ 25 ppm significantly reducing biofilm formation activity. XANES and EXAFS spectra analysis further confirmed the presence of ZnO in co-cultured B. subtilis cells, which suggests penetration of cell membranes by either ZnO NPs or toxic Zn+ ions from ionized ZnO NPs, the latter of which may be deionized to ZnO within bacterial cells. Together, these results demonstrate that ZnO NPs can affect B. subtilis viability through the inhibition of cell growth, cytosolic protein expression, and biofilm formation, and suggest that future ZnO-NP waste management strategies would do well to mitigate the potential environmental impact engendered by the disposal of these nanoparticles.

  19. Reconstruction of the Regulatory Network for Bacillus subtilis and Reconciliation with Gene Expression Data.

    PubMed

    Faria, José P; Overbeek, Ross; Taylor, Ronald C; Conrad, Neal; Vonstein, Veronika; Goelzer, Anne; Fromion, Vincent; Rocha, Miguel; Rocha, Isabel; Henry, Christopher S

    2016-01-01

    We introduce a manually constructed and curated regulatory network model that describes the current state of knowledge of transcriptional regulation of Bacillus subtilis. The model corresponds to an updated and enlarged version of the regulatory model of central metabolism originally proposed in 2008. We extended the original network to the whole genome by integration of information from DBTBS, a compendium of regulatory data that includes promoters, transcription factors (TFs), binding sites, motifs, and regulated operons. Additionally, we consolidated our network with all the information on regulation included in the SporeWeb and Subtiwiki community-curated resources on B. subtilis. Finally, we reconciled our network with data from RegPrecise, which recently released their own less comprehensive reconstruction of the regulatory network for B. subtilis. Our model describes 275 regulators and their target genes, representing 30 different mechanisms of regulation such as TFs, RNA switches, Riboswitches, and small regulatory RNAs. Overall, regulatory information is included in the model for ∼2500 of the ∼4200 genes in B. subtilis 168. In an effort to further expand our knowledge of B. subtilis regulation, we reconciled our model with expression data. For this process, we reconstructed the Atomic Regulons (ARs) for B. subtilis, which are the sets of genes that share the same "ON" and "OFF" gene expression profiles across multiple samples of experimental data. We show how ARs for B. subtilis are able to capture many sets of genes corresponding to regulated operons in our manually curated network. Additionally, we demonstrate how ARs can be used to help expand or validate the knowledge of the regulatory networks by looking at highly correlated genes in the ARs for which regulatory information is lacking. During this process, we were also able to infer novel stimuli for hypothetical genes by exploring the genome expression metadata relating to experimental conditions

  20. Reconstruction of the Regulatory Network for Bacillus subtilis and Reconciliation with Gene Expression Data

    PubMed Central

    Faria, José P.; Overbeek, Ross; Taylor, Ronald C.; Conrad, Neal; Vonstein, Veronika; Goelzer, Anne; Fromion, Vincent; Rocha, Miguel; Rocha, Isabel; Henry, Christopher S.

    2016-01-01

    We introduce a manually constructed and curated regulatory network model that describes the current state of knowledge of transcriptional regulation of Bacillus subtilis. The model corresponds to an updated and enlarged version of the regulatory model of central metabolism originally proposed in 2008. We extended the original network to the whole genome by integration of information from DBTBS, a compendium of regulatory data that includes promoters, transcription factors (TFs), binding sites, motifs, and regulated operons. Additionally, we consolidated our network with all the information on regulation included in the SporeWeb and Subtiwiki community-curated resources on B. subtilis. Finally, we reconciled our network with data from RegPrecise, which recently released their own less comprehensive reconstruction of the regulatory network for B. subtilis. Our model describes 275 regulators and their target genes, representing 30 different mechanisms of regulation such as TFs, RNA switches, Riboswitches, and small regulatory RNAs. Overall, regulatory information is included in the model for ∼2500 of the ∼4200 genes in B. subtilis 168. In an effort to further expand our knowledge of B. subtilis regulation, we reconciled our model with expression data. For this process, we reconstructed the Atomic Regulons (ARs) for B. subtilis, which are the sets of genes that share the same “ON” and “OFF” gene expression profiles across multiple samples of experimental data. We show how ARs for B. subtilis are able to capture many sets of genes corresponding to regulated operons in our manually curated network. Additionally, we demonstrate how ARs can be used to help expand or validate the knowledge of the regulatory networks by looking at highly correlated genes in the ARs for which regulatory information is lacking. During this process, we were also able to infer novel stimuli for hypothetical genes by exploring the genome expression metadata relating to experimental

  1. A report on extensive lateral genetic reciprocation between arsenic resistant Bacillus subtilis and Bacillus pumilus strains analyzed using RAPD-PCR.

    PubMed

    Khowal, Sapna; Siddiqui, Md Zulquarnain; Ali, Shadab; Khan, Mohd Taha; Khan, Mather Ali; Naqvi, Samar Husain; Wajid, Saima

    2017-02-01

    The study involves isolation of arsenic resistant bacteria from soil samples. The characterization of bacteria isolates was based on 16S rRNA gene sequences. The phylogenetic consanguinity among isolates was studied employing rpoB and gltX gene sequence. RAPD-PCR technique was used to analyze genetic similarity between arsenic resistant isolates. In accordance with the results Bacillus subtilis and Bacillus pumilus strains may exhibit extensive horizontal gene transfer. Arsenic resistant potency in Bacillus sonorensis and high arsenite tolerance in Bacillus pumilus strains was identified. The RAPD-PCR primer OPO-02 amplified a 0.5kb DNA band specific to B. pumilus 3ZZZ strain and 0.75kb DNA band specific to B. subtilis 3PP. These unique DNA bands may have potential use as SCAR (Sequenced Characterized Amplified Region) molecular markers for identification of arsenic resistant B. pumilus and B. subtilis strains. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy.

    PubMed

    Feng, Yue; Liu, Song; Jiao, Yun; Gao, Hui; Wang, Miao; Du, Guocheng; Chen, Jian

    2017-02-01

    L-asparaginase (EC 3.5.1.1, ASN) exhibits great commercial value due to its uses in the food and medicine industry. In this study, we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B. subtilis WB600 through a combined strategy. First, eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for ASN secretion in B. subtilis by using Hpa II promoter, respectively. The signal peptide wapA achieved the highest extracellular ASN activity (28.91 U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1 % enhanced ASN activity. By two rounds of error-prone PCR mutation, the P43 promoter variants with remarkably enhanced strength (D7, E2, H6, B2, and F3) were identified. B2 (-28: A → G, -13: A → G) achieved ASN activity up to 51.13 U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41 U/mL, which was 100 % higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6 U/mL (2.5 g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65 °C and its half-life at 65 °C was 61 min. The K m and k cat of the ASN were 5.29 mM and 54.4 s(-1), respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.

  3. Identical amino acid sequence of the aroA(G) gene products of Bacillus subtilis 168 and B. subtilis Marburg strain.

    PubMed

    Bolotin, A; Khazak, V; Stoynova, N; Ratmanova, K; Yomantas, Y; Kozlov, Y

    1995-09-01

    A DNA fragment containing the aroA(G) gene of Bacillus subtilis 168, encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase-chorismate mutase, was cloned and sequenced. The N-terminus of the protein encoded by aroA(G) showed homology with chorismate mutase encoded by aroH of B. subtilis and with the chorismate mutase parts of proteins encoded by the pheA and tyrA genes of Escherichia coli. The C-terminus of the aroA(G) product has sequence similarity with 3-deoxy-D-manno-octulosonate 8-phosphate synthase of E. coli. It was shown that the proteins encoded by the aroA(G) gene of B. subtilis 168 and the aroA gene of B. subtilis ATCC 6051 Marburg strain are identical, so the observed differences in DAHP synthase activity from these two strains must result from other changes.

  4. Biosorption of nickel by Pseudomonas cepacia 120S and Bacillus subtilis 117S.

    PubMed

    Abdel-Monem, M O; Al-Zubeiry, A H S; Al-Gheethi, A A S

    2010-01-01

    Biosorption of nickel by two bacterial species: Bacillus subtilis 117S and Pseudomonas cepacia 120S was studied. The maximum uptake of nickel was achieved at 234.4 microg Ni2+ ml(-1) by P. cepacia 120S (living and dead biomass) and at 117.2 and 351.6 microg Ni2+ ml(-1) by living and dead biomass of B. subtilis 117S. The increase in biomass concentration has shown an increase in the nickel uptake. The nickel removal increased significantly during contact time from 1 to 8 h then remained constant until 24 h where the equilibrium occurred. Biosorption efficiency of nickel increased with increasing pH from 2 to 7 for living and dead biomass of P. cepacia 120S and B. subtilis 117S. Temperature had an important role in nickel biosorption by both species. The nickel removal by living biomass was significantly disturbed after pretreatment of bacterial biomass with sodium azide, mercuric chloride and formaldehyde. Esterification of carboxyl groups, methylation of amino groups and extraction of lipid fraction of biomass by acetone and benzene significantly reduced the biosorption capacity of nickel. Repeated biosorption and desorption operations exhibited that the biosorption capacity of bacterial biomass regenerated with HNO3 and NaOH as desorbing medium increased significantly in cycle 4 for P. cepacia 120S and B. subtilis 117S. In case of regeneration with HNO3 and distilled water the biosorption capacity increased significantly in cycle 4 for B. subtilis 117S and did not differ significantly from cycle 1 to cycle 4 for P. cepacia 120S. The biosorption capacity of living and dead biomass of B. subtilis 117S and dead biomass of P cepacia 120S (155.5 as compared to 175.6 and 169.8 mg Ni2+ g(-1)) was higher than that of sludge, tea and saw dust (148.4, 52.7 and 44.6 mg Ni2+ g(-1)).

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

    PubMed Central

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

    2014-01-01

    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 inoculated to ginseng plants and the incidence and severity of disease was assessed to examine the efficacy of the bacterium as a biological control against C. panacicola. Results Inoculation of Panax ginseng plants with B. subtilis significantly suppressed the number of disease lesions of C. panacicola and was as effective as the chemical fungicide iminoctadine tris(albesilate). The antifungal activity of B. subtilis against C. panacicola was observed on a co-culture medium. Interestingly, treatment with B. subtilis did not significantly affect the diameter of the lesions, suggesting that the mechanism of protection was through the reduction in the incidence of infection related to the initial events of the infection cycle, including penetration and infection via spore germination and appressorium formation rather than by the inhibition of invasive growth after infection. Conclusion Our results suggest that B. subtilis HK-CSM-1 can be used as an effective and ecologically friendly biological control agent for anthracnose in P. ginseng. PMID:25378997

  6. Production of the polyketide 6-deoxyerythronolide B in the heterologous host Bacillus subtilis.

    PubMed

    Kumpfmüller, Jana; Methling, Karen; Fang, Lei; Pfeifer, Blaine A; Lalk, Michael; Schweder, Thomas

    2016-02-01

    Polyketides, such as erythromycin, are complex natural products with diverse therapeutic applications. They are synthesized by multi-modular megaenzymes, so-called polyketide synthases (PKSs). The macrolide core of erythromycin, 6-deoxyerythronolide B (6dEB), is produced by the deoxyerythronolide B synthase (DEBS) that consists of three proteins each with a size of 330-370 kDa. We cloned and investigated the expression of the corresponding gene cluster from Saccharopolyspora erythraea, which comprises more than 30 kb, in Bacillus subtilis. It is shown that the DEBS genes are functionally expressed in B. subtilis when the native eryAI-III operon was separated into three individual expression cassettes with optimized ribosomal binding sites. A synthesis of 6dEB could be detected by using the acetoin-inducible acoA promoter and a fed-batch simulating EnBase-cultivation strategy. B. subtilis was capable of the secretion of 6dEB into the medium. In order to improve the 6dEB production, several genomic modifications of this production strain were tested. This included the knockout of the native secondary metabolite clusters of B. subtilis for the synthesis of surfactin (26 kb), bacillaene (76 kb), and plipastatin (38 kb). It is revealed that the deletion of the prpBD operon, responsible for propionyl-CoA utilization, resulted in a significant increase of the 6dEB product yield when exogenous propionate is provided. Although the presented B. subtilis 6dEB production strain is not competitive with established Escherichia coli 6dEB production strains, the results of this study indicate that B. subtilis is a suitable heterologous host for the secretory production of a complex polyketide.

  7. Effects of Bacillus subtilis on the reduction of U(VI) by nano-Fe0

    NASA Astrophysics Data System (ADS)

    Ding, Congcong; Cheng, Wencai; Sun, Yubing; Wang, Xiangke

    2015-09-01

    The effects of Bacillus subtilis (B. subtilis, a typical model bacterium) on the reduction of U(VI) by nanoscale zero-valent iron (nano-Fe0) were investigated using batch techniques. The reaction products were analysed using spectroscopic techniques, and a kinetics model was developed to elucidate the mechanisms of U(VI) reduction by nano-Fe0. The presence of B. subtilis enhanced the U(VI) sorption rate at pH 3.5-9.5 but inhibited the reduction rate of U(VI) to U(IV) at pH > 4.5. According to the FTIR and XRD analysis, the reduction of U(VI) to U(IV) was inhibited due to the formation of inner-sphere surface complexes between the oxygen-containing functional groups of B. subtilis or extracellular polymeric substances with the Fe(II)/Fe(III) generated by nano-Fe0, which blocked electron transport from the Fe0 core to U(VI). Based on the EXAFS analysis, a fitting of U-Fe shell at ∼3.44 Å revealed inner-sphere bidentate complexes between uranyl and the oxide film of nano-Fe0. For the nano-Fe0 + B. subtilis system, the U-Fe shell (at ∼3.44 Å) and the U-C/P shell (at ∼2.90 Å) further indicated the formation of inner-sphere surface complexes. The kinetics model supported that U(VI) reduction was triggered by U(VI) sorption on the oxide shell of nano-Fe0. The XPS and XANES analyses showed that reductive precipitation was the main mechanism of U(VI) removal by nano-Fe0, whereas the sorption process dominated the removal of U(VI) in the presence of B. subtilis, which was further demonstrated by TEM images.

  8. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

  9. Display of native proteins on Bacillus subtilis spores.

    PubMed

    Pan, Jae-Gu; Choi, Soo-Keun; Jung, Heung-Chae; Kim, Eui-Joong

    2014-09-01

    In principle, protein display is enabled by fusing target proteins to naturally secreted, surface-anchored protein motifs. In this work, we developed a method of native protein display on the Bacillus spore surface that obviates the need to construct fusion proteins to display a motif. Spore coat proteins are expressed in the mother cell compartment and are subsequently assembled and deposited on the surface of spores. Therefore, target proteins overexpressed in the mother cell compartment during the late sporulation phase were expected to be targeted and displayed on the spore surface. As a proof of principle, we demonstrated the display of carboxymethylcellulase (CMCase) in its native form on the spore surface. The target protein, CMCase, was expressed under the control of the cry1Aa promoter, which is controlled by σ(E) and σ(K) and is expressed in the mother cell compartment. The correct display was confirmed using enzyme activity assays, flow cytometry, and immunogold electron microscopy. In addition, we demonstrated the display of a β-galactosidase tetramer and confirmed its correct display using enzyme activity assays and protein characterization. This native protein display system, combined with the robust nature of Bacillus spores, will broaden the range of displayable target proteins. Consequently, the applications of display technology will be expanded, including high-throughput screening, vaccines, biosensors, biocatalysis, bioremediation, and other innovative bioprocesses.

  10. [A study of the mechanisms of probiotic effect of Bacillus subtilis 8130 strain].

    PubMed

    Ushakova, N A; Kotenkova, E V; Kozlova, A A; Nifatov, A V

    2006-01-01

    The wild-type Bacillus subtilis strain 8130 secreted metabolites that stimulated two to three times the growth of the test cultures of lactic acid bacteria. It exhibited endoglucanase activity that depended on the composition of nutrient medium. The addition of the product of two-stage culturing of B. subtilis 8130 to the diet of pigs (0.2% of fodder weight) made it possible to increase the daily weight gain by 19% and decrease the consumption of mixed fodder by 10%. Digestion of protein, fat, and other organic compounds increased by 3-4% and cellulose by 12%. It was shown that B. subtilis 8130 is a probiotic with targeted action stimulating digestion (primarily the digestion of cellulose). The enrichment of a dry-beer pellet with the product of solid-phase fermentation by bacillus (1 x 10(8) cells per gram dry pellet) allowed the pellet to entered into the diet of a calf (6% of the weight of fodder with probiotic), causing additional weight gain by 12% and a 10% economy of fodder consumption.

  11. Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein.

    PubMed

    Jürgen, B; Hanschke, R; Sarvas, M; Hecker, M; Schweder, T

    2001-04-01

    Bacillus subtilis and related Bacillus species are frequently used as hosts for the industrial production of recombinant proteins. In this study the cellular response of B. subtilis to the overproduction of an insoluble heterologous protein was investigated. For this purpose PorA, an outer membrane protein from Neisseria meningitidis, which accumulates after overexpression in the cytoplasm of B. subtilis mainly in the form of inclusion bodies, was used. The molecular response to overexpression of porA has been analysed at the transcriptional level using the DNA macro array technique and at the translational level by two-dimensional polyacrylamide gel electrophoresis. It was found that the expression of the heat shock genes of class I (dnaK, groEL and grpE) and class III (clpP and clpC) are increased under overproducing conditions. Furthermore, the protein levels of the two ribosomal proteins RpsB and RplJ are increased in the PorA overproducing cells. The transcriptome analysis indicated that mRNA levels of genes encoding pyrimidine and purine synthesis enzymes but also from ribosomal protein genes have elevated levels under overproducing conditions. Finally, the association of the protease ClpP and its ATPase subunits ClpC and ClpX with the PorA inclusion bodies was demonstrated by means of the immunogold labelling technique.

  12. Influence of Glutamic Acid on the Endogenous Respiration of Bacillus subtilis

    PubMed Central

    Clifton, C. E.; Cherry, John

    1966-01-01

    Clifton, C. E. (Stanford University, Stanford, Calif.), and John Cherry. Influence of glutamic acid on the endogenous respiration of Bacillus subtilis. J. Bacteriol. 91:546–550. 1966.—Amino acids serve as the major initial endogenous substrate for Bacillus subtilis. The endogenous activity of freshly harvested washed cells is high and falls off rapidly with time of shaking at 30 C to lower but still significant levels. The rate of O2 consumption after the addition of glutamic acid also decreases as the cells age, but more slowly than noted for endogenous respiration. When cells were fed glutamate as soon as possible after harvesting, an apparent stimulation of endogenous respiration was noted. However, endogenous activity was inhibited if the cell suspensions were shaken for at least 1 hr before addition of the glutamate. Similar results were obtained with glycerol or glucose as exogenous substrates. Variation in rates of respiration with age of the cells, inherent instability of B. subtilis, and possible utilization of substances initially excreted by the cells appear to account for the variations noted regarding the influence of an exogenous substrate on endogenous respiration. PMID:4956754

  13. Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfpo and Escherichia coli entD genes.

    PubMed Central

    Grossman, T H; Tuckman, M; Ellestad, S; Osburne, M S

    1993-01-01

    In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin. We isolated two sets of B. subtilis DNA sequences that complemented the mutations of several E. coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes. One set contained DNA sequences that complemented only an entD mutation. The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations. The two sets of DNA sequences did not appear to overlap. AB. subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics. These data indicate that (i) at least five of the siderophore biosynthesis genes of B. subtilis can function in E. coli, (ii) the genetic organization of these siderophore genes in B. subtilis is similar to that in E. coli, and (iii) the B. subtilis entD homolog is required for efficient growth in low-iron medium. The nucleotide sequence of the B. subtilis DNA contained in plasmid pENTA22, a clone expressing the B. subtilis entD homolog, revealed the presence of at least two genes. One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B. subtilis and which is highly homologous to the E. coli entD gene. We present evidence that the E. coli entD and B. subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules. Images PMID:8407792

  14. Cytokine responses of human intestinal epithelial-like Caco-2 cells to the nonpathogenic bacterium Bacillus subtilis (natto).

    PubMed

    Hosoi, Tomohiro; Hirose, Rieko; Saegusa, Shizue; Ametani, Akio; Kiuchi, Kan; Kaminogawa, Shuichi

    2003-05-15

    Intestinal epithelial cells produce cytokines in response to pathogenic bacteria. However, cellular responses of these cells to nonpathogenic strains, such as Bacillus subtilis, are yet to be determined. In this study, we investigate whether epithelial-like human colon carcinoma Caco-2 cells produce cytokines in response to B. subtilis or B. subtilis (natto). The latter strain is utilized for manufacturing the fermented soy food "natto". Live cells of nonpathogenic B. subtilis JCM 1465(T), B. subtilis (natto) and E. coli JCM 1649(T), as well as pathogenic S. enteritidis JCM 1652 and P. aeruginosa JCM 5516 strains, induced secretion of interleukin-6 (IL-6) and/or IL-8, but not IL-7, IL-15 or tumor necrosis factor alpha (TNF-alpha). Transepithelial electrical resistance (TER) of Caco-2 cell monolayers cultured with E. coli, S. enteritidis or P. aeruginosa decreased more rapidly than that of cells cultured with B. subtilis or B. subtilis (natto). The amounts of cytokine induced by B. subtilis (natto) cells were strain-dependent. Moreover, B. subtilis (natto) cells subjected to hydrochloric acid treatment, but not autoclaving, induced a higher secretion of IL-6 and IL-8 than intact cells. Tyrosine kinase inhibitors, including AG126 and genistein, suppressed cytokine secretion. Our results suggest that the nonpathogenic B. subtilis (natto) bacterium induces cytokine responses in intestinal epithelial cells via activation of an intracellular signaling pathway, such as that of nuclear factor-kappa B (NF-kappaB).

  15. Toxicological assessment of nattokinase derived from Bacillus subtilis var. natto.

    PubMed

    Lampe, Bradley J; English, J Caroline

    2016-02-01

    Subtilisin NAT, commonly known as "nattokinase," is a fibrinolytic enzyme produced by the bacterial strain B. subtilis var. natto, which plays a central role in the fermentation of soybeans into the popular Japanese food natto. Recent studies have reported on the potential anticoagulatory and antihypertensive effects of nattokinase administration in humans, with no indication of adverse effects. To evaluate the safety of nattokinase in a more comprehensive manner, several GLP-compliant studies in rodents and human volunteers have been conducted with the enzyme product, NSK-SD (Japan Bio Science Laboratory Co., Ltd., Japan). Nattokinase was non-mutagenic and non-clastogenic in vitro, and no adverse effects were observed in 28-day and 90-day subchronic toxicity studies conducted in Sprague-Dawley rats at doses up to 167 mg/kg-day and 1000 mg/kg-day, respectively. Mice inoculated with 7.55 × 10(8) CFU of the enzyme-producing bacterial strain showed no signs of toxicity or residual tissue concentrations of viable bacteria. Additionally consumption of 10 mg/kg-day nattokinase for 4 weeks was well tolerated in healthy human volunteers. These findings suggest that the oral consumption of nattokinase is of low toxicological concern. The 90-day oral subchronic NOAEL for nattokinase in male and female Sprague-Dawley rats is 1000 mg/kg-day, the highest dose tested. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Antioxidation, angiotensin converting enzyme inhibition activity, nattokinase, and antihypertension of Bacillus subtilis (natto)-fermented pigeon pea.

    PubMed

    Lee, Bao-Hong; Lai, Yi-Syuan; Wu, She-Ching

    2015-12-01

    Because of the high incidence of cardiovascular diseases in Asian countries, traditional fermented foods from Asia have been increasingly investigated for antiatherosclerotic effects. This study investigated the production of nattokinase, a serine fibrinolytic enzyme, in pigeon pea by Bacillus subtilis fermentation. B. subtilis 14714, B. subtilis 14715, B. subtilis 14716, and B. subtilis 14718 were employed to produce nattokinase. The highest nattokinase activity in pigeon pea was obtained using B. subtilis 14715 fermentation for 32 hours. In addition, the levels of antioxidants (phenolics and flavonoids) and angiotensin converting enzyme inhibitory activity were increased in B. subtilis 14715-fermented pigeon pea, compared with those in nonfermented pigeon pea. In an animal model, we found that both water extracts of pigeon pea (100 mg/kg body weight) and water extracts of B. subtilis-fermented pigeon pea (100 mg/kg body weight) significantly improved systolic blood pressure (21 mmHg) and diastolic blood pressure (30 mmHg) in spontaneously hypertensive rats. These results suggest that Bacillus-fermented pigeon pea has benefits for cardiovascular health and can be developed as a new dietary supplement or functional food that prevents hypertension. Copyright © 2015. Published by Elsevier B.V.

  17. Bacteriocin from Bacillus subtilis as a novel drug against diabetic foot ulcer bacterial pathogens

    PubMed Central

    Joseph, Baby; Dhas, Berlina; Hena, Vimalin; Raj, Justin

    2013-01-01

    Objective To isolate and identify Bacillus subtilis (B. subtilis) from soil and to characterize and partially purify the bacteriocin. To evaluate the antimicrobial activity against four diabetic foot ulcer bacterial pathogens. Methods Genotypic identification was done based on Bergey's manual of systemic bacteriology. Antimicrobial susceptibility test was done by Kirby-Bauer disc diffusion method. Colonies were identified by colony morphology and biochemical characterization and also compared with MTCC 121 strain. Further identification was done by 16S rRNA sequencing. Inhibitory activities of partially purified bacteriocin on all the DFU isolates were done by agar well diffusion method. The strain was identified to produce bacteriocin by stab overlay assay. Bacteriocin was extracted by organic solvent extraction using chloroform, further purified by HPLC and physical, and chemical characterization was performed. Results The four isolates showed high level of resistance to amoxyclav and sensitivity to ciprofloxacin. HPLC purification revealed that the extracts are bacteriocin. The phylogenetic tree analysis results showed that the isolate was 99% related to B. subtilis BSF01. The results reveled activity to all the four isolates and high level of activity was seen in case of Klebsiella sp. Conclusions Partially purified bacteriocin was found to have antimicrobial activity against the four diabetic foot ulcer bacterial pathogens, which can thus be applied as a better drug molecule on further studies. The strain B. subtilis are found to be safe for use and these antimicrobial peptides can be used as an antimicrobial in humans to treat DFU bacterial pathogens. PMID:24093784

  18. Biochemical and Molecular Characterization of the Bacillus subtilis Acetoin Catabolic Pathway

    PubMed Central

    Huang, Min; Oppermann-Sanio, Fred Bernd; Steinbüchel, Alexander

    1999-01-01

    A recent study indicated that Bacillus subtilis catabolizes acetoin by enzymes encoded by the acu gene cluster (F. J. Grundy, D. A. Waters, T. Y. Takova, and T. M. Henkin, Mol. Microbiol. 10:259–271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by aco gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth. By hybridization with a DNA probe covering acoA and acoB of the AoDH ES from Clostridium magnum, genomic fragments from B. subtilis harboring acoA, acoB, acoC, acoL, and acoR homologous genes were identified, and some of them were functionally expressed in E. coli. Furthermore, acoA was inactivated in B. subtilis by disruptive mutagenesis; these mutants were impaired to express PPi-dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source. Therefore, acetoin is catabolized in B. subtilis by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described acu genes obscure. PMID:10368162

  19. Soluble Expression of (+)-γ-Lactamase in Bacillus subtilis for the Enantioselective Preparation of Abacavir Precursor.

    PubMed

    Xue, Tian-Yun; Xu, Guo-Chao; Han, Rui-Zhi; Ni, Ye

    2015-07-01

    Chiral Vince lactam (γ-lactam) is an important precursor of many carbocyclic nucleoside analogues and pharmaceuticals. Here, a (+)-γ-lactamase encoding gene delm from Delftia sp. CGMCC 5755 was identified through genome hunting. To achieve its soluble and functional expression, Escherichia coli and Bacillus subtilis expression systems were introduced. Compared with E. coli system, recombinant (+)-γ-lactamase showed improved protein solubility and catalytic activity in B. subtilis 168. Reaction conditions for enantioselective resolution of γ-lactam were optimized to be at 30 °C, pH 9.0, and 300 rpm when employing the recombinant B. subtilis 168/pMA5-delm whole cells. Kinetic analysis showed that the apparent V max and K m were 0.595 mmol/(min · gDCW) and 378 mmol/L, respectively. No obvious substrate inhibition was observed. In a 500-mL reaction system, enantioselective resolution of 100 g/L γ-lactam was achieved with 10 g/L dry cells, resulting in 55.2 % conversion and 99 % ee of (-)-γ-lactam. All above suggested that recombinant B. subtilis 168/pMA5-delm could potentially be applied in the preparation of optically pure (-)-γ-lactam.

  20. Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine.

    PubMed Central

    Rosenkrantz, M S; Dingman, D W; Sonenshein, A L

    1985-01-01

    The activity of aconitase in Bacillus subtilis is greatly reduced in cells cultured in media containing rapidly metabolized carbon sources (e.g., glucose). Thus, expression of this enzyme appears to be subject to a form of catabolite repression. Since the product of the citB gene of B. subtilis is required for aconitase activity, we cloned the wild-type allele of this gene and used this DNA as a probe for transcription of citB in cells grown in various media. The steady-state level of RNA that hybridized to this probe was about 10-fold higher in B. subtilis cells grown in citrate-glutamine medium than in cells grown in glucose-glutamine medium. This result correlates well with the steady-state levels of aconitase activity. Two transcripts were shown to initiate within the cloned DNA; the steady-state level of one of these transcripts varied in the same way as did aconitase activity when cells were grown in media containing different carbon sources. This is the first demonstration of regulation by the carbon source of the level of a vegatative-cell transcript in B. subtilis. Images PMID:2413006

  1. Cloning of thermostable cellulase genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis.

    PubMed

    Liu, Jian-Ming; Xin, Xiu-Juan; Li, Chun-Xiu; Xu, Jian-He; Bao, Jie

    2012-02-01

    Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.

  2. Metabolic pathway analysis and kinetic studies for production of nattokinase in Bacillus subtilis.

    PubMed

    Unrean, Pornkamol; Nguyen, Nhung H A

    2013-01-01

    We have constructed a reaction network model of Bacillus subtilis. The model was analyzed using a pathway analysis tool called elementary mode analysis (EMA). The analysis tool was used to study the network capabilities and the possible effects of altered culturing conditions on the production of a fibrinolytic enzyme, nattokinase (NK) by B. subtilis. Based on all existing metabolic pathways, the maximum theoretical yield for NK synthesis in B. subtilis under different substrates and oxygen availability was predicted and the optimal culturing condition for NK production was identified. To confirm model predictions, experiments were conducted by testing these culture conditions for their influence on NK activity. The optimal culturing conditions were then applied to batch fermentation, resulting in high NK activity. The EMA approach was also applied for engineering B. subtilis metabolism towards the most efficient pathway for NK synthesis by identifying target genes for deletion and overexpression that enable the cell to produce NK at the maximum theoretical yield. The consistency between experiments and model predictions proves the feasibility of EMA being used to rationally design culture conditions and genetic manipulations for the efficient production of desired products.

  3. Identification of a soluble diacylglycerol kinase required for lipoteichoic acid production in Bacillus subtilis.

    PubMed

    Jerga, Agoston; Lu, Ying-Jie; Schujman, Gustavo E; de Mendoza, Diego; Rock, Charles O

    2007-07-27

    Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.

  4. Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene.

    PubMed Central

    Helmann, J D; Márquez, L M; Chamberlin, M J

    1988-01-01

    Bacillus subtilis contains multiple forms of RNA polymerase holoenzyme, distinguished by the presence of different specificity determinants known as sigma factors. The sigma 28 factor was initially purified as a unique transcriptional activity in vegetatively growing B. subtilis cells. Purification of the sigma 28 protein has allowed tryptic peptides to be prepared and sequenced. The sequence of one tryptic peptide fragment was used to prepare an oligonucleotide probe specific for the sigma 28 structural gene, and the gene was isolated from a B. subtilis subgenomic library. The complete nucleotide sequence of the sigma 28 gene was determined, and the cloned sigma 28 gene was used to construct a mutant strain which does not express the sigma 28 protein. This strain also failed to synthesize flagellin protein and grew as long filaments. The predicted sigma 28 gene product is a 254-amino-acid polypeptide with a calculated molecular weight of 29,500. The sigma 28 protein sequence was similar to that of other sequenced sigma factors and to the flbB gene product of Escherichia coli. Since the flbB gene product is a positive regulator of flagellar synthesis in E. coli, it is likely that sigma 28 functions to regulate flagellar synthesis in B. subtilis. Images PMID:2832368

  5. Genetic modification in Bacillus subtilis for production of C30 carotenoids.

    PubMed

    Maeda, Isamu

    2012-01-01

    C30 carotenoids, which have shorter backbones than C40 carotenoids, are known to be produced in the pathogenic bacterium Staphylococcus aureus that causes opportunistic infection. The first committed enzyme in the C30 carotenoid synthetic pathway is dehydrosqualene synthase CrtM. CrtM converts farnesyl pyrophosphate to dehydrosqualene. Dehydrosqualene desaturase CrtN then converts dehydrosqualene to the yellow C30 carotenoid, 4,4'-diaponeurosporene. This chapter describes a method to synthesize C30 carotenoids in Bacillus subtilis, which is generally recognized as a safe (GRAS) organism. Introduction of S. aureus crtM and crtN genes into B. subtilis results in yellow pigmentation. The B. subtilis transformant accumulates two C30 carotenoids, 4,4'-diapolycopene and 4,4'-diaponeurosporene. Furthermore, together with crtMN, introduction of S. aureus crtP and crtQ genes, which encode mixed function oxidase and glycosyltransferase, respectively, donates the ability to produce glycosylated C30 carotenoic acid. Thus, carotenoid biosynthesis genes of S. aureus is applicable to genetically modify B. subtilis in order to construct a safe organism producing C30 carotenoids.

  6. Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis

    PubMed Central

    Julsing, Mattijs K.; Rijpkema, Michael; Woerdenbag, Herman J.; Quax, Wim J.

    2007-01-01

    In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis. PMID:17458547

  7. Bacillus subtilis Protects Porcine Intestinal Barrier from Deoxynivalenol via Improved Zonula Occludens-1 Expression

    PubMed Central

    Gu, Min Jeong; Song, Sun Kwang; Park, Sung Moo; Lee, In Kyu; Yun, Cheol-Heui

    2014-01-01

    Intestinal epithelial cells (IECs) forming the barrier for the first-line of protection are interconnected by tight junction (TJ) proteins. TJ alteration results in impaired barrier function, which causes potentially excessive inflammation leading to intestinal disorders. It has been suggested that toll-like receptor (TLR) 2 ligands and some bacteria enhance epithelial barrier function in humans and mice. However, no such study has yet to be claimed in swine. The aim of the present study was to examine whether Bacillus subtilis could improve barrier integrity and protection against deoxynivalenol (DON)-induced barrier disruption in porcine intestinal epithelial cell line (IPEC-J2). We found that B. subtilis decreased permeability of TJ and improved the expression of zonula occludens (ZO)-1 and occludin during the process of forming TJ. In addition, ZO-1 expression of IPEC-J2 cells treated with B. subtilis was up-regulated against DON-induced damage. In conclusion, B. subtilis may have potential to enhance epithelial barrier function and to prevent the cells from DON-induced barrier dysfunction. PMID:25049991

  8. Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

    PubMed Central

    Petricek, M; Rutberg, L; Schröder, I; Hederstedt, L

    1990-01-01

    A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon. Images PMID:2110138

  9. Partial biochemical characterization of crude extract extracellular chitinase enzyme from Bacillus subtilis B 298

    NASA Astrophysics Data System (ADS)

    Lestari, P.; Prihatiningsih, N.; Djatmiko, H. A.

    2017-02-01

    Extraction and characterization of extracellular chitinase from Bacillus subtilis B 298 have been done. Growth curve determination of B. subtilis B 298, production curve determination of crude extract chitinase from B. subtilis B 298, and partial biochemical characterization of crude extract chitinase have been achieved in this study. Optimum growth of B. subtilis B 298 was achieved at logarithmic phase within 9 hours incubation time, so it was used as inoculum for enzyme production. According to production curve of the enzyme, it was known that incubation time which gave the highest chitinase activity of 15 hours with activity of 6.937 U/mL respectively. Effect of various temperatures on chitinase activity showed that optimum activity was achieved at 40°C with an activity of 5.764 U/mL respectively. Meanwhile, the optimum pH for chitinase activity was achieved at pH of 5.0 with an activity of 6.813 U/mL respectively. This enzyme was then classified as metalloenzyme due to the decline of the activity by EDTA addition. All divalent cations tested acted as inhibitors.

  10. Mechanism of decay of the cry1Aa mRNA in Bacillus subtilis.

    PubMed Central

    Vázquez-Cruz, C; Olmedo-Alvarez, G

    1997-01-01

    We undertook the study of the decay process of the cry1Aa mRNA of Bacillus thuringiensis expressed in B. subtilis. The cry1Aa transcript is a 3.7-kb mRNA expressed during sporulation whose transcriptional control has previously been studied in both B. subtilis and B. thuringiensis. We found that the cry1Aa mRNA has a half-life of around 9 min and that its decay occurs through endoribonucleolytic cleavages which result in three groups of high-molecular-weight mRNA intermediates ranging in size from 2.7 to 0.5 kb. A comparative study carried out with Escherichia coli showed a similar pattern of degradation intermediates. Primer extension analysis carried out on RNA from B. subtilis revealed that most cleavages occur within two regions located toward the 5' and 3' ends of the mRNA. The most prominent processing site observed for the cry1Aa mRNA isolated from B. subtilis is only two bases away from that occurring on RNA isolated from E. coli. Most cleavage sites occur at seemingly single-stranded RNA segments rich in A and U nucleotides, suggesting that a common and conserved mechanism may process the cry1Aa mRNA. PMID:9335281

  11. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system.

    PubMed

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-06-16

    Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain.

  12. Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase.

    PubMed Central

    Fujita, Y; Freese, E

    1981-01-01

    A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes. Images PMID:6257649

  13. Identification and characterization of the vanillin dehydrogenase YfmT in Bacillus subtilis 3NA.

    PubMed

    Graf, Nadja; Wenzel, Marian; Altenbuchner, Josef

    2016-04-01

    With vanillin as one of the most important flavoring agents, many efforts have been made to optimize its biotechnological production from natural abundant substrates. However, its toxicity against the hosts results in rather low yields and product concentrations. Bacillus subtilis as a soil-dwelling bacterium is a possible lignin-derived compound-degrading microorganism. Therefore, its vanillin and ferulic acid metabolism was investigated. With a rather high tolerance for vanillin up to 20 mM, it is a promising candidate to produce natural vanillin. In this study, the well-studied phenolic acid decarboxylases PadC and BsdBCD could be ascribed to function as the only enzymes in B. subtilis 3NA converting ferulic acid to 4-vinylguaiacol and vanillic acid to guaiacol, respectively. As vanillin also becomes converted to guaiacol, a previous conversion to vanillic acid was assumed. Usage of bioinformatic tools revealed YfmT, which could be shown to function as the only vanillin dehydrogenase in B. subtilis 3NA. Thus, YfmT was further characterized regarding its temperature and pH optima as well as its substrate range. Vanillin and ferulic acid metabolic routes in the tested B. subtilis strain were revealed, a direct conversion of ferulic acid to vanillin, however, could not be found.

  14. Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions.

    PubMed

    Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Chen, Jian; Liu, Long

    By combining advanced omics technology and computational modeling, systems biologists have identified and inferred thousands of regulatory events and system-wide interactions of the bacterium Bacillus subtilis, which is commonly used both in the laboratory and in industry. This dissection of the multiple layers of regulatory networks and their interactions has provided invaluable information for unraveling regulatory mechanisms and guiding metabolic engineering. In this review, we discuss recent advances in the systems biology and metabolic engineering of B. subtilis and highlight current gaps in our understanding of global metabolism and global pathway engineering in this organism. We also propose future perspectives in the systems biology of B. subtilis and suggest ways that this approach can be used to guide metabolic engineering. Specifically, although hundreds of regulatory events have been identified or inferred via systems biology approaches, systematic investigation of the functionality of these events in vivo has lagged, thereby preventing the elucidation of regulatory mechanisms and further rational pathway engineering. In metabolic engineering, ignoring the engineering of multilayer regulation hinders metabolic flux redistribution. Post-translational engineering, allosteric engineering, and dynamic pathway analyses and control will also contribute to the modulation and control of the metabolism of engineered B. subtilis, ultimately producing the desired cellular traits. We hope this review will aid metabolic engineers in making full use of available systems biology datasets and approaches for the design and perfection of microbial cell factories through global metabolism optimization. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2016-08-01

    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.

  16. Inactivation of Bacillus subtilis spores by combined pulsed light and thermal treatments.

    PubMed

    Luz Artíguez, Mari; Martínez de Marañón, Iñigo

    2015-12-02

    The combined effect of pulsed light (PL) and heat processing was evaluated on the inactivation of Bacillus subtilis spores. Those processes were applied separately and the time between both treatments was modified to evaluate whether the effect of the first treatment is maintained for a long time. B. subtilis spores subjected to sublethal pre-treatments were more sensitive to subsequent treatments (PL or thermal treatments) than untreated spores. Heating followed by PL was the most effective combination in reducing B. subtilis counts. Bacterial spores remained sensitized to subsequent treatment for at least 24 h of storage in water, whatever the temperature was (4 or 30°C). Sensitivity of B. subtilis cells to PL or heat processing increased after germination in a nutrient broth, being equally sensitive from 3 to 24 h. Vegetative cells maintained their enhanced sensitivity to subsequent processing after spore germination. The results of this work demonstrate that the combination of heating and PL treatment is a promising preservation method for microbial inactivation. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed Central

    Hsueh, Yi-Huang; Lin, Kuen-Song; Ke, Wan-Ju; Hsieh, Chien-Te; Chiang, Chao-Lung; Tzou, Dong-Ying; Liu, Shih-Tung

    2015-01-01

    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. PMID:26669836

  18. The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA.

    PubMed

    Li, Y; Ferenci, T

    1996-07-01

    The complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined. surA was composed of 1338 bp and encoded 445 amino acid residues. The deduced polypeptide of M(r) 51519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the 'sucrose box' residues thought to be important for catalysis of the transfer of fructose from sucrose. The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA. SurA displayed an optimum temperature for sucrose hydrolysis of 55 degrees C and high stability. The M(r) of SurA determined by gel filtration was 105,000, which suggested that the active form of the enzyme is a dimer. SurA exhibited an apparent Km of 40 mM for sucrose but, unlike the homologous B. subtilis enzyme, had no detectable sucrose phosphate hydrolase activity.

  19. Antimicrobial and plant growth-promoting properties of the cacao endophyte Bacillus subtilis ALB629.

    PubMed

    Falcäo, L L; Silva-Werneck, J O; Vilarinho, B R; da Silva, J P; Pomella, A W V; Marcellino, L H

    2014-06-01

    To investigate the effects of the endophyte Bacillus subtilisALB629 on the growth of cacao seedlings at early developmental stage and to evaluate its antimicrobial properties. Germinating cacao seeds were inoculated with ALB629, and seedlings growth was evaluated 30 days later. Significant increase (P < 0·05) was observed in the root system (up to 30%), leaf area (14%) and stem height (7·6%). ALB629 colonized the entire plant, prevailing over indigenous micro-organisms. In addition, it was tested in vitro, by pairing assays, and showed antagonistic effect against the phytopathogenic fungi Moniliophthora perniciosa, Colletotrichum sp. and C. gossypii. When tested in cacao-grafting procedure in the field, ALB629 increased the grafting success rate (24%), indicating its protective effect. In addition, this Bacillus secretes an antagonist compound, as shown by the antifungal activity of the cell-free culture. Bacillus subtilisALB629 promotes cacao root growth, besides promoting growth of the aerial part of cacao seedlings. It has antimicrobial properties and produces an antifungal compound. ALB629 presented beneficial characteristics for cacao cultivation, being a good biological control agent candidate. Furthermore, it is a potential source of antifungal compound with potential for commercial exploitation. © 2014 The Society for Applied Microbiology.

  20. Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75.

    PubMed

    Shah, Ziaullah; Krumholz, Lee; Aktas, Deniz Fulya; Hasan, Fariha; Khattak, Mutiullah; Shah, Aamer Ali

    2013-11-01

    A polyurethane (PU) degrading bacterial strain MZA-75 was isolated from soil through enrichment technique. The bacterium was identified through 16S rRNA gene sequencing, the phylogenetic analysis indicated the strain MZA-75 belonged to genus Bacillus having maximum similarity with Bacillus subtilis strain JBE0016. The degradation of PU films by strain MZA-75 in mineral salt medium (MSM) was analyzed by scanning electron microscopy (SEM), fourier transform infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM revealed the appearance of widespread cracks on the surface. FTIR spectrum showed decrease in ester functional group. Increase in polydispersity index was observed in GPC, which indicates chain scission as a result of microbial treatment. CO2 evolution and cell growth increased when PU was used as carbon source in MSM in Sturm test. Increase in both cell associated and extracellular esterases was observed in the presence of PU indicated by p-Nitrophenyl acetate (pNPA) hydrolysis assay. Analysis of cell free supernatant by gas chromatography-mass spectrometry (GC-MS) revealed that 1,4-butanediol and adipic acid monomers were produced. Bacillus subtilis strain MZA-75 can degrade the soft segment of polyester polyurethane, unfortunately no information about the fate of hard segment could be obtained. Growth of strain MZA-75 in the presence of these metabolites indicated mineralization of ester hydrolysis products into CO2 and H2O.

  1. Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste.

    PubMed

    Cedrola, Sabrina Martins Lage; de Melo, Ana Cristina Nogueira; Mazotto, Ana Maria; Lins, Ulysses; Zingali, Russolina Benedeta; Rosado, Alexandre Soares; Peixoto, Raquel S; Vermelho, Alane Beatriz

    2012-03-01

    The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml(-1)). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.

  2. SwrAA activates poly-gamma-glutamate synthesis in addition to swarming in Bacillus subtilis.

    PubMed

    Osera, Cecilia; Amati, Giuseppe; Calvio, Cinzia; Galizzi, Alessandro

    2009-07-01

    Poly-gamma-glutamic acid (gamma-PGA) is an extracellular polymer produced by various strains of Bacillus. Iotat was first described as the component of the capsule in Bacillus anthracis, where it plays a relevant role in virulence. gamma-PGA is also a distinctive component of 'natto', a traditional Japanese food consisting of soybean fermented by Bacillus subtilis (natto). Domesticated B. subtilis strains do not synthesize gamma-PGA although they possess the functional biosynthetic pgs operon. In the present work we explore the correlation between the genetic determinants, swrAA and degU, which allow a derivative of the domestic strain JH642 to display a mucoid colony morphology on LB agar plates due to the production of gamma-PGA. Full activation of the pgs operon requires the co-presence of SwrAA and the phosphorylated form of DegU (DegU approximately P). The presence of either DegU approximately P or SwrAA alone has only marginal effects on pgs operon transcription and gamma-PGA production. Although SwrAA was identified as necessary for swarming and full swimming motility together with DegU, we show that motility is not involved in gamma-PGA production. Activation of gamma-PGA synthesis is therefore a motility-independent phenotype in which SwrAA and DegU approximately P display a cooperative effect.

  3. Unraveling the predator-prey relationship of Cupriavidus necator and Bacillus subtilis.

    PubMed

    Seccareccia, Ivana; Kovács, Ákos T; Gallegos-Monterrosa, Ramses; Nett, Markus

    2016-11-01

    Cupriavidus necator is a non-obligate bacterial predator of Gram-negative and Gram-positive bacteria. In this study, we set out to determine the conditions, which are necessary to observe predatory behavior of C. necator. Using Bacillus subtilis as a prey organism, we confirmed that the predatory performance of C. necator is correlated with the available copper level, and that the killing is mediated, at least in part, by secreted extracellular factors. The predatory activity depends on the nutrition status of C. necator, but does not require a quorum of predator cells. This suggests that C. necator is no group predator. Further analyses revealed that sporulation enables B. subtilis to avoid predation by C. necator. In contrast to the interaction with predatory myxobacteria, however, an intact spore coat is not required for resistance. Instead resistance is possibly mediated by quiescence. Copyright © 2016 Elsevier GmbH. All rights reserved.

  4. Adsorption of microbial esterases on Bacillus subtilis-templated cobalt oxide nanoparticles.

    PubMed

    Jang, Eunjin; Ryu, Bum Han; Shim, Hyun-Woo; Ju, Hansol; Kim, Dong-Wan; Kim, T Doohun

    2014-04-01

    Due to low diffusion rates and large surface areas, nanomaterials have received great interest as supporting materials for enzyme immobilization. Here, the preparation of a cobalt oxide nanoparticle using Bacillus subtilis as a biological template and use of the nanostructure for microbial esterase immobilization is described. Morphological features and size distributions were investigated using electron microscopy (EM) and dynamic light scattering (DLS). Catalytic properties of enzyme-coated nanostructures were investigated using 4-methylumbelliferyl acetate and p-nitrophenyl (PNP) acetate as model substrates. Enzyme-coated nanostructures were observed to retain ∼85% of the initial activity after 15 successive reaction cycles, and enzyme immobilization processes could be repeated four times without a loss of immobilization potential. The present work demonstrates that B. subtilis-templated cobalt oxide nanoparticles have the potential to be used as biocompatible immobilization materials, and are promising candidates for the preparation of effective nanobiocatalysts.

  5. Engineering of Bacillus subtilis for the Production of 2,3-Butanediol from Sugarcane Molasses.

    PubMed

    Deshmukh, Apoorva Nandkumar; Nipanikar-Gokhale, Padmaja; Jain, Rishi

    2016-05-01

    2,3-butanediol is known to be a platform chemical with several potential industrial applications. Sustainable industrial scale production can be attained by using a sugarcane molasses based fermentation process using Bacillus subtilis. However, the accumulation of acetoin needs to be reduced to improve process efficiency. In this work, B. subtilis was genetically modified in order to increase the yield of 2,3-butanediol. Metabolic engineering strategies such as cofactor engineering and overexpression of the key enzyme butanediol dehydrogenase were attempted. Both the strategies individually led to a statistically significant increase in the 2,3-butanediol yields for sugarcane molasses based fermentation. Cofactor engineering led to a 26 % increase in 2,3-butanediol yield and overexpression of bdhA led to a 11 % increase. However, the combination of the two strategies did not lead to a synergistic increase in 2,3-butanediol yield.

  6. [An Efficient Method for Genetic Certification of Bacillus subtilis strains, Prospective Producers of Biopreparations].

    PubMed

    Terletskiy, V P; Tyshenko, V I; Novikova, I I; Boikova, I V; Tyulebaev, S D; Shakhtamirov, I Ya

    2016-01-01

    Genetic certification of commercial strains of bacteria antagonistic to phytopathogenic microorganisms guarantees their unequivocal identification and confirmation of safety. In Russia, unlike EU countries, genetic certification of Bacillus subtilis strains is not used. Based on the previously proposed double digestion selective label (DDSL) fingerprinting, a method for genetic identification and certification of B. subtilis strains was proposed. The method was tested on several strains differing in their physiological and biochemical properties and in the composition of secondary metabolites responsible for the spectrum of antibiotic activity. High resolving power of this approach was shown. Optimal restriction endonucleases (SgsI and Eco32I) were determined and validated. A detailed protocol for genetic certification of this bacterial species was developed. DDSL is a universal method, which may be adapted for genetic identification and certification of other bacterial species.

  7. New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

    PubMed Central

    Price, Alan R.; Cook, Sandra J.

    1972-01-01

    The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection. PMID:4623224

  8. Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato.

    PubMed

    Ben Khedher, Saoussen; Kilani-Feki, Olfa; Dammak, Mouna; Jabnoun-Khiareddine, Hayfa; Daami-Remadi, Mejda; Tounsi, Slim

    2015-12-01

    The aim of this study is to evaluate the efficacy of the strain Bacillus subtilis V26, a local isolate from the Tunisian soil, to control potato black scurf caused by Rhizoctonia solani. The in vitro antifungal activity of V26 significantly inhibited R. solani growth compared to the untreated control. Microscopic observations revealed that V26 caused considerable morphological deformations of the fungal hyphae such as vacuolation, protoplast leakage and mycelia crack. The most effective control was achieved when strain V26 was applied 24h prior to inoculation (protective activity) in potato slices. The antagonistic bacterium V26 induced significant suppression of root canker and black scurf tuber colonization compared to untreated controls with a decrease in incidence disease of 63% and 81%, respectively, and promoted plant growth under greenhouse conditions on potato plants. Therefore, B. subtilis V26 has a great potential to be commercialized as a biocontrol agent against R. solani on potato crops.

  9. A novel Bacillus subtilis expression vector based on bacteriophage phi 105.

    PubMed

    Gibson, R M; Errington, J

    1992-11-02

    We have developed a novel expression vector based on the bacteriophage phi 105, and employed it for the production of mutant beta-lactamases in Bacillus subtilis. Expression of the beta-lactamase-encoding gene was low when cloned into the prophage under the control of its own promoter. However, expression was considerably elevated when the gene was inserted into the phage genome in the same orientation as phage transcription. A defective phi 105 vector was constructed with a deletion removing a region needed for cell lysis, and with a mutation in the immunity repressor, rendering it temperature sensitive. Production of beta-lactamase could then be induced by a shift in temperature and without concomitant cell lysis, facilitating purification of the protein from the culture supernatant. This phage has considerable potential for development as a vector for controllable production of heterologous proteins in B. subtilis.

  10. Crystallization and preliminary X-ray analysis of an arabinoxylan arabinofuranohydrolase from Bacillus subtilis

    SciTech Connect

    Vandermarliere, Elien; Bourgois, Tine M.; Van Campenhout, Steven; Strelkov, Sergei V.; Volckaert, Guido; Delcour, Jan A.; Courtin, Christophe M.; Rabijns, Anja

    2007-08-01

    The crystallization and preliminary X-ray analysis of the family 43 glycoside hydrolase arabinoxylan arabinofuranohydrolase from B. subtilis soaked with xylotriose is described in order to gain insight in the way the enzyme binds its substrates. Arabinoxylan arabinofuranohydrolases (AXH) are α-l-arabinofuranosidases (EC 3.2.1.55) that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl residues from arabinoxylan, hence their name. In this study, the crystallization and preliminary X-ray analysis of the AXH from Bacillus subtilis, a glycoside hydrolase belonging to family 43, is described. Purified recombinant AXH crystallized in the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 68.7, b = 73.7, c = 106.5 Å. X-ray diffraction data were collected to a resolution of 1.55 Å.

  11. Influence of Silica Nanoparticles on Antioxidant Potential of Bacillus subtilis IMV B-7023

    NASA Astrophysics Data System (ADS)

    Skorochod, Iryna O.; Roy, Alla O.; Kurdish, Ivan K.

    2016-03-01

    It was found that if introduced into a nutrient medium of 0.05-1 g/L nano-SiO2, the oxidant activity (OA) of the culture medium (CM) of bacilli increased by 43.2-60.1 % and the antioxidant activity (AA) decreased by 4.5-11.8 %. SiO2 nanoparticles had different effects on antiradical activity (ARA) of the CM of Bacillus subtilis IMV B-7023. In particular, nano-SiO2 had no significant effect on the ability of the CM of bacilli to inactivate the 2.2-diphenyl-1-picrylhydrazyl (DPPH·) free radical. However, for the content of the nanomaterial of 0.01-1 g/L decreased hydroxyl radical scavenging in the CM of B. subtilis IMV B-7023 on 7.2-17.6 % compared with a control. Low doses of silica nanoparticles stimulated the reducing power of the CM of bacteria and then highly suppressed it.

  12. Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis.

    PubMed

    Zamboni, Nicola; Mouncey, Nigel; Hohmann, Hans-Peter; Sauer, Uwe

    2003-01-01

    We present redirection of electron flow to more efficient proton pumping branches within respiratory chains as a generally applicable metabolic engineering strategy, which tailors microbial metabolism to the specific requirements of high cell density processes by improving product and biomass yields. For the example of riboflavin production by Bacillus subtilis, we reduced the rate of maintenance metabolism by about 40% in a cytochrome bd oxidase knockout mutant. Since the putative Yth and the caa(3) oxidases were of minor importance, the most likely explanation for this improvement is translocation of two protons per transported electron via the remaining cytochrome aa(3) oxidase, instead of only one proton via the bd oxidase. The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and B. subtilis biomass in fed-batch cultures.

  13. Surface charge and hydrodynamic coefficient measurements of Bacillus subtilis spore by optical tweezers.

    PubMed

    Pesce, Giuseppe; Rusciano, Giulia; Sasso, Antonio; Isticato, Rachele; Sirec, Teja; Ricca, Ezio

    2014-04-01

    In this work we report on the simultaneous measurement of the hydrodynamic coefficient and the electric charge of single Bacillus subtilis spores. The latter has great importance in protein binding to spores and in the adhesion of spores onto surfaces. The charge and the hydrodynamic coefficient were measured by an accurate procedure based on the analysis of the motion of single spores confined by an optical trap. The technique has been validated using charged spherical polystyrene beads. The excellent agreement of our results with the expected values demonstrates the quality of our procedure. We measured the charge of spores of B. subtilis purified from a wild type strain and from two isogenic mutants characterized by an altered spore surface. Our technique is able to discriminate the three spore types used, by their charge and by their hydrodynamic coefficient which is related to the hydrophobic properties of the spore surface. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Number of Deoxyribonucleic Acid Uptake Sites in Competent Cells of Bacillus subtilis

    PubMed Central

    Singh, R. N.

    1972-01-01

    Two direct methods are presented for estimating the average number of deoxyribonucleic acid (DNA) uptake sites in competent cells of Bacillus subtilis from measurement of 14C- or 3H-thymine-labeled DNA uptake by competent culture. Advantage is taken of two facts: (i) effective contact between competent cells and transforming DNA molecules is established within a short time after mixing them together, and (ii) DNA molecules enter the competent B. subtilis cells in a linear fashion at a finite speed. From the number of DNA molecules initially attached to competent cells by brief exposure to transforming DNA in the first method or from the rate of DNA uptake by competent culture in the second method, the average number of DNA uptake sites is calculated to be 20 to 53 per competent cell. PMID:4622899

  15. Differential Gene Expression to Investigate the Effects of Low-level Electrochemical Currents on Bacillus subtilis

    PubMed Central

    2011-01-01

    With the emergence and spread of multidrug resistant bacteria, effective methods to eliminate both planktonic bacteria and those embedded in surface-attached biofilms are needed. Electric currents at μA-mA/cm2 range are known to reduce the viability of bacteria. However, the mechanism of such effects is still not well understood. In this study, Bacillus subtilis was used as the model Gram-positive species to systematically investigate the effects of electrochemical currents on bacteria including the morphology, viability, and gene expression of planktonic cells, and viability of biofilm cells. The data suggest that weak electrochemical currents can effectively eliminate B. subtilis both as planktonic cells and in biofilms. DNA microarray results indicate that the genes associated with oxidative stress response, nutrient starvation, and membrane functions were induced by electrochemical currents. These findings suggest that ions and oxidative species generated by electrochemical reactions might be important for the killing effects of these currents. PMID:22078549

  16. Rice Seed Priming with Picomolar Rutin Enhances Rhizospheric Bacillus subtilis CIM Colonization and Plant Growth.

    PubMed

    Singh, Akanksha; Gupta, Rupali; Pandey, Rakesh

    2016-01-01

    The effect of rutin, a bioflavonoid on the growth and biofilm formation of Bacillus subtilis strain CIM was investigated. In addition to swimming, swarming, and twitching potentials of B. subtilis CIM (BS), one picomolar (1 pM) of rutin was also observed to boost the biofilm forming ability of the bacterium. Bio-priming of rice seeds with BS and rutin not only augmented root and shoot lengths but also the photosynthetic pigments like chlorophyll and carotenoid. Similarly, high accumulation of phenolic and flavonoid contents was observed in the leaves. Fluorescent microscopic images revealed that BS plus rutin enhanced callose deposition in the leaves. It was also established that the least formation of reactive oxygen species in BS plus rutin treated rice plants was due to higher free radicals scavenging activity and total antioxidant potential. The results highlight chemo attractant nature of BS towards rutin, which by enhancing biofilm formation and root colonization indirectly strengthened the plants' defensive state.

  17. Rice Seed Priming with Picomolar Rutin Enhances Rhizospheric Bacillus subtilis CIM Colonization and Plant Growth

    PubMed Central

    Singh, Akanksha; Gupta, Rupali; Pandey, Rakesh

    2016-01-01

    The effect of rutin, a bioflavonoid on the growth and biofilm formation of Bacillus subtilis strain CIM was investigated. In addition to swimming, swarming, and twitching potentials of B. subtilis CIM (BS), one picomolar (1 pM) of rutin was also observed to boost the biofilm forming ability of the bacterium. Bio-priming of rice seeds with BS and rutin not only augmented root and shoot lengths but also the photosynthetic pigments like chlorophyll and carotenoid. Similarly, high accumulation of phenolic and flavonoid contents was observed in the leaves. Fluorescent microscopic images revealed that BS plus rutin enhanced callose deposition in the leaves. It was also established that the least formation of reactive oxygen species in BS plus rutin treated rice plants was due to higher free radicals scavenging activity and total antioxidant potential. The results highlight chemo attractant nature of BS towards rutin, which by enhancing biofilm formation and root colonization indirectly strengthened the plants’ defensive state. PMID:26742102

  18. Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis.

    PubMed

    Sato, T; Yamada, Y; Ohtani, Y; Mitsui, N; Murasawa, H; Araki, S

    2001-03-01

    Efficient production of menaquinone (MK) by Bacillus subtilis was achieved. An edible strain of B. subtilis, isolated from the traditional Japanese food natto, was mutated to improve MK productivity. A menadione-resistant mutant producing 30% more MK than its parent strain was obtained. Soybean extract and glycerol were the best nitrogen and carbon sources, respectively, among the sources tested. Addition of yeast extract also increased MK productivity. The maximum concentration of MK reached about 35.0 mg/l after 4 days of culture in a jar fermenter. The pH of the medium decreased to 5.5 after the start of cultivation, then spontaneously increased to 7.7-8.0. This pH change might be important in the production of MK because only small amounts of MK were obtained when pH was controlled at 5.7, 6.0, 7.0, 7.5 or 8.0.

  19. Intracellular Biosynthesis of Fluorescent CdSe Quantum Dots in Bacillus subtilis: A Strategy to Construct Signaling Bacterial Probes for Visually Detecting Interaction Between Bacillus subtilis and Staphylococcus aureus.

    PubMed

    Yan, Zheng-Yu; Ai, Xiao-Xia; Su, Yi-Long; Liu, Xin-Ying; Shan, Xiao-Hui; Wu, Sheng-Mei

    2016-02-01

    In this work, fluorescent Bacillus subtilis (B. subtilis) cells were developed as probes for imaging applications and to explore behaviorial interaction between B. subtilis and Staphylococcus aureus (S. aureus). A novel biological strategy of coupling intracellular biochemical reactions for controllable biosynthesis of CdSe quantum dots by living B. subtilis cells was demonstrated, through which highly luminant and photostable fluorescent B. subtilis cells were achieved with good uniformity. With the help of the obtained fluorescent B. subtilis cells probes, S. aureus cells responded to co-cultured B. subtilis and to aggregate. The degree of aggregation was calculated and nonlinearly fitted to a polynomial model. Systematic investigations of their interactions implied that B. subtilis cells inhibit the growth of neighboring S. aureus cells, and this inhibition was affected by both the growth stage and the amount of surrounding B. subtilis cells. Compared to traditional methods of studying bacterial interaction between two species, such as solid culture medium colony observation and imaging mass spectrometry detection, the procedures were more simple, vivid, and photostable due to the efficient fluorescence intralabeling with less influence on the cells' surface, which might provide a new paradigm for future visualization of microbial behavior.

  20. The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNA(Asn).

    PubMed

    Nair, Nilendra; Raff, Hannah; Islam, Mohammed Tarek; Feen, Melanie; Garofalo, Denise M; Sheppard, Kelly

    2016-02-13

    Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code.

  1. Altered promoter selection by a novel form of Bacillus subtilis RNA polymerase.

    PubMed

    Jaehning, J A; Wiggs, J L; Chamberlin, M J

    1979-11-01

    Bacillus subtilis RNA polymerase holoenzyme prepared by several standard methods utilizes bacteriophage T7 DeltaD111 DNA as an efficient template. The major RNA products are specific transcripts from T7 promoters A(1) and C; these promoters are also efficiently utilized by RNA polymerases purified from a wide range of other bacterial species [Wiggs, J., Bush, J. & Chamberlin, M. (1979) Cell 16, 97-109]. In contrast, B. subtilis RNA polymerase preparations purified by a modification of the method of Burgess and Jendrisak (designated fraction 5) utilize T7 DeltaD111 promoters A(1) and C and an additional promoter site, J, which has been located at 90.6% on the standard T7 physical map. This promoter is not used by B. subtilis core RNA polymerase or by RNA polymerase from any other bacterial species we have tested. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of fraction 5 RNA polymerase shows that it contains B. subtilis components sigma and delta and a polypeptide of M(r) 92,000 in addition to the B. subtilis beta, beta', and alpha subunits. Chromatography of fraction 5 on single-stranded DNA-cellulose gives an enzyme fraction, Bs I, that is indistinguishable from B. subtilis RNA polymerase holoenzyme both in its peptide composition (betabeta'alpha(2)sigma) and in the selective transcription of only T7 RNAs A(1) and C. Chromatography of fraction 5 on phosphocellulose yields an enzyme fraction, Bs II, devoid of sigma subunit but containing the M(r) 92,000 peptide and traces of delta. This fraction synthesizes predominantly T7 J RNA in vitro together with traces of T7 A(1) and C RNAs. Hence, B. subtilis RNA polymerase fraction Bs II appears to contain a form of RNA polymerase that can transcribe selectively without detectable amounts of B. subtilis sigma subunit and that utilizes a promoter site not used by other known bacterial RNA polymerases. The structural basis for this specificity is not yet known.

  2. Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

    PubMed Central

    Westbrook, Adam W.; Moo-Young, Murray

    2016-01-01

    ABSTRACT The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for Escherichia coli and Saccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery. IMPORTANCE In this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in Bacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of B. subtilis strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application. PMID:27260361

  3. Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions.

    PubMed

    Ploss, Tina N; Reilman, Ewoud; Monteferrante, Carmine G; Denham, Emma L; Piersma, Sjouke; Lingner, Anja; Vehmaanperä, Jari; Lorenz, Patrick; van Dijl, Jan Maarten

    2016-03-29

    Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. Here we show that activity of the htrB promoter as induced by overproduction of AmyM was "noisy", which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

  4. Biodegradation of crude oil by a defined co-culture of indigenous bacterial consortium and exogenous Bacillus subtilis.

    PubMed

    Tao, Kaiyun; Liu, Xiaoyan; Chen, Xueping; Hu, Xiaoxin; Cao, Liya; Yuan, Xiaoyu

    2017-01-01

    The aim of this work was to study biodegradation of crude oil by defined co-cultures of indigenous bacterial consortium and exogenous Bacillus subtilis. Through residual oil analysis, it is apparent that the defined co-culture displayed a degradation ratio (85.01%) superior to indigenous bacterial consortium (71.32%) after 7days of incubation when ratio of inoculation size of indigenous bacterial consortium and Bacillus subtilis was 2:1. Long-chain n-alkanes could be degraded markedly by Bacillus subtilis. Result analysis of the bacterial community showed that a decrease in bacterial diversity in the defined co-culture and the enrichment of Burkholderiales order (98.1%) degrading hydrocarbons. The research results revealed that the promising potential of the defined co-culture for application to degradation of crude oil.

  5. Draft Genome Sequence of Bacillus subtilis subsp. natto Strain CGMCC 2108, a High Producer of Poly-γ-Glutamic Acid.

    PubMed

    Tan, Siyuan; Meng, Yonghong; Su, Anping; Zhang, Chen; Ren, Yuanyuan

    2016-05-26

    Here, we report the 4.1-Mb draft genome sequence of Bacillus subtilis subsp. natto strain CGMCC 2108, a high producer of poly-γ-glutamic acid (γ-PGA). This sequence will provide further help for the biosynthesis of γ-PGA and will greatly facilitate research efforts in metabolic engineering of B. subtilis subsp. natto strain CGMCC 2108. Copyright © 2016 Tan et al.

  6. Mixed culture models for predicting intestinal microbial interactions between Escherichia coli and Lactobacillus in the presence of probiotic Bacillus subtilis.

    PubMed

    Yang, J J; Niu, C C; Guo, X H

    2015-01-01

    Bacillus has been proposed as a probiotic due to its in vivo effectiveness in the gastrointestinal tract through antimicrobial activities. The present study investigates the effects of Lactobacillus alone or in the presence of Bacillus subtilis MA139 on the inhibition of pathogenic Escherichia coli K88. Mixed cultures were used to predict the possible interactions among these bacteria within the intestinal tract of animals. B. subtilis MA139 was first assayed for its inhibition against E. coli K88 both under shaking and static culture conditions. A co-culture assay was employed under static conditions to test the inhibitory effects of Lactobacillus reuteri on E. coli K88, with or without addition of B. subtilis MA139. The results showed that B. subtilis MA139 had marked inhibition against E. coli K88 under shaking conditions and weak inhibition under static conditions. Lactobacillus alone as well as in combination with B. subtilis MA139 spores exerted strong inhibition against E. coli K88 under static conditions. However, the inhibition by Lactobacillus in combination with B. subilis spores was much higher than that by Lactobacillus alone (P<0.01). B. subtilis MA139 significantly decreased the pH and oxidation-reduction potential values of the co-culture broth compared to that of Lactobacillus alone (P<0.05). The viability of Lactobacillus increased when co-cultured with B. subtilis MA139 because of significantly higher Lactobacillus counts and lower pH values in the broth (P<0.05). The role of Bacillus in the mixed culture models suggests that Bacillus may produce beneficial effects by increasing the viability of lactobacilli and subsequently inhibiting the growth of pathogenic E. coli. Therefore, the combination of Bacillus and Lactobacillus species as a probiotic is recommended.

  7. Comparative Genomic Analysis of Bacillus amyloliquefaciens and Bacillus subtilis Reveals Evolutional Traits for Adaptation to Plant-Associated Habitats

    PubMed Central

    Zhang, Nan; Yang, Dongqing; Kendall, Joshua R. A.; Borriss, Rainer; Druzhinina, Irina S.; Kubicek, Christian P.; Shen, Qirong; Zhang, Ruifu

    2016-01-01

    Bacillus subtilis and its sister species B. amyloliquefaciens comprise an evolutionary compact but physiologically versatile group of bacteria that includes strains isolated from diverse habitats. Many of these strains are used as plant growth-promoting rhizobacteria (PGPR) in agriculture and a plant-specialized subspecies of B. amyloliquefaciens—B. amyloliquefaciens subsp. plantarum, has recently been recognized, here we used 31 whole genomes [including two newly sequenced PGPR strains: B. amyloliquefaciens NJN-6 isolated from Musa sp. (banana) and B. subtilis HJ5 from Gossypium sp. (cotton)] to perform comparative analysis and investigate the genomic characteristics and evolution traits of both species in different niches. Phylogenomic analysis indicated that strains isolated from plant-associated (PA) habitats could be distinguished from those from non-plant-associated (nPA) niches in both species. The core genomes of PA strains are more abundant in genes relevant to intermediary metabolism and secondary metabolites biosynthesis as compared with those of nPA strains, and they also possess additional specific genes involved in utilization of plant-derived substrates and synthesis of antibiotics. A further gene gain/loss analysis indicated that only a few of these specific genes (18/192 for B. amyloliquefaciens and 53/688 for B. subtilis) were acquired by PA strains at the initial divergence event, but most were obtained successively by different subgroups of PA stains during the evolutional process. This study demonstrated the genomic differences between PA and nPA B. amyloliquefaciens and B. subtilis from different niches and the involved evolutional traits, and has implications for screening of PGPR strains in agricultural production. PMID:28066362

  8. Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis

    PubMed Central

    Watzlawick, Hildegard; Altenbuchner, Josef

    2016-01-01

    ABSTRACT Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the −35 box of the σA-type promoter of Pgan, which is located upstream of ganS. IMPORTANCE Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis. Nevertheless, the B. subtilis utilization system of galactan

  9. Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.

    PubMed

    Watzlawick, Hildegard; Morabbi Heravi, Kambiz; Altenbuchner, Josef

    2016-10-15

    Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is

  10. [Effect of carbon and nitrogen sources and complex B vitamins on the synthesis of alkaline protease by different strains of Bacillus mesentericus and Bacillus subtilis].

    PubMed

    Emtseva, T V

    1975-01-01

    The effect of different sources of carbon, nitrogen, amino acids and vitamins on the synthesis of alkaline proteases by the stock and mutant strains of Bacillus mesentericus and by the natural strain of Bacillus subtilis-12 has been investigated. The maximum synthesis of alkaline protease has been obtained in the media containing starch or its hydrolysates--dextrine and maltose as the carbon source. Ammonium phosphate and casein as the nitrogen source prove to be optimal for Bac. mesentericus and Bac. subtilis, respectively. Complex B vitamins added to the nutrient medium accelerate the enzyme synthesis 2.5-4-fold.

  11. Purine salvage pathways of Bacillus subtilis and effect of guanine on growth of GMP reductase mutants.

    PubMed Central

    Endo, T; Uratani, B; Freese, E

    1983-01-01

    We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic chart of the purine salvage pathway of Bacillus subtilis. PMID:6408059

  12. Specificity of Metal Sensing: Iron and Manganese Homeostasis in Bacillus subtilis*

    PubMed Central

    Helmann, John D.

    2014-01-01

    Metalloregulatory proteins allow cells to sense metal ions and appropriately adjust the expression of metal uptake, storage, and efflux pathways. Bacillus subtilis provides a model for the coordinate regulation of iron and manganese homeostasis that involves three key regulators: Fur senses iron sufficiency, MntR senses manganese sufficiency, and PerR senses the intracellular Fe/Mn ratio. Here, I review the structural and physiological bases of selective metal perception, the effects of non-cognate metals, and mechanisms that may serve to coordinate iron and manganese homeostasis. PMID:25160631

  13. [Anaerobic solid-phase fermentation of plant substrates by Bacillus subtilis].

    PubMed

    Ushakova, N A; Brodskiĭ, E S; Kozlova, A A; Nifatov, A V

    2009-01-01

    Solid-phase growth of Bacillus subtilis 8130 on cellulose-rich plant substrates (presscakes or pulp) under hypoxic conditions was accompanied by cellulose depolymerization, protein hydrolysis, and degradation of other plant components, including some processes of mixed-type carbohydrate fermentation. The bacterial fermentation yielded propionic, butyric, and hexanoic acids and butyric acid derivatives. The bacterial metabolism and fermentation degree can be characterized by the proportions of fatty acids in the reaction mixture. The product of sea buckthorn cake fermentation has a good sorption quality.

  14. Crystallization and preliminary X-ray analysis of an arabinoxylan arabinofuranohydrolase from Bacillus subtilis

    PubMed Central

    Vandermarliere, Elien; Bourgois, Tine M.; Van Campenhout, Steven; Strelkov, Sergei V.; Volckaert, Guido; Delcour, Jan A.; Courtin, Christophe M.; Rabijns, Anja

    2007-01-01

    Arabinoxylan arabinofuranohydrolases (AXH) are α-l-arabinofuranosidases (EC 3.2.1.55) that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl residues from arabinoxylan, hence their name. In this study, the crystallization and preliminary X-ray analysis of the AXH from Bacillus subtilis, a glycoside hydrolase belonging to family 43, is described. Purified recombinant AXH crystallized in the orthorhombic space group P212121, with unit-cell parameters a = 68.7, b = 73.7, c = 106.5 Å. X-ray diffraction data were collected to a resolution of 1.55 Å. PMID:17671370

  15. Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry.

    PubMed

    Gonzalez, David J; Haste, Nina M; Hollands, Andrew; Fleming, Tinya C; Hamby, Matthew; Pogliano, Kit; Nizet, Victor; Dorrestein, Pieter C

    2011-09-01

    Microbial competition exists in the general environment, such as soil or aquatic habitats, upon or within unicellular or multicellular eukaryotic life forms. The molecular actions that govern microbial competition, leading to niche establishment and microbial monopolization, remain undetermined. The emerging technology of imaging mass spectrometry (IMS) enabled the observation that there is directionality in the metabolic output of the organism Bacillus subtilis when co-cultured with Staphylococcus aureus. The directionally released antibiotic alters S. aureus virulence factor production and colonization. Therefore, IMS provides insight into the largely hidden nature of competitive microbial encounters and niche establishment, and provides a paradigm for future antibiotic discovery.

  16. Decontamination of Bacillus subtilis Spores in a Sealed Package Using a Non-thermal Plasma System

    NASA Astrophysics Data System (ADS)

    Keener, Kevin M.; Jensen, J. L.; Valdramidis, V. P.; Byrne, E.; Connolly, J.; Mosnier, J. P.; Cullen, P. J.

    The safety of packaged food and medical devices is a major concern to consumers and government officials. Recent inventions (PK-1 and PK-2) based on the principles of non-thermal, atmospheric plasma has shown significant reduction in bacterial contamination inside a sealed package. The objective of this study was to evaluate the PK-1 and PK-2 systems in the reduction of Bacillus subtilis spores using packages containing air or modified atmosphere (MA) gas (65% O2/30% CO2/5% N2). The experimental design consisted of the following parameters: (1) two voltage conditions: 13.5 kV with 1.0 cm electrode gap (PK-1) and 80 kV with 4.5 cm electrode gap (PK-2), (2) two treatment conditions: inside and outside the field of ionization, (3) PK-1 and PK-2 optimized treatment times: 300 and 120 s, respectively, and (4) two package gas types: air and modified atmosphere (MA) gas (65% O2/30% CO2/5% N2). Measurements included: (1) bacterial reductions of Bacillus subtilis var. niger (B. atrophaeus), (2) ozone, nitrous oxides (NOx), and carbon monoxide concentrations, and (3) relative humidity. Bacillus subtilis (1.7 × 106/strip) were loaded into sterile uncovered petri dishes and treated with ionization generated in packages using air or MA gas blend. Samples were treated for 300 s (PK-1) or 120 s (PK-2) and stored at room ­temperature for 24 h. Results documented relative humidity (RH) ranged from 20% to 30%. After 300 s of PK-1 treatment (13.5 kV/44 W/1.0 cm gap), ozone concentrations were 6,000 ppm (air) and 7,500 ppm (MA). After 120 s of PK-2 treatment (80 kV/150 W/4.5 cm), ozone concentrations were 7,500 ppm (air) and 12,000 ppm (MA). Ozone and NOx concentrations were non-detect (ND) after 24 h. PK-1 carbon monoxide levels were <20 ppm (air) and <100 ppm (MA) after 24 h. The PK-2 carbon monoxide levels were <20 ppm (air) and <400 ppm (MA) after 24 h. Treatments showed reductions in spores of greater than 6 log10 after 24 h. Reductions were maintained without additional re

  17. Thermal death of Bacillus subtilis spores in oil-water systems.

    PubMed

    Shigemoto, Mayumi; Nakagawa, Kayo; Sakamoto, Jin J; Tsuchidoi, Tetsuaki

    2010-03-01

    The thermal death of the spores of Bacillus subtilis 168 in oil-water systems including emulsions and separated layers consisting of phosphate buffer and soybean oil or n-hexadecane was investigated. The resultant survivor curve consisted of two phases, an initial rapid reduction followed by a slow reduction, possibly as reflected by the death in the water phase and the oil phase, respectively. The concentration of oil in the system strikingly affected the pattern of thermal death. These results suggest that the spore location in the oil-water system may be a critical factor in determining the heat resistance.

  18. Role of sugar uptake and metabolic intermediates on catabolite repression in Bacillus subtilis.

    PubMed Central

    Lopez, J M; Thoms, B

    1977-01-01

    Many phosphorylated intermediates exert catabolite repression on the enzyme acetoin dehydrogenase in Bacillus subtilis. This was shown with strains that are blocked at different positions in central metabolism when they receive sugars that cannot be metabolized past enzymatic block(s). In the case of sorbitol, transport events were not involved in catabolite repression, for this sugar cannot repress acetoin dehydrogenase in a strain lacking sorbitol dehydrogenase but otherwise able to take up sorbitol. The presence of glucose did not markedly influence the uptake of acetoin. PMID:401492

  19. Psoralen-plus-light damage and repair in transforming DNA of Bacillus subtilis

    SciTech Connect

    Hadden, C.T.

    1981-01-01

    The relative contributions of excision and recombination in the repair of damage by 8-methoxypsoralen (8-MOP) plus black light to Bacillus subtilis were studied. The results indicate that the pyrimidine dimer excision system and a recombination pathway are probably both involved in repair of lethal damage to cells exposed in vivo to 8-MOP plus black light, but repair is not very efficient. Transforming DNA exposed in vitro to 8-MOP plus black light was inactivated mainly by crosslinks rather than by monoadducts, and was repaired predominantly by an incision-dependent process. There was very little demonstrable damage-induced recombination in transforming DNA.

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

    NASA Technical Reports Server (NTRS)

    Mennigmann, Horst-Dieter

    1992-01-01

    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.

  1. Cell Wall Binding Properties of the Bacillus subtilis Autolysin(s)

    PubMed Central

    Fan, David P.

    1970-01-01

    Cell walls isolated from exponentially growing Bacillus subtilis have autolysin(s) attached to them. An autolysin can be released from the walls by incubation at 0 C with 3 m LiCl. The enzyme can reattach to walls when the salt concentration is reduced. The bound enzyme cannot be removed or destroyed by washing the walls with 8 m urea at 0 C. The binding of free enzyme to walls at 0 C can take place normally in the presence of 2 m urea. PMID:4988245

  2. Dry-heat Resistance of Bacillus Subtilis Var. Niger Spores on Mated Surfaces

    NASA Technical Reports Server (NTRS)

    Simko, G. J.; Devlin, J. D.; Wardle, M. D.

    1971-01-01

    Bacillus subtilis var. niger spores were placed on the surfaces of test coupons manufactured from typical spacecraft materials including stainless steel, magnesium, titanium, and aluminum. These coupons were then juxtaposed at the inoculated surfaces and subjected to test pressures of 0, 1000, 5000, and 10,000 psi. Tests were conducted in ambient, nitrogen, and helium atmospheres. While under the test pressure condition, the spores were exposed to 125 C for intervals of 5, 10, 20, 50, or 80 min. Survivor data were subjected to a linear regression analysis that calculated decimal reduction times.

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

    NASA Technical Reports Server (NTRS)

    Mennigmann, Horst-Dieter

    1992-01-01

    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.

  4. SURVIVAL OF MICROORGANISMS IN A SIMULATED MARTIAN ENVIRONMENT. I. BACILLUS SUBTILIS VAR. GLOBIGII.

    PubMed

    HAGEN, C A; HAWRYLEWICZ, E J; EHRLICH, R

    1964-05-01

    Survival of Bacillus subtilis var. globigii in a simulated Martian environment was demonstrated. Previous contact with the simulated Martian soil or atmosphere reduced germination or outgrowth of unheated spores, or both. Inoculation into simulated Martian soil and then flushing with a simulated Martian atmosphere were lethal to both vegetative cells and spores. After one diurnal temperature cycle (26 to -60 C), the majority of of cells present were spores. No further effect of the diurnal cycle on survival was noted in any of the experimental samples.

  5. Second stage production of iturin A by induced germination of Bacillus subtilis RB14.

    PubMed

    Rahman, Mohammad Shahedur; Ano, Takashi; Shoda, Makoto

    2006-10-01

    Bacillus subtilis RB14, a dual producer of lipopeptide antibiotics iturin A and surfactin undergoes sporulation in the submerged fermentation and the production of these secondary metabolites becomes halted. In this study, production of lipopeptide antibiotics was investigated by induced germination of the spores by heat-activation and nutrient supplementation. The induced spores became metabolically active vegetative state and produced lipopeptide antibiotic iturin A that added up the total production at the end of the fermentation. However, additional production of surfactin was not observed. This second time iturin A production by the germinated cells from the spores was defined as second stage production.

  6. Development of natto with germination-defective mutants of Bacillus subtilis (natto).

    PubMed

    Mitsui, Nobuo; Murasawa, Hisashi; Sekiguchi, Junichi

    2009-03-01

    The effects of cortex-lysis related genes with the pdaA, sleB, and cwlD mutations of Bacillus subtilis (natto) NAFM5 on sporulation and germination were investigated. Single or double mutations did not prevent normal sporulation, but did affect germination. Germination was severely inhibited by the double mutation of sleB and cwlD. The quality of natto made with the sleB cwlD double mutant was tested, and the amounts of glutamic acid and ammonia were very similar to those in the wild type. The possibility of industrial development of natto containing a reduced number of viable spores is presented.

  7. Sterilization of Bacillus subtilis Spores Using an Atmospheric Plasma Jet with Argon and Oxygen Mixture Gas

    NASA Astrophysics Data System (ADS)

    Shen, Jie; Cheng, Cheng; Fang, Shidong; Xie, Hongbing; Lan, Yan; Ni, Guohua; Meng, Yuedong; Luo, Jiarong; Wang, Xiangke

    2012-03-01

    To determine an efficient sterilization mechanism, Bacillus subtilis spore samples were exposed to an atmospheric plasma jet. By using argon/oxygen mixture gas, the decimal reduction value was reduced from 60 s (using argon gas) to 10 s. More dramatically, after 5 min treatment, the colony-forming unit (CFU) was reduced by six orders. To understand the underlying mechanism of the efficient sterilization by plasma, the contributions from heat, UV radiation, charged particles, ozone, and reactive oxygen radicals were distinguished in this work, showing that charged particles and ozone were the main killing factors. The shape changes of the spores were also discussed.

  8. Simultaneous and selective production of levan and poly(gamma-glutamic acid) by Bacillus subtilis.

    PubMed

    Shih, Ing-Lung; Yu, Yun-Ti

    2005-01-01

    Bacillus subtilis(natto) Takahashi, used to prepare the fermented soybean product natto, was grown in a basal medium containing 5% (w/w) sucrose and 1.5% (w/w) L-glutamate and produced 58% (w/w) poly(gamma-glutamic acid) and 42% (w/w) levan simultaneously. After 21 h, 40-50 mg levan ml-1 had been produced in medium containing 20% (w/w) sucrose but without L-glutamate. In medium containing L-glutamic acid but without sucrose, mainly poly(gamma-glutamic acid) was produced.

  9. Moderate expression of the transcriptional regulator ALsR enhances acetoin production by Bacillus subtilis.

    PubMed

    Zhang, Xian; Zhang, Rongzhen; Bao, Teng; Yang, Taowei; Xu, Meijuan; Li, Huazhong; Xu, Zhenghong; Rao, Zhiming

    2013-09-01

    Acetoin, a major extracellular catabolic product of Bacillus subtilis cultured on glucose, is widely used to add flavor to food and also serves as a precursor for chemical synthesis. The biosynthesis of acetoin from pyruvate requires the enzymes α-acetolactate synthase (ALS) and α-acetolactate decarboxylase (ALDC), both of which are encoded by the alsSD operon. The transcriptional regulator ALsR is essential for the expression of alsSD. Here we focused on enhancing the production of acetoin by B. subtilis using different promoters to express ALsR. The expression of reporter genes was much higher under the control of the HpaII promoter than under control of the P bdhA promoter. Although the HpaII promoter highly enhanced transcription of the alsSD operon through overexpression of ALsR, the production of acetoin was not significantly increased. In contrast, moderate enhancement of ALsR expression using the P bdhA promoter significantly improved acetoin production. Compared with the wild-type, the enzyme activities of ALS and ALDC in B. subtilis harboring P bdhA were increased by approximately twofold, and the molar yield of acetoin from glucose was improved by 62.9 % in shake flask fermentation. In a 5-L fermentor, the engineered B. subtilis ultimately yielded 41.5 g/L of acetoin. Based on these results, we conclude that enhanced expression of ALDC and ALS by moderately elevated expression of the transcriptional regulator ALsR could increase acetoin production in recombinant B. subtilis.

  10. A Novel Two-Gene Requirement for the Octanoyltransfer Reaction of Bacillus subtilis Lipoic Acid Biosynthesis

    PubMed Central

    Martin, Natalia; Christensen, Quin H.; Mansilla, María C.; Cronan, John E.; de Mendoza, Diego

    2011-01-01

    SUMMARY The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM, and ywfL which we have renamed lplJ, lipM and lipL, respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to E. coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulfur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilis ΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coli ΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion paper (Christensen et al., 2011) provide evidence that LipM and LipL catalyze sequential reactions in a novel pathway for lipoic acid biosynthesis. PMID:21338420

  11. Rhizosphere Inhibition of Cucumber Fusarium Wilt by Different Surfactin- excreting Strains of Bacillus subtilis.

    PubMed

    Jia, Ke; Gao, Yu-Han; Huang, Xiao-Qin; Guo, Rong-Jun; Li, Shi-Dong

    2015-06-01

    Bacillus subtilis B006 strain effectively suppresses the cucumber fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (Foc). The population dynamics of Foc, strain B006 and its surfactin over-producing mutant B841 and surfactin-deficient mutant B1020, in the rhizosphere were determined under greenhouse conditions to elucidate the importance of the lipopeptides excreted by these strains in suppressing Foc. Results showed that B. subtilis strain B006 effectively suppressed the disease in natural soil by 42.9%, five weeks after transplanting, whereas B841 and B1020 suppressed the disease by only 22.6% and 7.1%, respectively. Quantitative PCR assays showed that effective colonization of strain B006 in the rhizosphere suppressed Foc propagation by more than 10 times both in nursery substrate and in field-infected soil. Reduction of Foc population at the cucumber stems in a range of 0.96 log10 ng/g to 2.39 log10 ng/g was attained at the third and the fifth weeks of B006 treatment in nursery substrate. In field-infected soil, all three treatments with B. subtilis suppressed Foc infection, indicated by the reduction of Foc population at a range of 2.91 log10 ng/g to 3.36 log10 ng/g at the stem base, one week after transplanting. This study reveals that the suppression of fusarium wilt disease is affected by the effective colonization of the surfactin-producing B. subtilis strain in the rhizosphere. These results improved our understanding of the biocontrol mechanism of the B. subtilis strain B006 in the natural soil and facilitate its application as biocontrol agent in the field.

  12. Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology.

    PubMed

    Chen, Po Ting; Chiang, Chung-Jen; Chao, Yun-Peng

    2007-01-01

    Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.

  13. Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis.

    PubMed

    Chen, Po Ting; Chiang, Chung-Jen; Chao, Yun-Peng

    2007-01-01

    Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.

  14. Pathway engineering of Bacillus subtilis for microbial production of N-acetylglucosamine.

    PubMed

    Liu, Yanfeng; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-09-01

    Glucosamine (GlcN) and its acetylated derivative, N-acetylglucosamine (GlcNAc), are widely used in nutraceutical and pharmaceutical industries. Currently, GlcN and GlcNAc are mainly produced by hydrolysis from crab and shrimp shells, which can cause severe environmental pollution and carries the potential risk of allergic reactions. In this study, we attempted to achieve microbial production of GlcNAc by pathway engineering of Bacillus subtilis 168. Specifically, glmS (encoding GlcN-6-phosphate synthase) from B. subtilis 168 and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) from Saccharomyces cerevisiae S288C were firstly co-overexpressed in B. subtilis; the level of GlcNAc reached 240mg/L in shake flask culture. Next, nagP, encoding the GlcNAc-specific enzyme of phosphotransferase system, was deleted to block the importation of extracellular GlcNAC, thus improving GlcNAc production to 615mg/L in shake flask culture. Then, nagA (encoding GlcNAc-6-phosphate deacetylase), gamA (encoding GlcN-6-phosphate deaminase), and nagB (encoding GlcN-6-phosphate deaminase) were deleted to block the catabolism of intracellular GlcNAc, thereby further increasing the GlcNAc titer to 1.85g/L in shake flask culture. Finally, microbial production of GlcNAc by the engineered B. subtilis 168 was conducted in a 3-L fed-batch bioreactor, and the GlcNAc titer reached 5.19g/L, which was 2.8-fold of that in shake flask culture. This is the first report regarding the pathway engineering of B. subtilis for microbial production of GlcNAc, and provides a good starting point for further metabolic engineering to achieve the industrial production of GlcNAc by a generally regarded as safe strain. © 2013 Elsevier Inc. All rights reserved.

  15. Bio-remediation of acephate-Pb(II) compound contaminants by Bacillus subtilis FZUL-33.

    PubMed

    Lin, Wenting; Huang, Zhen; Li, Xuezhen; Liu, Minghua; Cheng, Yangjian

    2016-07-01

    Removal of Pb(2+) and biodegradation of organophosphorus have been both widely investigated respectively. However, bio-remediation of both Pb(2+) and organophosphorus still remains largely unexplored. Bacillus subtilis FZUL-33, which was isolated from the sediment of a lake, possesses the capability for both biomineralization of Pb(2+) and biodegradation of acephate. In the present study, both Pb(2+) and acephate were simultaneously removed via biodegradation and biomineralization in aqueous solutions. Batch experiments were conducted to study the influence of pH, interaction time and Pb(2+) concentration on the process of removal of Pb(2+). At the temperature of 25°C, the maximum removal of Pb(2+) by B.subtilis FZUL-33 was 381.31±11.46mg/g under the conditions of pH5.5, initial Pb(2+) concentration of 1300mg/L, and contact time of 10min. Batch experiments were conducted to study the influence of acephate on removal of Pb(2+) and the influence of Pb(2+) on biodegradation of acephate by B.subtilis FZUL-33. In the mixed system of acephate-Pb(2+), the results show that biodegradation of acephate by B.subtilis FZUL-33 released PO4(3+), which promotes mineralization of Pb(2+). The process of biodegradation of acephate was affected slightly when the concentration of Pb(2+) was below 100mg/L. Based on the results, it can be inferred that the B.subtilis FZUL-33 plays a significant role in bio-remediation of organophosphorus-heavy metal compound contamination.

  16. Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis.

    PubMed

    Jin, Peng; Zhang, Linpei; Yuan, Panhong; Kang, Zhen; Du, Guocheng; Chen, Jian

    2016-04-20

    Chondroitin and heparosan, important polysaccharides and key precursors of chondroitin sulfate and heparin/heparan sulfate, have drawn much attention due to their wide applications in many aspects. In this study, we designed two independent synthetic pathways of chondroitin and heparosan in food-grade Bacillus subtilis, integrating critical synthases genes derived from Escherichia coli into B. subtilis genome. By RT-PCR analysis, we confirmed that synthases genes transcripted an integral mRNA chain, suggesting co-expression. In shaken flask, chondroitin and heparosan were produced at a level of 1.83gL(-1) and 1.71gL(-1), respectively. Since B. subtilis endogenous tuaD gene encodes the limiting factor of biosynthesis, overexpressing tuaD resulted in enhanced chondroitin and heparosan titers, namely 2.54gL(-1) and 2.65gL(-1). Moreover, production reached the highest peaks of 5.22gL(-1) and 5.82gL(-1) in 3-L fed-batch fermentation, respectively, allowed to double the production that in shaken flask. The weight-average molecular weight of chondroitin and heparosan from B. subtilis E168C/pP43-D and E168H/pP43-D were 114.07 and 67.70kDa, respectively. This work provided alternative safer synthetic pathways for metabolic engineering of chondroitin and heparosan in B. subtilis and a useful approach for enhancing production, which can be optimized for further improvement.

  17. Fractionation of Natural Organic Matter Upon Adsorption to the Bacterium, Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Manecki, M.; Maurice, P. A.; Fein, J. B.

    2001-12-01

    High pressure size exclusion chromatography (HPSEC) was used to measure changes in molecular weight distribution and average molecular weight upon adsorption of fulvic acid onto Bacillus subtilis at pH 3-7. The FA was an XAD-8 extract from a stream in the New Jersey Pine Barrens (USA), and had a weight average molecular weight of 1890 Da. Adsorption of aqueous FA onto B.subtilis was relatively fast, with steady state attained within 2 hours. An adsorption isotherm at pH 4.5 revealed a strong affinity of FA for the B.subtilis surface. The maximum adsorption capacity of a 20g bacteria/L suspension was greater than 9 mg C/L of FA at pH 4.5. Adsorption of FA onto B.subtilis was strongly pH dependent, increasing markedly with decreasing pH over the pH range 3-7. Comparison of HPSEC analysis of control (FA not reacted with bacteria) versus reacted samples showed that in all experiments, the weight average molecular weight (Mw) of FA remaining in solution decreased by several hundred Da. The observed decrease in solution Mw upon adsorption indicated that the higher molecular weight FA components adsorbed preferentially to the bacterial surfaces, at all studied pH values (3-7). Additionally, there was a low molecular weight FA fraction that did not adsorb, even at low pH. Our results suggest that hydrophobic interactions may be important for FA sorption to B.subtilis and that low molecular weight, more hydrophilic components may thus be less likely to adsorb than higher molecular weight, more hydrophobic components.

  18. Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome.

    PubMed

    Itaya, M

    1995-07-22

    A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined. An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In the B. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in the B. subtilis chromosome and possible applications of this technique are discussed.

  19. Draft Genome Sequence of Commercial Textile Dye-Decolorizing and -Degrading Bacillus subtilis Strain C3 Isolated in India

    PubMed Central

    Kunadia, Khushbu; Nathani, Neelam M.; Kothari, Vishal; Kotadia, Rohit J.; Kothari, Charmy R.; Joshi, Anjali; Rank, Jalpa K.; Faldu, Priti R.; Shekar, M. Chandra; Viroja, Mitkumar J.; Patel, Priyank A.; Jadeja, Divyarajsinh; Reddy, Bhaskar; Pal Singh, Ravindra; Koringa, Prakash G.; Joshi, Chaitanya G.

    2016-01-01

    Bacillus subtilis C3, a commercial textile dye-decolorizing and -degrading bacterium, was isolated from the common effluent treatment plant (CEPT) of the Jetpur textile dyeing and printing industrial sector situated in the district of Rajkot, Gujarat, India. Here, we present the annotated 4.18-Mb draft genome sequence of B. subtilis C3, providing information about the metabolic pathways involved in decolorization and degradation of several commercial textile azo dyes. Thus, we confirm B. subtilis C3 as a potential candidate for bioremediation of textile effluents. PMID:26966205

  20. Draft Genome Sequence of Commercial Textile Dye-Decolorizing and -Degrading Bacillus subtilis Strain C3 Isolated in India.

    PubMed

    Kunadia, Khushbu; Nathani, Neelam M; Kothari, Vishal; Kotadia, Rohit J; Kothari, Charmy R; Joshi, Anjali; Rank, Jalpa K; Faldu, Priti R; Shekar, M Chandra; Viroja, Mitkumar J; Patel, Priyank A; Jadeja, Divyarajsinh; Reddy, Bhaskar; Pal Singh, Ravindra; Koringa, Prakash G; Joshi, Chaitanya G; Kothari, Ramesh K

    2016-03-10

    Bacillus subtilis C3, a commercial textile dye-decolorizing and -degrading bacterium, was isolated from the common effluent treatment plant (CEPT) of the Jetpur textile dyeing and printing industrial sector situated in the district of Rajkot, Gujarat, India. Here, we present the annotated 4.18-Mb draft genome sequence of B. subtilis C3, providing information about the metabolic pathways involved in decolorization and degradation of several commercial textile azo dyes. Thus, we confirm B. subtilis C3 as a potential candidate for bioremediation of textile effluents.

  1. Bioreduction of Cr(VI) by alkaliphilic Bacillus subtilis and interaction of the membrane groups

    PubMed Central

    Mary Mangaiyarkarasi, M.S.; Vincent, S.; Janarthanan, S.; Subba Rao, T.; Tata, B.V.R.

    2010-01-01

    Detoxification of Cr(VI) under alkaline pH requires attention due to the alkaline nature of many effluents. An alkaliphilic gram-positive Bacillus subtilis isolated from tannery effluent contaminated soil was found to grow and reduce Cr(VI) up to 100% at an alkaline pH 9. Decrease in pH to acidic range with growth of the bacterium signified the role played by metabolites (organic acids) in chromium resistance and reduction mechanism. The XPS and FT-IR spectra confirmed the reduction of Cr(VI) by bacteria into +3 oxidation state. Chromate reductase assay indicated that the reduction was mediated by constitutive membrane bound enzymes. The kinetics of Cr(VI) reduction activity derived using the monod equation proved (Ks = 0.00032) high affinity of the organism to the metal. This study thus helped to localize the reduction activity at subcellular level in a chromium resistant alkaliphilic Bacillus sp. PMID:23961119

  2. Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus.

    PubMed

    Shank, Elizabeth A; Klepac-Ceraj, Vanja; Collado-Torres, Leonardo; Powers, Gordon E; Losick, Richard; Kolter, Roberto

    2011-11-29

    Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis--the soil--were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain's relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.

  3. Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains

    PubMed Central

    2014-01-01

    Background Shikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104 against viral influenza. Microbial production of SA has been extensively investigated in Escherichia coli, and to a less extent in Bacillus subtilis. However, metabolic flux of the high SA-producing strains has not been explored. In this study, we constructed with genetic manipulation and further determined metabolic flux with 13C-labeling test of high SA-producing B. subtilis strains. Results B. subtilis 1A474 had a mutation in SA kinase gene (aroI) and accumulated 1.5 g/L of SA. Overexpression of plasmid-encoded aroA, aroB, aroC or aroD in B. subtilis revealed that aroD had the most significantly positive effects on SA production. Simultaneous overexpression of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroA) and SA dehydrogenase (aroD) in B. subtilis BSSA/pSAAroA/pDGSAAroD resulted in SA production of 3.2 g/L. 13C-Metabolic flux assay (MFA) on the two strains BSSA/pHCMC04/pDG148-stu and BSSA/pSAAroA/pDGSAAroD indicated the carbon flux from glucose to SA increased to 4.6% in BSSA/pSAAroA/pDGSAAroD from 1.9% in strain BSSA/pHCMC04/pDG148-stu. The carbon flux through tricarboxylic acid cycle significantly reduced, while responses of the pentose phosphate pathway and the glycolysis to high SA production were rather weak, in the strain BSSA/pSAAroA/pDGSAAroD. Based on the results from MFA, two potential targets for further optimization of SA production were identified. Experiments on genetic deletion of phosphoenoylpyruvate kinase gene confirmed its positive influence on SA production, while the overexpression of the transketolase gene did not lead to increase in SA production. Conclusion Of the genes involved in shikimate pathway in B. subtilis, aroD exerted most significant influence on SA accumulation. Overexpression of plasmid-encoded aroA and aroD doubled SA production than its parent strain. MFA revealed metabolic flux

  4. Evaluation of germination, distribution, and persistence of Bacillus subtilis spores through the gastrointestinal tract of chickens.

    PubMed

    Latorre, J D; Hernandez-Velasco, X; Kallapura, G; Menconi, A; Pumford, N R; Morgan, M J; Layton, S L; Bielke, L R; Hargis, B M; Téllez, G

    2014-07-01

    Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 10(6) spores/g of feed, whereas a second group was gavaged with a single dose of 10(6) spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75°C for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 10(5) spores per gram of digesta, which is about a 1-log10 reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10(2) spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative

  5. Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis

    PubMed Central

    2011-01-01

    Background Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase. Results The alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21. Conclusions This is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis. PMID:21978209

  6. [Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells].

    PubMed

    Znamenskaia, L V; Vershinina, O A; Vershinina, V I; Krasnov, S I; Kostrov, S V; Akimkina, T V; Leshchinskaia, I B; Hartley, R W

    1999-01-01

    Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate.

  7. In Bacillus subtilis, the SatA (formerly YyaR) acetyltransferase detoxifies streptothricin via lysine acetylation.

    PubMed

    Burckhardt, Rachel M; Escalante-Semerena, Jorge C

    2017-08-25

    Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA(+) restored streptothricin resistance to B. subtilis satA strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-CoA. A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA(+) in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.Importance Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The alluded enzyme is a member of a family of proteins that is broadly distributed in all domains of life, but poorly studied in B. subtilis and B. anthracis. The initial characterization of the enzyme provides insights into its mechanism of catalysis. Copyright © 2017 American Society for

  8. Enhanced dipicolinic acid production during the stationary phase in Bacillus subtilis by blocking acetoin synthesis.

    PubMed

    Toya, Yoshihiro; Hirasawa, Takashi; Ishikawa, Shu; Chumsakul, Onuma; Morimoto, Takuya; Liu, Shenghao; Masuda, Kenta; Kageyama, Yasushi; Ozaki, Katsuya; Ogasawara, Naotake; Shimizu, Hiroshi

    2015-01-01

    Bacterial bio-production during the stationary phase is expected to lead to a high target yield because the cells do not consume the substrate for growth. Bacillus subtilis is widely used for bio-production, but little is known about the metabolism during the stationary phase. In this study, we focused on the dipicolinic acid (DPA) production by B. subtilis and investigated the metabolism. We found that DPA production competes with acetoin synthesis and that acetoin synthesis genes (alsSD) deletion increases DPA productivity by 1.4-fold. The mutant showed interesting features where the glucose uptake was inhibited, whereas the cell density increased by approximately 50%, resulting in similar volumetric glucose consumption to that of the parental strain. The metabolic profiles revealed accumulation of pyruvate, acetyl-CoA, and the TCA cycle intermediates in the alsSD mutant. Our results indicate that alsSD-deleted B. subtilis has potential as an effective host for stationary-phase production of compounds synthesized from these intermediates.

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

    SciTech Connect

    O'Hara, M.B.; Hageman, J.H.

    1987-05-01

    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 ( UC)-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 CaS . When (CaS ) was < 1 x 10 W, rates of proteolysis in sporulating cells were reduced 4-8 times that in cells in calcium ion- sufficient medium. Further, omission of CaS 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 (UVCa). The authors concluded that B. subtilis cells required both metabolic energy and calcium ions for normal proteolysis.

  10. Microbial Activation of Bacillus subtilis-Immobilized Microgel Particles for Enhanced Oil Recovery.

    PubMed

    Son, Han Am; Choi, Sang Koo; Jeong, Eun Sook; Kim, Bohyun; Kim, Hyun Tae; Sung, Won Mo; Kim, Jin Woong

    2016-09-06

    Microbially enhanced oil recovery involves the use of microorganisms to extract oil remaining in reservoirs. Here, we report fabrication of microgel particles with immobilized Bacillus subtilis for application to microbially enhanced oil recovery. Using B. subtilis isolated from oil-contaminated soils in Myanmar, we evaluated the ability of this microbe to reduce the interfacial tension at the oil-water interface via production of biosurfactant molecules, eventually yielding excellent emulsification across a broad range of the medium pH and ionic strength. To safely deliver B. subtilis into a permeable porous medium, in this study, these bacteria were physically immobilized in a hydrogel mesh of microgel particles. In a core flooding experiment, in which the microgel particles were injected into a column packed with silica beads, we found that these particles significantly increased oil recovery in a concentration-dependent manner. This result shows that a mesh of microgel particles encapsulating biosurfactant-producing microorganisms holds promise for recovery of oil from porous media.

  11. Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis.

    PubMed

    Wolf, Diana; Domínguez-Cuevas, Patricia; Daniel, Richard A; Mascher, Thorsten

    2012-11-01

    L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to β-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. Our results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

  12. The prevalence and origin of exoprotease-producing cells in the Bacillus subtilis biofilm.

    PubMed

    Marlow, Victoria L; Cianfanelli, Francesca R; Porter, Michael; Cairns, Lynne S; Dale, J Kim; Stanley-Wall, Nicola R

    2014-01-01

    Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells.

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

    PubMed

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

    2011-12-01

    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 consisting of negatively charged phospholipids were detected in the rod-shaped bacterium Bacillus subtilis. It was also shown that the cardiolipin-specific dye, nonyl acridine orange (NAO), is preferentially distributed at the cell poles and in the septal regions in both Escherichia coli and B. subtilis. These results suggest that phosphatidylglycerol is the principal component of the observed spiral domains in B. subtilis. Here, using the fluorescent dyes FM4-64 and NAO, we examined whether these lipid domains are linked to the presence of cell wall peptidoglycan. We show that in protoplasted cells, devoid of the peptidoglycan layer, helix-like lipid structures are not preserved. Specific lipid domains are also missing in cells depleted of MurG, an enzyme involved in peptidoglycan synthesis, indicating a link between lipid domain formation and peptidoglycan synthesis.

  14. Disruption of Autolysis in Bacillus subtilis using TiO2 Nanoparticles

    PubMed Central

    McGivney, Eric; Han, Linchen; Avellan, Astrid; VanBriesen, Jeanne; Gregory, Kelvin B.

    2017-01-01

    In contrast to many nanotoxicity studies where nanoparticles (NPs) are observed to be toxic or reduce viable cells in a population of bacteria, we observed that increasing concentration of TiO2 NPs increased the cell survival of Bacillus subtilis in autolysis-inducing buffer by 0.5 to 5 orders of magnitude over an 8 hour exposure. Molecular investigations revealed that TiO2 NPs prevent or delay cell autolysis, an important survival and growth-regulating process in bacterial populations. Overall, the results suggest two potential mechanisms for the disruption of autolysis by TiO2 NPs in a concentration dependent manner: (i) directly, through TiO2 NP deposition on the cell wall, delaying the collapse of the protonmotive-force and preventing the onset of autolysis; and (ii) indirectly, through adsorption of autolysins on TiO2 NP, limiting the activity of released autolysins and preventing further lytic activity. Enhanced darkfield microscopy coupled to hyperspectral analysis was used to map TiO2 deposition on B. subtilis cell walls and released enzymes, supporting both mechanisms of autolysis interference. The disruption of autolysis in B. subtilis cultures by TiO2 NPs suggests the mechanisms and kinetics of cell death may be influenced by nano-scale metal oxide materials, which are abundant in natural systems. PMID:28303908

  15. Overexpression and characterization in Bacillus subtilis of a positionally nonspecific lipase from Proteus vulgaris.

    PubMed

    Lu, Yaping; Lin, Qian; Wang, Jin; Wu, Yufan; Bao, Wuyundalai; Lv, Fengxia; Lu, Zhaoxin

    2010-09-01

    A Proteus vulgaris strain named T6 which produced lipase (PVL) with nonpositional specificity had been isolated in our laboratory. To produce the lipase in large quantities, we cloned its gene, which had an opening reading frame of 864 base pairs and encoded a deduced 287-amino-acid protein. The PVL gene was inserted into the Escherichia coli expression vector pET-DsbA, and active lipase was expressed in E. coli BL21 cells. The secretive expression of PVL gene in Bacillus subtilis was examined. Three vectors, i.e., pMM1525 (xylose-inducible), pMMP43 (constitutive vector, derivative of pMM1525), and pHPQ (sucrose-inducible, constructed based on pHB201), were used to produce lipase in B. subtilis. Recombinant B. subtilis WB800 cells harboring the pHPQ-PVL plasmid could synthesize and secrete the PVL protein in high yield. The lipase activity reached 356.8 U/mL after induction with sucrose for 72 h in shake-flask culture, representing a 12-fold increase over the native lipase activity in P. vulgaris. The characteristics of the heterologously expressed lipase were identical to those of the native one.

  16. Bacillus subtilis attachment to Aspergillus niger hyphae results in mutually altered metabolism.

    PubMed

    Benoit, Isabelle; van den Esker, Marielle H; Patyshakuliyeva, Aleksandrina; Mattern, Derek J; Blei, Felix; Zhou, Miaomiao; Dijksterhuis, Jan; Brakhage, Axel A; Kuipers, Oscar P; de Vries, Ronald P; Kovács, Ákos T

    2015-06-01

    Interaction between microbes affects the growth, metabolism and differentiation of members of the microbial community. While direct and indirect competition, like antagonism and nutrient consumption have a negative effect on the interacting members of the population, microbes have also evolved in nature not only to fight, but in some cases to adapt to or support each other, while increasing the fitness of the community. The presence of bacteria and fungi in soil results in various interactions including mutualism. Bacilli attach to the plant root and form complex communities in the rhizosphere. Bacillus subtilis, when grown in the presence of Aspergillus niger, interacts similarly with the fungus, by attaching and growing on the hyphae. Based on data obtained in a dual transcriptome experiment, we suggest that both fungi and bacteria alter their metabolism during this interaction. Interestingly, the transcription of genes related to the antifungal and putative antibacterial defence mechanism of B. subtilis and A. niger, respectively, are decreased upon attachment of bacteria to the mycelia. Analysis of the culture supernatant suggests that surfactin production by B. subtilis was reduced when the bacterium was co-cultivated with the fungus. Our experiments provide new insights into the interaction between a bacterium and a fungus.

  17. Interactions between Streptomyces coelicolor and Bacillus subtilis: Role of Surfactants in Raising Aerial Structures

    PubMed Central

    Straight, Paul D.; Willey, Joanne M.; Kolter, Roberto

    2006-01-01

    Using mixed-species cultures, we have undertaken a study of interactions between two common spore-forming soil bacteria, Bacillus subtilis and Streptomyces coelicolor. Our experiments demonstrate that the development of aerial hyphae and spores by S. coelicolor is inhibited by surfactin, a lipopeptide surfactant produced by B. subtilis. Current models of aerial development by sporulating bacteria and fungi postulate a role for surfactants in reducing surface tension at air-liquid interfaces, thereby removing the major barrier to aerial growth. S. coelicolor produces SapB, an amphipathic peptide that is surface active and required for aerial growth on certain media. Loss of aerial hyphae in developmental mutants can be rescued by addition of purified SapB. While a surfactant from a fungus can substitute for SapB in a mutant that lacks aerial hyphae, not all surfactants have this effect. We show that surfactin is required for formation of aerial structures on the surface of B. subtilis colonies. However, in contrast to this positive role, our experiments reveal that surfactin acts antagonistically by arresting S. coelicolor aerial development and causing altered expression of developmental genes. Our observations support the idea that surfactants function specifically for a given organism regardless of their shared ability to reduce surface tension. Production of surfactants with antagonistic activity could provide a powerful competitive advantage during surface colonization and in competition for resources. PMID:16788200

  18. Investigating the Inactivation Mechanism of Bacillus subtilis Spores by High Pressure CO2

    PubMed Central

    Rao, Lei; Zhao, Feng; Wang, Yongtao; Chen, Fang; Hu, Xiaosong; Liao, Xiaojun

    2016-01-01

    The objective of this study was to investigate the inactivation mechanism of Bacillus subtilis spores by high pressure CO2 (HPCD) processing. The spores of B. subtilis were subjected to heat at 0.1 MPa or HPCD at 6.5-20 MPa, and 64-86°C for 0-120 min. The germination, the permeability of inner membrane (IM) and cortex, the release of pyridine-2, 6-dicarboxylic acid (DPA), and changes in the morphological and internal structures of spores were investigated. The HPCD-treated spores did not lose heat resistance and their DPA release was lower than the inactivation, suggesting that spores did not germinate during HPCD. The flow cytometry analysis suggested that the permeability of the IM and cortex of HPCD-treated spores was increased. Furthermore, the DPA of the HPCD-treated spores were released in parallel with their inactivation and the fluorescence photomicrographs showed that these treated spores were stained by propidium iodide, ensuring that the permeability of IM of spores was increased by HPCD. The scanning electron microscopy photomicrographs showed that spores were crushed into debris or exhibited a hollowness on the surface, and the transmission electron microscopy photomicrographs exhibited an enlarged core, ruptured and indistinguishable IM and a loss of core materials in the HPCD-treated spores, indicating that HPCD damaged the structures of the spores. These findings suggested that HPCD inactivated B. subtilis spores by directly damaging the structure of the spores, rather than inducing germination of the spores. PMID:27656175

  19. Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli

    PubMed Central

    Jameson, Katie H.; Wilkinson, Anthony J.

    2017-01-01

    Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis. PMID:28075389

  20. Production of nattokinase by batch and fed-batch culture of Bacillus subtilis.

    PubMed

    Cho, Young-Han; Song, Jae Yong; Kim, Kyung Mi; Kim, Mi Kyoung; Lee, In Young; Kim, Sang Bum; Kim, Hyeon Shup; Han, Nam Soo; Lee, Bong Hee; Kim, Beom Soo

    2010-09-30

    Nattokinase was produced by batch and fed-batch culture of Bacillus subtilis in flask and fermentor. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on the production of nattokinase. In flask culture, the highest cell growth and nattokinase activity were obtained with 50 g/L of peptone supplementation. In this condition, nattokinase activity was 630 unit/ml at 12 h. In batch culture of B. subtilis in fermentor, the highest nattokinase activity of 3400 unit/ml was obtained at 10h with 50 g/L of peptone supplementation. From the batch kinetics data, it was shown that nattokinase production was growth-associated and culture should be harvested before stationary phase for maximum nattokinase production. In fed-batch culture of B. subtilis using pH-stat feeding strategy, cell growth (optical density monitored at 600 nm) increased to ca. 100 at 22 h, which was 2.5 times higher than that in batch culture. The highest nattokinase activity was 7100 unit/ml at 19 h, which was also 2.1 times higher than that in batch culture.

  1. Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in clarified cashew apple juice.

    PubMed

    de Oliveira, Darlane Wellen Freitas; França, Italo Waldimiro Lima; Félix, Anne Kamilly Nogueira; Martins, João Jeferson Lima; Giro, Maria Estela Aparecida; Melo, Vânia Maria M; Gonçalves, Luciana Rocha Barros

    2013-01-01

    In this work a low cost medium for the production of a biosurfactant by Bacillus subtilis LAMI005 and the kinetics of surfactin production considering the effect of initial substrate concentration were investigated. First, cashew apple juice supplementation for optimal production of biosurfactant by B. subtilis LAMI005 was studied. The medium formulated with clarified cashew apple juice and distilled water, supplemented with 1.0 g/L of (NH(4))(2)SO(4), proved to be the best among the nutrients evaluated. The crude biosurfactant had the ability to decrease the surface tension of water to 30 dyne/cm, with a critical micelle concentration (CMC) of 63.0 mg/L. Emulsification experiments indicated that this biosurfactant effectively emulsified kerosene (IE(24)=67%) and soybean oil (IE(24)=64%). Furthermore, the emulsion stability was always very high. It was shown by biochemical analysis, IR spectra, that there is no qualitative differences in the composition of the crude biosurfactant from a standard sample of surfactin from B. subtilis. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli.

    PubMed

    Jameson, Katie H; Wilkinson, Anthony J

    2017-01-10

    Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis.

  3. Extracellular DNA Release by Undomesticated Bacillus subtilis Is Regulated by Early Competence

    PubMed Central

    de Figueras, Carolina González; González-Pastor, José Eduardo

    2012-01-01

    Extracellular DNA (eDNA) release is a widespread capacity described in many microorganisms. We identified and characterized lysis-independent eDNA production in an undomesticated strain of Bacillus subtilis. DNA fragments are released during a short time in late-exponential phase. The released eDNA corresponds to whole genome DNA, and does not harbour mutations suggesting that is not the result of error prone DNA synthesis. The absence of eDNA was linked to a spread colony morphology, which allowed a visual screening of a transposon library to search for genes involved in its production. Transposon insertions in genes related to quorum sensing and competence (oppA, oppF and comXP) and to DNA metabolism (mfd and topA) were impaired in eDNA release. Mutants in early competence genes such as comA and srfAA were also defective in eDNA while in contrast mutations in late competence genes as those for the DNA uptake machinery had no effect. A subpopulation of cells containing more DNA is present in the eDNA producing strains but absent from the eDNA defective strain. Finally, competent B. subtilis cells can be transformed by eDNA suggesting it could be used in horizontal gene transfer and providing a rationale for the molecular link between eDNA release and early-competence in B. subtilis that we report. PMID:23133654

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

    PubMed

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

    2012-10-01

    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 quantities. For future pharmaceutical applications, this study aimed to investigate the effects of levan produced by B. subtilis Natto, mainly as potential hypoglycemic agent, (previously optimized with a molecular weight equal to 72.37 and 4,146 kDa) in Wistar male rats with diabetes induced by streptozotocin and non-diabetic rats and to monitor their plasma cholesterol and triacylglycerol levels. After 15 days of experimentation, the animals were sacrificed, and their blood samples were analyzed. The results, compared using analysis of variance, demonstrated that for this type of levan, a hypoglycemic effect was not observed, as there was no improvement of diabetes symptoms during the experiment. However, levan did not affect any studied parameters in normal rats, indicating that the exopolysaccharide can be used for other purposes.

  5. A process for high-efficiency isoflavone deglycosylation using Bacillus subtilis natto NTU-18.

    PubMed

    Kuo, Lun-Cheng; Wu, Ren-Yu; Lee, Kung-Ta

    2012-06-01

    In order to produce isoflavone aglycosides effectively, a process of isoflavone hydrolysis by Bacillus subtilis natto NTU-18 (BCRC 80390) was established. This process integrates the three stages for the production of isoflavone aglycosides in one single fermenter, including the growth of B. subtilis natto, production of β-glucosidase, deglycosylation of fed isoflavone glycosides. After 8 h of batch culture of B. subtilis natto NTU-18 in 2 L of soy medium, a total of 3 L of soy isoflavone glucoside solution containing 3.0 mg/mL of daidzin and 1.0 mg/mL of genistin was fed continuously over 34 h. The percentage deglycosylation of daidzin and genistin was 97.7% and 94.6%, respectively. The concentration of daidzein and genistein in the broth reached 1,066.8 μg/mL (4.2 mM) and 351 μg/mL (1.3 mM), respectively, and no residual daidzin or genistin was detected. The productivity of the bioconversion of daidzein and genistein over the 42 h of culture was 25.6 mg/L/h and 8.5 mg/L/h, respectively. This showed that this is an efficient bioconversion process for selective estrogen receptor modulator production.

  6. Protective role of bacillithiol in superoxide stress and Fe–S metabolism in Bacillus subtilis

    PubMed Central

    Fang, Zhong; Dos Santos, Patricia C

    2015-01-01

    Glutathione (GSH) serves as the prime thiol in most organisms as its depletion increases antibiotic and metal toxicity, impairs oxidative stress responses, and affects Fe and Fe–S cluster metabolism. Many gram-positive bacteria lack GSH, but instead produce other structurally unrelated yet functionally equivalent thiols. Among those, bacillithiol (BSH) has been recently identified in several low G+C gram-positive bacteria. In this work, we have explored the link between BSH and Fe–S metabolism in Bacillus subtilis. We have identified that B. subtilis lacking BSH is more sensitive to oxidative stress (paraquat), and metal toxicity (Cu(I) and Cd(II)), but not H2O2. Furthermore, a slow growth phenotype of BSH null strain in minimal medium was observed, which could be recovered upon the addition of selected amino acids (Leu/Ile and Glu/Gln), supplementation of iron, or chemical complementation with BSH disulfide (BSSB) to the growth medium. Interestingly, Fe–S cluster containing isopropylmalate isomerase (LeuCD) and glutamate synthase (GOGAT) showed decreased activities in BSH null strain. Deficiency of BSH also resulted in decreased levels of intracellular Fe accompanied by increased levels of manganese and altered expression levels of Fe–S cluster biosynthetic SUF components. Together, this study is the first to establish a link between BSH and Fe–S metabolism in B. subtilis. PMID:25988368

  7. Plant growth promotion by spermidine-producing Bacillus subtilis OKB105.

    PubMed

    Xie, Shan-Shan; Wu, Hui-Jun; Zang, Hao-Yu; Wu, Li-Ming; Zhu, Qing-Qing; Gao, Xue-Wen

    2014-07-01

    The interaction between plants and plant-growth-promoting rhizobacteria (PGPR) is a complex, reciprocal process. On the one hand, plant compounds such as carbohydrates and amino acids serve as energy sources for PGPR. On the other hand, PGPR promote plant growth by synthesizing plant hormones and increasing mineral availability in the soil. Here, we evaluated the growth-promoting activity of Bacillus subtilis OKB105 and identified genes associated with this activity. The genes yecA (encoding a putative amino acid/polyamine permease) and speB (encoding agmatinase) are involved in the secretion or synthesis of polyamine in B. subtilis OKB105. Disruption of either gene abolished the growth-promoting activity of the bacterium, which was restored when polyamine synthesis was complemented. Moreover, high-performance liquid chromatography analysis of culture filtrates of OKB105 and its derivatives demonstrated that spermidine, a common polyamine, is the pivotal plant-growth-promoting compound. In addition, real-time polymerase chain reaction analysis revealed that treatment with B. subtilis OKB105 induced expansin gene (Nt-EXPA1 and Nt-EXPA2) expression and inhibited the expression of the ethylene biosynthesis gene ACO1. Furthermore, enzyme-linked immunosorbent assay analysis showed that the ethylene content in plant root cells decreased in response to spermidine produced by OKB105. Therefore, during plant interactions, OKB105 may produce and secrete spermidine, which induces expansin production and lowers ethylene levels.

  8. Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways

    PubMed Central

    Gómez-Marroquín, Martha; Martin, Holly A.; Pepper, Amber; Girard, Mary E.; Kidman, Amanda A.; Vallin, Carmen; Yasbin, Ronald E.; Pedraza-Reyes, Mario; Robleto, Eduardo A.

    2016-01-01

    In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu+ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu+ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome. PMID:27399782

  9. [Coculture of actinomycetes with Bacillus subtilis and its effect on the bioactive secondary metabolites].

    PubMed

    Huang, Bing; Liu, Ning; Huang, Ying; Chen, Jinchun

    2009-06-01

    To explore the effect of coculturing actinomycetes with Bacillus subtilis on the production of bioactive secondary metabolites, we studied the difference between fermentation products of monocultures and the corresponding cocultures of 22 actinomycetes by antimicrobial assay and HPLC-PDA analysis. We selected Streptomyces strain FXJ2.014 with high bioactivity for further analysis and found additional metabolites in fermentation extracts of cocultures of strains FXJ2.014, FXJ1.296 and AS 4.1252 respectively with B. subtilis. Quinomycin A was the main bioactive metabolite produced by the monoculture of strain FXJ2.014, while a new quinomycin-like component named FXJ2.014-HB was produced when strain FXJ2.014 was cocultured with B. subtilis. Further tests of antimicrobial and antitumor activities indicated that FXJ2.014-HB and Quinomycin A had significant differences in terms of bioactivity. Moreover, the inhibitory activity of FXJ2.014-HB to a variety of tumor cell lines was weaker than the highly toxic Quinomycin A, indicating its potential to be an antibiotic with low cell toxicity. In conclusion, coculture can be used as a promising approach to discover bioactive secondary metabolites from actinomycetes.

  10. Engineering Bacillus subtilis for acetoin production from glucose and xylose mixtures.

    PubMed

    Chen, Tao; Liu, Wei-xi; Fu, Jing; Zhang, Bo; Tang, Ya-jie

    2013-12-01

    As a vital flavor compound, acetoin is extensively used in dairy products and drinks industry. In this study, Bacillus subtilis was engineered to metabolize glucose and xylose as substrates for acetoin production. Initially, gene araE from B. subtilis, encoding the xylose transport protein AraE, was placed under the control of the constitutive promoter P43 for over-expression. Batch cultures showed that 10 g/L xylose was depleted completely in 32 h. Subsequently, genes xylA and xylB from Escherichia coli, encoding xylose isomerase and xylulokinase respectively, were introduced into B. subtilis, and the recombinant turned out to assimilate glucose and xylose without preference. In shake-flask fermentations, 5.5 g/L acetoin with a yield of 0.70 mol(mol sugar)(-1) was obtained by the optimum strain BSUL13 under microaerobic conditions, which offered a metabolic engineering strategy on engineering microbe as cell factory for the production of high-valued chemicals from renewable resource. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Further studies on the regulation of amino sugar metabolism in Bacillus subtilis

    PubMed Central

    Bates, C. J.; Pasternak, C. A.

    1965-01-01

    1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its Km is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (Km 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose. PMID:14343123

  12. Presence of Calcium Lowers the Expansion of Bacillus subtilis Colony Biofilms

    PubMed Central

    Mhatre, Eisha; Sundaram, Anandaroopan; Hölscher, Theresa; Mühlstädt, Mike; Bossert, Jörg; Kovács, Ákos T.

    2017-01-01

    Robust colony formation by Bacillus subtilis is recognized as one of the sessile, multicellular lifestyles of this bacterium. Numerous pathways and genes are responsible for the architecturally complex colony structure development. Cells in the biofilm colony secrete extracellular polysaccharides (EPS) and protein components (TasA and the hydrophobin BslA) that hold them together and provide a protective hydrophobic shield. Cells also secrete surfactin with antimicrobial as well as surface tension reducing properties that aid cells to colonize the solid surface. Depending on the environmental conditions, these secreted components of the colony biofilm can also promote the flagellum-independent surface spreading of B. subtilis, called sliding. In this study, we emphasize the influence of Ca2+ in the medium on colony expansion of B. subtilis. Interestingly, the availability of Ca2+ has no major impact on the induction of complex colony morphology. However, in the absence of this divalent ion, peripheral cells of the colony expand radially at later stages of development, causing colony size to increase. We demonstrate that the secreted extracellular compounds, EPS, BslA, and surfactin facilitate colony expansion after biofilm maturation. We propose that Ca2+ hinders biofilm colony expansion by modifying the amphiphilic properties of surfactin. PMID:28212310

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

    PubMed

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

    2015-10-01

    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.

  14. Presence and function of a thick mucous layer rich in polysaccharides around Bacillus subtilis spores.

    PubMed

    Faille, Christine; Ronse, Annette; Dewailly, Etienne; Slomianny, Christian; Maes, Emmanuel; Krzewinski, Frédéric; Guerardel, Yann

    2014-01-01

    This study was designed to establish the presence and function of the mucous layer surrounding spores of Bacillus subtilis. First, an external layer of variable thickness and regularity was often observed on B. subtilis spores. Further analyses were performed on B. subtilis 98/7 spores surrounded by a thick layer. The mechanical removal of the layer did not affect their resistance to heat or their ability to germinate but rendered the spore less hydrophilic, more adherent to stainless steel, and more resistant to cleaning. This layer was mainly composed of 6-deoxyhexoses, ie rhamnose, 3-O-methyl-rhamnose and quinovose, but also of glucosamine and muramic lactam, known also to be a part of the bacterial peptidoglycan. The specific hydrolysis of the peptidoglycan using lysozyme altered the structure of the required mucous layer and affected the physico-chemical properties of the spores. Such an outermost mucous layer has also been seen on spores of B. licheniformis and B. clausii isolated from food environments.

  15. Isolation, identification and characterization of Bacillus subtilis ZJB-063, a versatile nitrile-converting bacterium.

    PubMed

    Zheng, Yu-Guo; Chen, Jing; Liu, Zhi-Qiang; Wu, Ming-Huo; Xing, Liang-Ying; Shen, Yin-Chu

    2008-01-01

    Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of epsilon-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of epsilon-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.

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

    PubMed Central

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

    2012-01-01

    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 quantities. For future pharmaceutical applications, this study aimed to investigate the effects of levan produced by B. subtilis Natto, mainly as potential hypoglycemic agent, (previously optimized with a molecular weight equal to 72.37 and 4,146 kDa) in Wistar male rats with diabetes induced by streptozotocin and non-diabetic rats and to monitor their plasma cholesterol and triacylglycerol levels. After 15 days of experimentation, the animals were sacrificed, and their blood samples were analyzed. The results, compared using analysis of variance, demonstrated that for this type of levan, a hypoglycemic effect was not observed, as there was no improvement of diabetes symptoms during the experiment. However, levan did not affect any studied parameters in normal rats, indicating that the exopolysaccharide can be used for other purposes. PMID:24031993

  17. The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis.

    PubMed

    Pottathil, Mridula; Lazazzera, Beth A

    2003-01-01

    In the field of cell-cell communication, an emerging class of extracellular signaling peptides that function intracellularly has been identified in Gram-positive bacteria. One illustrative member of this group is the Phr family of extracellular signaling peptides of Bacillus subtilis. The Phr signaling peptides are secreted by the bacterium, and then, despite the presence of intracellular peptidases, they are actively transported into the cell where they interact with intracellular receptors to regulate gene expression. The intracellular receptors are members of a family of aspartyl-phosphate phosphatases, the Rap phosphatases. These phosphatases cause the dephosphorylation of response regulator proteins, ubiquitous regulatory proteins in bacteria. Immediately downstream of the genes for the Rap phosphatases are the genes for the Phr peptides, forming rap phr signaling cassettes. There are at least seven rap phr signaling cassettes in B. subtilis, and the genome sequence of other Gram-positive, endospore-forming bacteria suggests that similar cassettes may also function in these bacteria. In B. subtilis, the rap phr cassettes regulate sporulation, genetic competence, and genes comprising the quorum response (i.e. the response to high cell density). This review will address the mechanism of extracellular Phr signaling peptide production, transport, response, and their role in quorum sensing.

  18. Effect of carbon and nitrogen on the cannibalistic behavior of Bacillus subtilis.

    PubMed

    Nandy, Subir Kumar; Venkatesh, K V

    2008-12-01

    Bacillus subtilis is known to exhibit cannibalism under nutrient limitation to delay sporulation. Cells of B. subtilis in phosphate buffer solution (PBS) demonstrate an oscillatory behavior in cell number due to cannibalism. Since PBS did not contain any nutrients, the effect of carbon and nitrogen sources on the cannibalistic behavior is unclear. In this study, the effect of external carbon and nitrogen on the cannibalistic behavior of B. subtilis is presented. The studies demonstrated that when glucose as a carbon source was introduced into PBS in the absence of any other nutrients, the cannibalistic tendency was delayed. This delay increased with the increase in the amount of glucose present in the PBS. Thus, the cannibalism was observed to be very sensitive to the amount of carbon present in the medium. However, when the medium contained only ammonium sulfate as a nitrogen source and was devoid of any carbon, the effect on cannibalism was minimal. The study, therefore, demonstrated that cannibalism was more sensitive to carbon than nitrogen indicating that the phenomenon of cannibalism may be more dependent on the status of energy in the medium than on nitrogen assimilation.

  19. Greenhouse evaluation of Bacillus subtilis AP-01 and Trichoderma harzianum AP-001 in controlling tobacco diseases

    PubMed Central

    Maketon, Monchan; Apisitsantikul, Jirasak; Siriraweekul, Chatchai

    2008-01-01

    Two biological control agents, Bacillus subtilis AP-01 (Larminar™) and Trichoderma harzianum AP-001 (Trisan™) alone or/in combination were investigated in controlling three tobacco diseases, including bacterial wilt (Ralstonia solanacearum), damping-off (Pythium aphanidermatum), and frogeye leaf spot (Cercospora nicotiana). Tests were performed in greenhouse by soil sterilization prior to inoculation of the pathogens. Bacterial-wilt and damping off pathogens were drenched first and followed with the biological control agents and for comparison purposes, two chemical fungicides. But for frogeye leaf spot, which is an airborne fungus, a spraying procedure for every treatment including a chemical fungicide was applied instead of drenching. Results showed that neither B. subtilis AP-01 nor T harzianum AP-001 alone could control the bacterial wilt, but when combined, their controlling capabilities were as effective as a chemical treatment. These results were also similar for damping-off disease when used in combination. In addition, the combined B. subtilis AP-01 and T. harzianum AP-001 resulted in a good frogeye leaf spot control, which was not significantly different from the chemical treatment. PMID:24031219

  20. Proteomics Analyses of Bacillus subtilis after Treatment with Plumbagin, a Plant-Derived Naphthoquinone

    PubMed Central

    Reddy, Panga Jaipal; Ray, Sandipan; Sathe, Gajanan J.; Prasad, T.S. Keshava; Rapole, Srikanth; Panda, Dulal

    2015-01-01

    Abstract Infectious diseases and increasing antibiotic resistance among diverse classes of microbes are global health concerns and a prime focus of omics systems science applications in novel drug discovery. Plumbagin is a plant-derived naphthoquinone, a natural product that exhibits antibacterial activity against gram-positive bacteria. In the present study, we investigated the antimicrobial effects of plumbagin against Bacillus subtilis using two complementary proteomics techniques: two-dimensional electrophoresis (2-DE) and isobaric tag for relative and absolute quantification (iTRAQ). Comparative quantitative proteomics analysis of plumbagin treated and untreated control samples identified differential expression of 230 proteins (1% FDR, 1.5 fold-change and ≥2 peptides) in B. subtilis after plumbagin treatment. Pathway analysis involving the differentially expressed proteins suggested that plumbagin effectively increases heme and protein biosynthesis, whereas fatty acid synthesis was significantly reduced. Gene expression and metabolic activity assays further corroborated the proteomics findings. We anticipate that plumbagin blocks the cell division by altering the membrane permeability required for energy generation. This is the first report, to the best of our knowledge, offering new insights, at proteome level, for the putative mode(s) of action of plumbagin and attendant cellular targets in B. subtilis. The findings also suggest new ways forward for the modern omics-guided drug target discovery, building on traditional plant medicine. PMID:25562197