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Sample records for bacillus subtilis tyrs

  1. The Bacillus subtilis tyrZ Gene Encodes a Highly Selective Tyrosyl-tRNA Synthetase and Is Regulated by a MarR Regulator and T Box Riboswitch

    PubMed Central

    Williams-Wagner, Rebecca N.; Grundy, Frank J.; Raina, Medha; Ibba, Michael

    2015-01-01

    ABSTRACT Misincorporation of d-tyrosine (d-Tyr) into cellular proteins due to mischarging of tRNATyr with d-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation of Bacillus subtilis. Furthermore, many B. subtilis strains lack a functional gene encoding d-aminoacyl-tRNA deacylase, which prevents misincorporation of d-Tyr in most organisms. B. subtilis has two genes that encode tyrosyl-tRNA synthetase: tyrS is expressed under normal growth conditions, and tyrZ is known to be expressed only when tyrS is inactivated by mutation. We hypothesized that tyrZ encodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow when d-Tyr is present. We show that TyrZ is more selective for l-Tyr over d-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression of tyrZ is required for growth and biofilm formation in the presence of d-Tyr. Both tyrS and tyrZ are preceded by a T box riboswitch, but tyrZ is found in an operon with ywaE, which is predicted to encode a MarR family transcriptional regulator. Expression of tyrZ is repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression of tyrZ may allow growth when excess d-Tyr is present. IMPORTANCE Accurate protein synthesis requires correct aminoacylation of each tRNA with the cognate amino acid and discrimination against related compounds. Bacillus subtilis produces d-Tyr, an analog of l-Tyr that is toxic when incorporated into protein, during stationary phase. Most organisms utilize a d-aminoacyl-tRNA deacylase to prevent misincorporation of d-Tyr. This work demonstrates that the increased selectivity of the TyrZ form of tyrosyl-tRNA synthetase may provide a mechanism by which B. subtilis prevents misincorporation of d-Tyr in the absence of a functional d-aminoacyl-tRNA deacylase gene. PMID:25733610

  2. 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.

  3. 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

  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. 75 FR 862 - Bacillus subtilis; Registration Review Proposed Decision; Notice of Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-06

    ... AGENCY Bacillus subtilis; Registration Review Proposed Decision; Notice of Availability AGENCY... proposed registration review decision for the pesticide Bacillus subtilis (case 6012) and opens a public... EPA's proposed registration review decision Bacillus subtilis (case 6012). The Bacillus subtilis...

  7. 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).

  8. 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

  9. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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. 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

  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. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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...

  2. Architecture and Assembly of the Bacillus subtilis Spore Coat

    DTIC Science & Technology

    2014-09-26

    arrows). EM of ruthenium red stained B. subtilis spores demonstrated the presence of an outermost glycoprotein layer, and it was suggested that this layer...Rather the amorphous layer likely corresponds to the outer crust layer of B. subtilis spores that stains with ruthenium red and is glycoprotein rich...RL (2004) Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus

  3. 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

  4. 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.

  5. 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...

  6. 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.

  7. 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

  8. 77 FR 73934 - Bacillus subtilis Strain QST 713 Variant Soil; Amendment to an Exemption From the Requirement of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-12

    ... AGENCY 40 CFR Part 180 Bacillus subtilis Strain QST 713 Variant Soil; Amendment to an Exemption From the Requirement of a Tolerance for Bacillus subtilis Strain QST 713 To Include Residues of Bacillus subtilis... Bacillus subtilis strain QST 713 in or on all food commodities by including residues of Bacillus...

  9. 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

  10. [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.

  11. 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

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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)

  3. 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.

  4. 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

  5. [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.

  6. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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.

  9. 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.

  10. 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

    ... AGENCY 40 CFR Part 180 Bacillus Subtilis Strain CX-9060; Exemption From the Requirement of a Tolerance... an exemption from the requirement of a tolerance for residues of the microbial pesticide Bacillus... eliminates the need to establish a maximum permissible level for residues of Bacillus subtilis strain...

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. The dtd gene from Bacillus amyloliquefaciens encodes a putative D-tyrosyl-tRNATyr deacylase and is a selectable marker for Bacillus subtilis.

    PubMed

    Geraskina, Natalia V; Butov, Ivan A; Yomantas, Yurgis A V; Stoynova, Nataliya V

    2015-02-01

    Genetically engineered microbes are of high practical importance due to their cost-effective production of valuable metabolites and enzymes, and the search for new selectable markers for genetic manipulation is of particular interest. Here, we revealed that the soil bacterium Bacillus amyloliquefaciens A50 is tolerant to the non-canonical amino acid D-tyrosine (D-Tyr), in contrast to the closely related Bacillus strain B. subtilis 168, which is a widely used "domesticated" laboratory strain. The gene responsible for resistance to D-Tyr was identified. The resistance was associated with the activity of a potential D-tyrosyl-tRNA(Tyr) deacylase. Orthologs of this enzyme are capable of hydrolyzing the ester bond and recycling misacetylated D-aminoacyl-tRNA molecules into free tRNAs and D-amino acids. This gene, yrvI (dtd), is applicable as a convenient, small selectable marker for non-antibiotic resistance selection in experiments aimed at genome editing of D-Tyr-sensitive microorganisms.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

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

    PubMed

    Kim, Jin-Ha; Prabhu, Ponnandy; Jeya, Marimuthu; Tiwari, Manish Kumar; Moon, Hee-Jung; Singh, Raushan Kumar; Lee, Jung-Kul

    2010-02-01

    An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k(cat) of 14,504 min(-1) and a k(cat)/K(m) of 121 min(-1) mM(-1) for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. [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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 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...

  16. 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....

  17. 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...

  18. 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....

  19. 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...

  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 180.1309 - 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...

  2. 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....

  3. 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...

  4. 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...

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. [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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

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

    DTIC Science & Technology

    2011-12-01

    W-A. ; Pinyupa, P. Film coating of seeds with Bacillus cereus RS87 spores for early plant growth enhancement. Can. J. Microbial. 2008, 54, 861 - 867...Optimization and Characterization of Bacillus subtilis Spore Quality Sb. GRANT NUMBER Sc. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Sd. PROJECT NUMBER Smith...Environmental Protection Agency for the production of Bacillus subtilis spores. The cells require 12- 14 days to sporulate. Our goal was to conduct a study

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. High Pressure Germination of Bacillus subtilis Spores with Alterations in Levels and Types of Germination Proteins

    DTIC Science & Technology

    2014-01-01

    1ITLE AND SUBTITLE 5a CONTRACTNUMBER High pressure germination of Bacillus subtilis spores with W911NF-09-l-0286 alterations in levels and types of...A moderate high pressure (mHP) of 150 megaPascals (MPa) triggers germination of Bacillus subtilis spores via germinant receptors (GRs), while...germination by a very high pressure (vHP) of550 MPa is GR-independent. The mHP and vHP germination of Bacillus subtilis spores with different levels ofGRs

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  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 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. 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.

  6. 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

  7. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. 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.

  10. 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.

  11. 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

  12. 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.

  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.

  14. 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.

  15. Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC*

    PubMed Central

    Ravikumar, Vaishnavi; Shi, Lei; Krug, Karsten; Derouiche, Abderahmane; Jers, Carsten; Cousin, Charlotte; Kobir, Ahasanul; Mijakovic, Ivan; Macek, Boris

    2014-01-01

    Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates. PMID:24390483

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. [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.

  18. 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.

  19. 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.

  20. 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.

  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. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  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. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Crystallization and preliminary X-ray characterization of queD from Bacillus subtilis, an enzyme involved in queuosine biosynthesis

    SciTech Connect

    Cicmil, Nenad Shi, Lu

    2008-02-01

    B. subtilis queD was overexpressed, purified and crystallized. A diffraction data set was collected to a resolution of 3.6 Å. QueD (previously named ykvK) is one of several enzymes involved in the biosynthesis of the hypermodified nucleoside queuosine. Queuosine is incorporated into tRNA at position 34 of four tRNAs: tRNA{sup His}, tRNA{sup Asp}, tRNA{sup Asn} and tRNA{sup Tyr}. The crystallization and preliminary X-ray crystallographic studies of queD are described here. The recombinant protein from Bacillus subtilis was overproduced in Escherichia coli and crystallized using the hanging-drop vapor-diffusion method from 25% PEG 600, 100 mM NaCl and sodium citrate buffer pH 5.5 at 291 K. The crystals diffract to 3.6 Å resolution and belong to the cubic space group F4{sub 1}32, with unit-cell parameter a = 240.88 Å.

  4. 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

  5. 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

  6. 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

  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. 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.

  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. 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.

  12. 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

  13. 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.

  14. Cyclo(D-Tyr-D-Phe): a new antibacterial, anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode.

    PubMed

    Nishanth Kumar, S; Dileep, C; Mohandas, C; Nambisan, Bala; Ca, Jayaprakas

    2014-03-01

    A new microbial cyclic dipeptide (diketopiperazine), cyclo(D-Tyr-D-Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(D-Tyr-D-Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(D-Tyr-D-Phe) recorded significant antitumor activity against A549 cells (IC50 value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(D-Tyr-D-Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(D-Tyr-D-Phe). Flow cytometry analysis showed that the cyclo(D-Tyr-D-Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase-3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(D-Tyr-D-Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(D-Tyr-D-Phe) with synthetic cyclo(D-Tyr-D-Phe) and cyclo(L-Tyr-L-Phe). Natural and synthetic cyclo(D-Tyr-D-Phe) recorded similar pattern of activity. Although synthetic cyclo(L-Tyr-L-Phe) recorded lower activity. But in the case of reducing power activity

  15. Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis

    DTIC Science & Technology

    2012-11-01

    Kingdom. 35. Setlow B, et al. 2009. Characterization of Bacillus megaterium spores lacking enzymes that degrade the spore cortex. J. Appl. Microbiol...REPORT Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis 14. ABSTRACT 16. SECURITY CLASSIFICATION OF... Bacillus subtilis spores with a gerP mutation triggered spore germination via nutrient germinant receptors (GRs) slowly, although this defect was

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

    DTIC Science & Technology

    1993-01-01

    Vander Snoeck, P., Daneau, R.D., and Meunier, F. "Nosocomial Bacteremia Caused by Bacillus species", Clin. Micro bioi. Infect. Dis., 7, pp. 783...between B. cereus and B. subtilis existed in diagnostic laboratories before that time (Gordon 1973). B. subtilis, as well as other Bacillus species...or other interventions, which may have introduced the organism to sensitive tissue. Richard et al. (1988) described 11 cases of Bacillus bacteremias

  17. 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.

  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. 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

  20. 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

  1. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. [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.

  11. 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.

  12. 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…

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

    DTIC Science & Technology

    2014-06-01

    1ITLE AND SUBTITLE 5a CONTRACTNUMBER Function of the SpoVAEa and SpoVAF proteins of Bacillus W911NF-09-1-0286 subtilis spores 5b. GRANT NUMBER 5c...ABSTRACT The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developng spores as members of the spoVA operon that encodes proteins essential...8217\\ ;~ 1~~~4-~,.1. A\\ C’~~1T 1\\ D~ ~~,.1 C’~~1T 1\\ T’\\ ~-~ ,.1;~~1. •• 4-~,.1 ~:-:1~-1 •• ;~ ~~~~~~~ ~f:’ 15. SUBJECT TERMS Bacillus , spores SpoVA

  3. 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.

  4. A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation

    PubMed Central

    Flanagan, Kelly A.; Comber, Joseph D.; Mearls, Elizabeth; Fenton, Colleen; Wang Erickson, Anna F.

    2016-01-01

    ABSTRACT SpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell during Bacillus subtilis sporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor σG. Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of σG. The csfB gene is ordinarily transcribed in the forespore only by the early-acting sigma factor σF. However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28, csfB was also aberrantly transcribed later by σG, the very target of CsfB inhibition. This regulation of csfB by SpoIIQ Tyr-28 is specific, given that the expression of other σF-dependent genes was unaffected. Moreover, we identified a conserved element within the csfB promoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes σG activity in the forespore as a channel component but also specifically maximizes σG activity as part of a gene regulatory circuit that represses σG-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times. IMPORTANCE Cellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developing Bacillus subtilis spore is directed by the alternative sigma factor σG. The activity of σG requires a channel apparatus through which the adjacent mother cell provides

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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....

  15. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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).

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  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. 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.

  5. 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

  6. 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.

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

    PubMed

    Yi, Jun; Cheng, Jinping

    2017-04-04

    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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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)).

  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. 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.

  8. [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.

  9. 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.

  10. 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.

  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. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements.

    PubMed

    Zhang, Junjiao; Kang, Zhen; Ling, Zhenmin; Cao, Wenlong; Liu, Long; Wang, Miao; Du, Guocheng; Chen, Jian

    2013-10-01

    The present work aims to construct a robust recombinant Bacillus subtilis to achieve secretory production of alkaline polygalacturonate lyase (PGL). First, 6 signal peptides (amyX, bpr, vpr, yvgO, wapA and nprE) were screened with a semi-rational approach and comparatively investigated their effects on the production of PGL. The signal peptide bpr directed efficient PGL secretory expression and increased PGL titer to 313.7 U mL(-1). By optimizing and applying strong promoter P43 and Shine-Dalgarno sequence, higher titer of 446.3 U mL(-1) PGL was achieved. Finally, the capacity of the recombinant B. subtilis WB43CB was evaluated with a fed-batch strategy in 3 L fermentor. The PGL titer reached 632.6 U mL(-1) with a productivity of 17.6 U mL(-1) h(-1), which was the highest secretory production of PGL by the B. subtilis system. The recombinant B. subtilis strain WB43CB constructed in the present work has great potential in production of alkaline PGL.

  18. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

    PubMed Central

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-01-01

    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. PMID:27305971

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  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. Characterization of DegU-dependent expression of bpr in Bacillus subtilis.

    PubMed

    Tsukahara, Kensuke; Ogura, Mitsuo

    2008-03-01

    The response regulator DegU and its cognate histidine kinase DegS constitute a two-component system in the Gram-positive soil bacterium Bacillus subtilis. The phosphorylated form of DegU is known to activate transcription of more than 120 genes in B. subtilis, including the bpr gene encoding bacillopeptidase F. To characterize DegU-dependent regulation of bpr, the interaction of the bpr regulatory region with His-tagged DegU was analyzed using gel retardation and footprint analyses. This revealed that DegU bound three direct repeats of a motif that is known to be arranged as an inverted repeat in the comK promoter, to which DegU binds. Mutational analysis using a bpr-lacZ fusion revealed that the three direct repeats in bpr are needed for DegU-dependent transcription activation.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. [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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Unraveling the molecular basis for ligand binding in truncated hemoglobins: the trHbO Bacillus subtilis case.

    PubMed

    Boechi, Leonardo; Mañez, Pau Arroyo; Luque, F Javier; Marti, Marcelo A; Estrin, Dario A

    2010-03-01

    Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2-over-2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O(2) bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. [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.

  15. Co-production of surfactin and a novel bacteriocin by Bacillus subtilis subsp. subtilis H4 isolated from Bikalga, an African alkaline Hibiscus sabdariffa seed fermented condiment.

    PubMed

    Compaoré, Clarisse S; Nielsen, Dennis S; Ouoba, Labia I I; Berner, Torben S; Nielsen, Kristian F; Sawadogo-Lingani, Hagrétou; Diawara, Bréhima; Ouédraogo, Georges A; Jakobsen, Mogens; Thorsen, Line

    2013-04-01

    Bikalga is a Hibiscus sabdariffa seed fermented condiment widely consumed in Burkina Faso and neighboring countries. The fermentation is dominated by Bacillus subtilis group species. Ten B. subtilis subsp. subtilis (six isolates) and Bacillus licheniformis (four isolates) isolated from traditional Bikalga were examined for their antimicrobial activity against a panel of 36 indicator organisms including Gram-positive and Gram-negative bacteria and yeasts. The Bacillus spp. isolates showed variable inhibitory abilities depending on the method used. Both Gram-positive and Gram-negative bacteria were inhibited in the agar spot assay while only Gram-positive pathogens were inhibited in the agar well diffusion assay. Cell free supernatants (CFS) of pure cultures of 3 B. subtilis subsp. subtilis (G2, H4 and F1) strains inhibited growth of Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus and Bacillus cereus, while CFS of 2 B. licheniformis (E3 and F9) strains only inhibited M. luteus. The antimicrobial substance(s) produced by B. subtilis subsp. subtilis H4 was further characterized. The antimicrobial substance(s) produced by H4 was detected from mid-exponential growth phase. The activity was sensitive to protease and trypsin, but resistant to the proteolytic action of proteinase K and papain. Treatment with α-amylase and lipase II resulted in a complete loss of antimicrobial effect, indicating that a sugar moiety and lipid moiety are necessary for the activity. Treatment with mercapto-ethanol resulted in a significant loss, indicative of the presence of disulfide bridges. The antimicrobial activity of H4 was heat resistant and active at pH3-10. PCR detection of yiwB, sboA, spoX, albA and spaS, etnS genes and genes coding for surfactins and plipastatins (fengycins) indicated a potential for subtilosin, subtilin and lipopeptide production, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out and a single band

  16. 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.

  17. 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

  18. Localization of Components of the RNA-Degrading Machine in Bacillus subtilis

    PubMed Central

    Cascante-Estepa, Nora; Gunka, Katrin; Stülke, Jörg

    2016-01-01

    In bacteria, the control of mRNA stability is crucial to allow rapid adaptation to changing conditions. In most bacteria, RNA degradation is catalyzed by the RNA degradosome, a protein complex composed of endo- and exoribonucleases, RNA helicases, and accessory proteins. In the Gram-positive model organism Bacillus subtilis, the existence of a RNA degradosome assembled around the membrane-bound endoribonuclease RNase Y has been proposed. Here, we have studied the intracellular localization of the protein that have been implicated in the potential B. subtilis RNA degradosome, i.e., polynucleotide phosphorylase, the exoribonucleases J1 and J2, the DEAD-box RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase. Our data suggests that the bulk of these enzymes is located in the cytoplasm. The RNases J1 and J2 as well as the RNA helicase CshA were mainly localized in the peripheral regions of the cell where also the bulk of messenger RNA is localized. We were able to demonstrate active exclusion of these proteins from the transcribing nucleoid. Taken together, our findings suggest that the interactions of the enzymes involved in RNA degradation in B. subtilis are rather transient. PMID:27708634

  19. Functional Characterization of Core Components of the Bacillus subtilis Cyclic-Di-GMP Signaling Pathway

    PubMed Central

    Gao, Xiaohui; Mukherjee, Sampriti; Matthews, Paige M.; Hammad, Loubna A.; Kearns, Daniel B.

    2013-01-01

    Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems. PMID:23893111

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

    PubMed Central

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

    2016-01-01

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

  1. 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

  2. Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli.

    PubMed

    Kim, S I; Kim, H Y; Kwak, J H; Kwon, S H; Lee, S Y

    2000-02-29

    To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.

  3. DNA-Mediated Prophage Induction in Bacillus subtilis Lysogenic for φ105c4

    PubMed Central

    Garro, Anthony J.

    1973-01-01

    Prophage was induced when strains of Bacillus subtilis 168 lysogenic for φ105c4 were grown to competence and exposed to specific bacterial DNAs. The time course of phage production was similar to that observed for mitomycin C induction of wild-type prophage. Induction was directly dependent upon DNA concentration up to levels which were saturating for the transformation of bacterial auxotrophic markers. The extent of induction varied with the source of DNA. The burst of phage induced by DNA isolated from a W23 strain of B. subtilis was fivefold less than that induced by DNA from B. subtilis 168 strains, while B. licheniformis DNA was completely inactive. This order of inducing activity was correlated with the ability of the respective DNAs to transform auxotrophic markers carried by one of the φ105c4 lysogens. Differences in inducing activity also were observed for different forms of φ105 DNA. The DNAs isolated from φ105 phage particles and φ105c4 lysogens were inactive, whereas DNA from cells lysogenized by wild-type φ105 induced a burst of phage. When tested for transforming activity, however, both φ105c4 and φ105 lysogen DNAs were equally effective. An induction mechanism which involves recombination at the prophage insertion site is proposed to explain these differences. PMID:4199106

  4. DNA-damage-inducible (din) loci are transcriptionally activated in competent Bacillus subtilis

    SciTech Connect

    Love, P.E.; Lyle, M.J.; Yasbin, R.E.

    1985-09-01

    DNA damage-inducible (din) operon fusions were generated in Bacillus subtilis by transpositional mutagenesis. These YB886(din::Tn917-lacZ) fusion isolates produced increased ..beta..-galactosidase when exposed to mitomycin C, UV radiation, or ethyl methanesulfonate, indicating that the lacZ structural gene had inserted into host transcriptional units that are induced by a variety of DNA-damaging agents. One of the fusion strains was DNA-repair deficient and phenotypically resembled a UV-sensitive mutant of B. subtilis. Induction of ..beta..-galactosidase also occurred in the competent subpopulation of each of the din fusion strains, independent of exposure to DNA-damaging agents. Both the DNA-damage-inducible and competence-inducible components of ..beta..-galactosidase expression were abolished by the recE4 mutation, which inhibits SOS-like (SOB) induction but does not interfere with the development of the component state. The results indicate that gene expression is stimulated at specific loci within the B. subtilis chromosome both by DNA-damaging agents and by the development of competence and that this response is under the control of the SOB regulatory system. Furthermore, they demonstrate that at the molecular level SOB induction and the development of competence are interrelated cellular events.

  5. 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.

  6. 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.

  7. Production of Surfactant from Bacillus subtilis ATCC 21332 using Potato substrates

    SciTech Connect

    Fox, Sandra Lynn; Bala, Greg Alan

    2000-12-01

    Surfactin, a lipopeptide biosurfactant, produced by Bacillus subtilis is known to reduce the surface tension of water from 72 to 27 mN/m. Potato substrates were evaluated as a carbon source for surfactant production by B. subtilis ATCC 21332. An established potato medium, simulated liquid and solid potato waste media, and a commercially prepared potato starch in a mineral salts medium were evaluated in shake flask experiments to verify growth, surface tension reduction, and carbohydrate reduction capabilities. Total carbohydrate assays and glucose monitoring indicated that B. subtilis was able to degrade potato substrates to produce surfactant. Surface tensions dropped from 71.3±0.1 to 28.3±0.3 mN/m (simulated solid potato medium) and to 27.5±0.3 mN/m (mineral salts medium). A critical micelle concentration (CMC) of 0.10 g/l was obtained from a methylene chloride extract of the simulated solid potato medium.

  8. 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.

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

    PubMed Central

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

    2015-01-01

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

  11. 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

  12. 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

  13. Gut Adhesive Bacillus subtilis Spores as a Platform for Mucosal Delivery of Antigens

    PubMed Central

    Tavares Batista, Milene; Souza, Renata D.; Paccez, Juliano D.; Luiz, Wilson B.; Ferreira, Ewerton L.; Cavalcante, Rafael C. M.; Ferreira, Rita C. C.

    2014-01-01

    Bacillus subtilis spores have been used as safe and heat-resistant antigen delivery vectors. Nonetheless, the oral administration of spores typically induces weak immune responses to the passenger antigens, which may be attributed to the fast transit through the gastrointestinal tract. To overcome this limitation, we have developed B. subtilis spores capable of binding to the gut epithelium by means of expressing bacterial adhesins on the spore surface. The resulting spores bound to in vitro intestinal cells, showed a longer transit through the mouse intestinal tract, and interacted with Peyer's patch cells. The adhesive spores increased the systemic and secreted antibody responses to the Streptococcus mutans P1 protein, used as a model antigen, following oral, intranasal, and sublingual administration. Additionally, P1-specific antibodies efficiently inhibited the adhesion of the oral pathogen Streptococcus mutans to abiotic surfaces. These results support the use of gut-colonizing B. subtilis spores as a new platform for the mucosal delivery of vaccine antigens. PMID:24421038

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Purification and characterization of nattokinase from Bacillus subtilis natto B-12.

    PubMed

    Wang, Cong; Du, Ming; Zheng, Dongmei; Kong, Fandong; Zu, Guoren; Feng, Yibing

    2009-10-28

    Bacillus subtilis natto B-12 was isolated from natto, a traditional fermented soybean food in Japan. A fibrinolytic enzyme (B-12 nattokinase) was purified from the supernatant of B. subtilis natto B-12 culture broth and showed strong fibrinolytic activity. The enzyme was homogenously purified to 56.1-fold, with a recovery of 43.2% of the initial activity. B-12 nattokinase was demonstrated to be homogeneous by SDS-PAGE and was identified as a monomer of 29000 +/- 300 Da in its native state by SDS-PAGE and size exclusion methods. The optimal pH value and temperature were 8.0 and 40 degrees C, respectively. Purified nattokinase showed high thermostability at temperatures from 30 to 50 degrees C and alkaline stability within the range of pH 6.0-9.0. The enzyme activity was activated by Zn(2+) and obviously inhibited by Fe(3+) and Al(3+). This study provides some important information for the effect factors of fibrinolytic activity, the purification methods, and characterization of nattokinase from B. subtilis natto B-12, which enriches the theoretical information of nattokinase for the research and development of nattokinase as a functional additive of food.

  20. 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.

  1. 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

  2. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-03-01

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

  5. Preparation, crystallization and preliminary X-ray analysis of protein YtlP from Bacillus subtilis

    SciTech Connect

    Liu, Cong; Li, Dan; Hederstedt, Lars; Li, Lanfen; Liang, Yu-He Su, Xiao-Dong

    2006-10-01

    The crystallization and preliminary X-ray crystallographic analysis of protein YtlP from B. subtilis is reported. Bacillus subtilis YtlP is a protein that is predicted to belong to the bacterial and archael 2′-5′ RNA-ligase family. It contains 183 residues and two copies of the HXTX sequence motif conserved among proteins belonging to this family. In order to determine the structure of YtlP and to compare it with the paralogue YjcG and identified 2′-5′ RNA ligases, the gene ytlP was amplified from B. subtilis genomic DNA and cloned into expression vector pET-21a. The soluble protein was produced in Escherichia coli, purified to homogeneity and crystals suitable for X-ray analysis were obtained. The crystal diffracted to 2.0 Å and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 34.16, b = 48.54, c = 105.75 Å.

  6. Two distinct groups within the Bacillus subtilis group display significantly different spore heat resistance properties.

    PubMed

    Berendsen, Erwin M; Zwietering, Marcel H; Kuipers, Oscar P; Wells-Bennik, Marjon H J

    2015-02-01

    The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply.

  7. Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis.

    PubMed

    Nakayama, Motokazu; Shimatani, Kanami; Ozawa, Tadahiro; Shigemune, Naofumi; Tomiyama, Daisuke; Yui, Koji; Katsuki, Mao; Ikeda, Keisuke; Nonaka, Ai; Miyamoto, Takahisa

    2015-01-01

    Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.

  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. Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

    SciTech Connect

    Li, Dan; Chan, Chiomui; Liang, Yu-He Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

    2005-05-01

    B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°.

  10. Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in Bacillus subtilis

    PubMed Central

    Mielcarek, Andreas; Blauenburg, Bastian; Miethke, Marcus; Marahiel, Mohamed A.

    2015-01-01

    Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis. PMID:25826316

  11. The stringent response plays a key role in Bacillus subtilis survival of fatty acid starvation.

    PubMed

    Pulschen, André A; Sastre, Diego E; Machinandiarena, Federico; Crotta Asis, Agostina; Albanesi, Daniela; de Mendoza, Diego; Gueiros-Filho, Frederico J

    2017-02-01

    The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram-negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram-positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp-synthetases (RelBs , RelP and RelQ) or bearing a RelBs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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).

  17. 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.

  18. 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

  19. Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector.

    PubMed

    Zhang, Licong; Li, Xiaodan; Wei, Dandan; Wang, Jue; Shan, Anshan; Li, Zhongyu

    2015-10-01

    Plectasin, the first fungus defensin, is especially efficient against Gram-positive bacteria. To explore an effective approach for expressing plectasin in Bacillus subtilis, the sequence encoding plectasin fused with the small ubiquitin-like modifier (SUMO) gene, the 6 × His gene and the signal peptide of SacB were cloned into an E. coli-B. subtilis shuttle vector pGJ148 in which the maltose utilization operon promoter Pglv directed the expression. The fusion protein successfully secreted in culture and approximately, 41 mg of the recombinant fusion protein SUMO-plectasin was purified per liter of culture supernatant. After purification by Ni-NTA resin column and digestion by SUMO protease, 5.5 mg of plectasin with a purity of 94 % was obtained from 1 L fermentation culture. Recombinant plectasin was found inhibition activity against S. pneumoniae, S. aureus and S. epidermidis. These results indicate that the maltose-induced expression system may be a safe and efficient way for the large-scale production of soluble peptides in B. subtilis.

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

    PubMed Central

    Polka, Jessica K.

    2014-01-01

    Bacteria from several taxa, including Kurthia zopfii, Myxococcus xanthus, and Bacillus mycoides, have been reported to align growth of their colonies to small features on the surface of solid media, including anisotropies created by compression. While the function of this phenomenon is unclear, it may help organisms navigate on solid phases, such as soil. The origin of this behavior is also unknown: it may be biological (that is, dependent on components that sense the environment and regulate growth accordingly) or merely physical. Here we show that B. subtilis, an organism that typically does not respond to media compression, can be induced to do so with two simple and synergistic perturbations: a mutation that maintains cells in the swarming (chained) state, and the addition of EDTA to the growth media, which further increases chain length. EDTA apparently increases chain length by inducing defects in cell separation, as the treatment has only marginal effects on the length of individual cells. These results lead us to three conclusions. First, the wealth of genetic tools available to B. subtilis will provide a new, tractable chassis for engineering compression sensitive organisms. Second, the sensitivity of colony morphology to media compression in Bacillus can be modulated by altering a simple physical property of rod-shaped cells. And third, colony morphology under compression holds promise as a rapid, simple, and low-cost way to screen for changes in the length of rod-shaped cells or chains thereof. PMID:25289183

  1. Global Transcriptional Analysis of Virus-Host Interactions between Phage ϕ29 and Bacillus subtilis

    PubMed Central

    2016-01-01

    ABSTRACT The study of phage-host relationships is essential to understanding the dynamic of microbial systems. Here, we analyze genome-wide interactions of Bacillus subtilis and its lytic phage ϕ29 during the early stage of infection. Simultaneous high-resolution analysis of virus and host transcriptomes by deep RNA sequencing allowed us to identify differentially expressed bacterial genes. Phage ϕ29 induces significant transcriptional changes in about 0.9% (38/4,242) and 1.8% (76/4,242) of the host protein-coding genes after 8 and 16 min of infection, respectively. Gene ontology enrichment analysis clustered upregulated genes into several functional categories, such as nucleic acid metabolism (including DNA replication) and protein metabolism (including translation). Surprisingly, most of the transcriptional repressed genes were involved in the utilization of specific carbon sources such as ribose and inositol, and many contained promoter binding-sites for the catabolite control protein A (CcpA). Another interesting finding is the presence of previously uncharacterized antisense transcripts complementary to the well-known phage ϕ29 messenger RNAs that adds an additional layer to the viral transcriptome complexity. IMPORTANCE The specific virus-host interactions that allow phages to redirect cellular machineries and energy resources to support the viral progeny production are poorly understood. This study provides, for the first time, an insight into the genome-wide transcriptional response of the Gram-positive model Bacillus subtilis to phage ϕ29 infection. PMID:27489274

  2. Altered Sporulation and Respiratory Patterns in Mutants of Bacillus subtilis Induced by Acridine Orange

    PubMed Central

    Bott, K. F.; Davidoff-Abelson, R.

    1966-01-01

    Bott, K. F. (The University of Chicago, Chicago, Ill.), and R. Davidoff-Abelson. Altered sporulation and respiratory patterns in mutants of Bacillus subtilis induced by acridine orange. J. Bacteriol. 92:229–240. 1966.—The addition of acridine orange to vegetative cultures of Bacillus subtilis induces the formation of sporulation mutants at a frequency of 20% or greater. These mutants are grouped into seven categories which reflect their different morphological properties. They are altered in their vegetative metabolism, as indicated by abnormal growth on synthetic media. Sporulation of these mutants is impaired at several levels, all of which are stable upon repeated subculturing. The initial stages of sporulation which require no increased metabolic activity (proteolytic enzyme activity and antibiotic production) are functional in all strains, but glucose dehydrogenase activity, an enzyme associated with early synthetic functions in spore synthesis, is significantly reduced. Reduced nicotinamide adenine dinucleotide oxidase is slightly depressed. It is suggested that acridine orange interacts with a cellular constituent controlling respiration and consequently prevents an increased metabolic activity that may be associated with normal spore synthesis. Images PMID:4957434

  3. Characterization of Bacillus subtilis HC8, a novel plant‐beneficial endophytic strain from giant hogweed

    PubMed Central

    Malfanova, Natalia; Kamilova, Faina; Validov, Shamil; Shcherbakov, Andrey; Chebotar, Vladimir; Tikhonovich, Igor; Lugtenberg, Ben

    2011-01-01

    Summary Thirty endophytic bacteria were isolated from various plant species growing near Saint‐Petersburg, Russia. Based on a screening for various traits, including plant‐beneficial properties and DNA fragment patterns, potential siblings were removed. The remaining isolates were taxonomically identified using 16S rDNA sequences and potential human and plant pathogens were removed. The remaining strains were tested for their ability to promote radish root growth and to protect tomato plants against tomato foot and root rot. One strain, Bacillus subtilis HC8, isolated from the giant hogweed Heracleum sosnowskyi Manden, significantly promoted plant growth and protected tomato against tomato foot and root rot. Metabolites possibly responsible for these plant‐beneficial properties were identified as the hormone gibberellin and (lipo)peptide antibiotics respectively. The antibiotic properties of strain HC8 are similar to those of the commercially available plant‐beneficial strain Bacillus amyloliquefaciens FZB42. However, thin layer chromatography profiles of the two strains differ. It is speculated that endophytes such as B. subtilis HC8 contribute to the fast growth of giant hogweed. PMID:21366893

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  5. Production of Acetoin through Simultaneous Utilization of Glucose, Xylose, and Arabinose by Engineered Bacillus subtilis.

    PubMed

    Zhang, Bo; Li, Xin-Li; Fu, Jing; Li, Ning; Wang, Zhiwen; Tang, Ya-Jie; Chen, Tao

    2016-01-01

    Glucose, xylose and arabinose are the three most abundant monosaccharide found in lignocellulosic biomass. Effectively and simultaneously utilization of these sugars by microorganisms for production of the biofuels and bio-chemicals is essential toward directly fermentation of the lignocellulosic biomass. In our previous study, the recombinant Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain was already shown to efficiently utilize xylose for production of acetoin, with a yield of 0.36 g/g xylose. In the current study, the Bacillus subtilis168ARSRCPΔacoAΔbdhA strain was further engineered to produce acetoin from a glucose, xylose, and arabinose mixtures. To accomplish this, the endogenous xylose transport protein AraE, the exogenous xylose isomerase gene xylA and the xylulokinase gene xylB from E. coli were co-overexpressed in the Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain, which enabled the resulting strain, denoted ZB02, to simultaneously utilize glucose and xylose. Unexpectedly, the ZB02 strain could simultaneously utilize glucose and arabinose also. Further results indicated that the transcriptional inhibition of the arabinose transport protein gene araE was the main limiting factor for arabinose utilization in the presence of glucose. Additionally, the arabinose operon in B. subtilis could be activated by the addition of arabinose, even in the presence of glucose. Through fed-batch fermentation, strain ZB02 could simultaneously utilize glucose, xylose, and arabinose, with an average sugar consumption rate of 3.00 g/l/h and an average production of 62.2 g/l acetoin at a rate of 0.864 g/l/h. Finally, the strain produced 11.2 g/l acetoin from lignocellulosic hydrolysate (containing 20.6g/l glucose, 12.1 g/l xylose and 0.45 g/l arabinose) in flask cultivation, with an acetoin yield of 0.34 g/g total sugar. The result demonstrates that this strain has good potential for the utilization of lignocellulosic hydrolysate for production of acetoin.

  6. Production of Acetoin through Simultaneous Utilization of Glucose, Xylose, and Arabinose by Engineered Bacillus subtilis

    PubMed Central

    Fu, Jing; Li, Ning; Wang, Zhiwen; Tang, Ya-jie; Chen, Tao

    2016-01-01

    Glucose, xylose and arabinose are the three most abundant monosaccharide found in lignocellulosic biomass. Effectively and simultaneously utilization of these sugars by microorganisms for production of the biofuels and bio-chemicals is essential toward directly fermentation of the lignocellulosic biomass. In our previous study, the recombinant Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain was already shown to efficiently utilize xylose for production of acetoin, with a yield of 0.36 g/g xylose. In the current study, the Bacillus subtilis168ARSRCPΔacoAΔbdhA strain was further engineered to produce acetoin from a glucose, xylose, and arabinose mixtures. To accomplish this, the endogenous xylose transport protein AraE, the exogenous xylose isomerase gene xylA and the xylulokinase gene xylB from E. coli were co-overexpressed in the Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain, which enabled the resulting strain, denoted ZB02, to simultaneously utilize glucose and xylose. Unexpectedly, the ZB02 strain could simultaneously utilize glucose and arabinose also. Further results indicated that the transcriptional inhibition of the arabinose transport protein gene araE was the main limiting factor for arabinose utilization in the presence of glucose. Additionally, the arabinose operon in B. subtilis could be activated by the addition of arabinose, even in the presence of glucose. Through fed-batch fermentation, strain ZB02 could simultaneously utilize glucose, xylose, and arabinose, with an average sugar consumption rate of 3.00 g/l/h and an average production of 62.2 g/l acetoin at a rate of 0.864 g/l/h. Finally, the strain produced 11.2 g/l acetoin from lignocellulosic hydrolysate (containing 20.6g/l glucose, 12.1 g/l xylose and 0.45 g/l arabinose) in flask cultivation, with an acetoin yield of 0.34 g/g total sugar. The result demonstrates that this strain has good potential for the utilization of lignocellulosic hydrolysate for production of acetoin. PMID:27467131

  7. Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis.

    PubMed

    Duvall, E J; Williams, D M; Mongkolsuk, S; Lovett, P S

    1984-06-01

    Plasmid pPL603 is a promoter cloning vector for Bacillus subtilis and consists of a 1.1-kilobase fragment of Bacillus pumilus DNA inserted between the EcoRI and BamHI sites of pUB110. The gene cat-86, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, is located on the 1.1-kilobase cloned DNA. When pPL603 is present in B. subtilis, cat-86 is unexpressed during vegetative growth but expressed during sporulation. The regulation of cat-86 in pPL603 is due to sequences within two restriction fragments, designated P1 and R1, that precede the main coding portion of the gene. The P1 fragment promotes transcription of cat-86 only during sporulation, whereas the adjacent R1 fragment lacks promoter function but contains sequences essential to chloramphenicol inducibility. A second B. pumilus gene, cat-66, was cloned in B. subtilis and is expressed throughout the vegetative growth and sporulation cycle. The cat-66 coding region is preceded by two adjacent restriction fragments designated as P2 and R2. P1 and P2 are identical in size and share 95% conservation of base sequence. R1 and R2 are also identical in size and share 91% conservation of base sequence. Fragment substitution experiments demonstrate that R2 can functionally replace R1. The substitution of P2 for P1 promotes cat-86 expression throughout vegetative growth and sporulation. Analysis of a derivative of pPL603 in which P2 has replaced P1 demonstrates that P2 promotes transcription of cat-86 during vegetative growth and that P2 contains the start site for transcription of cat-86. Thus, P1 and P2 differ strikingly in vegetative promoter function, yet they differ by single-base substitutions at only 11 positions of 203.

  8. DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis.

    PubMed

    Winston, S; Sueoka, N

    1980-05-01

    We examined the effect of the inhibition of initiation of DNA replication on the membrane association of the chromosomal origin of replication of Bacillus subtilis and the Staphylococcus aureus-Bacillus pumilus chimeric plasmid pSL103, using temperature-sensitive mutants of B. subtilis that have specifically affected initiation. Inhibition of initiation of the chromosome and pSL103 in the initiation mutant dna-1 results in a decrease in the membrane association of both a marker near the chromosomal origin, purA16, and the plasmid pSL103. The membrane association of both purA16 and pSL103 can be recovered by allowing initiation to resume at the permissive temperature. In another initiation mutant, dnaB19, only the initiation and membrane association of the host chromosome are affected at the nonpermissive temperature, whereas both initiation and membrane association are not affected in the plasmid pSL103. In experiments in vitro, DNA containing the purA16 marker and pSL103 DNA molecules are both selectively released during incubation of purified DNA-membrane complexes prepared from dna-1 cells at the nonpermissive temperature. On the other hand, only purA16 DNA is released in vitro from the DNA-membrane complex prepared from dnaB19 cells. This consistent coupling between initiation and membrane association indicates that DNA-membrane association is critical for the initiation of the B. subtilis chromosome and the plasmid pSL103.

  9. Poly-γ-Glutamic Acids Contribute to Biofilm Formation and Plant Root Colonization in Selected Environmental Isolates of Bacillus subtilis.

    PubMed

    Yu, Yiyang; Yan, Fang; Chen, Yun; Jin, Christopher; Guo, Jian-Hua; Chai, Yunrong

    2016-01-01

    Bacillus subtilis is long known to produce poly-γ-glutamic acids (γ-PGA) as one of the major secreted polymeric substances. In B. subtilis, the regulation of γ-PGA production and its physiological role are still unclear. B. subtilis is also capable of forming structurally complex multicellular communities, or biofilms, in which an extracellular matrix consisting of secreted proteins and polysaccharides holds individual cells together. Biofilms were shown to facilitate B. subtilis-plant interactions. In this study, we show that different environmental isolates of B. subtilis, all capable of forming biofilms, vary significantly in γ-PGA production. This is possibly due to differential regulation of γ-PGA biosynthesis genes. In many of those environmental isolates, γ-PGA seems to contribute to robustness and complex morphology of the colony biofilms, suggesting a role of γ-PGA in biofilm formation. Our evidence further shows that in selected B. subtilis strains, γ-PGA also plays a role in root colonization by the bacteria, pinpointing a possible function of γ-PGA in B. subtilis-plant interactions. Finally, we found that several pathways co-regulate both γ-PGA biosynthesis genes and genes for the biofilm matrix in B. subtilis, but in an opposing fashion. We discussed potential biological significance of that.

  10. A gene at 333 degrees on the Bacillus subtilis chromosome encodes the newly identified sigma B-dependent general stress protein GspA.

    PubMed Central

    Antelmann, H; Bernhardt, J; Schmid, R; Hecker, M

    1995-01-01

    In Bacillus subtilis, general stress proteins (Gsps) are induced in response to different stresses (heat, salt, or ethanol) or after nutrient starvation. The majority of the genes for the Gsps are organized in a very large stationary-phase or stress regulon which is controlled by alternative sigma factor sigma B. The most striking spots on Coomassie-stained two-dimensional gels belong to GsiB and GspA, which are synthesized at extremely high levels in response to different stresses. Therefore, we determined the N-terminal protein sequence of GspA, which exhibited total identity to a hypothetical 33.5-kDa protein of B. subtilis encoded by open reading frame 2 (ipa-12d) in the sacY-tyrS1 intergenic region. The GspA-encoding gene gspA and the upstream and downstream regions were cloned with the aid of the PCR technique. By primer extension experiments, one sigma B-dependent promoter immediately upstream of the coding region was identified. A putative factor-independent terminator closely followed the coding region. By Northern (RNA) blot analysis, a 0.95-kb transcript was detected which indicates a monocistronic transcriptional unit. The gspA mRNA was strongly induced by different stimuli like heat or salt stress and starvation for glucose. Analysis of RNA isolated from a sigma B deletion mutant revealed that the transcription of gspA is sigma B dependent. Insertional inactivation of the B. subtilis chromosomal gspA gene confirmed that the gspA gene is not essential for either vegetative growth or growth under the influence of different stresses. In gspA mutant cells, the level of flagellin was increased severalfold over that in wild-type cells. PMID:7768864

  11. Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms.

    PubMed

    Kesel, Sara; Grumbein, Stefan; Gümperlein, Ina; Tallawi, Marwa; Marel, Anna-Kristina; Lieleg, Oliver; Opitz, Madeleine

    2016-04-01

    Many bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain,B. subtilis B-1, which forms biofilms containing mainly γ-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of γ-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms.

  12. Responses of Bacillus subtilis to hypotonic challenges: physiological contributions of mechanosensitive channels to cellular survival.

    PubMed

    Hoffmann, Tamara; Boiangiu, Clara; Moses, Susanne; Bremer, Erhard

    2008-04-01

    Mechanosensitive channels are thought to function as safety valves for the release of cytoplasmic solutes from cells that have to manage a rapid transition from high- to low-osmolarity environments. Subsequent to an osmotic down-shock of cells grown at high osmolarity, Bacillus subtilis rapidly releases the previously accumulated compatible solute glycine betaine in accordance with the degree of the osmotic downshift. Database searches suggest that B. subtilis possesses one copy of a gene for a mechanosensitive channel of large conductance (mscL) and three copies of genes encoding proteins that putatively form mechanosensitive channels of small conductance (yhdY, yfkC, and ykuT). Detailed mutational analysis of all potential channel-forming genes revealed that a quadruple mutant (mscL yhdY yfkC ykuT) has no growth disadvantage in high-osmolarity media in comparison to the wild type. Osmotic down-shock experiments demonstrated that the MscL channel is the principal solute release system of B. subtilis, and strains with a gene disruption in mscL exhibited a severe survival defect upon an osmotic down-shock. We also detected a minor contribution of the SigB-controlled putative MscS-type channel-forming protein YkuT to cellular survival in an mscL mutant. Taken together, our data revealed that mechanosensitive channels of both the MscL and MscS types play pivotal roles in managing the transition of B. subtilis from hyper- to hypo-osmotic environments.

  13. Interaction of protoplasts, L forms, and bacilli of Bacillus subtilis with 12 strains of bacteriophage.

    PubMed Central

    Jacobson, E D; Landman, O E

    1975-01-01

    The interaction of 12 phage strains with bacilli, protoplasts, and L forms of Bacillus subtilis 168 and with eight of its mutants and two of its lysogens is described qualitatively and quantitatively. After removal of the cell wall from B. subtilis 168, 11 of the 12 phage strains can still adsorb to the protoplasts, nine kill their wall-less host cells, and five multiply in the naked bacteria, forming plaques on L form lawns. Individual gene mutations can have similarly pleiotropic effects, strongly dependent upon the plating medium. Thus, the gta A mutation, which causes loss of glucosylation of the wall teichoic acid, results in loss of wall adsorption sites for phi (but not membrane sites) and for phi105. Phages phi25, SP82G and phie can still adsorb to gta A bacilli and plaque in unstabilized and sorbitol-stabilized lawns of this mutant, but they can not plaque in sucrose-stabilized lawns. The lysogenized wild type, B. subtilis 168 (SPO2), also exhibits a pleiotropic pattern, showing different levels of resistance to phages SPO2, phi1, phie, and phi25. Its resistance pattern is very similar to that of wild-type protoplasts. On the basis of such patterns, the bacterial mutants and strain B. subtilis 168 (SPO2) could be ordered into four classes and the phage strains classified into four to six groups. Together, they form four to six interaction complexes, based partly on adsorption sites and perhaps partly on metabolic blocks in phage development. Images PMID:809420

  14. Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms

    PubMed Central

    Kesel, Sara; Grumbein, Stefan; Gümperlein, Ina; Tallawi, Marwa; Marel, Anna-Kristina

    2016-01-01

    Many bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain, B. subtilis B-1, which forms biofilms containing mainly γ-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of γ-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms. PMID:26873313

  15. Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in Bacillus subtilis

    PubMed Central

    Schäffer, Christina; Wugeditsch, Thomas; Messner, Paul; Whitfield, Chris

    2002-01-01

    The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-α-d-14C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli. PMID:12324313

  16. Anaerobic transcription activation in Bacillus subtilis: identification of distinct FNR-dependent and -independent regulatory mechanisms.

    PubMed Central

    Cruz Ramos, H; Boursier, L; Moszer, I; Kunst, F; Danchin, A; Glaser, P

    1995-01-01

    Bacillus subtilis is able to grow anaerobically using alternative electron acceptors, including nitrate or fumarate. We characterized an operon encoding the dissimilatory nitrate reductase subunits homologous to the Escherichia coli narGHJI operon and the narK gene encoding a protein with nitrite extrusion activity. Downstream from narK and co-transcribed with it a gene (fnr) encoding a protein homologous to E.coli FNR was found. Disruption of fnr abolished both nitrate and fumarate utilization as electron acceptors and anaerobic induction of narK. Four putative FNR binding sites were found in B.subtilis sequences. The consensus sequence, centred at position -41.5, is identical to the consensus for the DNA site for E.coli CAP. Bs-FNR contained a four cysteine residue cluster at its C-terminal end. This is in contrast to Ec-FNR, where a similar cluster is present at the N-terminal end. It is possible that oxygen modulates the activity of both activators by a similar mechanism involving iron. Unlike in E.coli, where fnr expression is weakly repressed by anaerobiosis, fnr gene expression in B.subtilis is strongly activated by anaerobiosis. We have identified in the narK-fnr intergenic region a promotor activated by anaerobiosis independently of FNR. Thus induction of genes involved in anaerobic respiration requires in B.subtilis at least two levels of regulation: activation of fnr transcription and activation of FNR to induce transcription of FNR-dependent promoters. Images PMID:8846791

  17. CodY Regulates Expression of the Bacillus subtilis Extracellular Proteases Vpr and Mpr

    PubMed Central

    Barbieri, Giulia; Voigt, Birgit; Albrecht, Dirk; Hecker, Michael; Albertini, Alessandra M.; Sonenshein, Abraham L.; Ferrari, Eugenio

    2015-01-01

    ABSTRACT CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. IMPORTANCE CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular

  18. Two tandemly located promoters, artificially constructed, are active in a Bacillus subtilis alpha-amylase secretion vector.

    PubMed

    Furusato, T; Takano, J; Jigami, Y; Tanaka, H; Yamane, K

    1986-04-01

    An 85 bp DNA fragment, the nucleotide sequence of which had 84% homology with the sequence for the promoter, ribosome binding site and NH2-terminal five amino acids of the Bacillus amyloliquefaciens alpha-amylase gene, was chemically synthesized. In order to analyze the promoter activity of a Bacillus subtilis alpha-amylase secretion vector, the fragment was inserted between the promoter and signal peptide-coding region of Bacillus subtilis alpha-amylase gene. Both promoters, tandemly repeated, functioned in transcribing the B. subtilis alpha-amylase signal peptide-coding region followed by the Escherichia coli beta-lactamase structural gene. The transcription initiation sites were determined by the primer extension method. The extracellular production of beta-lactamase was stimulated by two promoters as compared with that by the plasmids containing either promoter region alone. The change of two amino acids in the NH2-terminal region of the B. subtilis alpha-amylase signal peptide had no effect on the secretion of beta-lactamase from B. subtilis cells.

  19. SpoVT: From Fine-Tuning Regulator in Bacillus subtilis to Essential Sporulation Protein in Bacillus cereus

    PubMed Central

    Eijlander, Robyn T.; Holsappel, Siger; de Jong, Anne; Ghosh, Abhinaba; Christie, Graham; Kuipers, Oscar P.

    2016-01-01

    Sporulation is a highly sophisticated developmental process adopted by most Bacilli as a survival strategy to withstand extreme conditions that normally do not support microbial growth. A complicated regulatory cascade, divided into various stages and taking place in two different compartments of the cell, involves a number of primary and secondary regulator proteins that drive gene expression directed toward the formation and maturation of an endospore. Such regulator proteins are highly conserved among various spore formers. Despite this conservation, both regulatory and phenotypic differences are observed between different species of spore forming bacteria. In this study, we demonstrate that deletion of the regulatory sporulation protein SpoVT results in a severe sporulation defect in Bacillus cereus, whereas this is not observed in Bacillus subtilis. Although spores are initially formed, the process is stalled at a later stage in development, followed by lysis of the forespore and the mother cell. A transcriptomic investigation of B. cereus ΔspoVT shows upregulation of genes involved in germination, potentially leading to premature lysis of prespores formed. Additionally, extreme variation in the expression of species-specific genes of unknown function was observed. Introduction of the B. subtilis SpoVT protein could partly restore the sporulation defect in the B. cereus spoVT mutant strain. The difference in phenotype is thus more than likely explained by differences in promoter targets rather than differences in mode of action of the conserved SpoVT regulator protein. This study stresses that evolutionary variances in regulon members of sporulation regulators can have profound effects on the spore developmental process and that mere protein homology is not a foolproof predictor of similar phenotypes. PMID:27790204

  20. phoR sequences as a phylogenetic marker to differentiate the species in the Bacillus subtilis group.

    PubMed

    Guo, Qinggang; Li, Shezeng; Lu, Xiuyun; Li, Baoqing; Stummer, Belinda; Dong, Weixin; Ma, Ping

    2012-11-01

    Bacillus subtilis and its closely related species are indistinguishable from one another by morphological characteristics and 16S rDNA sequences. In this study, the partial phoR sequence was tested to determine the phylogenetic relationship of species in the B. subtilis group. Degenerate primers were developed according to the relatively conserved nucleotide sequences of phoR and the linked gene phoP in the B. subtilis group. The primers amplified a 1100 bp phoR fragment from strains representative of 6 species in the B. subtilis group. Based on the sequenced fragments, 26 type strains comprising these 6 species were clearly distinguished. At the intraspecies level, the phoR sequence similarities were 90%-100%, but at the interspecies level, the phoR sequence similarities were 32.8%-75%. Compared with the gyrB sequence, the phoR sequences showed a larger divergence especially at the interspecies levels. Therefore, the phoR sequence may be an efficient alternative marker for phylogenetic and taxonomic analysis of species in the B. subtilis group. Twenty-three Bacillus undomesticated isolates were tested for identification and phylogenetic analysis based on the phoR and gyrB sequences. The 23 isolates could be clearly delineated into 4 distinct groups, 10 as B. subtilis, 3 as B. mojavensis, 2 as B. atrophaeus, and 8 as B. amyloliquefaciens.

  1. Protective role of spore structural components in determining Bacillus subtilis spore resistance to simulated mars surface conditions.

    PubMed

    Moeller, Ralf; Schuerger, Andrew C; Reitz, Günther; Nicholson, Wayne L

    2012-12-01

    Spores of wild-type and mutant Bacillus subtilis strains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants.

  2. Effect of Mono and Di-rhamnolipids on Biofilms Pre-formed by Bacillus subtilis BBK006.

    PubMed

    De Rienzo, Mayri A Díaz; Martin, Peter J

    2016-08-01

    Different microbial inhibition strategies based on the planktonic bacterial physiology have been known to have limited efficacy on the growth of biofilms communities. This problem can be exacerbated by the emergence of increasingly resistant clinical strains. Biosurfactants have merited renewed interest in both clinical and hygienic sectors due to their potential to disperse microbial biofilms. In this work, we explore the aspects of Bacillus subtilis BBK006 biofilms and examine the contribution of biologically derived surface-active agents (rhamnolipids) to the disruption or inhibition of microbial biofilms produced by Bacillus subtilis BBK006. The ability of mono-rhamnolipids (Rha-C10-C10) produced by Pseudomonas aeruginosa ATCC 9027 and the di-rhamnolipids (Rha-Rha-C14-C14) produced by Burkholderia thailandensis E264, and phosphate-buffered saline to disrupt biofilm of Bacillus subtilis BBK006 was evaluated. The biofilm produced by Bacillus subtilis BBK006 was more sensitive to the di-rhamnolipids (0.4 g/L) produced by Burkholderia thailandensis than the mono-rhamnolipids (0.4 g/L) produced by Pseudomonas aeruginosa ATCC 9027. Rhamnolipids are biologically produced compounds safe for human use. This makes them ideal candidates for use in new generations of bacterial dispersal agents and useful for use as adjuvants for existing microbial suppression or eradication strategies.

  3. Genetic heterogeneity in the cysA-fus region of the Bacillus subtilis chromosome: identification of the hos gene.

    PubMed Central

    Matsuzaki, S; Kobayashi, Y

    1985-01-01

    We identified a new gene, hos, which exerts different sporulation phenotypes in Bacillus subtilis strains with different genetic backgrounds. The hos+ gene showed normal sporulation in the genetic background of JH642 but showed temperature-sensitive sporulation in that of the Tano-oka W. The hos gene was mapped between cysA and rpoB. PMID:3922952

  4. 40 CFR 180.1209 - Bacillus subtilis strain QST 713 and strain QST 713 variant soil; exemption from the requirement...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... strain QST 713 variant soil; exemption from the requirement of a tolerance. 180.1209 Section 180.1209... strain QST 713 and strain QST 713 variant soil; exemption from the requirement of a tolerance. An... Bacillus subtilis strain QST 713 and strain QST 713 variant soil when used in or on all food commodities....

  5. 40 CFR 180.1209 - Bacillus subtilis strain QST 713 and strain QST 713 variant soil; exemption from the requirement...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... strain QST 713 variant soil; exemption from the requirement of a tolerance. 180.1209 Section 180.1209... strain QST 713 and strain QST 713 variant soil; exemption from the requirement of a tolerance. An... Bacillus subtilis strain QST 713 and strain QST 713 variant soil when used in or on all food commodities....

  6. [Growth peculiarities and properties of Bacillus subtilis IMV B-7023 cell surface in the medium with glycerophosphate].

    PubMed

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

    2009-01-01

    It is established that, depending on the amount of the basic elements of carbon and phosphorus nutrition in the cultivation medium, Bacillus subtilis IMV B-7023 can use glycerophosphate as a source of carbon, carbon and phosphorus, or phosphorus. The found differences in bacterium physiology correlate with the change of cell surface properties.

  7. Protective Role of Spore Structural Components in Determining Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions

    PubMed Central

    Schuerger, Andrew C.; Reitz, Günther; Nicholson, Wayne L.

    2012-01-01

    Spores of wild-type and mutant Bacillus subtilis strains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants. PMID:23064347

  8. Draft Genome Sequence of the Biofilm-Producing Bacillus subtilis Strain B-1, Isolated from an Oil Field

    PubMed Central

    Kesel, S.; Moormann, F.; Gümperlein, I.; Mader, A.; Morikawa, M.; Lieleg, O.

    2014-01-01

    We report here the draft genome sequence of the Bacillus subtilis strain B-1, a strain known to form biofilms. The biofilm matrix mainly consists of the biopolymer γ-polyglutamate (γ-PGA). The sequence of the genome of this strain allows the study of specific genes involved in biofilm formation. PMID:25502661

  9. Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

    PubMed Central

    Juhas, Mario; Wong, Christine; Ajioka, James W.

    2016-01-01

    The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis. PMID:27798678

  10. The Bacillus subtilis spore coat provides "eat resistance" during phagocytic predation by the protozoan Tetrahymena thermophila.

    PubMed

    Klobutcher, Lawrence A; Ragkousi, Katerina; Setlow, Peter

    2006-01-03

    Bacillus spores are highly resistant to many environmental stresses, owing in part to the presence of multiple "extracellular" layers. Although the role of some of these extracellular layers in resistance to particular stresses is known, the function of one of the outermost layers, the spore coat, is not completely understood. This study sought to determine whether the spore coat plays a role in resistance to predation by the ciliated protozoan Tetrahymena, which uses phagocytosis to ingest and degrade other microorganisms. Wild-type dormant spores of Bacillus subtilis were efficiently ingested by the protozoan Tetrahymena thermophila but were neither digested nor killed. However, spores with various coat defects were killed and digested, leaving only an outer shell termed a rind, and supporting the growth of Tetrahymena. A similar rind was generated when coat-defective spores were treated with lysozyme alone. The sensitivity of spores with different coat defects to predation by T. thermophila paralleled the spores' sensitivities to lysozyme. Spore killing by T. thermophila was by means of lytic enzymes within the protozoal phagosome, not by initial spore germination followed by killing. These findings suggest that a major function of the coat of spores of Bacillus species is to protect spores against predation. We also found that indigestible rinds were generated even from spores in which cross-linking of coat proteins was greatly reduced, implying the existence of a coat structure that is highly resistant to degradative enzymes.

  11. Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

    PubMed

    Reddy, Chinreddy Subramanyam; Achary, V Mohan Murali; Manna, Mrinalini; Singh, Jitender; Kaul, Tanushri; Reddy, Malireddy K

    2015-03-01

    The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.

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

    PubMed

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

    2015-06-01

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

  13. [Growth conditions and production 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; Rudenskaia, G N; Kostrov, S V; Sharipova, M R

    2006-01-01

    The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).

  14. Time Series Analysis of the Bacillus subtilis Sporulation Network Reveals Low Dimensional Chaotic Dynamics

    PubMed Central

    Lecca, Paola; Mura, Ivan; Re, Angela; Barker, Gary C.; Ihekwaba, Adaoha E. C.

    2016-01-01

    Chaotic behavior refers to a behavior which, albeit irregular, is generated by an underlying deterministic process. Therefore, a chaotic behavior is potentially controllable. This possibility becomes practically amenable especially when chaos is shown to be low-dimensional, i.e., to be attributable to a small fraction of the total systems components. In this case, indeed, including the major drivers of chaos in a system into the modeling approach allows us to improve predictability of the systems dynamics. Here, we analyzed the numerical simulations of an accurate ordinary differential equation model of the gene network regulating sporulation initiation in Bacillus subtilis to explore whether the non-linearity underlying time series data is due to low-dimensional chaos. Low-dimensional chaos is expectedly common in systems with few degrees of freedom, but rare in systems with many degrees of freedom such as the B. subtilis sporulation network. The estimation of a number of indices, which reflect the chaotic nature of a system, indicates that the dynamics of this network is affected by deterministic chaos. The neat separation between the indices obtained from the time series simulated from the model and those obtained from time series generated by Gaussian white and colored noise confirmed that the B. subtilis sporulation network dynamics is affected by low dimensional chaos rather than by noise. Furthermore, our analysis identifies the principal driver of the networks chaotic dynamics to be sporulation initiation phosphotransferase B (Spo0B). We then analyzed the parameters and the phase space of the system to characterize the instability points of the network dynamics, and, in turn, to identify the ranges of values of Spo0B and of the other drivers of the chaotic dynamics, for which the whole system is highly sensitive to minimal perturbation. In summary, we described an unappreciated source of complexity in the B. subtilis sporulation network by gathering

  15. Bacillus subtilis Class Ib Ribonucleotide Reductase Is a Dimanganese(III)-Tyrosyl Radical Enzyme†

    PubMed Central

    Zhang, Yan; Stubbe, JoAnne

    2011-01-01

    Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from E. coli. As isolated, recombinant (r) rNrdF contained a diferric-tyrosyl radical (Fe(III)2-Y•) cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O2. Apo-rNrdF loaded using 4 Mn(II)/β2, O2 and reduced NrdI (a flavodoxin), can form a dimanganese(III)-Y• (Mn(III)2-Y•) cofactor. In the presence of rNrdE/ATP/CDP, Mn(III)2-Y• and Fe(III)2-Y• rNrdF generate dCDP at 132 and 10 nmol min-1 mg-1 respectively (both normalized for 1 Y•/β2). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in LB medium, with levels of NrdE and NrdF elevated 35 fold relative to the wild type (wt) strain. NrdE and NrdF co-purified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)2-Y• center with 2 Mn and 0.5 Y•/β2 and activity of 318-363 nmol min-1 mg-1 (normalized for 1 Y•/β2). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)2-Y• enzyme. PMID:21561096

  16. Impact of a Bacterial Volatile 2,3-Butanediol on Bacillus subtilis Rhizosphere Robustness

    PubMed Central

    Yi, Hwe-Su; Ahn, Yeo-Rim; Song, Geun C.; Ghim, Sa-Youl; Lee, Soohyun; Lee, Gahyung; Ryu, Choong-Min

    2016-01-01

    Volatile compounds, such as short chain alcohols, acetoin, and 2,3-butanediol, produced by certain strains of root-associated bacteria (rhizobacteria) elicit induced systemic resistance in plants. The effects of bacterial volatile compounds (BVCs) on plant and fungal growth have been extensively studied; however, the impact of bacterial BVCs on bacterial growth remains poorly understood. In this study the effects of a well-characterized bacterial volatile, 2,3-butanediol, produced by the rhizobacterium Bacillus subtilis, were examined in the rhizosphere. The nature of 2,3-butanediol on bacterial cells was assessed, and the effect of the molecule on root colonization was also determined. Pepper roots were inoculated with three B. subtilis strains: the wild type, a 2,3-butanediol overexpressor, and a 2,3-butanediol null mutant. The B. subtilis null strain was the first to be eliminated in the rhizosphere, followed by the wild-type strain. The overexpressor mutant was maintained at roots for the duration of the experiment. Rhizosphere colonization by a saprophytic fungus declined from 14 days post-inoculation in roots treated with the B. subtilis overexpressor strain. Next, exudates from roots exposed to 2,3-butanediol were assessed for their impact on fungal and bacterial growth in vitro. Exudates from plant roots pre-treated with the 2,3-butanediol overexpressor were used to challenge various microorganisms. Growth was inhibited in a saprophytic fungus (Trichoderma sp.), the 2,3-butanediol null B. subtilis strain, and a soil-borne pathogen, Ralstonia solanacearum. Direct application of 2,3-butanediol to pepper roots, followed by exposure to R. solanacearum, induced expression of Pathogenesis-Related (PR) genes such as CaPR2, CaSAR8.2, and CaPAL. These results indicate that 2,3-butanediol triggers the secretion of root exudates that modulate soil fungi and rhizosphere bacteria. These data broaden our knowledge regarding bacterial volatiles in the rhizosphere and

  17. Influence of Bacillus subtilis cell walls and EDTA on calcite dissolution rates and crystal surface features.

    PubMed

    Friis, A K; Davis, T A; Figueira, M M; Paquette, J; Mucci, A

    2003-06-01

    This study investigates the influence of EDTA and the Gram-positive cell walls of Bacillus subtilis on the dissolution rates and development of morphological features on the calcite [1014] surface. The calcite dissolution rates are compared at equivalent saturation indicies (SI) and relative to its dissolution behavior in distilled water (DW). Results indicate that the presence of metabolically inactive B. subtilis does not affect the dissolution rates significantly. Apparent increases in dissolution rates in the presence of the dead bacterial cells can be accounted for by a decrease of the saturation state of the solution with respect to calcite resulting from bonding of dissolved Ca2+ by functional groups on the cell walls. In contrast, the addition of EDTA to the experimental solutions results in a distinct increase in dissolution rates relative to those measured in DW and the bacterial cell suspensions. These results are partly explained by the 6.5-8 orders of magnitude greater stability of the Ca-EDTA complex relative to the Ca-B. subtilis complexes as well as its free diffusion to and direct attack of the calcite surface. Atomic force microscopy images of the [1014] surface of calcite crystals exposed to our experimental solutions reveal the development of dissolution pits with different morphologies according to the nature and concentration of the ligand. Highly anisotropic dissolution pits develop in the early stages of the dissolution reaction at low B. subtilis concentrations (0.004 mM functional group sites) and in DW. In contrast, at high functional group concentrations (4.0 mM EDTA or equivalent B. subtilis functional group sites), dissolution pits are more isotropic. These results suggest that the mechanism of calcite dissolution is modified by the presence of high concentrations of organic ligands. Since all the pits that developed on the calcite surfaces display some degree of anisotropy and dissolution rates are strongly SI dependent, the rate

  18. HtrC Is Involved in Proteolysis of YpeB during Germination of Bacillus anthracis and Bacillus subtilis Spores

    PubMed Central

    Bernhards, Casey B.; Chen, Yan; Toutkoushian, Hannah

    2014-01-01

    Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn2+ or Ca2+ ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation. PMID:25384476

  19. HtrC is involved in proteolysis of YpeB during germination of Bacillus anthracis and Bacillus subtilis spores.

    PubMed

    Bernhards, Casey B; Chen, Yan; Toutkoushian, Hannah; Popham, David L

    2015-01-01

    Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn(2+) or Ca(2+) ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation.

  20. Mechanisms of induction of germination of Bacillus subtilis spores by high pressure.

    PubMed

    Paidhungat, Madan; Setlow, Barbara; Daniels, William B; Hoover, Dallas; Papafragkou, Efstathia; Setlow, Peter

    2002-06-01

    Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.

  1. Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis

    SciTech Connect

    Thoelke, M.S.; Casper, J.M.; Ordal, G.W. )

    1990-02-05

    The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it.

  2. Effects of solar ultraviolet radiations on Bacillus subtilis spores and T-7 bacteriophage

    NASA Technical Reports Server (NTRS)

    Spizizen, J.; Isherwood, J. E.; Taylor, G. R.

    1975-01-01

    Spores of Bacillus subtilis HA 101 and the DNA polymerase I-defective mutant HA 101 (59)F were exposed to selected wavelengths of solar ultraviolet light and space vacuum during the return of Apollo 16. In addition, coliphage T-7 suspensions were exposed to solar ultraviolet radiation as part of the Microbial Response to Space Environment Experiment. Optical filters were employed to provide different energy levels at wavelengths 254 nm and 280 nm. Dose-response curves for lethal and mutagenic effects were compared with ground-based data. A close parallel was observed between the results of solar radiation and ground tests with spores of the two strains. However, significantly greater inactivation of T-7 bacteriophage was observed after exposure to solar ultraviolet radiation.

  3. Action of Bacillus subtilis alpha-amylase on native wheat starch.

    PubMed

    Colonna, P; Buléon, A; Lemarié, F

    1988-06-05

    Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.

  4. Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight.

    PubMed

    Porras-Domínguez, Jaime R; Ávila-Fernández, Ángela; Miranda-Molina, Afonso; Rodríguez-Alegría, María Elena; Munguía, Agustín López

    2015-11-05

    Levan is a fructan polymer that offers a variety of applications in the chemical, health, cosmetic and food industries. Most of the levan applications depend on levan molecular weight, which in turn depends on the source of the synthesizing enzyme and/or on reaction conditions. Here we demonstrate that in the particular case of levansucrase from Bacillus subtilis 168, enzyme concentration is also a factor defining the molecular weight levan distribution. While a bimodal distribution has been reported at the usual enzyme concentrations (1 U/ml equivalent to 0.1 μM levansucrase) we found that a low molecular weight normal distribution is solely obtained al high enzyme concentrations (>5 U/ml equivalent to 0.5 μM levansucrase) while a high normal molecular weight distribution is synthesized at low enzyme doses (0.1 U/ml equivalent to 0.01 μM of levansucrase).

  5. Dry-heat resistance of Bacillus subtilis var. niger spores on mated surfaces.

    PubMed

    Simko, G J; Devlin, J D; Wardle, M D

    1971-10-01

    Bacillus subtilis var. niger spores were placed on the surfaces of test coupons manufactured from typical spacecraft materials (stainless steel, magnesium, titanium, and aluminum). These coupons were then juxtaposed at the inoculated surfaces and subjected to test pressures of 0, 1,000, 5,000, 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, with survivor data being subjected to a linear regression analysis that calculated decimal reduction times. Differences in the dry-heat resistance of the test organism resulting from pressure, atmosphere, and material were observed.

  6. Optimizing Carbon/Nitrogen Ratio for Biosurfactant Production by a Bacillus subtilis Strain

    NASA Astrophysics Data System (ADS)

    Fonseca, R. R.; Silva, A. J. R.; de Franca, F. P.; Cardoso, V. L.; Sérvulo, E. F. C.

    A Bacillus subtilis strain isolated from contaminated soil from a refinery has been screened for biosurfactant production in crystal sugar (sucrose) with different nitrogen sources (NaNO3' (NH4)2SO4' urea, and residual brewery yeast). The highest reduction in surface tension was achieved with a 48-h fermentation of crystal sugar and ammonium nitrate. Optimization of carbon/nitrogen ratio (3,9, and 15) and agitation rate (50, 150, and 250 rpm) for biosurfactant production was carried out using complete factorial design and response surface analysis. The condition of C/N 3 and 250 rpm allowed the maximum increase in surface activity of biosurfactant. A suitable model has been developed, having presented great accordance experimental data. Preliminary characterization of the bioproduct suggested it to be a lipopeptide with some isomers differing from those of a commercial surfactin.

  7. Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture.

    PubMed

    Toledo, F L; Gonzalez-Lopez, J; Calvo, C

    2008-11-01

    Three bacterial strains isolated from waste crude oil were selected due to their capacity of growing in the presence of hydrocarbons and production of bioemulsifier. The genetic identification (PCR of the 16S rDNA gene using fD1 and rD1 primers) of these strains showed their affiliation to Bacillus subtilis, Alcaligenes faecalis and Enterobacter sp. These strains were able to emulsify n-octane, toluene, xylene, mineral oils and crude oil, look promising for bioremediation application. Finally, chemical composition, emulsifying activity and surfactant activity of the biopolymers produced by the selected strains were studies under different culture conditions. Our results showed that chemical and functional properties of the bioemulsifiers were affected by the carbon source added to the growth media.

  8. Effectiveness of four chemical solutions in eliminating Bacillus subtilis spores on gutta-percha cones.

    PubMed

    Siqueira, J F; da Silva, C H; Cerqueira M das, D; Lopes, H P; de Uzeda, M

    1998-06-01

    Gutta-percha cones should be free of pathogenic micro-organisms before being used for root canal filling. This study was carried out to evaluate the effectiveness of four chemical agents in eliminating Bacillus subtilis spores from gutta-percha cones. The solutions tested were 5.25% sodium hypochlorite, 2% glutaraldehyde, 2% chlorhexidine digluconate, and 70% ethyl alcohol. The gutta-percha cones coated with spores were placed into contact with the chemical agents for 1, 3, 5 and 10 min. The results showed that 5.25% sodium hypochlorite was effective in destroying the spores after 1 min of contact. Glutaraldehyde, chlorhexidine and ethyl alcohol did not decontaminate the gutta-percha cones even after 10 min of contact.

  9. [Influence of silicon dioxide and saponite on growth of Bacillus subtilis IMV B-7023].

    PubMed

    Chobotar'ov, A Iu; Hordiienko, A S; Samchuk, A I; Kurdysh, I K

    2010-01-01

    Growth of Bacillus subtilis IMV B-7023 was studied under the presence of silicon dioxide and saponite cultivation in the medium. It was established that the adding ofthese materials into the medium affects significantly the bacteria growth activity. Efficiency and orientation of the process depends on the type of dispersed material and contents of phosphate in the cultivation medium. Thus, under the concentration of 0.6 g/l PO4(3-) saponite stimulates the growth of bacteria but at a lower concentration (0.1 g/l PO4(3-)) growth activity of microorganisms becomes lower. At the same time in the presence of silicon dioxide the stimulation of bacterial growth is observed at different concentrations of phosphate in the medium. It is shown that the present effect is not the result of the phosphate sorption on the surface of the investigated dispersed materials. The contact interaction of solid particles with bacterial cells was found.

  10. Bacillus subtilis extracytoplasmic function (ECF) sigma factors and defense of the cell envelope

    PubMed Central

    Helmann, John D.

    2016-01-01

    Summary Bacillus subtilis provides a model for investigation of the bacterial cell envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the envelope. Although their individual regulons overlap, σW is most closely associated with membrane-active agents, σX with cationic antimicrobial peptide resistance, and σV with resistance to lysozyme. Here, I highlight the role of the σM regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of envelope synthesis and cell division, as well as stress-inducible alternative enzymes. Studies of these cell envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics. PMID:26901131

  11. Early stages in Bacillus subtilis transformation: association between homologous DNA and surface structures.

    PubMed Central

    Garcia, E; Lopez, P; Ureña, M T; Espinosa, M

    1978-01-01

    The addition of ethylenediaminetetraacetate to competent cultures of Bacillus subtilis irreversibly inhibited the transformability as well as the cellular binding of DNA. Our results show that the inhibition of DNA binding by ethylenediaminetetraacetate in whole cells, protoplasts, and membrane vesicles is mainly due to a permanent alteration of the DNA receptors. Transformation absolutely requires free magnesium ions, whereas DNA binding is a magnesium-independent step. In contrast to ethylenediaminetetraacetate, the absence of Mg2+ does not irreversibly affect the capacity of the competent cells to be transformed DNA-binding receptors located at the cell surface remain associated with the plasma membrane after protoplasting and after isolation of membrane vesicles. A Mg2+-dependent endonucleolytic activity associated with the membrane appears to be responsible for the lower levels of binding by protoplasts in the presence of this ion. PMID:99433

  12. Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis.

    PubMed

    Wiedermannová, Jana; Sudzinová, Petra; Kovaľ, Tomaš; Rabatinová, Alžbeta; Šanderova, Hana; Ramaniuk, Olga; Rittich, Šimon; Dohnálek, Jan; Fu, Zhihui; Halada, Petr; Lewis, Peter; Krásny, Libor

    2014-04-01

    Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

  13. Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family.

    PubMed

    Obuchowski, M; Madec, E; Delattre, D; Boël, G; Iwanicki, A; Foulger, D; Séror, S J

    2000-10-01

    We cloned the yloO gene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC. This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure. PrpC has phosphatase activity in vitro against a synthetic substrate, p-nitrophenol phosphate, and endogenous Bacillus subtilis proteins. The prkC and prpC genes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC. These findings suggest that PrkC and PrpC may function as a couple in vivo.

  14. Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

    PubMed Central

    Shevtsov, Mikhail B.; Chen, Yanling; Gollnick, Paul; Antson, Alfred A.

    2005-01-01

    In Bacillus subtilis the anti-TRAP protein (AT) is produced in response to the accumulation of uncharged tRNATrp. AT regulates expression of genes involved in tryptophan biosynthesis and transport by binding to the tryptophan-activated trp RNA-binding attenuation protein (TRAP) and preventing its interaction with several mRNAs. Here, we report the x-ray structure of AT at 2.8 Å resolution, showing that the protein subunits assemble into tight trimers. Four such trimers are further associated into a 12-subunit particle in which individual trimers are related by twofold and threefold symmetry axes. Twelve DnaJ-like, cysteine-rich zinc-binding domains form spikes on the surface of the dodecamer. Available data suggest several possible ways for AT to interact with the 11-subunit TRAP. Interaction between the two symmetry-mismatching molecules could be assisted by the flexible nature of AT zinc-binding domains. PMID:16306262

  15. Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis.

    PubMed

    Kwon, Eun-Yeong; Kim, Kyung Mi; Kim, Mi Kyoung; Lee, In Young; Kim, Beom Soo

    2011-09-01

    Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.

  16. Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology.

    PubMed

    Deepak, V; Kalishwaralal, K; Ramkumarpandian, S; Babu, S Venkatesh; Senthilkumar, S R; Sangiliyandi, G

    2008-11-01

    Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of Nattokinase by Bacillus subtilis at pH 7.5. The four variables involved in this study were Glucose, Peptone, CaCl2, and MgSO4. The statistical analysis of the results showed that, in the range studied; only peptone had a significant effect on Nattokinase production. The optimized medium containing (%) Glucose: 1, Peptone: 5.5, MgSO4: 0.2 and CaCl2: 0.5 resulted in 2-fold increased level of Nattokinase (3194.25U/ml) production compared to initial level (1599.09U/ml) after 10h of fermentation. Nattokinase production was checked with fibrinolytic activity.

  17. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto.

    PubMed

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-12-01

    Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27,724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a=74.3, b=49.9, c=56.3 Å, β=95.2°. Diffraction images were processed to a resolution of 1.74 Å with an Rmerge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.

  18. Chemical structure and biological activity of a quorum sensing pheromone from Bacillus subtilis subsp. natto.

    PubMed

    Okada, Masahiro; Nakamura, Yuta; Hayashi, Shunsuke; Ozaki, Koki; Usami, Syohei

    2015-10-01

    Bacillus subtilis subsp. natto secrets a peptide pheromone, named ComXnatto pheromone, as an inducer for biofilm formation containing poly-γ-glutamic acid. Recently, the ComXnatto pheromone was identified to be a hexapeptide with an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu, and the tryptophan residue was post-translationally modified with a farnesyl group. In order to determine the precise modification of the tryptophan residue, ComXnatto pheromone was synthesized using solid-phase peptide synthesis. Biological activity of the ComXnatto pheromone was then investigated. It was demonstrated that poly-γ-glutamic acid production were accelerated by ComXnatto pheromone at more than 1 nM in natto.

  19. Radiobiological results from the Bacillus subtilis Biostack experiments within the Apollo and the ASTP space flights.

    PubMed

    Facius, R; Bucker, H; Hildebrand, D; Horneck, G; Holtz, G; Reitz, G; Schafer, M; Toth, B

    1978-01-01

    In order to check the results of earlier Biostack experiments, new experimental techniques were developed for the Biostack III experiment in the Apollo-Soyuz test project (ASTP). These techniques resulted in an increased accuracy of localization down to 0.2 micrometers for the determination of the impact parameter, accompanied by an increase in the sample size available for biological investigation. In addition, colony forming ability, metabolic mutations, and mutations affecting UV- and x-ray sensitivity were rendered observable by these methods. The biological and physical results obtained so far from the evaluation of the Bacillus subtilis experiment within Biostack III confirm and extend the findings of the previous Biostack experiments. They also add to the questions about the mechanisms of action of the radiation field under investigation, since the observed effects cannot be interpreted in terms of standard concepts.

  20. Enhancing Production of Alkaline Polygalacturonate Lyase from Bacillus subtilis by Fed-Batch Fermentation

    PubMed Central

    Zou, Mouyong; Guo, Fenfen; Li, Xuezhi; Zhao, Jian; Qu, Yinbo

    2014-01-01

    Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) is an enzyme used in many industries. We developed a fed-batch fermentation process that combines the enzymatic pretreatment of the carbon source with controlling the pH of the fermentative broth to enhance the PGL production from Bacillus subtilis 7-3-3 to decrease the production cost. Maintaining the fermentation broth at pH 6.5 prior to feeding with ammonia and at pH 6.0 after feeding significantly improved PGL activity (743.5 U mL−1) compared with the control (202.5 U mL−1). The average PGL productivity reached 19.6 U mL−1 h−1 after 38 h of fermentation. The crude PGL was suitable for environmentally friendly ramie enzymatic degumming. PMID:24603713

  1. Bacillus subtilis extracytoplasmic function (ECF) sigma factors and defense of the cell envelope.

    PubMed

    Helmann, John D

    2016-04-01

    Bacillus subtilis provides a model for investigation of the bacterial cell envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the envelope. Although their individual regulons overlap, σ(W) is most closely associated with membrane-active agents, σ(X) with cationic antimicrobial peptide resistance, and σ(V) with resistance to lysozyme. Here, I highlight the role of the σ(M) regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of envelope synthesis and cell division, as well as stress-inducible alternative enzymes. Studies of these cell envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics.

  2. Structure of YqgQ Protein from Bacillus subtilis, a Conserved Hypothetical Protein

    SciTech Connect

    Lakshminarasimhan, D.; Eswaramoorthy, S; Burley, S; Swaminathan, S

    2010-01-01

    The crystal structure of the hypothetical protein YqgQ from Bacillus subtilis has been determined to 2.1 {angstrom} resolution. The crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 51.85, b = 41.25, c = 55.18 {angstrom}, {beta} = 113.4{sup o}, and contained three protein molecules in the asymmetric unit. The structure was determined by the single-wavelength anomalous dispersion method using selenium-labeled protein and was refined to a final R factor of 24.7% (R{sub free} = 28.0%). The protein molecule mainly comprises a three-helical bundle. Its putative function is inferred to be single-stranded nucleic acid binding based on sequence and structural homology.

  3. The Bacillus Subtilis RNA Helicase YxiN Is Distended in Solution

    SciTech Connect

    Wang, S.; Overgaard, M.T.; Hu, Y.; McKay, D.B.

    2009-05-26

    The Bacillus subtilis YxiN protein is a modular three-domain RNA helicase of the DEx(D/H)-box protein family. The first two domains form the highly conserved helicase core, and the third domain confers RNA target binding specificity. Small angle x-ray scattering on YxiN and two-domain fragments thereof shows that the protein has a distended structure in solution, in contrast to helicases involved in replication processes. These data are consistent with a chaperone activity in which the carboxy-terminal domain of YxiN tethers the protein to the vicinity of its targets and the helicase core is free to transiently interact with RNA duplexes, possibly to melt out misfolded elements of secondary structure.

  4. Crystallographic Snapshots of the Complete Catalytic Cycle of the Unregulated Aspartate Transcarbamoylase from Bacillus subtilis

    SciTech Connect

    K Harris; G Cockrell; D Puleo; E Kantrowitz

    2011-12-31

    Here, we report high-resolution X-ray structures of Bacillus subtilis aspartate transcarbamoylase (ATCase), an enzyme that catalyzes one of the first reactions in pyrimidine nucleotide biosynthesis. Structures of the enzyme have been determined in the absence of ligands, in the presence of the substrate carbamoyl phosphate, and in the presence of the bisubstrate/transition state analog N-phosphonacetyl-L-aspartate. Combining the structural data with in silico docking and electrostatic calculations, we have been able to visualize each step in the catalytic cycle of ATCase, from the ordered binding of the substrates, to the formation and decomposition of the tetrahedral intermediate, to the ordered release of the products from the active site. Analysis of the conformational changes associated with these steps provides a rationale for the lack of cooperativity in trimeric ATCases that do not possess regulatory subunits.

  5. Biocontrol Activity of Bacillus subtilis Isolated from Agaricus bisporus Mushroom Compost Against Pathogenic Fungi.

    PubMed

    Liu, Can; Sheng, Jiping; Chen, Lin; Zheng, Yanyan; Lee, David Yue Wei; Yang, Yang; Xu, Mingshuang; Shen, Lin

    2015-07-08

    Bacillus subtilis strain B154, isolated from Agaricus bisporus mushroom compost infected by red bread mold, exhibited antagonistic activities against Neurospora sitophila. Antifungal activity against phytopathogenic fungi was also observed. The maximum antifungal activity was reached during the stationary phase. This antifungal activity was stable over a wide pH and temperature range and was not affected by proteases. Assay of antifungal activity in vitro indicated that a purified antifungal substance could strongly inhibit mycelia growth and spore germination of N. sitophila. In addition, treatment with strain B154 in A. bisporus mushroom compost infected with N. sitophila significantly increased the yield of bisporus mushrooms. Ultraviolet scan spectroscopy, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-associated laser desorption ionization time-of-flight mass spectrometry, and electrospray ionization tandem mass spectrometry analyses revealed a molecular weight consistent with 1498.7633 Da. The antifungal compound might belong to a new type of lipopeptide fengycin.

  6. Genetic analysis of a pleiotropic deletion mutation (delta igf) in Bacillus subtilis.

    PubMed Central

    Fujita, Y; Fujita, T

    1983-01-01

    A delta igf mutation of Bacillus subtilis (formerly called fdpAl) is a large deletion causing pleiotropic defects. The mapping of the delta igf deletion by phage PBS1 transduction revealed the following map order: sacA, thiC, hsrE, delta igf, ts199, purA. To analyze the pleiotropic nature of the delta igf mutation, mutants affected in each property of the pleiotropic mutation were isolated, and the mutations were mapped. iol and gnt mutants could not grow on inositol and gluconate, respectively, and fdp mutants were affected only in fructose-bisphosphatase. The map order from sacA to purA was as follows: sacA, thiC, hsrE, iol-6, gnt-4, fdp-74, hsrB, ts199, purA. The delta igf deletion covered loci from iol-6 to hsrB. PMID:6302085

  7. Exploring the protein stability landscape: Bacillus subtilis lipase A as a model for detergent tolerance.

    PubMed

    Fulton, Alexander; Frauenkron-Machedjou, Victorine Josiane; Skoczinski, Pia; Wilhelm, Susanne; Zhu, Leilei; Schwaneberg, Ulrich; Jaeger, Karl-Erich

    2015-04-13

    A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/β-hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.

  8. Specific RNA Interference in Caenorhabditis elegans by Ingested dsRNA Expressed in Bacillus subtilis

    PubMed Central

    Lezzerini, Marco; van de Ven, Koen; Veerman, Martijn; Brul, Stanley; Budovskaya, Yelena V.

    2015-01-01

    In nematodes, genome-wide RNAi-screening has been widely used as a rapid and efficient method to identify genes involved in the aging processes. By far the easiest way of inducing RNA interference (RNAi) in Caenorhabditis elegans is by feeding Escherichia coli that expresses specific double stranded RNA (dsRNA) to knockdown translation of targeted mRNAs. However, it has been shown that E. coli is mildly pathogenic to C. elegans and this pathogenicity might influence aging and the accuracy of the RNAi-screening during aging may as well be affected. Here, we describe a novel system that utilizes the non-pathogenic bacterium Bacillus subtilis, to express dsRNA and therefore eliminates the effects of bacterial pathogenicity from the genetic analysis of aging. PMID:25928543

  9. Exo-polygalacturonase production by Bacillus subtilis CM5 in solid state fermentation using cassava bagasse

    PubMed Central

    Swain, Manas R.; Kar, Shaktimay; Ray, Ramesh C.

    2009-01-01

    The purpose of this investigation was to study the effect of Bacillus subtilis CM5 in solid state fermentation using cassava bagasse for production of exo-polygalacturonase (exo-PG). Response surface methodology was used to evaluate the effect of four main variables, i.e. incubation period, initial medium pH, moisture holding capacity (MHC) and incubation temperature on enzyme production. A full factorial Central Composite Design was applied to study these main factors that affected exo-PG production. The experimental results showed that the optimum incubation period, pH, MHC and temperature were 6 days, 7.0, 70% and 50°C, respectively for optimum exo-PG production. PMID:24031409

  10. Cooperative Recruitment of FtsW to the Division Site of Bacillus subtilis

    PubMed Central

    Gamba, Pamela; Hamoen, Leendert W.; Daniel, Richard A.

    2016-01-01

    Five essential proteins are known to assemble at the division site of Bacillus subtilis. However, the recruitment of the FtsW homolog is still unclear. Here, we take advantage of spore germination to facilitate the depletion of essential proteins and to study the divisome assembly in the absence of previous division events. We show that, unlike what has been shown for the Escherichia coli divisome, the assembly of FtsW is interdependent with the localization of PBP 2B and FtsL, which are key components of the membrane bound division complex. Interestingly, the Z-ring appeared to disassemble upon prolonged depletion of late division proteins. Nevertheless, we could restore Z-ring formation and constriction by re-inducing FtsW, which suggests that the stability of the Z-ring is stimulated by the assembly of a functional division complex. PMID:27895631

  11. Numerical simulation of wrinkle morphology formation and the evolution of different Bacillus subtilis biofilms.

    PubMed

    Wang, Xiaoling; Hao, Mudong; Wang, Guoqing

    2016-01-01

    Wrinkle morphology is a distinctive phenomenon observed in mature biofilms that are produced by a great number of bacteria. The wrinkle pattern depends on the mechanical properties of the agar substrate and the biofilm itself, governed by the extracellular matrix (ECM). Here we study the macroscopic structures and the evolution of Bacillus subtilis biofilm wrinkles using the commercial finite element software ABAQUS. A mechanical model and simulation are set up to analyze and evaluate bacteria biofilm's wrinkle characteristics. We uncover the wrinkle formation mechanism and enumerate the quantitative relationship between wrinkle structure and mechanical properties of biofilm and its substrate. Our work can be used to modify the wrinkle pattern and control the biofilm size.

  12. Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis.

    PubMed

    Fedorova, Ksenia; Kayumov, Airat; Woyda, Kathrin; Ilinskaja, Olga; Forchhammer, Karl

    2013-05-02

    The Bacillus subtilis glutamine synthetase (GS) plays a dual role in cell metabolism by functioning as catalyst and regulator. GS catalyses the ATP-dependent synthesis of glutamine from glutamate and ammonium. Under nitrogen-rich conditions, GS becomes feedback-inhibited by high intracellular glutamine levels and then binds transcription factors GlnR and TnrA, which control the genes of nitrogen assimilation. While GS-bound TnrA is no longer able to interact with DNA, GlnR-DNA binding is shown to be stimulated by GS complex formation. In this paper we show a new physiological feature of the interaction between glutamine synthetase and TnrA. The transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in vivo and in vitro, while the GlnR protein does not affect the activity of the enzyme.

  13. Bacillus subtilis Swarmer Cells Lead the Swarm, Multiply, and Generate a Trail of Quiescent Descendants

    PubMed Central

    Hamouche, Lina; Laalami, Soumaya; Daerr, Adrian; Song, Solène; Holland, I. Barry; Séror, Simone J.; Hamze, Kassem

    2017-01-01

    ABSTRACT Bacteria adopt social behavior to expand into new territory, led by specialized swarmers, before forming a biofilm. Such mass migration of Bacillus subtilis on a synthetic medium produces hyperbranching dendrites that transiently (equivalent to 4 to 5 generations of growth) maintain a cellular monolayer over long distances, greatly facilitating single-cell gene expression analysis. Paradoxically, while cells in the dendrites (nonswarmers) might be expected to grow exponentially, the rate of swarm expansion is constant, suggesting that some cells are not multiplying. Little attention has been paid to which cells in a swarm are actually multiplying and contributing to the overall biomass. Here, we show in situ that DNA replication, protein translation and peptidoglycan synthesis are primarily restricted to the swarmer cells at dendrite tips. Thus, these specialized cells not only lead the population forward but are apparently the source of all cells in the stems of early dendrites. We developed a simple mathematical model that supports this conclusion. PMID:28174308

  14. Self-regulation of exopolysaccharide production in Bacillus subtilis by a tyrosine kinase.

    PubMed

    Elsholz, Alexander K W; Wacker, Sarah A; Losick, Richard

    2014-08-01

    We report that the Bacillus subtilis exopolysaccharide (EPS) is a signaling molecule that controls its own production. EPS synthesis depends on a tyrosine kinase that consists of a membrane component (EpsA) and a kinase component (EpsB). EPS interacts with the extracellular domain of EpsA, which is a receptor, to control kinase activity. In the absence of EPS, the kinase is inactivated by autophosphorylation. The presence of EPS inhibits autophosphorylation and instead promotes the phosphorylation of a glycosyltransferase in the biosynthetic pathway, thereby stimulating the production of EPS. Thus, EPS production is subject to a positive feedback loop that ties its synthesis to its own concentration. Tyrosine kinase-mediated self-regulation could be a widespread feature of the control of exopolysaccharide production in bacteria.

  15. Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis.

    PubMed

    Gilbert, Charlie; Howarth, Mark; Harwood, Colin R; Ellis, Tom

    2017-03-07

    The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics, and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis. Using this approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling, and we show that cellular consortia can be engineered to self-assemble functional protein-protein conjugates with tunable composition. This novel genetically encoded modular system provides a flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.

  16. Early Extracellular Events in Infection of Competent Bacillus subtilis by DNA of Bacteriophage SP82G

    PubMed Central

    Williams, Gordon L.; Green, D. Macdonald

    1972-01-01

    Analysis, by the recovery of specific genetic “markers,” of the effects of DNase I, physical shear, and temperature shock on DNA-cell complexes demonstrates that sequential attachment of both ends of bacteriophage SP82G DNA to Bacillus subtilis precedes entry of the DNA molecule into the cell, and that each attachment is end-and time-specific. The first attachment involves an initial reversible phase, followed by irreversible binding. After a latent period, the second end then attaches to the cell. Entry of the molecule begins immediately after binding of the second end has occurred, and entry is complete within 3 min. The polarity of entry, as judged by attainment of resistance to DNase I, is the reverse of that observed in normal phage injection. PMID:4624760

  17. INCORPORATION OF DEOXYRIBONUCLEIC ACID IN THE BACILLUS SUBTILIS TRANSFORMATION SYSTEM1

    PubMed Central

    Young, F. E.; Spizizen, John

    1963-01-01

    Young, F. E. (Western Reserve University Cleveland, Ohio) and John Spizizen. Incorporation of deoxyribonucleic acid in the Bacillus subtilis transformation system. J. Bacteriol. 86:392–400. 1963.—The optimal conditions for the incorporation of deoxyribonucleic acid (DNA) were studied. In competent cells, the irreversible binding of DNA was influenced by temperature, hydrogen ion concentration, and aeration. Divalent cations, such as barium, strontium, calcium, or magnesium, were required. Under suboptimal environmental conditions and with metabolic inhibitors, the process of transformation was decreased to a greater extent than was incorporation of DNA. Under conditions of phosphate depletion, the incorporation of P32 increased. However, the frequency of transformation decreased. This inducible process was not related to competence. PMID:14066414

  18. [Influence of vermiculite particles on antioxidant properties of cultural medium of Bacillus subtilis IMV V-7023].

    PubMed

    Skorokhod, I A; Kudrish, I K

    2014-01-01

    It is shown that in the process of cultivation of Bacillus subtilis IMV V-7023 in the medium with vermiculite (1.5-5.0 g/l) one can observe the oppressing of some indexes of antioxidant properties of cultural medium of bacteria. In particular, a decline of hydroxyl radical scavenging activity in the Fenton reaction by 2.8-11.6%, ability to inhibit formation of malondialdehyde - by 4.4-13.1% and inactivation of 2,2'-Diphenyl-l-picrylhydrazyl (DPPH·) radical - by 3.1-8.5% were observed. Thus oxidant activity increased substantially. Besides oppressing influence of particles of vermiculite on protector properties of the cultural medium of bacilli it is found out that with the increase of the content of dispersible material in the nutrient medium the reducing power of cultural medium of these bacteria increased.

  19. Use of organic acids for prevention and removal of Bacillus subtilis biofilms on food contact surfaces.

    PubMed

    Akbas, Meltem Yesilcimen; Cag, Seyda

    2016-10-01

    The efficacies of organic acid (citric, malic, and gallic acids) treatments at 1% and 2% concentrations on prevention and removal of Bacillus subtilis biofilms were investigated in this study. The analyses were conducted on microtitration plates and stainless steel coupons. The biofilm removal activities of these organic acids were compared with chlorine on both surfaces. The results showed that citric acid treatments were as powerful as chlorine treatments for prevention and removal of biofilms. The antibiofilm effects of malic acid treatments were higher than gallic acid and less than citric acid treatment. When the antibiofilm effects of these acids and chlorine on the two surfaces were compared, the prevention and removal of biofilms were measured higher on microtitration plates than those on stainless steel coupons. Higher reductions were obtained by increasing concentrations of sanitizers on 24-hour biofilm with 20-minute sanitizer treatments for removal of biofilms.

  20. Enantioselective bioconversion using Escherichia coli cells expressing Saccharomyces cerevisiae reductase and Bacillus subtilis glucose dehydrogenase.

    PubMed

    Park, Hyun Joo; Jung, Jihye; Choi, Hyejeong; Uhm, Ki-Nam; Kim, Hyung Kwoun

    2010-09-01

    Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.

  1. Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700

    PubMed Central

    Ji, Shengyue; Li, Weili; Baloch, Abdul Rasheed; Wang, Meng; Li, Hengxin; Cao, Binyun; Zhang, Hongfu

    2017-01-01

    The efficient production of antimicrobial peptides (AMPs) for clinical applications has attracted the attention of the scientific community. To develop a novel microbial cell factory for the efficient biosynthesis of a cecropin A-melittin mutant (CAM-W), a recombinant Bacillus subtilis WB700 expression system was genetically modified with a novel vector, including a fusion gene encoding CAM-W, the autoprotease EDDIE and the signal peptide SacB under the control of the maltose-inducible promoter Pglv. A total of 159 mg of CAM-W was obtained from 1 L of fermentation supernatant. The purified CAM-W showed a consistent size with the expected molecular weight of 3.2 kDa. Our findings suggest that this novel expression system can be used as a powerful tool for the efficient production of CAM-W. PMID:28071737

  2. Responses of Bacillus subtilis spores to space environment: results from experiments in space.

    PubMed

    Horneck, G

    1993-02-01

    Onboard of several spacecrafts (Apollo 16, Spacelab 1, LDEF), spores of Bacillus subtilis were exposed to selected parameters of space, such as space vacuum, different spectral ranges of solar UV-radiation and cosmic rays, applied separately or in combination, and we have studied their survival and genetic changes after retrieval. The spores survive extended periods of time in space--up to several years--, if protected against the high influx of solar UV-radiation. Water desorption caused by the space vacuum leads to structural changes of the DNA; the consequences are an increased mutation frequency and altered photobiological properties of the spores. UV-effects, such as killing and mutagenesis, are augmented, if the spores are in space vacuum during irradiation. Vacuum-specific photoproducts which are different from the 'spore photoproduct' may cause the synergistic response of spores to the simultaneous action of UV and vacuum. The experiments provide an experimental test of certain steps of the panspermia hypothesis.

  3. Early-blocked asporogenous mutants of Bacillus subtilis are lysogenized at reduced frequency by temperate bacteriophages.

    PubMed Central

    Ikeuchi, T; Kurahashi, K

    1978-01-01

    The establishment of lysogeny in early-blocked asporogenous (Spo-) mutants of Bacillus subtilis 168, which were also defective in the production of antibiotics (Abs-), by temperate phage phi105 or SPO2 was studied. It was found that the frequency of lysogenization of Spo-Abs-mutants was 10 to 20% that of the wild-type bacteria. There was no difference in the efficiency of plating and the burst size of phi105 between wild-type and mutant strains. Phi105 lysogens of mutant strains were as stable as those of the wild type. Several rifampin-resistant mutants defective in the production of antibiotics were isolated. They were also defective in spore formation and lysogenized by phi105 at reduced frequency. PMID:96089

  4. A dual control mechanism synchronizes riboflavin and sulphur metabolism in Bacillus subtilis.

    PubMed

    Pedrolli, Danielle Biscaro; Kühm, Christian; Sévin, Daniel C; Vockenhuber, Michael P; Sauer, Uwe; Suess, Beatrix; Mack, Matthias

    2015-11-10

    Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for biosynthesis and/or transport of riboflavin (rib genes). Cytoplasmic riboflavin is rapidly and almost completely converted to FMN by flavokinases. When cytoplasmic levels of FMN are sufficient ("high levels"), FMN binding to FMN riboswitches leads to a reduction of rib gene expression. We report here that the protein RibR counteracts the FMN-induced "turn-off" activities of both FMN riboswitches in Bacillus subtilis, allowing rib gene expression even in the presence of high levels of FMN. The reason for this secondary metabolic control by RibR is to couple sulfur metabolism with riboflavin metabolism.

  5. Synthesis, Release, and Recapture of Compatible Solute Proline by Osmotically Stressed Bacillus subtilis Cells

    PubMed Central

    Hoffmann, Tamara; von Blohn, Carsten; Stanek, Agnieszka; Moses, Susanne; Barzantny, Helena

    2012-01-01

    Bacillus subtilis synthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, an opuE mutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of the opuE mutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of the opuE mutant was considerably lower than that of its opuE+ parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels of B. subtilis participated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesized de novo and subsequently released by salt-stressed B. subtilis cells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered. PMID:22685134

  6. Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore

    SciTech Connect

    Zawadzka, A. M.; Kim, Y.; Maltseva, N; Nichiporuk, R; Fan, Y; Joachimiak, A; Raymond, KN

    2009-12-22

    Iron deprivation activates the expression of components of the siderophore-mediated iron acquisition systems in Bacillus subtilis, including not only the synthesis and uptake of its siderophore bacillibactin but also expression of multiple ABC transporters for iron scavenging using xenosiderophores. The yclNOPQ operon is shown to encode the complete transporter for petrobactin (PB), a photoreactive 3,4-catecholate siderophore produced by many members of the B. cereus group, including B. anthracis. Isogenic disruption mutants in the yclNOPQ transporter, including permease YclN, ATPase YclP, and a substrate-binding protein YclQ, are unable to use either PB or the photoproduct of FePB (FePB{sup {nu}}) for iron delivery and growth, in contrast to the wild-type B. subtilis. Complementation of the mutations with the copies of the respective genes restores this capability. The YclQ receptor binds selectively iron-free and ferric PB, the PB precursor, 3,4-dihydroxybenzoic acid (3,4-DHB), and FePB{sup {nu}} with high affinity; the ferric complexes are seen in ESI-MS, implying strong electrostatic interaction between the protein-binding pocket and siderophore. The first structure of a Gram-positive siderophore receptor is presented. The 1.75-{angstrom} crystal structure of YclQ reveals a bilobal periplasmic binding protein (PBP) fold consisting of two {alpha}/{beta}/{alpha} sandwich domains connected by a long {alpha}-helix with the binding pocket containing conserved positively charged and aromatic residues and large enough to accommodate FePB. Orthologs of the B. subtilis PB-transporter YclNOPQ in PB-producing Bacilli are likely contributors to the pathogenicity of these species and provide a potential target for antibacterial strategies.

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

    PubMed Central

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

    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 xynC genes, respectively. Both of these enzymes have been defined with respect to structure and function. In this study, the effects of deletion of the xynA and xynC genes, individually and in combination, were evaluated for xylan utilization and formation of acidic xylooligosaccharides. Parent strain 168 depolymerizes methylglucuronoxylans (MeGXn), releasing the xylobiose and xylotriose utilized for growth and accumulating the aldouronate methylglucuronoxylotriose (MeGX3) with some methylglucuronoxylotetraose (MeGX4). The combined GH11 and GH30 activities process the products generated by their respective actions on MeGXn to release a maximal amount of neutral xylooligosaccharides for assimilation and growth, at the same time forming MeGX3 in which the internal xylose is substituted with methylglucuronate (MeG). Deletion of xynA results in the accumulation of β-1,4-xylooligosaccharides with degrees of polymerization ranging from 4 to 18 and an average degree of substitution of 1 in 7.2, each with a single MeG linked α-1,2 to the xylose penultimate to the xylose at the reducing terminus. Deletion of the xynC gene results in the accumulation of aldouronates comprised of 4 or more xylose residues in which the MeG may be linked α-1,2 to the xylose penultimate to the nonreducing xylose. These B. subtilis lines may be used for the production of acidic xylooligosaccharides with applications in human and veterinary medicine. PMID:24271172

  8. Optimization of medium composition for the production of antimicrobial activity by Bacillus subtilis B38.

    PubMed

    Tabbene, Olfa; Slimene, Imen Ben; Djebali, Kais; Mangoni, Maria-Luisa; Urdaci, Maria-Camino; Limam, Ferid

    2009-01-01

    An antimicrobial activity produced by Bacillus subtilis B38 was found to be effective against several bacteria, including pathogenic and spoilage microorganisms such as, Listeria monocytogenes, Salmonella enteridis, and clinical isolates of methicillin-resistant Staphylococcus species. Nutrients such as carbon, nitrogen sources, and inorganic salts enhanced the production level of the antibacterial activity by B. subtilis B38. A first screening step showed that lactose, ammonium succinate, and manganese most influenced both cell growth and antibacterial activity production. These three factors varied at two levels in eight experiments using full factorial design. Results indicated that maximum cell growth (OD = 10.2) and maximum production of antibacterial activity (360 AU/mL) were obtained in a modified medium containing 1.5% (w/v) lactose, 0.15% (w/v) ammonium succinate, and 0.3 mg/L manganese. Depending on the indicator strain used, the antibacterial activity was 2- to 4-fold higher in the modified culture medium than in TSB medium under the same conditions. Thin layer chromatography-bioautography assay showed the presence of three active spots with R(f) values of 0.47, 0.7, and 0.82 in TSB medium. However, the inhibition zone of two spots (R(f) values of 0.7 and 0.82) was slightly larger in the modified medium. Moreover, a large zone of inhibition with an R(f) value of 0.3, was observed in this modified medium, instead of the spot having an R(f) value of 0.47. These results suggest that the nutrients act as environmental factors, quantitatively and qualitatively affecting the production of antibacterial compounds by B. subtilis B38.

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

    PubMed Central

    Raga-Carbajal, Enrique; Olvera, Clarita; Rodríguez-Alegría, Maria Elena; Carrillo-Nava, Ernesto; Costas, Miguel; López Munguía, Agustín

    2015-01-01

    Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final

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

    PubMed

    Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J; Chatterjee, Aditi; Prasad, T S Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

    2015-01-01

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

  11. Phenotypic characterization and complementation analysis of Bacillus subtilis 6S RNA single and double deletion mutants.

    PubMed

    Hoch, Philipp G; Burenina, Olga Y; Weber, Michael H W; Elkina, Daria A; Nesterchuk, Mikhail V; Sergiev, Petr V; Hartmann, Roland K; Kubareva, Elena A

    2015-10-01

    6S RNA, a global regulator of transcription in bacteria, binds to housekeeping RNA polymerase (RNAP) holoenzymes to competitively inhibit transcription from DNA promoters. Bacillus subtilis encodes two 6S RNA homologs whose differential functions are as yet unclear. We constructed derivative strains of B. subtilis PY79 lacking 6S-1 RNA (ΔbsrA), 6S-2 RNA (ΔbsrB) or both (ΔbsrAB) to study the physiological role of the two 6S RNAs. We observed two growth phenotypes of mutant strains: (i) accelerated decrease of optical density toward extended stationary phase and (ii) faster outgrowth from stationary phase under alkaline stress conditions (pH 9.8). The first phenotype was observed for bacteria lacking bsrA, and even more pronounced for ΔbsrAB bacteria, but not for those lacking bsrB. The magnitude of the second phenotype was relatively weak for ΔbsrB, moderate for ΔbsrA and again strongest for ΔbsrAB bacteria. Whereas ΔbsrAB bacteria complemented with bsrB or bsrA (strains ΔbsrAB + B and ΔbsrAB + A) mimicked the phenotypes of the ΔbsrA and ΔbsrB strains, respectively, complementation with the gene ssrS encoding Escherichia coli 6S RNA failed to cure the "low stationary optical density" phenotype of the double mutant, despite ssrS expression, in line with previous findings. Finally, proteomics (two-dimensional differential gel electrophoresis, 2D-DIGE) of B. subtilis 6S RNA deletion strains unveiled a set of proteins that were expressed at higher levels particularly during exponential growth and preferentially in mutant strains lacking 6S-2 RNA. Several of these proteins are involved in metabolism and stress responses.

  12. Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture

    PubMed Central

    Dauner, Michael; Storni, Tazio; Sauer, Uwe

    2001-01-01

    The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing Bacillus subtilis. Based on [U-13C6]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited B. subtilis is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B. subtilis, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATP-dependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes. PMID:11717290

  13. A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

    PubMed Central

    Parashar, Vijay; Konkol, Melissa A.; Kearns, Daniel B.

    2013-01-01

    Bacillus subtilis biofilm formation is tightly regulated by elaborate signaling pathways. In contrast to domesticated lab strains of B. subtilis which form smooth, essentially featureless colonies, undomesticated strains such as NCIB 3610 form architecturally complex biofilms. NCIB 3610 also contains an 80-kb plasmid absent from laboratory strains, and mutations in a plasmid-encoded homolog of a Rap protein, RapP, caused a hyperrugose biofilm phenotype. Here we explored the role of rapP phrP in biofilm formation. We found that RapP is a phosphatase that dephosphorylates the intermediate response regulator Spo0F. RapP appears to employ a catalytic glutamate to dephosphorylate the Spo0F aspartyl phosphate, and the implications of the RapP catalytic glutamate are discussed. In addition to regulating B. subtilis biofilm formation, we found that RapP regulates sporulation and genetic competence as a result of its ability to dephosphorylate Spo0F. Interestingly, while rap phr gene cassettes routinely form regulatory pairs; i.e., the mature phr gene product inhibits the activity of the rap gene product, the phrP gene product did not inhibit RapP activity in our assays. RapP activity was, however, inhibited by PhrH in vivo but not in vitro. Additional genetic analysis suggests that RapP is directly inhibited by peptide binding. We speculate that PhrH could be subject to posttranslational modification in vivo and directly inhibit RapP activity or, more likely, PhrH upregulates the expression of a peptide that, in turn, directly binds to RapP and inhibits its Spo0F phosphatase activity. PMID:23524609

  14. The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains▿ †

    PubMed Central

    Zeigler, Daniel R.; Prágai, Zoltán; Rodriguez, Sabrina; Chevreux, Bastien; Muffler, Andrea; Albert, Thomas; Bai, Renyuan; Wyss, Markus; Perkins, John B.

    2008-01-01

    Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three—NCIB 3610T, PY79, and SMY—we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more “W23 islands” in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains. PMID:18723616

  15. Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis

    SciTech Connect

    Newman, Joseph A.; Das, Sanjan K.; Sedelnikova, Svetlana E.; Rice, David W.

    2006-10-01

    A putative pyridoxal kinase from B. subtilis has been cloned, overexpressed, purified and crystallized and data have been collected to 2.8 Å resolution. Pyridoxal kinases (PdxK) are able to catalyse the phosphorylation of three vitamin B{sub 6} precursors, pyridoxal, pyridoxine and pyridoxamine, to their 5′-phosphates and play an important role in the vitamin B{sub 6} salvage pathway. Recently, the thiD gene of Bacillus subtilis was found to encode an enzyme which has the activity expected of a pyridoxal kinase despite its previous assignment as an HMPP kinase owing to higher sequence similarity. As such, this enzyme would appear to represent a new class of ‘HMPP kinase-like’ pyridoxal kinases. B. subtilis thiD has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a binary complex with ADP and Mg{sup 2+}. X-ray diffraction data have been collected from crystals to 2.8 Å resolution at 100 K. The crystals belong to a primitive tetragonal system, point group 422, and analysis of the systematic absences suggest that they belong to one of the enantiomorphic pair of space groups P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. Consideration of the space-group symmetry and unit-cell parameters (a = b = 102.9, c = 252.6 Å, α = β = γ = 90°) suggest that the crystals contain between three and six molecules in the asymmetric unit. A full structure determination is under way to provide insights into aspects of the enzyme mechanism and substrate specificity.

  16. Bacillus subtilis 6S-2 RNA serves as a template for short transcripts in vivo.

    PubMed

    Hoch, Philipp G; Schlereth, Julia; Lechner, Marcus; Hartmann, Roland K

    2016-04-01

    The global transcriptional regulator 6S RNA is abundant in a broad range of bacteria. The RNA competes with DNA promoters for binding to the housekeeping RNA polymerase (RNAP) holoenzyme. When bound to RNAP, 6S RNA serves as a transcription template for RNAP in an RNA-dependent RNA polymerization reaction. The resulting short RNA transcripts (so-called product RNAs = pRNAs) can induce a stable structural rearrangement of 6S RNA when reaching a certain length. This rearrangement leads to the release of RNAP and thus the recovery of transcription at DNA promoters. While most bacteria express a single 6S RNA, some harbor a second 6S RNA homolog (termed 6S-2 RNA in Bacillus subtilis). Bacillus subtilis 6S-2 RNA was recently shown to exhibit essentially all hallmark features of a bona fide 6S RNA in vitro, but evidence for the synthesis of 6S-2 RNA-derived pRNAs in vivo has been lacking so far. This raised the question of whether the block of RNAP by 6S-2 RNA might be lifted by a mechanism other than pRNA synthesis. However, here we demonstrate that 6S-2 RNA is able to serve as a template for pRNA synthesis in vivo. We verify this finding by using three independent approaches including a novel primer extension assay. Thus, we demonstrate the first example of an organism that expresses two distinct 6S RNAs that both exhibit all mechanistic features defined for this type of regulatory RNA.

  17. Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis

    PubMed Central

    Obuchowski, Michał; Nidzworski, Dawid

    2016-01-01

    Effective vaccination against influenza virus infection is a serious problem mainly due to antigenic variability of the virus. Among many of investigated antigens, the extracellular domain of the M2 protein (M2e) features high homology in all strains of influenza A viruses and antibodies against M2e and is protective in animal models; this makes it a potential candidate for generation of a universal influenza vaccine. However, due to the low immunogenicity of the M2e, formulation of a vaccine based on this antigen requires some modification to induce effective immune responses. In this work we evaluated the possible use of Bacillus subtilis spores as a carrier of the Influenza A M2e antigen in mucosal vaccination. A tandem repeat of 4 consensus sequences coding for human—avian—swine—human M2e (M2eH-A-S-H) peptide was fused to spore coat proteins and stably exposed on the spore surface, as demonstrated by the immunostaining of intact, recombinant spores. Oral immunization of mice with recombinant endospores carrying M2eH-A-S-H elicited specific antibody production without the addition of adjuvants. Bacillus subtilis endospores can serve as influenza antigen carriers. Recombinant spores constructed in this work showed low immunogenicity although were able to induce antibody production. The System of influenza antigen administration presented in this work is attractive mainly due to the omitting time-consuming and cost-intensive immunogen production and purification. Therefore modification should be made to increase the immunogenicity of the presented system. PMID:27902762

  18. Cloning of a fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli.

    PubMed

    Ghasemi, Younes; Dabbagh, Fatemeh; Ghasemian, Abdollah

    2012-09-01

    Several investigations are being pursued to enhance the efficacy and specificity of fibrinolytic therapy. In this regard, microbial fibrinolytic enzymes attracted much more medical interests during these decades. Subtilisin, a member of subtilases (the superfamily of subtilisin-like serine proteases) and also a fibrinolytic enzyme is quite common in Gram-positive bacteria, and Bacillus species stand out in particular, as many extracellular and even intracellular variants have been identified. In the present work, the subtilisin gene from Bacillus subtilis PTCC 1023 was cloned into the vector pET-15b and expressed in Escherichia coli strain BL21 (DE3). Total genomic DNA were isolated and used for PCR amplification of the subtilisin gene by means of the specific primers. SDS-PAGE and enzyme assay were done for characterizing the expressed protein. A ~1,100 bp of the structural subtilisin gene was amplified. The DNA and amino acid sequence alignments resulting from the BLAST search of subtilisin showed high sequence identity with the other strains of B. subtilis, whereas significantly lower identity was observed with other bacterial subtilisins. The recombinant enzyme had the same molecular weight as other reported subtilisins and the E. coli transformants showed high subtilisin activity. This study provides evidence that subtilisin can be actively expressed in E. coli. The commercial availability of subtilisin is of great importance for industrial applications and also pharmaceutical purposes as thrombolytic agent. Thus, the characterization of new recombinant subtilisin and the development of rapid, simple, and effective production methods are not only of academic interest, but also of practical importance.

  19. Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

    PubMed Central

    Cermak, Nathan; Feijó Delgado, Francisco; Setlow, Barbara; Setlow, Peter

    2015-01-01

    ABSTRACT We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl3 to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability. IMPORTANCE Spores of Bacillus species cause food spoilage and disease and are extremely resistant to standard decontamination methods. This hardiness is partly due to spores' extremely low permeability to chemicals, including water. We present a method to directly monitor the uptake of molecules into B. subtilis spores by weighing spores in fluid. The results demonstrate the exchange of core water with subsecond resolution and show a correlation between water permeability and the rate at which small molecules can initiate or inhibit germination in coat-damaged spores. The ability to directly measure the uptake of molecules in the context of spores with known structural or genetic deficiencies is expected to provide insight into the determinants of spores' extreme resistance. PMID:26483518

  20. Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

    PubMed Central

    Gao, Xiaohui; Dong, Xiao; Subramanian, Sundharraman; Matthews, Paige M.; Cooper, Caleb A.; Kearns, Daniel B.

    2014-01-01

    Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis. PMID:25085482

  1. TEMPERATURE-SENSITIVE MUTANTS OF BACILLUS SUBTILIS BACTERIOPHAGE SP3 II.

    PubMed Central

    Nishihara, Mutsuko; Romig, W. R.

    1964-01-01

    Nishihara, Mutsuko (University of California, Los Angeles), and W. R. Romig. Temperature-sensitive mutants of Bacillus subtilis bacteriophage SP3. II. In vivo complementation studies. J. Bacteriol. 88:1230–1239. 1964.—A plate-spotting procedure was used in initial attempts to group the temperature-sensitive Bacillus subtilis phage SP3 mutants by complementation. The results obtained did not show any clear patterns of reactions among the mutants. Crosses were, therefore, repeated in broth at a temperature of 49 C, which greatly reduced the extent of replication of each mutant type alone. The data on mixed infections indicated that there was a minimum of six complementation groups. Of the 12 isolates, 7 did not seem to complement with each other; the rest complemented with each other and with the seven noncomplementing mutants. There was a positive correlation between the complementation reaction of a pair and the recovery of wild-phenotype phages from a 49 C broth lysate. The relative proportion of phages capable of forming wild-phenotype plaques on plates incubated at 46 C to the total number of plaque-forming units was higher in a lysate of a mixed infection with two mutants than in lysates of each mutant alone. Moreover, this frequency was higher for a mixed lysate made at 49 C than for a lysate of the same two mutants made at 37 C. These observations suggested that genetic recombination might occur at 49 C, and that the increased recovery of wild-phenotype phages in lysates made at this temperature might be due to a selective advantage for these phages. Recombination experiments at 37 C with some complementing pairs gave frequencies of 2.0 to 4.8%. The ratio of wild-phenotype revertants to total phages in the stock lysates used for these crosses at 37 C was less than 10−6. The noncomplementing mutants were not conclusively shown to be nonidentical. PMID:14234775

  2. Degradation of soil cyanide by single and mixed cultures of Pseudomonas stutzeri and Bacillus subtilis.

    PubMed

    Nwokoro, Ogbonnaya; Dibua, Marie Esther Uju

    2014-03-01

    The aim of this investigation was to study whether certain bacteria could be used for cyanide degradation in soil. The bacteria Pseudomonas stutzeri and Bacillus subtilis were selected based on their good growth in a minimal medium containing 0.8 mg mL-1 potassium cyanide (KCN). In this study we tested their ability to reduce cyanide levels in a medium containing 1.5 mg mL-1 of KCN. Although both microorganisms reduced cyanide levels, Pseudomonas stutzeri was the more effective test organism. Later on, the selected cultures were grown, diluted and their various cell concentrations were used individually and in combination to test their ability of cyanide degradation in soil samples collected around a cassava processing mill. Bacillus subtilis caused degradation of soil cyanide from 0.218 mg g-1 soil immediately with an inoculum concentration of 0.1 (OD600nm) to 0.072 mg g-1 soil after 10 days with an inoculum concentration of 0.6 (OD600nm) implying a 66.9 % reduction. Pseudomonas stutzeri cell concentration of 0.1 (OD600nm) decreased soil cyanide from 0.218 mg g-1 soil initially to 0.061 mg g-1 soil after 10 days with an inoculum concentration of 0.6 (OD600nm) (72 % reduction). The mixed culture of the two bacteria produced the best degradation of soil cyanide from 0.218 mg g-1 soil sample with a combined inoculum concentration of 0.1 (OD600nm) initially to 0.025 mg g-1 soil with a combined inoculum concentration of 0.6 (OD600nm) after 10 days incubation resulting in an 88.5 % degradation of soil cyanide. The analysed bacteria displayed high cyanide degradation potential and may be useful for efficient decontamination of cyanide contaminated sites.

  3. Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering.

    PubMed

    Wang, Xiaoyue; Wang, Guanglu; Li, Xinli; Fu, Jing; Chen, Tao; Wang, Zhiwen; Zhao, Xueming

    2016-08-10

    Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis.

  4. A Novel Function of δ Factor from Bacillus subtilis as a Transcriptional Repressor.

    PubMed

    Prajapati, Ranjit Kumar; Sur, Runa; Mukhopadhyay, Jayanta

    2016-11-11

    δ, a small protein found in most Gram-positive bacteria was, for a long time, thought to be a subunit of RNA polymerase (RNAP) and was shown to be involved in recycling of RNAP at the end of each round of transcription. However, how δ participates in both up-regulation and down-regulation of genes in vivo remains unclear. We have recently shown, in addition to the recycling of RNAP, δ functions as a transcriptional activator by binding to an A-rich sequence located immediately upstream of the -35 element, consequently facilitating the open complex formation. The result had explained the mechanism of up-regulation of the genes by δ. Here, we show that Bacillus subtilis δ could also function as a transcriptional repressor. Our results demonstrate that δ binds to an A-rich sequence located near the -35 element of the spo0B promoter, the gene involved in the regulatory cascade of bacterial sporulation and inhibits the open complex formation due to steric clash with σ region 4.2. We observed a significant increase in the mRNA level of the spo0B gene in a δ-knock-out strain of B. subtilis compared with the wild-type. Thus, the results report a novel function of δ, and suggest the mechanism of down-regulation of genes in vivo by the protein.

  5. Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium

    PubMed Central

    Gudiña, Eduardo J.; Fernandes, Elisabete C.; Rodrigues, Ana I.; Teixeira, José A.; Rodrigues, Lígia R.

    2015-01-01

    In this work, biosurfactant production by Bacillus subtilis #573 was evaluated using corn steep liquor (CSL) as culture medium. The best results were obtained in a culture medium consisting of 10% (v/v) of CSL, with a biosurfactant production of about 1.3 g/l. To the best of our knowledge, this is the first report describing biosurfactant production by B. subtilis using CSL as culture medium. Subsequently, the effect of different metals (iron, manganese, and magnesium) on biosurfactant production was evaluated using the medium CSL 10%. It was found that for all the metals tested, the biosurfactant production was increased (up to 4.1, 4.4, and 3.5 g/l for iron, manganese, and magnesium, respectively). When the culture medium was supplemented with the optimum concentration of the three metals simultaneously, the biosurfactant production was increased up to 4.8 g/l. Furthermore, the biosurfactant exhibited a good performance in oil recovery assays when compared with chemical surfactants, which suggests its possible application in microbial enhanced oil recovery or bioremediation. PMID:25705209

  6. Adenylosuccinate lyase of Bacillus subtilis regulates the activity of the glutamyl-tRNA synthetase.

    PubMed Central

    Gendron, N; Breton, R; Champagne, N; Lapointe, J

    1992-01-01

    In Bacillus subtilis, the glutamyl-tRNA synthetase [L-glutamate:tRNA(Glu) ligase (AMP-forming), EC 6.1.1.17] is copurified with a polypeptide of M(r) 46,000 that influences its affinity for its substrates and increases its thermostability. The gene encoding this regulatory factor was cloned with the aid of a 41-mer oligonucleotide probe corresponding to the amino acid sequence of an NH2-terminal segment of this factor. The nucleotide sequence of this gene and the physical map of the 1475-base-pair fragment on which it was cloned are identical to those of purB, which encodes the adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4.3.2.2), an enzyme involved in the de novo synthesis of purines. This gene complements the purB mutation of Escherichia coli JK268, and its presence on a multicopy plasmid behind the trc promoter in the purB- strain gives an adenylosuccinate lyase level comparable to that in wild-type B. subtilis. A complex between the adenylosuccinate lyase and the glutamyl-tRNA synthetase was detected by centrifugation on a density gradient. The interaction between these enzymes may play a role in the coordination of purine metabolism and protein biosynthesis. Images PMID:1608947

  7. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide.

    PubMed

    You, L; Arnold, F H

    1996-01-01

    Sequential rounds of error-prone PCR to introduce random mutations and screening of the resultant mutant libraries have been used to enhance the total catalytic activity of subtilisin E significantly in a non-natural environment, aqueous dimethylformamide (DMF). Seven DNA substitutions coding for three new amino acid substitutions were identified in a mutant isolated after two additional generations of directed evolution carried out on 10M subtilisin E, previously "evolved' to increase its specific activity in DMF. A Bacillus subtilis-Escherichia coli shuttle vector was developed in order to increase the size of the mutant library that could be established in B.subtilis and the stringency of the screening process was increased to reflect total as well as specific activity. This directed evolution approach has been extremely effective for improving enzyme activity in a non-natural environment: the resulting-evolved 13M subtilisin exhibits specific catalytic efficiency towards the hydrolysis of a peptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in 60% DMF solution that is three times that of the parent 10M and 471 times that of wild type subtilisin E. The total activity of the 13M culture supernatant is enhanced 16-fold over that of the parent 10M.

  8. Response of Bacillus subtilis to nitric oxide and the nitrosating agent sodium nitroprusside.

    PubMed

    Moore, Charles M; Nakano, Michiko M; Wang, Tao; Ye, Rick W; Helmann, John D

    2004-07-01

    We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.

  9. Electrical wiring of live, metabolically enhanced Bacillus subtilis cells with flexible osmium-redox polymers.

    PubMed

    Coman, Vasile; Gustavsson, Tobias; Finkelsteinas, Arnonas; von Wachenfeldt, Claes; Hägerhäll, Cecilia; Gorton, Lo

    2009-11-11

    The present study explores genetic engineering of the respiratory chain and the application of two different flexible osmium redox polymers to achieve efficient electric communication between the gram-positive organism Bacillus subtilis and an electrode. Poly(1-vinylimidazole)(12)-[Os-(4,4'-dimethyl-2,2'-bipyridyl)(2)Cl(2)](+/2+) (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N'-methylated-2,2'-biimidazole)(3)](2+/3+) (osmium redox polymer II) were investigated for efficient electrical "wiring" of viable gram-positive bacterial cells to electrodes. Using a B. subtilis strain that overproduces succinate/quinone oxidoreductase (respiratory complex II), we were able to improve the current response several fold using succinate as substrate, in both batch and flow analysis modes, and using gold and graphite electrodes. The efficiency of the osmium redox polymer, working as electron transfer mediator between the cells and the electrode, was compared with that of a soluble mediator (hexacyanoferrate). The results demonstrated that mediators did not have to pass the cytosolic membrane to bring about an efficient electronic communication between bacterial cells with a thick cell wall and electrodes.

  10. PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

    PubMed Central

    Nanninga, N.

    1969-01-01

    The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis. PMID:4979363

  11. Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis

    SciTech Connect

    Rathinaswamy, Priya; Pundle, Archana V.; Prabhune, Asmita A.; SivaRaman, Hepzibah; Brannigan, James A. Dodson, Guy G.; Suresh, C. G.

    2005-07-01

    An unannotated protein reported from B. subtilis has been expressed in E. coli and identified as possessing penicillin V acylase activity. The crystallization and preliminary crystallographic analysis of this penicillin V acylase is presented. Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 Å. The crystals belonged to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 Å. The estimated Matthews coefficient was 3.23 Å{sup 3} Da{sup −1}, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.

  12. Generation of deletions in pneumococcal mal genes cloned in Bacillus subtilis.

    PubMed Central

    Lopez, P; Espinosa, M; Greenberg, B; Lacks, S A

    1984-01-01

    The pneumococcal recombinant plasmid pLS70, which contains two strong promoters for transcription of the malM and malX genes, is unstable when transferred to Bacillus subtilis, and it gives rise to deleted derivatives. Analysis of proteins produced by the deleted plasmids and restriction mapping of 29 different deletions showed that stabilization in B. subtilis was accompanied by deletions affecting both promoters. Plasmids containing even a single strong promoter were at a selective disadvantage. Nucleotide sequences surrounding the deletions in 10 plasmids were determined. Six different deletions occurred between directly repeated sequences of 3-13 base pairs in length, presumably by a recombination mechanism involving short homologies. Four deletions occurred between sites not contained within repeated sequences. A weak but significant similarity of an 11-base sequence was found surrounding these deletions and the corresponding points of junction in the progenitor plasmids. It is suggested that this sequence may be the recognition site for a topoisomerase-like enzyme that can produce deletions. Images PMID:6089185

  13. Enhanced production of poly-γ-glutamic acid by a newly-isolated Bacillus subtilis.

    PubMed

    Ju, Wan-Taek; Song, Yong-Su; Jung, Woo-Jin; Park, Ro-Dong

    2014-11-01

    Application of poly-gamma-glutamic acid (γ-PGA), an unusual macromolecular anionic polypeptide, is limited due to the high cost associated with its low productivity. Screening bacterial strains to find a more efficient producer is one approach to overcome this limitation. Strain MJ80 was isolated as a γ-PGA producer among 1,500 bacterial colonies obtained from soil samples. It was identified as Bacillus subtilis, based on the biochemical and morphological properties and 16S rDNA gene sequencing. It produced γ-PGA from both glutamic acid and soybean powder, identifying it as a facultative glutamic acid-metabolizing bacterium. After optimization of its culture conditions, B. subtilis MJ80 showed γ-PGA productivity of 75.5 and 68.7 g/l in 3 and 300 l jar fermenters for 3 days cultivation, respectively, the highest productivity reported to date, suggesting MJ80 to be a promising strain for γ-PGA production.

  14. Long-Term Dosimetry of Solar UV Radiation in Antarctica with Spores of Bacillus subtilis

    PubMed Central

    Puskeppeleit, Monika; Quintern, Lothar E.; el Naggar, Saad; Schott, Jobst-Ulrich; Eschweiler, Ute; Horneck, Gerda; Bücker, Horst

    1992-01-01

    The main objective was to assess the influence of the seasonal stratospheric ozone depletion on the UV climate in Antarctica by using a biological test system. This method is based on the UV sensitivity of a DNA repair-deficient strain of Bacillus subtilis (TKJ 6321). In our field experiment, dried layers of B. subtilis spores on quartz discs were exposed in different seasons in an exposure box open to solar radiation at the German Antarctic Georg von Neumayer Station (70°37′S, 8°22′W). The UV-induced loss of the colony-forming ability was chosen as the biological end point and taken as a measure for the absorbed biologically harmful UV radiation. Inactivation constants were calculated from the resulting dose-response curves. The results of field experiments performed in different seasons indicate a strongly season-dependent trend of the daily UV-B level. Exposures performed at extremely depleted ozone concentrations (October 1990) gave higher biologically harmful UV-B levels than expected from the calculated season-dependent trend, which was determined at normal ozone values. These values were similar to values which were measured during the Antarctic summer, indicating that the depleted ozone column thickness has an extreme influence on the biologically harmful UV climate on ground. PMID:16348742

  15. Biological Activities of a Mixture of Biosurfactant from Bacillus subtilis and Alkaline Lipase from Fusarium oxysporum

    PubMed Central

    Pereira de Quadros, Cedenir; Cristina Teixeira Duarte, Marta; Maria Pastore, Gláucia

    2011-01-01

    In this study, we investigate the antimicrobial effects of a mixture of a biosurfactant from Bacillus subtilis and an alkaline lipase from Fusarium oxysporum (AL/BS mix) on several types of microorganisms, as well as their abilities to remove Listeria innocua ATCC 33093 biofilm from stainless steel coupons. The AL/BS mix had a surface tension of around 30 mN.m-1, indicating that the presence of alkaline lipase did not interfere in the surface activity properties of the tensoactive component. The antimicrobial activity of the AL/BS mix was determined by minimum inhibitory concentration (MIC) micro-assays. Among all the tested organisms, the presence of the mixture only affected the growth of B. subtilis CCT 2576, B. cereus ATCC 10876 and L. innocua. The most sensitive microorganism was B. cereus (MIC 0.013 mg.mL-1). In addition, the effect of the sanitizer against L. innocua attached to stainless steel coupons was determined by plate count after vortexing. The results showed that the presence of the AL/BS mix improved the removal of adhered cells relative to treatment done without the sanitizer, reducing the count of viable cells by 1.72 log CFU.cm-2. However, there was no significant difference between the sanitizers tested and an SDS detergent standard (p<0.05). PMID:24031642

  16. Effect of nanocomposite packaging containing ZnO on growth of Bacillus subtilis and Enterobacter aerogenes.

    PubMed

    Esmailzadeh, Hakimeh; Sangpour, Parvaneh; Shahraz, Farzaneh; Hejazi, Jalal; Khaksar, Ramin

    2016-01-01

    Recent advances in nanotechnology have opened new windows in active food packaging. Nano-sized ZnO is an inexpensive material with potential antimicrobial properties. The aim of the present study is to evaluate the antibacterial effect of low density Polyethylene (LDPE) containing ZnO nanoparticles on Bacillus subtilis and Enterobacter aerogenes. ZnO nanoparticles have been synthesized by facil molten salt method and have been characterized by X-ray diffraction (XRD), and scanning electron microscopy (SEM). Nanocomposite films containing 2 and 4 wt.% ZnO nanoparticles were prepared by melt mixing in a twin-screw extruder. The growth of both microorganisms has decreased in the presence of ZnO containing nanocomposites compared with controls. Nanocomposites with 4 wt.% ZnO nanoparticles had stronger antibacterial effect against both bacteria in comparison with the 2 wt.% ZnO containing nanocomposites. B. subtilis as Gram-positive bacteria were more sensitive to ZnO containing nanocomposite films compared with E. aerogenes as Gram-negative bacteria. There were no significant differences between the migration of Zn ions from 2 and 4 wt.% ZnO containing nanocomposites and the released Zn ions were not significantly increased in both groups after 14 days compared with the first. Regarding the considerable antibacterial effects of ZnO nanoparticles, their application in active food packaging can be a suitable solution for extending the shelf life of food.

  17. FlgN Is Required for Flagellum-Based Motility by Bacillus subtilis

    PubMed Central

    Cairns, Lynne S.; Marlow, Victoria L.; Kiley, Taryn B.; Birchall, Christopher; Ostrowski, Adam; Aldridge, Phillip D.

    2014-01-01

    The assembly of the bacterial flagellum is exquisitely controlled. Flagellar biosynthesis is underpinned by a specialized type III secretion system that allows export of proteins from the cytoplasm to the nascent structure. Bacillus subtilis regulates flagellar assembly using both conserved and species-specific mechanisms. Here, we show that YvyG is essential for flagellar filament assembly. We define YvyG as an orthologue of the Salmonella enterica serovar Typhimurium type III secretion system chaperone, FlgN, which is required for the export of the hook-filament junction proteins, FlgK and FlgL. Deletion of flgN (yvyG) results in a nonmotile phenotype that is attributable to a decrease in hag translation and a complete lack of filament polymerization. Analyses indicate that a flgK-flgL double mutant strain phenocopies deletion of flgN and that overexpression of flgK-flgL cannot complement the motility defect of a ΔflgN strain. Furthermore, in contrast to previous work suggesting that phosphorylation of FlgN alters its subcellular localization, we show that mutation of the identified tyrosine and arginine FlgN phosphorylation sites has no effect on motility. These data emphasize that flagellar biosynthesis is differentially regulated in B. subtilis from classically studied Gram-negative flagellar systems and questions the biological relevance of some posttranslational modifications identified by global proteomic approaches. PMID:24706744

  18. Study of the tensile properties of individual multicellular fibres generated by Bacillus subtilis

    PubMed Central

    Ye, Xuan; Zhao, Liang; Liang, Jiecun; Li, Xide; Chen, Guo-Qiang

    2017-01-01

    Multicellular fibres formed by Bacillus subtilis (B. subtilis) are attracting interest because of their potential application as degradable biomaterials. However, mechanical properties of individual fibres remain unknown because of their small dimensions. Herein, a new approach is developed to investigate the tensile properties of individual fibres with an average diameter of 0.7 μm and a length range of 25.7–254.3 μm. Variations in the tensile strengths of fibres are found to be the result of variable interactions among pairs of microbial cells known as septa. Using Weibull weakest-link model to study this mechanical variability, we predict the length effect of the sample. Moreover, the mechanical properties of fibres are found to depend highly on relative humidity (RH), with a brittle–ductile transition occurring around RH = 45%. The elastic modulus is 5.8 GPa in the brittle state, while decreases to 62.2 MPa in the ductile state. The properties of fibres are investigated by using a spring model (RH < 45%) for its elastic behaviour, and the Kelvin–Voigt model (RH > 45%) for the time-dependent response. Loading-unloading experiments and numerical calculations demonstrate that necking instability comes from structural changes (septa) and viscoelasticity dominates the deformation of fibres at high RH. PMID:28378797

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

    PubMed

    Gunter, E A; Melekhin, A K

    2015-01-01

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

  20. Plant Growth Promotion by Volatile Organic Compounds Produced by Bacillus subtilis SYST2

    PubMed Central

    Tahir, Hafiz A. S.; Gu, Qin; Wu, Huijun; Raza, Waseem; Hanif, Alwina; Wu, Liming; Colman, Massawe V.; Gao, Xuewen

    2017-01-01

    Bacterial volatiles play a significant role in promoting plant growth by regulating the synthesis or metabolism of phytohormones. In vitro and growth chamber experiments were conducted to investigate the effect of volatile organic compounds (VOCs) produced by the plant growth promoting rhizobacterium Bacillus subtilis strain SYST2 on hormone regulation and growth promotion in tomato plants. We observed a significant increase in plant biomass under both experimental conditions; we observed an increase in photosynthesis and in the endogenous contents of gibberellin, auxin, and cytokinin, while a decrease in ethylene levels was noted. VOCs emitted by SYST2 were identified through gas chromatography-mass spectrometry analysis. Of 11 VOCs tested in glass jars containing plants in test tubes, only two, albuterol and 1,3-propanediole, were found to promote plant growth. Furthermore, tomato plants showed differential expression of genes involved in auxin (SlIAA1. SlIAA3), gibberellin (GA20ox-1), cytokinin (SlCKX1), expansin (Exp2, Exp9. Exp 18), and ethylene (ACO1) biosynthesis or metabolism in roots and leaves in response to B. subtilis SYST2 VOCs. Our findings suggest that SYST2-derived VOCs promote plant growth by triggering growth hormone activity, and provide new insights into the mechanism of plant growth promotion by bacterial VOCs. PMID:28223976