Inhibition of biofilm formation in Bacillus subtilis by new halogenated furanones.

PubMed

Kayumov, Airat R; Khakimullina, Elvina N; Sharafutdinov, Irshad S; Trizna, Elena Y; Latypova, Lilia Z; Thi Lien, Hoang; Margulis, Anna B; Bogachev, Mikhail I; Kurbangalieva, Almira R

2015-05-01

Gram-positive bacteria can cause various infections including hospital-acquired infections. While in the biofilm, the resistance of bacteria to both antibiotics and the human immune system is increased causing difficulties in the treatment. Bacillus subtilis, a non-pathogenic Gram-positive bacterium, is widely used as a model organism for studying biofilm formation. Here we investigated the effect of novel synthesized chloro- and bromo-containing 2(5H)-furanones on biofilm formation by B. subtilis. Mucobromic acid (3,4-dibromo-5-hydroxy-2(5H)-furanone) and the two derivatives of mucochloric acid (3,4-dichloro-5-hydroxy-2(5H)-furanone)-F8 and F12-were found to inhibit the growth and to efficiently prevent biofilm formation by B. subtilis. Along with the low production of polysaccharide matrix and repression of the eps operon, strong repression of biofilm-related yqxM also occurred in the presence of furanones. Therefore, our data confirm that furanones affect significantly the regulatory pathway(s) leading to biofilm formation. We propose that the global regulator, Spo0A, is one of the potential putative cellular targets for these compounds.

RNases and Helicases in Gram-Positive Bacteria.

PubMed

Durand, Sylvain; Condon, Ciaran

2018-04-01

RNases are key enzymes involved in RNA maturation and degradation. Although they play a crucial role in all domains of life, bacteria, archaea, and eukaryotes have evolved with their own sets of RNases and proteins modulating their activities. In bacteria, these enzymes allow modulation of gene expression to adapt to rapidly changing environments. Today, >20 RNases have been identified in both Escherichia coli and Bacillus subtilis , the paradigms of the Gram-negative and Gram-positive bacteria, respectively. However, only a handful of these enzymes are common to these two organisms and some of them are essential to only one. Moreover, although sets of RNases can be very similar in closely related bacteria such as the Firmicutes Staphylococcus aureus and B. subtilis , the relative importance of individual enzymes in posttranscriptional regulation in these organisms varies. In this review, we detail the role of the main RNases involved in RNA maturation and degradation in Gram-positive bacteria, with an emphasis on the roles of RNase J1, RNase III, and RNase Y. We also discuss how other proteins such as helicases can modulate the RNA-degradation activities of these enzymes.

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation.

PubMed

Burckhardt, Rachel M; Escalante-Semerena, Jorge C

2017-11-01

Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for s treptothricin a ce t yltransferase A , formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA + restored streptothricin resistance to B. subtilis satA ( Bs SatA) strains. Purified Bs SatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity ( K d [dissociation constant] = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA + in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil. IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis The initial characterization of the enzyme provides insights into its

In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation

PubMed Central

Burckhardt, Rachel M.

2017-01-01

ABSTRACT Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA+ restored streptothricin resistance to B. subtilis satA (BsSatA) strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd [dissociation constant] = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA+ in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil. IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis. This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis. The initial characterization of the enzyme provides insights into its

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

PubMed

Koo, Byoung-Mo; Kritikos, George; Farelli, Jeremiah D; Todor, Horia; Tong, Kenneth; Kimsey, Harvey; Wapinski, Ilan; Galardini, Marco; Cabal, Angelo; Peters, Jason M; Hachmann, Anna-Barbara; Rudner, David Z; Allen, Karen N; Typas, Athanasios; Gross, Carol A

2017-03-22

A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

[New antibiotics produced by Bacillus subtilis strains].

PubMed

Malanicheva, I A; Kozlov, D G; Efimenko, T A; Zenkova, V A; Kastrukha, G S; Reznikova, M I; Korolev, A M; Borshchevskaia, L N; Tarasova, O D; Sineokiĭ, S P; Efremenkova, O V

2014-01-01

Two Bacillus subtilis strains isolated from the fruiting body of a basidiomycete fungus Pholiota squarrosa exhibited a broad range of antibacterial activity, including those against methicillin-resistant Staphylococcus aureus INA 00761 (MRSA) and Leuconostoc mes6nteroides VKPM B-4177 resistant to glycopep-> tide antibiotics, as well as antifungal activity. The strains were identified as belonging to the "B. subtilis" com- plex based on their morphological and physiological characteristics, as well as by sequencing of the 16S rRNA gene fragments. Both strains (INA 01085 and INA 01086) produced insignificant amounts of polyene antibiotics (hexaen and pentaen, respectively). Strain INA 01086 produced also a cyclic polypeptide antibiotic containing Asp, Gly, Leu, Pro, Tyr, Thr, Trp, and Phe, while the antibiotic of strain INA 01085 contained, apart from these, two unidentified nonproteinaceous amino acids. Both polypeptide antibiotics were new compounds efficient against gram-positive bacteria and able to override the natural bacterial antibiotic resistance.

The poly-gamma-glutamate of Bacillus subtilis interacts specifically with silver nanoparticles

PubMed Central

Eymard-Vernain, Elise; Coute, Yohann; Adrait, Annie; Rabilloud, Thierry; Sarret, Géraldine

2018-01-01

For many years, silver nanoparticles, as with other antibacterial nanoparticles, have been extensively used in manufactured products. However, their fate in the environment is unclear and raises questions. We studied the fate of silver nanoparticles in the presence of bacteria under growth conditions that are similar to those found naturally in the environment (that is, bacteria in a stationary phase with low nutrient concentrations). We demonstrated that the viability and the metabolism of a gram-positive bacteria, Bacillus subtilis, exposed during the stationary phase is unaffected by 1 mg/L of silver nanoparticles. These results can be partly explained by a physical interaction of the poly-gamma-glutamate (PGA) secreted by Bacillus subtilis with the silver nanoparticles. The coating of the silver nanoparticles by the secreted PGA likely results in a loss of the bioavailability of nanoparticles and, consequently, a decrease of their biocidal effect. PMID:29813090

Advances and prospects of Bacillus subtilis cellular factories: From rational design to industrial applications.

PubMed

Gu, Yang; Xu, Xianhao; Wu, Yaokang; Niu, Tengfei; Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Liu, Long

2018-05-15

Bacillus subtilis is the most characterized gram-positive bacterium that has significant attributes, such as growing well on cheap carbon sources, possessing clear inherited backgrounds, having mature genetic manipulation methods, and exhibiting robustness in large-scale fermentations. Till date, B. subtilis has been identified as attractive hosts for the production of recombinant proteins and chemicals. By applying various systems and synthetic biology tools, the productivity features of B. subtilis can be thoroughly analyzed and further optimized via metabolic engineering. In the present review, we discussed why B. subtilis is the primary organisms used for metabolic engineering and industrial applications. Additionally, we summarized the recent advances in systems and synthetic biology, engineering strategies for improving cellular performances, and metabolic engineering applications of B. subtilis. In particular, we proposed emerging opportunities and essential strategies to enable the successful development of B. subtilis as microbial cell factories. Copyright © 2018. Published by Elsevier Inc.

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.

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

Disinfection of gram-negative and gram-positive bacteria using DynaJets® hydrodynamic cavitating jets.

PubMed

Loraine, Gregory; Chahine, Georges; Hsiao, Chao-Tsung; Choi, Jin-Keun; Aley, Patrick

2012-05-01

Cavitating jet technologies (DynaJets®) were investigated as a means of disinfection of gram-negative Escherichia coli, Klebsiellapneumoniae, Pseudomonas syringae, and Pseudomonas aeruginosa, and gram-positive Bacillus subtilis. The hydrodynamic cavitating jets were found to be very effective in reducing the concentrations of all of these species. In general, the observed rates of disinfection of gram-negative species were higher than for gram-positive species. However, different gram-negative species also showed significant differences (P. syringae 6-log(10) reduction, P. aeruginosa 2-log(10) reduction) under the same conditions. Disinfection of E. coli repeatedly showed five orders of magnitude reduction in concentration within 45-60-min at low nozzle pressure (2.1 bar). Optimization of nozzle design and operating pressures increased disinfection rates per input energy by several orders of magnitude. The power efficiencies of the hydrodynamic cavitating jets were found to be 10-100 times greater than comparable ultrasonic systems. Copyright © 2011 Elsevier B.V. All rights reserved.

Generation of multiple cell types in Bacillus subtilis.

PubMed

Lopez, Daniel; Vlamakis, Hera; Kolter, Roberto

2009-01-01

Bacillus subtilis is a Gram-positive bacterium that is well known for its ability to differentiate into metabolically inactive spores that are highly resistant to environmental stresses. In fact, populations of genetically identical B. subtilis comprise numerous distinct cell types. In addition to spores, cells can become genetically competent, motile, produce extracellular matrix or degradative enzymes, or secrete toxins that allow them to cannibalize their neighbors. Many of the cell fates listed above appear to be mutually exclusive. In this review, we discuss how individual cells within a population control their gene expression to ensure that proper regulation of differentiation occurs. These different cell fates are regulated by an intricate network that relies primarily on the activity of three major transcriptional regulators: Spo0A, DegU, and ComK. While individual cells must choose distinct cell fates, the population as a whole exhibits a spectrum of phenotypes whose diversity may increase fitness.

Complete sequence of the first chimera genome constructed by cloning the whole genome of Synechocystis strain PCC6803 into the Bacillus subtilis 168 genome.

PubMed

Watanabe, Satoru; Shiwa, Yuh; Itaya, Mitsuhiro; Yoshikawa, Hirofumi

2012-12-01

Genome synthesis of existing or designed genomes is made feasible by the first successful cloning of a cyanobacterium, Synechocystis PCC6803, in Gram-positive, endospore-forming Bacillus subtilis. Whole-genome sequence analysis of the isolate and parental B. subtilis strains provides clues for identifying single nucleotide polymorphisms (SNPs) in the 2 complete bacterial genomes in one cell.

Influence of heterologous MreB proteins on cell morphology of Bacillus subtilis.

PubMed

Schirner, Kathrin; Errington, Jeff

2009-11-01

The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB-like genes, mreB, mbl and mreBH, the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreB(B. licheniformis) (mreB(Bl)) or mreB(C. perfringens) (mreB(Cp)) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCD(Bl) could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod-shaped cells. In contrast, expression of mreBCD(Cp) was not sufficient to confer a rod shape to B. subtilis Delta mreBCD, indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis.

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX.

PubMed

Omardien, Soraya; Ter Beek, Alexander; Vischer, Norbert; Montijn, Roy; Schuren, Frank; Brul, Stanley

2018-06-14

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

Antibiotic Stimulation of a Bacillus subtilis Migratory Response

PubMed Central

Liu, Yongjin; Kyle, Steven

2018-01-01

ABSTRACT Competitive interactions between bacteria reveal physiological adaptations that benefit fitness. Bacillus subtilis is a Gram-positive species with several adaptive mechanisms for competition and environmental stress. Biofilm formation, sporulation, and motility are the outcomes of widespread changes in a population of B. subtilis. These changes emerge from complex, regulated pathways for adapting to external stresses, including competition from other species. To identify competition-specific functions, we cultured B. subtilis with multiple species of Streptomyces and observed altered patterns of growth for each organism. In particular, when plated on agar medium near Streptomyces venezuelae, B. subtilis initiates a robust and reproducible mobile response. To investigate the mechanistic basis for the interaction, we determined the type of motility used by B. subtilis and isolated inducing metabolites produced by S. venezuelae. Bacillus subtilis has three defined forms of motility: swimming, swarming, and sliding. Streptomyces venezuelae induced sliding motility specifically in our experiments. The inducing agents produced by S. venezuelae were identified as chloramphenicol and a brominated derivative at subinhibitory concentrations. Upon further characterization of the mobile response, our results demonstrated that subinhibitory concentrations of chloramphenicol, erythromycin, tetracycline, and spectinomycin all activate a sliding motility response by B. subtilis. Our data are consistent with sliding motility initiating under conditions of protein translation stress. This report underscores the importance of hormesis as an early warning system for potential bacterial competitors and antibiotic exposure. IMPORTANCE Antibiotic resistance is a major challenge for the effective treatment of infectious diseases. Identifying adaptive mechanisms that bacteria use to survive low levels of antibiotic stress is important for understanding pathways to

Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma

NASA Astrophysics Data System (ADS)

Mai-Prochnow, Anne; Clauson, Maryse; Hong, Jungmi; Murphy, Anthony B.

2016-12-01

Cold atmospheric-pressure plasma (CAP) is a relatively new method being investigated for antimicrobial activity. However, the exact mode of action is still being explored. Here we report that CAP efficacy is directly correlated to bacterial cell wall thickness in several species. Biofilms of Gram positive Bacillus subtilis, possessing a 55.4 nm cell wall, showed the highest resistance to CAP, with less than one log10 reduction after 10 min treatment. In contrast, biofilms of Gram negative Pseudomonas aeruginosa, possessing only a 2.4 nm cell wall, were almost completely eradicated using the same treatment conditions. Planktonic cultures of Gram negative Pseudomonas libanensis also had a higher log10 reduction than Gram positive Staphylococcus epidermidis. Mixed species biofilms of P. aeruginosa and S. epidermidis showed a similar trend of Gram positive bacteria being more resistant to CAP treatment. However, when grown in co-culture, Gram negative P. aeruginosa was more resistant to CAP overall than as a mono-species biofilm. Emission spectra indicated OH and O, capable of structural cell wall bond breakage, were present in the plasma. This study indicates that cell wall thickness correlates with CAP inactivation times of bacteria, but cell membranes and biofilm matrix are also likely to play a role.

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

PubMed

Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

2017-02-15

The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

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

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.

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.

A major protein component of the Bacillus subtilis biofilm matrix.

PubMed

Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

2006-02-01

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

First steps of bacteriophage SPP1 entry into Bacillus subtilis.

PubMed

Jakutytė, Lina; Lurz, Rudi; Baptista, Catarina; Carballido-Lopez, Rut; São-José, Carlos; Tavares, Paulo; Daugelavičius, Rimantas

2012-01-20

The mechanism of genome transfer from the virion to the host cytoplasm is critical to understand and control the beginning of viral infection. The initial steps of bacteriophage SPP1 infection of the Gram-positive bacterium Bacillus subtilis were monitored by following changes in permeability of the cytoplasmic membrane (CM). SPP1 leads to a distinctively faster CM depolarization than the one caused by podovirus ϕ29 or myovirus SP01 during B. subtilis infection. Depolarization requires interaction of SPP1 infective virion to its receptor protein YueB. The amplitude of depolarization depends on phage input and concentration of YueB at the cell surface. Sub-millimolar concentrations of Ca(2+) are necessary and sufficient for SPP1 reversible binding to the host envelope and thus to trigger depolarization while DNA delivery to the cytoplasm depends on millimolar concentrations of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-11-01

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

Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1.

PubMed

Jakutyte-Giraitiene, Lina; Gasiunas, Giedrius

2016-08-01

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes.

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.

Discovery of Novel Cell Wall-Active Compounds Using PywaC, a Sensitive Reporter of Cell Wall Stress, in the Model Gram-Positive Bacterium Bacillus subtilis

PubMed Central

Czarny, T. L.; Perri, A. L.; French, S.

2014-01-01

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds. PMID:24687489

Subtilomycin: A New Lantibiotic from Bacillus subtilis Strain MMA7 Isolated from the Marine Sponge Haliclona simulans

PubMed Central

Phelan, Robert W.; Barret, Matthieu; Cotter, Paul D.; O’Connor, Paula M.; Chen, Rui; Morrissey, John P.; Dobson, Alan D. W.; O’Gara, Fergal; Barbosa, Teresa M.

2013-01-01

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications. PMID:23736764

Structural and biochemical analyses of YvgN and YtbE from Bacillus subtilis

PubMed Central

Lei, Jian; Zhou, Yan-Feng; Li, Lan-Fen; Su, Xiao-Dong

2009-01-01

Bacillus subtilis is one of the most studied gram-positive bacteria. In this work, YvgN and YtbE from B. subtilis, assigned as AKR5G1 and AKR5G2 of aldo-keto reductase (AKR) superfamily. AKR catalyzes the NADPH-dependent reduction of aldehyde or aldose substrates to alcohols. YvgN and YtbE were studied by crystallographic and enzymatic analyses. The apo structures of these proteins were determined by molecular replacement, and the structure of holoenzyme YvgN with NADPH was also solved, revealing the conformational changes upon cofactor binding. Our biochemical data suggest both YvgN and YtbE have preferential specificity for derivatives of benzaldehyde, such as nitryl or halogen group substitution at the 2 or 4 positions. These proteins also showed broad catalytic activity on many standard substrates of AKR, such as glyoxal, dihydroxyacetone, and DL-glyceraldehyde, suggesting a possible role in bacterial detoxification. PMID:19585557

Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase.

PubMed

Park, Jung-Ho; Yamaguchi, Yoshihiro; Inouye, Masayori

2011-08-04

MazF is an mRNA interferase which cleaves mRNAs at a specific sequence. Here, we show that in contrast to MazF-ec from Escherichia coli, which specifically cleaves ACA sequences, MazF-bs from Bacillus subtilis is an mRNA interferase that specifically cleaves a five-base sequence, UACAU. MazF homologues widely prevailing in Gram-positive bacteria were found to be highly homologous to MazF-bs, suggesting that they may also have similar cleavage specificity. This cleavage site is over-represented in the B. subtilis genes associated with biosynthesis of secondary metabolites, suggesting that MazF-bs may be involved in the regulation of the production of secondary metabolites. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Biodegradation of naphthalene and phenanthren by Bacillus subtilis 3KP

NASA Astrophysics Data System (ADS)

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

2017-06-01

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

CdSe quantum dot internalization by Bacillus subtilis and Escherichia coli

NASA Astrophysics Data System (ADS)

Kloepfer, Jeremiah A.; Mielke, Randall E.; Nadeau, Jay L.

2004-06-01

Biological labeling has been demonstrated with CdSe quantum dots in a variety of animal cells, but bacteria are harder to label because of their cell walls. We discuss the challenges of using minimally coated, bare CdSe quantum dots as luminescent internal labels for bacteria. These quantum dots were solubilized with mercaptoacetic acid and conjugated to adenine. Significant evidence for the internal staining of Bacillus subtilis (Gram positive) and Escherichia coli (Gram negative) using these structures is presented via steady-state emission, epifluorescence microscopy, transmission electron microscopy, and energy dispersive spectroscopy. In particular, the E. coli adenine auxotroph, and not the wild type, took up adenine coated quantum dots, and this only occurred in adenine deficient growth media. Labeling strength was enhanced by performing the incubation under room light. This process was examined with steady-state emission spectra and time-resolved luminescence profiles obtained from time-correlated-single-photon counting.

A pathway closely related to the (D)-tagatose pathway of gram-negative enterobacteria identified in the gram-positive bacterium Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M; Galleni, Moreno; Joris, Bernard

2013-06-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

Hydraphiles enhance antimicrobial potency against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis.

PubMed

Patel, Mohit B; Garrad, Evan C; Stavri, Ariel; Gokel, Michael R; Negin, Saeedeh; Meisel, Joseph W; Cusumano, Zachary; Gokel, George W

2016-06-15

Hydraphiles are synthetic amphiphiles that form ion-conducting pores in liposomal membranes. These pores exhibit open-close behavior when studied by planar bilayer conductance techniques. In previous work, we showed that when co-administered with various antibiotics to the DH5α strain of Escherichia coli, they enhanced the drug's potency. We report here potency enhancements at low concentrations of hydraphiles for the structurally and mechanistically unrelated antibiotics erythromycin, kanamycin, rifampicin, and tetracycline against Gram negative E. coli (DH5α and K-12) and Pseudomonas aeruginosa, as well as Gram positive Bacillus subtilis. Earlier work suggested that potency increases correlated to ion transport function. The data presented here comport with the function of hydraphiles to enhance membrane permeability in addition to, or instead of, their known function as ion conductors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modeling the acid-base properties of bacterial surfaces: A combined spectroscopic and potentiometric study of the gram-positive bacterium Bacillus subtilis.

PubMed

Leone, Laura; Ferri, Diego; Manfredi, Carla; Persson, Per; Shchukarev, Andrei; Sjöberg, Staffan; Loring, John

2007-09-15

In this study, macroscopic and spectroscopic data were combined to develop a surface complexation model that describes the acid-base properties of Bacillus subtilis. The bacteria were freeze-dried and then resuspended in 0.1 M NaCl ionic medium. Macroscopic measurements included potentiometric acid-base titrations and electrophoretic mobility measurements. In addition, ATR-FTIR spectra of wet pastes from suspensions of Bacillus subtilis at different pH values were collected. The least-squares program MAGPIE was used to generate a surface complexation model that takes into account the presence of three acid-base sites on the surface: tripple bond COOH, tripple bond NH+, and tripple bond PO-, which were identified previously by XPS measurements. Both potentiometric titration data and ATR-FTIR spectra were used quantitatively, and electrostatic effects at the charged bacterial surface were accounted for using the constant capacitance model. The model was calculated using two different approaches: in the first one XPS data were used to constrain the ratio of the total concentrations of all three surface sites. The capacitance of the double layer, the total buffer capacity, and the deprotonation constants of the tripple bond NH+, tripple bond POH, and tripple bond COOH species were determined in the fit. A second approach is presented in which the ratio determined by XPS of the total concentrations of tripple bond NH+ to tripple bond PO- sites is relaxed. The total concentration of tripple bond PO- sites was determined in the fit, while the deprotonation constant for tripple bond POH was manually varied until the minimization led to a model which predicted an isoelectric point that resulted in consistency with electrophoretic mobility data. The model explains well the buffering capacity of Bacillus subtilis suspensions in a wide pH range (between pH=3 and pH=9) which is of considerable environmental interest. In particular, a similar quantitative use of the IR data

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.

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

A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

PubMed Central

Van der Heiden, Edwige; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M.; Galleni, Moreno; Joris, Bernard

2013-01-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. PMID:23524682

Bottleneck in secretion of α-amylase in Bacillus subtilis.

PubMed

Yan, Shaomin; Wu, Guang

2017-07-19

Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However, the secretion stress limits the high yield of α-amylase in B. subtilis although huge efforts have been made to address this secretion bottleneck. In this question-oriented review, every effort is made to answer the following questions, which look simple but are long-standing, through reviewing of literature: (1) Does α-amylase need a specific and dedicated chaperone? (2) What signal sequence does CsaA recognize? (3) Does CsaA require ATP for its operation? (4) Does an unfolded α-amylase is less soluble than a folded one? (5) Does α-amylase aggregate before transporting through Sec secretion system? (6) Is α-amylase sufficient stable to prevent itself from misfolding? (7) Does α-amylase need more disulfide bonds to be stabilized? (8) Which secretion system does PrsA pass through? (9) Is PrsA ATP-dependent? (10) Is PrsA reused after folding of α-amylase? (11) What is the fate of PrsA? (12) Is trigger factor (TF) ATP-dependent? The literature review suggests that not only the most of those questions are still open to answers but also it is necessary to calculate ATP budget in order to better understand how B. subtilis uses its energy for production and secretion.

Discovery of novel cell wall-active compounds using P ywaC, a sensitive reporter of cell wall stress, in the model gram-positive bacterium Bacillus subtilis.

PubMed

Czarny, T L; Perri, A L; French, S; Brown, E D

2014-06-01

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

B. subtilis as a Model for Studying the Assembly of Fe-S Clusters in Gram-Positive Bacteria.

PubMed

Dos Santos, Patricia C

2017-01-01

Complexes of iron and sulfur (Fe-S clusters) are widely distributed in nature and participate in essential biochemical reactions. The biological formation of Fe-S clusters involves dedicated pathways responsible for the mobilization of sulfur, the assembly of Fe-S clusters, and the transfer of these clusters to target proteins. Genomic analysis of Bacillus subtilis and other Gram-positive bacteria indicated the presence of only one Fe-S cluster biosynthesis pathway, which is distinct in number of components and organization from previously studied systems. B. subtilis has been used as a model system for the characterization of cysteine desulfurases responsible for sulfur mobilization reactions in the biogenesis of Fe-S clusters and other sulfur-containing cofactors. Cysteine desulfurases catalyze the cleavage of the C-S bond from the amino acid cysteine and subsequent transfer of sulfur to acceptor molecules. These reactions can be monitored by the rate of alanine formation, the first product in the reaction, and sulfide formation, a byproduct of reactions performed under reducing conditions. The assembly of Fe-S clusters on protein scaffolds and the transfer of these clusters to target acceptors are determined through a combination of spectroscopic methods probing the rate of cluster assembly and transfer. This chapter provides a description of reactions promoting the assembly of Fe-S clusters in bacteria as well as methods used to study functions of each biosynthetic component and identify mechanistic differences employed by these enzymes across different pathways. © 2017 Elsevier Inc. All rights reserved.

Effect of Bacillus subtilis on Granite Weathering: A Laboratory Experiment

NASA Astrophysics Data System (ADS)

Song, W.; Ogawa, N.; Oguchi, C. T.; Hatta, T.; Matsukura, Y.

2006-12-01

We performed a comparative experiment to investigate how the ubiquitous soil bacterium Bacillus subtilis weathers granite and which granite-forming minerals weather more rapidly via biological processes. Batch type experiments (granite specimen in a 500 ml solution including NaCl, glucose, yeast extract and bacteria Bacillus subtilis at 27°E C) were carried out for 30 days. Granite surfaces were observed by SEM before and after the experiment. Bacillus subtilis had a strong influence on granite weathering by forming pits. There were 2.4 times as many pits and micropores were 2.3 times wider in granite exposed to Bacillus subtilis when compared with bacteria-free samples. Bacillus subtilis appear to preferentially select an optimum place to adhere to the mineral and dissolve essential elements from the mineral to live. Plagioclase was more vulnerable to bacterial weathering than biotite among the granite composing minerals.

Chitosan nanocomposite films based on Ag-NP and Au-NP biosynthesis by Bacillus Subtilis as packaging materials.

PubMed

Youssef, Ahmed M; Abdel-Aziz, Mohamed S; El-Sayed, Samah M

2014-08-01

Chitosan-silver (CS-Ag) and Chitosan-gold (CS-Au) nanocomposites films were synthesized by a simple chemical method. A local bacterial isolate identified as Bacillus subtilis ss subtilis was found to be capable to synthesize both silver nanoparticles (Ag-NP) and gold nanoparticles (Au-NP) from silver nitrate (AgNO3) and chloroauric acid (AuCl(4-)) solutions, respectively. The biosynthesis of both Ag-NP and Au-NP characterize using UV/vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD), and then added to chitosan by different ratios (0.5, 1 and 2%). The prepared chitosan nanocomposites films were characterize using UV, XRD, SEM and TEM. Moreover, the antibacterial activity of the prepared films was evaluated against gram positive (Staphylococcus aureus) and gram negative bacteria (Pseudomonas aerugenosa), fungi (Aspergillus niger) and yeast (Candida albicans). Therefore, these materials can be potential used as antimicrobial agents in packaging applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Peptidoglycan turnover and recycling in Gram-positive bacteria.

PubMed

Reith, Jan; Mayer, Christoph

2011-10-01

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

[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.

Inducible error-prone repair in B. subtilis. Final report, September 1, 1979-June 30, 1981

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yasbin, R. E.

1981-06-01

The research performed under this contract has been concentrated on the relationship between inducible DNA repair systems, mutagenesis and the competent state in the gram positive bacterium Bacillus subtilis. The following results have been obtained from this research: (1) competent Bacillus subtilis cells have been developed into a sensitive tester system for carcinogens; (2) competent B. subtilis cells have an efficient excision-repair system, however, this system will not function on bacteriophage DNA taken into the cell via the process of transfection; (3) DNA polymerase III is essential in the mechanism of the process of W-reactivation; (4) B. subtilis strains curedmore » of their defective prophages have been isolated and are now being developed for gene cloning systems; (5) protoplasts of B. subtilis have been shown capable of acquiring DNA repair enzymes (i.e., enzyme therapy); and (6) a plasmid was characterized which enhanced inducible error-prone repair in a gram positive organism.« less

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

PubMed Central

Overkamp, Wout; Beilharz, Katrin; Detert Oude Weme, Ruud; Solopova, Ana; Karsens, Harma; Kovács, Ákos T.; Kok, Jan

2013-01-01

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria. PMID:23956387

Gene position in a long operon governs motility development in Bacillus subtilis

PubMed Central

Cozy, Loralyn M.; Kearns, Daniel B.

2010-01-01

Growing cultures of Bacillus subtilis bifurcate into subpopulations of motile individuals and non-motile chains of cells that are differentiated at the level of gene expression. The motile cells are ON and the chaining cells are OFF for transcription that depends on RNA polymerase and the alternative sigma factor σD. Here we show that chaining cells were OFF for σD-dependent gene expression because σD levels fell below a threshold, and σD activity was inhibited by the anti-sigma factor FlgM. The probability that σD exceeded the threshold was governed by the position of the sigD genes. The proportion of ON cells increased when sigD was artificially moved forward in the 27kb fla/che operon. In addition, we identified a new σD-dependent promoter that increases sigD expression and may provide positive feedback to stabilize the ON state. Finally, we demonstrate that ON/OFF motility states in B. subtilis are a form of development because mosaics of stable and differentiated epigenotypes were evident when the normally dispersed bacteria were forced to grow in one dimension. PMID:20233303

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

Non-HACEK gram-negative bacillus endocarditis.

PubMed

Morpeth, Susan; Murdoch, David; Cabell, Christopher H; Karchmer, Adolf W; Pappas, Paul; Levine, Donald; Nacinovich, Francisco; Tattevin, Pierre; Fernández-Hidalgo, Núria; Dickerman, Stuart; Bouza, Emilio; del Río, Ana; Lejko-Zupanc, Tatjana; de Oliveira Ramos, Auristela; Iarussi, Diana; Klein, John; Chirouze, Catherine; Bedimo, Roger; Corey, G Ralph; Fowler, Vance G

2007-12-18

Infective endocarditis caused by non-HACEK (species other than Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, or Kingella species) gram-negative bacilli is rare, is poorly characterized, and is commonly considered to be primarily a disease of injection drug users. To describe the clinical characteristics and outcomes of patients with non-HACEK gram-negative bacillus endocarditis in a large, international, contemporary cohort of patients. Observations from the International Collaboration on Infective Endocarditis Prospective Cohort Study (ICE-PCS) database. 61 hospitals in 28 countries. Hospitalized patients with definite endocarditis. Characteristics of non-HACEK gram-negative bacillus endocarditis cases were described and compared with those due to other pathogens. Among the 2761 case-patients with definite endocarditis enrolled in ICE-PCS, 49 (1.8%) had endocarditis (20 native valve, 29 prosthetic valve or device) due to non-HACEK, gram-negative bacilli. Escherichia coli (14 patients [29%]) and Pseudomonas aeruginosa (11 patients [22%]) were the most common pathogens. Most patients (57%) with non-HACEK gram-negative bacillus endocarditis had health care-associated infection, whereas injection drug use was rare (4%). Implanted endovascular devices were frequently associated with non-HACEK gram-negative bacillus endocarditis compared with other causes of endocarditis (29% vs. 11%; P < 0.001). The in-hospital mortality rate of patients with endocarditis due to non-HACEK gram-negative bacilli was high (24%) despite high rates of cardiac surgery (51%). Because of the small number of patients with non-HACEK gram-negative bacillus endocarditis in each treatment group and the lack of long-term follow-up, strong treatment recommendations are difficult to make. In this large, prospective, multinational cohort, more than one half of all cases of non-HACEK gram-negative bacillus endocarditis were associated with

The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

PubMed Central

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

2003-01-01

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

Biofilms affecting progression of mild steel corrosion by Gram positive Bacillus sp.

PubMed

Lin, Johnson; Madida, Bafana B

2015-10-01

The biodeterioration of metals have detrimental effects on the environment with economic implications. The deterioration of metals is of great concern to industry. In this study, mild steel coupons which were immersed in a medium containing Gram-positive Bacillus spp. and different nutrient sources were compared with the control in sterile deionized water. The weight loss of the coupons in the presence of Bacillus spp. alone was lower than the control and was further reduced when additional carbon sources, especially fructose, were added. The level of metal corrosion was significantly increased in the presence of nitrate with or without bacteria. There was a significant strong correlation between the weight loss and biofilm level (r =  0.64; p < 0.05). The addition of nitrate and Bacillus spp. produced more biofilms on the coupons and resulted in greater weight loss compared to that with Bacillus spp. only under the same conditions. However, Bacillus spp. enriched with carbon sources formed less biofilms and results in lower weight loss compared to that with Bacillus spp. only. The production of biofilm by Bacillus spp. influences the level of metal corrosion under different environmental conditions, thereby, supporting the development of a preventive strategy against corrosion. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-level iron mitigates fusaricidin-induced membrane damage and reduces membrane fluidity leading to enhanced drug resistance in Bacillus subtilis.

PubMed

Yu, Wen-Bang; Ye, Bang-Ce

2016-05-01

Fusaricidins are a class of cyclic lipopeptide antibiotics that have strong antifungal activities against plant pathogenic fungi and excellent bactericidal activities against Gram-positive bacteria. The mechanism through which fusaricidin exerts its action is not yet entirely clear. To investigate the mode of action of fusaricidin, we determined the physiological and transcriptional responses of Bacillus subtilis to fusaricidin treatment by using a systems-level approach. Our data show that fusaricidin rapidly induced the expression of σ(W) regulon and caused membrane damage in B. subtilis. We further demonstrated that ferric ions play multiple roles in the action of fusaricidin on B. subtilis. Iron deprivation blocked the formation of hydroxyl radical in the cells and significantly inhibited the bactericidal activity of fusaricidin. Conversely, high levels of iron (>2 mM) repressed the expression of BkdR regulon, resulting in a smaller cellular pool of branched-chain precursors for iso- and anteiso-branched fatty acids, which in turn led to a decrease in the proportion of branched-chain fatty acids in the membrane of B. subtilis. This change in membrane composition reduced its bilayer fluidity and increased its resistance to antimicrobial agents. In conclusion, our experiments uncovered some novel interactions and a synergism between cellular iron levels and drug resistance in Gram-positive bacteria. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antioxidant activity via DPPH, gram-positive and gram-negative antimicrobial potential in edible mushrooms.

PubMed

Ahmad, Nisar; Mahmood, Fazal; Khalil, Shahid Akbar; Zamir, Roshan; Fazal, Hina; Abbasi, Bilal Haider

2014-10-01

Edible mushrooms (EMs) are nutritionally rich source of proteins and essential amino acids. In the present study, the antioxidant activity via 1,1-diphenyl-2-picrylhydrazyl (DPPH) and antimicrobial potential in EMs (Pleurotus ostreatus, Morchella esculenta, P. ostreatus (Black), P. ostreatus (Yellow) and Pleurotus sajor-caju) were investigated. The DPPH radical scavenging activity revealed that the significantly higher activity (66.47%) was observed in Morchella esculenta at a maximum concentration. Similarly, the dose-dependent concentrations (200, 400, 600, 800 and 1000 µg) were also used for other four EMs. Pleurotus ostreatus exhibited 36.13% activity, P. ostreatus (Black (B)) exhibited 30.64%, P. ostreatus (Yellow (Y)) exhibited 40.75% and Pleurotus sajor-caju exhibited 47.39% activity at higher concentrations. Furthermore, the antimicrobial potential were investigated for its toxicity against gram-negative bacterial strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Klebsiella pneumonia, Erwinia carotovora and Agrobacterium tumifaciens), gram-positive bacterial strains (Bacillus subtilis, Bacillus atrophaeus and Staphylococcus aureus) and a fungal strain (Candida albicans) in comparison with standard antibiotics. Antimicrobial screening revealed that the ethanol extract of P. ostreatus was active against all microorganism tested except E. coli. Maximum zone of inhibition (13 mm) was observed against fungus and A. tumifaciens. P. sajor-caju showed best activities (12.5 mm) against B. subtilis, B. atrophaeus and K. pneumonia. P. ostreatus (Y) showed best activities against P. aeroginosa (21.83 mm), B. atrophaeus (20 mm) and C. albicans (21 mm). P. ostreatus (B) exhibited best activities against C. albicans (16 mm) and slightly lower activities against all other microbes except S. typhi. M. esculenta possess maximum activities in terms of inhibition zone against all microorganisms tested except S. typhi. © The Author(s) 2012.

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

PubMed

Pan, Jieru; Huang, Tianpei; Yao, Fan; Huang, Zhipeng; Powell, Charles A; Qiu, Sixin; Guan, Xiong

2008-01-01

AHL-lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specifically hydrolyzes N-acyl-homoserine lactones (AHLs) secreted by Gram-negative bacteria and thereby attenuates the symptoms caused by plant pathogens. In this study, an aiiA gene was cloned from Bacillus subtilis BS-1 by PCR with a pair of degenerate primers. The deduced 250 amino acid sequence contained two small conserved regions, 103SHLHFDH109 and 166TPGHTPGH173, which are characteristic of the metallo-beta-lactamase family. Homology comparison revealed that the deduced amino acid sequence had a high degree of similarity with those of the known AiiA proteins in the B. cereus group. Additionally, the aiiA gene was expressed in Escherichia coli BL21 (DE3) pLysS and the expressed AiiA protein could attenuate the soft rot symptoms caused by Erwinia carotovora var. carotovora.

Ecology and genomics of Bacillus subtilis.

PubMed

Earl, Ashlee M; Losick, Richard; Kolter, Roberto

2008-06-01

Bacillus subtilis is a remarkably diverse bacterial species that is capable of growth within many environments. Recent microarray-based comparative genomic analyses have revealed that members of this species also exhibit considerable genomic diversity. The identification of strain-specific genes might explain how B. subtilis has become so broadly adapted. The goal of identifying ecologically adaptive genes could soon be realized with the imminent release of several new B. subtilis genome sequences. As we embark upon this exciting new era of B. subtilis comparative genomics we review what is currently known about the ecology and evolution of this species.

Molecular study of a squalene cyclase homolog gene in Bacillus subtilis

NASA Astrophysics Data System (ADS)

Bosak, T.; Pearson, A.; Losick, R.

2005-12-01

Polycyclic triterpenoids such as hopanes and steranes are formed by enzymatic cyclization of linear isoprenoid precursors by squalene cyclases and oxidosqualene cyclases. Due to their amazing preservation potential, polycyclic triterpenoids have been used to indicate the source of organic matter in oils and sediments for decades, although many cannot be attributed to known organisms and genes. To bridge the gap between the genomic database and the geochemical record, we are using molecular tools to study the expression, intracellular localization, and products of a squalene cyclase homolog found in Bacillus subtilis, a Gram-positive soil bacterium. We find that the gene is expressed during sporulation and is localized to the spore coat. Our results may help to understand the source of some previously unassigned natural products, and they may also provide clues to the physiological role of triterpenoids in the Bacillales.

In Silico Evidence for the Horizontal Transfer of gsiB, a σΒ-Regulated Gene in Gram-Positive Bacteria, to Lactic Acid Bacteria ▿

PubMed Central

Asteri, Ioanna-Areti; Boutou, Effrossyni; Anastasiou, Rania; Pot, Bruno; Vorgias, Constantinos E.; Tsakalidou, Effie; Papadimitriou, Konstantinos

2011-01-01

gsiB, coding for glucose starvation-inducible protein B, is a characteristic member of the σΒ stress regulon of Bacillus subtilis and several other Gram-positive bacteria. Here we provide in silico evidence for the horizontal transfer of gsiB in lactic acid bacteria that are devoid of the σΒ factor. PMID:21421783

Adaptation of Bacillus subtilis to Life at Extreme Potassium Limitation.

PubMed

Gundlach, Jan; Herzberg, Christina; Hertel, Dietrich; Thürmer, Andrea; Daniel, Rolf; Link, Hannes; Stülke, Jörg

2017-07-05

Potassium is the most abundant metal ion in every living cell. This ion is essential due to its requirement for the activity of the ribosome and many enzymes but also because of its role in buffering the negative charge of nucleic acids. As the external concentrations of potassium are usually low, efficient uptake and intracellular enrichment of the ion is necessary. The Gram-positive bacterium Bacillus subtilis possesses three transporters for potassium, KtrAB, KtrCD, and the recently discovered KimA. In the absence of the high-affinity transporters KtrAB and KimA, the bacteria were unable to grow at low potassium concentrations. However, we observed the appearance of suppressor mutants that were able to overcome the potassium limitation. All these suppressor mutations affected amino acid metabolism, particularly arginine biosynthesis. In the mutants, the intracellular levels of ornithine, citrulline, and arginine were strongly increased, suggesting that these amino acids can partially substitute for potassium. This was confirmed by the observation that the supplementation with positively charged amino acids allows growth of B. subtilis even at the extreme potassium limitation that the bacteria experience if no potassium is added to the medium. In addition, a second class of suppressor mutations allowed growth at extreme potassium limitation. These mutations result in increased expression of KtrAB, the potassium transporter with the highest affinity and therefore allow the acquisition and accumulation of the smallest amounts of potassium ions from the environment. IMPORTANCE Potassium is essential for every living cell as it is required for the activity for many enzymes and for maintaining the intracellular pH by buffering the negative charge of the nucleic acids. We have studied the adaptation of the soil bacterium Bacillus subtilis to life at low potassium concentrations. If the major high-affinity transporters are missing, the bacteria are unable to grow

Differential antimicrobial activity of silver nanoparticles to bacteria Bacillus subtilis and Escherichia coli, and toxicity to crop plant Zea mays and beneficial B. subtilis-inoculated Z. mays

NASA Astrophysics Data System (ADS)

Doody, Michael A.; Wang, Dengjun; Bais, Harsh P.; Jin, Yan

2016-10-01

As silver nanoparticles (AgNPs) have become increasingly used in commercial antimicrobial agents and industrial and military products, concerns are increasing over their broad environmental and health impacts and risks because they are finding their way to the environment. This study was designed to quantify the antimicrobial activity of citrate-coated AgNPs (c-AgNPs; transmission electron microscope size of 44.9 ± 7.2 nm) to two species of bacteria, i.e., Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and toxicity to a major crop plant Zea mays and beneficial bacteria-inoculated plant (i.e., B. subtilis-inoculated Z. mays symbiont). Our results reveal that the exposure of c-AgNPs significantly inhibited bacteria growth and altered their growth kinetics. Z. mays experienced significant sublethal effects including reduced root length and biomass, and hyper-accumulation of Ag in roots. The beneficial interactions between B. subtilis and Z. mays were weakened as well because both species suffered sublethal effects. Potential mechanisms leading to the antimicrobial activity and toxicity of c-AgNPs to the bacteria, plant, and plant-bacteria symbiont examined in this study were discussed. Taken together, our findings advance the current knowledge of AgNPs antimicrobial property or toxicity to bacteria, crop plant, and beneficial plant-bacteria symbiotic interaction, which is a critical component for NPs environmental impact and risk assessment.

The actin-like MreB proteins in Bacillus subtilis: a new turn.

PubMed

Chastanet, Arnaud; Carballido-Lopez, Rut

2012-06-01

A decade ago, two breakthrough descriptions were reported: 1) the first helix-like protein localization pattern of MreB and its paralog Mbl in Bacillus subtilis and 2) the crystal structure of Thermotoga maritima MreB1, which was remarkably similar to that of actin. These discoveries strongly stimulated the field of bacterial development, leading to the identification of many new cytoskeletal proteins (1) and the publication of many studies describing the helical patterns of protein, DNA and even lipid domains. However, today, new breakthroughs are shaking up what had become a dogma. Instead of helical structures, MreBs appear to form discrete patches that move circumferentially around the cell, questioning the idea of MreB cables forming an actin-like cytoskeleton. Furthermore, increasing evidence of biochemical properties that are unlike the properties of actin suggest that the molecular behavior of MreB proteins may be different. The aim of this review is to summarize the current knowledge of the so-called "actin-like" MreB cytoskeleton through a discussion of the model Gram-positive bacterium B. subtilis and the most recent findings in this rapidly evolving research field.

Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

PubMed Central

Borisova, Marina; Gaupp, Rosmarie; Duckworth, Amanda; Schneider, Alexander; Dalügge, Désirée; Mühleck, Maraike; Deubel, Denise; Unsleber, Sandra; Yu, Wenqi; Muth, Günther; Bischoff, Markus; Götz, Friedrich

2016-01-01

ABSTRACT Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival. PMID:27729505

Transformation of gram positive bacteria by sonoporation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yunfeng; Li, Yongchao

The present invention provides a sonoporation-based method that can be universally applied for delivery of compounds into Gram positive bacteria. Gram positive bacteria which can be transformed by sonoporation include, for example, Bacillus, Streptococcus, Acetobacterium, and Clostridium. Compounds which can be delivered into Gram positive bacteria via sonoporation include nucleic acids (DNA or RNA), proteins, lipids, carbohydrates, viruses, small organic and inorganic molecules, and nano-particles.

Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

PubMed

Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

2016-01-01

Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

Real-time Attack of LL-37 on Single Bacillus subtilis Cells

PubMed Central

Barns, Kenneth J.; Weisshaar, James C.

2013-01-01

Time-lapse fluorescence microscopy of single, growing Bacillus subtilis cells with 2-12 s time resolution reveals the mechanisms of antimicrobial peptide (AMP) action on a Gram-positive species with unprecedented detail. For the human cathelicidin LL-37 attacking B. subtilis, the symptoms of antimicrobial stress differ dramatically depending on the bulk AMP concentration. At 2 μM LL-37, the mean single-cell growth rate decreases, but membrane permeabilization does not occur. At 4 μM LL-37, cells abruptly shrink in size at the same time that Sytox Green enters the cytoplasm and stains the nucleoids. We interpret the shrinkage event as loss of turgor pressure (and presumably the membrane potential) due to permeabilization of the membrane. Movies of Sytox Green staining at 0.5 frame/s show that nucleoid staining is initially local, more consistent with pore formation than with global permeabilization models. In a novel “growth recovery” assay, cells are incubated with LL-37 for a variable period and then rinsed with fresh growth medium lacking LL-37. The growth rate attenuation observed at 2 μM LL-37 is a recoverable symptom, while the abrupt cell shrinkage observed at 4 μM LL-37 is not. PMID:23454084

The Role of Functional Amyloids in Multicellular Growth and Development of Gram-Positive Bacteria.

PubMed

Dragoš, Anna; Kovács, Ákos T; Claessen, Dennis

2017-08-07

Amyloid fibrils play pivotal roles in all domains of life. In bacteria, these fibrillar structures are often part of an extracellular matrix that surrounds the producing organism and thereby provides protection to harsh environmental conditions. Here, we discuss the role of amyloid fibrils in the two distant Gram-positive bacteria, Streptomyces coelicolor and Bacillus subtilis . We describe how amyloid fibrils contribute to a multitude of developmental processes in each of these systems, including multicellular growth and community development. Despite this variety of tasks, we know surprisingly little about how their assembly is organized to fulfill all these roles.

The Na+ transport in gram-positive bacteria defect in the Mrp antiporter complex measured with 23Na nuclear magnetic resonance.

PubMed

Górecki, Kamil; Hägerhäll, Cecilia; Drakenberg, Torbjörn

2014-01-15

(23)Na nuclear magnetic resonance (NMR) has previously been used to monitor Na(+) translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na NMR method was adapted for measurements of internal Na(+) concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na(+) concentration than an external one. Copyright © 2013 Elsevier Inc. All rights reserved.

Abh and AbrB Control of Bacillus subtilis Antimicrobial Gene Expression▿

PubMed Central

Strauch, Mark A.; Bobay, Benjamin G.; Cavanagh, John; Yao, Fude; Wilson, Angelo; Le Breton, Yoann

2007-01-01

The Bacillus subtilis abh gene encodes a protein whose N-terminal domain has 74% identity to the DNA-binding domain of the global regulatory protein AbrB. Strains with a mutation in abh showed alterations in the production of antimicrobial compounds directed against some other Bacillus species and gram-positive microbes. Relative to its wild-type parental strain, the abh mutant was found deficient, enhanced, or unaffected for the production of antimicrobial activity. Using lacZ fusions, we examined the effects of abh upon the expression of 10 promoters known to be regulated by AbrB, including five that transcribe well-characterized antimicrobial functions (SdpC, SkfA, TasA, sublancin, and subtilosin). For an otherwise wild-type background, the results show that Abh plays a negative regulatory role in the expression of four of the promoters, a positive role for the expression of three, and no apparent regulatory role in the expression of the other three promoters. Binding of AbrB and Abh to the promoter regions was examined using DNase I footprinting, and the results revealed significant differences. The transcription of abh is not autoregulated, but it is subject to a degree of AbrB-afforded negative regulation. The results indicate that Abh is part of the complex interconnected regulatory system that controls gene expression during the transition from active growth to stationary phase. PMID:17720793

Silver-doped manganese dioxide and trioxide nanoparticles inhibit both gram positive and gram negative pathogenic bacteria.

PubMed

Kunkalekar, R K; Prabhu, M S; Naik, M M; Salker, A V

2014-01-01

Palladium, ruthenium and silver-doped MnO2 and silver doped Mn2O3 nanoparticles were synthesized by simple co-precipitation technique. SEM-TEM analysis revealed the nano-size of these synthesized samples. XPS data illustrates that Mn is present in 4+ and 3+ oxidation states in MnO2 and Mn2O3 respectively. Thermal analysis gave significant evidence for the phase changes with increasing temperature. Antibacterial activity of these synthesized nanoparticles on three Gram positive bacterial cultures (Staphylococcus aureus ATCC 6538, Streptococcus epidermis ATCC 12228, Bacillus subtilis ATCC 6633) and three Gram negative cultures (Escherichia coli ATCC 8739, Salmonella abony NCTC 6017 and Klebsiella pneumoniae ATCC 1003) was investigated using a disc diffusion method and live/dead assay. Only Ag-doped MnO2 and Ag-doped Mn2O3 nanoparticles showed antibacterial property against all six-test bacteria but Ag-doped MnO2 was found to be more effective than Ag-doped Mn2O3. Copyright © 2013 Elsevier B.V. All rights reserved.

A time resolved metabolomics study: the influence of different carbon sources during growth and starvation of Bacillus subtilis.

PubMed

Meyer, Hanna; Weidmann, Hendrikje; Mäder, Ulrike; Hecker, Michael; Völker, Uwe; Lalk, Michael

2014-07-01

In its natural environment, the soil, the Gram-positive model bacterium Bacillus subtilis frequently encounters nutrient limitation and other stress factors. Efficient adaptation mechanisms are necessary to cope with this wide range of environmental challenges. The ability to utilize diverse carbon sources represents a key adaptation process that allows B. subtilis to thrive in its natural habitat. To gain a comprehensive insight into the metabolism of B. subtilis, global metabolite analyses were performed during growth with glucose alone or glucose with either malate, fumarate or citrate as carbon/energy sources. Furthermore, to achieve a comprehensive coverage of a wide range of chemically different metabolites, complementary GC-MS, LC-MS and (1)H-NMR analyses were applied. This study reveals that the availability of different carbon sources results in different extracellular metabolite profiles whereas a regulated intracellular metabolite equilibrium was observed. In addition, the typical energy-starvation induced activation of the general stress sigma factor σ(B) was only observed upon entry into the stationary phase with glucose or glucose and malate as carbon sources.

An application of polymer-enhanced capillary transient isotachophoresis with an emissive boronic acid functionalized squarylium dye as an on-capillary labeling agent for gram-positive bacteria.

PubMed

Saito, Shingo; Maeda, Takeshi; Nakazumi, Hiroyuki; Colyer, Christa L

2013-01-01

In this paper, the characterization and application of the "PectI" (polymer-enhanced capillary transient isotachophoresis) technique for the separation and detection of same genus, gram-positive bacteria, Bacillus globigii (Bg) and Bacillus subtilis, is demonstrated by employing a boronic acid-functionalized squarylium dye (SQ-BA) as an on-capillary labeling agent, including the quantitative performance and applicability to crude samples. The effect of borate in the separation buffer was also investigated, which revealed that borate strongly affects the separation behavior of bacteria.

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 post...

Understanding the ontogeny and succession of Bacillus velezensis and B. subtilis subsp. subtilis by focusing on kimchi fermentation.

PubMed

Cho, Min Seok; Jin, Yong Ju; Kang, Bo Kyoung; Park, Yu Kyoung; Kim, ChangKug; Park, Dong Suk

2018-05-04

Bacillus subtilis and B. velezensis are frequently isolated from various niches, including fermented foods, water, and soil. Within the Bacillus subtilis group, B. velezensis and B. subtilis subsp. subtilis have received significant attention as biological resources for biotechnology-associated industries. Nevertheless, radical solutions are urgently needed to identify microbes during their ecological succession to accurately confirm their action at the species or subspecies level in diverse environments, such as fermented materials. Thus, in this study, previously published genome data of the B. subtilis group were compared to exploit species- or subspecies-specific genes for use as improved qPCR targets to detect B. velezensis and B. subtilis subsp. subtilis in kimchi samples. In silico analyses of the selected genes and designed primer sequences, in conjunction with SYBR Green real-time PCR, confirmed the robustness of this newly developed assay. Consequently, this study will allow for new insights into the ontogeny and succession of B. velezensis and B. subtilis subsp. subtilis in various niches. Interestingly, in white kimchi without red pepper powder, neither B. subtilis subsp. subtilis nor B. velezensis was detected.

Self-sensing in Bacillus subtilis quorum-sensing systems

PubMed Central

Bareia, Tasneem; Pollak, Shaul; Eldar, Avigdor

2017-01-01

Bacterial cell-cell signaling, or quorum sensing, is characterized by the secretion and group-wide detection of small diffusible signal molecules called autoinducers. This mechanism allows cells to coordinate their behavior in a density-dependent manner. A quorum-sensing cell may directly respond to the autoinducers it produces in a cell-autonomous and quorum-independent manner, but the strength of such self-sensing effect and its impact on bacterial physiology are unclear. Here, we explored the existence and impact of self-sensing in the Bacillus subtilis ComQXP and Rap-Phr quorum-sensing systems. By comparing the quorum-sensing response of autoinducer-secreting and non-secreting cells in co-culture, we found that secreting cells consistently showed a stronger response than non-secreting cells. Combining genetic and quantitative analyses, we demonstrated this effect to be a direct result of self-sensing and ruled out an indirect regulatory effect of the autoinducer production genes on response sensitivity. In addition, self-sensing in the ComQXP system affected persistence to antibiotic treatment. Together, these findings indicate the existence of self-sensing in the two most common designs of quorum-sensing systems of Gram-positive bacteria. PMID:29038467

Visualization of tandem repeat mutagenesis in Bacillus subtilis.

PubMed

Dormeyer, Miriam; Lentes, Sabine; Ballin, Patrick; Wilkens, Markus; Klumpp, Stefan; Kohlheyer, Dietrich; Stannek, Lorena; Grünberger, Alexander; Commichau, Fabian M

2018-03-01

Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation. Copyright © 2018 Elsevier B.V. All rights reserved.

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).

Cell Envelope Stress Response in Cell Wall-Deficient L-Forms of Bacillus subtilis

PubMed Central

Wolf, Diana; Domínguez-Cuevas, Patricia; Daniel, Richard A.

2012-01-01

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). PMID:22964256

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.

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 on...

Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis.

PubMed

Ulvatne, Hilde; Samuelsen, Ørjan; Haukland, Hanne H; Krämer, Manuela; Vorland, Lars H

2004-08-15

Most antimicrobial peptides have an amphipathic, cationic structure, and an effect on the cytoplasmic membrane of susceptible bacteria has been postulated as the main mode of action. Other mechanisms have been reported, including inhibition of cellular functions by binding to DNA, RNA and proteins, and the inhibition of DNA and/or protein synthesis. Lactoferricin B (Lfcin B), a cationic peptide derived from bovine lactoferrin, exerts slow inhibitory and bactericidal activity and does not lyse susceptible bacteria, indicating a possible intracellular target. In the present study incorporation of radioactive precursors into DNA, RNA and proteins was used to demonstrate effects of Lfcin B on macromolecular synthesis in bacteria. In Escherichia coli UC 6782, Lfcin B induces an initial increase in protein and RNA synthesis and a decrease in DNA synthesis. After 10 min, the DNA-synthesis increases while protein and RNA-synthesis decreases significantly. In Bacillus subtilis, however, all synthesis of macromolecules is inhibited for at least 20 min. After 20 min RNA-synthesis increases. The results presented here show that Lfcin B at concentrations not sufficient to kill bacterial cells inhibits incorporation of radioactive precursors into macromolecules in both Gram-positive and Gram-negative bacteria.

Programmed Pathogen Sense and Destroy Circuits

DTIC Science & Technology

2009-02-18

detection and the peptide-mediated Com QS system of Bacillus subtilis for gram-positive detection. Together these two prototype sentinel circuits cover a...and E. coli. We are currently in the process of constructing receivers for a gram-positive pathogen, Bacillus subtilis . Gram-negative...QS signals. Figure 11: Gram positive QS systems. Agr QS of Staphylococcus aureus (left) and Com QS of Bacillus subtilis . Following the successful

Pirated Siderophores Promote Sporulation in Bacillus subtilis.

PubMed

Grandchamp, Gabrielle M; Caro, Lews; Shank, Elizabeth A

2017-05-15

In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis- produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA , for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilis IMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis The interaction is mediated by the E

Pirated Siderophores Promote Sporulation in Bacillus subtilis

PubMed Central

Grandchamp, Gabrielle M.; Caro, Lews

2017-01-01

ABSTRACT In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis. Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis-produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA, for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis. Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilis. IMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis. The interaction is

Bacillus subtilis strain specificity affects performance improvement in broilers.

PubMed

Rhayat, L; Jacquier, V; Brinch, K S; Nielsen, P; Nelson, A; Geraert, P-A; Devillard, E

2017-07-01

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

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...

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. [65...

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.

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.

Antibacterial and anticancer activity of ε-poly-L-lysine (ε-PL) produced by a marine Bacillus subtilis sp.

PubMed

El-Sersy, Nermeen A; Abdelwahab, Abeer E; Abouelkhiir, Samia S; Abou-Zeid, Dunja-Manal; Sabry, Soraya A

2012-10-01

A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using 16S rDNA sequence analysis. The bacterium produced a compound active against a number of gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS product is ε-poly-L-lysine. To achieve maximum production, Plackett-Burman experimental design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in optimized medium composed of g/l: (NH(4))(2) SO(4), 15; K(2)HPO(4), 0.3; KH(2)PO(4), 2; MgSO(4) · 7 H(2)O, 1; ZnSO(4) · 7 H(2)O, 0; FeSO(4) · 7 H(2)O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized culture condition, a product value of 76.3 mg/l could be obtained. According to available literature, this is the first announcement for the production of ε-poly-L-lysine (ε-PL) by a member of genus Bacillus. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cytoplasmic Acidification and the Benzoate Transcriptome in Bacillus subtilis

PubMed Central

Kitko, Ryan D.; Cleeton, Rebecca L.; Armentrout, Erin I.; Lee, Grace E.; Noguchi, Ken; Berkmen, Melanie B.; Jones, Brian D.; Slonczewski, Joan L.

2009-01-01

Background Bacillus subtilis encounters a wide range of environmental pH. The bacteria maintain cytoplasmic pH within a narrow range. Response to acid stress is a poorly understood function of external pH and of permeant acids that conduct protons into the cytoplasm. Methods and Principal Findings Cytoplasmic acidification and the benzoate transcriptome were observed in Bacillus subtilis. Cytoplasmic pH was measured with 4-s time resolution using GFPmut3b fluorimetry. Rapid external acidification (pH 7.5 to 6.0) acidified the B. subtilis cytoplasm, followed by partial recovery. Benzoate addition up to 60 mM at external pH 7 depressed cytoplasmic pH but left a transmembrane ΔpH permitting growth; this robust adaptation to benzoate exceeds that seen in E. coli. Cytoplasmic pH was depressed by 0.3 units during growth with 30 mM benzoate. The transcriptome of benzoate-adapted cells was determined by comparing 4,095 gene expression indices following growth at pH 7, +/− 30 mM benzoate. 164 ORFs showed ≥2-fold up-regulation by benzoate (30 mM benzoate/0 mM), and 102 ORFs showed ≥2-fold down-regulation. 42% of benzoate-dependent genes are regulated up or down, respectively, at pH 6 versus pH 7; they are candidates for cytoplasmic pH response. Acid-stress genes up-regulated by benzoate included drug resistance genes (yhbI, yhcA, yuxJ, ywoGH); an oligopeptide transporter (opp); glycine catabolism (gcvPA-PB); acetate degradation (acsA); dehydrogenases (ald, fdhD, serA, yrhEFG, yjgCD); the TCA cycle (citZ, icd, mdh, sucD); and oxidative stress (OYE-family yqjM, ohrB). Base-stress genes down-regulated by benzoate included malate metabolism (maeN), sporulation control (spo0M, spo0E), and the SigW alkali shock regulon. Cytoplasmic pH could mediate alkali-shock induction of SigW. Conclusions B. subtilis maintains partial pH homeostasis during growth, and withstands high concentrations of permeant acid stress, higher than for gram-negative neutralophile E. coli. The benzoate

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 strain...

Not so simple, not so subtle: the interspecies competition between Bacillus simplex and Bacillus subtilis and its impact on the evolution of biofilms

PubMed Central

Rosenberg, Gili; Steinberg, Nitai; Oppenheimer-Shaanan, Yaara; Olender, Tsvia; Doron, Shany; Ben-Ari, Julius; Sirota-Madi, Alexandra; Bloom-Ackermann, Zohar; Kolodkin-Gal, Ilana

2016-01-01

Bacillus subtilis biofilms have a fundamental role in shaping the soil ecosystem. During this process, they unavoidably interact with neighbour bacterial species. We studied the interspecies interactions between biofilms of the soil-residing bacteria B. subtilis and related Bacillus species. We found that proximity between the biofilms triggered recruitment of motile B. subtilis cells, which engulfed the competing Bacillus simplex colony. Upon interaction, B. subtilis secreted surfactin and cannibalism toxins, at concentrations that were inert to B. subtilis itself, which eliminated the B. simplex colony, as well as colonies of Bacillus toyonensis. Surfactin toxicity was correlated with the presence of short carbon-tail length isomers, and synergistic with the cannibalism toxins. Importantly, during biofilm development and interspecies interactions a subpopulation in B. subtilis biofilm lost its native plasmid, leading to increased virulence against the competing Bacillus species. Overall, these findings indicate that genetic programs and traits that have little effect on biofilm development when each species is grown in isolation have a dramatic impact when different bacterial species interact. PMID:28721238

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

... 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 Strain QST 713 Variant Soil... in or on all food commodities by including residues of Bacillus subtilis strain QST 713 variant soil...

Molecular mechanisms involved in Bacillus subtilis biofilm formation

PubMed Central

Mielich-Süss, Benjamin; Lopez, Daniel

2014-01-01

Summary Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil-dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programs of cellular specialization and cell-cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis. In particular, a special emphasis is placed on summarizing the most recent discoveries in the field and integrating them into the general view of these truly sophisticated microbial communities. PMID:24909922

Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon.

PubMed Central

Meijer, W J; de Boer, A J; van Tongeren, S; Venema, G; Bron, S

1995-01-01

A 3.1 kb fragment of the large (approximately 55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced. In contrast to the parental plasmid, derived minireplicons were unstably maintained. Using deletion analysis the fragment essential and sufficient for replication was delineated to 1.1 kb. This 1.1 kb fragment is located between two divergently transcribed genes, denoted orfA and orfB, neither of which is required for replication. orfA shows homology to the B.subtilis chromosomal genes rapA (spoOL, gsiA) and rapB (spoOP). The 1.1 kb fragment, which is characterized by the presence of several regions of dyad symmetry, contains no open reading frames of more than 85 codons and shows no similarity with other known plasmid replicons. The structural organization of the pLS20 minimal replicon is entirely different from that of typical rolling circle plasmids from Gram-positive bacteria. The pLS20 minireplicons replicate in polA5 and recA4 B.subtilis strains. Taken together, these results strongly suggest that pLS20 belongs to a new class of theta replicons. PMID:7667098

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

Microbiological efficacy of superheated steam. I. Communication: results with spores of Bacillus subtilis and Bacillus stearothermophilus and with spore earth.

PubMed

Spicher, G; Peters, J; Borchers, U

1999-02-01

For the spores of Bacillus subtilis and Bacillus stearothermophilus as well as for spore earth (acc. DIN 58,946 Part 4 of August 1982), the dependence of resistance on the superheating of the steam used to kill germs was determined. A material (glass fibre fleece) was used as the germ carrier which does not superheat on contact with steam. The temperature of the saturated steam was 100 degrees C (B. subtilis) and 120 degrees C (B. stearothermophilus and spore earth). The yardstick for the resistance of the spores or bioindicators was the exposure period of the saturated or superheated steam at which 50% of the treated test objects no longer showed any viable test germs. The spores of Bacillus subtilis were far more sensitive to superheating of steam and reacted far more than the spores of Bacillus stearothermophilus and the germs in the spore earth. When superheating by 4 Kelvin the spores of Bacillus subtilis were approximately 2.5 times more resistant than they were to saturated steam. The resistance of Bacillus stearothermophilus and spore earth was only slightly higher up to superheating by 10 Kelvin. The spores of Bacillus subtilis had the highest resistance during superheating by 29 Kelvin; they were 119 times more resistant than they were to saturated steam. The resistance maximum of the spores of Bacillus stearothermophilus was at an superheating by around 22 Kelvin. However, the spores were only 4.1 times more resistant than they were to saturated steam. When using steam to kill germs, we must expect superheated steam. This raises the question whether the spores of Bacillus stearothermophilus, with their weaker reaction to the superheating of steam, are suitable as test germs for sterilisation with steam in all cases.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zawadzka, A. M.; Kim, Y.; Maltseva, N

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 ironmore » 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.« less

Construction of probe of the plant growth-promoting bacteria Bacillus subtilis useful for fluorescence in situ hybridization.

PubMed

Posada, Luisa F; Alvarez, Javier C; Hu, Chia-Hui; de-Bashan, Luz E; Bashan, Yoav

2016-09-01

Strains of Bacillus subtilis are plant growth-promoting bacteria (PGPB) of many crops and are used as inoculants. PGPB colonization is an important trait for success of a PGPB on plants. A specific probe, based on the 16 s rRNA of Bacillus subtilis, was designed and evaluated to distinguishing, by fluorescence in situ hybridization (FISH), between this species and the closely related Bacillus amyloliquefaciens. The selected target for the probe was between nucleotides 465 and 483 of the gene, where three different nucleotides can be identified. The designed probe successfully hybridized with several strains of Bacillus subtilis, but failed to hybridize not only with B. amyloliquefaciens, but also with other strains such as Bacillus altitudinis, Bacillus cereus, Bacillus gibsonii, Bacillus megaterium, Bacillus pumilus; and with the external phylogenetic strains Azospirillum brasilense Cd, Micrococcus sp. and Paenibacillus sp. The results showed the specificity of this molecular probe for B. subtilis.

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. Copyright © 2015. Published by Elsevier B.V.

The potent antimicrobial properties of cell penetrating peptide-conjugated silver nanoparticles with excellent selectivity for gram-positive bacteria over erythrocytes.

PubMed

Liu, Lihong; Yang, Jun; Xie, Jianping; Luo, Zhentao; Jiang, Jiang; Yang, Yi Yan; Liu, Shaomin

2013-05-07

Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (gram-positive Bacillus subtilis, gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects.

Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain.

PubMed

Khochamit, Nalisa; Siripornadulsil, Surasak; Sukon, Peerapol; Siripornadulsil, Wilailak

2015-01-01

The antimicrobial activity and probiotic properties of Bacillus subtilis strain KKU213, isolated from local soil, were investigated. The cell-free supernatant (CFS) of a KKU213 culture containing crude bacteriocins exhibited inhibitory effects on Gram-positive bacteria, including Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, and Staphylococcus aureus. The antibacterial activity of the CFS precipitated with 40% ammonium sulfate (AS) remained even after treatment at 60 and 100 °C, at pH 4 and 10 and with proteolytic enzymes, detergents and heavy metals. When analyzed by SDS-PAGE and overlaid with the indicator strains B. cereus and S. aureus, the 40% AS precipitate exhibited inhibitory activity on proteins smaller than 10 kDa. However, proteins larger than 25 kDa and smaller than 10 kDa were still observed on a native protein gel. Purified subtilosin A was prepared by Amberlite XAD-16 bead extraction and HPLC and analyzed by Nano-LC-QTOF-MS. Its molecular mass was found to be 3.4 kDa, and it retained its antibacterial activity. These results are consistent with the detection of the anti-listerial subtilosin A gene of the sbo/alb cluster in the KKU213 strain, which is 100% identical to that of B. subtilis subsp. subtilis 168. In addition to stable and cyclic subtilosin A, a mixture of many extracellular antibacterial peptides was also detected in the KKU213 culture. The KKU213 strain produced extracellular amylase, cellulase, lipase and protease, is highly acid-resistant (pH 2) when cultured in inulin and promotes health and reduces infection of intestinally colonized broiler chickens. Therefore, we propose that bacteriocin-producing B. subtilis KKU213 could be used as a potential probiotic strain or protective culture. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

Fe-S cluster biogenesis in Gram-positive bacteria: SufU is a zinc-dependent sulfur transfer protein.

PubMed

Selbach, Bruna P; Chung, Alexander H; Scott, Aubrey D; George, Simon J; Cramer, Stephen P; Dos Santos, Patricia C

2014-01-14

The biosynthesis of Fe-S clusters in Bacillus subtilis and other Gram-positive bacteria is catalyzed by the SufCDSUB system. The first step in this pathway involves the sulfur mobilization from the free amino acid cysteine to a sulfur acceptor protein SufU via a PLP-dependent cysteine desulfurase SufS. In this reaction scheme, the formation of an enzyme S-covalent intermediate is followed by the binding of SufU. This event leads to the second half of the reaction where a deprotonated thiol of SufU promotes the nucleophilic attack onto the persulfide intermediate of SufS. Kinetic analysis combined with spectroscopic methods identified that the presence of a zinc atom tightly bound to SufU (Ka = 10(17) M(-1)) is crucial for its structural and catalytic competency. Fe-S cluster assembly experiments showed that despite the high degree of sequence and structural similarity to the ortholog enzyme IscU, the B. subtilis SufU does not act as a standard Fe-S cluster scaffold protein. The involvement of SufU as a dedicated agent of sulfur transfer, rather than as an assembly scaffold, in the biogenesis of Fe-S clusters in Gram-positive microbes indicates distinct strategies used by bacterial systems to assemble Fe-S clusters.

The putative hydrolase YycJ (WalJ) affects the coordination of cell division with DNA replication in Bacillus subtilis and may play a conserved role in cell wall metabolism.

PubMed

Biller, Steven J; Wayne, Kyle J; Winkler, Malcolm E; Burkholder, William F

2011-02-01

Bacteria must accurately replicate and segregate their genetic information to ensure the production of viable daughter cells. The high fidelity of chromosome partitioning is achieved through mechanisms that coordinate cell division with DNA replication. We report that YycJ (WalJ), a predicted member of the metallo-β-lactamase superfamily found in most low-G+C Gram-positive bacteria, contributes to the fidelity of cell division in Bacillus subtilis. B. subtilis ΔwalJ (ΔwalJ(Bsu)) mutants divide over unsegregated chromosomes more frequently than wild-type cells, and this phenotype is exacerbated when DNA replication is inhibited. Two lines of evidence suggest that WalJ(Bsu) and its ortholog in the Gram-positive pathogen Streptococcus pneumoniae, WalJ(Spn) (VicX), play a role in cell wall metabolism: (i) strains of B. subtilis and S. pneumoniae lacking walJ exhibit increased sensitivity to a narrow spectrum of cephalosporin antibiotics, and (ii) reducing the expression of a two-component system that regulates genes involved in cell wall metabolism, WalRK (YycFG), renders walJ essential for growth in B. subtilis, as observed previously with S. pneumoniae. Together, these results suggest that the enzymatic activity of WalJ directly or indirectly affects cell wall metabolism and is required for accurate coordination of cell division with DNA replication.

Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase

PubMed Central

Afonso, José P.; Chintakayala, Kiran; Suwannachart, Chatrudee; Sedelnikova, Svetlana; Giles, Kevin; Hoyes, John B.; Soultanas, Panos; Rafferty, John B.; Oldham, Neil J.

2013-01-01

The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ′ Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ′ complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ′ binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg2+ conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3. PMID:23525462

A Bacillus subtilis malate dehydrogenase gene.

PubMed Central

Jin, S; De Jesús-Berríos, M; Sonenshein, A L

1996-01-01

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation. PMID:8550482

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...

Expanding the Use of a Fluorogenic Method to Determine Activity and Mode of Action of Bacillus thuringiensis Bacteriocins Against Gram-Positive and Gram-Negative Bacteria

PubMed Central

de la Fuente-Salcido, Norma M.; Barboza-Corona, J. Eleazar; Espino Monzón, A. N.; Pacheco Cano, R. D.; Balagurusamy, N.; Bideshi, Dennis K.; Salcedo-Hernández, Rubén

2012-01-01

Previously we described a rapid fluorogenic method to measure the activity of five bacteriocins produced by Mexican strains of Bacillus thuringiensis against B. cereus 183. Here we standardize this method to efficiently determine the activity of bacteriocins against both Gram-positive and Gram-negative bacteria. It was determined that the crucial parameter required to obtain reproducible results was the number of cells used in the assay, that is, ~4 × 108 cell/mL and ~7 × 108 cell/mL, respectively, for target Gram-positive and Gram-negative bacteria. Comparative analyses of the fluorogenic and traditional well-diffusion assays showed correlation coefficients of 0.88 to 0.99 and 0.83 to 0.99, respectively, for Gram-positive and Gram-negative bacteria. The fluorogenic method demonstrated that the five bacteriocins of B. thuringiensis have bacteriolytic and bacteriostatic activities against all microorganisms tested, including clinically significant bacteria such as Listeria monocytogenes, Proteus vulgaris, and Shigella flexneri reported previously to be resistant to the antimicrobials as determined using the well-diffusion protocol. These results demonstrate that the fluorogenic assay is a more sensitive, reliable, and rapid method when compared with the well-diffusion method and can easily be adapted in screening protocols for bacteriocin production by other microorganisms. PMID:22919330

Sequence-specific sup 1 H NMR resonance assignments of Bacillus subtilis HPr: Use of spectra obtained from mutants to resolve spectral overlap

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wittekind, M.; Klevit, R.E.; Reizer, J.

1990-08-07

On the basis of an analysis of two-dimensional {sup 1}H NMR spectra, the complete sequence-specific {sup 1}H NMR assignments are presented for the phosphocarrier protein HPr from the Gram-positive bacterium Bacillus subtilis. During the assignment procedure, extensive use was made of spectra obtained from point mutants of HPr in order to resolve spectral overlap and to provide verification of assignments. Regions of regular secondary structure were identified by characteristic patterns of sequential backbone proton NOEs and slowly exchanging amide protons. B subtilis HPr contains four {beta}-strands that form a single antiparallel {beta}-sheet and two well-defined {alpha}-helices. There are two stretchesmore » of extended backbone structure, one of which contains the active site His{sub 15}. The overall fold of the protein is very similar to that of Escherichia coli HPr determined by NMR studies.« less

The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network.

PubMed

Radeck, Jara; Fritz, Georg; Mascher, Thorsten

2017-02-01

The cell envelope stress response (CESR) encompasses all regulatory events that enable a cell to protect the integrity of its envelope, an essential structure of any bacterial cell. The underlying signaling network is particularly well understood in the Gram-positive model organism Bacillus subtilis. It consists of a number of two-component systems (2CS) and extracytoplasmic function σ factors that together regulate the production of both specific resistance determinants and general mechanisms to protect the envelope against antimicrobial peptides targeting the biogenesis of the cell wall. Here, we summarize the current picture of the B. subtilis CESR network, from the initial identification of the corresponding signaling devices to unraveling their interdependence and the underlying regulatory hierarchy within the network. In the course of detailed mechanistic studies, a number of novel signaling features could be described for the 2CSs involved in mediating CESR. This includes a novel class of so-called intramembrane-sensing histidine kinases (IM-HKs), which-instead of acting as stress sensors themselves-are activated via interprotein signal transfer. Some of these IM-HKs are involved in sensing the flux of antibiotic resistance transporters, a unique mechanism of responding to extracellular antibiotic challenge.

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed Central

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-01-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus. PMID:9097439

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-04-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Immobilization of Cellulase from Bacillus subtilis UniMAP-KB01 on Multi-walled Carbon Nanotubes for Biofuel Production

NASA Astrophysics Data System (ADS)

Naresh, Sandrasekaran; Hoong Shuit, Siew; Kunasundari, Balakrishnan; Hoo Peng, Yong; Qi, Hwa Ng; Teoh, Yi Peng

2018-03-01

Bacillus subtilis UniMAP-KB01, a cellulase producer was isolated from Malaysian mangrove soil. Through morphological identification it was observed that the B. subtilis appears to be in rod shaped and identified as a gram positive bacterium. Growth profile of isolated B. subtilis was established by measuring optical density (OD) at 600 nm for every 1 hour intervals. Polymath software was employed to plot the growth profile and the non-linear plot established gave the precision value of linear regression, R2 of 0.9602, root mean square deviation (RMSD) of 0.0176 and variance of 0.0025. The hydrolysis capacity testing revealed the cellulolytic index of 2.83 ± 0.46 after stained with Gram’s Iodine. The harvested crude enzyme after 24 hours incubation in carboxymethylcellulose (CMC) broth at 45°C and 100 RPM, was tested for enzyme activity. Through Filter Paper Assay (FPA), the cellulase activity was calculated to be 0.05 U/mL. The hydrolysis capacity testing and FPA shown an acceptable value for thermophilic bacterial enzyme activity. Thus, this isolated strain reasoned to be potential for producing thermostable cellulase which will be immobilized onto multi-walled carbon nanotubes and the cellulolytic activity will be characterized for biofuel production.

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

DTIC Science & Technology

2012-11-01

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...gerP, Bacillus subtilis , dipicolinic acid Xuan Y. Butzin, Anthony J. Troiano, William H. Coleman, Keren K. Griffiths, Christopher J. Doona, Florence E

Screen for agents that induce autolysis in Bacillus subtilis.

PubMed

Lacriola, Christopher J; Falk, Shaun P; Weisblum, Bernard

2013-01-01

The growing prevalence of antibiotic-resistant infections underscores the need to discover new antibiotics and to use them with maximum effectiveness. In response to these needs, we describe a screening protocol for the discovery of autolysis-inducing agents that uses two Bacillus subtilis reporter strains, SH-536 and BAU-102. To screen chemical libraries, autolysis-inducing agents were first identified with a BAU-102-based screen and then subdivided with SH-536 into two major groups: those that induce autolysis by their direct action on the cell membrane and those that induce autolysis secondary to inhibition of cell wall synthesis. SH-536 distinguishes between the two groups of autolysis-inducing agents by synthesizing and then releasing β-galactosidase (β-Gal) in late stationary phase at a time that cells have nearly stopped growing and are therefore tolerant of cell wall synthesis inhibitors. Four hits, named compound 2, compound 3, compound 5, and compound 24, obtained previously as inducers of autolysis by screening a 10,080-compound discovery library with BAU-102, were probed with SH-536 and found to release β-Gal, indicating that their mode of action was to permeabilize the B. subtilis cell membrane. The four primary hits inhibited growth in Staphylococcus aureus, Enterococcus faecium, Bacillus subtilis, and Bacillus anthracis, with MICs in the 12.5- to 25-μg/ml (20 to 60 μM) range. The four primary hits were further used to probe B. subtilis, and their action was partially characterized with respect to the dependence of induced autolysis on specific autolysins.

Screen for Agents That Induce Autolysis in Bacillus subtilis

PubMed Central

Lacriola, Christopher J.; Falk, Shaun P.

2013-01-01

The growing prevalence of antibiotic-resistant infections underscores the need to discover new antibiotics and to use them with maximum effectiveness. In response to these needs, we describe a screening protocol for the discovery of autolysis-inducing agents that uses two Bacillus subtilis reporter strains, SH-536 and BAU-102. To screen chemical libraries, autolysis-inducing agents were first identified with a BAU-102-based screen and then subdivided with SH-536 into two major groups: those that induce autolysis by their direct action on the cell membrane and those that induce autolysis secondary to inhibition of cell wall synthesis. SH-536 distinguishes between the two groups of autolysis-inducing agents by synthesizing and then releasing β-galactosidase (β-Gal) in late stationary phase at a time that cells have nearly stopped growing and are therefore tolerant of cell wall synthesis inhibitors. Four hits, named compound 2, compound 3, compound 5, and compound 24, obtained previously as inducers of autolysis by screening a 10,080-compound discovery library with BAU-102, were probed with SH-536 and found to release β-Gal, indicating that their mode of action was to permeabilize the B. subtilis cell membrane. The four primary hits inhibited growth in Staphylococcus aureus, Enterococcus faecium, Bacillus subtilis, and Bacillus anthracis, with MICs in the 12.5- to 25-μg/ml (20 to 60 μM) range. The four primary hits were further used to probe B. subtilis, and their action was partially characterized with respect to the dependence of induced autolysis on specific autolysins. PMID:23089762

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. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Bacillus subtilis as a Platform for Molecular Characterisation of Regulatory Mechanisms of Enterococcus faecalis Resistance against Cell Wall Antibiotics

PubMed Central

Fang, Chong; Stiegeler, Emanuel; Cook, Gregory M.; Mascher, Thorsten; Gebhard, Susanne

2014-01-01

To combat antibiotic resistance of Enterococcus faecalis, a better understanding of the molecular mechanisms, particularly of antibiotic detection, signal transduction and gene regulation is needed. Because molecular studies in this bacterium can be challenging, we aimed at exploiting the genetically highly tractable Gram-positive model organism Bacillus subtilis as a heterologous host. Two fundamentally different regulators of E. faecalis resistance against cell wall antibiotics, the bacitracin sensor BcrR and the vancomycin-sensing two-component system VanSB-VanRB, were produced in B. subtilis and their functions were monitored using target promoters fused to reporter genes (lacZ and luxABCDE). The bacitracin resistance system BcrR-BcrAB of E. faecalis was fully functional in B. subtilis, both regarding regulation of bcrAB expression and resistance mediated by the transporter BcrAB. Removal of intrinsic bacitracin resistance of B. subtilis increased the sensitivity of the system. The lacZ and luxABCDE reporters were found to both offer sensitive detection of promoter induction on solid media, which is useful for screening of large mutant libraries. The VanSB-VanRB system displayed a gradual dose-response behaviour to vancomycin, but only when produced at low levels in the cell. Taken together, our data show that B. subtilis is a well-suited host for the molecular characterization of regulatory systems controlling resistance against cell wall active compounds in E. faecalis. Importantly, B. subtilis facilitates the careful adjustment of expression levels and genetic background required for full functionality of the introduced regulators. PMID:24676422

Bacillus subtilis as a platform for molecular characterisation of regulatory mechanisms of Enterococcus faecalis resistance against cell wall antibiotics.

PubMed

Fang, Chong; Stiegeler, Emanuel; Cook, Gregory M; Mascher, Thorsten; Gebhard, Susanne

2014-01-01

To combat antibiotic resistance of Enterococcus faecalis, a better understanding of the molecular mechanisms, particularly of antibiotic detection, signal transduction and gene regulation is needed. Because molecular studies in this bacterium can be challenging, we aimed at exploiting the genetically highly tractable Gram-positive model organism Bacillus subtilis as a heterologous host. Two fundamentally different regulators of E. faecalis resistance against cell wall antibiotics, the bacitracin sensor BcrR and the vancomycin-sensing two-component system VanSB-VanRB, were produced in B. subtilis and their functions were monitored using target promoters fused to reporter genes (lacZ and luxABCDE). The bacitracin resistance system BcrR-BcrAB of E. faecalis was fully functional in B. subtilis, both regarding regulation of bcrAB expression and resistance mediated by the transporter BcrAB. Removal of intrinsic bacitracin resistance of B. subtilis increased the sensitivity of the system. The lacZ and luxABCDE reporters were found to both offer sensitive detection of promoter induction on solid media, which is useful for screening of large mutant libraries. The VanSB-VanRB system displayed a gradual dose-response behaviour to vancomycin, but only when produced at low levels in the cell. Taken together, our data show that B. subtilis is a well-suited host for the molecular characterization of regulatory systems controlling resistance against cell wall active compounds in E. faecalis. Importantly, B. subtilis facilitates the careful adjustment of expression levels and genetic background required for full functionality of the introduced regulators.

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

PubMed

Rosenberg, Jonathan; Müller, Peter; Lentes, Sabine; Thiele, Martin J; Zeigler, Daniel R; Tödter, Dominik; Paulus, Henry; Brantl, Sabine; Stülke, Jörg; Commichau, Fabian M

2016-09-01

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria. © 2016 John Wiley & Sons Ltd.

Streptococcus mutans: a new Gram-positive paradigm?

PubMed Central

Quivey, Robert G.; Koo, Hyun; Abranches, Jacqueline

2013-01-01

Despite the enormous contributions of the bacterial paradigms Escherichia coli and Bacillus subtilis to basic and applied research, it is well known that no single organism can be a perfect representative of all other species. However, given that some bacteria are difficult, or virtually impossible, to cultivate in the laboratory, that some are recalcitrant to genetic and molecular manipulation, and that others can be extremely dangerous to manipulate, the use of model organisms will continue to play an important role in the development of basic research. In particular, model organisms are very useful for providing a better understanding of the biology of closely related species. Here, we discuss how the lifestyle, the availability of suitable in vitro and in vivo systems, and a thorough understanding of the genetics, biochemistry and physiology of the dental pathogen Streptococcus mutans have greatly advanced our understanding of important areas in the field of bacteriology such as interspecies biofilms, competence development and stress responses. In this article, we provide an argument that places S. mutans, an organism that evolved in close association with the human host, as a novel Gram-positive model organism. PMID:23393147

A report on extensive lateral genetic reciprocation between arsenic resistant Bacillus subtilis and Bacillus pumilus strains analyzed using RAPD-PCR.

PubMed

Khowal, Sapna; Siddiqui, Md Zulquarnain; Ali, Shadab; Khan, Mohd Taha; Khan, Mather Ali; Naqvi, Samar Husain; Wajid, Saima

2017-02-01

The study involves isolation of arsenic resistant bacteria from soil samples. The characterization of bacteria isolates was based on 16S rRNA gene sequences. The phylogenetic consanguinity among isolates was studied employing rpoB and gltX gene sequence. RAPD-PCR technique was used to analyze genetic similarity between arsenic resistant isolates. In accordance with the results Bacillus subtilis and Bacillus pumilus strains may exhibit extensive horizontal gene transfer. Arsenic resistant potency in Bacillus sonorensis and high arsenite tolerance in Bacillus pumilus strains was identified. The RAPD-PCR primer OPO-02 amplified a 0.5kb DNA band specific to B. pumilus 3ZZZ strain and 0.75kb DNA band specific to B. subtilis 3PP. These unique DNA bands may have potential use as SCAR (Sequenced Characterized Amplified Region) molecular markers for identification of arsenic resistant B. pumilus and B. subtilis strains. Copyright © 2016 Elsevier Inc. All rights reserved.

Sticking together: building a biofilm the Bacillus subtilis way.

PubMed

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

2013-03-01

Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials.

PubMed

Rogers, J V; Choi, Y W; Richter, W R; Rudnicki, D C; Joseph, D W; Sabourin, C L K; Taylor, M L; Chang, J C S

2007-10-01

To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.

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.

Biodegradation of furfural by Bacillus subtilis strain DS3.

PubMed

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

2015-07-01

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

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

PubMed

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

2014-11-01

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

Genomics of Methylotrophy in Gram-Positive Methylamine-Utilizing Bacteria

PubMed Central

McTaggart, Tami L.; Beck, David A. C.; Setboonsarng, Usanisa; Shapiro, Nicole; Woyke, Tanja; Lidstrom, Mary E.; Kalyuzhnaya, Marina G.; Chistoserdova, Ludmila

2015-01-01

Gram-positive methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of methylotrophy pathways/enzymes within this group. However, genome-based knowledge of methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive methylotrophs limits our global understanding of methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of methylotrophy in Gram-positive methylotrophic bacteria. This study is a major contribution to the field of methylotrophy, aimed at closing the gap in the genomic knowledge of methylotrophy within this diverse group of bacteria. PMID:27682081

[Asymmetric biosynthesis of d-pseudoephedrine by recombinant Bacillus subtilis].

PubMed

Peng, Yanhong; Zhang, Liang; Ding, Zhongyang; Wang, Zhengxiang; Shi, Guiyang

2011-07-01

In order to successfully express the carbonyl reductase gene mldh in Bacillus subtilis and complete coenzyme regeneration by B. subtilis glucose dehydrogenase, the promoter PrpsD and the terminator TrpsD from B. subtilis rpsD gene were used as the expression cassette to be a recombinant plasmid pHY300plk-PrpsD-TrpsD. After that, the carbonyl reductase gene mldh was inserted into the previous plasmid and a plasmid pHY300plk-PrpsD-mldh-TrpsD was achieved, followed by transformed into B. subtilis Wb600 to obtain a recombinant B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD). Subsequently, the results for whole-cell biotransformation from recombinant B. subtilis showed that it could be used to catalyze MAK (1-phenyl- 1-keto-2-methylaminopropane) to d-pseudoephedrine in the presence of glucose. The yield of d-pseudoephedrine could be up to 97.5 mg/L and the conversion rate of MAK was 24.1%. This study indicates the possibility of biotransformation production of d-pseudoephedrine from recombinant B. subtilis.

A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

PubMed

Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

2017-01-01

Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

Antimicrobial gageomacrolactins characterized from the fermentation of the marine-derived bacterium Bacillus subtilis under optimum growth conditions.

PubMed

Tareq, Fakir Shahidullah; Kim, Ji Hye; Lee, Min Ah; Lee, Hyi-Seung; Lee, Jong-Seok; Lee, Yeon-Ju; Shin, Hee Jae

2013-04-10

Marine bacteria are a potential source of structurally diversified bioactive secondary metabolites that are not found in terrestrial sources. In our continuous effort to search for new antimicrobial agents from marine-derived bacteria, we isolated bacterial strain 109GGC020 from a marine sediment sample collected from Gageocho, Republic of Korea. The strain was identified as Bacillus subtilis based on a 16s rRNA sequence analysis. After a 7-day fermentation of the B. subtilis strain under optimum growth conditions three new and four known secondary metabolites were discovered using chromatographic procedures, and their biological activities were evaluated against both bacteria and crop-devastating fungi. The discovered metabolites were confirmed by extensive 2D NMR and high-resolution ESI-MS data analyses to have the structures of new macrolactin derivatives gageomacrolactins 1-3 and known macrolactins A (4), B (5), F (6), and W (7). The stereoconfigurations of 1-3 were assigned based on coupling constant values, chemical derivatization studies, and a literature review. The coupling constants were very crucial to determine the relative geometries of olefins in 1-3 because of overlap of the ¹H NMR signals. The NMR data of these compounds were recorded in different solvents to overcome this problem and obtain accurate coupling constant values. The new macrolactin derivatives 1-3 displayed good antibiotic properties against both Gram-positive (S. aureus, B. subtilis, and B. cereus) and Gram-negative (E. coli, S. typhi, and P. aeruginosa) bacteria with minimum inhibitory concentration (MIC) values of 0.02-0.05 μM. Additionally, the antifungal activities of 1-7 were evaluated against pathogenic fungi and found to inhibit mycelial growth of A. niger, B. cinerea, C. acutatum, C. albicans, and R. solani with MIC values of 0.04-0.3 μM, demonstrating that these compounds were good fungicides.

Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

PubMed

Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

2017-06-01

Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

PubMed

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

2011-09-01

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

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.

Extracellular signaling and multicellularity in Bacillus subtilis.

PubMed

Shank, Elizabeth Anne; Kolter, Roberto

2011-12-01

Bacillus subtilis regulates its ability to differentiate into distinct, co-existing cell types in response to extracellular signaling molecules produced either by itself, or present in its environment. The production of molecules by B. subtilis cells, as well as their response to these signals, is not uniform across the population. There is specificity and heterogeneity both within genetically identical populations as well as at the strain-level and species-level. This review will discuss how extracellular signaling compounds influence B. subtilis multicellularity with regard to matrix-producing cannibal differentiation, germination, and swarming behavior, as well as the specificity of the quorum-sensing peptides ComX and CSF. It will also highlight how imaging mass spectrometry can aid in identifying signaling compounds and contribute to our understanding of the functional relationship between such compounds and multicellular behavior. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacillus subtilis genome diversity.

PubMed

Earl, Ashlee M; Losick, Richard; Kolter, Roberto

2007-02-01

Microarray-based comparative genomic hybridization (M-CGH) is a powerful method for rapidly identifying regions of genome diversity among closely related organisms. We used M-CGH to examine the genome diversity of 17 strains belonging to the nonpathogenic species Bacillus subtilis. Our M-CGH results indicate that there is considerable genetic heterogeneity among members of this species; nearly one-third of Bsu168-specific genes exhibited variability, as measured by the microarray hybridization intensities. The variable loci include those encoding proteins involved in antibiotic production, cell wall synthesis, sporulation, and germination. The diversity in these genes may reflect this organism's ability to survive in diverse natural settings.

Screening beneficial rhizobacteria from Spartina maritima for phytoremediation of metal polluted salt marshes: comparison of gram-positive and gram-negative strains.

PubMed

Paredes-Páliz, Karina I; Caviedes, Miguel A; Doukkali, Bouchra; Mateos-Naranjo, Enrique; Rodríguez-Llorente, Ignacio D; Pajuelo, Eloísa

2016-10-01

The aim of our work was the isolation and characterization of bacteria from the rhizosphere of Spartina maritima in the metal contaminated Odiel estuary (Huelva, SW Spain). From 25 strains, 84 % were identified as gram-positive, particularly Staphylococcus and Bacillus. Gram-negative bacteria were represented by Pantoea and Salmonella. Salt and heavy metal tolerance, metal bioabsorption, plant growth promoting (PGP) properties, and biofilm formation were investigated in the bacterial collection. Despite the higher abundance of gram-positive bacteria, gram-negative isolates displayed higher tolerance toward metal(loid)s (As, Cu, Zn, and Pb) and greater metal biosorption, as deduced from ICP-OES and SEM-EDX analyses. Besides, they exhibited better PGP properties, which were retained in the presence of metals and the ability to form biofilms. Gram-negative strains Pantoea agglomerans RSO6 and RSO7, together with gram-positive Bacillus aryabhattai RSO25, were selected for a bacterial consortium aimed to inoculate S. maritima plants in metal polluted estuaries for phytoremediation purposes.

Antibacterial activity of oregano (Origanum vulgare Linn.) against gram positive bacteria.

PubMed

Saeed, Sabahat; Tariq, Perween

2009-10-01

The present investigation is focused on antibacterial potential of infusion, decoction and essential oil of oregano (Origanum vulgare) against 111 Gram-positive bacterial isolates belonging to 23 different species related to 3 genera. Infusion and essential oil exhibited antibacterial activity against Staphylococcus saprophyticus, S. aureus, Micrococcus roseus, M. kristinae, M. nishinomiyaensis, M. lylae, M. luteus, M. sedentarius, M. varians, Bacillus megaterium, B. thuringiensis, B. alvei, B. circulans, B. brevis, B. coagulans, B. pumilus, B. laterosporus, B. polymyxa, B. macerans, B. subtilis, B. firmus, B. cereus and B. lichiniformis. The infusion exhibited maximum activity against B. laterosporus (17.5 mm mean zone of inhibition+/-1.5 Standard deviation) followed by B. polymyxa (17.0 mm+/-2.0 SD) and essential oil of oregano exhibited maximum activity against S. saprophyticus (16.8 mm+/-1.8 SD) followed by B. circulans (14.5 mm+/-0.5 SD). While all these tested isolates were found resistant to decoction of oregano.

The difference in in vivo sensitivity between Bacillus licheniformis PerR and Bacillus subtilis PerR is due to the different cellular environments.

PubMed

Kim, Jung-Hoon; Won, Young-Bin; Ji, Chang-Jun; Yang, Yoon-Mo; Ryu, Su-Hyun; Ju, Shin-Yeong; Kwon, Yumi; Lee, Yeh-Eun; Lee, Jin-Won

2017-02-26

PerR, a member of Fur family of metal-dependent regulators, is a major peroxide sensor in many Gram positive bacteria, and controls the expression of genes involved in peroxide resistance. Bacillus licheniformis, a close relative to the well-studied model organism Bacillus subtilis, contains three PerR-like proteins (PerR BL , PerR2 and PerR3) in addition to Fur and Zur. In the present study, we characterized the role of PerR BL in B. licheniformis. In vitro and in vivo studies indicate that PerR BL , like PerR BS , uses either Fe 2+ or Mn 2+ as a corepressor and only the Fe 2+ -bound form of PerR BL senses low levels of H 2 O 2 by iron-mediated histidine oxidation. Interestingly, regardless of the difference in H 2 O 2 sensitivity, if any, between PerR BL and PerR BS , B. licheniformis expressing PerR BL or PerR BS could sense lower levels of H 2 O 2 and was more sensitive to H 2 O 2 than B. subtilis expressing PerR BL or PerR BS . This result suggests that the differences in cellular milieu between B. subtilis and B. licheniformis, rather than the intrinsic differences in PerR BS and PerR BL per se, affect the H 2 O 2 sensing ability of PerR inside the cell and the H 2 O 2 resistance of cell. In contrast, B. licheniformis and B. subtilis expressing Staphylococcus aureus PerR (PerR SA ), which is more sensitive to H 2 O 2 than PerR BL and PerR BS , were more resistant to H 2 O 2 than those expressing either PerR BL or PerR BS . This result indicates that the sufficient difference in H 2 O 2 susceptibility of PerR proteins can override the difference in cellular environment and affect the resistance of cell to H 2 O 2 . Copyright © 2017 Elsevier Inc. All rights reserved.

Whole-genome sequencing of Bacillus subtilis XF-1 reveals mechanisms for biological control and multiple beneficial properties in plants.

PubMed

Guo, Shengye; Li, Xingyu; He, Pengfei; Ho, Honhing; Wu, Yixin; He, Yueqiu

2015-06-01

Bacillus subtilis XF-1 is a gram-positive, plant-associated bacterium that stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. In particular, it is especially highly efficient at controlling the clubroot disease of cruciferous crops. Its 4,061,186-bp genome contains an estimated 3853 protein-coding sequences and the 1155 genes of XF-1 are present in most genome-sequenced Bacillus strains: 3757 genes in B. subtilis 168, and 1164 in B. amyloliquefaciens FZB42. Analysis using the Cluster of Orthologous Groups database of proteins shows that 60 genes control bacterial mobility, 221 genes are related to cell wall and membrane biosynthesis, and more than 112 are genes associated with secondary metabolites. In addition, the genes contributed to the strain's plant colonization, bio-control and stimulation of plant growth. Sequencing of the genome is a fundamental step for developing a desired strain to serve as an efficient biological control agent and plant growth stimulator. Similar to other members of the taxon, XF-1 has a genome that contains giant gene clusters for the non-ribosomal synthesis of antifungal lipopeptides (surfactin and fengycin), the polyketides (macrolactin and bacillaene), the siderophore bacillibactin, and the dipeptide bacilysin. There are two synthesis pathways for volatile growth-promoting compounds. The expression of biosynthesized antibiotic peptides in XF-1 was revealed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Comprehensive Analysis of Temporal Alterations in Cellular Proteome of Bacillus subtilis under Curcumin Treatment

PubMed Central

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. PMID:25874956

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.

Heme inhibition of ferrisiderophore reductase in Bacillus subtilis.

PubMed

Lodge, J S; Gaines, C G; Arceneaux, J E; Byers, B R

1982-11-01

Heme was a noncompetitive inhibitor (apparent K(i) and K'(i) = 0.043 mM) of a ferrisiderophore reductase purified from Bacillus subtilis; protoporphyrin IX had no effect. The cellular level of heme may partly regulate the function of this reductase to yield a controlled flow of iron into metabolism.

Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator.

PubMed

Rogers, J V; Sabourin, C L K; Choi, Y W; Richter, W R; Rudnicki, D C; Riggs, K B; Taylor, M L; Chang, J

2005-01-01

To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Bacillus anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to > or =1000 ppm hydrogen peroxide gas for 20 min. Hydrogen peroxide exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials except G. stearothermophilus on industrial carpet. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with both surrogates. The effectiveness of gaseous hydrogen peroxide on the growth of biological indicators and spore strips was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days postexposure, decontaminated biological indicators and spore strips exhibited no growth, while the nondecontaminated samples displayed growth. Significant differences in decontamination efficacy of hydrogen peroxide gas on porous and nonporous surfaces were observed when comparing the mean log reduction in B. anthracis spores with B. subtilis and G. stearothermophilus spores. These results provide comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using hydrogen peroxide gas.

Bacillus subtilis vegetative isolate surviving chlorine dioxide exposure: an elusive mechanism of resistance.

PubMed

Martin, D J H; Wesgate, R L; Denyer, S P; McDonnell, G; Maillard, J-Y

2015-12-01

Oxidizing agents such as chlorine dioxide are widely used microbicides, including for disinfection of medical equipment. We isolated a Bacillus subtilis isolate from a washer-disinfector whose vegetative form demonstrated unique resistance to chlorine dioxide (0·03%) and hydrogen peroxide (7·5%). The aim of this study was to understand the mechanisms of resistance expressed by this isolate. A range of resistance mechanisms were investigated in the B. subtilis isolate and a reference B. subtilis strain (ATCC 6051) to include bacterial cell aggregation, the presence of profuse exopolysaccharide (EPS), and the expression of detoxification enzymes. The basis of resistance of the isolate to high concentrations of oxidizing agents was not linked to the presence of endospores. Although, the presence of EPS, aggregation and expression of detoxification enzymes may play a role in bacterial survival to low concentrations of chlorine dioxide, it is unlikely that the mechanisms helped tested to survive the bactericidal effect of higher oxidizer concentrations. Overall, the mechanisms conferring resistance to chlorine dioxide and hydrogen peroxide remains elusive. Based on recent advances in the mode of action of oxidizing agents and notably hydrogen peroxide, we postulate that additional efficient intracellular mechanisms may be involved to explain significant resistance to in-use concentrations of commonly used high-level disinfectants. The isolation of a highly resistant vegetative Gram-positive bacterium to a highly reactive oxidizing agent is worrying. Understanding the mechanisms conferring such resistance is essential to effectively control such bacterial isolates. Here, we postulate that there are still mechanisms of bacterial resistance that have not been fully characterized. © 2015 The Authors published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.

Biosynthesis of o-succinylbenzoic acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the alpha-ketoglutarate dehydrogenase complex.

PubMed Central

Palaniappan, C; Taber, H; Meganathan, R

1994-01-01

The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity. PMID:8169214

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.

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

Production of mannanase from Bacillus Subtilis LBF-005 and its potential for manno-oligosaccharides production

NASA Astrophysics Data System (ADS)

Yopi, Rahmani, Nanik; Jannah, Alifah Mafatikhul; Nugraha, Irfan Pebi; Ramadana, Roni Masri

2017-11-01

Endo-β-1, 4-mannanase is the key enzymes for randomly hydrolyzing the β-1,4-linkages within the mannan backbone releasing manno-oligosaccharides (MOS). A marine bacterium of Bacillus subtilis LBF-005 was reported have ability to produce endo-type mannanase. The aims of this research were to compare commercial biomass Locust Bean Gum (LBG) and raw biomass contaning mannan as carbon source for mannanase production from Bacillus subtilis LBF-005, to analyze the optimum condition of mannanase production, and to find out the potential of the mannanase for MOS production. Bacillus subtilis LBF-005 was cultivated in Artificial Sea Water (ASW) medium contain NaCl and various mannan biomass as carbon source for mannanase production. The cells were grown in submerged fermentation. The maximum enzyme activity was obtained with porang potato as a substrate with concentration 1%, pH medium 8, and incubation temperature 50°C with an enzyme activity of 37.7 U/mL. The mainly MOS product released by crude mannanase produced by Bacillus subtilis LBF-005 were mannobiose (M2), mannotriose (M3), mannotetraose (M4), and mannopentaose (M5).

The Bacillus subtilis GntR family repressor YtrA responds to cell wall antibiotics.

PubMed

Salzberg, Letal I; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D

2011-10-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σ(M). Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses.

[Expression of N domain of chromogranin A in Bacillus subtilis and its antifungal activity].

PubMed

Li, Rui-Fang; Lou, Jin-Xian; Zhang, Tian-Yuan

2004-03-01

Chromogranin A (CGA) is a soluble protein existed in most secreted cells and neurons. It was recently found that the bovine CGA N terminal region has vasoinhibitory, antibacterial and antifungal activities. Since the need for effective antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for fungal infections, it becomes imperative to find antifungal compounds with low toxicity toward mammalian cells. To study the antifungal activity of CGA N terminal region, the DNA fragment encoding for the N terminal 1-76 amino acid sequence (CGA1-76) of human CGA was amplified by PCR technique. After DNA sequence analysis, the amplified DNA fragment was cloned into the Bacillus subtilis inducible and expression vector pSBPTQ constructed in this study and the resultant plasmid pSVTQ was then transformed into triple-protease deficient Bacillus subtilis strain DB403 competent cells. The transformants was screened on LB plates containing 10 microg/mL kanamycin. The positive transformant DB403 (pSVTQ) was grown on kanmycin containing 2 x MSR medium and sucrose was added to 2% final concentration for induction after 2h cultivation. The culture supernatant was used to run SDS-PAGE. The result of SDS-PAGE showed that the CGA1-76 was expressed by sucrose induction and the expressed product secreted into the medium with a yield of 5 mg/L. The expressed product reacts specifically with mouse anti CGA47-68 monoclonal antibody. The antifungal activity of the expressed product was examined by adding the culture supernatant to the fungal spore or Candida albican suspensions at appropriate proportion and found that the recombinant human CGA1-76 produced in Bacillus subtilis inhibits the growth of Fusarium sp. Alternaria sp. and Candida albican at the concerntration of 4 micromol/L. These results demonstrate that human CGA1-76 has expressed in Bacillus subtilis and the expressed product is immunogenic and has the antifungal activity.

Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6.

PubMed

Man, Li-Li; Xiang, Dian-Jun; Zhang, Chun-Lan

2018-02-06

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

Three phase partitioning and spectroscopic characterization of bioactive constituent from halophilic Bacillus subtilis EMB M15.

PubMed

Yadav, Neerja; Gupta, Munishwar Nath; Khare, Sunil K

2017-10-01

In the present study, a halophilic Bacillus subtilis subsp. spizizenii (NCBI GenBank accession number KX109607) was isolated from the Sambhar Salt Lake, Rajasthan India. This organism exhibited significance antibacterial and antifungal activity against Proteus vulgaris, Bacillus subtilis, Aspergillus niger, Rhizopus oligosporus and Penicillium chrysogenum respectively. The bioactive constituent responsible for it was extracted by three phase partitioning and purified by column chromatography. The purified compound was further characterized by FTIR-ATR, NMR and Mass spectrometry. The mass spectra show a molecular ion of m/z 301.14. The compound has very high antimicrobial activity showing 35mm zone of inhibition against Bacillus subtilis. Copyright © 2017 Elsevier Ltd. All rights reserved.

The comER Gene Plays an Important Role in Biofilm Formation and Sporulation in both Bacillus subtilis and Bacillus cereus.

PubMed

Yan, Fang; Yu, Yiyang; Wang, Luyao; Luo, Yuming; Guo, Jian-Hua; Chai, Yunrong

2016-01-01

Bacteria adopt alternative cell fates during development. In Bacillus subtilis, the transition from planktonic growth to biofilm formation and sporulation is controlled by a complex regulatory circuit, in which the most important event is activation of Spo0A, a transcription factor and a master regulator for genes involved in both biofilm formation and sporulation. In B. cereus, the regulatory pathway controlling biofilm formation and cell differentiation is much less clear. In this study, we show that a novel gene, comER, plays a significant role in biofilm formation as well as sporulation in both B. subtilis and B. cereus. Mutations in the comER gene result in defects in biofilm formation and a delay in spore formation in the two Bacillus species. Our evidence supports the idea that comER may be part of the regulatory circuit that controls Spo0A activation. comER likely acts upstream of sda, a gene encoding a small checkpoint protein for both sporulation and biofilm formation, by blocking the phosphor-relay and thereby Spo0A activation. In summary, our studies outlined a conserved, positive role for comER, a gene whose function was previously uncharacterized, in the regulation of biofilm formation and sporulation in the two Bacillus species.

The comER Gene Plays an Important Role in Biofilm Formation and Sporulation in both Bacillus subtilis and Bacillus cereus

PubMed Central

Yan, Fang; Yu, Yiyang; Wang, Luyao; Luo, Yuming; Guo, Jian-hua; Chai, Yunrong

2016-01-01

Bacteria adopt alternative cell fates during development. In Bacillus subtilis, the transition from planktonic growth to biofilm formation and sporulation is controlled by a complex regulatory circuit, in which the most important event is activation of Spo0A, a transcription factor and a master regulator for genes involved in both biofilm formation and sporulation. In B. cereus, the regulatory pathway controlling biofilm formation and cell differentiation is much less clear. In this study, we show that a novel gene, comER, plays a significant role in biofilm formation as well as sporulation in both B. subtilis and B. cereus. Mutations in the comER gene result in defects in biofilm formation and a delay in spore formation in the two Bacillus species. Our evidence supports the idea that comER may be part of the regulatory circuit that controls Spo0A activation. comER likely acts upstream of sda, a gene encoding a small checkpoint protein for both sporulation and biofilm formation, by blocking the phosphor-relay and thereby Spo0A activation. In summary, our studies outlined a conserved, positive role for comER, a gene whose function was previously uncharacterized, in the regulation of biofilm formation and sporulation in the two Bacillus species. PMID:27446060

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

PubMed

Bower, S; Perkins, J B; Yocum, R R; Howitt, C L; Rahaim, P; Pero, J

1996-07-01

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

PubMed Central

Bower, S; Perkins, J B; Yocum, R R; Howitt, C L; Rahaim, P; Pero, J

1996-01-01

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene. PMID:8763940

Cloning and strong expression of a Bacillus subtilis WL-3 mannanase gene in B. subtilis.

PubMed

Yoon, Ki-Hong; Lim, Byung-Lak

2007-10-01

A gene encoding the mannanase of Bacillus subtilis WL-3, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and the nucleotide sequence of a 2.7-kb DNA fragment containing the mannanase gene was subsequently determined. The mannanase gene, designated manA, consisted of 1,080 nucleotides encoding polypeptide of 360 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to glycosyl hydrolase family 26. The manA gene was strongly expressed in B. subtilis 168 by cloning the gene downstream of a strong B. subtilis promoter of plasmid pJ27Delta 88U. In flask cultures, the production of mannanase by recombinant B. subtilis 168 reached maximum levels of 300 units/ml and 450 units/ml in LB medium and LB medium containing 0.3% locust bean gum, respectively. Based on the zymogram of the mannanase, it was found that the mannanase produced by recombinant B. subtilis could be maintained stably without proteolytic degradation during the culture time.

Removing Bacillus subtilis from fermentation broth using alumina nanoparticles.

PubMed

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

2015-12-01

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

Complete Genome Sequence of the Poly-γ-Glutamate-Synthesizing Bacterium Bacillus subtilis Bs-115.

PubMed

Wang, Fengqing; Gong, Lijuan; Zhou, Lihong; Liang, Jinzhong

2018-04-19

Bacillus subtilis Bs-115 was isolated from the soil of a corn field in Yutai County, Jinan City, Shandong Province, People's Republic of China, and is characterized by the efficient synthesis of poly-γ-glutamate (γ-PGA), with corn saccharification liquid as the sole energy and carbon source during the process of γ-PGA formation. Here, we report the complete genome sequence of Bacillus subtilis Bs-115 and the genes associated with poly-γ-glutamate synthesis. Copyright © 2018 Wang et al.

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

Treesearch

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

2014-01-01

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

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.

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

PubMed Central

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

2011-01-01

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

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.

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

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

PubMed

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.

1-(2-aminophenyl)-1H-1,2,3-triazole-4-carboxylic acid: activity against Gram-positive and Gram-negative pathogens including Vibrio cholerae

NASA Astrophysics Data System (ADS)

Maji, Krishnendu; Haldar, Debasish

2017-10-01

We report a new synthetic aromatic ε-amino acid containing a triazole moiety with antimicrobial potential against Gram-positive, Gram-negative and pathogenic bacteria including Vibrio cholerae. Structure-property relationship studies revealed that all the functional groups are essential to enhance the antimicrobial activity. The 1-(2-aminophenyl)-1H-1,2,3-triazole-4-carboxylic acid was synthesized by click chemistry. From X-ray crystallography, the amino acid adopts a kink-like structure where the phenyl and triazole rings are perpendicular to each other and the amine and acid groups maintain an angle of 60°. The agar diffusion test shows that the amino acid has significant antibacterial activity. The liquid culture test exhibits that the minimum inhibitory concentration (MIC) value for Bacillus subtilis and Vibrio cholerae is 59.5 µg ml-1. FE-SEM experiments were performed to study the morphological changes of bacterial shape after treatment with compound 1. The antimicrobial activity of the amino acid was further studied by DNA binding and degradation study, protein binding, dye-binding assay and morphological analysis. Moreover, the amino acid does not have any harmful effect on eukaryotes.

Engineering genome-reduced Bacillus subtilis for acetoin production from xylose.

PubMed

Yan, Panpan; Wu, Yuanqing; Yang, Li; Wang, Zhiwen; Chen, Tao

2018-02-01

To investigate the capacity of a genome-reduced Bacillus subtilis strain as chassis cell for acetoin production from xylose. To endow the genome-reduced Bacillus subtilis strain BSK814 with the ability to utilize xylose, we inserted a native xyl operon into its genome and deleted the araR gene. The resulting strain BSK814A2 produced 2.94 g acetoin/l from 10 g xylose/l, which was 39% higher than control strain BSK19A2. The deletion of the bdhA and acoA genes further improved xylose utilization efficiency and increased acetoin production to 3.71 g/l in BSK814A4. Finally, BSK814A4 produced up to 23.3 g acetoin/l from 50 g xylose/l, with a yield of 0.46 g/g xylose. Both the titer and yield were 39% higher than those of control strain BSK19A4. As a chassis cell, genome-reduced B. subtilis showed significantly improved capacity for the production of the overflow product acetoin from xylose compared with wild-type strain.

Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis.

PubMed

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

2008-09-01

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

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

PubMed

Lyons, Nicholas A; Kolter, Roberto

2017-07-05

Kin discrimination systems are found in numerous communal contexts like multicellularity and are theorized to prevent exploitation of cooperative behaviors. The kin discrimination system in Bacillus subtilis differs from most other such systems because it excludes nonkin cells rather than including kin cells. Because nonkin are the target of the system, B. subtilis can potentially distinguish degrees of nonkin relatedness, not just kin versus nonkin. We examined this by testing a large strain collection of diverse Bacillus species against B. subtilis in different multicellular contexts. The effects of kin discrimination extend to nearby species, as the other subtilis clade species were treated with the same antagonism as nonkin. Species in the less-related pumilus clade started to display varied phenotypes but were mostly still discriminated against, while cereus clade members and beyond were no longer subject to kin discrimination. Seeking a reason why other species are perceived as antagonistic nonkin, we tested the ability of B. subtilis to steal communally produced surfactant from these species. We found that the species treated as nonkin were the only ones that made a surfactant that B. subtilis could utilize and that nonkin antagonism prevented such stealing when the two strains were mixed. The nonkin exclusion kin discrimination method thus allows effective protection of the cooperative behaviors prevalent in multicellularity while still permitting interactions with more distant species that are not a threat. IMPORTANCE Multicellular systems like bacterial biofilms and swarms rely on cooperative behaviors that could be undermined by exploitative invaders. Discriminating kin from nonkin is one way to help guard against such exploitation but has thus far been examined only intraspecifically, so the phylogenetic range of this important trait is unknown. We tested whether Bacillus subtilis treats other species as nonkin by testing a single strain against a

Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance.

PubMed

Jeyaram, Kumaraswamy; Romi, Wahengbam; Singh, Thangjam Anand; Adewumi, Gbenga Adedeji; Basanti, Khundrakpam; Oguntoyinbo, Folarin Anthony

2011-11-01

PCR amplification of 16S rRNA gene by universal primers followed by restriction fragment length polymorphism analysis using RsaI, CfoI and HinfI endonucleases, distinctly differentiated closely related Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus from Bacillus subtilis sensu stricto. This simple, economical, rapid and reliable protocol could be an alternative to misleading phenotype-based grouping of these closely related species. Copyright © 2011 Elsevier B.V. All rights reserved.

The Bacillus subtilis GntR Family Repressor YtrA Responds to Cell Wall Antibiotics▿§

PubMed Central

Salzberg, Letal I.; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D.

2011-01-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σM. Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses. PMID:21856850

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

USDA-ARS?s Scientific Manuscript database

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

[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.

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

PubMed Central

2012-01-01

Background Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. Conclusions The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available

Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis

PubMed Central

Stannek, Lorena; Gunka, Katrin; Care, Rachel A.; Gerth, Ulf; Commichau, Fabian M.

2015-01-01

The Gram-positive model bacterium Bacillus subtilis contains two glutamate dehydro genase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (gudBCR) in the laboratory strain 168 due to a perfect 18 bp-long direct repeat that renders the GudB enzyme inactive and unstable. Although constitutively expressed the GudBCR protein can hardly be detected in B. subtilis as it is rapidly degraded within stationary growth phase. Its high instability qualifies GudBCR as a model substrate for studying protein turnover in B. subtilis. Recently, we have developed a visual screen to monitor the GudBCR stability in the cell using a GFP-GudBCR fusion. Using fluorescent microscopy we found that the GFP protein is simultaneously degraded together with GudBCR. This allows us to analyze the stability of GudBCR in living cells. By combining the visual screen with a transposon mutagenesis approach we looked for mutants that show an increased fluorescence signal compared to the wild type indicating a stabilized GFP-GudBCR fusion. We observed, that disruption of the arginine kinase encoding gene mcsB upon transposon insertion leads to increased amounts of the GFP-GudBCR fusion in this mutant. Deletion of the cognate arginine phosphatase YwlE in contrast results in reduced levels of the GFP-GudBCR fusion. Recently, it was shown that the kinase McsB is involved in phosphorylation of GudBCR on arginine residues. Here we show that selected arginine-lysine point mutations of GudBCR exhibit no influence on degradation. The activity of McsB and YwlE, however, are crucial for the activation and inhibition, respectively, of a proteolytic machinery that efficiently degrades the unstable GudBCR protein in B. subtilis. PMID:25610436

High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris.

PubMed

Tang, Wei; Li, Zhezhe; Li, Chunhua; Yu, Xianhong; Wang, Fei; Wan, Xin; Wang, Yaping; Ma, Lixin

2016-06-01

Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B. subtilis strains 168 and zj016. The yield (3.81 g/l) and specific activity (788 U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83 mg of the purified enzyme (4089.72 U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B. subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B. subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B. subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B. subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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

2009-01-01

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

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. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Construction of a Food Grade Recombinant Bacillus subtilis Based on Replicative Plasmids with an Auxotrophic Marker for Biotransformation of d-Fructose to d-Allulose.

PubMed

He, Weiwei; Mu, Wanmeng; Jiang, Bo; Yan, Xin; Zhang, Tao

2016-04-27

A food grade recombinant Bacillus subtilis that produces d-psicose 3-epimerase (DPEase; EC 5.1.3.30) was constructed by transforming a replicative multicopy plasmid with a d-alanine racemase gene marker into B. subtilis 1A751 with the d-alanine racemase gene knocked out. The DPEase was expressed in B. subtilis without antibiotic resistance genes and without adding antibiotics during fermentation. Whole cells of the food grade recombinant B. subtilis were used to biotransform d-fructose to d-allulose. The two tandem promoters, including the HpaII and P43 promoters, increased expression levels compared to the use of one promoter, HpaII. For large-scale d-allulose production, the optimal enzyme dose was 40 enzyme activity units of dry cells per gram of d-fructose, which produced a 28.5% turnover yield in 60 min. The recombinant plasmid exhibited stability over 100 generations. This food grade recombinant B. subtilis may be used for large-scale d-allulose production in the food industry.

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis

PubMed Central

Nicolas, Pierre; Repoila, Francis; Bardowski, Jacek; Aymerich, Stéphane

2017-01-01

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks. PMID:28723971

CotA of Bacillus subtilis Is a Copper-Dependent Laccase

PubMed Central

Hullo, Marie-Françoise; Moszer, Ivan; Danchin, Antoine; Martin-Verstraete, Isabelle

2001-01-01

The spore coat protein CotA of Bacillus subtilis displays similarities with multicopper oxidases, including manganese oxidases and laccases. B. subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with ΔcotA spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light. PMID:11514528

Evaluation of dermal wound healing and in vitro antioxidant efficiency of Bacillus subtilis SPB1 biosurfactant.

PubMed

Zouari, Raida; Moalla-Rekik, Dorsaf; Sahnoun, Zouheir; Rebai, Tarek; Ellouze-Chaabouni, Semia; Ghribi-Aydi, Dhouha

2016-12-01

Lipopeptide microbial surfactants are endowed with unique surface properties as well as antimicrobial, anti-wrinkle, moisturizing and free radical scavenging activities. They were introduced safely in dermatological products, as long as they present low cytotoxicity against human cells. The present study was undertaken to evaluate the in vitro antioxidant activities and the wound healing potential of Bacillus subtilis SPB1 lipopeptide biosurfactant on excision wounds induced in experimental rats. The scavenging effect of Bacillus subtilis SPB1 biosurfactant on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 1mg/mL was 70.4% (IC 50 =0.55mg/mL). The biosurfactant produced by Bacillus subtilis SPB1 also showed good reducing power and significant effects in terms of the β-carotene test (IC 50 =2.26mg/mL) when compared to BHA as a reference standard. Moreover, an interesting ferrous ion chelating activity (80.32%) was found for SPB1 biosurfactant at 1mg/mL. Furthermore, the topical application of Bacillus subtilis SPB1 biosurfactant based gel on the wound site in a rat model every two days, increased significantly the percentage of wound closure over a period of 13days, when compared to the untreated and CICAFLORA™-treated groups. Wound healing effect of SPB1 biosurfactant based gel was confirmed by histological study. Biopsies treated with SPB1 lipopeptides showed wholly re-epithelialized wound with a perfect epidermal regeneration. The present study provides justification for the use of Bacillus subtilis SPB1 lipopeptide biosurfactant based gel for the treatment of normal and complicated wounds as well as skin diseases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Antioxidation, angiotensin converting enzyme inhibition activity, nattokinase, and antihypertension of Bacillus subtilis (natto)-fermented pigeon pea.

PubMed

Lee, Bao-Hong; Lai, Yi-Syuan; Wu, She-Ching

2015-12-01

Because of the high incidence of cardiovascular diseases in Asian countries, traditional fermented foods from Asia have been increasingly investigated for antiatherosclerotic effects. This study investigated the production of nattokinase, a serine fibrinolytic enzyme, in pigeon pea by Bacillus subtilis fermentation. B. subtilis 14714, B. subtilis 14715, B. subtilis 14716, and B. subtilis 14718 were employed to produce nattokinase. The highest nattokinase activity in pigeon pea was obtained using B. subtilis 14715 fermentation for 32 hours. In addition, the levels of antioxidants (phenolics and flavonoids) and angiotensin converting enzyme inhibitory activity were increased in B. subtilis 14715-fermented pigeon pea, compared with those in nonfermented pigeon pea. In an animal model, we found that both water extracts of pigeon pea (100 mg/kg body weight) and water extracts of B. subtilis-fermented pigeon pea (100 mg/kg body weight) significantly improved systolic blood pressure (21 mmHg) and diastolic blood pressure (30 mmHg) in spontaneously hypertensive rats. These results suggest that Bacillus-fermented pigeon pea has benefits for cardiovascular health and can be developed as a new dietary supplement or functional food that prevents hypertension. Copyright © 2015. Published by Elsevier B.V.

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.

PubMed

Guan, Chengran; Cui, Wenjing; Cheng, Jintao; Zhou, Li; Guo, Junling; Hu, Xu; Xiao, Guoping; Zhou, Zhemin

2015-09-21

Bacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA). The promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the -10 and -35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level. The auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression

Thermal Inactivation of Aerosolized Bacillus subtilis var. niger Spores

PubMed Central

Mullican, Charles L.; Buchanan, Lee M.; Hoffman, Robert K.

1971-01-01

A hot-air sterilizer capable of exposing airborne microorganisms to elevated temperatures with an almost instantaneous heating time was developed and evaluated. With this apparatus, aerosolized Bacillus subtilis var. niger spores were killed in about 0.02 sec when exposed to temperatures above 260 C. This is about 500 times faster than killing times reported by others. Extrapolation and comparison of data on the time and temperature required to klll B. subtilis var. niger spores on surfaces show that approximately the same killing time is required as is necessary for spores in air, if corrections are made for the heating time of the surface. PMID:5002138

High-and low-affinity binding sites for Cd on the bacterial cell walls of Bacillus subtilis and Shewanella oneidensis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mishra, B.; Boyanov, M.; Bunker, B. A.

2010-08-01

Bulk Cd adsorption isotherm experiments, thermodynamic equilibrium modeling, and Cd K edge EXAFS were used to constrain the mechanisms of proton and Cd adsorption to bacterial cells of the commonly occurring Gram-positive and Gram-negative bacteria, Bacillus subtilis and Shewanella oneidensis, respectively. Potentiometric titrations were used to characterize the functional group reactivity of the S. oneidensis cells, and we model the titration data using the same type of non-electrostatic surface complexation approach as was applied to titrations of B. subtilis suspensions by Fein et al. (2005). Similar to the results for B. subtilis, the S. oneidensis cells exhibit buffering behavior frommore » approximately pH 3-9 that requires the presence of four distinct sites, with pK{sub a} values of 3.3 {+-} 0.2, 4.8 {+-} 0.2, 6.7 {+-} 0.4, and 9.4 {+-} 0.5, and site concentrations of 8.9({+-}2.6) x 10{sup -5}, 1.3({+-}0.2) x 10{sup -4}, 5.9({+-}3.3) x 10{sup -5}, and 1.1({+-}0.6) x 10{sup -4} moles/g bacteria (wet mass), respectively. The bulk Cd isotherm adsorption data for both species, conducted at pH 5.9 as a function of Cd concentration at a fixed biomass concentration, were best modeled by reactions with a Cd:site stoichiometry of 1:1. EXAFS data were collected for both bacterial species as a function of Cd concentration at pH 5.9 and 10 g/L bacteria. The EXAFS results show that the same types of binding sites are responsible for Cd sorption to both bacterial species at all Cd loadings tested (1-200 ppm). Carboxyl sites are responsible for the binding at intermediate Cd loadings. Phosphoryl ligands are more important than carboxyl ligands for Cd binding at high Cd loadings. For the lowest Cd loadings studied here, a sulfhydryl site was found to dominate the bound Cd budgets for both species, in addition to the carboxyl and phosphoryl sites that dominate the higher loadings. The EXAFS results suggest that both Gram-positive and Gram-negative bacterial cell walls

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

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.

Characterization of biochemical properties of Bacillus subtilis RecQ helicase.

PubMed

Qin, Wei; Liu, Na-Nv; Wang, Lijun; Zhou, Min; Ren, Hua; Bugnard, Elisabeth; Liu, Jie-Lin; Zhang, Lin-Hu; Vendôme, Jeremie; Hu, Jin-Shan; Xi, Xu Guang

2014-12-01

RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg(2+)-dependent DNA helicase activity with a 3' → 5' polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3'-overhang but is inactive on blunt-ended dsDNA and 5'-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5'-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]'s possible functions in preserving genomic stability. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

PubMed Central

Saida, H.; Ytow, N.; Seki, H.

1998-01-01

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment. PMID:9464416

Effect of Bromouracil-containing Deoxyribonucleic Acid on Bacillus subtilis

PubMed Central

Gimlin, Dixie M.; Hardman, Sue D.; Kelley, Betty N.; Butler, Grace C.; Leach, Franklin R.

1966-01-01

Gimlin, Dixie M. (Oklahoma State University, Stillwater), Sue D. Hardman, Betty N. Kelley, Grace C. Butler, and Franklin R. Leach. Effect of bromouracil-containing deoxyribonucleic acid on Bacillus subtilis. J. Bacteriol. 92:366–374. 1966.—Replacement of one-half of the thymine with bromouracil in Bacillus subtilis transforming deoxyribonucleic acid (DNA) resulted in a slight decrease in transforming activity, but, when used at high concentrations, this DNA preparation inhibited cell growth. Acid-hydrolyzed DNA, or addition of equivalent concentrations of the free base bromouracil in a transforming mixture, was without effect on cell growth. Treatment of the DNA preparation with deoxyribonuclease completely destroyed transforming activity and killing effect, whereas treatments with ribonuclease and trypsin were without effect on either transformation or killing activity. Growth of competent B. subtilis cells in test tubes was inhibited by high concentrations of both normal and bromouracil-containing DNA, with the bromouracil-containing DNA being significantly more inhibitory. This type of inhibition was also reflected in the time of division of the cells. The inhibitory effect was not due to viscosity, or to mutagenicity. The time course of killing paralleled transformation, and competency was required. These results can be interpreted as being due to uptake of homologous but imperfect DNA (containing bromouracil instead of thymine) by means of the systems involved in transformation, followed by either integration (resulting in lethal transformation, activation of a defective, nonlytic but lethal prophage) or interference with the recombination mechanism. PMID:16562122

Logarithmic sensing in Bacillus subtilis aerotaxis.

PubMed

Menolascina, Filippo; Rusconi, Roberto; Fernandez, Vicente I; Smriga, Steven; Aminzare, Zahra; Sontag, Eduardo D; Stocker, Roman

2017-01-01

Aerotaxis, the directed migration along oxygen gradients, allows many microorganisms to locate favorable oxygen concentrations. Despite oxygen's fundamental role for life, even key aspects of aerotaxis remain poorly understood. In Bacillus subtilis, for example, there is conflicting evidence of whether migration occurs to the maximal oxygen concentration available or to an optimal intermediate one, and how aerotaxis can be maintained over a broad range of conditions. Using precisely controlled oxygen gradients in a microfluidic device, spanning the full spectrum of conditions from quasi-anoxic to oxic (60 n mol/l-1 m mol/l), we resolved B. subtilis' 'oxygen preference conundrum' by demonstrating consistent migration towards maximum oxygen concentrations ('monotonic aerotaxis'). Surprisingly, the strength of aerotaxis was largely unchanged over three decades in oxygen concentration (131 n mol/l-196 μ mol/l). We discovered that in this range B. subtilis responds to the logarithm of the oxygen concentration gradient, a rescaling strategy called 'log-sensing' that affords organisms high sensitivity over a wide range of conditions. In these experiments, high-throughput single-cell imaging yielded the best signal-to-noise ratio of any microbial taxis study to date, enabling the robust identification of the first mathematical model for aerotaxis among a broad class of alternative models. The model passed the stringent test of predicting the transient aerotactic response despite being developed on steady-state data, and quantitatively captures both monotonic aerotaxis and log-sensing. Taken together, these results shed new light on the oxygen-seeking capabilities of B. subtilis and provide a blueprint for the quantitative investigation of the many other forms of microbial taxis.

Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800

PubMed Central

2013-01-01

Background Nattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production. Results A gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65°C and pH 9. The nattokinase was stable at temperature up to 50°C and in pH range of 5–11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity

Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800.

PubMed

Nguyen, Thao Thi; Quyen, Thi Dinh; Le, Hoang Thanh

2013-09-10

Nattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production. A gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65 °C and pH 9. The nattokinase was stable at temperature up to 50 °C and in pH range of 5-11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of

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

PubMed

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

2016-11-29

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

Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens

PubMed Central

Powers, Matthew J.; Sanabria-Valentín, Edgardo; Bowers, Albert A.

2015-01-01

ABSTRACT Interspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain (phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria. IMPORTANCE Biofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that

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

PubMed Central

2017-01-01

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

The methionine salvage pathway in Bacillus subtilis

PubMed Central

Sekowska, Agnieszka; Danchin, Antoine

2002-01-01

Background Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR). Very little was known about MTR recycling for methionine salvage in Bacillus subtilis. Results Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling. Conclusions A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane). PMID:12022921

Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evaluation of its antibacterial activity.

PubMed

Velmurugan, Palanivel; Iydroose, Mahudunan; Mohideen, Mohmed Hanifa Abdul Kader; Mohan, Thankiah Selva; Cho, Min; Oh, Byung-Taek

2014-08-01

This study highlights the ability of nitrate-reducing Bacillus subtilis EWP-46 cell-free extract used for preparation of silver nanoparticles (AgNPs) by reduction of silver ions into nano silver. The production of AgNPs was optimized with several parameters such as hydrogen ion concentration, temperature, silver ion (Ag(+) ion) and time. The maximum AgNPs production was achieved at pH 10.0, temperature 60 °C, 1.0 mM Ag(+) ion and 720 min. The UV-Vis spectrum showed surface plasmon resonance peak at 420 nm, energy-dispersive X-ray spectroscopy (SEM-EDX) spectra showed the presence of element silver in pure form. Atomic force microscopy (AFM) and transmission electron microscopy images illustrated the nanoparticle size, shape, and average particle size ranging from 10 to 20 nm. Fourier transform infrared spectroscopy provided the evidence for the presence of biomolecules responsible for the reduction of silver ion, and X-ray diffraction analysis confirmed that the obtained nanoparticles were in crystalline form. SDS-PAGE was performed to identify the proteins and its molecular mass in the purified nitrate reductase from the cell-free extract. In addition, the minimum inhibitory concentration and minimum bactericidal concentration of AgNPs were investigated against gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus aureus) bacteria.

Characterization of high hydrostatic pressure-injured Bacillus subtilis cells.

PubMed

Inaoka, Takashi; Kimura, Keitarou; Morimatsu, Kazuya; Yamamoto, Kazutaka

2017-06-01

High hydrostatic pressure (HHP) affects various cellular processes. Using a sporulation-deficient Bacillus subtilis strain, we characterized the properties of vegetative cells subjected to HHP. When stationary-phase cells were exposed to 250 MPa of HHP for 10 min at 25 °C, approximately 50% of cells were viable, although they exhibited a prolonged growth lag. The HHP-injured cells autolyzed in the presence of NaCl or KCl (at concentrations ≥100 mM). Superoxide dismutase slightly protected the viability of HHP-treated cells, whereas vegetative catalases had no effect. Thus, unlike HHP-injured Escherichia coli, oxidative stress only slightly affected vegetative B. subtilis subjected to HHP.

SporeWeb: an interactive journey through the complete sporulation cycle of Bacillus subtilis.

PubMed

Eijlander, Robyn T; de Jong, Anne; Krawczyk, Antonina O; Holsappel, Siger; Kuipers, Oscar P

2014-01-01

Bacterial spores are a continuous problem for both food-based and health-related industries. Decades of scientific research dedicated towards understanding molecular and gene regulatory aspects of sporulation, spore germination and spore properties have resulted in a wealth of data and information. To facilitate obtaining a complete overview as well as new insights concerning this complex and tightly regulated process, we have developed a database-driven knowledge platform called SporeWeb (http://sporeweb.molgenrug.nl) that focuses on gene regulatory networks during sporulation in the Gram-positive bacterium Bacillus subtilis. Dynamic features allow the user to navigate through all stages of sporulation with review-like descriptions, schematic overviews on transcriptional regulation and detailed information on all regulators and the genes under their control. The Web site supports data acquisition on sporulation genes and their expression, regulon network interactions and direct links to other knowledge platforms or relevant literature. The information found on SporeWeb (including figures and tables) can and will be updated as new information becomes available in the literature. In this way, SporeWeb offers a novel, convenient and timely reference, an information source and a data acquisition tool that will aid in the general understanding of the dynamics of the complete sporulation cycle.

Hyperexpression of the gene for a Bacillus alpha-amylase in Bacillus subtilis cells: enzymatic properties and crystallization of the recombinant enzyme.

PubMed

Ikawa, K; Araki, H; Tsujino, Y; Hayashi, Y; Igarashi, K; Hatada, Y; Hagihara, H; Ozawa, T; Ozaki, K; Kobayashi, T; Ito, S

1998-09-01

We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).

RNA-seq analysis of antibiotic-producing Bacillus subtilis SC-8 in response to signal peptide PapR of Bacillus cereus.

PubMed

Yeo, In-Cheol; Lee, Nam Keun; Yang, Byung Wook; Hahm, Young Tae

2014-01-01

Bacillus subtilis SC-8 produces an antibiotic that has narrow antagonistic activity against bacteria in the Bacillus cereus group. In B. cereus group bacteria, peptide-activating PlcR (PapR) plays a significant role in regulating the transcription of virulence factors. When B. subtilis SC-8 and B. cereus are co-cultured, PapR is assumed to stimulate antibiotic production by B. subtilis SC-8. To better understand the effect of PapR on this interspecies interaction, the global transcriptome profile of B. subtilis SC-8 was analyzed in the presence of PapR. Significant changes were detected in 12.8 % of the total transcripts. Genes related to amino acid transport and metabolism (16.5 %) and transcription (15 %) were mainly upregulated, whereas genes involved in carbohydrate transport and metabolism (12.7 %) were markedly downregulated. The expression of genes related to transcription, including several transcriptional regulators and proteins involved in tRNA biosynthesis, was increased. The expression levels of genes associated with several transport systems, such as antibiotic, cobalt, and iron complex transporters, was also significantly altered. Among the downregulated genes were transcripts associated with spore formation, the subtilosin A gene cluster, and nitrogen metabolism.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sporulation during growth in a gut isolate of Bacillus subtilis.

PubMed

Serra, Cláudia R; Earl, Ashlee M; Barbosa, Teresa M; Kolter, Roberto; Henriques, Adriano O

2014-12-01

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Sporulation during Growth in a Gut Isolate of Bacillus subtilis

PubMed Central

Serra, Cláudia R.; Earl, Ashlee M.; Barbosa, Teresa M.; Kolter, Roberto

2014-01-01

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment. PMID:25225273

The sigma factors of Bacillus subtilis.

PubMed Central

Haldenwang, W G

1995-01-01

The specificity of DNA-dependent RNA polymerase for target promotes is largely due to the replaceable sigma subunit that it carries. Multiple sigma proteins, each conferring a unique promoter preference on RNA polymerase, are likely to be present in all bacteria; however, their abundance and diversity have been best characterized in Bacillus subtilis, the bacterium in which multiple sigma factors were first discovered. The 10 sigma factors thus far identified in B. subtilis directly contribute to the bacterium's ability to control gene expression. These proteins are not merely necessary for the expression of those operons whose promoters they recognize; in many instances, their appearance within the cell is sufficient to activate these operons. This review describes the discovery of each of the known B. subtilis sigma factors, their characteristics, the regulons they direct, and the complex restrictions placed on their synthesis and activities. These controls include the anticipated transcriptional regulation that modulates the expression of the sigma factor structural genes but, in the case of several of the B. subtilis sigma factors, go beyond this, adding novel posttranslational restraints on sigma factor activity. Two of the sigma factors (sigma E and sigma K) are, for example, synthesized as inactive precursor proteins. Their activities are kept in check by "pro-protein" sequences which are cleaved from the precursor molecules in response to intercellular cues. Other sigma factors (sigma B, sigma F, and sigma G) are inhibited by "anti-sigma factor" proteins that sequester them into complexes which block their ability to form RNA polymerase holoenzymes. The anti-sigma factors are, in turn, opposed by additional proteins which participate in the sigma factors' release. The devices used to control sigma factor activity in B, subtilis may prove to be as widespread as multiple sigma factors themselves, providing ways of coupling sigma factor activation to

Characterization of a bacteriocin-like substance produced from a novel isolated strain of Bacillus subtilis SLYY-3

NASA Astrophysics Data System (ADS)

Li, Junfeng; Li, Hongfang; Zhang, Yuanyuan; Duan, Xiaohui; Liu, Jie

2014-12-01

In the present research, the strain SLYY-3 was isolated from sediments of Jiaozhou Bay, Qingdao, China. The strain SLYY-3, which produced a bacteriocin-like substance (BLS), was characterized to be a strain of Bacillus subtillis by biochemical profiling and 16S rDNA sequence analysis. It is the first time to report that Bacillus subtilis from Jiaozhou Bay sediments could produce a BLS. The BLS of B. subtillis SLYY-3 exhibited strong inhibitory activity against gram-positive bacteria (including Staphylococcus aureus and B. subtillis) and some fungi (including Penicillium glaucum, Aspergillus niger and Aspergillus flavus). The antimicrobial activity was detected from culture in the exponential growth phase and reached its maximum when culture entered into stationary growth phase. It was thermo-tolerant even when being kept at 100°C for 60 min without losing any activity and stable over a wide pH range from 1.0 to 12.0 while being inactivated by proteolytic enzyme and trypsin, indicating the proteinaceous nature of the BLS. The BLS was purified by precipitation with hydrochloric acid (HCl) and gel filteration (Sephadex G-100). SDS-PAGE analysis of the extracellular peptides of SLYY-3 revealed a bacteriocin-like protein with a molecular mass of 66 kDa. Altogether, these characteristics indicate the potential of the BLS for food industry as a protection against pathogenic and spoilage microorganisms.

Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A.

PubMed

Sánchez, Marta; Prim, Núria; Rández-Gil, Francisca; Pastor, F I Javier; Diaz, Pilar

2002-05-05

Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive. Copyright 2002 Wiley Periodicals, Inc.

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.

The essential nature of YqfG, a YbeY homologue required for 3' maturation of Bacillus subtilis 16S ribosomal RNA is suppressed by deletion of RNase R.

PubMed

Baumgardt, Kathrin; Gilet, Laetitia; Figaro, Sabine; Condon, Ciarán

2018-06-05

Ribosomal RNAs are processed from primary transcripts containing 16S, 23S and 5S rRNAs in most bacteria. Maturation generally occurs in a two-step process, consisting of a first crude separation of the major species by RNase III during transcription, followed by precise trimming of 5' and 3' extensions on each species upon accurate completion of subunit assembly. The various endo- and exoribonucleases involved in the final processing reactions are strikingly different in Escherichia coli and Bacillus subtilis, the two best studied representatives of Gram-negative and Gram-positive bacteria, respectively. Here, we show that the one exception to this rule is the protein involved in the maturation of the 3' end of 16S rRNA. Cells depleted for the essential B. subtilis YqfG protein, a homologue of E. coli YbeY, specifically accumulate 16S rRNA precursors bearing 3' extensions. Remarkably, the essential nature of YqfG can be suppressed by deleting the ribosomal RNA degrading enzyme RNase R, i.e. a ΔyqfG Δrnr mutant is viable. Our data suggest that 70S ribosomes containing 30S subunits with 3' extensions of 16S rRNA are functional to a degree, but become substrates for degradation by RNase R and are eliminated.

Global transcriptional responses of Bacillus subtilis to xenocoumacin 1.

PubMed

Zhou, T; Zeng, H; Qiu, D; Yang, X; Wang, B; Chen, M; Guo, L; Wang, S

2011-09-01

To determine the global transcriptional response of Bacillus subtilis to an antimicrobial agent, xenocoumacin 1 (Xcn1). Subinhibitory concentration of Xcn1 applied to B. subtilis was measured according to Hutter's method for determining optimal concentrations. cDNA microarray technology was used to study the global transcriptional response of B. subtilis to Xcn1. Real-time RT-PCR was employed to verify alterations in the transcript levels of six genes. The subinhibitory concentration was determined to be 1 μg ml(-1). The microarray data demonstrated that Xcn1 treatment of B. subtilis led to more than a 2.0-fold up-regulation of 480 genes and more than a 2.0-fold down-regulation of 479 genes (q ≤ 0.05). The transcriptional responses of B. subtilis to Xcn1 were determined, and several processes were affected by Xcn1. Additionally, cluster analysis of gene expression profiles after treatment with Xcn1 or 37 previously studied antibiotics indicated that Xcn1 has similar mechanisms of action to protein synthesis inhibitors. These microarray data showed alterations of gene expression in B. subtilis after exposure to Xcn1. From the results, we identified various processes affected by Xcn1. This study provides a whole-genome perspective to elucidate the action of Xcn1 as a potential antimicrobial agent. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Cannibalism enhances biofilm development in Bacillus subtilis.

PubMed

López, Daniel; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2009-11-01

Cannibalism is a mechanism to delay sporulation in Bacillus subtilis. Cannibal cells express the skf and sdp toxin systems to lyse a fraction of their sensitive siblings. The lysed cells release nutrients that serve to feed the community, effectively delaying spore formation. Here we provide evidence that the subpopulation of cells that differentiates into cannibals is the same subpopulation that produces the extracellular matrix that holds cells together in biofilms. Cannibalism and matrix formation are both triggered in response to the signalling molecule surfactin. Nutrients released by the cannibalized cells are preferentially used by matrix-producing cells, as they are the only cells expressing resistance to the Skf and Sdp toxins. As a result this subpopulation increases in number and matrix production is enhanced when cannibalism toxins are produced. The cannibal/matrix-producing subpopulation is also generated in response to antimicrobials produced by other microorganisms and may thus constitute a defense mechanism to protect B. subtilis from the action of antibiotics in natural settings.

Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.

PubMed

Meyer, Frederik M; Jules, Matthieu; Mehne, Felix M P; Le Coq, Dominique; Landmann, Jens J; Görke, Boris; Aymerich, Stéphane; Stülke, Jörg

2011-12-01

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

Identification of Bacillus spp. from Bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization.

PubMed

Ouoba, L I I; Parkouda, C; Diawara, B; Scotti, C; Varnam, A H

2008-01-01

To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. ITS-PCR allowed typing mainly at species level. Rep-PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria.

Genetic map of the Bacillus stearothermophilus NUB36 chromosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vallier, H.; Welker, N.E.

1990-02-01

A circular genetic map of Bacillus stearothermophilus NUB36 was constructed by transduction with bacteriophage TP-42C and protoplast fusion. Sixty-four genes were tentatively assigned a cognate Bacillus subtilis gene based on growth response to intermediates or end products of metabolism, cross-feeding, accumulation of intermediates, or their relative order in a linkage group. Although the relative position of many genes on the Bacillus subtilis genetic map appears to be similar, some differences were detected. The tentative order of the genes in the Bacillus stearothermophilus aro region is aspB-aroBAFEC-tyra-hisH-(trp), whereas it is aspB-aroE-tyrA-hisH-(trp)-aroHBF in Bacillus subtilis. The aroA, aroC, and aroG genes inmore » Bacillus subtilis are located in another region. The tentative order of genes in the trp operon of Bacillus stearothermophilus is trpFCDABE, whereas it is trpABFCDE in Bacillus subtilis.« less

Isolation, evaluation and characterization of Bacillus subtilis from cotton rhizospheric soil with biocontrol activity against Fusarium oxysporum.

PubMed

Gajbhiye, Archana; Rai, Alok R; Meshram, Sudhir U; Dongre, A B

2010-07-01

Present investigation is based on the isolation of Bacillus subtilis from cotton rhizosphere and their evaluation as biocontrol agent against Fusarium oxysporum. The production of extracellular hydrolytic enzyme was studied for determining the antagonism. 43% of 21 isolates were identified under the B. subtilis group on the basis of biochemical characterization. 38% isolates showed competitive activity against Fusarium oxysporum and exhibit more than 50% mycelial inhibition in dual culture bioassay. The pot assay of cotton by seed treatment and soil amendment technique under green house condition showed the competent activity of the isolates in preventing the wilting of cotton seedlings due to F. oxysporum infection. SVI values of 30 day old seedlings indicated that the soil inoculation with B. subtilis BP-2 and seed treatment with B. subtilis BP-9 significantly promoted the growth of cotton seedlings. RAPD profiling revealed the diversity in the Bacillus subtilis group, ranging from 10 to 32%. The discriminative pattern among the isolates belonging to the same species was validated by 16S rDNA partial sequencing which identified them into four different strains of B. subtilis.

Fluoro-Phenyl-Styrene-Sulfonamide, a Novel Inhibitor of σB Activity, Prevents the Activation of σB by Environmental and Energy Stresses in Bacillus subtilis

PubMed Central

Ringus, Daina L.; Gaballa, Ahmed; Helmann, John D.; Wiedmann, Martin

2013-01-01

Sigma B (σB) is an alternative sigma factor that regulates the general stress response in Bacillus subtilis and in many other Gram-positive organisms. σB activity in B. subtilis is tightly regulated via at least three distinct pathways within a complex signal transduction cascade in response to a variety of stresses, including environmental stress, energy stress, and growth at high or low temperatures. We probed the ability of fluoro-phenyl-styrene-sulfonamide (FPSS), a small-molecule inhibitor of σB activity in Listeria monocytogenes, to inhibit σB activity in B. subtilis through perturbation of signal transduction cascades under various stress conditions. FPSS inhibited the activation of σB in response to multiple categories of stress known to induce σB activity in B. subtilis. Specifically, FPSS prevented the induction of σB activity in response to energy stress, including entry into stationary phase, phosphate limitation, and azide stress. FPSS also inhibited chill induction of σB activity in a ΔrsbV strain, suggesting that FPSS does not exclusively target the RsbU and RsbP phosphatases or the anti–anti-sigma factor RsbV, all of which contribute to posttranslational regulation of σB activity. Genetic and biochemical experiments, including artificial induction of σB, analysis of the phosphorylation state of the anti–anti-sigma factor RsbV, and in vitro transcription assays, indicate that while FPSS does not bind directly to σB to inhibit activity, it appears to prevent the release of B. subtilis σB from its anti-sigma factor RsbW. PMID:23524614

Selective heterogeneity in exoprotease production by Bacillus subtilis.

PubMed

Davidson, Fordyce A; Seon-Yi, Chung; Stanley-Wall, Nicola R

2012-01-01

Bacteria have elaborate signalling mechanisms to ensure a behavioural response that is most likely to enhance survival in a changing environment. It is becoming increasingly apparent that as part of this response, bacteria are capable of cell differentiation and can generate multiple, mutually exclusive co-existing cell states. These cell states are often associated with multicellular processes that bring benefit to the community as a whole but which may be, paradoxically, disadvantageous to an individual subpopulation. How this process of cell differentiation is controlled is intriguing and remains a largely open question. In this paper, we consider an important aspect of cell differentiation that is known to occur in the gram-positive bacterium Bacillus subtilis: we investigate the role of two master regulators DegU and Spo0A in the control of extra-cellular protease production. Recent work in this area focussed the on role of DegU in this process and suggested that transient effects in protein production were the drivers of cell-response heterogeneity. Here, using a combination of mathematical modelling, analysis and stochastic simulations, we provide a complementary analysis of this regulatory system that investigates the roles of both DegU and Spo0A in extra-cellular protease production. In doing so, we present a mechanism for bimodality, or system heterogeneity, without the need for a bistable switch in the underlying regulatory network. Moreover, our analysis leads us to conclude that this heterogeneity is in fact a persistent, stable feature. Our results suggest that system response is divided into three zones: low and high signal levels induce a unimodal or undifferentiated response from the cell population with all cells OFF and ON, respectively for exoprotease production. However, for intermediate levels of signal, a heterogeneous response is predicted with a spread of activity levels, representing typical "bet-hedging" behaviour.

Selective Heterogeneity in Exoprotease Production by Bacillus subtilis

PubMed Central

Davidson, Fordyce A.; Seon-Yi, Chung; Stanley-Wall, Nicola R.

2012-01-01

Bacteria have elaborate signalling mechanisms to ensure a behavioural response that is most likely to enhance survival in a changing environment. It is becoming increasingly apparent that as part of this response, bacteria are capable of cell differentiation and can generate multiple, mutually exclusive co-existing cell states. These cell states are often associated with multicellular processes that bring benefit to the community as a whole but which may be, paradoxically, disadvantageous to an individual subpopulation. How this process of cell differentiation is controlled is intriguing and remains a largely open question. In this paper, we consider an important aspect of cell differentiation that is known to occur in the Gram-positive bacterium Bacillus subtilis: we investigate the role of two master regulators DegU and Spo0A in the control of extra-cellular protease production. Recent work in this area focussed the on role of DegU in this process and suggested that transient effects in protein production were the drivers of cell-response heterogeneity. Here, using a combination of mathematical modelling, analysis and stochastic simulations, we provide a complementary analysis of this regulatory system that investigates the roles of both DegU and Spo0A in extra-cellular protease production. In doing so, we present a mechanism for bimodality, or system heterogeneity, without the need for a bistable switch in the underlying regulatory network. Moreover, our analysis leads us to conclude that this heterogeneity is in fact a persistent, stable feature. Our results suggest that system response is divided into three zones: low and high signal levels induce a unimodal or undifferentiated response from the cell population with all cells OFF and ON, respectively for exoprotease production. However, for intermediate levels of signal, a heterogeneous response is predicted with a spread of activity levels, representing typical “bet-hedging” behaviour. PMID:22745669

Identification and Characterization of Mutations Conferring Resistance to d-Amino Acids in Bacillus subtilis

PubMed Central

Leiman, Sara A.; Richardson, Charles; Foulston, Lucy; Elsholz, Alexander K. W.; First, Eric A.

2015-01-01

ABSTRACT Bacteria produce d-amino acids for incorporation into the peptidoglycan and certain nonribosomally produced peptides. However, d-amino acids are toxic if mischarged on tRNAs or misincorporated into protein. Common strains of the Gram-positive bacterium Bacillus subtilis are particularly sensitive to the growth-inhibitory effects of d-tyrosine due to the absence of d-aminoacyl-tRNA deacylase, an enzyme that prevents misincorporation of d-tyrosine and other d-amino acids into nascent proteins. We isolated spontaneous mutants of B. subtilis that survive in the presence of a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine. Whole-genome sequencing revealed that these strains harbored mutations affecting tRNATyr charging. Three of the most potent mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNA synthetase. In particular, resistance was conferred by mutations that destabilized the terminator hairpin of the tyrS riboswitch, as well as by a mutation that transformed a tRNAPhe into a tyrS riboswitch ligand. The most potent mutation, a substitution near the tyrosine recognition site of tyrosyl-tRNA synthetase, improved enzyme stereoselectivity. We conclude that these mutations promote the proper charging of tRNATyr, thus facilitating the exclusion of d-tyrosine from protein biosynthesis in cells that lack d-aminoacyl-tRNA deacylase. IMPORTANCE Proteins are composed of l-amino acids. Mischarging of tRNAs with d-amino acids or the misincorporation of d-amino acids into proteins causes toxicity. This work reports on mutations that confer resistance to d-amino acids and their mechanisms of action. PMID:25733611

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.

Genetic Competence Drives Genome Diversity in Bacillus subtilis

PubMed Central

Chevreux, Bastien; Serra, Cláudia R; Schyns, Ghislain; Henriques, Adriano O

2018-01-01

Abstract Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43% of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them. PMID:29272410

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

USDA-ARS?s Scientific Manuscript database

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

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

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

PubMed

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

2010-04-16

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

New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis.

PubMed

Hao, Xin; Nguyen, Tam; Kearns, Daniel B; Arpin, Carolynn C; Fall, Ray; Sammakia, Tarek

2011-09-15

We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacterials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures, p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming, was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoisomers showed that the 1R-anti-isomer was the most active with an average IC(50) of 16 μM (3-5 μg mL(-1)). For B. anthracis, the colony-spreading IC(50) values for 1R-anti-SPG and 1R-anti-NPG are 12 μM (2-4 μg mL(-1)) and >150 μM, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures. Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead to an understanding of the mechanism(s) of colony spreading as well as design and development of more potent inhibitors for the control of B. anthracis surface cultures. Copyright © 2011 Elsevier Ltd. All rights reserved.

Secreted production of Renilla luciferase in Bacillus subtilis.

PubMed

Chiang, Chung-Jen; Chen, Po Ting; Chao, Yun-Peng

2010-01-01

Luciferase (Rluc) from the soft coral Renilla reniformis has been widely used as a bioluminescent reporter, and its secreted production has been solely performed in mammalian cells thus far. To make the production more efficient, a series of approaches was attempted to overproduce Rluc extracellularly in Bacillus subtilis. First, Cys124 in the Rluc gene was substituted with Ala. The mutant gene was subsequently incorporated into a pUB110/R6K-based plasmid, consequently, fusing with the P43 promoter and the sacB signal peptide. With the nitrogen-rich medium, B. subtilis strain bearing the plasmid became able to secret a detectable amount of Rluc. Moreover, the secretion signal for the Rluc gene was replaced by the aprN leader peptide with or without the propeptide. The result led to a more than twofold increase in the secreted Rluc. Finally, by enhancing the transcription of the Rluc gene implementing the P43 and spac tandem promoter, it resulted in the secreted Rluc with a yield of 100 mg/L. Overall, this study illustrates a potential strategy for improving the secretion efficiency of heterologous proteins in B. subtilis.

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.

A family 30 glucurono-xylanase from Bacillus subtilis LC9: Expression, characterization and its application in Chinese bread making.

PubMed

Guo, Yalan; Gao, Zhen; Xu, Jiaxing; Chang, Siyuan; Wu, Bin; He, Bingfang

2018-05-22

A GH30-8 endoxylanase was identified from an environmental Bacillus subtilis isolate following growth selection on aspen wood glucuronoxylan. The putative endoxylanase was cloned for protein expression and characterization in the Gram-positive protease deficient protein expression host B. subtilis WB800. The extracellular activity obtained was 55 U/mL, which was 14.5-fold higher than that obtained with the native species. The apparent molecular mass of BsXyn30 was estimated as 43 kDa by SDS-PAGE. BsXyn30 showed an optimal activity at pH 7.0 and 60 °C. Recombinant BsXyn30 displayed maximum activity against aspen wood xylan, followed by beechwood xylan but showed no catalytic activity on arabinose-substituted xylans. Analysis of hydrolyzed products of beechwood xylan by thin-layer chromatography and mass spectroscopy revealed the presence of xylooligosaccharides with a single methyl-glucuronic acid residue. BsXyn30 exhibited very low activity for hydrolysis xylotetraose and xylopentaose, but had no detectable activity against xylobiose and xylotriose. Using BsXyn30 as an additive in breadmaking, a decrease in water-holding capacity, an increase in dough expansion as well as improvements in volume and specific volume of the bread were recorded. Thus, the present study provided the basis for the application of GH30 xylanase in breadmaking. Copyright © 2018 Elsevier B.V. All rights reserved.

An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

NASA Technical Reports Server (NTRS)

Jensen, R. A.; d'Amato, T. A.; Hochstein, L. I.

1988-01-01

The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

A love affair with Bacillus subtilis.

PubMed

Losick, Richard

2015-01-30

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

Assembly properties of the Bacillus subtilis actin, MreB.

PubMed

Mayer, Joshua A; Amann, Kurt J

2009-02-01

The bacterial actin MreB has been implicated in a variety of cellular roles including cell shape determination, cell wall synthesis, chromosome condensation and segregation, and the establishment and maintenance of cell polarity. Toward elucidating a clearer understanding of how MreB functions inside the bacterial cell, we investigated biochemically the polymerization of MreB from Bacillus subtilis. Light scattering and sedimentation assays revealed pH-, ionic-, cationic-, and temperature-dependent behavior. B. subtilis MreB polymerizes in the presence of millimolar divalent cations in a protein concentration-dependent manner. Polymerization is favored by decreasing pH and inhibited by monovalent salts and low temperatures. Although B. subtilis MreB binds and hydrolyzes both ATP and GTP, it does not require a bound nucleotide for assembly and polymerizes indistinguishably regardless of the nucleotide species bound, with a critical concentration of approximately 900 nM. A number of the presently reported properties of B. subtilis MreB differ significantly from those of T. maritima MreB1 (Bean and Amann [2008]: Biochemistry 47: 826-835), including the nucleotide requirements and temperature and ionic effects on polymerization state. These observations collectively suggest that additional factors interact with MreB to account for its complex dynamic behavior in cells.

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

USDA-ARS?s Scientific Manuscript database

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

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.

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

PubMed

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

2016-08-01

Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed pullulanase was 3.9 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular pullulanase activity of 24.5 U ml(-1). Therefore, promoter and host had an impact on pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition of Bacillus cereus growth by bacteriocin producing Bacillus subtilis isolated from fermented baobab seeds (maari) is substrate dependent.

PubMed

Kaboré, Donatien; Nielsen, Dennis Sandris; Sawadogo-Lingani, Hagrétou; Diawara, Bréhima; Dicko, Mamoudou Hama; Jakobsen, Mogens; Thorsen, Line

2013-03-01

Maari is a spontaneously alkaline fermented food condiment made from baobab tree seeds. Due to the spontaneous nature of maari fermentations growth of the opportunistic human pathogen Bacillus cereus is occasionally observed. Bacillus subtilis strains are important for alkaline seed fermentations because of their enzymatic activities contributing to desirable texture, flavor and pH development. Some B. subtilis strains have antimicrobial properties against B. cereus. In the present work, three bacteriocin producing B. subtilis strains (B3, B122 and B222) isolated from maari were tested. The production of antimicrobial activity by the three strains was found to be greatly influenced by the substrate. All three B. subtilis strains produced antimicrobial activity against B. cereus NVH391-98 in BHI broth as determined by the agar well diffusion assay, whereas no antimicrobial activity was detected in whole cooked baobab seeds and in 10% (w/v) grinded baobab seeds. Incorporation of BHI with up to 5% (w/w) grinded baobab seeds enhanced the antimicrobial activity of B. subtilis compared with pure BHI in a strain dependent manner. Incorporation of BHI with 50% (w/w) baobab grinded seeds decreased the antimicrobial activity. Addition of the inorganic salts FeCl₃, MgSO₄ and MnSO₄ has previously been reported to increase bacteriocin production of B. subtilis, but the addition of these salts to 10% (w/v) grinded baobab seed broth did not cause antimicrobial activity. Survival of B. cereus NVH391-98 in co-culture with B. subtilis was tested in BHI broth, 10% (w/v) grinded baobab seed based broth and during baobab seed fermentation to produce maari. B. cereus NVH391-98 grew well in all three substrates in mono-culture. All the 3 B. subtilis strains were able to decrease B. cereus NVH391-98 to levels below the detection limit (<10 CFU/ml) in BHI, but not in baobab seed based substrates, even though the outgrowth of B. cereus NVH391-98 was delayed by up to 40 h. In

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovach, Alexander R.; Hoff, Kirsten E.; Canty, John T.

Hfq is a post-transcriptional regulator that binds U- and A-rich regions of sRNAs and their target mRNAs to stimulate their annealing in order to effect translation regulation and, often, to alter their stability. The functional importance of Hfq and its RNA-binding properties are relatively well understood in Gram-negative bacteria, whereas less is known about the RNAbinding properties of this riboregulator in Gram-positive species. Here, we describe the structure of Hfq from the Grampositive pathogen Listeria monocytogenes in its RNA-free form and in complex with a U 6 oligoribonucleotide. As expected, the protein takes the canonical hexameric toroidal shape of allmore » other known Hfq structures. The U 6 RNA binds on the “proximal face” in a pocket formed by conserved residues Q9, N42, F43, and K58. Additionally residues G5 and Q6 are involved in protein-nucleic and inter-subunit contacts that promote uracil specificity. Unlike Staphylococcus aureus (Sa) Hfq, Lm Hfq requires magnesium to bind U 6 with high affinity. In contrast, the longer oligo-uridine, U 16, binds Lm Hfq tightly in the presence or absence of magnesium, thereby suggesting the importance of additional residues on the proximal face and possibly the lateral rim in RNA interaction. Lastly, intrinsic tryptophan fluorescence quenching (TFQ) studies reveal, surprisingly, that Lm Hfq can bind (GU) 3G and U6 on its proximal and distal faces, indicating a less stringent adenine-nucleotide specificity site on the distal face as compared to the Gram-positive Hfq proteins from Sa and Bacillus subtilis and suggesting as yet uncharacterized RNA-binding modes on both faces.« less

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes

DOE PAGES

Kovach, Alexander R.; Hoff, Kirsten E.; Canty, John T.; ...

2014-08-22

Hfq is a post-transcriptional regulator that binds U- and A-rich regions of sRNAs and their target mRNAs to stimulate their annealing in order to effect translation regulation and, often, to alter their stability. The functional importance of Hfq and its RNA-binding properties are relatively well understood in Gram-negative bacteria, whereas less is known about the RNAbinding properties of this riboregulator in Gram-positive species. Here, we describe the structure of Hfq from the Grampositive pathogen Listeria monocytogenes in its RNA-free form and in complex with a U 6 oligoribonucleotide. As expected, the protein takes the canonical hexameric toroidal shape of allmore » other known Hfq structures. The U 6 RNA binds on the “proximal face” in a pocket formed by conserved residues Q9, N42, F43, and K58. Additionally residues G5 and Q6 are involved in protein-nucleic and inter-subunit contacts that promote uracil specificity. Unlike Staphylococcus aureus (Sa) Hfq, Lm Hfq requires magnesium to bind U 6 with high affinity. In contrast, the longer oligo-uridine, U 16, binds Lm Hfq tightly in the presence or absence of magnesium, thereby suggesting the importance of additional residues on the proximal face and possibly the lateral rim in RNA interaction. Lastly, intrinsic tryptophan fluorescence quenching (TFQ) studies reveal, surprisingly, that Lm Hfq can bind (GU) 3G and U6 on its proximal and distal faces, indicating a less stringent adenine-nucleotide specificity site on the distal face as compared to the Gram-positive Hfq proteins from Sa and Bacillus subtilis and suggesting as yet uncharacterized RNA-binding modes on both faces.« less

Application of Bacillus subtilis 168 as a multifunctional agent for improvement of the durability of cement mortar.

PubMed

Park, Sung-Jin; Park, Jong-Myong; Kim, Wha-Jung; Ghim, Sa-Youl

2012-11-01

Microbiological calcium carbonate precipitation (MCCP) has been investigated for its ability to improve the durability of cement mortar. However, very few strains have been applied to crack remediation and strengthening of cementitious materials. In this study, we report the biodeposition of Bacillus subtilis 168 and its ability to enhance the durability of cement material. B. subtilis 168 was applied to the surface of cement specimens. The results showed a new layer of deposited organic-inorganic composites on the surface of the cement paste. In addition, the water permeability of the cement paste treated with B. subtilis 168 was lower than that of non-treated specimens. Furthermore, artificial cracks in the cement paste were completely remediated by the biodeposition of B. subtilis 168. The compressive strength of cement mortar treated with B. subtilis 168 increased by about 19.5% when compared with samples completed with only B4 medium. Taken together, these findings suggest that the biodeposition of B. subtilis 168 could be used as a sealing and coating agent to improve the strength and water resistance of concrete. This is the first paper to report the application of Bacillus subtilis 168 for its ability to improve the durability of cement mortar through calcium carbonate precipitation.

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

Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum.

PubMed

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.

Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis

PubMed Central

2011-01-01

Background Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase. Results The alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21. Conclusions This is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis. PMID:21978209

Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism in Bacillus subtilis Requires Spx

PubMed Central

Erwin, Kyle N.; Nakano, Shunji; Zuber, Peter

2005-01-01

Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions. PMID:15937167

Genetic Mapping of a Mutant Defective in d, l-Alanine Racemase in Bacillus subtilis 168

PubMed Central

Dul, Michael J.; Young, Frank E.

1973-01-01

Genetic analysis of a d-alanine requiring mutant (dal) of Bacillus subtilis reveals that the gene that codes for d,l-alanine racemase is linked to purB. The order of genes in this region of the chromosome is purB, pig, tsi, dal. Thus there are at least two clusters of genes that regulate cell wall biosynthesis in B. subtilis. PMID:4199510

Bacillus pumilus KatX2 confers enhanced hydrogen peroxide resistance to a Bacillus subtilis PkatA::katX2 mutant strain.

PubMed

Handtke, Stefan; Albrecht, Dirk; Zühlke, Daniela; Otto, Andreas; Becher, Dörte; Schweder, Thomas; Riedel, Kathrin; Hecker, Michael; Voigt, Birgit

2017-04-26

Bacillus pumilus cells exhibit a significantly higher resistance to hydrogen peroxide compared to closely related Bacilli like Bacillus subtilis. In this study we analyzed features of the catalase KatX2 of B. pumilus as one of the most important parts of the cellular response to hydrogen peroxide. KatX2, the vegetative catalase expressed in B. pumilus, was compared to the vegetative catalase KatA of B. subtilis. Data of our study demonstrate that B. pumilus can degrade toxic concentrations of hydrogen peroxide faster than B. subtilis. By replacing B. subtilis katA gene by katX2 we could significantly enhance its resistance to H 2 O 2 and its potential to eliminate this toxic compound. Mutant cells showed a 1.5- to 2-fold higher survival to toxic concentrations of hydrogen peroxide compared to wild type cells. Furthermore, we found reversible but also irreversible oxidations of the KatX2 protein which, in contrast to KatA, contains several cysteine residues. Our study indicates that the catalase KatX2 plays a major role in the increased resistance of B. pumilus to oxidative stress caused by hydrogen peroxide. Resistance to hydrogen peroxide of other Bacilli can be enhanced by exchanging the native catalase in the cells with katX2.

Novel Keratinase from Bacillus subtilis S14 Exhibiting Remarkable Dehairing Capabilities

PubMed Central

Macedo, Alexandre J.; da Silva, Walter O. Beys; Gava, Renata; Driemeier, David; Henriques, João Antonio Pêgas; Termignoni, Carlos

2005-01-01

We report the isolation of a keratinolytic-producing Bacillus subtilis strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase. This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it. Its unique nonactivity upon collagen enhances its industrial potential. PMID:15640244

Gram-positive Rod Surveillance for Early Anthrax Detection

PubMed Central

Begier, Elizabeth M.; Barrett, Nancy L.; Mshar, Patricia A.; Johnson, David G.

2005-01-01

Connecticut established telephone-based gram-positive rod (GPR) reporting primarily to detect inhalational anthrax cases more quickly. From March to December 2003, annualized incidence of blood isolates was 21.3/100,000 persons; reports included 293 Corynebacterium spp., 193 Bacillus spp., 73 Clostridium spp., 26 Lactobacillus spp., and 49 other genera. Around-the-clock GPR reporting has described GPR epidemiology and enhanced rapid communication with clinical laboratories. PMID:16229790

Use of Bacillus Subtilis PB6 as a potential antibiotic growth promoter replacement in improving performance of broiler birds.

PubMed

Jayaraman, Sathishkumar; Das, Partha Pratim; Saini, Prakash Chandra; Roy, Barun; Chatterjee, Paresh Nath

2017-08-01

The intestinal gut health is one of the primary determinants of broiler growth and performance. Among the various enteric diseases, necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens, which can result in severe economic losses in poultry farming. Antibiotics like bacitracin methylene disalicylate (BMD) and avilamycin (AVL) are commonly used antibiotic growth promoters (AGP) in poultry feed to control necrotic enteritis in birds. Bacillus subtilis PB6 was reported to prevent necrotic enteritis and improve performance in birds. This paper investigated the influence of Bacillus subtilis PB6 in improving the performance of broiler birds in comparison with BMD and avilamycin. A 35 day trial was conducted with 240 day-old commercial broiler chicks (VenCobb 400), which were divided into four treatment groups, where each treatment group was composed of 6 replicates each containing 10 birds, for a total of 60 birds per treatment. The treatment groups included a negative control (no AGP), Bacillus subtilis PB6, BMD, and avilamycin. The parameters analyzed included body weight, feed conversion ratio (FCR), mortality, villus histomorphometry, and European efficiency factor (EEF). Bacillus subtilis PB6 significantly (P < 0.05) improved body weight and FCR (8 points) compared to the control. The group supplemented with B. subtilis PB6 or BMD had higher (P < 0.05) body weight compared to all other treatment groups. The supplementation of B. subtilis PB6 significantly improved the villus height (P < 0.05) compared to control and other AGP groups. The EEF was found to be the highest in the B. subtilis PB6 supplemented group at 35th day as compared to other treatment groups. The combined data from this study indicate that supplementation of B. subtilis PB6 improves overall performance of broilers compared to BMD and avilamycin, and can be used as potential AGP replacement in poultry farming. © 2017 Poultry Science Association Inc.

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.

A molecular model for illegitimate recombination in Bacillus subtilis.

PubMed

Temeyer, K B; Hopkins, K M; Chapman, L F

1991-01-01

The recombinant DNA junctions at which pUB110 and Bacillus subtilis chromosomal DNA were joined to form the plasmid pKBT1 were cloned and sequenced. From the sequencing data we conclude that the pUB110 sequence is intact in the pair of cloned pKBT1 fragments and pTL12 sequences are not present. A molecular model for the formation of pKBT1 based on structural motifs characteristic of the joint sites is presented.

Five new amicoumacins isolated from a marine-derived bacterium Bacillus subtilis.

PubMed

Li, Yongxin; Xu, Ying; Liu, Lingli; Han, Zhuang; Lai, Pok Yui; Guo, Xiangrong; Zhang, Xixiang; Lin, Wenhan; Qian, Pei-Yuan

2012-02-01

Four novel amicoumacins, namely lipoamicoumacins A-D (1-4), and one new bacilosarcin analog (5) were isolated from culture broth of a marine-derived bacterium Bacillus subtilis, together with six known amicoumacins. Their structures were elucidated on the basis of extensive spectroscopic (2D NNR, IR, CD and MS) analysis and in comparison with data in literature.

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. Copyright © 2017 Elsevier B.V. All rights reserved.

[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.

Dynamic sporulation gene co-expression networks for Bacillus subtilis 168 and the food-borne isolate Bacillus amyloliquefaciens: a transcriptomic model

PubMed Central

Omony, Jimmy; de Jong, Anne; Krawczyk, Antonina O.; Eijlander, Robyn T.; Kuipers, Oscar P.

2018-01-01

Sporulation is a survival strategy, adapted by bacterial cells in response to harsh environmental adversities. The adaptation potential differs between strains and the variations may arise from differences in gene regulation. Gene networks are a valuable way of studying such regulation processes and establishing associations between genes. We reconstructed and compared sporulation gene co-expression networks (GCNs) of the model laboratory strain Bacillus subtilis 168 and the food-borne industrial isolate Bacillus amyloliquefaciens. Transcriptome data obtained from samples of six stages during the sporulation process were used for network inference. Subsequently, a gene set enrichment analysis was performed to compare the reconstructed GCNs of B. subtilis 168 and B. amyloliquefaciens with respect to biological functions, which showed the enriched modules with coherent functional groups associated with sporulation. On basis of the GCNs and time-evolution of differentially expressed genes, we could identify novel candidate genes strongly associated with sporulation in B. subtilis 168 and B. amyloliquefaciens. The GCNs offer a framework for exploring transcription factors, their targets, and co-expressed genes during sporulation. Furthermore, the methodology described here can conveniently be applied to other species or biological processes. PMID:29424683

Dynamic sporulation gene co-expression networks for Bacillus subtilis 168 and the food-borne isolate Bacillus amyloliquefaciens: a transcriptomic model.

PubMed

Omony, Jimmy; de Jong, Anne; Krawczyk, Antonina O; Eijlander, Robyn T; Kuipers, Oscar P

2018-02-09

Sporulation is a survival strategy, adapted by bacterial cells in response to harsh environmental adversities. The adaptation potential differs between strains and the variations may arise from differences in gene regulation. Gene networks are a valuable way of studying such regulation processes and establishing associations between genes. We reconstructed and compared sporulation gene co-expression networks (GCNs) of the model laboratory strain Bacillus subtilis 168 and the food-borne industrial isolate Bacillus amyloliquefaciens. Transcriptome data obtained from samples of six stages during the sporulation process were used for network inference. Subsequently, a gene set enrichment analysis was performed to compare the reconstructed GCNs of B. subtilis 168 and B. amyloliquefaciens with respect to biological functions, which showed the enriched modules with coherent functional groups associated with sporulation. On basis of the GCNs and time-evolution of differentially expressed genes, we could identify novel candidate genes strongly associated with sporulation in B. subtilis 168 and B. amyloliquefaciens. The GCNs offer a framework for exploring transcription factors, their targets, and co-expressed genes during sporulation. Furthermore, the methodology described here can conveniently be applied to other species or biological processes.

[Effect of carbon and nitrogen sources and complex B vitamins on the synthesis of alkaline protease by different strains of Bacillus mesentericus and Bacillus subtilis].

PubMed

Emtseva, T V

1975-01-01

The effect of different sources of carbon, nitrogen, amino acids and vitamins on the synthesis of alkaline proteases by the stock and mutant strains of Bacillus mesentericus and by the natural strain of Bacillus subtilis-12 has been investigated. The maximum synthesis of alkaline protease has been obtained in the media containing starch or its hydrolysates--dextrine and maltose as the carbon source. Ammonium phosphate and casein as the nitrogen source prove to be optimal for Bac. mesentericus and Bac. subtilis, respectively. Complex B vitamins added to the nutrient medium accelerate the enzyme synthesis 2.5-4-fold.

The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

PubMed

Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

2015-10-01

One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization, kinetic, and thermodynamic studies of marine pectinase from Bacillus subtilis.

PubMed

Joshi, Manasi; Nerurkar, Madhura; Adivarekar, Ravindra

2015-01-01

Characterization, kinetic and thermodynamic parameters of purified pectinase from Bacillus subtilis, isolated from a marine sediment sample collected from Chinchani beach at Tarapore, India, were studied. Marine pectinase produced under submerged growth conditions was purified by ammonium sulfate precipitation followed by gel filtration chromatography using DEAE cellulose. Partial characterization of the marine pectinase was carried out in terms of effect of pH, temperature, substrate concentration, and metal ions. It was found that pectinase from marine B. subtilis showed maximal activity in alkaline buffer at pH 9.0 and at 40°C. It was also found that metal ions, namely, Mn(2+) and Fe(2+), stimulate pectinase activity. Marine pectinase followed Michaelis-Menten kinetics. The kinetics and thermodynamic parameters of the purified marine pectinase from B. subtilis were studied as the characterization of the enzyme is vital for its use in industrial processes.

From the genome sequence to the protein inventory of Bacillus subtilis.

PubMed

Becher, Dörte; Büttner, Knut; Moche, Martin; Hessling, Bernd; Hecker, Michael

2011-08-01

Owing to the low number of proteins necessary to render a bacterial cell viable, bacteria are extremely attractive model systems to understand how the genome sequence is translated into actual life processes. One of the most intensively investigated model organisms is Bacillus subtilis. It has attracted world-wide research interest, addressing cell differentiation and adaptation on a molecular scale as well as biotechnological production processes. Meanwhile, we are looking back on more than 25 years of B. subtilis proteomics. A wide range of methods have been developed during this period for the large-scale qualitative and quantitative proteome analysis. Currently, it is possible to identify and quantify more than 50% of the predicted proteome in different cellular subfractions. In this review, we summarize the development of B. subtilis proteomics during the past 25 years. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

How Changes in Anti-SD Sequences Would Affect SD Sequences in Escherichia coli and Bacillus subtilis.

PubMed

Abolbaghaei, Akram; Silke, Jordan R; Xia, Xuhua

2017-05-05

The 3' end of the small ribosomal RNAs (ssu rRNA) in bacteria is directly involved in the selection and binding of mRNA transcripts during translation initiation via well-documented interactions between a Shine-Dalgarno (SD) sequence located upstream of the initiation codon and an anti-SD (aSD) sequence at the 3' end of the ssu rRNA. Consequently, the 3' end of ssu rRNA (3'TAIL) is strongly conserved among bacterial species because a change in the region may impact the translation of many protein-coding genes. Escherichia coli and Bacillus subtilis differ in their 3' ends of ssu rRNA, being GAUC ACCUCCUUA 3' in E. coli and GAUC ACCUCCUU UCU3' or GAUC ACCUCCUU UCUA3' in B. subtilis Such differences in 3'TAIL lead to species-specific SDs (designated SD Ec for E. coli and SD Bs for B. subtilis ) that can form strong and well-positioned SD/aSD pairing in one species but not in the other. Selection mediated by the species-specific 3'TAIL is expected to favor SD Bs against SD Ec in B. subtilis , but favor SD Ec against SD Bs in E. coli Among well-positioned SDs, SD Ec is used more in E. coli than in B. subtilis , and SD Bs more in B. subtilis than in E. coli Highly expressed genes and genes of high translation efficiency tend to have longer SDs than lowly expressed genes and genes with low translation efficiency in both species, but more so in B. subtilis than in E. coli Both species overuse SDs matching the bolded part of the 3'TAIL shown above. The 3'TAIL difference contributes to the host specificity of phages. Copyright © 2017 Abolbaghaei et al.

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

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

PubMed

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

2006-09-01

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

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

USDA-ARS?s Scientific Manuscript database

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

Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides

PubMed Central

Zaprasis, Adrienne; Brill, Jeanette; Thüring, Marietta; Wünsche, Guido; Heun, Magnus; Barzantny, Helena; Hoffmann, Tamara

2013-01-01

Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute l-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (Dpp, Opp, App, and DtpT) operating in B. subtilis and relies on their hydrolysis to liberate proline. The effectiveness with which proline-containing peptides confer osmoprotection varies considerably, and this can be correlated with the amount of the liberated and subsequently accumulated free proline by the osmotically stressed cell. Through gene disruption experiments, growth studies, and the quantification of the intracellular proline pool, we have identified the PapA (YqhT) and PapB (YkvY) peptidases as responsible for the hydrolysis of various types of Xaa-Pro dipeptides and Xaa-Pro-Xaa tripeptides. The PapA and PapB peptidases possess overlapping substrate specificities. In contrast, osmoprotection by peptides of various lengths and compositions with a proline residue positioned at their N terminus was not affected by defects in the PapA and PapB peptidases. Taken together, our data provide new insight into the physiology of the osmotic stress response of B. subtilis. They illustrate the flexibility of this ubiquitously distributed microorganism to effectively exploit environmental resources in its acclimatization to sustained high-osmolarity surroundings through the accumulation of compatible solutes. PMID:23144141

Gram-Positive Bacteria with Probiotic Potential for the Apis mellifera L. Honey Bee: The Experience in the Northwest of Argentina.

PubMed

Audisio, Marcela Carina

2017-03-01

Apis mellifera L. is one of the most important natural pollinators of significant crops and flowers around the world. It can be affected by different types of illnesses: american foulbrood, nosemosis, varroasis, viruses, among others. Such infections mainly cause a reduction in honey production and in extreme situations, the death of the colony. Argentina is the world's second largest honey exporter and the third largest honey producer, after China and Turkey. Given both the prominence of the honey bee in nature and the economic importance of apiculture in Argentina and the world, it is crucial to develop efficient and sustainable strategies to control honey bee diseases and to improve bee colony health. Gram-positive bacteria, such as lactic acid bacteria, mainly Lactobacillus, and Bacillus spp. are promising options. In the Northwest of Argentina, several Lactobacillus and Bacillus strains from the honey bee gut and honey were isolated by our research group and characterized by using in vitro tests. Two strains were selected because of their potential probiotic properties: Lactobacillus johnsonii CRL1647 and Bacillus subtilis subsp. subtilis Mori2. Under independent trials with both experimental and commercial hives, it was determined that each strain was able to elicit probiotic effects on bee colonies reared in the northwestern region of Argentina. One result was the increase in egg-laying by the queen which therefore produced an increase in bee number and, consequently, a higher honey yield. Moreover, the beneficial bacteria reduced the incidence of two important bee diseases: nosemosis and varroosis. These results are promising and extend the horizon of probiotic bacteria to the insect world, serving beekeepers worldwide as a natural tool that they can administer as is, or combine with other disease-controlling methods.

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

Potassium sensing histidine kinase in Bacillus subtilis.

PubMed

López, Daniel; Gontang, Erin A; Kolter, Roberto

2010-01-01

The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

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

Pulsed dielectric barrier discharge for Bacillus subtilis inactivation in water

NASA Astrophysics Data System (ADS)

Hernández-Arias, A. N.; Rodríguez-Méndez, B. G.; López-Callejas, R.; Valencia-Alvarado, R.; Mercado-Cabrera, A.; Peña-Eguiluz, R.; Barocio, S. R.; Muñoz-Castro, A. E.; de la Piedad Beneitez, A.

2012-06-01

The inactivation of Bacillus subtilis bacteria in water has been experimentally studied by means of a pulsed dielectric barrier discharge (PDBD) in a coaxial reactor endowed with an alumina dielectric. The plasma source is capable of operating at atmospheric pressure with gas, water or hybrid gas-liquid media at adjustable 25 kV pulses, 30 μs long and at a 500 Hz frequency. In order to evaluate the inactivation efficiency of the system, a set of experiments were designed on the basis of oxygen flow control. The initial data have showed a significant bacterial rate reduction of 103-107 CFU/mL. Additional results proved that applying an oxygen flow for a few seconds during the PDBD treatment inactivates the Bacillus subtilis population with 99.99% effectiveness. As a reference, without gas flow but with the same exposure times, this percentage is reduced to ~90%. The analysis of the relationship between inactivation rate and chemical species in the discharge has been carried out using optical emission spectroscopy as to identifying the main reactive species. Reactive oxygen species such as atomic oxygen and ozone tuned out to be the dominant germicidal species. Some proposed inactivation mechanisms of this technique are discussed.

A Computational Study of Phenotype Switching in Bacillus Subtilis Biofilm

NASA Astrophysics Data System (ADS)

Smith, Howard; Wang, Xiaoling; Jiang, Yi

Bacillus Subtilis (B. Subtilis), is known to differentiate into three main phenotypes during biofilm growth. Novel techniques to track the spatial and temporal evolution of the three main phenotypes exhibited by B. Subtilis have been developed. However, the techniques do not explain the environmental causes of the phenotype switching and how this leads to the spatiotemporal organization of the biofilm. We hypothesize that cells switch their phenotype according to nutrients and autoinducer levels. We test the hypothesis using a hybrid agent-based and continuous model. The bacteria in our model are individual cells that can (i) grow and divide by the intake of nutrients, (ii) produce and secrete EPS, (iii) form spores and (iv) produce an auto inducer. Using a threshold for nutrient and thresholds for autoinducers, we were able to reproduce the experimental spatiotemporal dynamics. From our simulations we observed that in order to reproduce experimental results, two different autoinducers were necessary. The results also suggest that low-EPS producing biofilms generally obtained higher cell populations. Furthermore, most of the cells that become spore forming cells arise from matrix producing cells.

Galactose Metabolism Plays a Crucial Role in Biofilm Formation by Bacillus subtilis

PubMed Central

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

2012-01-01

ABSTRACT Galactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. In Bacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since the galE mutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to a galE null mutant in the presence of galactose, we identified mutations in sinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role in B. subtilis biofilm formation and that the expressions of both the gal and eps genes are interrelated. Finally, we propose that B. subtilis and other members of the Bacillus genus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources. PMID:22893383

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... FOOD Exemptions From Tolerances § 180.1111 Bacillus subtilis GB03; exemption from the requirement of a... all raw agricultural commodities when used in accordance with good agricultural practices. [73 FR...

Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis.

PubMed

López, Daniel; Fischbach, Michael A; Chu, Frances; Losick, Richard; Kolter, Roberto

2009-01-06

We report a previously undescribed quorum-sensing mechanism for triggering multicellularity in Bacillus subtilis. B. subtilis forms communities of cells known as biofilms in response to an unknown signal. We discovered that biofilm formation is stimulated by a variety of small molecules produced by bacteria--including the B. subtilis nonribosomal peptide surfactin--that share the ability to induce potassium leakage. Natural products that do not cause potassium leakage failed to induce multicellularity. Small-molecule-induced multicellularity was prevented by the addition of potassium, but not sodium or lithium. Evidence is presented that potassium leakage stimulates the activity of a membrane protein kinase, KinC, which governs the expression of genes involved in biofilm formation. We propose that KinC responds to lowered intracellular potassium concentration and that this is a quorum-sensing mechanism that enables B. subtilis to respond to related and unrelated bacteria.

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

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.

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.

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

Bacillus subtilis as potential producer for polyhydroxyalkanoates.

PubMed

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

2009-07-20

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.

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

Regulation of the spoVM gene of Bacillus subtilis.

PubMed

Le, Ai Thi Thuy; Schumann, Wolfgang

2008-11-01

The spoVM gene of Bacillus subtilis codes for a 26 amino-acid peptide that is essential for sporulation. Analysis of the expression of the spoVM gene revealed that wild-type cells started to synthesize a spoVM-specific transcript at t2, whereas the SpoVM peptide accumulated at t4. Both the transcript and the peptide were absent from an spoVM knockout strain. The 5' untranslated region of the spoVM transcript increased expression of SpoVM. Possible regulation mechanisms are discussed.

Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling.

PubMed

Singh, Praveen K; Ramachandran, Gayetri; Ramos-Ruiz, Ricardo; Peiró-Pastor, Ramón; Abia, David; Wu, Ling J; Meijer, Wilfried J J

2013-10-01

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

Activity of Kaempferia pandurata (Roxb.) rhizome ethanol extract against MRSA, MRCNS, MSSA, Bacillus subtilis and Salmonella typhi.

PubMed

Sukandar, Elin Yulinah; Sunderam, Nethiyakalyani; Fidrianny, Irda

2014-01-01

Temu kunci (Kaempferia pandurata (Roxb.)) has a number of benefits and one of these is antibacterial. The rhizome is said to have antibacterial activity against Streptococcus mutans, Lactocillus sp. and Candida albicans. The aim of the study is to test the antibacterial activity of Kaempferia pandurata (Roxb.) rhizome ethanol extract on methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase negative Staphylococci (MRCNS), methicillin-sensitive Staphylococcus aureus (MSSA), Bacillus subtilis and Salmonella typhi. Antimicrobial activity of the extract was assayed by the microdilution method using Mueller Hinton Broth with sterilized 96 round-bottomed microwells to determine the Minimum Inhibitory Concentration (MIC) as well as to determine the time-kill activity. The MIC of the extract was 16 ppm for both Bacillus subtilis and MRSA; 8 ppm for both MSSA and Salmonella typhi and 4 ppm for MRCNS. Ethanol extract of Kaempferia pandurata (Roxb.) showed antibacterial activity against all the tested bacteria and was the most potent against MRCNS, with MIC 4 ppm. The killing profile test of the extract displayed bactericidal activity at 8-16 ppm against MRSA, MSSA, Bacillus subtilis and Salmonella typhi and bacteriostatic activity at 4 ppm towards MRCNS.

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.

Analysis of the Function of a Putative 2,3-Diphosphoglyceric Acid-Dependent Phosphoglycerate Mutase from Bacillus subtilis

PubMed Central

Pearson, Claire L.; Loshon, Charles A.; Pedersen, Lotte B.; Setlow, Barbara; Setlow, Peter

2000-01-01

A Bacillus subtilis gene termed yhfR encodes the only B. subtilis protein with significant sequence similarity to 2,3-diphosphoglycerate-dependent phosphoglycerate mutases (dPGM). This gene is expressed at a low level during growth and sporulation, but deletion of yhfR had no effect on growth, sporulation, or spore germination and outgrowth. YhfR was expressed in and partially purified from Escherichia coli but had little if any PGM activity and gave no detectable PGM activity in B. subtilis. These data indicate that B. subtilis does not require YhfR and most likely does not require a dPGM. PMID:10869096

Crystal Structure of Bacillus subtilis α-Amylase in Complex with Acarbose

PubMed Central

Kagawa, Masayuki; Fujimoto, Zui; Momma, Mitsuru; Takase, Kenji; Mizuno, Hiroshi

2003-01-01

The crystal structure of Bacillus subtilis α-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process. PMID:14617662

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

USDA-ARS?s Scientific Manuscript database

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

Differences in the roles of a glutamine amidotransferase subunit of pyridoxal 5'-phosphate synthase between Bacillus circulans and Bacillus subtilis.

PubMed

Itagaki, Shiori; Haga, Minami; Oikawa, Yuji; Sakoda, Ayaka; Ohke, Yoshie; Sawada, Hiroshi; Eguchi, Tadashi; Tamegai, Hideyuki

2013-01-01

BtrC2 of the butirosin producer Bacillus circulans is a non-catalytic subunit of 2-deoxy-scyllo-inosose (DOI) synthase that is involved in butirosin biosynthesis, and also a homolog of glutamine amidotransferase subunit (PdxT) of pyridoxal 5'-phosphate (PLP) synthase of Bacillus subtilis. BtrC2 has been found to have functions in B. circulans both in primary and secondary metabolism. In this study, we investigated the properties of PdxT of B. subtilis in order to determine whether the property of enzyme stabilization is universal among PdxT homologs. Complementation with PdxT in the btrC2 disruptant of B. circulans restored the growth and short-term production of antibiotics, but long-term production of antibiotics cannot be restored. Additionally, PdxT did not bind physically with or stabilize BtrC. Our results indicate that the function of BtrC2 in secondary metabolism is specific properties, not universal among PdxT homologs.

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

σI from Bacillus subtilis: Impact on Gene Expression and Characterization of σI-dependent Transcription that Requires New Types of Promoters with Extended -35 and -10 Elements.

PubMed

Ramaniuk, Olga; Převorovský, Martin; Pospíšil, Jiří; Vítovská, Dragana; Kofroňová, Olga; Benada, Oldřich; Schwarz, Marek; Šanderová, Hana; Hnilicová, Jarmila; Krásný, Libor

2018-06-18

σ I from Bacillus subtilis is a σ factor associating with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here we provide a comprehensive characterization of this transcriptional regulator. By RNA-seq of wt and σ I -null strains at 37°C and 52°C we identified ∼130 genes affected by the absence of σ I Further analysis revealed that the majority of these genes were affected by σ I indirectly. The σ I regulon, i.e., the genes directly regulated by σ I , consists of 16 genes of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σ I in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σ I -dependent promoters are relatively information-rich in comparison with most other promoters. In summary, this study supplies information about the least explored σ factor from the industrially important model organism B. subtilis Importance In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons ( i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σ I regulon from the industrially important model Gram-positive bacterium - Bacillus subtilis We reveal that σ I affects expression of ∼ 130 genes, of which 16 are directly regulated by σ I , including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σ I -dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription

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

Galactose metabolism plays a crucial role in biofilm formation by Bacillus subtilis.

PubMed

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

2012-01-01

Galactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. In Bacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since the galE mutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to a galE null mutant in the presence of galactose, we identified mutations in sinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role in B. subtilis biofilm formation and that the expressions of both the gal and eps genes are interrelated. Finally, we propose that B. subtilis and other members of the Bacillus genus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources. Bacteria switch from unicellular to multicellular states by producing extracellular matrices that contain exopolysaccharides. In such aggregates, known as biofilms, bacteria are more resistant to antibiotics. This makes biofilms a serious problem in clinical settings. The resilience of biofilms makes them very useful in industrial settings. Thus, understanding the production of biofilm matrices is an important problem in microbiology. In studying the synthesis of the biofilm matrix of Bacillus subtilis, we provide further understanding of a long-standing microbiological observation that certain mutants defective in the utilization of galactose became sensitive to it. In this

Mechanisms of adaptation to nitrosative stress in Bacillus subtilis.

PubMed

Rogstam, Annika; Larsson, Jonas T; Kjelgaard, Peter; von Wachenfeldt, Claes

2007-04-01

Bacteria use a number of mechanisms for coping with the toxic effects exerted by nitric oxide (NO) and its derivatives. Here we show that the flavohemoglobin encoded by the hmp gene has a vital role in an adaptive response to protect the soil bacterium Bacillus subtilis from nitrosative stress. We further show that nitrosative stress induced by the nitrosonium cation donor sodium nitroprusside (SNP) leads to deactivation of the transcriptional repressor NsrR, resulting in derepression of hmp. Nitrosative stress induces the sigma B-controlled general stress regulon. However, a sigB null mutant did not show increased sensitivity to SNP, suggesting that the sigma B-dependent stress proteins are involved in a nonspecific protection against stress whereas the Hmp flavohemoglobin plays a central role in detoxification. Mutations in the yjbIH operon, which encodes a truncated hemoglobin (YjbI) and a predicted 34-kDa cytosolic protein of unknown function (YjbH), rendered B. subtilis hypersensitive to SNP, suggesting roles in nitrosative stress management.

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis.

PubMed

Kawai, Yoshikazu; Asai, Kei; Errington, Jeffery

2009-08-01

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.

Study of the Bacillus flora of Nigerian spices.

PubMed

Antai, S P

1988-05-01

Bacteriological examination of 230 samples of five different unprocessed spices (aligator pepper, red pepper, black pepper, thyme and curry powder) collected randomly from Port Harcourt main markets revealed that the spices were highly contaminated, with bacterial counts ranging from 1.8 x 10(4) to 1.1 x 10(8) per gram. Bacillus cereus was isolated in high numbers in the majority of the 230 samples examined. It was also observed that other Bacillus spp. including B. subtilis, B. polymyxa and B. coagulans occurred in significant numbers.

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

Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

PubMed

Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

2006-01-01

An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.

Malate-Mediated Carbon Catabolite Repression in Bacillus subtilis Involves the HPrK/CcpA Pathway ▿ §

PubMed Central

Meyer, Frederik M.; Jules, Matthieu; Mehne, Felix M. P.; Le Coq, Dominique; Landmann, Jens J.; Görke, Boris; Aymerich, Stéphane; Stülke, Jörg

2011-01-01

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate. PMID:22001508

Biochemical characterization of Gram-positive and Gram-negative plant-associated bacteria with micro-Raman spectroscopy.

PubMed

Paret, Mathews L; Sharma, Shiv K; Green, Lisa M; Alvarez, Anne M

2010-04-01

Raman spectra of Gram-positive and Gram-negative plant bacteria have been measured with micro-Raman spectrometers equipped with 785 and 514.5 nm lasers. The Gram-positive bacteria Microbacterium testaceum, Paenibacillus validus, and Clavibacter michiganensis subsp. michiganensis have strong carotenoid bands in the regions 1155-1157 cm(-1) and 1516-1522 cm(-1) that differentiate them from other tested Gram-negative bacteria. In the Raman spectrum of Gram-positive bacteria Bacillus megaterium excited with 785 nm laser, the Raman bands at 1157 and 1521 cm(-1) are weak in intensity compared to other Gram-positive bacteria, and these bands did not show significant resonance Raman enhancement in the spectrum recorded with 514.5 nm laser excitation. The Gram-positive bacteria could be separated from each other based on the bands associated with the in-phase C=C (v(1)) vibrations of the polyene chain of carotenoids. None of the Gram-negative bacteria tested had carotenoid bands. The bacteria in the genus Xanthomonas have a carotenoid-like pigment, xanthomonadin, identified in Xanthomonas axonopodis pv. dieffenbachiae, and it is a unique Raman marker for the bacteria. The representative bands for xanthomonadin were the C-C stretching (v(2)) vibrations of the polyene chain at 1135-1136 cm(-1) and the in-phase C=C (v(1)) vibrations of the polyene chain at 1529-1531 cm(-1), which were distinct from the carotenoid bands of other tested bacteria. The tyrosine peak in the region 1170-1175 cm(-1) was the only other marker present in Gram-negative bacteria that was absent in all tested Gram-positives. A strong-intensity exopolysaccharide-associated marker at 1551 cm(-1) is a distinguishable feature of Enterobacter cloacae. The Gram-negative Agrobacterium rhizogenes and Ralstonia solanacearum were differentiated from each other and other tested bacteria on the basis of presence or absence and relative intensities of peaks. The principal components analysis (PCA) of the spectra

Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

PubMed

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

2016-08-11

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

Analysis of Host-Takeover During SPO1 Infection of Bacillus subtilis.

PubMed

Stewart, Charles R

2018-01-01

When Bacillus subtilis is infected by bacteriophage SPO1, the phage directs the remodeling of the host cell, converting it into a factory for phage reproduction. Much synthesis of host DNA, RNA, and protein is shut off, and cell division is prevented. Here I describe the protocols by which we have demonstrated those processes, and identified the roles played by specific SPO1 gene products in causing those processes.

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

USDA-ARS?s Scientific Manuscript database

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

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

USDA-ARS?s Scientific Manuscript database

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

[Use of antagonistic Bacillus subtilis bacteria for treatment of nosocomial urinary tract infections].

PubMed

Pushkarev, A M; Tuĭgunova, V G; Zaĭnullin, R R; Kuznetsova, T N; Gabidullin, Iu Z

2007-01-01

Effect of Bactisporin--a probiotic, containing spores of aerobic Bacillus subtilis 3H bacterium--for complex treatment of patients with nosocomial urinary tract infections was studied. 68 Cultures of different species of conditionally pathogenic bacteria were isolated from urine of the patients. Susceptibility of the isolated cultures to antibiotics before and after application of B. subtilis 3H metabolites was determined. The metabolites were accumulated on potato-glucose agar (PGA) while bacterium was cultivated on kapron membranes placed on surface of the medium. Influence of obtained metabolites on isolated strains was assessed by cultivation of each strain in metabolites-rich PGA during 24 h. Metabolites of B. subtilis led to decrease in resistance of isolated uropathogenic microflora to antibiotics. Use of Bactisporin in complex treatment of nosocomial urinary tract infections resulted in accelerated elimination of causative microorganism.

Simple, Inexpensive, and Rapid Way to Produce Bacillus subtilis Spores for the Guthrie Bioassay

PubMed Central

Franklin, Martha L.; Clark, William A.

1981-01-01

Esculin agar has been found to be a simple, inexpensive, rapid, and reliable means to promote production of spores of inhibitor-sensitive clones of Bacillus subtilis strains ATCC 6051 and 6633 for use in the Guthrie bioassay screening tests for genetic metabolic disorders. Images PMID:6790564

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.

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.

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

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.

DNA gyrase inhibitors block development of Bacillus subtilis bacteriophage SP01.

PubMed Central

Alonso, J C; Sarachu, A N; Grau, O

1981-01-01

SP01 development was inhibited by nalidixic acid and novobiocin in the sensitive host Bacillus subtilis 168M. Inhibition by novobiocin was prevented by a Novr mutation in the cellular DNA gyrase gene. Nalidixic acid inhibition persisted in hosts carrying a Nalr gyrase, but could be overcome by phage mutation. We conclude that SP01 requires for its development subunit B of the host DNA gyrase, but replaces or modifies subunit A. PMID:6270354

Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and trp Operon Expression in Bacillus licheniformis▿

PubMed Central

Levitin, Anastasia; Yanofsky, Charles

2010-01-01

Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNATrp. Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNATrp deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis. PMID:20061467

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Properties of Thermosensitive Extracellular α-Amylases of Bacillus subtilis

PubMed Central

Yamane, Kunio; Maruo, Bunji

1974-01-01

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

Sticking together: building a biofilm the Bacillus subtilis way

PubMed Central

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

2014-01-01

Preface Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long-served as a robust model organism to examine the molecular mechanisms of biofilm formation and a number of studies have revealed that this process is subject to a number of integrated regulatory pathways. In this Review, we focus on the molecular mechanisms controlling biofilm assembly and briefly summarize the current state of knowledge regarding their disassembly. We also discuss recent progress that has expanded our understanding of biofilm formation on plant roots, which are a natural habitat for this soil bacterium. PMID:23353768

Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

PubMed

Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

2014-09-02

A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

Folic acid and the methylation of homocysteine by Bacillus subtilis

PubMed Central

Salem, A. R.; Pattison, J. R.; Foster, M. A.

1972-01-01

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C1 transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B12. 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes. PMID:4627401

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

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.

Improvement of iturin A production in Bacillus subtilis ZK0 by overexpression of the comA and sigA genes.

PubMed

Zhang, Z; Ding, Z T; Zhong, J; Zhou, J Y; Shu, D; Luo, D; Yang, J; Tan, H

2017-06-01

Bacillus subtilis ZK0, which was isolated from cotton, produces a type of lipopeptide antibiotic iturin A that inhibits the growth of pathogenic fungi on agricultural crops. However, the low level of iturin A production by B. subtilis ZK0 does not support its large-scale application. In this study, B. subtilis ZK0 was subjected to genetic manipulation to improve iturin A production. By the independent or simultaneous overexpression of two regulatory genes (comA and sigA), iturin A production by B. subtilis ZK0 was significantly increased. When both genes were simultaneously overexpressed under optimal conditions, iturin A production increased up to 215 mg l -1 (an approximate 43-fold increase compared with B. subtilis ZK0). Moreover, overexpression of both genes was unexpectedly found to inhibit biofilm formation by B. subtilis ZK0, indicating that the phenomenon of 'stuck fermentation' would be avoided during B. subtilis ZK0 fermentation. In conclusion, a genetic manipulation method that improves iturin A production and inhibits biofilm formation in B. subtilis ZK0 is reported for the first time and this method has the potential to be widely applied in B. subtilis ZK0 commercial fermentation. This study provides new perspectives on improving iturin A production by Bacillus subtilis. Our newly engineered strains could be applied to commercial fermentation by enhancing yields of iturin A and reducing the rate of 'stuck fermentation'. Increased production would facilitate more widespread application of this powerful antibiotic. © 2017 The Society for Applied Microbiology.

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

USDA-ARS?s Scientific Manuscript database

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

Experimental Research on the Sterilization of Escherichia Coli and Bacillus Subtilis in Drinking Water by Dielectric Barrier Discharge

NASA Astrophysics Data System (ADS)

Li, Yang; Yi, Chengwu; Li, Jingjing; Yi, Rongjie; Wang, Huijuan

2016-02-01

The bactericidal effect on the representative type of Gram-negative Escherichia coli (E. coli) and Gram-positive Bacillus subtilis in drinking water was investigated in this paper by using dielectric barrier discharge (DBD) advanced oxidation technology. The sterilizing rates under different conditions of reaction time t, input voltage V, pH value, and initial concentration of bacteria C0 were investigated to figure out the optimum sterilization conditions. Our observations and comparisons of cell morphology alteration by scanning electron microscopy and transmission electron microscopy revealed the sterilization mechanisms. The results showed that the sterilizing rate increased obviously with the extension of reaction time t and the rise of input voltage V. The optimal sterilization effect was achieved when the pH value was 7.1. As the initial concentration of bacteria rose, the sterilizing rate decreased. When the input voltage was 2.2 kV and the initial concentration of bacteria was relatively low, the sterilizing rate almost reached 100% after a certain treatment time in neutral aqueous solution. The reasons for the great damage of cell structure and the killing of bacteria are the oxidation of O3, OH and the accumulation of active species produced by DBD. The article provides a certain theoretical and experimental basis for DBD application in water pollution treatment. supported by the Science and Technology Support Project Plan and Social Development of Jiangsu Province, China (No. BE2011732), the Science and Technology Support Project Plan and Social Development of Zhenjiang, Jiangsu Province, China (No. SH2012013)

Antimicrobial and plant growth-promoting properties of the cacao endophyte Bacillus subtilis ALB629.

PubMed

Falcäo, L L; Silva-Werneck, J O; Vilarinho, B R; da Silva, J P; Pomella, A W V; Marcellino, L H

2014-06-01

To investigate the effects of the endophyte Bacillus subtilisALB629 on the growth of cacao seedlings at early developmental stage and to evaluate its antimicrobial properties. Germinating cacao seeds were inoculated with ALB629, and seedlings growth was evaluated 30 days later. Significant increase (P < 0·05) was observed in the root system (up to 30%), leaf area (14%) and stem height (7·6%). ALB629 colonized the entire plant, prevailing over indigenous micro-organisms. In addition, it was tested in vitro, by pairing assays, and showed antagonistic effect against the phytopathogenic fungi Moniliophthora perniciosa, Colletotrichum sp. and C. gossypii. When tested in cacao-grafting procedure in the field, ALB629 increased the grafting success rate (24%), indicating its protective effect. In addition, this Bacillus secretes an antagonist compound, as shown by the antifungal activity of the cell-free culture. Bacillus subtilisALB629 promotes cacao root growth, besides promoting growth of the aerial part of cacao seedlings. It has antimicrobial properties and produces an antifungal compound. ALB629 presented beneficial characteristics for cacao cultivation, being a good biological control agent candidate. Furthermore, it is a potential source of antifungal compound with potential for commercial exploitation. © 2014 The Society for Applied Microbiology.

Successful Conversion of the Bacillus subtilis BirA Group II Biotin Protein Ligase into a Group I Ligase

PubMed Central

Henke, Sarah K.; Cronan, John E.

2014-01-01

Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity. PMID:24816803

Inhibitory effects of Bacillus subtilis on plant pathogens of conservatory in high latitudes

NASA Astrophysics Data System (ADS)

Xue, Chun-Mei; Wang, Xue; Yang, Jia-Li; Zhang, Yue-Hua

2018-03-01

Researching the effect of three kinds of Bacillus and their mixed strains inhibitory on common fungal diseases of conservatory vegetables. The results showed that B. megaterium culture medium had a significant inhibition effect on Cucumber Fusarium wilt, and the inhibition rate was up to 84.36%; B. mucilaginosus and B. megaterium sterile superna-tant had an obvious inhibitory effect on brown disease of eggplant, and the inhibition rate as high as 85.49%; B. subtilis sterile supernatant had a good inhibitory effect on the spore germination of C. Fusarium wilt, and the inhibition rate was 76.83%. The results revealed that Bacillus had a significant inhibitory effect on five common fungal pathogens. Three kinds of Bacillus can be used for the prevention and control of common fungal diseases in conservatory vegetables.

Purine biosynthesis is the bottleneck in trimethoprim-treated Bacillus subtilis.

PubMed

Stepanek, Jennifer Janina; Schäkermann, Sina; Wenzel, Michaela; Prochnow, Pascal; Bandow, Julia Elisabeth

2016-10-01

Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Suitability of Bacillus subtilis and Bacillus stearothermophilus spores as test organism bioindicators for detecting superheating of steam].

PubMed

Spicher, G; Peters, J

1997-02-01

Biological indicators used to test sterilisation procedures for their efficacy consist of a so-called germ carrier to which the microorganisms used as test organisms adhere. In previous papers we demonstrated that carriers made of filter paper on contact with saturated steam show superheating while carriers made of glass fibre fleece as well as wetted filter paper do not. Using spores of Bacillus subtilis and Bacillus stearothermophilus as test organisms we have now investigated whether and to what extent carrier superheating affects the characteristic values (t50%) of these biological indicators. The indicators were exposed to saturated steam at 100 degrees C (B. subtilis) or 120 degrees C (B. stearothermophilus) under three different exposure conditions: 1. dry (i.e. conditioned to 45% relative humidity before introduction into the sterilising chamber), freely accessible; 2. dry with a substratum and a cover of filter card-board; 3. wet (moistened with twice distilled water before introduction into the sterilising chamber), freely accessible. For previously selected exposure periods, the incidence of indicators with surviving test organisms was determined. The reaction pattern of bioindicators with spores of B. stearothermophilus was different from that of bioindicators with spores of B. subtilis. For B. subtilis, the incidence of bioindicators exhibiting surviving test organisms depended on the nature of the carries as well as on the exposure conditions. On filter paper carriers, t50% increased in the order "wet, freely accessible", "dry, freely accessible", "dry, between filter card-board". On dry and wetted glass fibre fleece, resistance was approximately the same; when the indicators were sandwiched between layers of filter card-board, t50% increased. For B. stearothermophilus, t50% was largely dependent on the carrier material alone. The values obtained for filter paper were invariably much lower than those for glass fibre fleece. As the results show, using

Inhibitory activity of surfactin, produced by different Bacillus subtilis subsp. subtilis strains, against Listeria monocytogenes sensitive and bacteriocin-resistant strains.

PubMed

Sabaté, Daniela C; Audisio, M Carina

2013-03-30

Three surfactin-producing Bacillus subtilis strains, C4, M1 and G2III, previously isolated from honey and intestines from the Apis mellifera L. bee, were phylogenetically characterized at sub-species level as B. subtilis subsp. subtilis using gyrA gene sequencing. The antagonistic effect of surfactin was studied against seven different Listeria monocytogenes strains, 6 of which were resistant to bacteriocins. Surfactin showed anti-Listeria activity against all 7 strains and a dose of 0.125 mg/mL of surfactin was enough to inhibit this pathogen. Surfactin sintetized by B. subtilis subsp. subtilis C4 inhibited the pathogen in lower concentrations, 0.125 mg/mL, followed by G2III and M1 with 0.25 and 1mg/mL, respectively. In particular, a dose of 0.125 mg/mL reduced the viability of L. monocytogenes 99/287 RB6, a bacteriocin-resistant strain, to 5 log orders. Surfactin assayed maintained anti-Listeria activity within a pH range of between 2 and 10, after heat treatment (boiling for 10 min and autoclaving at 121 °C for 15 min) and after treatment with proteolytic enzymes. These results suggest that surfactin can be used as a new tool for prevention and the control of L. monocytogenes in different environments, for example, in the food industry. Copyright © 2012 Elsevier GmbH. All rights reserved.

Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action

PubMed Central

de Souza, Renata Damásio; Batista, Milene Tavares; Luiz, Wilson Barros; Cavalcante, Rafael Ciro Marques; Amorim, Jaime Henrique; Bizerra, Raíza Sales Pereira; Martins, Eduardo Gimenes; de Souza Ferreira, Luís Carlos

2014-01-01

Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains. PMID:24475289

The Highly Conserved Asp23 Family Protein YqhY Plays a Role in Lipid Biosynthesis in Bacillus subtilis

PubMed Central

Tödter, Dominik; Gunka, Katrin; Stülke, Jörg

2017-01-01

In most bacteria, fatty acid biosynthesis is an essential process that must be controlled by the availability of precursors and by the needs of cell division. So far, no mechanisms controlling synthesis of malonyl-coenzyme A (CoA), the committed step in fatty acid synthesis, have been identified in the Gram-positive model bacterium Bacillus subtilis. We have studied the localization and function of two highly expressed proteins of unknown function, YqhY and YloU. Both proteins are members of the conserved and widespread Asp23 family. While the deletion of yloU had no effect, loss of the yqhY gene induced the rapid acquisition of suppressor mutations. The vast majority of these mutations affect subunits of the acetyl-CoA carboxylase (ACCase) complex, the enzyme that catalyzes the formation of malonyl-CoA. Moreover, lack of yqhY is accompanied by the formation of lipophilic clusters in the polar regions of the cells indicating an increased activity of ACCase. Our results suggest that YqhY controls the activity of ACCase and that this control results in inhibition of ACCase activity. Hyperactivity of the enzyme complex in the absence of YqhY does then provoke mutations that cause reduced ACCase activity. PMID:28579978

Interactions between Streptomyces coelicolor and Bacillus subtilis: Role of surfactants in raising aerial structures.

PubMed

Straight, Paul D; Willey, Joanne M; Kolter, Roberto

2006-07-01

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.

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.

MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis.

PubMed

Ito, Masahiro; Hicks, David B; Henkin, Tina M; Guffanti, Arthur A; Powers, Benjamin D; Zvi, Lior; Uematsu, Katsuyuki; Krulwich, Terry A

2004-08-01

The stator-force generator that drives Na+-dependent motility in alkaliphilic Bacillus pseudofirmus OF4 is identified here as MotPS, MotAB-like proteins with genes that are downstream of the ccpA gene, which encodes a major regulator of carbon metabolism. B. pseudofirmus OF4 was only motile at pH values above 8. Disruption of motPS resulted in a non-motile phenotype, and motility was restored by transformation with a multicopy plasmid containing the motPS genes. Purified and reconstituted MotPS from B. pseudofirmus OF4 catalysed amiloride analogue-sensitive Na+ translocation. In contrast to B. pseudofirmus, Bacillus subtilis contains both MotAB and MotPS systems. The role of the motPS genes from B. subtilis in several motility-based behaviours was tested in isogenic strains with intact motAB and motPS loci, only one of the two mot systems or neither mot system. B. subtilis MotPS (BsMotPS) supported Na+-stimulated motility, chemotaxis on soft agar surfaces and biofilm formation, especially after selection of an up-motile variant. BsMotPS also supported motility in agar soft plugs immersed in liquid; motility was completely inhibited by an amiloride analogue. BsMotPS did not support surfactin-dependent swarming on higher concentration agar surfaces. These results indicate that BsMotPS contributes to biofilm formation and motility on soft agar, but not to swarming, in laboratory strains of B. subtilis in which MotAB is the dominant stator-force generator. BsMotPS could potentially be dominant for motility in B. subtilis variants that arise in particular niches.

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions.

PubMed

Ploss, Tina N; Reilman, Ewoud; Monteferrante, Carmine G; Denham, Emma L; Piersma, Sjouke; Lingner, Anja; Vehmaanperä, Jari; Lorenz, Patrick; van Dijl, Jan Maarten

2016-03-29

Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. Here we show that activity of the htrB promoter as induced by overproduction of AmyM was "noisy", which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

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…

Variation of gene expression in Bacillus subtilis samples of fermentation replicates.

PubMed

Zhou, Ying; Yu, Wen-Bang; Ye, Bang-Ce

2011-06-01

The application of comprehensive gene expression profiling technologies to compare wild and mutated microorganism samples or to assess molecular differences between various treatments has been widely used. However, little is known about the normal variation of gene expression in microorganisms. In this study, an Agilent customized microarray representing 4,106 genes was used to quantify transcript levels of five-repeated flasks to assess normal variation in Bacillus subtilis gene expression. CV analysis and analysis of variance were employed to investigate the normal variance of genes and the components of variance, respectively. The results showed that above 80% of the total variation was caused by biological variance. For the 12 replicates, 451 of 4,106 genes exhibited variance with CV values over 10%. The functional category enrichment analysis demonstrated that these variable genes were mainly involved in cell type differentiation, cell type localization, cell cycle and DNA processing, and spore or cyst coat. Using power analysis, the minimal biological replicate number for a B. subtilis microarray experiment was determined to be six. The results contribute to the definition of the baseline level of variability in B. subtilis gene expression and emphasize the importance of replicate microarray experiments.

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.

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. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Cortex content of asporogenous mutants of Bacillus subtilis.

PubMed Central

Imae, Y; Strominger, J L

1976-01-01

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none. PMID:1262319

A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

PubMed

Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

2017-08-01

To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

The sensitivity of Bacillus subtilis to diverse antimicrobial compounds is influenced by Abh.

PubMed

Murray, Ewan J; Stanley-Wall, Nicola R

2010-12-01

Abh is a transition state regulator of Bacillus subtilis that controls biofilm formation and the production of several diverse antimicrobial compounds. Using a high-throughput non-biased technique, we show for the first time that Abh influences the sensitivity of B. subtilis to diverse antimicrobial compounds. Following up on these findings with a combination of classical genetics and antibiotic susceptibility assays, we demonstrate that Abh influences cellular processes such as the remodelling of the cell wall. We present data demonstrating that the extracytoplasmic function sigma factor σ(X) controls resistance to β-lactam antibiotics by activating abh transcription. Downstream from Abh, activation of slrR expression by Abh is responsible for controlling the sensitivity of B. subtilis to such antibiotics due to the role that SlrR plays in regulating autolysin biosynthesis. The abh mutant additionally exhibits increased resistance to aminoglycoside antimicrobials. We confirm that aminoglycoside killing of B. subtilis is likely to be caused by oxidative damage but rule out the possibility that the increased resistance of the abh mutant to aminoglycosides is due to a general increase in resistance to oxidative stress.

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.

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

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.

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism*

PubMed Central

Murray, David S.; Chinnam, Nagababu; Tonthat, Nam Ky; Whitfill, Travis; Wray, Lewis V.; Fisher, Susan H.; Schumacher, Maria A.

2013-01-01

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg62, from an adjacent subunit. Notably, Arg62 must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens. PMID:24158439

Comparative evaluation of extracellular β-D-fructofuranosidase in submerged and solid-state fermentation produced by newly identified Bacillus subtilis strain.

PubMed

Lincoln, Lynette; More, Sunil S

2018-04-17

To screen and identify a potential extracellular β-D-fructofuranosidase or invertase producing bacterium from soil, and comparatively evaluate the enzyme biosynthesis under submerged and solid-state fermentation. Extracellular invertase producing bacteria were screened from soil. Identification of the potent bacterium was performed based on microscopic examinations and 16S rDNA molecular sequencing. Bacillus subtilis LYN12 invertase secretion was surplus with wheat bran humidified with molasses medium (70%), with elevated activity at 48 h and 37 °C under solid-state fermentation, whereas under submerged conditions increased activity was observed at 24 h and 45 °C in the molasses medium. The study revealed a simple fermentative medium for elevated production of extracellular invertase from a fast growing Bacillus strain. Bacterial invertases are scarce and limited reports are available. By far, this is the first report on the comparative analysis of optimization of extracellular invertase synthesis from Bacillus subtilis strain by submerged and solid-state fermentation. The use of agricultural residues increased yields resulting in development of a cost-effective and stable approach. Bacillus subtilis LYN12 invertase possesses excellent fermenting capability to utilize agro-industrial residues under submerged and solid-state conditions. This could be a beneficial candidate in food and beverage processing industries. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

PubMed Central

Yocum, R R; Perkins, J B; Howitt, C L; Pero, J

1996-01-01

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli. PMID:8755891

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

PubMed

Yocum, R R; Perkins, J B; Howitt, C L; Pero, J

1996-08-01

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.

Fungal Competitors Affect Production of Antimicrobial Lipopeptides in Bacillus subtilis Strain B9-5.

PubMed

DeFilippi, Stefanie; Groulx, Emma; Megalla, Merna; Mohamed, Rowida; Avis, Tyler J

2018-04-01

Bacillus subtilis has shown success in antagonizing plant pathogens where strains of the bacterium produce antimicrobial cyclic lipopeptides (CLPs) in response to microbial competitors in their ecological niche. To gain insight into the inhibitory role of these CLPs, B. subtilis strain B9-5 was co-cultured with three pathogenic fungi. Inhibition of mycelial growth and spore germination was assessed and CLPs produced by B. subtilis B9-5 were quantified over the entire period of microbial interaction. B. subtilis B9-5 significantly inhibited mycelial growth and spore germination of Fusarium sambucinum and Verticillium dahliae, but not Rhizopus stolonifer. LC-MS analysis revealed that B. subtilis differentially produced fengycin and surfactin homologs depending on the competitor. CLP quantification suggested that the presence of Verticillium dahliae, a fungus highly sensitive to the compounds, caused an increase followed by a decrease in CLP production by the bacterium. In co-cultures with Fusarium sambucinum, a moderately sensitive fungus, CLP production increased more gradually, possibly because of its slower rate of spore germination. With co-cultures of the tolerant fungus Rhizopus stolonifer, B. subtilis produced high amounts of CLPs (per bacterial cell) for the duration of the interaction. Variations in CLP production could be explained, in part, by the pathogens' overall sensitivities to the bacterial lipopeptides and/or the relative growth rates between the plant pathogen and B. subtilis. CLP production varied substantially temporally depending on the targeted fungus, which provides valuable insight concerning the effectiveness of B. subtilis B9-5 protecting its ecological niche against the ingress of these pathogens.

Inhibition of quorum sensing-mediated virulence in Serratia marcescens by Bacillus subtilis R-18.

PubMed

Devi, Kannan Rama; Srinivasan, Subramaniyan; Ravi, Arumugam Veera

2018-04-13

Serratia marcescens is an opportunistic human pathogen causing various nosocomial infections, most importantly urinary tract infections (UTIs). It exhibits increased resistance towards the conventional antibiotics. This study was aimed to evaluate the anti-virulence effect of a rhizosphere soil bacterium Bacillus subtilis strain R-18 against the uropathogen S. marcescens. First, the bacterial cell-free culture supernatant (CFCS) of B. subtilis strain R-18 was evaluated for its quorum sensing inhibitory (QSI) potential against biomarker strain Chromobacterium violaceum and the test pathogen S. marcescens. The B. subtilis R-18 CFCS effectively inhibited the quorum sensing (QS)-mediated violacein pigment production in C. violaceum and prodigiosin pigment production in S. marcescens. Furthermore, B. subtilis R-18 CFCS was successively extracted with different solvent systems. Of these solvents, B. subtilis R-18 petroleum ether (PE) extract showed inhibition in biofilm formation, protease, lipase, and hemolysin productions in S. marcescens. Fourier transform infrared spectroscopic (FT-IR) analysis revealed the alterations in the cellular components of bacterial cell pellets obtained from B. subtilis R-18 PE extract treated and untreated S. marcescens. The differential gene expression study further validated the downregulation of virulence-associated genes. Characterization of the active principle in B. subtilis R-18 PE extract by gas chromatography-mass spectrometry (GC-MS) analysis showed the presence of multiple compounds with therapeutic values, which could possibly reduce the QS-dependent phenotypes in S. marcescens. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy-d-Xylulose-5-Phosphate Pathway in Bacillus subtilis▿ †

PubMed Central

Xue, Junfeng; Ahring, Birgitte K.

2011-01-01

To enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacterium Bacillus subtilis, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed in B. subtilis DSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H2O2) stress, on isoprene production were further examined. Heat, salt, and H2O2 induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria. PMID:21296950

Effects of microbial loading and sporulation temperature on atmospheric plasma inactivation of Bacillus subtilis spores

NASA Astrophysics Data System (ADS)

Deng, X. T.; Shi, J. J.; Shama, G.; Kong, M. G.

2005-10-01

Current inactivation studies of Bacillus subtilis spores using atmospheric-pressure glow discharges (APGD) do not consider two important factors, namely microbial loading at the surface of a substrate and sporulation temperature. Yet these are known to affect significantly microbial resistance to heat and hydrogen peroxide. This letter investigates effects of microbial loading and sporulation temperature on spore resistance to APGD. It is shown that microbial loading can lead to a stacking structure as a protective shield against APGD treatment and that high sporulation temperature increases spore resistance by altering core water content and cross-linked muramic acid content of B. subtilis spores.

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

Foam separation of Pseudomonas fluorescens and Bacillus subtilis var. niger.

PubMed

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 mueq/ml of NaCl, KCl, Na(2)SO(4), K(2)SO(4), CaCl(2), CaSO(4), MgCl(2), or MgSO(4) produced increases in the cell concentration in the residual suspension (not carried into the foam) from 2.9 x 10(5) up to 1.6 x 10(6) to 2.8 x 10(7) cells per milliliter (initial suspensions contain from 3.3 x 10(7) to 4.8 x 10(7) 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 mueq/ml of any of the eight salts increased the residual cell concentration by one order of magnitude from 1.2 x 10(4) to about 4.0 x 10(5) 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.

Fitness Trade-Offs in Competence Differentiation of Bacillus subtilis

PubMed Central

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

2016-01-01

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

Fitness Trade-Offs in Competence Differentiation of Bacillus subtilis.

PubMed

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

2016-01-01

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

Characterization of a Stable L-Form of Bacillus subtilis 168

PubMed Central

Gilpin, Richard W.; Young, Frank E.; Chatterjee, Anadi N.

1973-01-01

A stable L-form of Bacillus subtilis 168 (sal-1) has been isolated which grows and divides logarithmically in liquid medium with a generation time of 60 min. This mutant does not synthesize cell wall as evidenced by chemical, biochemical, and morphological analyses. Antibiotics which specifically inhibit cell wall biosynthesis do not affect the growth of the L-form. Significant differences exist between the membrane proteins of the bacillary form and the L-form. The relative profile of membrane proteins varies with the salt concentration of the medium in both the L-form and the bacillary form. Images PMID:4631836

ANTIMICROBIAL ACTION OF THE LEAF EXTRACT OF MORINGA OLEIFERA LAM.

PubMed Central

Pal, Saroj K.; Mukherjee, Pulok K.; Saha, Kakali; Pal, M.; Saha, B.P.

1995-01-01

The ethnolic extract of the leaves of Moringa oleifera Lam. (Fam. Moringaceae) was tested for antimicrobial activities against Gram Positive – Bacillus cereus, Bacillus subtilis Staphylococcus aureus, Sarcina lutea: Gram negative – Escherichia coli and Acid fast Mycobacterium phlei. Significant antimicrobial activity of the extract was found in this study. PMID:22556699

An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA.

PubMed

Vivian, J P; Porter, C J; Wilce, J A; Wilce, M C J

2007-07-13

In Bacillus subtilis, the termination of DNA replication via polar fork arrest is effected by a specific protein:DNA complex formed between the replication terminator protein (RTP) and DNA terminator sites. We report the crystal structure of a replication terminator protein homologue (RTP.C110S) of B. subtilis in complex with the high affinity component of one of its cognate DNA termination sites, known as the TerI B-site, refined at 2.5 A resolution. The 21 bp RTP:DNA complex displays marked structural asymmetry in both the homodimeric protein and the DNA. This is in contrast to the previously reported complex formed with a symmetrical TerI B-site homologue. The induced asymmetry is consistent with the complex's solution properties as determined using NMR spectroscopy. Concomitant with this asymmetry is variation in the protein:DNA binding pattern for each of the subunits of the RTP homodimer. It is proposed that the asymmetric "wing" positions, as well as other asymmetrical features of the RTP:DNA complex, are critical for the cooperative binding that underlies the mechanism of polar fork arrest at the complete terminator site.

Hematite enhances the removal of Cr(VI) by Bacillus subtilis BSn5 from aquatic environment.

PubMed

Ma, Shuai; Song, Chang-Shun; Chen, Yuefang; Wang, Fei; Chen, Hui-Lun

2018-06-05

In the present study, we investigated the removal of Cr(VI) and the associated bacterial activity in the systems containing Bacillus subtilis BSn5 (B. subtilis BSn5) and hematite. The microcalorimetry was used to study the effect of hematite on the normal physiological functions of B. subtilis BSn5 towards the removal of Cr(VI) for the first time. The results of the heat flux and the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that hematite does not affect the normal physiological functions of B. subtilis BSn5, and can help the strains maintain their activity in the presence of Cr(VI). More importantly, the relative capacity and intensity of Cr(VI) and total Cr removal by B. subtilis BSn5 in the presence of hematite were higher than that in the absence of hematite. The enhancement effect could be associated with their mineral adsorption, biosorption, Fe(II) reduction, bioreduction and immobilization functions. This study demonstrates the possibility of reducing the toxicity of Cr(VI) and enhancing the Cr(VI) removal efficiency in contaminated environments using a combination of hematite and B. subtilis BSn5. Copyright © 2018 Elsevier Ltd. All rights reserved.

A singular enzymatic megacomplex from Bacillus subtilis.

PubMed

Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

2007-01-02

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

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

Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization.

PubMed

Defeu Soufo, Hervé Joël; Graumann, Peter L

2005-03-03

Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds. In this work, we show that Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell. Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner.

Dry Heat Inactivation of Bacillus subtilis var. niger Spores as a Function of Relative Humidity

PubMed Central

Brannen, J. P.; Garst, D. M.

1972-01-01

Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations. Images PMID:4625341

Immune responses induced by recombinant Bacillus subtilis expressing the spike protein of transmissible gastroenteritis virus in pigs.

PubMed

Mou, Chunxiao; Zhu, Liqi; Xing, Xianping; Lin, Jian; Yang, Qian

2016-07-01

Transmissible gastroenteritis (TGE) causes severe diarrhea in suckling piglets, results in enormous economic loss in swine-producing areas of the world. To develop an effective, safe, and convenient vaccine for the prevention of TGE, we have constructed a recombinant Bacillus subtilis strain (B. subtilis CotGSG) displaying the transmissible gastroenteritis virus (TGEV) spike (S) protein and discussed its immune function to intestinal submucosal dendritic cells (DCs). Our results showed that the recombinant B. subtilis had the ability to recruit more DCs to sample B. subtilis CotGSG, migrate to MLNs, and induce immune responses. Immunized piglets with B. subtilis CotGSG could significantly elevate the specific SIgA titers in feces, IgG titers and neutralizing antibodies in serum. Collectively, our results suggested that recombinant B. subtilis CotGSG expressing the TGEV S protein could effectively induce immune responses via DCs, and provided a perspective on potential novel strategy and approach that may be applicable to the development of the next generation of TGEV vaccines. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacillus subtilis Fur represses one of two paralogous haem-degrading monooxygenases

PubMed Central

Gaballa, Ahmed

2011-01-01

Identification of genes regulated by the ferric uptake regulator (Fur) protein has provided insights into the diverse mechanisms of adaptation to iron limitation. In the soil bacterium Bacillus subtilis, Fur senses iron sufficiency and represses genes that enable iron uptake, including biosynthetic and transport genes for the siderophore bacillibactin and uptake systems for siderophores produced by other organisms. We here demonstrate that Fur regulates hmoA (formerly yetG), which encodes a haem monooxygenase. HmoA is the first characterized member of a divergent group of putative monooxygenases that cluster separately from the well-characterized IsdG family. B. subtilis also encodes an IsdG family protein designated HmoB (formerly YhgC). Unlike hmoA, hmoB is constitutively expressed and not under Fur control. HmoA and HmoB both bind haemin in vitro with approximately 1 : 1 stoichiometry and degrade haemin in the presence of an electron donor. Mutational and spectroscopic analyses indicate that HmoA and HmoB have distinct active site architectures and interact differently with haem. We further show that B. subtilis can use haem as an iron source, but that this ability is independent of HmoA and HmoB. PMID:21873409

Antimicrobial Activities of Leaf Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and Gram-Positive Bacteria

PubMed Central

Biswas, Bipul; Rogers, Kimberly; McLaughlin, Fredrick; Yadav, Anand

2013-01-01

Aim. To determine the antimicrobial potential of guava (Psidium guajava) leaf extracts against two gram-negative bacteria (Escherichia coli and Salmonella enteritidis) and two gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) which are some of foodborne and spoilage bacteria. The guava leaves were extracted in four different solvents of increasing polarities (hexane, methanol, ethanol, and water). The efficacy of these extracts was tested against those bacteria through a well-diffusion method employing 50 μL leaf-extract solution per well. According to the findings of the antibacterial assay, the methanol and ethanol extracts of the guava leaves showed inhibitory activity against gram-positive bacteria, whereas the gram-negative bacteria were resistant to all the solvent extracts. The methanol extract had an antibacterial activity with mean zones of inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of 6.11 and 11.0 mm against B. cereus and S. aureus, respectively. On the basis of the present finding, guava leaf-extract might be a good candidate in the search for a natural antimicrobial agent. This study provides scientific understanding to further determine the antimicrobial values and investigate other pharmacological properties. PMID:24223039

Isolation and Characterization of Phages Infecting Bacillus subtilis

PubMed Central

Biegalska, Anna; Łoś, Marcin; Richert, Malwina

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

Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene.

PubMed Central

Recorbet, G; Robert, C; Givaudan, A; Kudla, B; Normand, P; Faurie, G

1993-01-01

The sacB gene from Bacillus subtilis confers sucrose sensitivity upon gram-negative bacteria. The gene was investigated for use as a potential conditional suicide system for Escherichia coli released into soil. To ensure against the loss of the cell death function encoded under nonselective conditions, the nptI-sacR-B suicide cassette was inserted into the E. coli chromosome by using a circular nonreplicative integration vector. Stability studies yielded no loss of the suicide cassette in the integrated E. coli EL1026 strain. sacB induction in the absence of a selective pressure resulted in a lysis efficiency of up to 99.9%. The microcosm experiments confirmed the ability of the suicide cassette to limit the growth and reduce the survival of E. coli strains released into soil. Sucrose addition to sterile soil resulted in a 10(-3)-fold reduction of the final E. coli population density. sacB induction prevented the proliferation and triggered the rapid disappearance of E. coli from natural soil. Mutation to sucrose tolerance occurred at a frequency of 10(-5), making E. coli EL1026 a potential counterselectable donor strain for gene transfer studies. Specificity and potential adaptability to a wide range of gram-negative bacteria are additional conveniences of this conditional suicide system for the containment and counterselection of engineered microorganisms. PMID:8517732

Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene.

PubMed

Recorbet, G; Robert, C; Givaudan, A; Kudla, B; Normand, P; Faurie, G

1993-05-01

The sacB gene from Bacillus subtilis confers sucrose sensitivity upon gram-negative bacteria. The gene was investigated for use as a potential conditional suicide system for Escherichia coli released into soil. To ensure against the loss of the cell death function encoded under nonselective conditions, the nptI-sacR-B suicide cassette was inserted into the E. coli chromosome by using a circular nonreplicative integration vector. Stability studies yielded no loss of the suicide cassette in the integrated E. coli EL1026 strain. sacB induction in the absence of a selective pressure resulted in a lysis efficiency of up to 99.9%. The microcosm experiments confirmed the ability of the suicide cassette to limit the growth and reduce the survival of E. coli strains released into soil. Sucrose addition to sterile soil resulted in a 10(-3)-fold reduction of the final E. coli population density. sacB induction prevented the proliferation and triggered the rapid disappearance of E. coli from natural soil. Mutation to sucrose tolerance occurred at a frequency of 10(-5), making E. coli EL1026 a potential counterselectable donor strain for gene transfer studies. Specificity and potential adaptability to a wide range of gram-negative bacteria are additional conveniences of this conditional suicide system for the containment and counterselection of engineered microorganisms.

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

A novel expression vector for the secretion of abaecin in Bacillus subtilis.

PubMed

Li, Li; Mu, Lan; Wang, Xiaojuan; Yu, Jingfeng; Hu, Ruiping; Li, Zhen

This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5μM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5μM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

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. Copyright 2010 Elsevier B.V. All rights reserved.

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.

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

PubMed

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

2016-07-19

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.

Metabolic engineering of Bacillus subtilis for production of D-lactic acid.

PubMed

Awasthi, Deepika; Wang, Liang; Rhee, Mun S; Wang, Qingzhao; Chauliac, Diane; Ingram, Lonnie O; Shanmugam, Keelnatham T

2018-02-01

Poly lactic acid (PLA) based plastics is renewable, bio-based, and biodegradable. Although present day PLA is composed of mainly L-LA, an L- and D- LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D-LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D-lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D-LDHs were purified and biochemically characterized. Among these, D-LDH from L. delbrueckii subspecies bulgaricus supported the highest D-LA titer (about 1M) and productivity (2 g h -1  g cells -1 ) at 37°C (B. subtilis strain DA12). The D-LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D-LA g -1 glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g -1 glucose and the D-lactate titer was 180 ± 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost. © 2017 Wiley Periodicals, Inc.

Sensitivity of bacteria to diamond nanoparticles of various size differs in gram-positive and gram-negative cells.

PubMed

Beranová, Jana; Seydlová, Gabriela; Kozak, Halyna; Benada, Oldřich; Fišer, Radovan; Artemenko, Anna; Konopásek, Ivo; Kromka, Alexander

2014-02-01

In this study, the influence of the size and surface termination of diamond nanoparticles (DNPs) on their antibacterial activity against Escherichia coli and Bacillus subtilis was assessed. The average size and distribution of DNPs were determined by dynamic light scattering and X-ray diffraction techniques. The chemical composition of the DNPs studied by X-ray photoelectron spectroscopy showed that DNPs > 5 nm and oxidized particles have a higher oxygen content. The antibacterial potential of DNPs was assessed by the viable count method. In general, E. coli exhibited a higher sensitivity to DNPs than B. subtilis. However, in the presence of all the DNPs tested, the B. subtilis colonies exhibited altered size and morphology. Antibacterial activity was influenced not only by DNP concentration but also by DNP size and form. Whereas untreated 5-nm DNPs were the most effective against E. coli, the antibacterial activity of 18-50-nm DNPs was higher against B. subtilis. Transmission electron microscopy showed that DNPs interact with the bacterial surface, probably affecting vital cell functions. We propose that DNPs interfere with the permeability of the bacterial cell wall and/or membrane and hinder B. subtilis colony spreading. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75.

PubMed

Shah, Ziaullah; Krumholz, Lee; Aktas, Deniz Fulya; Hasan, Fariha; Khattak, Mutiullah; Shah, Aamer Ali

2013-11-01

A polyurethane (PU) degrading bacterial strain MZA-75 was isolated from soil through enrichment technique. The bacterium was identified through 16S rRNA gene sequencing, the phylogenetic analysis indicated the strain MZA-75 belonged to genus Bacillus having maximum similarity with Bacillus subtilis strain JBE0016. The degradation of PU films by strain MZA-75 in mineral salt medium (MSM) was analyzed by scanning electron microscopy (SEM), fourier transform infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM revealed the appearance of widespread cracks on the surface. FTIR spectrum showed decrease in ester functional group. Increase in polydispersity index was observed in GPC, which indicates chain scission as a result of microbial treatment. CO2 evolution and cell growth increased when PU was used as carbon source in MSM in Sturm test. Increase in both cell associated and extracellular esterases was observed in the presence of PU indicated by p-Nitrophenyl acetate (pNPA) hydrolysis assay. Analysis of cell free supernatant by gas chromatography-mass spectrometry (GC-MS) revealed that 1,4-butanediol and adipic acid monomers were produced. Bacillus subtilis strain MZA-75 can degrade the soft segment of polyester polyurethane, unfortunately no information about the fate of hard segment could be obtained. Growth of strain MZA-75 in the presence of these metabolites indicated mineralization of ester hydrolysis products into CO2 and H2O.

Selection and evaluation of Malaysian Bacillus spp. strains as potential probiotics in cultured tiger grouper (Epinephelus fuscoguttatus).

PubMed

Yasin, Ina-salwany Md; Razak, Nabilah Fatin; Natrah, F M I; Harmin, Sharr Azni

2016-07-01

A total of 58 Gram-positive bacteria strains were isolated from the marine environment and screened for potential probiotics for disease prevention and improving the productivity of tiger grouper Epinephelus fuscoguttatus larvae and juveniles. The bacteria were identified as Bacillus licheniformis, B. subtilis, B. circulans, B. sphaericus, B. cereus, Brevibacillus brevis, Corynebacterium propinquum, Leifsonia aquatica and Paenibacillus macerans. Only 24 strains showed antagonistic activities against four pathogenic strains; Vibrio alginolyticus, V. harveyi, V. parahaemolyticus and Aeromonas hydrophila, where two of the Bacillus strains, B12 and B45 demonstrated intermediate to highest level of inhibitory activity against these pathogenic strains, respectively. Further assessment by co-culture assay showed that Bacillus strain B12 exhibited a total inhibition of V. alginolyticus, while B45 strain displayed no inhibitory activity. Mixed culture of Bacillus B12 and B45 strains to outcompete V. alginolyticus was observed at a cell density of 10(7) CFU ml(-1). Molecular identification and phylogenetic tree analysis have categorized Bacillus strain B12 to the reference strains GQ340480 and JX290193 of? B. amyloliquafaciens, and Bacillus strain B45 with a reference strain JF496522 of B. subtilis. Safety tests of probionts by intraperitoneal administration of B12 and B45 strains at cell densities of 103, 105 and 10(7) CFU ml(-1) revealed no abnormalities and cent percent survival for healthy Epinephelus fuscoguttatus juveniles within 15 days of experimental period. Overall, the study revealed that Bacillus B12 strain possesses tremendous probiotic potential that could be used as a feed supplement in tiger grouper diets. ?

Measurement of Metabolic Activity in Dormant Spores of Bacillus Species

DTIC Science & Technology

2015-01-14

SECURITY CLASSIFICATION OF: Spores of Bacillus megaterium and Bacillus subtilis were harvested shortly after release from sporangia, incubated under...Measurement of Metabolic Activity in Dormant Spores of Bacillus Species Report Title Spores of Bacillus megaterium and Bacillus subtilis were...ribosomal RNA when newly harvested Bacillus subtilis spores are incubated at physiological temperatures, as well as some evidence for transcription in

Comparative evaluation of agroindustrial byproducts for the production of alkaline protease by wild and mutant strains of Bacillus subtilis in submerged and solid state fermentation.

PubMed

Mukhtar, Hamid; Haq, Ikramul

2013-01-01

The present study describes the screening of different agroindustrial byproducts for enhanced production of alkaline protease by a wild and EMS induced mutant strain of Bacillus subtilis IH-72(EMS8). During submerged fermentation, different agro-industrial byproducts were tested which include defatted seed meals of rape, guar, sunflower, gluten, cotton, soybean, and gram. In addition to these meals, rice bran, wheat bran, and wheat flour were also evaluated for protease production. Of all the byproducts tested, soybean meal at a concentration of 20 g/L gave maximum production of the enzyme, that is, 5.74  ±  0.26 U/mL from wild and 11.28  ±  0.45 U/mL from mutant strain, during submerged fermentation. Different mesh sizes (coarse, medium, and fine) of the soybean meal were also evaluated, and a finely ground soybean meal (fine mesh) was found to be the best. In addition to the defatted seed meals, their alkali extracts were also tested for the production of alkaline protease by Bacillus subtilis, but these were proved nonsignificant for enhanced production of the enzyme. The production of the enzyme was also studied in solid state fermentation, and different agro-industrial byproducts were also evaluated for enzyme production. Wheat bran partially replaced with guar meal was found as the best substrate for maximum enzyme production under solid state fermentation conditions.

Comparative Evaluation of Agroindustrial Byproducts for the Production of Alkaline Protease by Wild and Mutant Strains of Bacillus subtilis in Submerged and Solid State Fermentation

PubMed Central

Haq, Ikramul

2013-01-01

The present study describes the screening of different agroindustrial byproducts for enhanced production of alkaline protease by a wild and EMS induced mutant strain of Bacillus subtilis IH-72EMS8. During submerged fermentation, different agro-industrial byproducts were tested which include defatted seed meals of rape, guar, sunflower, gluten, cotton, soybean, and gram. In addition to these meals, rice bran, wheat bran, and wheat flour were also evaluated for protease production. Of all the byproducts tested, soybean meal at a concentration of 20 g/L gave maximum production of the enzyme, that is, 5.74  ±  0.26 U/mL from wild and 11.28  ±  0.45 U/mL from mutant strain, during submerged fermentation. Different mesh sizes (coarse, medium, and fine) of the soybean meal were also evaluated, and a finely ground soybean meal (fine mesh) was found to be the best. In addition to the defatted seed meals, their alkali extracts were also tested for the production of alkaline protease by Bacillus subtilis, but these were proved nonsignificant for enhanced production of the enzyme. The production of the enzyme was also studied in solid state fermentation, and different agro-industrial byproducts were also evaluated for enzyme production. Wheat bran partially replaced with guar meal was found as the best substrate for maximum enzyme production under solid state fermentation conditions. PMID:24294129

Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species

PubMed Central

de Hoon, Michiel J. L.; Makita, Yuko; Nakai, Kenta; Miyano, Satoru

2005-01-01

In prokaryotes, genes belonging to the same operon are transcribed in a single mRNA molecule. Transcription starts as the RNA polymerase binds to the promoter and continues until it reaches a transcriptional terminator. Some terminators rely on the presence of the Rho protein, whereas others function independently of Rho. Such Rho-independent terminators consist of an inverted repeat followed by a stretch of thymine residues, allowing us to predict their presence directly from the DNA sequence. Unlike in Escherichia coli, the Rho protein is dispensable in Bacillus subtilis, suggesting a limited role for Rho-dependent termination in this organism and possibly in other Firmicutes. We analyzed 463 experimentally known terminating sequences in B. subtilis and found a decision rule to distinguish Rho-independent transcriptional terminators from non-terminating sequences. The decision rule allowed us to find the boundaries of operons in B. subtilis with a sensitivity and specificity of about 94%. Using the same decision rule, we found an average sensitivity of 94% for 57 bacteria belonging to the Firmicutes phylum, and a considerably lower sensitivity for other bacteria. Our analysis shows that Rho-independent termination is dominant for Firmicutes in general, and that the properties of the transcriptional terminators are conserved. Terminator prediction can be used to reliably predict the operon structure in these organisms, even in the absence of experimentally known operons. Genome-wide predictions of Rho-independent terminators for the 57 Firmicutes are available in the Supporting Information section. PMID:16110342

Assessment of the Requirements for Magnesium Transporters in Bacillus subtilis

PubMed Central

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

2014-01-01

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

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

PubMed

Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

2016-01-15

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Bacillus subtilis spreads by surfing on waves of surfactant

PubMed Central

Angelini, Thomas E.; Roper, Marcus; Kolter, Roberto; Weitz, David A.; Brenner, Michael P.

2009-01-01

The bacterium Bacillus subtilis produces the molecule surfactin, which is known to enhance the spreading of multicellular colonies on nutrient substrates by lowering the surface tension of the surrounding fluid, and to aid in the formation of aerial structures. Here we present experiments and a mathematical model that demonstrate how the differential accumulation rates induced by the geometry of the bacterial film give rise to surfactant waves. The spreading flux increases with increasing biofilm viscosity. Community associations are known to protect bacterial populations from environmental challenges such as predation, heat, or chemical stresses, and enable digestion of a broader range of nutritive sources. This study provides evidence of enhanced dispersal through cooperative motility, and points to nonintuitive methods for controlling the spread of biofilms. PMID:19826092

Silver nanocrystallites: Facile biofabrication using Shewanella oneidensis, and an evaluation of their comparative toxicity on Gram-negative and Gram-positive bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suresh, Anil K; Wang, Wei; Pelletier, Dale A

Microorganisms have long been known to develop resistance to metal ions either by sequestering metals inside the cell or by effluxing them into the extracellular media. Here we report the biosynthesis of extracellular silver based single nanocrystallites of well-defined composition and homogeneous morphology utilizing the -proteobacterium, Shewanella oneidensis strain MR-1, upon incubation with an aqueous solution of silver nitrate. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the size range 2 11 nm (with an average of 4 1.5 nm). The bactericidal effect of these biologically synthesized silver nanoparticles (biogenic-Ag) are comparedmore » to similar chemically synthesized nanoparticles (colloidal silver [colloidal-Ag] and oleate capped silver [oleate-Ag]). The determination of the bactericidal effect of these different silver nanoparticles was assessed using both Gram-negative (E. coli) and Gram-positive (B. subtilis) bacteria and based on the diameter of the inhibition zone in disc diffusion tests, minimum inhibitory concentrations, Live/Dead staining assays, and atomic force microscopy. From a toxicity perspective, a clear synthesis procedure, and a surface coat- and strain-dependent inhibition were observed for silver nanoparticles. Biogenic-Ag was found to be of higher toxicity when compared to colloidal-Ag for both E. coli and B. subtilis. E. coli was found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, Oleate-Ag was not toxic to either of the bacteria. These findings have important implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.« less

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

PubMed

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

2015-11-01

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

Bacillus subtilis and yeast cell wall improve the intestinal health of broilers challenged by Clostridium perfringens.

PubMed

Li, Z; Wang, W; Lv, Z; Liu, D; Guo, Y

2017-12-01

1. The objective was to investigate the effects of Bacillus subtilis, yeast cell wall (YCW) and their combination on intestinal health of broilers challenged by Clostridium perfringens over a 21-d period. 2. Using a 5 × 2 factorial arrangement of treatments, 800 1-d-old male Cobb 500 broilers were used to study the effects of feed additives (without additive or with zinc bacitracin, B. subtilis, YCW, and the combination of B. subtilis and YCW), pathogen challenge (without or with Clostridium perfringens challenge), and their interactive effects. 3. C. perfringens infection increased intestinal lesions scores, damaged intestinal histomorphology, increased serum endotoxin concentration, cytokine mRNA expression and intestinal population of C. perfringens and Escherichia coli and decreased ileal bifidobacteria numbers. The 4 additives decreased serum endotoxin. Zinc bacitracin tended to decrease cytokine mRNA expression and the intestinal number of C. perfringens and E. coli. B. subtilis, YCW and their combination increased cytokine mRNA expression. B. subtilis and YCW decreased the number of C. perfringens and E. coli in the ileum, and their combination decreased pathogens numbers in the ileum and caecum. 4. In conclusion, B. subtilis, YCW and their combination improved the intestinal health of NE-infected broilers, and could be potential alternatives to antibiotics.

Effects of a Potential Autochthonous Probiotic Bacillus subtilis 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber, Apostichopus japonicus Selenka

NASA Astrophysics Data System (ADS)

Zhao, Yancui; Yuan, Lei; Wan, Junli; Sun, Hushan; Wang, Yiyan; Zhang, Qin

2018-04-01

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber ( Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0 (control), 105, 107, and 109 CFU g-1 for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 109 CFU g-1 ( P < 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber ( P > 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 105 and 107 CFU g-1 inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 109 CFU g-1 not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.

Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR.

PubMed

Sajitha, K L; Dev, Suma Arun; Maria Florence, E J

2016-07-01

Bacillus subtilis is a potent biocontrol agent producing a wide array of antifungal lipopeptides for the inhibition of fungal growth. B. subtilis B1 isolated from market-available compost provided an efficient control of rubberwood sapstain fungus, Lasiodiplodia theobromae. The current study is aimed to identify and characterize the lipopeptides responsible for the biocontrol of rubberwood sapstain fungus by Bacillus subtilis B1. The bacterial whole-cell surface extract from the dual culture of B. subtilis B1 and sapstain fungus (L. theobromae) was analysed using MALDI-TOF-MS. The protonated as well as sodium, potassium adducts of homologues of iturin C, surfactin, bacillomycin D and fengycin A and B were identified and expression of the lipopeptide biosynthetic genes could be confirmed through RT-PCR. This is the first report of mycobacillin and trimethylsilyl derivative of bacilysin during antagonism through MALDI-TOF-MS. MALDI-TOF-MS with RT-PCR offered easy platforms to characterize the antifungal lipopeptides. The identification of antifungal lipopeptides can lead to the formulation of prospective biocontrol by-products which have wide-scale utility.

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

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

PubMed

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 Δ spoVT 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.

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

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

PubMed

Yi, Jun; Cheng, Jinping

2017-07-01

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

Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

PubMed

Hosseini-Abari, Afrouzossadat; Kim, Byung-Gee; Lee, Sang-Hyuk; Emtiazi, Giti; Kim, Wooil; Kim, June-Hyung

2016-12-01

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO 4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of chromium-enriched bacillus subtilis KT260179 supplementation on chicken growth performance, plasma lipid parameters, tissue chromium levels, cecal bacterial composition and breast meat quality.

PubMed

Yang, Jiajun; Qian, Kun; Zhang, Wei; Xu, Yayuan; Wu, Yijing

2016-11-08

Both chromium (Cr) and probiotic bacillus own the virtues of regulating animal metabolism and meat quality. Purpose of this study was to evaluate the efficiency of supplemental Cr and bacillus in the form of chromium-enriched Bacillus subtilis KT260179 (CEBS) on chicken growth performance, plasma lipid parameters, tissue chromium levels, cecal bacterial composition and breast meat quality. Six hundred of 1-day-old Chinese Huainan Partridge chickens were divided into four groups randomly: Control, inorganic Cr, Bacillus subtilis, and CEBS. The feed duration was 56 days. After 28 days of treatment, broiler feed CEBS or normal B. subtilis had higher body weights than control broiler, and after 56 days, chickens given either CEBS or B. subtilis had greater body weights than control broiler or those given inorganic Cr. Plasma total cholesterol, triglycerides, and low density lipoprotein cholesterol levels declined significantly in the CEBS group compared with the control, whereas plasma high density lipoprotein cholesterol levels increased significantly. The concentration of Cr in blood and breast muscle increased after CEBS and inorganic Cr supplementation. B. subtilis and CEBS supplementation caused a significant increase in the numbers of Lactobacillus and Bifidobacterium in the caecum, while the numbers of Escherichia coli and Salmonella decreased significantly compared to the control. Feed adding CEBS increased the lightness, redness, and yellowness of breast meat, improved the water-holding capacity, decreased the shear force and cooking loss. In all, CEBS supplementation promoted body growth, improved plasma lipid parameters, increased tissue Cr concentrations, altered cecal bacterial composition and improved breast meat quality.

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. Copyright © 2016. Published by Elsevier B.V.

Survival of Bacillus subtilis endospores on ultraviolet-irradiated rover wheels and Mars regolith under simulated Martian conditions.

PubMed

Kerney, Krystal R; Schuerger, Andrew C

2011-06-01

Endospores of Bacillus subtilis HA101 were applied to a simulated Mars Exploration Rover (MER) wheel and exposed to Mars-normal UV irradiation for 1, 3, or 6 h. The experiment was designed to simulate a contaminated rover wheel sitting on its landing platform before rolling off onto the martian terrain, as was encountered during the Spirit and Opportunity missions. When exposed to 1 h of Mars UV, a reduction of 81% of viable endospores was observed compared to the non-UV irradiated controls. When exposed for 3 or 6 h, reductions of 94.6% and 96.6%, respectively, were observed compared to controls. In a second experiment, the contaminated rover wheel was rolled over a bed of heat-sterilized Mars analog soil; then the analog soil was exposed to full martian conditions of UV irradiation, low pressure (6.9 mbar), low temperature (-10°C), and an anaerobic CO(2) martian atmosphere for 24 h to determine whether endospores of B. subtilis on the contaminated rover wheel could be transferred to the surface of the analog soil and survive martian conditions. The experiment simulated conditions in which a rover wheel might come into contact with martian regolith immediately after landing, such as is designed for the upcoming Mars Science Laboratory (MSL) rover. The contaminated rover wheel transferred viable endospores of B. subtilis to the Mars analog soil, as demonstrated by 31.7% of samples showing positive growth. However, when contaminated soil samples were exposed to full martian conditions for 24 h, only 16.7% of samples exhibited positive growth-a 50% reduction in the number of soil samples positive for the transferred viable endospores.

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.

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

PubMed

Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

2017-01-01

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

Heterologous expression and characterization of a new heme-catalase in Bacillus subtilis 168.

PubMed

Philibert, Tuyishime; Rao, Zhiming; Yang, Taowei; Zhou, Junping; Huang, Genshu; Irene, Komera; Samuel, Niyomukiza

2016-06-01

Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784 U/mg expressed in Bacillus subtilis 168 and 98.097 µmol/min/mg peroxidatic activity, the apparent K m of catalytic activity was 59.6 ± 13 mM with higher turnover rate (K cat = 322.651 × 10(3) s(-1)). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40 °C and the recombinant heme-catalase exhibited a strong Fe(2+) preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.

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.

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

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed