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Sample records for recombinant bacillus anthracis

  1. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    International Nuclear Information System (INIS)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui; Leppla, Stephen H.

    2013-01-01

    Highlights: ► Non-infectious and protease-deficient Bacillus anthracis protein expression system. ► Successful expression and purification of a tumor-targeted fusion protein drug. ► Very low endotoxin contamination of purified protein. ► Efficient protein secretion simplifies purification. ► Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  2. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Leppla, Stephen H., E-mail: sleppla@niaid.nih.gov [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  3. Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

    Science.gov (United States)

    Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

    2018-05-01

    Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

  4. Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

    Science.gov (United States)

    2011-09-01

    future predictive modeling toolkits. 1 1. Introduction The use of Bacillus anthracis as a bio - weapon in the United States in 2001 affirmed the need...for improved sensing and detection of biological weapons of mass destruction (WMD). Protective Antigen (PA) protein of Bacillus anthracis is the...Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

  5. Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

    International Nuclear Information System (INIS)

    Midha, Shuchi; Mishra, Rajeev; Aziz, M.A.; Sharma, Meenakshi; Mishra, Ashish; Khandelwal, Puneet; Bhatnagar, Rakesh

    2005-01-01

    Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N ω -hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein

  6. Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Pieper Rembert

    2011-05-01

    Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

  7. Characterization of 21 Strains of Bacillus Anthracis

    National Research Council Canada - National Science Library

    Kournikakis, B

    2000-01-01

    Twenty-one strains of Bacillus anthracis currently held in the culture collection at DRES were characterized by colonial morphology, antibiotic sensitivity and BiologTM metabolic identification profiles...

  8. Method for screening inhibitors of the toxicity of Bacillus anthracis

    Science.gov (United States)

    Cirino, Nick M.; Jackson, Paul J.; Lehnert, Bruce E.

    2001-01-01

    The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

  9. Dendritic Cells Endocytose Bacillus Anthracis Spores: Implications for Anthrax Pathogenesis

    National Research Council Canada - National Science Library

    Brittingham, Katherine C; Ruthel, Gordon; Panchal, Rekha G; Fuller, Claudette L; Ribot, Wilson J

    2005-01-01

    Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhaled anthrax because they initiate germination and dissemination of spores...

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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...

  12. Improvements to a Markerless Allelic Exchange System for Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Roger D Plaut

    Full Text Available A system was previously developed for conducting I-SceI-mediated allelic exchange in Bacillus anthracis. In this system, recombinational loss of a chromosomally-integrated allelic exchange vector is stimulated by creation of a double-stranded break within the vector by the homing endonuclease I-SceI. Although this system is reasonably efficient and represents an improvement in the tools available for allelic exchange in B. anthracis, researchers are nonetheless required to "pick and patch" colonies in order to identify candidate "exchangeants." In the present study, a number of improvements have been made to this system: 1 an improved I-SceI-producing plasmid includes oriT so that both plasmids can now be introduced by conjugation, thus avoiding the need for preparing electro-competent cells of each integration intermediate; 2 antibiotic markers have been changed to allow the use of the system in select agent strains; and 3 both plasmids have been marked with fluorescent proteins, allowing the visualization of plasmid segregation on a plate and obviating the need for "picking and patching." These modifications have made the process easier, faster, and more efficient, allowing for parallel construction of larger numbers of mutant strains. Using this improved system, the genes encoding the tripartite anthrax toxin were deleted singly and in combination from plasmid pXO1 of Sterne strain 34F2. In the course of this study, we determined that DNA transfer to B. anthracis could be accomplished by conjugation directly from a methylation-competent E. coli strain.

  13. Genetic Characterization of Bacillus anthracis 17 JB strain.

    Science.gov (United States)

    Seyed-Mohamadi, Sakineh; Moradi Bidhendi, Soheila; Tadayon, Keyvan; Ghaderi, Rainak

    2015-06-01

    Bacillus anthracis is one of the most homogenous bacteria ever described. Some level of diversity. Bacillus anthracis 17JB is a laboratory strain It is broadly used as a challenge strain in guinea pigs for potency test of anthrax vaccine. This work describes genetic characterization of B. anthracis 17 JB strain using the SNPs and MLVA genotyping. In SNPs typing, the originally French 17JB strain represented the A.Br. 008/009 subgroup. In Levy's genotyping method, 843, 451 and 864 bp long fragments were identified at AA03, AJ03 and AA07 loci, respectively. In the vaccine manufacturer perspective these findings are much valuable on their own account, but similar research is required to extend molecular knowledge of B. anthracis epidemiology in Persia.

  14. Functional Comparison of the Two Bacillus anthracis Glutamate Racemases▿

    OpenAIRE

    Dodd, Dylan; Reese, Joseph G.; Louer, Craig R.; Ballard, Jimmy D.; Spies, M. Ashley; Blanke, Steven R.

    2007-01-01

    Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because l-glutamate stereoisomerization to d-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and rac...

  15. Computational Fluid Dynamics Modeling of Bacillus anthracis ...

    Science.gov (United States)

    Journal Article Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. Four different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Despite the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways of the human at the same air concentration of anthrax spores. This greater deposition of spores in the upper airways in the human resulted in lower penetration and deposition in the tracheobronchial airways and the deep lung than that predict

  16. Decontamination of Soil Contaminated with Bacillus anthracis ...

    Science.gov (United States)

    Technical Brief This technical summary will provide decontamination personnel rapid access to information on which decontamination approaches are most effective for soils contaminated with B anthracis.

  17. Novel giant siphovirus from Bacillus anthracis features unusual genome characteristics.

    Directory of Open Access Journals (Sweden)

    Holly H Ganz

    Full Text Available Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales, featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.

  18. Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

    Science.gov (United States)

    2004-12-01

    13061 Neisseria lactamica .............................................................. 23970 Bacillus coagulans ...NEG Bacillus coagulane 7050 NEG NEG Bacillus cereus 13472 NEG NEG Bacillus licheniforms 12759 NEG NEG Bacillus cereus 13824 NEG NEG Bacillus ...Assay for a Unique Chromosomal Sequence of Bacillus anthracis Elizabeth Bode,1 William Hurtle,2† and David Norwood1* United States Army Medical

  19. The search and identification of the new immunodiagnostic targets of bacillus anthracis spore

    International Nuclear Information System (INIS)

    Biketov, S.; Dunaytsev, I.; Baranova, E.; Marinin, L.; Dyatlov, I.

    2009-01-01

    Spores of Bacillus anthracis have been used as bio warfare agent to bio terrorize purposes. As efficiency of anti-epidemic measures included urgent prevention and treatment is determined by terms within which the bio agent is identified. Direct and rapid spore detection by antibodies based detection system is very attractive alternative to current PCR-based assays or routine phenotyping which are the most accurate but are also complex, time-consumption and expensive. The main difficulty with respect to such kind of anthrax spores detection is a cross-reaction with spores of closely related bacteria. For development of species-specific antibodies to anthrax spores recombinant scFvs or hybridoma technique were used. In both case surface spore antigens contained species-specific epitopes are need. Among exosporium proteins only ExsF(BxpB), ExsK and SoaA are specific to B.cereus group. On the surface of B. anthracis spores, a unique tetrasaccharides containing an novel monosaccharide - anthrose, was discovered. It was shown that anthrose can be serving as species-specific target for B. anthracis spores detection. We have revealed that EA1 isolated from spore of Russians strain STI-1 contain carbohydrate which formed species-specific epitopes and determine immunogenicity of this antigen. Antibodies to this antigen specifically recognized the surface target of B. anthracis spores and do not reacted with others Bacillus spore. Based on these antibodies we developed the test-systems in different formats for rapid direct detection and identification of B. anthracis spores. The results of trial these test-systems with using more than 50 different Bacillus strains were indicated that carbohydrate of EA1 isolated from spore is effective immunodiagnostic target for anthrax spores bio detection.(author)

  20. Identification of Bacillus anthracis by Using Monoclonal Antibody to Cell Wall Galactose-N-Acetylglucosamine Polysaccharide

    Science.gov (United States)

    1990-02-01

    Bacillus circulans ATCC 4513 b - - NR NT NT NT NT Bacillus coagulans ATCC 7050 b - - NR NT NT NT NT Bacillus eugilitis B-61 f - - NR NT NT NT NT...American Society for Microbiology W Identification of Bacillus anthracis by-U-sing Monoclonal Antibody CC to Cell Wall Galactose-N-Acetylglucosamine...Received 22 June 1989/Accepted 31 October 1989 ’ Guanidine extracts of crude Bacillus anthracis cell wall were used to vaccinate BALB/c mice and to

  1. Assembly and Function of the Bacillus anthracis S-Layer.

    Science.gov (United States)

    Missiakas, Dominique; Schneewind, Olaf

    2017-09-08

    Bacillus anthracis, the anthrax agent, is a member of the Bacillus cereus sensu lato group, which includes invasive pathogens of mammals or insects as well as nonpathogenic environmental strains. The genes for anthrax pathogenesis are located on two large virulence plasmids. Similar virulence plasmids have been acquired by other B. cereus strains and enable the pathogenesis of anthrax-like diseases. Among the virulence factors of B. anthracis is the S-layer-associated protein BslA, which endows bacilli with invasive attributes for mammalian hosts. BslA surface display and function are dependent on the bacterial S-layer, whose constituents assemble by binding to the secondary cell wall polysaccharide (SCWP) via S-layer homology (SLH) domains. B. anthracis and other pathogenic B. cereus isolates harbor genes for the secretion of S-layer proteins, for S-layer assembly, and for synthesis of the SCWP. We review here recent insights into the assembly and function of the S-layer and the SCWP.

  2. Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

    Science.gov (United States)

    Salard, Isabelle; Mercey, Emilie; Rekka, Eleni; Boucher, Jean-Luc; Nioche, Pierre; Mikula, Ivan; Martasek, Pavel; Raman, C S; Mansuy, Daniel

    2006-12-01

    Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.

  3. Historical distribution and molecular diversity of Bacillus anthracis, Kazakhstan.

    Science.gov (United States)

    Aikembayev, Alim M; Lukhnova, Larissa; Temiraliyeva, Gulnara; Meka-Mechenko, Tatyana; Pazylov, Yerlan; Zakaryan, Sarkis; Denissov, Georgiy; Easterday, W Ryan; Van Ert, Matthew N; Keim, Paul; Francesconi, Stephen C; Blackburn, Jason K; Hugh-Jones, Martin; Hadfield, Ted

    2010-05-01

    To map the distribution of anthrax outbreaks and strain subtypes in Kazakhstan during 1937-2005, we combined geographic information system technology and genetic analysis by using archived cultures and data. Biochemical and genetic tests confirmed the identity of 93 archived cultures in the Kazakhstan National Culture Collection as Bacillus anthracis. Multilocus variable number tandem repeat analysis genotyping identified 12 genotypes. Cluster analysis comparing these genotypes with previously published genotypes indicated that most (n = 78) isolates belonged to the previously described A1.a genetic cluster, 6 isolates belonged to the A3.b cluster, and 2 belonged to the A4 cluster. Two genotypes in the collection appeared to represent novel genetic sublineages; 1 of these isolates was from Krygystan. Our data provide a description of the historical, geographic, and genetic diversity of B. anthracis in this Central Asian region.

  4. Encapsulated Bacillus anthracis interacts closely with liver endothelium.

    Science.gov (United States)

    Piris-Gimenez, Alejandro; Corre, Jean-Philippe; Jouvion, Gregory; Candela, Thomas; Khun, Huot; Goossens, Pierre L

    2009-11-01

    The Bacillus anthracis poly-gamma-D-glutamate capsule is essential for virulence. It impedes phagocytosis and protects bacilli from the immune system, thus promoting systemic dissemination. To further define the virulence mechanisms brought into play by the capsule, we characterized the interactions between encapsulated nontoxinogenic B. anthracis and its host in vivo through histological analysis, perfusion, and competition experiments with purified capsule. Clearance of encapsulated bacilli from the blood was rapid (>90% clearance within 5 min), with 75% of the bacteria being trapped in the liver. Competition experiments with purified capsule polyglutamate inhibited this interaction. At the septicemic phase of cutaneous infection with spores, the encapsulated bacilli were trapped in the vascular spaces of the liver and interacted closely with the liver endothelium in the sinusoids and terminal and portal veins. They often grow as microcolonies containing capsular material shed by the bacteria. We show that, in addition to its inhibitory effect on the interaction with the immune system, the capsule surrounding B. anthracis plays an active role in mediating the trapping of the bacteria within the liver and may thus contribute to anthrax pathogenesis. Because other microorganisms produce polyglutamate, it may also represent a general mechanism of virulence or in vivo survival.

  5. Survival of Bacillus anthracis spores in fruit juices and wine.

    Science.gov (United States)

    Leishman, Oriana N; Johnson, Miranda J; Labuza, Theodore P; Diez-Gonzalez, Francisco

    2010-09-01

    Foods have been identified as a potential target for bioterrorism due to their essential nature and global distribution. Foods produced in bulk have the potential to have large batches of product intentionally contaminated, which could affect hundreds or thousands of individuals. Bacillus anthracis spores are one potential bioterrorism agent that may survive pasteurization and remain viable throughout the shelf life of fruit juices and cause disease if consumed. This project examined B. anthracis spore survival in orange, apple, and grape juices, as well as wine. Samples of beverages were inoculated with spores of two nonpathogenic B. anthracis strains at approximately 10(6) CFU/ml, and the spore count was determined periodically during storage for 30 days at 4°C. After this time, the counts of survival spores never declined more than 1 log CFU/ml in any of the beverage types. These results indicate that spores can survive, with little to no loss in viability, for at least a month in fruit juices and wine.

  6. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

    Science.gov (United States)

    Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

    2016-01-01

    In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

  7. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

    Directory of Open Access Journals (Sweden)

    Farzana Islam Rume

    Full Text Available In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA with the analysis of 15 Variable Number Tandem Repeats (VNTR, demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

  8. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

    Science.gov (United States)

    Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

  9. Literature Review of DNA-Based Subspecies Analysis of Bacillus Anthracis Burkholderia Pseudomallel Burkholderia Mallei, and Yersinia Pestis

    National Research Council Canada - National Science Library

    Harvey, Steven

    1999-01-01

    ...; Bacillus anthracis, Burkholderia pseudomallei, Burkholderia mallei, and Yersinia pestis. Considerable research has been accomplished for the identification of polymorphisms from the strains B. anthracis and B. pseudomallei. The B...

  10. Hal Is a Bacillus anthracis Heme Acquisition Protein

    Science.gov (United States)

    Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

    2012-01-01

    The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

  11. Efficacy of Oritavancin in a Murine Model of Bacillus anthracis Spore Inhalation Anthrax

    National Research Council Canada - National Science Library

    Heine, H. S; Bassett, J; Miller, L; Bassett, A; Ivins, B. E; Lehous, D; Arhin, F. F; Parr, Jr., T. R; Moeck, G

    2008-01-01

    The inhaled form of Bacillus anthracis infection may be fatal to humans. The current standard of care for inhalational anthrax postexposure prophylaxis is ciprofloxacin therapy twice daily for 60 days...

  12. Lethality of Bacillus Anthracis Spores Due to Short Duration Heating Measured Using Infrared Spectroscopy

    National Research Council Canada - National Science Library

    Goetz, Kristina M

    2005-01-01

    In this research, Bacillus anthracis spores were subjected to bursts of heat lasting on the order of one second in duration using a laser system to simulate the explosive environment from an agent defeat weapon...

  13. Bacillus anthracis genome organization in light of whole transcriptome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Zhu, Wenhan; Passalacqua, Karla D.; Bergman, Nicholas; Borodovsky, Mark

    2010-03-22

    Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity.

  14. Functional and Immunological Analyses of Superoxide Dismutases and Other Spore-Associated Proteins of Bacillus anthracis

    Science.gov (United States)

    2008-08-20

    L., S. Hibbs, P. Tsai, G. L. Cao, and G. M. Rosen . 2005. Role of superoxide in the germination of Bacillus anthracis endospores. FEMS Microbiol...178:7994-8001. 42. Cohen, S., I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak ...K. W. Raines, G. L. Cao, S. Hibbs, P. Tsai, L. Baillie, G. M. Rosen , and A. S. Cross. 2007. Protective role of Bacillus anthracis exosporium in

  15. A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

    Directory of Open Access Journals (Sweden)

    Hirohito Ogawa

    Full Text Available Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

  16. Decontamination Options for Drinking Water Contaminated with Bacillus anthracis Spores

    Energy Technology Data Exchange (ETDEWEB)

    Raber, E; Burklund, A

    2010-02-16

    Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination options for use in a contaminated drinking water supply. The parameters were: (1) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus); (2) spore concentration in suspension (10{sup 2} to 10{sup 6} spores/ml); (3) chemical characteristics of decontaminant [sodium dicholor-s-triazinetrione dihydrate (Dichlor), hydrogen peroxide, potassium peroxymonosulfate (Oxone), sodium hypochlorite, and VirkonS{reg_sign}]; (4) decontaminant concentration (0.01% to 5%); and (5) decontaminant exposure time (10 min to 24 hr). Results from 162 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5%, and Dichlor and sodium hypochlorite at a concentration of 2%, were effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting EPA's biocide standard of greater than a 6 log kill after a 10-minute exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS{reg_sign} and Oxone were less effective decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for biocides. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.

  17. Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Redkar Rajendra

    2004-02-01

    Full Text Available Abstract Background Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH was used to identify specific chromosomal sequences unique to B. anthracis. Results Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. Conclusions Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

  18. Distribution and molecular evolution of bacillus anthracis genotypes in Namibia.

    Directory of Open Access Journals (Sweden)

    Wolfgang Beyer

    Full Text Available The recent development of genetic markers for Bacillus anthracis has made it possible to monitor the spread and distribution of this pathogen during and between anthrax outbreaks. In Namibia, anthrax outbreaks occur annually in the Etosha National Park (ENP and on private game and livestock farms. We genotyped 384 B. anthracis isolates collected between 1983-2010 to identify the possible epidemiological correlations of anthrax outbreaks within and outside the ENP and to analyze genetic relationships between isolates from domestic and wild animals. The isolates came from 20 animal species and from the environment and were genotyped using a 31-marker multi-locus-VNTR-analysis (MLVA and, in part, by twelve single nucleotide polymorphism (SNP markers and four single nucleotide repeat (SNR markers. A total of 37 genotypes (GT were identified by MLVA, belonging to four SNP-groups. All GTs belonged to the A-branch in the cluster- and SNP-analyses. Thirteen GTs were found only outside the ENP, 18 only within the ENP and 6 both inside and outside. Genetic distances between isolates increased with increasing time between isolations. However, genetic distance between isolates at the beginning and end of the study period was relatively small, indicating that while the majority of GTs were only found sporadically, three genetically close GTs, accounting for more than four fifths of all the ENP isolates, appeared dominant throughout the study period. Genetic distances among isolates were significantly greater for isolates from different host species, but this effect was small, suggesting that while species-specific ecological factors may affect exposure processes, transmission cycles in different host species are still highly interrelated. The MLVA data were further used to establish a model of the probable evolution of GTs within the endemic region of the ENP. SNR-analysis was helpful in correlating an isolate with its source but did not elucidate

  19. Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Chad W Stratilo

    Full Text Available Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin, compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.

  20. Bacillus anthracis infections – new possibilities of treatment

    Directory of Open Access Journals (Sweden)

    Dorota Żakowska

    2015-05-01

    Full Text Available [b]Introduction and objective[/b]. [i]Bacillus anthracis[/i] is one of biological agents which may be used in bioterrorism attacks. The aim of this study a review of the new treatment possibilities of anthrax, with particular emphasis on the treatment of pulmonary anthrax. [b]Abbreviated description of the state of knowledge[/b]. Pulmonary anthrax, as the most dangerous clinical form of the disease, is also extremely difficult to treat. Recently, considerable progress in finding new drugs and suitable therapy for anthrax has been achieved, for example, new antibiotics worth to mentioning, levofloxacin, daptomycin, gatifloxacin and dalbavancin. However, alternative therapeutic options should also be considered, among them the antimicrobial peptides, characterized by lack of inducible mechanisms of pathogen resistance. Very promising research considers bacteriophages lytic enzymes against selected bacteria species, including antibiotic-resistant strains. [b]Results[/b]. Interesting results were obtained using monoclonal antibodies: raxibacumab, cAb29 or cocktails of antibodies. The application of CpG oligodeoxynucleotides to boost the immune response elicited by Anthrax Vaccine Adsorbed and CMG2 protein complexes, also produced satisfying therapy results. Furthermore, the IFN-α and IFN-β, PA-dominant negative mutant, human inter-alpha inhibitor proteins and LF inhibitors in combination with ciprofloxacin, also showed very promising results. [b]Conclusions[/b]. Recently, progress has been achieved in inhalation anthrax treatment. The most promising new possibilities include: new antibiotics, peptides and bacteriophages enzymes, monoclonal antibodies, antigen PA mutants, and inter alpha inhibitors applications. In the case of the possibility of bioterrorist attacks, the examination of inhalation anthrax treatment should be intensively continued.

  1. Caenorhabditis elegans Predation on Bacillus anthracis: Decontamination of Spore Contaminated Soil with Germinants and Nematodes.

    Science.gov (United States)

    Schelkle, Bettina; Choi, Young; Baillie, Leslie W; Richter, William; Buyuk, Fatih; Celik, Elif; Wendling, Morgan; Sahin, Mitat; Gallagher, Theresa

    2017-01-01

    Remediation of Bacillus anthracis -contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms.

  2. Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

    Directory of Open Access Journals (Sweden)

    AH ahmadi

    2015-02-01

    Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

  3. The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

    Directory of Open Access Journals (Sweden)

    Sozhamannan Shanmuga

    2006-04-01

    Full Text Available Abstract Background Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. Results More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 × 10-5 - 8 × 10-8/cell. Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. Conclusion The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production.

  4. Identification of Proteins in the Exosporium of Bacillus Anthracis

    National Research Council Canada - National Science Library

    Redmond, Caroline; Baillie, Leslie W. J; Hibbs, Stephen; Moir, Arthur J. G; Moir, Anne

    2004-01-01

    .... The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis...

  5. Bacillus Collagen Like Protein of Anthracis: Immunological and Functional Analyses

    Science.gov (United States)

    2007-09-21

    factors such as heat, radiation, desiccation, pH extremes, and toxic chemicals (87). Cultivation of B. anthracis can be accomplished on non...obtained from the Naval Medical Research Center. B. anthracis was induced to sporulate on Leighton-Doi Medium (LD) (75). The broth was inoculated with an...heated at 65°C for 30 minutes, diluted, and plated on trypticase soy agar ( TSA ) to obtain viable counts. Since heat treatment kills the vegetative

  6. Differences in Susceptibility of Inbred Mice to Bacillus anthracis

    Science.gov (United States)

    1985-04-26

    dilutions of the mixture were prepared and injected into A/J and CBA/J mice via the tail vein, as described by Ezzell et al. (9). Five mice per strain were...xylazine (Rompun, Miles Laboratories, Shawnee, Kansas) in 50 pl, and were dissected iwnmediately. Gross pathological changes were noted, heart blood and...anthracis; a histopathological study of skin lesions produced by B. anthracis in susceptible and resistant animal species. J. Infect. Dis. 80:1-13. 9. Ezzell

  7. The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations.

    Directory of Open Access Journals (Sweden)

    Raymond Schuch

    2009-08-01

    Full Text Available Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

  8. Novel Sample Preparation Method for Safe and Rapid Detection of Bacillus anthracis Spores in Environmental Powders and Nasal Swabs

    OpenAIRE

    Luna, Vicki A.; King, Debra; Davis, Carisa; Rycerz, Tony; Ewert, Matthew; Cannons, Andrew; Amuso, Philip; Cattani, Jacqueline

    2003-01-01

    Bacillus anthracis spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects B. anthracis in samples containing

  9. Ecological Niche Modelling of the Bacillus anthracis A1.a sub-lineage in Kazakhstan

    Science.gov (United States)

    2011-01-01

    Background Bacillus anthracis, the causative agent of anthrax, is a globally distributed zoonotic pathogen that continues to be a veterinary and human health problem in Central Asia. We used a database of anthrax outbreak locations in Kazakhstan and a subset of genotyped isolates to model the geographic distribution and ecological associations of B. anthracis in Kazakhstan. The aims of the study were to test the influence of soil variables on a previous ecological niche based prediction of B. anthracis in Kazakhstan and to determine if a single sub-lineage of B. anthracis occupies a unique ecological niche. Results The addition of soil variables to the previously developed ecological niche model did not appreciably alter the limits of the predicted geographic or ecological distribution of B. anthracis in Kazakhstan. The A1.a experiment predicted the sub-lineage to be present over a larger geographic area than did the outbreak based experiment containing multiple lineages. Within the geographic area predicted to be suitable for B. anthracis by all ten best subset models, the A1.a sub-lineage was associated with a wider range of ecological tolerances than the outbreak-soil experiment. Analysis of rule types showed that logit rules predominate in the outbreak-soil experiment and range rules in the A1.a sub-lineage experiment. Random sub-setting of locality points suggests that models of B. anthracis distribution may be sensitive to sample size. Conclusions Our analysis supports careful consideration of the taxonomic resolution of data used to create ecological niche models. Further investigations into the environmental affinities of individual lineages and sub-lineages of B. anthracis will be useful in understanding the ecology of the disease at large and small scales. With model based predictions serving as approximations of disease risk, these efforts will improve the efficacy of public health interventions for anthrax prevention and control. PMID:22152056

  10. Intranasal immunization with protective antigen of Bacillus anthracis induces a long-term immunological memory response.

    Science.gov (United States)

    Woo, Sun-Je; Kang, Seok-Seong; Park, Sung-Moo; Yang, Jae Seung; Song, Man Ki; Yun, Cheol-Heui; Han, Seung Hyun

    2015-10-01

    Although intranasal vaccination has been shown to be effective for the protection against inhalational anthrax, establishment of long-term immunity has yet to be achieved. Here, we investigated whether intranasal immunization with recombinant protective antigen (rPA) of Bacillus anthracis induces immunological memory responses in the mucosal and systemic compartments. Intranasal immunization with rPA plus cholera toxin (CT) sustained PA-specific antibody responses for 6 months in lung, nasal washes, and vaginal washes as well as serum. A significant induction of PA-specific memory B cells was observed in spleen, cervical lymph nodes (CLNs) and lung after booster immunization. Furthermore, intranasal immunization with rPA plus CT remarkably generated effector memory CD4(+) T cells in the lung. PA-specific CD4(+) T cells preferentially increased the expression of Th1- and Th17-type cytokines in lung, but not in spleen or CLNs. Collectively, the intranasal immunization with rPA plus CT promoted immunologic memory responses in the mucosal and systemic compartments, providing long-term immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dan [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Rands, Anthony D.; Losee, Scott C. [Torion Technologies, American Fork, UT 84003 (United States); Holt, Brian C. [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Williams, John R. [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Lammert, Stephen A. [Torion Technologies, American Fork, UT 84003 (United States); Robison, Richard A. [Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602 (United States); Tolley, H. Dennis [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Lee, Milton L., E-mail: milton_lee@byu.edu [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States)

    2013-05-02

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  12. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    International Nuclear Information System (INIS)

    Li, Dan; Rands, Anthony D.; Losee, Scott C.; Holt, Brian C.; Williams, John R.; Lammert, Stephen A.; Robison, Richard A.; Tolley, H. Dennis; Lee, Milton L.

    2013-01-01

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  13. Detection of Bacillus anthracis in the air, soil and animal tissue

    Directory of Open Access Journals (Sweden)

    Kušar D.

    2012-01-01

    Full Text Available The objective of the present work was to establish effective and rapid diagnostic methods for the detection of Bacillus anthracis, a highly virulent zoonotic pathogen, in the air, soil and animal (or human tissue samples. Liquid culture of B. anthracis was aerosolized and four air sampling procedures were employed. Detection of B. anthracis in the air samples was successful with RCS High Flow sampler (culturebased detection and when sampling through the air filter (molecular detection using SmartHelix Complex Samples DNA Extraction Kit. Liquid B. anthracis culture was also employed for spiking the homogenised bovine lymphatic gland tissue and soil samples. DNA extraction was performed using three different commercial kits for each sample type. High Pure PCR Template Preparation Kit was the most effective for DNA extraction from animal tissue samples. Detection in the soil was successful when PowerSoil DNA Isolation Kit was used. Our results indicate that B. anthracis can be monitored in different matrices by rapid molecular methods when appropriate sampling and DNA extraction procedures are employed prior to PCR assay. The selected rapid protocols can be implemented in specialized veterinary or human diagnostic laboratories with moderate costs.

  14. Toxin-independent virulence of Bacillus anthracis in rabbits.

    Directory of Open Access Journals (Sweden)

    Haim Levy

    Full Text Available The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation.

  15. Laboratory Studies on Surface Sampling of Bacillus anthracis Contamination: Summary, Gaps, and Recommendations

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Amidan, Brett G.; Hu, Rebecca

    2011-11-28

    This report summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing, and analyzing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe, and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the (1) estimates of Bacillus anthracis contamination, as well as the bias and uncertainties in the estimates, and (2) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed and recommendations are given for future studies.

  16. Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis and Other Bacteria

    Science.gov (United States)

    2016-09-01

    Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis And Other Bacteria Thomas Brown, Salwa Shan, Teresa...infection can be detected as early as one hour after exposing as few as 105 CFU bacteria to the stressor. We predicted that similar responses could be used... bacteria to form confluent growth and for phage-induced plaques to appear. Techniques that permit faster detection of species-specific bacteria /phage

  17. Evaluation of immunoradiometric and ELISA versions of a microtitre plate assay for Bacillus anthracis spores

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, A P; Martin, K L; Cross, N L [Chemical Defence Experimental Establishment, Porton (UK); Drake, R G [Glasgow Univ. (UK). Inst. of Biochemistry

    1984-05-11

    Solid-phase indirectly-labelled antibody assays for Bacillus anthracis spores heat-fixed on polystyrene microtitre plates were compared as immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA) versions. Signal-to-noise ratios were usually higher in the IRMA than in the ELISA performed under parallel conditions but replicates were more varied in the IRMA. The antigen detection threshold and resolution limit calculated after regression analysis were broadly comparable in the 2 types of assay.

  18. Storage Effects on Sample Integrity of Environmental Surface Sampling Specimens with Bacillus anthracis Spores.

    Science.gov (United States)

    Perry, K Allison; O'Connell, Heather A; Rose, Laura J; Noble-Wang, Judith A; Arduino, Matthew J

    The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis . Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T 0 ) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 10 2 , p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.

  19. [Survival of Bacillus anthracis spores in various tannery baths].

    Science.gov (United States)

    Mendrycka, M; Mierzejewski, J

    2000-01-01

    The influence of tannery baths: liming, deliming, bating, pickling, tanning, retannage on the survival and on the germination dynamism of B. anthracis spores (Sterne strain) was investigated. The periods and the conditions of this influence were established according to technological process of cow hide tannage. Practically after every bath some part of the spores remained vital. The most effective killing of spores occurred after pickling, liming and deliming. Inversely, the most viable spores remained after bating and retannage process. The lack of correlation that was observed between survival and germination of spores after retannage bath can be explained by different mechanism of spores germination inhibition and their killing.

  20. Characterization of the sortase repertoire in Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Willy Aucher

    Full Text Available LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss, bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.

  1. Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, P.J.; Walthers, E.A.; Richmond, K.L. [Los Alamos National Lab., NM (United States)] [and others

    1997-04-01

    PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five Polymorphisms differed by the presence Of two to six copies of the 12-bp tandem repeat 5{prime}-CAATATCAACAA-3{prime}. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations. 22 refs., 4 figs., 3 tabs.

  2. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    Directory of Open Access Journals (Sweden)

    Ma’ayan Israeli

    2016-08-01

    Full Text Available Edema Factor (EF, the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP, and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.

  3. clpC operon regulates cell architecture and sporulation in Bacillus anthracis.

    Science.gov (United States)

    Singh, Lalit K; Dhasmana, Neha; Sajid, Andaleeb; Kumar, Prasun; Bhaduri, Asani; Bharadwaj, Mitasha; Gandotra, Sheetal; Kalia, Vipin C; Das, Taposh K; Goel, Ajay K; Pomerantsev, Andrei P; Misra, Richa; Gerth, Ulf; Leppla, Stephen H; Singh, Yogendra

    2015-03-01

    The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Inactivation of Bacillus anthracis Spores during Laboratory-Scale Composting of Feedlot Cattle Manure

    Science.gov (United States)

    Xu, Shanwei; Harvey, Amanda; Barbieri, Ruth; Reuter, Tim; Stanford, Kim; Amoako, Kingsley K.; Selinger, Leonard B.; McAllister, Tim A.

    2016-01-01

    Anthrax outbreaks in livestock have social, economic and health implications, altering farmer’s livelihoods, impacting trade and posing a zoonotic risk. Our study investigated the survival of Bacillus thuringiensis and B. anthracis spores sporulated at 15, 20, or 37°C, over 33 days of composting. Spores (∼7.5 log10 CFU g-1) were mixed with manure and composted in laboratory scale composters. After 15 days, the compost was mixed and returned to the composter for a second cycle. Temperatures peaked at 71°C on day 2 and remained ≥55°C for an average of 7 days in the first cycle, but did not exceed 55°C in the second. For B. thuringiensis, spores generated at 15 and 21°C exhibited reduced (P composting for spores generated at 15, 21, and 37°C, respectively. For both species, spore viability declined more rapidly (P composting cycle. Our findings suggest that the duration of thermophilic exposure (≥55°C) is the main factor influencing survival of B. anthracis spores in compost. As sporulation temperature did not influence survival of B. anthracis, composting may lower the viability of spores associated with carcasses infected with B. anthracis over a range of sporulation temperatures. PMID:27303388

  5. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely...... on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S...... targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European...

  6. Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

    International Nuclear Information System (INIS)

    Voss, Jarrod E.; Scally, Stephen W.; Taylor, Nicole L.; Dogovski, Con; Alderton, Malcolm R.; Hutton, Craig A.; Gerrard, Juliet A.; Parker, Michael W.; Dobson, Renwick C. J.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyses an important step in lysine biosynthesis. Here, the expression, purification, crystallization and preliminary diffraction analysis to 2.15 Å resolution of DHDPS from B. anthracis soaked with the substrate pyruvate are reported. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 Å are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  7. Bacillus anthracis: una mirada molecular a un patógeno célebre Bacillus anthracis: a molecular look at a famous pathogen

    Directory of Open Access Journals (Sweden)

    María E Pavan

    2011-12-01

    Full Text Available Bacillus anthracis es un bacilo gram positivo del grupo Bacillus cereus, que posee un genoma extremadamente monomórfco y comparte gran similitud fsiológica y de estructura genética con B. cereus y Bacillus thuringiensis. En este artículo se describen nuevos métodos moleculares para la identifcación y tipifcación de B. anthracis, basados en repeticiones en tándem de número variable o en diferencias genéticas detectadas por secuenciación, desarrollados en los últimos años. Los aspectos moleculares de los factores de virulencia tradicionales, cápsula, antígeno protector, factor letal y factor edema se describen en profundidad, junto con factores de virulencia recientemente propuestos, como los sideróforos, petrobactina y bacilibactina, la adhesina de la capa S y la lipoproteína MntA. También se detalla la organización molecular de los megaplásmidos pXO1 y pXO2, incluyendo la isla de patogenicidad de pXO1. El esqueleto genético de estos plásmidos se ha encontrado en otras especies relacionadas, probablemente debido a eventos de transferencia lateral. Finalmente, se presentan los dos receptores celulares del antígeno protector, ANTXR1/TEM8 y ANTXR2/CMG2, esenciales en la interacción del patógeno con el hospedador. Los estudios moleculares realizados en los últimos años han permitido aumentar enormemente el conocimiento de los diferentes aspectos de este microorganismo y su relación con el hospedador, pero a la vez han abierto nuevos interrogantes sobre este notorio patógeno.Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identifcation and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The

  8. The Pathogenomic Sequence Analysis of B. cereus and B.thuringiensis Isolates Closely Related to Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Han, Cliff S.; Xie, Gary; Challacombe, Jean F.; Altherr, MichaelR.; Smriti, B.; Bruce, David; Campbell, Connie S.; Campbell, Mary L.; Chen, Jin; Chertkov, Olga; Cleland, Cathy; Dimitrijevic-Bussod, M.; Doggett, Norman A.; Fawcett, John J.; Glavina, Tijana; Goodwin, Lynne A.; Hill, Karen K.; Hitchcock, Penny; Jackson, Paul J.; Keim, Paul; Kewalramani, Avinash Ramesh; Longmire, Jon; Lucas, Susan; Malfatti,Stephanie; McMurry, Kim; Meincke, Linda J.; Misra, Monica; Moseman,Bernice L.; Mundt, Mark; Munk, A. Christine; Okinaka, Richard T.; Parson-Quintana, B.; Reilly, Lee P.; Richardson, Paul; Robinson, DonnaL.; Rubin, Eddy; Saunders, Elizabeth; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Ticknor, Lawrence O.; Wills, Patti L.; Gilna, Payl; Brettin, Thomas S.

    2005-08-18

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B.cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including Banthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  9. The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.

    Science.gov (United States)

    Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

    2016-01-01

    Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites.

  10. The use of germinants to potentiate the sensitivity of Bacillus anthracis spores to peracetic acid

    Directory of Open Access Journals (Sweden)

    Ozgur eCelebi

    2016-01-01

    Full Text Available Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM and inosine (5 mM to reduce the concentration of peracetic acid (PAA required to inactivate B.anthracis spores. While L-alanine significantly enhanced (p=0.0085 the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p=0.0009. To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B.anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed one hour later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B.anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p<0.0001 in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B.anthracis spores contaminated sites.

  11. Modeling the potential distribution of Bacillus anthracis under multiple climate change scenarios for Kazakhstan.

    Directory of Open Access Journals (Sweden)

    Timothy Andrew Joyner

    Full Text Available Anthrax, caused by the bacterium Bacillus anthracis, is a zoonotic disease that persists throughout much of the world in livestock, wildlife, and secondarily infects humans. This is true across much of Central Asia, and particularly the Steppe region, including Kazakhstan. This study employed the Genetic Algorithm for Rule-set Prediction (GARP to model the current and future geographic distribution of Bacillus anthracis in Kazakhstan based on the A2 and B2 IPCC SRES climate change scenarios using a 5-variable data set at 55 km(2 and 8 km(2 and a 6-variable BioClim data set at 8 km(2. Future models suggest large areas predicted under current conditions may be reduced by 2050 with the A2 model predicting approximately 14-16% loss across the three spatial resolutions. There was greater variability in the B2 models across scenarios predicting approximately 15% loss at 55 km(2, approximately 34% loss at 8 km(2, and approximately 30% loss with the BioClim variables. Only very small areas of habitat expansion into new areas were predicted by either A2 or B2 in any models. Greater areas of habitat loss are predicted in the southern regions of Kazakhstan by A2 and B2 models, while moderate habitat loss is also predicted in the northern regions by either B2 model at 8 km(2. Anthrax disease control relies mainly on livestock vaccination and proper carcass disposal, both of which require adequate surveillance. In many situations, including that of Kazakhstan, vaccine resources are limited, and understanding the geographic distribution of the organism, in tandem with current data on livestock population dynamics, can aid in properly allocating doses. While speculative, contemplating future changes in livestock distributions and B. anthracis spore promoting environments can be useful for establishing future surveillance priorities. This study may also have broader applications to global public health surveillance relating to other diseases in addition to B

  12. Bacillus anthracis in China and its relationship to worldwide lineages

    Directory of Open Access Journals (Sweden)

    Schupp James M

    2009-04-01

    Full Text Available Abstract Background The global pattern of distribution of 1033 B. anthracis isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP. These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different B. anthracis isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP and multiple locus VNTR analysis (MLVA typing has been used to examine this archival collection of isolates. Results The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China. Conclusion B. anthracis isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads

  13. Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2006-04-01

    Full Text Available Abstract Background The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the

  14. Protein profiles of field isolates ofBacillus anthracis from different endemic areas of Indonesia

    Directory of Open Access Journals (Sweden)

    M Bhakti Poerwadikarta

    1998-03-01

    Full Text Available Sonicated cell-free extract proteins of 14 field isolates ofBacillus anthracis from six different endemic areas of Indonesia were analyzed by the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE methods . The protein profiles of each field isolate tested demonstrated slightly different at the protein bands with molecular weights of 18, 37, 52, 65 and 70 kDa, and varied between the field isolates and vaccine strains. The variation could provide clues to the source of anthrax transmission whether it was originated from similar strain or not.

  15. The human-bacterial pathogen protein interaction networks of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Matthew D Dyer

    2010-08-01

    Full Text Available Bacillus anthracis, Francisella tularensis, and Yersinia pestis are bacterial pathogens that can cause anthrax, lethal acute pneumonic disease, and bubonic plague, respectively, and are listed as NIAID Category A priority pathogens for possible use as biological weapons. However, the interactions between human proteins and proteins in these bacteria remain poorly characterized leading to an incomplete understanding of their pathogenesis and mechanisms of immune evasion.In this study, we used a high-throughput yeast two-hybrid assay to identify physical interactions between human proteins and proteins from each of these three pathogens. From more than 250,000 screens performed, we identified 3,073 human-B. anthracis, 1,383 human-F. tularensis, and 4,059 human-Y. pestis protein-protein interactions including interactions involving 304 B. anthracis, 52 F. tularensis, and 330 Y. pestis proteins that are uncharacterized. Computational analysis revealed that pathogen proteins preferentially interact with human proteins that are hubs and bottlenecks in the human PPI network. In addition, we computed modules of human-pathogen PPIs that are conserved amongst the three networks. Functionally, such conserved modules reveal commonalities between how the different pathogens interact with crucial host pathways involved in inflammation and immunity.These data constitute the first extensive protein interaction networks constructed for bacterial pathogens and their human hosts. This study provides novel insights into host-pathogen interactions.

  16. Optimization of a sample processing protocol for recovery of Bacillus anthracis spores from soil

    Science.gov (United States)

    Silvestri, Erin E.; Feldhake, David; Griffin, Dale; Lisle, John T.; Nichols, Tonya L.; Shah, Sanjiv; Pemberton, A; Schaefer III, Frank W

    2016-01-01

    Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14 spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

  17. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Morgunova, Ekaterina [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden); Illarionov, Boris; Saller, Sabine [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Popov, Aleksander [European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble CEDEX 09 (France); Sambaiah, Thota [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Bacher, Adelbert [Chemistry Department, Technical University of Munich, 85747 Garching (Germany); Cushman, Mark [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Fischer, Markus [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Ladenstein, Rudolf, E-mail: rudolf.ladenstein@ki.se [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden)

    2010-09-01

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

  18. Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation.

    Science.gov (United States)

    Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

    2006-01-01

    This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

  19. Rap Phosphatase of Virulence Plasmid pXO1 Inhibits Bacillus anthracis Sporulation†

    Science.gov (United States)

    Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

    2006-01-01

    This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis. PMID:16385039

  20. Measurements of the Ultraviolet Fluorescence Cross Sections and Spectra of Bacillus Anthracis Simulants

    Energy Technology Data Exchange (ETDEWEB)

    Stephens, J.R.

    1998-09-01

    Measurements of the ultraviolet autofluorescence spectra and absolute cross sections of the Bacillus anthracis (Ba) simulants Bacillus globigii (Bg), Bacillus megaterium (Bm), Bacillus subtilis (Bs), and Bacillus cereus (Bc) were measured. Fluorescence spectra and cross sections of pine pollen (Pina echinata) were measured for comparison. Both dried vegetative cells and spores separated from the sporulated vegetative material were studied. The spectra were obtained by suspending a small number (<10) of particles in air in our Single Particle Spectroscopy Apparatus (SPSA), illuminating the particles with light from a spectrally filtered arc lamp, and measuring the fluorescence spectra of the particles. The illumination was 280 nm (20 nm FWHM) and the fluorescence spectra was measured between 300 and 450 nm. The fluorescence cross section of vegetative Bg peaks at 320 nm with a maximum cross section of 5 X 10{sup -14} cm{sup 2}/sr-nm-particle while the Bg spore fluorescence peaks at 310 nm with peak fluorescence of 8 X 10{sup -15} cm{sup 2}/sr-nm-particle. Pine pollen particles showed a higher fluorescence peaking at 355 nm with a cross section of 1.7 X 10{sup -13} cm{sup 2}/sr-nm-particle. Integrated cross sections ranged from 3.0 X 10{sup -13} for the Bg spores through 2.25 X 10{sup -12} (cm{sup 2}/sr-particle) for the vegetative cells.

  1. Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry

    Directory of Open Access Journals (Sweden)

    David F. Waller

    2016-12-01

    Full Text Available Portable detection and quantitation methods for Bacillus anthracis (anthrax spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid. Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL, while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min result in short sample-to-signal times (less than an hour. With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field.

  2. The role of DNA restriction-modification systems in the biology of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Ramakrishnan eSitaraman

    2016-01-01

    Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

  3. [An historical, sociocultural view and in the fiction literature of Bacillus anthracis cases by shaving brushes].

    Science.gov (United States)

    Vázquez-Espinosa, E; Laganà, C; Vazquez, F

    2018-06-01

    In the period from 1915 to 1924 anthrax outbreaks were described by Bacillus anthracis due to the contamination of razor brushes that reached Europe and the United States from areas such as Japan, China or Russia. The brushes were made with badger hair, and then, to reduce the cost with horse hair and other animals. World War I supoosed that the traffics of these brushes, that passed through Europe, changed and the processes of sterilization of the same were deficient giving rise to these outbreaks, that in a percentage of 20% produced the death of the users. The impact of the fashion of wearing a beard, the presence of these cases in the press, in the society of that period, and literature are studied through the work of Agatha Christie who wrote, in 1936, the Hercules Poirot´s novel Cards on the table, and where she describes the murder of one of the characters with the shaving brush contaminated with Bacillus anthracis spores. ©The Author 2018. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).

  4. Pilot-scale crossflow-microfiltration and pasturization to remove spores of Bacillus anthracis (Sterne) from milk

    Science.gov (United States)

    HTST pasteurization of milk is generally ineffective against spore-forming bacteria such as Bacillus anthracis (BA) but is lethal to its vegetative cells. Crossflow microfiltration (MF), using ceramic membranes with a pore diameter of 1.4 um, has been shown to physically remove somatic cells, vegeta...

  5. Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa-Chromosomal Monophyly and Broad Geographic Distribution.

    Directory of Open Access Journals (Sweden)

    Kym S Antonation

    2016-09-01

    Full Text Available Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats in West and Central Africa (Côte d'Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo. The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans.

  6. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

    Directory of Open Access Journals (Sweden)

    Whittington Jessica

    2007-07-01

    Full Text Available Abstract Background The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2- during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. Results A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites, lipoprotein signal peptides (13 have SpII sites, and N-terminal membrane helices (9 have transmembrane helices. The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa of protective antigen (PA were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. Conclusion This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and

  7. A strain-variable bacteriocin in Bacillus anthracis and Bacillus cereus with repeated Cys-Xaa-Xaa motifs

    Directory of Open Access Journals (Sweden)

    Haft Daniel H

    2009-04-01

    Full Text Available Abstract Bacteriocins are peptide antibiotics from ribosomally translated precursors, produced by bacteria often through extensive post-translational modification. Minimal sequence conservation, short gene lengths, and low complexity sequence can hinder bacteriocin identification, even during gene calling, so they are often discovered by proximity to accessory genes encoding maturation, immunity, and export functions. This work reports a new subfamily of putative thiazole-containing heterocyclic bacteriocins. It appears universal in all strains of Bacillus anthracis and B. cereus, but has gone unrecognized because it is always encoded far from its maturation protein operon. Patterns of insertions and deletions among twenty-four variants suggest a repeating functional unit of Cys-Xaa-Xaa. Reviewers This article was reviewed by Andrei Osterman and Lakshminarayan Iyer.

  8. Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples.

    Science.gov (United States)

    Létant, Sonia E; Murphy, Gloria A; Alfaro, Teneile M; Avila, Julie R; Kane, Staci R; Raber, Ellen; Bunt, Thomas M; Shah, Sanjiv R

    2011-09-01

    In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

  9. Modeling the Ecological Niche of Bacillus anthracis to Map Anthrax Risk in Kyrgyzstan.

    Science.gov (United States)

    Blackburn, Jason K; Matakarimov, Saitbek; Kozhokeeva, Sabira; Tagaeva, Zhyldyz; Bell, Lindsay K; Kracalik, Ian T; Zhunushov, Asankadyr

    2017-03-01

    AbstractAnthrax, caused by the environmental bacterium Bacillus anthracis , is an important zoonosis nearly worldwide. In Central Asia, anthrax represents a major veterinary and public health concern. In the Republic of Kyrgyzstan, ongoing anthrax outbreaks have been reported in humans associated with handling infected livestock and contaminated animal by-products such as meat or hides. The current anthrax situation has prompted calls for improved insights into the epidemiology, ecology, and spatial distribution of the disease in Kyrgyzstan to better inform control and surveillance. Disease control for both humans and livestock relies on annual livestock vaccination ahead of outbreaks. Toward this, we used a historic database of livestock anthrax reported from 1932 to 2006 mapped at high resolution to develop an ecological niche model-based prediction of B. anthracis across Kyrgyzstan and identified spatial clusters of livestock anthrax using a cluster morphology statistic. We also defined the seasonality of outbreaks in livestock. Cattle were the most frequently reported across the time period, with the greatest number of cases in late summer months. Our niche models defined four areas as suitable to support pathogen persistence, the plateaus near Talas and Bishkek, the valleys of western Kyrgyzstan along the Fergana Valley, and the low-lying areas along the shore of Lake Isyk-Kul. These areas should be considered "at risk" for livestock anthrax and subsequent human cases. Areas defined by the niche models can be used to prioritize anthrax surveillance and inform efforts to target livestock vaccination campaigns.

  10. Identification of the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.

  11. A Study on molecular characterization of Razi Bacillus anthracis Sterne 34F2 substrain in Iran

    Directory of Open Access Journals (Sweden)

    Tadayon, K.

    2016-07-01

    Full Text Available Anthrax, a zoonotic disease caused by Bacillus anthracis, has affected humans since ancient times. For genomic characterization of Razi B. anthracis Sterne 34F2 substrain, single nucleotide polymorphism (SNP genotyping method developed by Van Erth, variable-number tandem-repeat (VNTR-8 analysis proposed by Keim, and multiple-locus VNTR analysis (MLVA-3 introduced by Levy were employed. In the SNPs typing system, where the nucleotide content of the genome at 13 evolutionary canonical loci was collectively analyzed, the originally South African 34F2 substrain was categorized in the A.Br.001/002 subgroup. In the VNTR-8 analysis, fragments with lengths of 314, 229, 162, 580, 532, 158, and 137 bp were identified at the following loci: vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, and pxO1, respectively. In addition, application of Levy's MLVA-3 genotyping method revealed that the genome of this strain carried 941, 451, and 864 bp fragments at AA03, AJ03, and AA07 loci, respectively. The present findings are undoubtedly helpful in meeting the requirements set by the World Organization for Animal Health (OIE and World Health Organization (WHO for anthrax vaccine manufacturers including Razi Institute. However, further similar studies are required to promote the current epidemiological knowledge of anthrax in Iran.

  12. Differential Binding of Co(II) and Zn(II) to Metallo-beta-Lactamase Bla2 from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Hawk, M.; Breece, R; Hajdin, C; Bender, K; Hu, Z; Costello, A; Bennett, B; Tierney, D; Crowder, M

    2009-01-01

    In an effort to probe the structure, mechanism, and biochemical properties of metallo-{beta}-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.

  13. Physical Sequestration of Bacillus anthracis in the Pulmonary Capillaries in Terminal Infection.

    Science.gov (United States)

    Jouvion, Gregory; Corre, Jean-Philippe; Khun, Huot; Moya-Nilges, Marie; Roux, Pascal; Latroche, Claire; Tournier, Jean-Nicolas; Huerre, Michel; Chrétien, Fabrice; Goossens, Pierre L

    2016-07-15

    The lung is the terminal target of Bacillus anthracis before death, whatever the route of infection (cutaneous, inhalational, or digestive). During a cutaneous infection in absence of toxins, we observed encapsulated bacteria colonizing the alveolar capillary network, bacteria and hemorrhages in alveolar and bronchiolar spaces, and hypoxic foci in the lung (endothelial cells) and brain (neurons and neuropil). Circulating encapsulated bacteria were as chains of approximately 13 µm in length. Bacteria of such size were immediately trapped within the lung capillary network, but bacteria of shorter length were not. Controlling lung-targeted pathology would be beneficial for anthrax treatment. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  14. Esterase activity as a novel parameter of spore germination in Bacillus anthracis

    International Nuclear Information System (INIS)

    Ferencko, Linda; Cote, Mindy A.; Rotman, Boris

    2004-01-01

    Spores of Bacillus anthracis were shown to produce esterase activity about 4 min after exposure to conventional germinants such as combinations of amino acids and purine ribosides. Neither amino acids nor ribosides alone induce germination and esterase activity. Expression of esterase activity was chloramphenicol resistant, and correlated with loss of spore refractivity, a traditional parameter of early germination. Based on these observations, we hypothesized that esterase activity could be used as a novel parameter for quantifying early events during spore germination. To test this hypothesis, we measured expression of esterase activity under a variety of germinating conditions. Using diacetyl fluorescein as fluorogenic substrate of esterases, we demonstrated that esterase activity was invariably induced whenever spores were triggered by known germinants. Moreover, D-alanine, an inhibitor of L-alanine-mediated germination, was found to significantly inhibit expression of esterase activity. In terms of molecular mechanisms, esterase expression could represent activation of proteases at the onset of spore germination

  15. Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

    Science.gov (United States)

    Terwilliger, Austen; Swick, Michelle C.; Pflughoeft, Kathryn J.; Pomerantsev, Andrei; Lyons, C. Rick; Koehler, Theresa M.

    2015-01-01

    ABSTRACT Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed “nutritional immunity.” Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCE The mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood—hemoglobin and serum albumin—can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for

  16. Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

    Science.gov (United States)

    Felker, Daniel L.; Burggraf, Larry W.

    2014-01-01

    Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating. PMID:24375142

  17. A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production.

    Science.gov (United States)

    Dale, Jennifer L; Raynor, Malik J; Ty, Maureen C; Hadjifrangiskou, Maria; Koehler, Theresa M

    2018-01-01

    Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075 , an atxA -regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 " skiA " for "sporulation kinase inhibitor." Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.

  18. In vitro evaluation of the effect of linezolid and levofloxacin on Bacillus anthracis toxin production, spore formation and cell growth.

    Science.gov (United States)

    Head, Breanne M; Alfa, Michelle; Sitar, Daniel S; Rubinstein, Ethan; Meyers, Adrienne F A

    2017-02-01

    Owing to its ability to form spores and toxins, Bacillus anthracis is considered a bioterror agent. Although current therapeutic strategies can be effective, treatment does not prevent sporulation and toxin production. To quantify the combined effect of a protein synthesis inhibitor and a bactericidal agent on B. anthracis toxin production, sporulation and cell growth. Susceptibility and synergy titrations were conducted on B. anthracis Sterne and 03-0191 strains using linezolid and levofloxacin. The effect of antibiotic exposure on cell viability was evaluated using a continuous medium replacement model. In vitro static models were used to study the effect of linezolid and levofloxacin on sporulation and toxin production. Spores were quantified using the heat shock method. Toxin was quantified via commercial ELISA. Synergy titrations indicated that the combination was synergistic or indifferent; however, in all models antagonism was observed. In the spore model, linezolid resulted in the lowest sporulation rates, while combination therapy resulted in the highest. In the toxin model, linezolid prevented toxin production altogether. This study advances our understanding of the effects of combination therapy on B. anthracis infection. Used alone, linezolid therapy abolishes toxin production and reduces sporulation. These results suggest that studies using a step-wise approach using linezolid initially to stop sporulation and toxin production followed by levofloxacin to rapidly kill vegetative B. anthracis can be recommended. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years.

    Science.gov (United States)

    Lekota, K E; Hassim, A; Rogers, P; Dekker, E H; Last, R; de Klerk-Lorist, L; van Heerden, H

    2018-05-02

    Anthrax is a disease with an age old history in Africa caused by the Gram-positive endospore forming soil bacterium Bacillus anthracis. Epizootics of wild ungulates occur annually in the enzootic region of Pafuri, Kruger National Park (KNP) in the Limpopo Province of South Africa. Rigorous routine surveillance and diagnostics in KNP, has not revealed these rare isolates since the 1990s, despite unabated annual outbreaks. In 2011 a cheetah was diagnosed as anthrax positive from a private game reserve in Limpopo Province and reported to State Veterinary Services for further investigation. Isolation, molecular diagnostics, whole genome sequencing and comparative genomics were carried out for B. anthracis KC2011. Bacteriological and molecular diagnostics confirmed the isolate as B. anthracis. Subsequent typing and whole genome single nucleotide polymorphisms analysis indicated it clustered alongside B. anthracis SA A0091 in the B.Br.010 SNP branch. Unlike B. anthracis KrugerB strain, KC2011 strain has unique SNPs and represents a new branch in the B-clade. The isolation and genotypic characterisation of KC2011 demonstrates a gap in the reporting of anthrax outbreaks in the greater Limpopo province area. The identification of vulnerable and susceptible cheetah mortalities due to this strain has implications for conservation measures and disease control.

  20. Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope.

    Science.gov (United States)

    Hutchison, Janine R; Erikson, Rebecca L; Sheen, Allison M; Ozanich, Richard M; Kelly, Ryan T

    2015-09-21

    Bacillus anthracis is the causative agent of anthrax and can be contracted by humans and herbivorous mammals by inhalation, ingestion, or cutaneous exposure to bacterial spores. Due to its stability and disease potential, B. anthracis is a recognized biothreat agent and robust detection and viability methods are needed to identify spores from unknown samples. Here we report the use of smartphone-based microscopy (SPM) in combination with a simple microfluidic incubation device (MID) to detect 50 to 5000 B. anthracis Sterne spores in 3 to 5 hours. This technique relies on optical monitoring of the conversion of the ∼1 μm spores to the filamentous vegetative cells that range from tens to hundreds of micrometers in length. This distinguishing filament formation is unique to B. anthracis as compared to other members of the Bacillus cereus group. A unique feature of this approach is that the sample integrity is maintained, and the vegetative biomass can be removed from the chip for secondary molecular analysis such as PCR. Compared with existing chip-based and rapid viability PCR methods, this new approach reduces assay time by almost half, and is highly sensitive, specific, and cost effective.

  1. The regulated synthesis of a Bacillus anthracis spore coat protein that affects spore surface properties.

    Science.gov (United States)

    Aronson, A; Goodman, B; Smith, Z

    2014-05-01

    Examine the regulation of a spore coat protein and the effects on spore properties. A c. 23 kDa band in coat/exosporial extracts of Bacillus anthracis Sterne spores varied in amount depending upon the conditions of sporulation. It was identified by MALDI as a likely orthologue of ExsB of Bacillus cereus. Little if any was present in an exosporial preparation with a location to the inner coat/cortex region established by spore fractionation and immunogold labelling of electron micrograph sections. Because of its predominant location in the inner coat, it has been renamed Cotγ. It was relatively deficient in spores produced at 37°C and when acidic fermentation products were produced a difference attributable to transcriptional regulation. The deficiency or absence of Cotγ resulted in a less robust exosporium positioned more closely to the coat. These spores were less hydrophobic and germinated somewhat more rapidly. Hydrophobicity and appearance were rescued in the deletion strain by introduction of the cotγ gene. The deficiency or lack of a protein largely found in the inner coat altered spore hydrophobicity and surface appearance. The regulated synthesis of Cotγ may be a paradigm for other spore coat proteins with unknown functions that modulate spore properties in response to environmental conditions. © 2014 The Society for Applied Microbiology.

  2. Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

    Science.gov (United States)

    2015-06-19

    decontamination strategies>> Maryline DEFEZ 1𔃼, Melissa HUNTER3J Susan WELKOS :~J Christopher COTE3 1 University Grenoble-Alpes, Grenoble, France. 1...inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores potential spore decontamination strategies 5a...8217 • Accidentally in Humans • Natural reservoir is soil • Anthrax Disease Cycle: - animals infected by soilborne spores in food and water or bites from certain

  3. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

    OpenAIRE

    Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-01-01

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-bin...

  4. Impact of spores on the comparative efficacies of five antibiotics for treatment of Bacillus anthracis in an in vitro hollow fiber pharmacodynamic model.

    Science.gov (United States)

    Louie, Arnold; VanScoy, Brian D; Brown, David L; Kulawy, Robert W; Heine, Henry S; Drusano, George L

    2012-03-01

    Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the "gold standards," doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compared. Against spore-forming B. anthracis, ciprofloxacin, moxifloxacin, linezolid, and meropenem reduced the B. anthracis population by 4 log(10) CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log(10) CFU/ml (analysis of variance [ANOVA] P = 0.01 versus other drugs). Against an isogenic non-spore-forming strain, meropenem killed the vegetative B. anthracis the fastest, followed by moxifloxacin and ciprofloxacin and then doxycycline. Linezolid offered the lowest bacterial kill rate. Heat shock studies using the spore-producing B. anthracis strain showed that with moxifloxacin, ciprofloxacin, and meropenem therapies the total population was mostly spores, while the population was primarily vegetative bacteria with linezolid and doxycycline therapies. Spores have a profound impact on the rate and extent of killing of B. anthracis. Against spore-forming B. anthracis, the five antibiotics killed the total (spore and vegetative) bacterial population at similar rates (within 1 log(10) CFU/ml of each other). However, bactericidal antibiotics killed vegetative B. anthracis faster than bacteriostatic drugs. Since only vegetative-phase B. anthracis produces the toxins that may kill the infected host, the rate and mechanism of killing of an antibiotic may determine its overall in vivo efficacy. Further studies are needed to examine this important observation.

  5. Proteomic signatures differentiating Bacillus anthracis Sterne sporulation on soil relative to laboratory media.

    Science.gov (United States)

    Wunschel, D S; Hutchison, J R; Deatherage Kaiser, B L; Merkley, E D; Hess, B M; Lin, A; Warner, M G

    2017-12-18

    The process of sporulation is vital for the stability and infectious cycle of Bacillus anthracis. The spore is the infectious form of the organism and therefore relevant to biodefense. While the morphological and molecular events occurring during sporulation have been well studied, the influence of growth medium and temperature on the proteins expressed in sporulated cultures is not well understood. Understanding the features of B. anthracis sporulation specific to natural vs. laboratory production will address an important question in microbial forensics. In an effort to bridge this knowledge gap, a system for sporulation on two types of agar-immobilized soils was used for comparison to cultures sporulated on two common types of solid laboratory media, and one liquid sporulation medium. The total number of proteins identified as well as their identity differed between samples generated in each medium and growth temperature, demonstrating that sporulation environment significantly impacts the protein content of the spore. In addition, a subset of proteins common in all of the soil-cultivated samples was distinct from the expression profiles in laboratory medium (and vice versa). These differences included proteins involved in thiamine and phosphate metabolism in the sporulated cultures produced on soils with a notable increase in expression of ATP binding cassette (ABC) transporters annotated to be for phosphate and antimicrobial peptides. A distinct set of ABC transporters for amino acids, sugars and oligopeptides were found in cultures produced on laboratory media as well as increases in carbon and amino acid metabolism-related proteins. These protein expression changes indicate that the sporulation environment impacts the protein profiles in specific ways that are reflected in the metabolic and membrane transporter proteins present in sporulated cultures.

  6. Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

    Energy Technology Data Exchange (ETDEWEB)

    Schnicker, Nicholas J. [Department; Razzaghi, Mortezaali [Department; Guha Thakurta, Sanjukta [Department; Chakravarthy, Srinivas [Biophysics; Dey, Mishtu [Department

    2017-10-17

    Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.

  7. Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

    Science.gov (United States)

    Amoako, Kingsley K; Janzen, Timothy W; Shields, Michael J; Hahn, Kristen R; Thomas, Matthew C; Goji, Noriko

    2013-08-01

    The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  8. Micro-Etched Platforms for Thermal Inactivation of Bacillus Anthracis and Bacillus Thuringiensis Spores

    Science.gov (United States)

    2008-03-01

    slips was first coated with a detergent wash. Commercially available Ivory soap shavings were diluted with sterile Millipore® water in a...environments. This removed controllable variability between the Bacillus species and increased the confidence in continued use of such surrogacy

  9. [Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

    Science.gov (United States)

    Gao, Zhiqi; Wang, Dongshu; Feng, Erling; Wang, Bingxiang; Hui, Yiming; Han, Shaobo; Jiao, Lei; Liu, Xiankai; Wang, Hengliang

    2014-11-04

    To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. We did not find any polymorphism of CRISPR in B. anthracis. The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

  10. Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase and bactericidal antibacterials targeting Bacillus species, Staphylococcus aureus and Mycobacterium tuberculosis.

    Science.gov (United States)

    Gustafsson, Tomas N; Osman, Harer; Werngren, Jim; Hoffner, Sven; Engman, Lars; Holmgren, Arne

    2016-06-01

    Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. The most potent compounds in the series gave IC(50) values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 μM (0.12 μg/ml) for B. subtilis, 1.5 μM (0.64 μg/ml) for S. aureus, 2 μM (0.86 μg/ml) for B. cereus and 10 μg/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. In Silico Genomic Fingerprints of the Bacillus anthracis Group Obtained by Virtual Hybridization

    Directory of Open Access Journals (Sweden)

    Hueman Jaimes-Díaz

    2015-02-01

    Full Text Available In this study we evaluate the capacity of Virtual Hybridization to identify between highly related bacterial strains. Eight genomic fingerprints were obtained by virtual hybridization for the Bacillus anthracis genome set, and a set of 15,264 13-nucleotide short probes designed to produce genomic fingerprints unique for each organism. The data obtained from each genomic fingerprint were used to obtain hybridization patterns simulating a DNA microarray. Two virtual hybridization methods were used: the Direct and the Extended method to identify the number of potential hybridization sites and thus determine the minimum sensitivity value to discriminate between genomes with 99.9% similarity. Genomic fingerprints were compared using both methods and phylogenomic trees were constructed to verify that the minimum detection value is 0.000017. Results obtained from the genomic fingerprints suggest that the distribution in the trees is correct, as compared to other taxonomic methods. Specific virtual hybridization sites for each of the genomes studied were also identified.

  12. Regulation of expression of a select group of Bacillus anthracis spore coat proteins.

    Science.gov (United States)

    Aronson, Arthur

    2018-04-01

    The spore coat of Bacilli is a relatively complex structure comprised of about 70 species of proteins in 2 or 3 layers. While some are involved in assembly or protection, the regulation of many are not well defined so lacZ transcriptional fusions were constructed to six Bacillus anthracis spore coat genes in order to gain insight into their possible functions. The genes were selected on the basis of the location of the encoded proteins within the coat and distribution among spore forming species. Conditions tested were temperature and media either as solid or liquid. The most extensive differences were for the relatively well expressed fusions to the cotH and cotM genes, which were greatest at 30°C on plates of a nutrient rich medium. The cotJ operon was moderately expressed under all conditions although somewhat higher on enriched plates at 30°C. Cot S was low under all conditions except for a substantial increase in biofilm medium. Cot∝ and cotF were essentially invariant with a somewhat greater expression in the more enriched medium. The capacity of a subset of coat genes to respond to various conditions reflects a flexibility in spore coat structure that may be necessary for adaptation to environmental challenges. This could account, at least in part, for the complexity of this structure.

  13. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  14. Bacillus anthracis Co-Opts Nitric Oxide and Host Serum Albumin for Pathogenicity in Hypoxic Conditions

    Directory of Open Access Journals (Sweden)

    Stephen eSt John

    2013-05-01

    Full Text Available Bacillus anthracis is a dangerous pathogen of humans and many animal species. Its virulence has been mainly attributed to the production of Lethal and Edema toxins as well as the antiphagocytic capsule. Recent data indicate that the nitric oxide (NO synthase (baNOS plays an important pathogenic role at the early stage of disease by protecting bacteria from the host reactive species and S-nytrosylating the mitochondrial proteins in macrophages. In this study we for the first time present evidence that bacteria-derived NO participates in the generation of highly reactive oxidizing species which could be abolished by the NOS inhibitor L-NAME, free thiols, and superoxide dismutase but not catalase. The formation of toxicants is likely a result of the simultaneous formation of NO and superoxide leading to a labile peroxynitrite and its stable decomposition product, nitrogen dioxide. The toxicity of bacteria could be potentiated in the presence of bovine serum albumin. This effect is consistent with the property of serum albumin to serves as a trap of a volatile NO accelerating its reactions. Our data suggest that during infection in the hypoxic environment of pre-mortal host the accumulated NO is expected to have a broad toxic impact on host cell functions.

  15. First detection of Bacillus anthracis in feces of free-ranging raptors from central Argentina.

    Science.gov (United States)

    Saggese, Miguel D; Noseda, Ramón P; Uhart, Marcela M; Deem, Sharon L; Ferreyra, Hebe; Romano, Marcelo C; Ferreyra-Armas, María C; Hugh-Jones, Martin

    2007-01-01

    Prevalence of anthrax spores in feces of raptors was determined from samples collected in November-December 2000 and April-May 2001 in an agricultural region of Santa Fé province, Argentina. Feces were tested from 48 birds of six raptor species. One of 14 chimango caracaras (Milvago chimango) and one of eight road-side hawks (Buteo magnirostris) tested positive. The prevalence of Bacillus anthracis spores in feces for the six species was 4% (n=48). The prevalence was 7% (n=14) for chimango caracaras, 13% for road-side hawks (n=8), and 0% for the remaining species (Burrowing owl [Speotyto cunicularia] [n=17], Swainson's hawk [Buteo swainsoni] [n=3], Aplomado falcon [Falco femoralis] [n=2], and American kestrel [Falco sparverius] [n=4]). Grouped by their feeding habits, prevalence for scavenger species was not significantly different than for predators (7% vs. 3%, P>0.999). This study provides evidence that in central Argentina scavenger and non-scavenger raptors may have a role in the epidemiology of anthrax. Long-term studies to determine the extent of this potential involvement in the epidemiology of anthrax in central Argentina are required.

  16. [Efficacy of enterocin S760 in treatment of mice with anthrax infection due to Bacillus anthracis M-71].

    Science.gov (United States)

    Svetoch, E A; Borzilov, A I; Eruslanov, B V; Korobova, O V; Kombarova, T I; Levchuk, V P; Teĭmurazov, M G; Stepanshin, Iu G; Marinin, L I; Diatlov, I A

    2011-01-01

    The therapeutic efficacy of enterocin S760, a broad spectrum antimicrobial peptide produced by Enterococcus faecium LWP760 was tested on mice infected with Bacillus anthracis M-71 to induce anthrax (second Tsenkovsky's vaccine). Intraperitoneal four-, two- or one-fold administration of the peptide in a dose of 25 mg/kg for 10 days for prophylactic (1 hour after the contamination) and therapeutic (24 hours after the contamination) purposes prevented or cured the infection in 90-100% of the mice versus the 100-percent lethality in the control (untreated animals). The antimicrobial activity of enterocin S760 against B. anthracis M-71 in vivo correlated with activity in vitro. Enterocin S760 is considered a novel promising antimicrobial for the treatment of grampositive and gramnegative infections.

  17. Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis, Yersinia pestis, and Burkholderia pseudomallei by Use of Laser Light Scattering Technology.

    Science.gov (United States)

    Bugrysheva, Julia V; Lascols, Christine; Sue, David; Weigel, Linda M

    2016-06-01

    Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or postexposure prophylaxis in the aftermath of the release of bacterial biothreat agents such as Bacillus anthracis, Yersinia pestis, or Burkholderia pseudomallei Conventional susceptibility tests require 16 to 48 h of incubation, depending on the bacterial species. We evaluated a method that is based on laser light scattering technology that measures cell density in real time. We determined that it has the ability to rapidly differentiate between growth (resistant) and no growth (susceptible) of several bacterial threat agents in the presence of clinically relevant antimicrobials. Results were available in 10 h of incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% to 75% for B. anthracis, Y. pestis, and B. pseudomallei compared to conventional methods. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

    Science.gov (United States)

    Hutchison, J R; Piepel, G F; Amidan, B G; Hess, B M; Sydor, M A; Deatherage Kaiser, B L

    2018-05-01

    We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD 95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD 95 results. Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD 95 was lowest for glass and highest for vinyl tile. LOD 95 values overall were lower for mRV-PCR than for the culture method. This study adds to the limited data on FNR and LOD 95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis. © 2018 The Society for Applied Microbiology.

  19. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids.

    Directory of Open Access Journals (Sweden)

    Silke R Klee

    Full Text Available Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as "B. cereus variety (var. anthracis".

  20. Poly(3-hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation.

    Science.gov (United States)

    Sadykov, Marat R; Ahn, Jong-Sam; Widhelm, Todd J; Eckrich, Valerie M; Endres, Jennifer L; Driks, Adam; Rutkowski, Gregory E; Wingerd, Kevin L; Bayles, Kenneth W

    2017-06-01

    Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species. © 2017 John Wiley & Sons Ltd.

  1. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  2. Recombination-deficient mutants of Bacillus subtilis

    International Nuclear Information System (INIS)

    Sadaie, Y.; Kada, T.

    1976-01-01

    Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (uv), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation, SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr + capacity for uv-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 tranduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The uv impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene product whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strains NIG43 and NIG45 were not inducible, indicating involvement of rec-43 + or rec-45 + gene product in the development of SPO2 prophage to a vegetative form. The uv-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains

  3. Composite sampling of a Bacillus anthracis surrogate with cellulose sponge surface samplers from a nonporous surface.

    Directory of Open Access Journals (Sweden)

    Jenia A M Tufts

    Full Text Available A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas, larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261. Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720 for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001. The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

  4. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, S.; Suffield, S. R.; Recknagle, K. P.; Jacob, R. E.; Einstein, D. R.; Kuprat, A. P.; Carson, J. P.; Colby, S. M.; Saunders, J. H.; Hines, S. A.; Teeguarden, J. G.; Straub, T. M.; Moe, M.; Taft, S. C.; Corley, R. A.

    2016-09-01

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation–exhalation breathing conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology for deposition.

  5. Computational Fluid Dynamics Modeling of Bacillus anthracis Spore Deposition in Rabbit and Human Respiratory Airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, Senthil; Suffield, Sarah R.; Recknagle, Kurtis P.; Jacob, Rick E.; Einstein, Daniel R.; Kuprat, Andrew P.; Carson, James P.; Colby, Sean M.; Saunders, James H.; Hines, Stephanie; Teeguarden, Justin G.; Straub, Tim M.; Moe, M.; Taft, Sarah; Corley, Richard A.

    2016-09-30

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.

  6. Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid.

    Science.gov (United States)

    Buhr, T L; Wells, C M; Young, A A; Minter, Z A; Johnson, C A; Payne, A N; McPherson, D C

    2013-08-01

    To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  7. Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

    Directory of Open Access Journals (Sweden)

    Marcellene A Gates-Hollingsworth

    Full Text Available Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA, the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation, whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

  8. Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

    Science.gov (United States)

    Somani, Vikas K; Aggarwal, Somya; Singh, Damini; Prasad, Tulika; Bhatnagar, Rakesh

    2016-01-01

    Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated

  9. Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "Road Closure".

    Science.gov (United States)

    Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Fleming, Joy; Bi, Li-Jun; Yang, Rui-Fu; Zhang, Xian-En

    2015-05-15

    Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

    Directory of Open Access Journals (Sweden)

    Sunil K Joshi

    2009-09-01

    Full Text Available Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC stimulates TCR signaling and activation of type-1 natural killer-like T (NKT cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA-mediated intracellular delivery of lethal factor (LF, a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8 and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

  11. The Efficiency of Methionine as a Radioprotectant of Bacillus anthracis for Cell Viability and Outgrowth Time after UVC and Gamma Irradiation

    Science.gov (United States)

    2015-03-01

    Qiagen, "EndoFree Plasmid Purification Handbook ," 2012. [Online]. Available: http://www.qiagen.com/resources/resourcedetail?id=f8ed5bab-15c3-4211 - bfa8...115] L. J. Hoffman, "Thermogravimetric Analysis of Bacillus anthracis Spores of DNA by Spectroscopy and Chromatography of Pyrolysis Products," M.S

  12. Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa

    Directory of Open Access Journals (Sweden)

    P. C.B. Turnbull

    2008-08-01

    Full Text Available TURNBULLP, P.C.B. DIEKMANNM,M., KILIAN, J.W., VERSFELDW, W.,DE VOS, V., ARNTZENL, L.,WOLTER, K., BARTELS, P. & KOTZE, A. 2008.N aturally acquired antibodies to Bacillusa nthracisp rotective antigeni n vultureso f southern Africa. Onderstepoort Journal of Veterinary Research, T5:95-102 Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories in North West Province, South Africa, were examined by an enzyme-linked immunosorbenats say( ELISAf or antibodiesto the Bacillus anthracis toxin protective antigen (PA. As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63% wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a hole and the other groups (P 0.05. Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheress, six out of ten Whitebacked Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypiust racheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. lt is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and the issue of the role of vultures in transmission of anthrax.

  13. CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

    Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

  14. Development and field testing of a mobile chlorine dioxide generation system for the decontamination of buildings contaminated with Bacillus anthracis

    International Nuclear Information System (INIS)

    Wood, Joseph P.; Blair Martin, G.

    2009-01-01

    The numerous buildings that became contaminated with Bacillus anthracis (the bacterium causing the disease anthrax) in 2001, and more recent B. anthracis - related events, point to the need to have effective decontamination technologies for buildings contaminated with biological threat agents. The U.S. Government developed a portable chlorine dioxide (ClO 2 ) generation system to decontaminate buildings contaminated with B. anthracis spores, and this so-called mobile decontamination trailer (MDT) prototype was tested through a series of three field trials. The first test of the MDT was conducted at Fort McClellan in Anniston, AL. during October 2004. Four test attempts occurred over two weekends; however, a number of system problems resulted in termination of the activity prior to any ClO 2 introduction into the test building. After making several design enhancements and equipment changes, the MDT was subjected to a second test. During this test, extensive leak checks were made using argon and nitrogen in lieu of chlorine gas; each subsystem was checked for functionality, and the MDT was operated for 24 h. This second test demonstrated the MDT flow and control systems functioned satisfactorily, and thus it was decided to proceed to a third, more challenging field trial. In the last field test, ClO 2 was generated and routed directly to the scrubber in a 12-h continuous run. Measurement of ClO 2 levels at the generator outlet showed that the desired production rate was not achieved. Additionally, only one of the two scrubbers performed adequately with regard to maintaining ClO 2 emissions below the limit. Numerous lessons were learned in the field trials of this ClO 2 decontamination technology.

  15. Genetic evidence for the involvement of the S-layer protein gene sap and the sporulation genes spo0A, spo0B, and spo0F in Phage AP50c infection of Bacillus anthracis.

    Science.gov (United States)

    Plaut, Roger D; Beaber, John W; Zemansky, Jason; Kaur, Ajinder P; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A; Mokashi, Vishwesh; Hannah, Ryan M; Pope, Robert K; Read, Timothy D; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

    2014-03-01

    In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.

  16. Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

    Science.gov (United States)

    Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

    2017-10-03

    Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

  17. Green-Tea and Epigallocatechin-3-Gallate are Bactericidal against Bacillus anthracis

    Science.gov (United States)

    2017-06-13

    strategies against B. anthracis (3). 60 After water, tea is the most consumed beverage in the world. Although containing little 61 caloric value, teas...Civilian B. 2002. Anthrax as a biological weapon, 2002: updated 270 recommendations for management . JAMA 287:2236-52. 271 4. Cabrera C, Artacho R...Sharma A, Gupta S, Sarethy IP, Dang S, Gabrani R. 2012. Green tea extract: possible mechanism 285 and antibacterial activity on skin pathogens. Food

  18. Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

    International Nuclear Information System (INIS)

    Jeong, Youngsu; Lee, Jonghee; Kim, Seongsoo

    2013-01-01

    The rapid and accurate identification of biological agents is a critical step in the case of bio-terror and biological warfare attacks. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been widely used for the identification of microorganisms. In this study, we describe a method for the rapid and accurate discrimination of Bacillus anthracis spores using MALDI-TOF MS. Our direct in-situ analysis of MALDI-TOF MS does not involve subsequent high-resolution mass analyses and sample preparation steps. This method allowed the detection of species-specific biomarkers from each Bacillus spores. Especially, B. anthracis spores had specific biomarker peaks at 2503, 3089, 3376, 6684, 6698, 6753, and 6840 m/z. Cluster and PCA analyses of the mass spectra of Bacillus spores revealed distinctively separated clusters and within-groups similarity. Therefore, we believe that this method is effective in the real-time identification of biological warfare agents such as B. anthracis as well as other microorganisms in the field

  19. A survey of the occurrence of Bacillus anthracis in North American soils over two long-range transects and within post-Katrina New Orleans

    Science.gov (United States)

    Griffin, Dale W.; Petrosky, Terry; Morman, Suzette A.; Luna, Vicki A.

    2009-01-01

    Soil samples were collected along a north-south transect extending from Manitoba, Canada, to the US-Mexico border near El Paso, Texas in 2004 (104 samples), a group of sites within New Orleans, Louisiana following Hurricane Katrina in 2005 (19 samples), and a Gulf Coast transect extending from Sulphur, Louisiana, to DeFuniak Springs, Florida, in 2007 (38 samples). Samples were collected from the top 40 cm of soil and were screened for the presence of total Bacillus species and Bacillus anthracis (anthrax), specifically using multiplex-polymerase chain reaction (PCR). Using an assay with a sensitivity of ~170 equivalent colony-forming units (CFU) g-1 field moist soil, the prevalence rate of Bacillus sp./B. anthracis in the north-south transect and the 2005 New Orleans post-Katrina sample set were 20/5% and 26/26%, respectively. Prevalence in the 2007 Gulf Coast sample set using an assay with a sensitivity of ~4 CFU g-1 of soil was 63/0%. Individual transect-set data indicate a positive relation between occurrences of species and soil moisture or soil constituents (i.e., Zn and Cu content). The 2005 New Orleans post-Katrina data indicated that B. anthracis is readily detectable in Gulf Coast soils following flood events. The data also indicated that occurrence, as it relates to soil chemistry, may be confounded by flood-induced dissemination of germinated cells and the mixing of soil constituents for short temporal periods following an event.

  20. Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Youngsu; Lee, Jonghee; Kim, Seongsoo [Agency for Defense Development, Daejeon (Korea, Republic of)

    2013-09-15

    The rapid and accurate identification of biological agents is a critical step in the case of bio-terror and biological warfare attacks. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been widely used for the identification of microorganisms. In this study, we describe a method for the rapid and accurate discrimination of Bacillus anthracis spores using MALDI-TOF MS. Our direct in-situ analysis of MALDI-TOF MS does not involve subsequent high-resolution mass analyses and sample preparation steps. This method allowed the detection of species-specific biomarkers from each Bacillus spores. Especially, B. anthracis spores had specific biomarker peaks at 2503, 3089, 3376, 6684, 6698, 6753, and 6840 m/z. Cluster and PCA analyses of the mass spectra of Bacillus spores revealed distinctively separated clusters and within-groups similarity. Therefore, we believe that this method is effective in the real-time identification of biological warfare agents such as B. anthracis as well as other microorganisms in the field.

  1. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

    Science.gov (United States)

    Hammerstrom, Troy G; Horton, Lori B; Swick, Michelle C; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M

    2015-02-01

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism. © 2014 John Wiley & Sons Ltd.

  2. The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

    Directory of Open Access Journals (Sweden)

    Haim Levy

    Full Text Available Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

  3. Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

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    Philippe Thullier

    Full Text Available The lethal toxin (LT of Bacillus anthracis, composed of the protective antigen (PA and the lethal factor (LF, plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF to form the edema toxin (ET, which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236, of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260 was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

  4. Pathology of wild-type and toxin-independent Bacillus anthracis meningitis in rabbits.

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    Assa Sittner

    Full Text Available Hemorrhagic meningitis is considered a complication of anthrax and was reported in about 50% of deadly cases in humans and non-human primates (NHP. Recently we demonstrated in Guinea pigs and rabbits that 100% of the B. anthracis-infected animals presented histopathology of meningitis at the time of death, some without any sign of hemorrhage. A similar pathology was observed in animals that succumbed following infection with the toxin deficient mutant, thus indicating that anthrax meningitis is a toxin-independent phenomenon. In this manuscript we describe a histopathological study of the B. anthracis infection of the central nervous system (CNS. Though we could find sporadic growth of the bacteria around blood vessels in the cortex, we report that the main infiltration route is the choroid plexus. We found massive destruction of entire sections of the choroid plexus coupled with massive aggregation of bacilli in the ventricles, in close proximity to the parenchyma. The choroid plexus also contained significant amounts of intravascular bacterial aggregates, often enclosed in what appear to be fibrin-like clots. The high concentration of these aggregates in areas of significant tissue destruction combined with the fact that capsular B. anthracis bacteria have a low tendency to adhere to endothelial cells, might suggest that these clots are used as an adherence mechanism by the bacteria. The major histopathological finding is meningitis. We find massive bacterial growth in the meninges without evidence of encephalitis, even when the bacteria emerge from a parenchymal blood vessel. Erythrocytes were present within the meningeal space but no clear vasculitis could be detected. Histology of the brain stem indicates meningitis, edema and hemorrhages that might explain death from suffocation due to direct damage to the respiratory center. All of these processes are toxin-independent, since they were observed following infection with either the wild

  5. The Poly-γ-D-Glutamic Acid Capsule of Bacillus licheniformis, a Surrogate of Bacillus anthracis Capsule Induces Interferon-Gamma Production in NK Cells through Interactions with Macrophages.

    Science.gov (United States)

    Lee, Hae-Ri; Jeon, Jun Ho; Rhie, Gi-Eun

    2017-05-28

    The poly-γ- D -glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis , provides protection of the bacterium from phagocytosis and allows its unimpeded growth in the host. We investigated crosstalk between murine natural killer (NK) cells and macrophages stimulated with the PGA capsule of Bacillus licheniformis , a surrogate of the B. anthracis capsule. PGA induced interferon-gamma production from NK cells cultured with macrophages. This effect was dependent on macrophage-derived IL-12 and cell-cell contact interaction with macrophages through NK cell receptor NKG2D and its ligand RAE-1. The results showed that PGA could enhance NK cell activation by inducing IL-12 production in macrophages and a contact-dependent crosstalk with macrophages.

  6. Obiltoxaximab Prevents Disseminated Bacillus anthracis Infection and Improves Survival during Pre- and Postexposure Prophylaxis in Animal Models of Inhalational Anthrax

    Science.gov (United States)

    Yamamoto, Brent J.; Shadiack, Annette M.; Carpenter, Sarah; Sanford, Daniel; Henning, Lisa N.; Gonzales, Nestor; O'Connor, Edward; Casey, Leslie S.

    2016-01-01

    The Centers for Disease Control and Prevention recommend adjunctive antitoxins when systemic anthrax is suspected. Obiltoxaximab, a monoclonal antibody against protective antigen (PA), is approved for treatment of inhalational anthrax in combination with antibiotics and for prophylaxis when alternative therapies are not available. The impact of toxin neutralization with obiltoxaximab during pre- and postexposure prophylaxis was explored, and efficacy results that supported the prophylaxis indication are presented here. New Zealand White rabbits and cynomolgus macaques received obiltoxaximab as a single intramuscular or intravenous dose of 2 to 16 mg/kg of body weight at various times relative to Bacillus anthracis aerosol spore challenge. The primary endpoint was survival, and effect of treatment timing was explored. In rabbits, obiltoxaximab administration 9 h postchallenge singly or combined with a 5-day levofloxacin regimen protected 89% to 100% of animals compared to 33% with levofloxacin monotherapy. In cynomolgus macaques, a single intramuscular dose of 16 mg/kg obiltoxaximab led to 100% survival when given 1 to 3 days preexposure and 83% to 100% survival when given 18 to 24 h postexposure and prior to systemic bacteremia onset. Obiltoxaximab administration after bacteremia onset resulted in lower (25% to 50%) survival rates reflective of treatment setting. Prophylactic administration of obiltoxaximab before spore challenge or to spore-challenged animals before systemic bacterial dissemination is efficacious in promoting survival, ameliorating toxemia, and inhibiting bacterial spread to the periphery. PMID:27431219

  7. BLACK-BACKED JACKAL EXPOSURE TO RABIES VIRUS, CANINE DISTEMPER VIRUS, AND BACILLUS ANTHRACIS IN ETOSHA NATIONAL PARK, NAMIBIA

    Science.gov (United States)

    Bellan, Steve E.; Cizauskas, Carrie A.; Miyen, Jacobeth; Ebersohn, Karen; Küsters, Martina; Prager, Katie; Van Vuuren, Moritz; Sabeta, Claude; Getz, Wayne M.

    2017-01-01

    Canine distemper virus (CDV) and rabies virus (RABV) occur worldwide in wild carnivore and domestic dog populations and pose threats to wildlife conservation and public health. In Etosha National Park (ENP), Namibia, anthrax is endemic and generates carcasses frequently fed on by an unusually dense population of black-backed jackals (Canis mesomelas). Using serology and phylogenetic analyses (on samples obtained from February, 2009 to July, 2010), and historical mortality records (1975–2011), we assessed jackal exposure to Bacillus anthracis (BA; the causal bacterial agent of anthrax), CDV, and RABV. Seroprevalence to all three pathogens was relatively high with 95% (n = 86), 73% (n = 86), and 9% (n = 81) of jackals exhibiting antibodies to BA, CDV, and RABV, respectively. Exposure to BA, as assessed with an anti-Protective Antigen ELISA test, increased significantly with age and all animals >1 yr old tested positive. Seroprevalence of exposure to CDV also increased significantly with age, with similar age-specific trends during both years of the study. No significant effect of age was found on RABV seroprevalence. Three of the seven animals exhibiting immunity to RABV were monitored for more than one year after sampling and did not succumb to the disease. Mortality records revealed that rabid animals are destroyed nearly every year inside the ENP tourist camps. Phylogenetic analyses demonstrated that jackal RABV in ENP is part of the same transmission cycle as other dog-jackal RABV cycles in Namibia. PMID:22493112

  8. Biochemical and Structural Analysis of an Eis Family Aminoglycoside Acetyltransferase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Green, Keith D.; Biswas, Tapan; Chang, Changsoo; Wu, Ruiying; Chen, Wenjing; Janes, Brian K.; Chalupska, Dominika; Gornicki, Piotr; Hanna, Philip C.; Tsodikov, Oleg V.; Joachimiak, Andrzej; Garneau-Tsodikova, Sylvie

    2015-05-26

    Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

  9. Failure of Sterne- and Pasteur-like strains of Bacillus anthracis to replicate and survive in the urban bluebottle blow fly Calliphora vicina under laboratory conditions.

    Directory of Open Access Journals (Sweden)

    Britta von Terzi

    Full Text Available This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4 known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7-9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.

  10. Microevolution of Anthrax from a Young Ancestor (M.A.Y.A. Suggests a Soil-Borne Life Cycle of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Peter Braun

    Full Text Available During an anthrax outbreak at the Pollino National Park (Basilicata, Italy in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA and next generation Whole Genome Sequencing (WGS. PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

  11. Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate.

    Science.gov (United States)

    Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D

    2008-11-25

    o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase

  12. Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate †

    Science.gov (United States)

    Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D.

    2009-01-01

    O-succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered Bi Uni Uni Bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct-binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first-half reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the ‘F’ form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered Bi Uni Uni Bi Iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogs with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogs tested as none were active except 4-(2-trifluoromethylphenyl)-4-oxobutyric acid which exhibited a 100-fold decrease in kcat/Km. Based on an understanding of OSB-CoA synthetase’s kinetic mechanism and substrate specificity, a reaction intermediate analog of OSB-AMP, 5’-O-(N-(2-trifluoromethylphenyl)-4-oxobutyl) adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of

  13. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, J. R. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Piepel, G. F. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Amidan, B. G. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Hess, B. M. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Sydor, M. A. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Deatherage Kaiser, B. L. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA

    2018-03-13

    Aims: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials, and assay methods on false-negative rate (FNR) and limit of detection (LOD95) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Methods and Results: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2 – 500 coupon-1) onto glass, stainless steel, vinyl tile, and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. Conclusions: Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. Significance and Impact of Study: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.

  14. Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth.

    Science.gov (United States)

    Bensman, M D; Mackie, R S; Minter, Z A; Gutting, B W

    2012-08-01

     The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species.  Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria.  The data suggested sera can have a significant impact on germination and growth of Sterne bacteria.  These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  15. Bacterial spores as possible contaminants of biomedical materials and devices. [Bacillus anthracis, clostridium botulinum, C. perfringens, C. tetani

    Energy Technology Data Exchange (ETDEWEB)

    Grecz, N; Kang, T

    1973-01-01

    Destruction of spores on biomedical devices in drugs, and biologicals is essential for prevention of infection of patients with pathogenic sporeformers. Of particular concern are Clostridium tetani, C. perfringens, C. botulinum, Bacillus anthracis and other sporeforming pathogens. Spores are ubiquitous in nature and contamination of biomedical devices varies depending on manufacturing process, handling, raw materials and other variables. In the last 20 years the number of cases per year of specific notifiable diseases in the United States was as follows: tetanus, 120 to 500 cases, botulism, 7 to 47 cases, and anthrax, 2 to 10 cases. Gas gangrene is caused by a mixed flora consisting predominantly of sporeformers. C botulinum, which usually acts as saprophytic agent of food poisoning, may also initiate pathogenic processes; there are nine cases on record in the United States of botulism wound infections almost half of which ended in death. The spores of these organisms are distinguished by high radiation resistance and their erradication often requires severe radiation treatments. Representative bacterial spores in various suspending media show D/sub 10/ values (dose necessary to destroy 90 percent of a given population) ranging from approximately 0.1 to 0.4 Mrad. Some viruses show D/sub 10/ values up to greater than 1 Mrad. The D/sub 10/-values of spores vary depending on physical, chemical and biological factors. This variability is important in evaluation and selection of biological indicator organisms. Radiation sterilization of biomedical devices and biomedical materials must provide safety from infectious microorganisms including radiation resistant spores and viruses.

  16. Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

    Science.gov (United States)

    vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

    2011-07-01

    A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

  17. Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (l-Ala-P)

    International Nuclear Information System (INIS)

    Au, Kinfai; Ren, Jingshan; Walter, Thomas S.; Harlos, Karl; Nettleship, Joanne E.; Owens, Raymond J.; Stuart, David I.; Esnouf, Robert M.

    2008-01-01

    Structures of BA0252, an alanine racemase from B. anthracis, in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) and determined by X-ray crystallography to resolutions of 2.1 and 1.47 Å, respectively, are described. Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) have determined by X-ray crystallo@@graphy to resolutions of 2.1 and 1.47 Å, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is d-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of l-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies

  18. Experimental Design for a Macrofoam-Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2014-12-05

    This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam-swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (culture and polymerase chain reaction) will be used. Only one previous study has investigated how the false negative rate depends on test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completing gaps in the available information on the performance of macrofoam-swab sampling at low concentrations.

  19. Experimental Design for a Macrofoam Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Hutchison, Janine R.

    2014-04-16

    This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (plating/counting and polymerase chain reaction) will be used. Only one previous study has investigated false negative as a function of affecting test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completing gaps in the available information on the performance of macrofoam swab sampling at low concentrations.

  20. False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kaiser, Brooke L.D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-01

    Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, and plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm2). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD95 was lowest for glass (0.429 CFU/cm2 with BAS and 0.341 CFU/cm2 with BG) and highest for vinyl tile (0.919 CFU/cm2 with BAS and 0.917 CFU/cm2 with BG). These mRV-PCR LOD95 values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm2 and BG: 0.820 to 1.489 CFU/cm2). The FNR and LOD95 values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

  1. [Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

    Science.gov (United States)

    Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

    2015-01-01

    To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance

  2. Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727

    Directory of Open Access Journals (Sweden)

    Mahillon Jacques

    2005-07-01

    Full Text Available Abstract Background Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first conjugative plasmid from the B. cereus group to be completely sequenced. Results The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI containing the anthrax capsule genes. Conclusion The complete sequence determination of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis

  3. DNA repair and its relation to recombination-deficient and other mutations in Bacillus subtilis

    International Nuclear Information System (INIS)

    Ganesan, A.T.

    1975-01-01

    DNA repair processes operating in Bacillus subtilis are similar to other transformable bacterial systems. Radiation-sensitive, recombination-deficient mutants are blocked in distinct steps leading to recombination. DNA polymerase I is essential for the repair of x-ray-induced damage to DNA but not for recombination

  4. Sporulation and germination gene expression analysis of Bacillus anthracis Sterne spores in skim milk under heat and different intervention techniques

    Science.gov (United States)

    To investigate how B. anthracis Stene spores survive in milk under heat (80 degree C, 10 minutes), pasteurization (72 degree C, 15 seconds) and pasteurization plus microfiltration, the expression levels of genes that related to sporulation and germination were tested using real-time PCR assays. Tw...

  5. The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Xiaofeng [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Walter, Michael H. [Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614 (United States); Paredes, Angel [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Morais, Marc C., E-mail: mcmorais@utmb.edu [Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States); Liu, Jun, E-mail: Jun.Liu.1@uth.tmc.edu [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States)

    2011-12-20

    The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T = 16 icosahedron attached to a contractile tail via a head-tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.

  6. Comparison of French and Worldwide Bacillus anthracis Strains Favors a Recent, Post-Columbian Origin of the Predominant North-American Clade.

    Science.gov (United States)

    Vergnaud, Gilles; Girault, Guillaume; Thierry, Simon; Pourcel, Christine; Madani, Nora; Blouin, Yann

    2016-01-01

    Bacillus anthracis, the highly dangerous zoonotic bacterial pathogen species is currently composed of three genetic groups, called A, B and C. Group A is represented worldwide whereas group B is present essentially in Western Europe and Southern Africa. Only three strains from group C have been reported. This knowledge is derived from the genotyping of more than 2000 strains collected worldwide. Strains from both group A and group B are present in France. Previous investigations showed that the majority of sporadic French strains belong to the so-called A.Br.011/009 group A clade and define a very remarkable polytomy with six branches. Here we explore the significance of this polytomy by comparing the French B. anthracis lineages to worldwide lineages. We take advantage of whole genome sequence data previously determined for 122 French strains and 45 strains of various origins. A total of 6690 SNPs was identified among the available dataset and used to draw the phylogeny. The phylogeny of the French B group strains which belongs to B.Br.CNEVA indicates an expansion from the south-east part of France (the Alps) towards the south-west (Massif-Central and Pyrenees). The relatively small group A strains belonging to A.Br.001/002 results from at least two independent introductions. Strikingly, the data clearly demonstrates that the currently predominant B. anthracis lineage in North America, called WNA for Western North American, is derived from one branch of the A.Br.011/009 polytomy predominant in France. The present work extends the range of observed substitution rate heterogeneity within B. anthracis, in agreement with its ecology and in contrast with some other pathogens. The population structure of the six branches A.Br.011/009 polytomy identified in France, diversity of branch length, and comparison with the WNA lineage, suggests that WNA is of post-Columbian and west European origin, with France as a likely source. Furthermore, it is tempting to speculate that

  7. Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk.

    Science.gov (United States)

    Tomasula, P M; Mukhopadhyay, S; Datta, N; Porto-Fett, A; Call, J E; Luchansky, J B; Renye, J; Tunick, M

    2011-09-01

    High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log(10) BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91±0.05 log(10) BA spores/mL of milk and the 1.4-μm membrane removed 4.50±0.35 log(10) BA spores/mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log(10) spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no

  8. False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Deatherage Kaiser, Brooke L [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-05-01

    Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, and plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm²). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD₉₅ was lowest for glass (0.429 CFU/cm² with BAS and 0.341 CFU/cm² with BG) and highest for vinyl tile (0.919 CFU/cm² with BAS and 0.917 CFU/cm² with BG). These mRV-PCR LOD₉₅ values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm² and BG: 0.820 to 1.489 CFU/cm²). The FNR and LOD₉₅ values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

  9. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    Science.gov (United States)

    1990-02-01

    procaryotic systems (12. 45). Certain eucaryotic ically cleaved by a trypsin-like proteas: ito produce a recep- viruses are currently being explored as...19847. Proteolytic activation of anthrax toxin bound to cellular recep- ACKN()WEIX;NMNTS tor%.. p. 111-112. In F. Fehrenbach et al. ifed.). Bacterial

  10. In Vitro and In Vivo Activity of Omadacycline Against Two Biothreat Pathogens: Bacillus Anthracis and Yersinia Pestis

    Science.gov (United States)

    2013-02-27

    visually at 18-24 hours (B. anthracis) or 42-48 hours (Y. pestis) and also by absorbance at 600 nm (SpectroMax M2, Molecular Devices). Thirty...certain uncomplicated infections; warns about disabling side effects that can occur together. May 12, 2016. Accessed August 29, 2016. Flamm RK, Rhomberg... odontology in the management of bioterrorism. In Evidence-Based Forensic Dentistry. Springer-Verlag Berlin Heidelberg 2013. pp. 149-152. Rotz LD

  11. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    Science.gov (United States)

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  12. Immunological correlates for protection against intranasal challenge of Bacillus anthracis spores conferred by a protective antigen-based vaccine in rabbits.

    Science.gov (United States)

    Weiss, Shay; Kobiler, David; Levy, Haim; Marcus, Hadar; Pass, Avi; Rothschild, Nili; Altboum, Zeev

    2006-01-01

    Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 x 10(5) conferred protection, whereas lower titers (between 10(4) and 10(5)) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.

  13. Development of Protective Immunity in New Zealand White Rabbits Challenged with Bacillus anthracis Spores and Treated with Antibiotics and Obiltoxaximab, a Monoclonal Antibody against Protective Antigen.

    Science.gov (United States)

    Henning, Lisa N; Carpenter, Sarah; Stark, Gregory V; Serbina, Natalya V

    2018-02-01

    The recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response. Copyright © 2018 Henning et al.

  14. Inactivation of Bacillus anthracis Spores by a Combination of Biocides and Heating under High-Temperature Short-Time Pasteurization Conditions ▿

    Science.gov (United States)

    Xu, Sa; Labuza, Theodore P.; Diez-Gonzalez, Francisco

    2008-01-01

    The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85°C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80°C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80°C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80°C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80°C, respectively. TTI6-log of less than 20 s were also achieved at 80°C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process

  15. Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions.

    Science.gov (United States)

    Xu, Sa; Labuza, Theodore P; Diez-Gonzalez, Francisco

    2008-06-01

    The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be

  16. Comparative genomics of Bacillus anthracis from the wool industry highlights polymorphisms of lineage A.Br.Vollum.

    Science.gov (United States)

    Derzelle, Sylviane; Aguilar-Bultet, Lisandra; Frey, Joachim

    2016-12-01

    With the advent of affordable next-generation sequencing (NGS) technologies, major progress has been made in the understanding of the population structure and evolution of the B. anthracis species. Here we report the use of whole genome sequencing and computer-based comparative analyses to characterize six strains belonging to the A.Br.Vollum lineage. These strains were isolated in Switzerland, in 1981, during iterative cases of anthrax involving workers in a textile plant processing cashmere wool from the Indian subcontinent. We took advantage of the hundreds of currently available B. anthracis genomes in public databases, to investigate the genetic diversity existing within the A.Br.Vollum lineage and to position the six Swiss isolates into the worldwide B. anthracis phylogeny. Thirty additional genomes related to the A.Br.Vollum group were identified by whole-genome single nucleotide polymorphism (SNP) analysis, including two strains forming a new evolutionary branch at the basis of the A.Br.Vollum lineage. This new phylogenetic lineage (termed A.Br.H9401) splits off the branch leading to the A.Br.Vollum group soon after its divergence to the other lineages of the major A clade (i.e. 6 SNPs). The available dataset of A.Br.Vollum genomes were resolved into 2 distinct groups. Isolates from the Swiss wool processing facility clustered together with two strains from Pakistan and one strain of unknown origin isolated from yarn. They were clearly differentiated (69 SNPs) from the twenty-five other A.Br.Vollum strains located on the branch leading to the terminal reference strain A0488 of the lineage. Novel analytic assays specific to these new subgroups were developed for the purpose of rapid molecular epidemiology. Whole genome SNP surveys greatly expand upon our knowledge on the sub-structure of the A.Br.Vollum lineage. Possible origin and route of spread of this lineage worldwide are discussed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights

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

    Science.gov (United States)

    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.

  18. Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Deatherage Kaiser, Brooke L [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Barrett, Christopher A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-03-31

    The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in. × 2 in.) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest for vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent report.

  19. Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kaiser, Brooke L. D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Barrett, Christopher A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-16

    The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest for vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results are discussed in a separate report.

  20. Structure of the Bacillus anthracis dTDP- L -rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Jackson; Lee, Jesi; Halavaty, Andrei S.; Minasov, George; Anderson, Wayne F.; Kuhn, Misty L. (NWU); (SFSU)

    2017-10-30

    L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), fromBacillus anthraciswas determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.

  1. Mini review: Recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives.

    Science.gov (United States)

    Lakowitz, Antonia; Godard, Thibault; Biedendieck, Rebekka; Krull, Rainer

    2018-05-01

    Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing. Their broad product portfolio of pharmaceutically relevant recombinant proteins described in research include antibody fragments, growth factors, interferons and interleukins, insulin, penicillin G acylase, streptavidin and different kinases produced in various cultivation systems like microtiter plates, shake flasks and bioreactor systems in batch, fed-batch and continuous mode. To further improve production and secretion performance of Bacillus, bottlenecks and limiting factors concerning proteases, chaperones, secretion machinery or feedback mechanisms can be identified on different cell levels from genomics and transcriptomics via proteomics to metabolomics and fluxomics. For systematical identification of recurring patterns characteristic of given regulatory systems and key genetic targets, systems biology and omics-technology provide suitable and promising approaches, pushing Bacillus further towards industrial application for recombinant pharmaceutical protein production. Copyright © 2017. Published by Elsevier B.V.

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    Chen, Po Ting; Chao, Yun-Peng

    2006-10-01

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

  4. The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants.

    Science.gov (United States)

    March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A

    2015-10-01

    This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat

  5. Inactivation of Vegetative Cells, but Not Spores, of Bacillus anthracis, B. cereus, and B. subtilis on Stainless Steel Surfaces Coated with an Antimicrobial Silver- and Zinc-Containing Zeolite Formulation

    Science.gov (United States)

    Galeano, Belinda; Korff, Emily; Nicholson, Wayne L.

    2003-01-01

    Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25°C and 80% relative humidity), the zeolite coating produced approximately 3 log10 inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected. PMID:12839825

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

    NARCIS (Netherlands)

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

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

  7. Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

    Science.gov (United States)

    Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

    2012-01-01

    We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate–protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate–spacer–carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate. PMID:22012665

  8. Decontamination efficacy of three commercial-off-the-shelf (COTS sporicidal disinfectants on medium-sized panels contaminated with surrogate spores of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Jason M Edmonds

    Full Text Available In the event of a wide area release and contamination of a biological agent in an outdoor environment and to building exteriors, decontamination is likely to consume the Nation's remediation capacity, requiring years to cleanup, and leading to incalculable economic losses. This is in part due to scant body of efficacy data on surface areas larger than those studied in a typical laboratory (5×10-cm, resulting in low confidence for operational considerations in sampling and quantitative measurements of prospective technologies recruited in effective cleanup and restoration response. In addition to well-documented fumigation-based cleanup efforts, agencies responsible for mitigation of contaminated sites are exploring alternative methods for decontamination including combinations of disposal of contaminated items, source reduction by vacuuming, mechanical scrubbing, and low-technology alternatives such as pH-adjusted bleach pressure wash. If proven effective, a pressure wash-based removal of Bacillus anthracis spores from building surfaces with readily available equipment will significantly increase the readiness of Federal agencies to meet the daunting challenge of restoration and cleanup effort following a wide-area biological release. In this inter-agency study, the efficacy of commercial-of-the-shelf sporicidal disinfectants applied using backpack sprayers was evaluated in decontamination of spores on the surfaces of medium-sized (∼1.2 m2 panels of steel, pressure-treated (PT lumber, and brick veneer. Of the three disinfectants, pH-amended bleach, Peridox, and CASCAD evaluated; CASCAD was found to be the most effective in decontamination of spores from all three panel surface types.

  9. Inhibition of the adenylyl cyclase toxin, edema factor, from Bacillus anthracis by a series of 18 mono- and bis-(M)ANT-substituted nucleoside 5'-triphosphates.

    Science.gov (United States)

    Taha, Hesham; Dove, Stefan; Geduhn, Jens; König, Burkhard; Shen, Yuequan; Tang, Wei-Jen; Seifert, Roland

    2012-01-01

    Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins, i.e. lethal factor, protective antigen and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). Conventional antibiotic treatment is ineffective against either toxaemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Our previous studies showed that EF is differentially inhibited by various purine and pyrimidine nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl (ANT) groups at the 2'(3')-O-ribosyl position, with the unique preference for the base cytosine (Taha et al., Mol Pharmacol 75:693 (2009)). MANT-CTP was the most potent EF inhibitor (K (i), 100 nM) among 16 compounds studied. Here, we examined the interaction of EF with a series of 18 2',3'-O-mono- and bis-(M)ANT-substituted nucleotides, recently shown to be very potent inhibitors of the AC toxin from Bordetella pertussis, CyaA (Geduhn et al., J Pharmacol Exp Ther 336:104 (2011)). We analysed purified EF and EF mutants in radiometric AC assays and in fluorescence spectroscopy studies and conducted molecular modelling studies. Bis-MANT nucleotides inhibited EF competitively. Propyl-ANT-ATP was the most potent EF inhibitor (K (i), 80 nM). In contrast to the observations made for CyaA, introduction of a second (M)ANT-group decreased rather than increased inhibitor potency at EF. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to bis-MANT-ATP, but FRET to bis-MANT-CTP was only small. Mutations N583Q, K353A and K353R differentially altered the inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP. The nucleotide binding site of EF accommodates bulky bis-(M)ANT-substituted purine and pyrimidine nucleotides, but the fit is suboptimal compared to CyaA. These data provide a basis

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

    Science.gov (United States)

    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.

  11. Production of Xylanase by Recombinant Bacillus subtilis DB104 Cultivated in Agroindustrial Waste Medium

    Directory of Open Access Journals (Sweden)

    Is Helianti

    2016-07-01

    Full Text Available A recombinant Bacillus subtilis DB104 strain harbouring recombinant plasmid pSKE194 containing an Open Reading Frame (ORF of endoxylanase and its indigenous promoter from the wild-type B. subtilis AQ1 strain was constructed. This recombinant B. subtilis DB104 strain had higher endoxylanase activity than the nonrecombinant B. subtilis DB104 strain in standard media, such as Luria Bertani (LB and LB with xylan. The agroindustrial wastes corncobs and tofu liquid waste were chosen as cost-effective carbon and nitrogen sources, respectively, to test the economics of xylanase production using the recombinant B. subtilis DB104 at a larger scale. Submerged fermentation using a 4.5 L working volume fermentor with tofu liquid waste and 4% corncobs produced maximum xylanase activity of 1296 ± 1.2 U/mg (601.7 ± 0.6 U/mL after 48-hour fermentation at 37°C with 150 rpm agitation; this is more than twofold higher than the activity produced in an Erlenmeyer flask. This is the first report of high xylanase activity produced from recombinant B. subtilis using inexpensive medium. During fermentation, the xylanase degrades corncobs into xylooligosaccharides, showing its potential as an enzyme feed additive or in xylooligosaccharide production.

  12. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    Science.gov (United States)

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  13. A new potential secretion pathway for recombinant proteins in Bacillus subtilis.

    Science.gov (United States)

    Wang, Guangqiang; Xia, Yongjun; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Haiqin; Ai, Lianzhong; Chen, Wei

    2015-11-10

    Secretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable 'cell factory' for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium. All four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides. Our results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.

  14. Construction and application of recombinant strain for the production of an alkaline protease from Bacillus licheniformis.

    Science.gov (United States)

    Lin, Songyi; Zhang, Meishuo; Liu, Jingbo; Jones, Gregory S

    2015-03-01

    The alkaline protease gene, Apr, from Bacillus licheniformis 2709 was cloned into an expression vector pET - 28b (+), to yield the recombinant plasmid pET-28b (+) - Apr. The pET-28b (+) - Apr was expressed in a high expression strain E. coli BL21. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 2709. Sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE) was used to access the protein expression. SDS-PAGE analysis indicated a protein of Mr of 38.8 kDa. The medium components and condition of incubation were optimized for the growth state of a recombinant strain. The optimal composition of production medium was composed of glucose 8 g/L, peptone 8 g/L and salt solution 10 mL. The samples were incubated on a rotary shaker of 180 r/min at 37°C for 24 h. Copyright © 2014. Published by Elsevier B.V.

  15. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

    Directory of Open Access Journals (Sweden)

    Shi Gui-Yang

    2009-10-01

    Full Text Available Abstract Background Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable. Results A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis α-amylase (BLA production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained. Conclusion This production level of BLA by B. licheniformis CBBD302(pHY-amyL is amongst the highest levels in Gram-positive bacteria reported so far.

  16. Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium

    Directory of Open Access Journals (Sweden)

    Nie Weijia

    2008-11-01

    Full Text Available Abstract Background Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce. Results The toxin genes tcdA and tcdB were amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of both tcdA and tcdB genes in the vector have been verified by DNA sequencing. The constructs were transformed into B. megaterium protoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination. Conclusion We have generated the full length and active recombinant TcdA and TcdB in Bacillus megaterium.

  17. Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology.

    Science.gov (United States)

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

    2007-01-01

    Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.

  18. Identification of Ciprofloxacin Resistance by SimpleProbe (trademark), High Resolution Melt and Pyrosequencing (trademark) Nucleic Acid Analysis in Biothreat Agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis

    Science.gov (United States)

    2010-01-01

    tularensis strain Schu4 following standardmicrobiological techniques to develop antibiotic resistance. Colonies from an original chocolate agar culture...35 C for two weeks. Growth in broth containing 16 mg/ml ciprofloxacin was transferred to chocolate agar plates containing 64 mg/ml ciprofloxacin and...expeditious therapy , the three PCR technologies described in this study were evaluated for the ability to accurately differentiate wt B. anthracis, Y

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

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    Tomasz Łęga

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

  20. The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease.

    Science.gov (United States)

    Tang, Zeli; Shang, Mei; Chen, Tingjin; Ren, Pengli; Sun, Hengchang; Qu, Hongling; Lin, Zhipeng; Zhou, Lina; Yu, Jinyun; Jiang, Hongye; Zhou, Xinyi; Li, Xuerong; Huang, Yan; Xu, Jin; Yu, Xinbing

    2016-12-19

    Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes. We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome. The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores. Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic

  1. Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis.

    Science.gov (United States)

    Wang, Yi; Chen, Zhenmin; Zhao, Ruili; Jin, Tingting; Zhang, Xiaoming; Chen, Xiangdong

    2014-08-31

    Bacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. Five genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually skfA, sdpC, xpf, and lytC. We constructed the multiple deletion mutant LM2531 (skfA sdpC lytC xpf) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical B. subtilis 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (β-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular β-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively). Our novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by B. subtilis. These findings show promise for guiding efforts to manipulate the genomes of other B. subtilis strains that are used for industrial purposes.

  2. Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis.

    Science.gov (United States)

    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.

  3. Construction of a recombinant Bacillus velezensis strain as an integrated control agent against plant diseases and insect pests.

    Science.gov (United States)

    Roh, Jong Yul; Liu, Qin; Choi, Jae Young; Wang, Yong; Shim, Hee Jin; Xu, Hong Guang; Choi, Gyung Ja; Kim, Jin-Cheol; Je, Yeon Ho

    2009-10-01

    To construct a new recombinant strain of Bacillus velezensis that has antifungal and insecticidal activity via the expression of the insecticidal Bacillus thuringiensis crystal protein, a B. thuringiensis expression vector (pHT1K-1Ac) was generated that contained the B. thuringiensis cry1Ac gene under the control of its endogenous promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K). This vector was introduced into a B. velezensis isolate that showed high antifungal activities against several plant diseases, including rice blast (Magnaporthe grisea), rice sheath blight (Rhizotonia solani), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans), and wheat leaf rust (Puccinia recondita), by electroporation. The recombinant B. velezensis strain was confirmed by PCR using cry1Ac-specific primers. Additionally, the recombinant strain produced a protein approximately 130 kDa in size and parasporal inclusion bodies similar to B. thuringiensis. The in vivo antifungal activity assay demonstrated that the activity of the recombinant B. velezensis strain was maintained at the same level as that of wild-type B. velezensis. Furthermore, it exhibited high insecticidal activity against a lepidopteran pest, Plutella xylostella, although its activity was lower than that of a recombinant B. thuringiensis strain, whereas wild-type B. velezensis strain did not show any insecticidal activity. These results suggest that this recombinant B. velezensis strain can be used to control harmful insect pests and fungal diseases simultaneously in one crop.

  4. Mob/oriT, a mobilizable site-specific recombination system for unmarked genetic manipulation in Bacillus thuringiensis and Bacillus cereus.

    Science.gov (United States)

    Wang, Pengxia; Zhu, Yiguang; Zhang, Yuyang; Zhang, Chunyi; Xu, Jianyi; Deng, Yun; Peng, Donghai; Ruan, Lifang; Sun, Ming

    2016-06-10

    Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed. We present a novel genetic manipulation method for B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis. The Mob/oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.

  5. Surface-Layer (S-Layer) Proteins Sap and EA1 Govern the Binding of the S-Layer-Associated Protein BslO at the Cell Septa of Bacillus anthracis

    Science.gov (United States)

    Kern, Valerie J.; Kern, Justin W.; Theriot, Julie A.; Schneewind, Olaf

    2012-01-01

    The Gram-positive pathogen Bacillus anthracis contains 24 genes whose products harbor the structurally conserved surface-layer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated Bacillus S-layer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the sap structural gene results in elongated chains of bacilli, as observed with a bslO mutant. The chain length of the sap mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in trans can be explained by an increased deposition of BslO onto the surface of sap mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings. PMID:22609927

  6. Evaluation of the ability of N-terminal fragment of lethal factor of Bacillus anthracis for delivery of Mycobacterium T cell antigen ESAT-6 into cytosol of antigen presenting cells to elicit effective cytotoxic T lymphocyte response

    International Nuclear Information System (INIS)

    Chandra, Subhash; Kaur, Manpreet; Midha, Shuchi; Bhatnagar, Rakesh; Banerjee-Bhatnagar, Nirupama

    2006-01-01

    We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine

  7. Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223

    Directory of Open Access Journals (Sweden)

    Yonghua Wang

    2011-11-01

    Full Text Available Experiments were carried out to investigate the effects of various factors on the activity and conformation of recombinant leucine aminopeptidase of Bacillus kaustophilus CCRC 11223 (BkLAP and potential utilization of BkLAP in the hydrolysis of anchovy protein. Optimal temperature and pH of BkLAP were 70 °C and 8.0 in potassium-phosphate buffer, respectively, and the activity was strongly stimulated by Ni2+, followed by Mn2+ and Co2+. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of BkLAP to different extents and further induce the changes in enzymatic activity. The secondary structure of BkLAP was slightly modified by Ni2+ at the concentration of 1×10−4 M, however, significant changes on the secondary structures of the enzyme were observed when Hg2+ was added to the concentration of 1×10−4 M. The potential application of BkLAP was evaluated through combination with the commercial or endogenous enzyme to hydrolysis the anchovy protein. Results showed that combining the BkLAP with other enzymes could significantly increase the degree of hydrolysis and amino acid component of hydrolysate. In this regard, BkLAP is a potential enzyme that can be used in the protein hydrolysate industry.

  8. CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers.

    Science.gov (United States)

    Schwartz, Jennifer K; Liu, Xiaofeng S; Tosha, Takehiko; Diebold, Adrienne; Theil, Elizabeth C; Solomon, Edward I

    2010-12-14

    DNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. The data show a single 5- or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30× more stable than Dps1; and the lower limit of K(D) for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.

  9. Germination of Bacillus cereus spores : the role of germination receptors

    NARCIS (Netherlands)

    Hornstra, L.M.

    2007-01-01

    The Bacillus cereus sensu lato group forms a highly homogeneous subdivision of the genus Bacillus and comprises several species that are relevant for humans. Notorious is Bacillus anthracis, the cause of the often-lethal disease anthrax, while the insect pathogen Bacillus

  10. Roles of Bacillus subtilis DprA and SsbA in RecA-mediated genetic recombination.

    Science.gov (United States)

    Yadav, Tribhuwan; Carrasco, Begoña; Serrano, Ester; Alonso, Juan C

    2014-10-03

    Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP · Mg(2+)-bound form (RecA · ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA · ATP. This work demonstrates that RecA · ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA · dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. In vitro Ca(2+)-dependent maturation of milk-clotting recombinant Epr: minor extracellular protease: from Bacillus licheniformis.

    Science.gov (United States)

    Ageitos, José Manuel; Vallejo, Juan Andrés; Serrat, Manuel; Sánchez-Pérez, Angeles; Villa, Tomás G

    2013-06-01

    The minor extracellular protease (Epr) is secreted into the culture medium during Bacillus licheniformis, strain USC13, stationary phase of growth. Whereas, B. subtilis Epr has been reported to be involved in swarming; the B. licheniformis protease is also involved in milk-clotting as shown by the curd forming ability of culture broths expressing this protein. The objectives of this study are the characterization of recombinant B. licheniformis Epr (minor extracellular protease) and the determination of its calcium-dependent activation process. In this work, we have cloned and expressed B. licheniformis Epr in Escherichia coli. We were also able to construct a tridimensional model for Epr based on its homology to Thermococcus kodakarensis pro-tk-subtilisin 2e1p, fervidolysin from Fervidobacterium pennivorans 1rv6, and B. lentus 1GCI subtilisin. Recombinant Epr was accumulated into inclusion bodies; after protein renaturation, Epr undergoes an in vitro calcium-dependent activation, similar to that described for tk protease. The recombinant Epr is capable of producing milk curds with the same clotting activity previously described for the native B. licheniformis Epr enzyme although further rheological and industrial studies should be carried out to confirm its real applicability. This work represents for the first time that Epr may be successfully expressed in a non-bacilli microorganism.

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

    Science.gov (United States)

    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.

  13. A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium Ions

    Science.gov (United States)

    Nielsen, Anders D.; Pusey, Marc L.; Fuglsang, Claus C.; Westh, Peter

    2003-01-01

    The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.

  14. Cloning and expression of the sucrose phosphorylase gene in Bacillus subtilis and synthesis of kojibiose using the recombinant enzyme.

    Science.gov (United States)

    Wang, Miaomiao; Wu, Jing; Wu, Dan

    2018-02-15

    Kojibiose as a prebiotic and inhibitor of α-glucosidase exhibits potential for a wide range of applications in the food and medicine fields; however, large-scale separation and extraction of kojibiose from nature is difficult. Sucrose phosphorylase (SPase) can be used for the production of kojibiose, and currently, SPase is only heterologously expressed in E. coli, making it unsuitable for use in the food industry. However, Bacillus subtilis is generally considered to be a safe organism potentially useful for SPase expression. Here, for the first time, we heterologously expressed Bifidobacterium adolescentis SPase in a food-grade B. subtilis strain. The results showed that SPase was efficiently secreted into the extracellular medium in the absence of a signal peptide. After culturing the recombinant strain in a 3-L bioreactor, crude SPase yield and activity reached 7.5 g/L and 5.3 U/mL, respectively, the highest levels reported to date. The optimal reaction conditions for kojibiose synthesis catalyzed by recombinant SPase were as follows: 0.5 M sucrose, 0.5 M glucose, 0.02 U enzyme /mg all_substrates , pH 7.0, 50 °C, and 30 h. Furthermore, the substrate-conversion rate reached 40.01%, with kojibiose accounting for 104.45 g/L and selectivity for kojibiose production at 97%. Here, we successfully expressed SPase in B. subtilis in the absence of a signal peptide and demonstrated its secretion into the extracellular medium. Our results indicated high levels of recombinant enzyme expression, with a substrate-conversion rate of 40.01%. These results provide a basis for large-scale preparation of kojibiose by the recombinant SPase.

  15. One-step production of biocommodities from lignocellulosic biomass by recombinant cellulolytic bacillus subtilis: opportunities and challenges

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiao-Zhou [Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); Zhang, Yi-Heng P. [Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); Institute for Critical Technology and Applied Science, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); BioEnergy Science Center of Department of Energy, Oak Ridge, TN (United States)

    2010-10-15

    One-step consolidated bioprocessing that integrates cellulase production, cellulose hydrolysis, and product fermentation into a single step for decreasing costly cellulase use, increasing volumetric productivity, and reducing capital investment is widely accepted for low-cost production of biofuels or other value-added biochemicals. Considering the narrow margins between biomass and low-value biocommodities, good physiological performance of industrial microbes is crucial for economically viable production. Bacillus subtilis, the best-characterized Gram-positive microorganism, is a major industrial microorganism with numerous valuable features such as hexose and pentose utilization, low-nutrient needs, fast growth rate, high protein secretion capacity, industrial safety, etc. As compared with other potential consolidated bioprocessing microorganisms such as Clostridium spp., Escherichia coli, and the yeast Saccharomyces cerevisiae, recombinant cellulolytic B. subtilis strains would be a potential platform for biocommodity production from nonfood biomass. Here, we review the advances in recombinant cellulolytic B. subtilis development and metabolic engineering for biocommodity production, and discuss the opportunities and challenges of cellulolytic B. subtilis for biocommodity production. (Copyright copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  16. Disinfection of Vegetative Cells of Bacillus anthracis

    Science.gov (United States)

    2016-03-01

    Peterson, A.; Donlan, R.M.; Arduino , M.J. Chlorine Inactivation of Bacterial Select Agents. Appl. and Environ. Microbial. 2005, 71, 5669–5689...Rose, L.J.; Rice, E.W.; Hodges, L.; Peterson, A.; Arduino , M.J. 2007. Monochloramine Inactivation of Bacterial Select Agents. Appl. and Environ

  17. Systematic Evaluation of Aggressive Air Sampling for Bacillus ...

    Science.gov (United States)

    Report The primary objectives of this project were to evaluate the Aggressive Air Sampling (AAS) method compared to currently used surface sampling methods and to determine if AAS is a viable option for sampling Bacillus anthracis spores.

  18. Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity.

    Science.gov (United States)

    Ami, Yasushi; Izumi, Yasuyuki; Matsuo, Kazuhiro; Someya, Kenji; Kanekiyo, Masaru; Horibata, Shigeo; Yoshino, Naoto; Sakai, Koji; Shinohara, Katsuaki; Matsumoto, Sohkichi; Yamada, Takeshi; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

    2005-10-01

    Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

  19. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  20. High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

    Directory of Open Access Journals (Sweden)

    Yao Nie

    Full Text Available Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3/pET-20b(+-pul and E. coli BL21(DE3/pET-22b(+-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3/pET-20b(+-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3/pET-22b(+-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3/pET-22b(+-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The

  1. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.

    1983-01-01

    Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

  2. Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant Bacillus sp. strain BRC1 with increased inulinase activity.

    Science.gov (United States)

    Park, Jang Min; Oh, Baek-Rock; Kang, In Yeong; Heo, Sun-Yeon; Seo, Jeong-Woo; Park, Seung-Moon; Hong, Won-Kyung; Kim, Chul Ho

    2017-07-01

    A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L -1 . Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L -1 , showing a high theoretical yield of 92.3%.

  3. Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant Bacillus subtilis and Candida maltosa

    Science.gov (United States)

    2011-01-01

    Background Xylose mother liquor has high concentrations of xylose (35%-40%) as well as other sugars such as L-arabinose (10%-15%), galactose (8%-10%), glucose (8%-10%), and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem. Results We designed a novel strategy in which Bacillus subtilis and Candida maltosa were combined and used to convert xylose in this mother liquor to xylitol, a product of higher value. First, the xylose mother liquor was detoxified with the yeast C. maltosa to remove furfural and 5-hydromethylfurfural (HMF), which are inhibitors of B. subtilis growth. The glucose present in the mother liquor was also depleted by this yeast, which was an added advantage because glucose causes carbon catabolite repression in B. subtilis. This detoxification treatment resulted in an inhibitor-free mother liquor, and the C. maltosa cells could be reused as biocatalysts at a later stage to reduce xylose to xylitol. In the second step, a recombinant B. subtilis strain with a disrupted xylose isomerase gene was constructed. The detoxified xylose mother liquor was used as the medium for recombinant B. subtilis cultivation, and this led to L-arabinose depletion and xylose enrichment of the medium. In the third step, the xylose was further reduced to xylitol by C. maltosa cells, and crystallized xylitol was obtained from this yeast transformation medium. C. maltosa transformation of the xylose-enriched medium resulted in xylitol with 4.25 g L-1·h-1 volumetric productivity and 0.85 g xylitol/g xylose specific productivity. Conclusion In this study, we developed a biological method for the purification of xylose from xylose mother liquor and subsequent preparation of xylitol by C. maltosa-mediated biohydrogenation of xylose

  4. Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant Bacillus subtilis and Candida maltosa

    Directory of Open Access Journals (Sweden)

    Jiang Mingguo

    2011-02-01

    Full Text Available Abstract Background Xylose mother liquor has high concentrations of xylose (35%-40% as well as other sugars such as L-arabinose (10%-15%, galactose (8%-10%, glucose (8%-10%, and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem. Results We designed a novel strategy in which Bacillus subtilis and Candida maltosa were combined and used to convert xylose in this mother liquor to xylitol, a product of higher value. First, the xylose mother liquor was detoxified with the yeast C. maltosa to remove furfural and 5-hydromethylfurfural (HMF, which are inhibitors of B. subtilis growth. The glucose present in the mother liquor was also depleted by this yeast, which was an added advantage because glucose causes carbon catabolite repression in B. subtilis. This detoxification treatment resulted in an inhibitor-free mother liquor, and the C. maltosa cells could be reused as biocatalysts at a later stage to reduce xylose to xylitol. In the second step, a recombinant B. subtilis strain with a disrupted xylose isomerase gene was constructed. The detoxified xylose mother liquor was used as the medium for recombinant B. subtilis cultivation, and this led to L-arabinose depletion and xylose enrichment of the medium. In the third step, the xylose was further reduced to xylitol by C. maltosa cells, and crystallized xylitol was obtained from this yeast transformation medium. C. maltosa transformation of the xylose-enriched medium resulted in xylitol with 4.25 g L-1·h-1 volumetric productivity and 0.85 g xylitol/g xylose specific productivity. Conclusion In this study, we developed a biological method for the purification of xylose from xylose mother liquor and subsequent preparation of xylitol by C. maltosa

  5. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.

    1979-01-01

    Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

  6. Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline α-amylase as a case study.

    Science.gov (United States)

    Ma, Yingfang; Yang, Haiquan; Chen, Xianzhong; Sun, Bo; Du, Guocheng; Zhou, Zhemin; Song, Jiangning; Fan, You; Shen, Wei

    2015-10-01

    In this study, atmospheric and room temperature plasma (ARTP), a promising mutation breeding technique, was successfully applied to generate Bacillus subtilis mutants that yielded large quantities of recombinant protein. The high throughput screening platform was implemented to select those mutants with the highest yield of recombinant alkaline α-amylase (AMY), including the preferred mutant B. subtilis WB600 mut-12#. The yield and productivity of recombinant AMY in B. subtilis WB600 mut-12# increased 35.0% and 8.8%, respectively, the extracellular protein concentration of which increased 37.9%. B. subtilis WB600 mut-12# exhibited good genetic stability. Cells from B. subtilis WB600 mut-12# became shorter and wider than those from the wild-type. This study is the first to report a novel powerful mutagenesis tool (ARTP) that significantly improves the yield of recombinant proteins in B. subtilis and may therefore play an important role in the high expression level of proteins in recombinant microbial hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group

    DEFF Research Database (Denmark)

    Agersø, Yvonne; Jensen, Lars Bogø; Givskov, Michael Christian

    2002-01-01

    In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure...

  8. Recombiner

    International Nuclear Information System (INIS)

    Saalfrank, H.

    1985-01-01

    Air containing hydrogen can be oxidized by heating in a container called a recombiner, in order to avoid the collection of hydrogen. The container is long and a large number of straight heating bars are arranged in parallel in it and they are flanged to a lid. The heating bars are surrounded by tubes, in order to obtain good heat transfer by a narrow annular gap. (orig.) [de

  9. Primary and secondary oxidative stress in Bacillus

    NARCIS (Netherlands)

    Mols, Maarten; Abee, Tjakko

    Coping with oxidative stress originating from oxidizing compounds or reactive oxygen species (ROS), associated with the exposure to agents that cause environmental stresses, is one of the prerequisites for an aerobic lifestyle of Bacillus spp. such as B. subtilis, B. cereus and B. anthracis. This

  10. Primary and secondary oxidative stress in Bacillus

    NARCIS (Netherlands)

    Mols, J.M.; Abee, T.

    2011-01-01

    Coping with oxidative stress originating from oxidizing compounds or reactive oxygen species (ROS), associated with the exposure to agents that cause environmental stresses, is one of the prerequisites for an aerobic lifestyle of Bacillus spp. such as B. subtilis, B. cereus and B. anthracis. This

  11. Recombinant protective antigen 102 (rPA102): profile of a second-generation anthrax vaccine.

    Science.gov (United States)

    Keitel, Wendy A

    2006-08-01

    Recent terrorist attacks involving the use of Bacillus anthracis spores have stimulated interest in the development of new vaccines for anthrax prevention. Studies of the pathogenesis of anthrax and of the immune responses following infection and immunization underscore the pivotal role that antibodies to the protective antigen play in protection. The most promising vaccine candidates contain purified recombinant protective antigen. Clinical trials of one of these, recombinant protective antigen (rPA)102, are underway. Initial results suggest that rPA102 is well tolerated and immunogenic. Additional trials are necessary to identify optimal formulations and immunization regimens for pre- and postexposure prophylaxis. Future licensure of these and other candidate vaccines will depend on their safety and immunogenicity profiles in humans, and their ability to confer protection in animal models of inhalational anthrax.

  12. Comparative analysis of immune effects in mice model: Clonorchis sinensis cysteine protease generated from recombinant Escherichia coli and Bacillus subtilis spores.

    Science.gov (United States)

    Wu, Zhanshuai; Tang, Zeli; Shang, Mei; Zhao, Lu; Zhou, Lina; Kong, Xiangzhan; Lin, Zhipeng; Sun, Hengchang; Chen, Tingjin; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2017-07-01

    Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.

  13. Extracellular secretion of a recombinant therapeutic peptide by Bacillus halodurans utilizing a modified flagellin type III secretion system

    CSIR Research Space (South Africa)

    Berger, E

    2011-08-01

    Full Text Available further 3.5-fold increase in the secretion of recombinant peptide fusions. Conclusions: The type III flagellar secretion system of B. halodurans has been shown to successfully secrete a therapeutic peptide as a heterologous flagellin fusion. Improvements...

  14. Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions.

    Science.gov (United States)

    González-Ponce, Karen S; Casados-Vázquez, Luz E; Salcedo-Hernández, Rubén; Bideshi, Dennis K; Del Rincón-Castro, María C; Barboza-Corona, José E

    2017-05-01

    In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry - B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74∆sp produced amorphous inclusions in both Cry - B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74∆sp chimeric inclusions of recombinant HD73 strain showed LC 50 s of 396.86 and 290.25 ng/cm 2 , respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.

  15. Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium.

    Science.gov (United States)

    Gerber, Adrian; Kleser, Michael; Biedendieck, Rebekka; Bernhardt, Rita; Hannemann, Frank

    2015-07-29

    Cholesterol, the precursor of all steroid hormones, is the most abundant steroid in vertebrates and exhibits highly hydrophobic properties, rendering it a difficult substrate for aqueous microbial biotransformations. In the present study, we developed a Bacillus megaterium based whole-cell system that allows the side-chain cleavage of this sterol and investigated the underlying physiological basis of the biocatalysis. CYP11A1, the side-chain cleaving cytochrome P450, was recombinantly expressed in the Gram-positive soil bacterium B. megaterium combined with the required electron transfer proteins. By applying a mixture of 2-hydroxypropyl-β-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol β-sitosterol could be efficiently converted to pregnenolone or 7-dehydropregnenolone. Fluorescence-microscopic analysis revealed that cholesterol accumulates in the carbon and energy storage-serving poly(3-hydroxybutyrate) (PHB) bodies and that the membrane proteins CYP11A1 and its redox partner adrenodoxin reductase (AdR) are likewise localized to their surrounding phospholipid/protein monolayer. The capacity to store cholesterol was absent in a mutant strain devoid of the PHB-producing polymerase subunit PhaC, resulting in a drastically decreased cholesterol conversion rate, while no effect on the expression of the recombinant proteins could be observed. We established a whole-cell system based on B. megaterium, which enables the conversion of the steroid hormone precursor cholesterol to pregnenolone in substantial quantities. We demonstrate that the microorganism's PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer, are crucial for the high conversion rate by serving as substrate storage. This microbial system opens the way for an industrial conversion of the abundantly available cholesterol to any type of steroid hormones, which represent one of the

  16. [PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

    Science.gov (United States)

    Popova, P Yu; Mikshis, N I

    2016-01-01

    Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses.

  17. Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells.

    Science.gov (United States)

    Pennacchio, Angela; Rossi, Mosè; Raia, Carlo A

    2013-07-01

    The synthesis of the aroma chemical cinnamyl alcohol (CMO) by means of enzymatic reduction of cinnamaldehyde (CMA) was investigated using NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus both as an isolated enzyme, and in recombinant Escherichia coli whole cells. The influence of parameters such as reaction time and cofactor, substrate, co-substrate 2-propanol and biocatalyst concentrations on the bioreduction reaction was investigated and an efficient and sustainable one-phase system developed. The reduction of CMA (0.5 g/L, 3.8 mmol/L) by the isolated enzyme occurred in 3 h at 50 °C with 97% conversion, and yielded high purity CMO (≥98%) with a yield of 88% and a productivity of 50 g/genzyme. The reduction of 12.5 g/L (94 mmol/L) CMA by whole cells in 6 h, at 37 °C and no requirement of external cofactor occurred with 97% conversion, 82% yield of 98% pure alcohol and a productivity of 34 mg/gwet cell weight. The results demonstrate the microbial system as a practical and efficient method for larger-scale synthesis of CMO.

  18. Th1 Cytokine-Secreting Recombinant Mycobacterium bovis Bacillus Calmette-Guérin and Prospective Use in Immunotherapy of Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Yi Luo

    2011-01-01

    Full Text Available Intravesical instillation of Mycobacterium bovis bacillus Calmette-Guérin (BCG has been used for treating bladder cancer for 3 decades. However, BCG therapy is ineffective in approximately 30–40% of cases. Since evidence supports the T helper type 1 (Th1 response to be essential in BCG-induced tumor destruction, studies have focused on enhancing BCG induction of Th1 immune responses. Although BCG in combination with Th1 cytokines (e.g., interferon-α has demonstrated improved efficacy, combination therapy requires multiple applications and a large quantity of cytokines. On the other hand, genetic manipulation of BCG to secrete Th1 cytokines continues to be pursued with considerable interest. To date, a number of recombinant BCG (rBCG strains capable of secreting functional Th1 cytokines have been developed and demonstrated to be superior to BCG. This paper discusses current rBCG research, concerns, and future directions with an intention to inspire the development of this very promising immunotherapeutic modality for bladder cancer.

  19. Observations on the migration of bacillus spores outside a contaminated facility during a decontamination efficacy study

    Science.gov (United States)

    Silvestri, Erin E.; Perkins, Sarah; Lordo, Robert; Kovacik, William; Nichols, Tonya L.; Bowling, Charlena Yoder; Griffin, Dale W.; Schaefer, Frank W.

    2015-01-01

    The potential for an intentional wide-area or indoor release of Bacillus anthracis spores remains a concern, but the fate and transport of B. anthracis spores in indoor and outdoor environments are not well understood. Some studies have examined the possibility of spore transport within ventilation systems and in buildings and transport into a building following an outdoor release. Little research exists regarding the potential for spores to migrate to the outside of a building following an indoor release.

  20. Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

    Science.gov (United States)

    Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

    2014-03-01

    Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Regioselective biooxidation of (+-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system

    Directory of Open Access Journals (Sweden)

    Schmid Rolf D

    2009-07-01

    Full Text Available Abstract Background (+-Nootkatone (4 is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+-valencene (1 provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+-nootkatone (4 from (+-valencene (1 involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+-valencene (1 at allylic C2-position to produce (+-nootkatone (4 via cis- (2 or trans-nootkatol (3. The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR and putidaredoxin (Pdx from Pseudomonas putida in Escherichia coli. Results Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+-valencene (1 yielding nootkatol (2 and 3 and (+-nootkatone (4. However, when the in vivo biooxidation of (+-valencene (1 with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents – isooctane, n-octane, dodecane or hexadecane – were set up, resulting in accumulation of nootkatol (2 and 3 and (+-nootkatone (4 of up to 97% of the total product. The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane. Conclusion This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+-nootkatone (4, as it is safe and can easily be

  2. Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system.

    Science.gov (United States)

    Girhard, Marco; Machida, Kazuhiro; Itoh, Masashi; Schmid, Rolf D; Arisawa, Akira; Urlacher, Vlada B

    2009-07-10

    (+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli. Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+)-valencene (1) yielding nootkatol (2 and 3) and (+)-nootkatone (4). However, when the in vivo biooxidation of (+)-valencene (1) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents - isooctane, n-octane, dodecane or hexadecane - were set up, resulting in accumulation of nootkatol (2 and 3) and (+)-nootkatone (4) of up to 97% of the total product. The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane. This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up.

  3. Identification, cloning and lactonase activity of recombinant protein of N-acyl homoserine lactonase (AiiA from Bacillus thuringiensis 147-115-16 strain.

    Directory of Open Access Journals (Sweden)

    Alvaro Mauricio Florez Escobar

    2014-01-01

    Short title: N-acyl homoserine lactonase (AiiA from Bacillus thuringiensis  Abstract: The quorum-quenching N-acyl homoserine lactonases are a family of bacterial metalloenzymes that participate in degradation of N-acyl homoserine lactones (AHLs, disrupting the quorum sensing system of gram negative bacterial species. From a collection of Bacillus thuringiensis strains isolated in Colombia from plants and exhibiting toxic activity against lepidopteran insects, 310 bacterial isolates were tested to determine lactonase activity by using biosensor systems in presence of synthetic N-hexanoyl-L-homoserine lactone (C6-HSL and N-octanoyl-L-homoserine lactone (C8-HSL. From them, 251 strains showed degrading activity to both C6-HSL and C8-HSL, 57% exhibited degrading activity to C6-HSL and 43% to C8-HSL. One B. thuringiensis strain, denoted as 147-115-16, that exhibit high degrading activity to C6-HSL and C8-HSL, was able to attenuate soft rot symptoms in infected potato slices with Pectobacterium carotovorum. This strain contains an homologous of the aiiA gene that was cloned, sequenced and expressed in Esherichia coli DE3. The recombinant protein AiiA147-11516 displays activity to C6-HSL, C8-HSL, N-(β-ketocaproyl (3-O-C6-HSL and N-3-oxo-dodecanoyl (3-O-C12-HSL. The recombinant strain in the presence of P. caratovorum cultures was able to attenuate the infection, suggesting that it interferes either with the accumulation or with the response to the AHLs signals. Acording to this data and based on previous report from recombinant AiiA147-11516, this enzyme exhibits activity to a wide range of catalytic substrates suggesting its industrial application in the disease control programs through plants transformation. Key words: lactones, Quorum sensing, Quorum quenching, Lactonases, Pectobacterium caratovorum Resumen: Las N-acíl homoserina lactonasas son una familia de metaloenzimas bacterianas que participan en la degradación de N-acil homoserina lactonas (AHLs

  4. Thermal Inactivation of Bacillus anthracis Spores Using Rapid Resistive Heating

    Science.gov (United States)

    2016-03-24

    agents. There is motivation for using thermal decontamination of B.a. spores for agent defeat scenarios. Spore-forming microorganisms are much...the top soil on Gruinard Island for over 40 years after the British detonated experimental anthrax bombs on the island during World War II (U.S

  5. Glyconanobiotics: Novel carbohydrated nanoparticle antibiotics for MRSA and Bacillus anthracis

    OpenAIRE

    Abeylath, Sampath C.; Turos, Edward; Dickey, Sonja; Limb, Daniel V.

    2007-01-01

    This report describes the synthesis and evaluation of glycosylated polyacrylate nanoparticles that have covalently-bound antibiotics within their framework. The requisite glycosylated drug monomers were prepared from one of three known antibiotics, an N-sec-butylthio β-lactam, ciprofloxacin, and a penicillin, by acylation with 3-O-acryloyl-1,2-O-isopropylidene-5,6 bis((chlorosuccinyl)oxy)-D-glucofuranose (7) or 6-O-acetyl-3-O-acryloyl-1,2-O-isopropylidene-5-(chlorosuccinyl)oxy-α-D-glucofurano...

  6. Bacillus anthracis infections – new possibilities of treatment

    Directory of Open Access Journals (Sweden)

    Dorota Żakowska

    2015-05-01

    Recently, progress has been achieved in inhalation anthrax treatment. The most promising new possibilities include: new antibiotics, peptides and bacteriophages enzymes, monoclonal antibodies, antigen PA mutants, and inter alpha inhibitors applications. In the case of the possibility of bioterrorist attacks, the examination of inhalation anthrax treatment should be intensively continued.

  7. Composite Sampling Approaches for Bacillus anthracis Surrogate Extracted from Soil.

    Directory of Open Access Journals (Sweden)

    Brian France

    Full Text Available Any release of anthrax spores in the U.S. would require action to decontaminate the site and restore its use and operations as rapidly as possible. The remediation activity would require environmental sampling, both initially to determine the extent of contamination (hazard mapping and post-decon to determine that the site is free of contamination (clearance sampling. Whether the spore contamination is within a building or outdoors, collecting and analyzing what could be thousands of samples can become the factor that limits the pace of restoring operations. To address this sampling and analysis bottleneck and decrease the time needed to recover from an anthrax contamination event, this study investigates the use of composite sampling. Pooling or compositing of samples is an established technique to reduce the number of analyses required, and its use for anthrax spore sampling has recently been investigated. However, use of composite sampling in an anthrax spore remediation event will require well-documented and accepted methods. In particular, previous composite sampling studies have focused on sampling from hard surfaces; data on soil sampling are required to extend the procedure to outdoor use. Further, we must consider whether combining liquid samples, thus increasing the volume, lowers the sensitivity of detection and produces false negatives. In this study, methods to composite bacterial spore samples from soil are demonstrated. B. subtilis spore suspensions were used as a surrogate for anthrax spores. Two soils (Arizona Test Dust and sterilized potting soil were contaminated and spore recovery with composites was shown to match individual sample performance. Results show that dilution can be overcome by concentrating bacterial spores using standard filtration methods. This study shows that composite sampling can be a viable method of pooling samples to reduce the number of analysis that must be performed during anthrax spore remediation.

  8. Draft Genome Sequences from a Novel Clade of Bacillus cereus Sensu Lato Strains, Isolated from the International Space Station

    NARCIS (Netherlands)

    Venkateswaran, Kasthuri; Checinska Sielaff, Aleksandra; Ratnayake, Shashikala; Pope, Robert K; Blank, Thomas E; Stepanov, Victor G; Fox, George E; van Tongeren, Sandra P; Torres, Clinton; Allen, Jonathan; Jaing, Crystal; Pierson, Duane; Perry, Jay; Koren, Sergey; Phillippy, Adam; Klubnik, Joy; Treangen, Todd J; Rosovitz, M J; Bergman, Nicholas H

    2017-01-01

    The draft genome sequences of six Bacillus strains, isolated from the International Space Station and belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group, are presented here. These strains were isolated from the Japanese Experiment Module (one strain), U.S. Harmony Node 2 (three

  9. Whole-Genome Sequences of 94 Environmental Isolates of Bacillus cereus Sensu Lato

    Science.gov (United States)

    Feldgarden, Michael; Kolter, Roberto; Mahillon, Jacques

    2013-01-01

    Bacillus cereus sensu lato is a species complex that includes the anthrax pathogen Bacillus anthracis and other bacterial species of medical, industrial, and ecological importance. Their phenotypes of interest are typically linked to large plasmids that are closely related to the anthrax plasmids pXO1 and pXO2. Here, we present the draft genome sequences of 94 isolates of B. cereus sensu lato, which were chosen for their plasmid content and environmental origins. PMID:24092776

  10. Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters

    OpenAIRE

    Calfee, M. Worth; Rose, Laura J.; Tufts, Jenia; Morse, Stephen; Clayton, Matt; Touati, Abderrahmane; Griffin-Gatchalian, Nicole; Slone, Christina; McSweeney, Neal

    2013-01-01

    The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37 mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50...

  11. Bacillus Coagulans

    Science.gov (United States)

    Bacillus coagulans is a type of bacteria. It is used similarly to lactobacillus and other probiotics as "beneficial" bacteria. People take Bacillus coagulans for diarrhea, including infectious types such as rotaviral ...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-09-14

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

  13. Bacillus cereus and related species.

    Science.gov (United States)

    Drobniewski, F A

    1993-10-01

    Bacillus cereus is a gram-positive aerobic or facultatively anaerobic spore-forming rod. It is a cause of food poisoning, which is frequently associated with the consumption of rice-based dishes. The organism produces an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxin, respectively. Other toxins are produced during growth, including phospholipases, proteases, and hemolysins, one of which, cereolysin, is a thiol-activated hemolysin. These toxins may contribute to the pathogenicity of B. cereus in nongastrointestinal disease. B. cereus isolated from clinical material other than feces or vomitus was commonly dismissed as a contaminant, but increasingly it is being recognized as a species with pathogenic potential. It is now recognized as an infrequent cause of serious nongastrointestinal infection, particularly in drug addicts, the immunosuppressed, neonates, and postsurgical patients, especially when prosthetic implants such as ventricular shunts are inserted. Ocular infections are the commonest types of severe infection, including endophthalmitis, panophthalmitis, and keratitis, usually with the characteristic formation of corneal ring abscesses. Even with prompt surgical and antimicrobial agent treatment, enucleation of the eye and blindness are common sequelae. Septicemia, meningitis, endocarditis, osteomyelitis, and surgical and traumatic wound infections are other manifestations of severe disease. B. cereus produces beta-lactamases, unlike Bacillus anthracis, and so is resistant to beta-lactam antibiotics; it is usually susceptible to treatment with clindamycin, vancomycin, gentamicin, chloramphenicol, and erythromycin. Simultaneous therapy via multiple routes may be required.

  14. Xylitol production from waste xylose mother liquor containing miscellaneous sugars and inhibitors: one-pot biotransformation by Candida tropicalis and recombinant Bacillus subtilis.

    Science.gov (United States)

    Wang, Hengwei; Li, Lijuan; Zhang, Lebin; An, Jin; Cheng, Hairong; Deng, Zixin

    2016-05-16

    The process of industrial xylitol production is a massive source of organic pollutants, such as waste xylose mother liquor (WXML), a viscous reddish-brown liquid. Currently, WXML is difficult to reuse due to its miscellaneous low-cost sugars, high content of inhibitors and complex composition. WXML, as an organic pollutant of hemicellulosic hydrolysates, accumulates and has become an issue of industrial concern in China. Previous studies have focused only on the catalysis of xylose in the hydrolysates into xylitol using one strain, without considering the removal of other miscellaneous sugars, thus creating an obstacle to subsequent large-scale purification. In the present study, we aimed to develop a simple one-pot biotransformation to produce high-purity xylitol from WXML to improve its economic value. In the present study, we developed a procedure to produce xylitol from WXML, which combines detoxification, biotransformation and removal of by-product sugars (purification) in one bioreactor using two complementary strains, Candida tropicalis X828 and Bacillus subtilis Bs12. At the first stage of micro-aerobic biotransformation, the yeast cells were allowed to grow and metabolized glucose and the inhibitors furfural and hydroxymethyl furfural (HMF), and converted xylose into xylitol. At the second stage of aerobic biotransformation, B. subtilis Bs12 was activated and depleted the by-product sugars. The one-pot process was successfully scaled up from shake flasks to 5, 150 L and 30 m(3) bioreactors. Approximately 95 g/L of pure xylitol could be obtained from the medium containing 400 g/L of WXML at a yield of 0.75 g/g xylose consumed, and the by-product sugars glucose, L-arabinose and galactose were depleted simultaneously. Our results demonstrate that the one-pot procedure is a viable option for the industrial application of WXML to produce value-added chemicals. The integration of complementary strains in the biotransformation of hemicellulosic hydrolysates is

  15. Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

    Science.gov (United States)

    Lei, Hao; Jones, Christopher; Zhu, Tian; Patel, Kavankumar; Wolf, Nina M; Fung, Leslie W-M; Lee, Hyun; Johnson, Michael E

    2016-02-15

    The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme. Published by Elsevier Ltd.

  16. Alleged B. anthracis exposure claims in a workers' compensation setting.

    Science.gov (United States)

    Jewell, Gregory; Dunning, Kari; Lockey, James E

    2006-01-01

    Workers' compensation insurance in some states may not provide coverage for medical evaluation costs of workplace exposures related to potential bioterrorism acts if there is no diagnosed illness or disease. Personal insurance also may not provide coverage for these exposures occurring at the workplace. Governmental entities, insurers, and employers need to consider how to address such situations and the associated costs. The objective of this study was to examine characteristics of workers and total costs associated with workers' compensation claims alleging potential exposure to the bioterrorism organism B. anthracis. We examined 192 claims referred for review to the Ohio Bureau of Workers' Compensation (OBWC) from October 10, 2001, through December 20, 2004. Although some cases came from out-of-state areas where B. anthracis exposure was known to exist, no Ohio claim was associated with true B. anthracis exposure or B. anthracis-related illness. Of the 155 eligible claims, 126 included medical costs averaging dollar 219 and ranging from dollar 24 to dollar 3,126. There was no difference in mean cost for government and non-government employees (p = 0.202 Wilcoxon). The number of claims and associated medical costs for evaluation and treatment of potential workplace exposure to B. anthracis were relatively small. These results can be attributed to several factors, including no documented B. anthracis exposures and disease in Ohio and prompt transmission of recommended diagnostic and prophylactic treatment protocols to physicians. How employers, insurers, and jurisdictions address payment for evaluation and treatment of potential or documented exposures resulting from a potential terrorism-related event should be addressed proactively.

  17. Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults.

    Science.gov (United States)

    Brown, Bruce K; Cox, Josephine; Gillis, Anita; VanCott, Thomas C; Marovich, Mary; Milazzo, Mark; Antonille, Tanya Santelli; Wieczorek, Lindsay; McKee, Kelly T; Metcalfe, Karen; Mallory, Raburn M; Birx, Deborah; Polonis, Victoria R; Robb, Merlin L

    2010-11-05

    The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA). A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA. The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. ClinicalTrials.gov NCT00057525.

  18. The Saccharomyces boulardii CNCM I-745 Strain Shows Protective Effects against the B. anthracis LT Toxin

    Directory of Open Access Journals (Sweden)

    Rodolphe Pontier-Bres

    2015-10-01

    Full Text Available The probiotic yeast Saccharomyces boulardii (S. boulardii has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

  19. The Saccharomyces boulardii CNCM I-745 strain shows protective effects against the B. anthracis LT toxin.

    Science.gov (United States)

    Pontier-Bres, Rodolphe; Rampal, Patrick; Peyron, Jean-François; Munro, Patrick; Lemichez, Emmanuel; Czerucka, Dorota

    2015-10-30

    The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

  20. Recombinant Programming

    OpenAIRE

    Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman

    2004-01-01

    This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

  1. Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates.

    Directory of Open Access Journals (Sweden)

    Francesco Celandroni

    Full Text Available The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.

  2. Allelic Variation on Murine Chromosome 11 Modifies Host Inflammatory Responses and Resistance to Bacillus anthracis

    Science.gov (United States)

    2011-12-01

    by an additional gene. Of note, this model does not rule out the possibility that multiple or different genes contribute to the host response to MDP...Immunity 35: 34–44. 62. Franchi L, Eigenbrod T, Munoz-Planillo R, Nunez G (2009) The inflamma- some: a caspase-1-activation platform that regulates

  3. Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples ▿

    OpenAIRE

    Létant, Sonia E.; Murphy, Gloria A.; Alfaro, Teneile M.; Avila, Julie R.; Kane, Staci R.; Raber, Ellen; Bunt, Thomas M.; Shah, Sanjiv R.

    2011-01-01

    In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real...

  4. Inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from Bacillus anthracis

    OpenAIRE

    Phelps, Jamie P.; Dang, Nghiep; Rasochova, Lada

    2007-01-01

    Chimeric cowpea mosaic virus (CPMV) particles displaying foreign peptide antigens on the particle surface are suitable for development of peptide-based vaccines. However, commonly used PEG precipitation-based purification methods are not sufficient for production of high quality vaccine candidates because they do not allow for separation of chimeric particles from cleaved contaminating species. Moreover, the purified particles remain infectious to plants. To advance the CPMV technology furthe...

  5. Indirect Detection Of Bacillus Anthracis (Anthrax) Using Amplified Gamma Phage-Based Assays

    Science.gov (United States)

    2007-11-01

    enter 26 Figure 3.0 Model of anthrax toxin entry into eukaryotic cells 27 the lungs and into the mucus membrane...extract from porcine and mixture meat and milk peptones, 2.0 g D(+) glucose, 5.0 g NaCl and 2.5 g disodium phosphate) and TSB (g/L: 17.0 pancreatic...are present in blood serum, lymph fluid, gastric secretions, milk , and saliva. Serum antibody concentrations are commonly determined using the

  6. Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis Toxin

    Science.gov (United States)

    1988-01-21

    concentration. Radial immmurnodiffusion plates were prepared with rabbit anti-mouse IgG ( Miles Scientific, Naperville, I) or rabbit anti-mouse IgM (Kirkegaard...6. Ezzell , J. W., B. E. Ivins, and S. H. Leppla. 1964. Immunoelectrophoretic analysis, toxicity, and kinetics o4 in 16 vitro production of the

  7. Feasibility of Wide-Area Decontamination of Bacillus anthracis Spores Using a Germination-Lysis Approach

    Science.gov (United States)

    2011-11-16

    Security, LLC 2011 CBD S& T Conference November 16, 2011 LLNL-PRES-508394 Lawrence Livermore National Laboratory LLNL-PRES-  Background...PRES-  Gruinard Island 5% formaldehyde  Sverdlosk Release UNKNOWN: but washing, chloramines , soil disposal believed to have been used...508394 Lawrence Livermore National Laboratory LLNL-PRES- 4 Disinfectant >6 Log Reduction on Materials (EPA, 2010a,b; Wood et al., 2011

  8. Anthrax Lethal Toxin Impairs Innate Immune Functions of Alveolar Macrophages and Facilitates Bacillus anthracis Survival

    National Research Council Canada - National Science Library

    Ribot, Wilson J; Panchal, Rekha G; Brittingham, Katherine C; Ruthel, Gordon; Kenny, Tara A; Lane, Douglas; Curry, Bob; Hoover, Timothy A; Friedlander, Arthur M; Bavari, Sina

    2006-01-01

    Alveolar macrophages (AM) are very important for pulmonary innate immune responses against invading inhaled pathogens because they directly kill the organisms and initiate a cascade of innate and adaptive immune responses...

  9. Modeling Rabbit Responses to Single and Multiple Aerosol Exposures of Bacillus anthracis Spores Data Set

    Data.gov (United States)

    U.S. Environmental Protection Agency — The two excel files contain all of the raw data that was modeled in the R code. The 6 word documents contain all of the R code that can be used in R to model the raw...

  10. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro

    Science.gov (United States)

    2013-03-11

    sterile screw-capped tube containing 5 ml of sterile PBS. New sporulation medium (3 grams of Tryptone, 3 grams of yeast extract, 2 grams of agar, 23...United States. Emerg Infect Dis 7: 933-944. 3. Penn CC, Klotz SA (1997) Anthrax pneumonia. Semin Respir Infect 12: 28-30. 4. Sweeney DA, Hicks CW, Cui X...Li Y, Eichacker PQ (2011) Anthrax infection. Am J Respir Crit Care Med 184: 1333-1341. 5. Roy CJ, Reed DS, Hutt JA (2010) Aerobiology and inhalation

  11. Measurements of DNA Damage and Repair in Bacillus anthracis Sterne Spores by UV Radiation

    Science.gov (United States)

    2014-09-18

    tightly into chromosomal structure, which serves to protect the DNA. The DNA is coiled several times and wrapped tightly around proteins. The wrapping...helps the DNA keep its chromatin structure. When DNA is to be replicated, enzymes unwind the chromosomal DNA. Therefore, DNA is not specifically used...2 See Appendix for Plasmid Information sheet. 30 the ice crystals disappeared . The tube was gently mixed by hand

  12. Bacillus anthracis Edema Toxin Inhibits Staphylococcus aureus Enterotoxin B Effects in Vitro: A Potential Protein Therapeutic?

    Science.gov (United States)

    2005-10-01

    5). Inherent characteristics of edema toxin and other procaryotic adenylate cyclases from Bordetella pertussis, Pseudomonas aeruginosa, and Yersinia...by mouse peritoneal macrophages: the role of cellular cyclic AMP. Immunology 64:719–724. 12. Krakauer, T. 1999. Induction of CC chemokines in human

  13. Host-Pathogen Coupled Networks: Model for Bacillus Anthracis Interaction with Host Macrophages

    Science.gov (United States)

    2015-09-01

    circles). Data points represent absorbance values at 570 nm following the MTT assay. Fitted values are KX2 = 10-6 nM-1s-1 and R = 0.21 nM s-1. (B...represent absorbance values at 570 nm following MTT assay. Here cell “death”, as indicated by the diminution of the signal (corrected for residual...cleaves the N-terminus of MAPKKs and induces tyrosine /threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun. 248(3):706

  14. Differentiation of strains from the Bacillus cereus group by RFLP-PFGE genomic fingerprinting.

    Science.gov (United States)

    Otlewska, Anna; Oltuszak-Walczak, Elzbieta; Walczak, Piotr

    2013-11-01

    Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus belong to the B. cereus group. The last three species are characterized by different phenotype features and pathogenicity spectrum, but it has been shown that these species are genetically closely related. The macrorestriction analysis of the genomic DNA with the NotI enzyme was used to generate polymorphism of restriction profiles for 39 food-borne isolates (B. cereus, B. mycoides) and seven reference strains (B. mycoides, B. thuringiensis, B. weihenstephanensis, and B. cereus). The PFGE method was applied to differentiate the examined strains of the B. cereus group. On the basis of the unweighted pair group method with the arithmetic mean method and Dice coefficient, the strains were divided into five clusters (types A-E), and the most numerous group was group A (25 strains). A total of 21 distinct pulsotypes were observed. The RFLP-PFGE analysis was successfully used for the differentiation and characterization of B. cereus and B. mycoides strains isolated from different food products. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Efforts to identify spore forming bacillus

    Energy Technology Data Exchange (ETDEWEB)

    Zuleiha, M.S.; Hilmy, N. (National Atomic Energy Agency, Jakarta (Indonesia). Pasar Djumat Research Centre)

    1982-04-01

    Efforts to identify 47 species of radioresistant spore forming bacillus sp. isolated from locally produced medical devices have been carried out. The identifications was conducted using 19 kinds of biochemical tests and compared to species to bacillus subtilis W. T.; bacillus pumilus E 601 and bacillus sphaericus Csub(I)A. The results showed that bacillus sp. examined could be divided into 6 groups, i.e. bacillus cereus; bacillus subtilis; bacillus stearothermophylus; bacillus coagulans; bacillus sphaericus and bacillus circulans.

  16. Efforts to identify spore forming bacillus

    International Nuclear Information System (INIS)

    Zuleiha, M.S.; Hilmy, Nazly

    1982-01-01

    Efforts to identify 47 species of radioresistant spore forming bacillus sp. isolated from locally produced medical devices have been carried out. The identifications was conducted using 19 kinds of biochemical tests and compared to species to bacillus subtilis W. T.; bacillus pumilus E 601 and bacillus sphaericus Csub(I)A. The results showed that bacillus sp. examined could be divided into 6 groups, i.e. bacillus cereus; bacillus subtilis; bacillus stearothermophylus; bacillus coagulans; bacillus sphaericus and bacillus circulans. (author)

  17. Mapping of Proteomic Composition on the Surfaces of Bacillus spores by Atomic Force Microscopy-based Immunolabeling

    Energy Technology Data Exchange (ETDEWEB)

    Plomp, M; Malkin, A J

    2008-06-02

    Atomic force microscopy provides a unique capability to image high-resolution architecture and structural dynamics of pathogens (e.g. viruses, bacteria and bacterial spores) at near molecular resolution in native conditions. Further development of atomic force microscopy in order to enable the correlation of pathogen protein surface structures with specific gene products is essential to understand the mechanisms of the pathogen life cycle. We have applied an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures through the visualization of the binding of antibodies, conjugated with nanogold particles, to specific epitopes on Bacillus spore surfaces. This information is generated while simultaneously acquiring the surface morphology of the pathogen. The immunospecificity of this labeling method was established through the utilization of specific polyclonal and monoclonal antibodies that target spore coat and exosporium epitopes of Bacillus atrophaeus and Bacillus anthracis spores.

  18. Genetic Recombination

    Science.gov (United States)

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  19. Factors affecting the solubility of Bacillus halmapalus alpha-amylase

    DEFF Research Database (Denmark)

    Faber, Cornelius; Hobley, Timothy John; Mollerup, Jørgen

    2008-01-01

    A detailed study of the solubility of recombinant Bacillus halmapalus alpha-amylase has been conducted. A semi-purified preparation from a bulk crystallisation was chos en that contained six isoforms with pI-values of between 5.5 and 6.1. The solubility was strongly affected by pH and could...

  20. Heat and desiccation are the predominant factors affecting inactivation of Bacillus licheniformis and Bacillus thuringiensis spores during simulated composting.

    Science.gov (United States)

    Stanford, K; Harvey, A; Barbieri, R; Xu, S; Reuter, T; Amoako, K K; Selinger, L B; McAllister, T A

    2016-01-01

    The suitability of composting for disposal of livestock mortalities due to Bacillus anthracis was assessed by measuring viability of surrogate spores from two strains each of Bacillus licheniformis and Bacillus thuringiensis after a heating cycle modelled on a cattle composting study. Sporulation was attempted from 10 to 37°C, but poor yields at lower temperatures resulted in 25, 30 and 37°C being selected to generate sufficient spores (8 log10  CFU ml(-1) ) for experiments. Spores were inoculated into 3 g autoclaved dried-ground compost rehydrated with 6 ml water or silica beads in a factorial design for each strain, sporulation temperature, matrix and sampling day (0, 25, 50, 100, 150). Maximum incubation temperature was 62°C, but spores were maintained at ≥55°C for 78 of 150 days. Although significant differences existed among Bacillus strains and sporulation temperatures, numbers of viable spores after 150 days averaged 1·3 log10  CFU g(-1) , a 5·2 log10 reduction from day 0. Spore inactivation was likely due to heat and desiccation as matrices were autoclaved prior to incubation, negating impacts of microflora. Results support composting for disposal of anthrax mortalities, provided long-term thermophillic heating is achieved. Due to limited sporulation at 10°C, livestock mortalities from anthrax at this or lower ambient temperatures would likely be of lower risk for disease transmission. © 2015 The Society for Applied Microbiology.

  1. BacillusRegNet

    DEFF Research Database (Denmark)

    Misirli, Goksel; Hallinan, Jennifer; Röttger, Richard

    2014-01-01

    As high-throughput technologies become cheaper and easier to use, raw sequence data and corresponding annotations for many organisms are becoming available. However, sequence data alone is not sufficient to explain the biological behaviour of organisms, which arises largely from complex molecular...... the associated BacillusRegNet website (http://bacillus.ncl.ac.uk)....

  2. Host organisms: Bacillus subtilis

    NARCIS (Netherlands)

    Hohman, Hans-Peter; van Dijl, Jan; Krishnappa, Laxmi; Pragai, Zoltan

    2016-01-01

    Bacillus subtilis and its close Bacillus relatives are important bacterial platforms for industrial production of enzymes and fine chemicals such as vitamin B2 and nucleotides. B. subtilis is an attractive bacterial organism for industrial use mainly because of its straightforward genetic

  3. Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

    Directory of Open Access Journals (Sweden)

    Ingrid de Souza Freire

    2014-09-01

    Full Text Available The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually.

  4. Comparative transcriptional profiling of Bacillus cereus sensu lato strains during growth in CO2-bicarbonate and aerobic atmospheres.

    Directory of Open Access Journals (Sweden)

    Karla D Passalacqua

    Full Text Available Bacillus species are spore-forming bacteria that are ubiquitous in the environment and display a range of virulent and avirulent phenotypes. This range is particularly evident in the Bacillus cereus sensu lato group; where closely related strains cause anthrax, food-borne illnesses, and pneumonia, but can also be non-pathogenic. Although much of this phenotypic range can be attributed to the presence or absence of a few key virulence factors, there are other virulence-associated loci that are conserved throughout the B. cereus group, and we hypothesized that these genes may be regulated differently in pathogenic and non-pathogenic strains.Here we report transcriptional profiles of three closely related but phenotypically unique members of the Bacillus cereus group--a pneumonia-causing B. cereus strain (G9241, an attenuated strain of B. anthracis (Sterne 34F(2, and an avirulent B. cereus strain (10987--during exponential growth in two distinct atmospheric environments: 14% CO(2/bicarbonate and ambient air. We show that the disease-causing Bacillus strains undergo more distinctive transcriptional changes between the two environments, and that the expression of plasmid-encoded virulence genes was increased exclusively in the CO(2 environment. We observed a core of conserved metabolic genes that were differentially expressed in all three strains in both conditions. Additionally, the expression profiles of putative virulence genes in G9241 suggest that this strain, unlike Bacillus anthracis, may regulate gene expression with both PlcR and AtxA transcriptional regulators, each acting in a different environment.We have shown that homologous and even identical genes within the genomes of three closely related members of the B. cereus sensu lato group are in some instances regulated very differently, and that these differences can have important implications for virulence. This study provides insights into the evolution of the B. cereus group, and

  5. Study of UV-induced mutagenesis in Bacillus subtilis

    International Nuclear Information System (INIS)

    Filippov, V.D.; Lotareva, O.V.

    1978-01-01

    The mechanism of UV-induced mutagenesis was studied in Bacillus subtilis departing from the assumption that a lower yield of UV-induced mutations should be found in mutants deficient in the recombination if production of mutations is coupled with the recombination process. Three recombination-deficient strains were used: two (recA and recF) with defects in different recombination pathways and the third (recB) has a block at a stage common for both of them. UV light induced reversions to prototrophy in recB cells and did not in recA and recF strains. Direct mutations, which confer to the cell additional growth requirements, were induced by UV light in recA and recF mutants. It is concluded that UV-induced mutagenesis in B subtilis is independent of the two known recombination mechanisms

  6. Adjuvant-Associated Peripheral Blood mRNA Profiles and Kinetics Induced by the Adjuvanted Recombinant Protein Candidate Tuberculosis Vaccine M72/AS01 in Bacillus Calmette–Guérin-Vaccinated Adults

    Directory of Open Access Journals (Sweden)

    Robert A. van den Berg

    2018-03-01

    Full Text Available Systems biology has the potential to identify gene signatures associated with vaccine immunogenicity and protective efficacy. The main objective of this study was to identify optimal postvaccination time points for evaluating peripheral blood RNA expression profiles in relation to vaccine immunogenicity and potential efficacy in recipients of the candidate tuberculosis vaccine M72/AS01. In this phase II open-label study (NCT01669096; https://clinicaltrials.gov/, healthy Bacillus Calmette–Guérin-primed, HIV-negative adults were administered two doses (30 days apart of M72/AS01. Twenty subjects completed the study and 18 subjects received two doses. Blood samples were collected pre-dose 1, pre-dose 2, and 1, 7, 10, 14, 17, and 30 days post-dose 2. RNA expression in whole blood (WB and peripheral blood mononuclear cells (PBMCs was quantified using microarray technology. Serum interferon-gamma responses and M72-specific CD4+ T cell responses to vaccination, and the observed safety profile were similar to previous trials. Two different approaches were utilized to analyze the RNA expression data. First, a kinetic analysis of RNA expression changes using blood transcription modules revealed early (1 day post-dose 2 activation of several pathways related to innate immune activation, both in WB and PBMC. Second, using a previously identified gene signature as a classifier, optimal postvaccination time points were identified. Since M72/AS01 efficacy remains to be established, a PBMC-derived gene signature associated with the protective efficacy of a similarly adjuvanted candidate malaria vaccine was used as a proxy for this purpose. This approach was based on the assumption that the AS01 adjuvant used in both studies could induce shared innate immune pathways. Subjects were classified as gene signature positive (GS+ or gene signature negative (GS−. Assignments of subjects to GS+ or GS− groups were confirmed by significant differences in RNA

  7. Phosphorescence In Bacillus Spores

    National Research Council Canada - National Science Library

    Reinisch, Lou; Swartz, Barry A; Bronk, Burt V

    2003-01-01

    .... Our present work attempts to build on this approach for environmental applications. We have measured a change in the fluorescence spectra of suspensions of Bacillus bacteria between the vegetative bacteria and their spores at room temperature...

  8. Screen for agents that induce autolysis in Bacillus subtilis.

    Science.gov (United States)

    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.

  9. Spectrum Recombination.

    Science.gov (United States)

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  10. Functional Analysis of the ComK Protein of Bacillus coagulans

    OpenAIRE

    Kovacs, Akos; Eckhardt, Thomas; van Kranenburg, Richard; Kuipers, Oscar

    2013-01-01

    The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own promoter region into Bacillus subtilis comK strain results in low transcriptional induction of the late competence gene comGA, but lacking bistable expression. The promoter regions of B. coagulans co...

  11. High-Resolution Spore Coat Architecture and Assembly of Bacillus Spores

    Energy Technology Data Exchange (ETDEWEB)

    Malkin, A J; Elhadj, S; Plomp, M

    2011-03-14

    Elucidating the molecular architecture of bacterial and cellular surfaces and its structural dynamics is essential to understanding mechanisms of pathogenesis, immune response, physicochemical interactions, environmental resistance, and provide the means for identifying spore formulation and processing attributes. I will discuss the application of in vitro atomic force microscopy (AFM) for studies of high-resolution coat architecture and assembly of several Bacillus spore species. We have demonstrated that bacterial spore coat structures are phylogenetically and growth medium determined. We have proposed that strikingly different species-dependent coat structures of bacterial spore species are a consequence of sporulation media-dependent nucleation and crystallization mechanisms that regulate the assembly of the outer spore coat. Spore coat layers were found to exhibit screw dislocations and two-dimensional nuclei typically observed on inorganic and macromolecular crystals. This presents the first case of non-mineral crystal growth patterns being revealed for a biological organism, which provides an unexpected example of nature exploiting fundamental materials science mechanisms for the morphogenetic control of biological ultrastructures. We have discovered and validated, distinctive formulation-specific high-resolution structural spore coat and dimensional signatures of B. anthracis spores (Sterne strain) grown in different formulation condition. We further demonstrated that measurement of the dimensional characteristics of B. anthracis spores provides formulation classification and sample matching with high sensitivity and specificity. I will present data on the development of an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures on the B. anthracis surfaces. These studies demonstrate that AFM can probe microbial surface architecture, environmental dynamics and the life cycle of bacterial and cellular systems at near

  12. Smallpox and pan-Orthodox Virus Detection by Real-Time 3’-Minor Groove Binder TaqMan Assays Oil the Roche LightCycler and the Cepheid Smart Cycler Platforms

    Science.gov (United States)

    2003-11-08

    Bacillus anthracis BA0068 Ames Sterne SPS 97.13.213 Bacillus cereus Bacillus coagulans Bacillus licheniformis Bacillus macerans Bacillus ...megaterium Bacillus polymyxa Bacillus sphaericus Bacillus stearothermophilus Bacillus subtilis subsp. niger Bacillus thuringiensis Bacillus popilliae...varicella- zoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving:

  13. The effect of growth medium on B. anthracis Sterne spore carbohydrate content.

    Science.gov (United States)

    Colburn, Heather A; Wunschel, David S; Antolick, Kathryn C; Melville, Angela M; Valentine, Nancy B

    2011-06-01

    The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides. We have investigated the carbohydrate composition of B. anthracis Sterne spores produced using four different medium types formulated with different sources of medium components. The amount of rhamnose, 3-O-methyl rhamnose and galactosamine was found to vary significantly between spores cultured using different medium formulations. The relative abundance of these monosaccharides compared to other monosaccharides such as mannosamine was also found to vary with medium type. Specific medium components were also found to impact the carbohydrate profile. Xylose has not been previously described in B. anthracis spores but was detected at low levels in two media. This may represent residual material from the brewery yeast extract used to formulate these two media. These results illustrate the utility of this method to capture the impact of growth medium on carbohydrate variation in spores. Detecting carbohydrate profiles in B. anthracis evidentiary material may provide useful forensic information on the growth medium used for sporulation. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  15. Evaluation of surface sampling method performance for Bacillus Spores on clean and dirty outdoor surfaces.

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Mollye C.; Einfeld, Wayne; Boucher, Raymond M.; Brown, Gary Stephen; Tezak, Matthew Stephen

    2011-06-01

    Recovery of Bacillus atrophaeous spores from grime-treated and clean surfaces was measured in a controlled chamber study to assess sampling method performance. Outdoor surfaces investigated by wipe and vacuum sampling methods included stainless steel, glass, marble and concrete. Bacillus atrophaeous spores were used as a surrogate for Bacillus anthracis spores in this study designed to assess whether grime-coated surfaces significantly affected surface sampling method performance when compared to clean surfaces. A series of chamber tests were carried out in which known amounts of spores were allowed to gravitationally settle onto both clean and dirty surfaces. Reference coupons were co-located with test coupons in all chamber experiments to provide a quantitative measure of initial surface concentrations of spores on all surfaces, thereby allowing sampling recovery calculations. Results from these tests, carried out under both low and high humidity conditions, show that spore recovery from grime-coated surfaces is the same as or better than spore recovery from clean surfaces. Statistically significant differences between method performance for grime-coated and clean surfaces were observed in only about half of the chamber tests conducted.

  16. Fate of pathogenic Bacillus cereus spores after ingestion by protist grazers

    DEFF Research Database (Denmark)

    Winding, Anne; Santos, Susana; Hendriksen, Niels Bohse

    The aim of this study is to understand the symbiosis between bacterivorous protists and pathogenic bacterial spores, in order to gain insight on survival and dispersal of pathogenic bacteria in the environment. It is generally accepted that resistance to grazing by protists has contributed...... to the evolution of Bacillus cereus group bacteria (e.g. B. cereus, B. anthracis, B. thuringiensis) as a pathogen. It has been hypothesized that the spore stage protects against digestion by predating protists. Indeed, B. thuringiensis spores have been shown to be readily ingested by ciliated protists but failed...... to be digested (Manasherob et al 1998 AEM 64:1750-). Here we report how diverse protist grazers grow on both vegetative cells and spores of B. cereus and how the bacteria survive ingestion and digestion, and even proliferate inside the digestive vacuoles of ciliated protists. The survival ability of B. cereus...

  17. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp plantarum and ‘Bacillus oryzicola’ are later heterotypic synonyms of Bacillus

    Science.gov (United States)

    The rhizosphere isolated bacteria belonging to the Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus clades are an important group of strains that are used as plant growth promoters and antagonists of plant pathogens. These properties have made these strains the focus of comm...

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

    Science.gov (United States)

    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.

  19. Microarray Analysis of Transposon Insertion Mutants in Bacillus Anthracis: Global Identification of Genes Required for Sporulation and Germination

    National Research Council Canada - National Science Library

    Day , Jr., William A; Rasmussen, Suzanne L; Carpenter, Beth M; Peterson, Scott N; Friedlander, Arthur M

    2007-01-01

    .... The system, used to identify genes required for generation of the infectious anthrax spore, spore germination and optimal growth on rich medium, was predictive of the contribution of two conserved...

  20. Bacillus anthracis-derived edema toxin (ET counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway

    Directory of Open Access Journals (Sweden)

    Nguyen Chinh

    2012-01-01

    Full Text Available Abstract Background A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET, which is formed by the combination of two proteins produced by the organism, edema factor (EF, which is an adenyl cyclase, and protective antigen (PA. Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse. Results Pretreatment of human microvascular endothelial cell(ECs of the lung (HMVEC-L with ET decreased interleukin (IL-8-driven transendothelial migration (TEM of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP. Conclusions We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.

  1. Whole Genome Sequencing and Phylogenetic Analysis of a Historical Collection of Bacillus anthracis Strains from Danish Cattle

    DEFF Research Database (Denmark)

    Derzelle, Sylviane; Girault, Guillaume; Kokotovic, Branko

    2015-01-01

    of such lineage in Europe is demonstrated for the first time, filling an historical gap within the phylogeography of the lineage. Comparative genome analyses of these strains with 41 isolates from other parts of the world revealed that the two Danish A.Br.008/011 strains were related to the heroin...

  2. Bacillus Anthracis Spore Interactions with Mammalian Cells: Relationship Between Germination State and the Outcome of in Vitro Infections

    Science.gov (United States)

    2011-02-28

    unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Report Documentation Page Form ApprovedOMB... Peacock S, Belton FC: Observations on the prophylaxis of experimental pulmonary anthrax in the monkey. J Hyg (Lond) 1956, 54(1):28-36. 8. Cleret A

  3. Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

    Science.gov (United States)

    2010-08-25

    in honey bee colony collapse disorder. Science 318: 283–287. 39. Towner JS, Sealy TK, Khristova ML, Albarino CG, Conlan S, et al. (2008) Newly...utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations...detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel

  4. Antimicrobial Effects of Gold/Copper Sulphide (Au/Cus) Core/Shell Nanoparticles on Bacillus Anthracis Spores and Cells

    Science.gov (United States)

    2013-01-01

    On tryptose broth, it flocculates and eventually settles to the bottom leaving a clear liquid above. It does not cause hemolysis on blood agar as...host through shock [3]. Symptoms of inhalational anthrax include fever, rapid and faint pulses, cyanosis, tachycardia and pleural effusion [3]. 10...in liquid LB medium was measured by optical density readings at 600 nm. They found that, 10, 50, and 100 µg/cm3 silver NPs slowed growth of 107E. coli

  5. A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

    DEFF Research Database (Denmark)

    Thierry, Simon; Hamidjaja, Raditijo A.; Girault, Guillaume

    2013-01-01

    been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR...

  6. Evaluation of the Efficacy of Methyl Bromide in the Decontamination of Building and Interior Materials Contaminated with Bacillus anthracis Spores

    Data.gov (United States)

    U.S. Environmental Protection Agency — Spreadsheets containing data for recovery of spores from different materials. Data on the fumigation parameters are also included. This dataset is associated with...

  7. DESTRUCTION OF FRANCISELLA TULARENSIS AND YERSINIA PESTIS PERSISTENCE OF BACILLUS ANTHRACIS SPORES AND CLOSTRIDIUM BOTULINUM IN MUNICIPAL SOLID LANDFILL LEACHATES

    Science.gov (United States)

    The United States Environmental Protection Agency Office of Research and Development National Homeland Security Research Center (NHSRC) in collaboration with the Department of Defense Edgewood Chemical Biological Center (ECBC) are evaluating the permanence of biological and chemi...

  8. A comparison of the immune response between early exposed and 1 year post exposure to Bacillus anthracis in Indonesia

    Science.gov (United States)

    Redhono, D.; Kusumawardani, A.; Dirgahayu, P.

    2018-03-01

    Anthrax is one of the zoonotic diseases that usually affects animals and can be transmitted to humans. Immune response of the body during an infection is the presence of antibodies as an effort to defend the body and it will survive for some time in the blood. The aim study is to find out how the initial response to the formation of antibodies and how these antibodies survive after one year. This study is cohort to people exposed to anthrax and found 130 people exposed to anthrax. The most risk factor was direct contact and consumed infected animal meat, which was 34.6%. Clinical manifestations of the skin were 12.3% and all respondents showed positive IgG. While 87.7% did not show any anthrax symptoms. IgG serum examination after 1 year of exposure to anthrax obtained 3.8% still detected antibodies in the body. The relationship between IgG titers with clinical manifestations of anthrax at one year post-outbreak is highly significant p 0.028. In conclusion a significant association between the clinical manifestation with antibody serum anthrax and it still detected after one-year post outbreaks of anthrax.

  9. Recombinant EXLX1 from Bacillus subtilis for enhancing enzymatic ...

    African Journals Online (AJOL)

    AJL

    2012-06-21

    Jun 21, 2012 ... enhancing enzymatic hydrolysis of corn stover with low cellulase loadings. Zhang Yan1, He Ming-Xiong2,3*, Wu Bo1, ... University, Chengdu 610064, China. 2Biogas Institute of Ministry of Agriculture, Biomass Energy Technology Research Centre, Section 4-13, Renming Nanlu,. Chengdu 610041, China.

  10. Plasmids replicatable in Bacillus subtilis, E. coli and lactic acid streptococcus bacteria

    NARCIS (Netherlands)

    Kok, Jan; Maat, Jan; van der Vossen, Josephus Mauritius; Venema, Gerard

    1997-01-01

    The claimed invention is drawn to a recombinant plasmid which can replicate in Bacillus subtilis, Escherichia coli, and lactic acid Streptococcus bacteria comprising the replication of origin from Streptococcus cremoris plasmid pWV01 as its origin of replication, in addition to coding marker genes

  11. Mode of action of Bacillus licheniformis pectin methylesterase on highly methylesterified and acetylated pectins

    NARCIS (Netherlands)

    Remoroza, C.A.; Wagenknecht, M.; Buchholt, H.C.; Moerschbacher, B.M.; Gruppen, H.; Schols, H.A.

    2015-01-01

    A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme (BliPME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50 °C. BliPME is able to

  12. Bacillus halodurans RecA-DNA binding and RecAmediated ...

    African Journals Online (AJOL)

    Abstract. In Escherichia coli, RecA protein catalyzes DNA pairing and strand exchange activities essential for genetic recombination. This is critical for normal cellular function under conditions that lead to altered. DNA metabolism and DNA damage. The RecA proteins of E. coli and Bacillus halodurans both can bind to DNA ...

  13. A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3

    NARCIS (Netherlands)

    Remoroza, C.A.; Wagenknecht, M.; Buchholt, H.C.; Moerschbacher, B.M.; Schols, H.A.; Gruppen, H.

    2014-01-01

    A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional

  14. Functional analysis of the ComK protein of Bacillus coagulans

    NARCIS (Netherlands)

    Kovács, Á.T.; Eckhardt, T.H.; Hartskamp, van M.; Kranenburg, van R.; Kuipers, O.P.

    2013-01-01

    The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own

  15. Functional Analysis of the ComK Protein of Bacillus coagulans

    NARCIS (Netherlands)

    Kovacs, Akos; Eckhardt, Thomas; van Kranenburg, Richard; Kuipers, Oscar

    2013-01-01

    The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own

  16. Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hong-geller, Elizabeth [Los Alamos National Laboratory; Valdez, Yolanda E [Los Alamos National Laboratory; Shou, Yulin [Los Alamos National Laboratory; Yoshida, Thomas M [Los Alamos National Laboratory; Marrone, Babetta L [Los Alamos National Laboratory; Dunbar, John [Los Alamos National Laboratory

    2009-01-01

    Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

  17. A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.

    Science.gov (United States)

    Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

    2017-05-05

    Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity

    Energy Technology Data Exchange (ETDEWEB)

    Hammerstrom, Troy G.; Lori, Horton B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-12-30

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activity in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

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

    Science.gov (United States)

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

    2015-03-01

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

  20. Bacillus subtilis genome diversity.

    Science.gov (United States)

    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.

  1. Impacts of sporulation temperature, exposure to compost matrix and temperature on survival of Bacillus cereus spores during livestock mortality composting.

    Science.gov (United States)

    Stanford, K; Reuter, T; Gilroyed, B H; McAllister, T A

    2015-04-01

    To investigate impact of sporulation and compost temperatures on feasibility of composting for disposal of carcasses contaminated with Bacillus anthracis. Two strains of B. cereus, 805 and 1391, were sporulated at either 20 or 37°C (Sporulation temperature, ST) and 7 Log10 CFU g(-1) spores added to autoclaved manure in nylon bags (pore size 50 μm) or in sealed vials. Vials and nylon bags were embedded into compost in either a sawdust or manure matrix each containing 16 bovine mortalities (average weight 617 ± 33 kg), retrieved from compost at intervals over 217 days and survival of B. cereus spores assessed. A ST of 20°C decreased spore survival by 1·4 log10 CFU g(-1) (P Compost temperatures >55°C reduced spore survival (P compost temperatures were key factors influencing survival of B. cereus spores in mortality compost. Composting may be most appropriate for the disposal of carcasses infected with B. anthracis at ambient temperatures ≤20°C under thermophillic composting conditions (>55°C). © 2015 The Society for Applied Microbiology.

  2. Heat activation and stability of amylases from Bacillus species

    African Journals Online (AJOL)

    Administrator

    2007-05-16

    May 16, 2007 ... as Bacillus macerans, Bacillus coagulans Bacillus licheniformis, Bacillus circulans, Bacillus megaterium, Bacillus polymyxa and Bacillus subtilis. Heat treatment at 70oC denatured the β-amylase component of the amylase source while α-amylase retained its potency at this temperature. Calcium.

  3. Rapid filtration separation-based sample preparation method for Bacillus spores in powdery and environmental matrices.

    Science.gov (United States)

    Isabel, Sandra; Boissinot, Maurice; Charlebois, Isabelle; Fauvel, Chantal M; Shi, Lu-E; Lévesque, Julie-Christine; Paquin, Amélie T; Bastien, Martine; Stewart, Gale; Leblanc, Eric; Sato, Sachiko; Bergeron, Michel G

    2012-03-01

    Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

  4. Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters.

    Science.gov (United States)

    Calfee, M Worth; Rose, Laura J; Tufts, Jenia; Morse, Stephen; Clayton, Matt; Touati, Abderrahmane; Griffin-Gatchalian, Nicole; Slone, Christina; McSweeney, Neal

    2014-01-01

    The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p≤0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p>0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident. Published by Elsevier B.V.

  5. Laser induced fluorescence lifetime characterization of Bacillus endospore species using time correlated single photon counting analysis with the multi-exponential fit method

    Science.gov (United States)

    Smith, Clint; Edwards, Jarrod; Fisher, Andmorgan

    2010-04-01

    Rapid detection of biological material is critical for determining presence/absence of bacterial endospores within various investigative programs. Even more critical is that if select material tests positive for bacillus endospores then tests should provide data at the species level. Optical detection of microbial endospore formers such as Bacillus sp. can be heavy, cumbersome, and may only identify at the genus level. Data provided from this study will aid in characterization needed by future detection systems for further rapid breakdown analysis to gain insight into a more positive signature collection of Bacillus sp. Literature has shown that fluorescence spectroscopy of endospores could be statistically separated from other vegetative genera, but could not be separated among one another. Results of this study showed endospore species separation is possible using laser-induce fluorescence with lifetime decay analysis for Bacillus endospores. Lifetime decays of B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain were investigated. Using the Multi-Exponential fit method data showed three distinct lifetimes for each species within the following ranges, 0.2-1.3 ns; 2.5-7.0 ns; 7.5-15.0 ns, when laser induced at 307 nm. The four endospore species were individually separated using principle component analysis (95% CI).

  6. Cytotoxic Potential of Bacillus cereus Strains ATCC 11778 and 14579 Against Human Lung Epithelial Cells Under Microaerobic Growth Conditions

    Directory of Open Access Journals (Sweden)

    Kathleen eKilcullen

    2016-02-01

    Full Text Available Bacillus cereus, a food poisoning bacterium closely related to Bacillus anthracis, secretes a multitude of virulence factors including enterotoxins, hemolysins, and phospholipases. However, the majority of the in vitro experiments evaluating the cytotoxic potential of B. cereus were carried out in the conditions of aeration, and the impact of the oxygen limitation in conditions encountered by the microbe in natural environment such as gastrointestinal tract remains poorly understood. This research reports comparative analysis of ATCC strains 11778 (BC1 and 14579 (BC2 in aerated and microaerobic (static cultures with regard to their toxicity for human lung epithelial cells. We showed that BC1 increased its toxicity upon oxygen limitation while BC2 was highly cytotoxic in both growth conditions. The combined effect of the pore-forming, cholesterol-dependent hemolysin, cereolysin O (CLO, and metabolic product(s such as succinate produced in microaerobic conditions provided substantial contribution to the toxicity of BC1 but not BC2 which relied mainly on other toxins. This mechanism is shared between CB1 and B. anthracis. It involves the permeabilization of the cell membrane which facilitates transport of toxic bacterial metabolites into the cell. The toxicity of BC1was potentiated in the presence of bovine serum albumin which appeared to serve as reservoir for bacteria-derived nitric oxide participating in the downstream production of reactive oxidizing species with the properties of peroxynitrite. In agreement with this the BC1cultures demonstrated the increased oxidation of the indicator dye Amplex Red catalyzed by peroxidase as well as the increased toxicity in the presence of externally added ascorbic acid.

  7. Essential Bacillus subtilis genes

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  8. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  9. Enzyme-driven Bacillus spore coat degradation leading to spore killing.

    Science.gov (United States)

    Mundra, Ruchir V; Mehta, Krunal K; Wu, Xia; Paskaleva, Elena E; Kane, Ravi S; Dordick, Jonathan S

    2014-04-01

    The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable. © 2013 Wiley Periodicals, Inc.

  10. Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing

    Directory of Open Access Journals (Sweden)

    Pruckler James M

    2008-11-01

    Full Text Available Abstract Background Bacillus cereus is most commonly associated with foodborne illness (diarrheal and emetic but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST schemes have recently been developed to genotype B. cereus and analysis has suggested a clonal or weakly clonal population structure for B. cereus and its close relatives B. anthracis and B. thuringiensis. In this study we used MLST to determine if B. cereus isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI illness were clonal or formed clonal complexes. Results A retrospective analysis of 55 clinical B. cereus isolates submitted to the Centers for Disease Control and Prevention between 1954 and 2004 was conducted. Clinical isolates from severe infections (n = 27, gastrointestinal (GI illness (n = 18, and associated isolates from food (n = 10 were selected for analysis using MLST. The 55 isolates were diverse and comprised 38 sequence types (ST in two distinct clades. Of the 27 isolates associated with serious illness, 13 clustered in clade 1 while 14 were in clade 2. Isolates associated with GI illness were also found throughout clades 1 and 2, while no isolates in this study belonged to clade 3. All the isolates from this study belonging to the clade 1/cereus III lineage were associated with severe disease while isolates belonging to clade1/cereus II contained isolates primarily associated with severe disease and emetic illness. Only three STs were observed more than once for epidemiologically distinct isolates. Conclusion STs of clinical B. cereus isolates were phylogenetically diverse and distributed among two of three previously described clades. Greater numbers of strains will need to be analyzed to confirm if specific lineages or clonal complexes are more likely to contain clinical isolates or be associated with specific illness, similar to B. anthracis and

  11. ORF Sequence: NC_005945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available Bacillus anthracis str. Sterne] MSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGP...TGDTGTTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGP...TGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTGATGLTGPTGPTGPS

  12. ORF Sequence: NC_007530 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available [Bacillus anthracis str. 'Ames Ancestor'] MSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGP...TGDTGTTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGP...TGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTGATGLTGPTGPTGPSGLGLPAGL

  13. ORF Sequence: NC_003995 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available Bacillus anthracis str. A2012] MSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGP...TGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGP...TGDTGTTGPTGPTGPTGPTGPTGPTGPTGATGLTGPTGPTGPSGLGLPAGLYAFNSGGISLD

  14. Fluorene biodegradation potentials of Bacillus strains isolated from ...

    African Journals Online (AJOL)

    Fluorene biodegradation potentials of Bacillus strains isolated from tropical ... Bacillus strains, putatively identified as Bacillus subtilis BM1 and Bacillus amyloliquefaciens BR1 were ... African Journal of Biotechnology, Vol 13(14), 1554-1559 ...

  15. Photoionization and Recombination

    Science.gov (United States)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  16. Rapid Methods for the Laboratory Identification of Pathogenic Microorganisms.

    Science.gov (United States)

    1982-09-01

    coli Hemophilus influenzae Bacillus anthracis Bacillus circulans Bacillus coagulans Bacillus cereus T Candida albicans Cryptococcus neoformans Legionel...reveree aide If neceeeary and Identify by block number) Lectins: Rapid Identification, Bacillus anthracisjCryptococcus " neoformans. Neisseria...field-type kit for the rapid identification of Bacillus anthracis. We have shown that certain lectins will selectively interact with B. anthracis

  17. Structure of Bacillus halmapalus alpha-amylase crystallized with and without the substrate analogue acarbose and maltose

    DEFF Research Database (Denmark)

    Lyhne-Iversen, Louise; Hobley, Timothy John; Kaasgaard, Svend G.

    2006-01-01

    Recombinant Bacillus halmapalus alpha-amylase (BHA) was studied in two different crystal forms. The first crystal form was obtained by crystallisation of BHA at room temperature in the presence of acarbose and maltose - data was collected at cryogenic temperatures to a resolution of 1.9 Å...

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

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    1982-01-01

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

  20. Recombination of cluster ions

    Science.gov (United States)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  1. Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens.

    Science.gov (United States)

    Wang, Li-Ting; Lee, Fwu-Ling; Tai, Chun-Ju; Kuo, Hsiao-Ping

    2008-03-01

    Strain BCRC 14193, isolated from soil, shared more than 99 % 16S rRNA gene sequence similarity with Bacillus amyloliquefaciens BCRC 11601(T) and Bacillus velezensis BCRC 17467(T). This strain was previously identified as B. amyloliquefaciens, based on DNA-DNA hybridization, but its DNA relatedness value with B. velezensis BCRC 17467(T) was 89 %. To investigate the relatedness of strain BCRC 14193, B. amyloliquefaciens and B. velezensis, the partial sequence of the gene encoding the subunit B protein of DNA gyrase (gyrB) was determined. B. velezensis BCRC 17467(T) shared high gyrB gene sequence similarity with B. amyloliquefaciens BCRC 14193 (98.4 %) and all of the B. amyloliquefaciens strains available (95.5-95.6 %). DNA-DNA hybridization experiments revealed high relatedness values between B. velezensis BCRC 17467(T) and B. amyloliquefaciens BCRC 11601(T) (74 %) and the B. amyloliquefaciens reference strains (74-89 %). Based on these data and the lack of phenotypic distinctive characteristics, we propose Bacillus velezensis as a later heterotypic synonym of Bacillus amyloliquefaciens.

  2. Impacts of Bacillus thuringiensis var. israelensis and Bacillus ...

    African Journals Online (AJOL)

    The study assessed the impact of bio-larvicides- Bacillus thuringiensis var. israelensis (Bti) and B. sphaericus (Bs) on anopheline mosquito larval densities in four selected areas of Lusaka urban district. Larval densities were determined using a standard WHO protocol at each study area prior to and after larviciding.

  3. Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis.

    Science.gov (United States)

    Beuchat, Larry R; Pettigrew, Charles A; Tremblay, Mario E; Roselle, Brian J; Scouten, Alan J

    2004-08-01

    Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.

  4. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  5. Hadron correlations from recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    Quark recombination is a successful model to describe the hadronization of a deconfined quark gluon plasma. Jet-like dihadron correlations measured at RHIC provide a challenge for this picture. We discuss how correlations between hadrons can arise from correlations between partons before hadronization. An enhancement of correlations through the recombination process, similar to the enhancement of elliptic flow is found. Hot spots from completely or partially quenched jets are a likely source of such parton correlations.

  6. Bacillus cereus and related species.

    OpenAIRE

    Drobniewski, F A

    1993-01-01

    Bacillus cereus is a gram-positive aerobic or facultatively anaerobic spore-forming rod. It is a cause of food poisoning, which is frequently associated with the consumption of rice-based dishes. The organism produces an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxin, respectively. Other toxins are produced during growth, including phospholipases, proteases, and hemolysins, one of which, cereolysin, is a thiol-activated hemolysin. These toxins may contribute to the pa...

  7. Carbohydrate metabolism in Bacillus subtilis

    International Nuclear Information System (INIS)

    Riedel, K.

    1980-01-01

    The glucose metabolism via the glycolytic pathway as well as via the oxidative and inoxidative hexose monophosphate pathways in Bacillus subtilis was studied applying 1- 14 C- and 6- 14 C-glucose, respectively, and determining labelled CO 2 and RNA. A method for calculating the catabolic pathways was developed. In nonproliferating cultures glucose is catabolized to 62% via the glycolytic pathway, to 20% via the oxidative, and to 18% via the inoxidative pathway

  8. Central carbon metabolism influences cellulase production in Bacillus licheniformis.

    Science.gov (United States)

    Wang, J; Liu, S; Li, Y; Wang, H; Xiao, S; Li, C; Liu, B

    2018-01-01

    Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13 C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13 C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering. Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering. © 2017 The Society for Applied Microbiology.

  9. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  10. Characterization of microsatellite loci in the stick insects Bacillus rossius rossius, Bacillus rossius redtenbacheri and Bacillus whitei (Insecta : Phasmatodea)

    DEFF Research Database (Denmark)

    Andersen, DH; Pertoldi, C; Loeschcke, V

    2005-01-01

    Five microsatellite markers were obtained from a dinucleotide enriched genomic library of the stick insect Bacillus rossius rossius. The markers were tested in three species of Bacillus. All loci were polymorphic when tested across species. The number of alleles at each locus was low (maximum four...

  11. Recombinational repair: workshop summary

    International Nuclear Information System (INIS)

    Howard-Flanders, P.

    1983-01-01

    Recombinational repair may or may not be synonymous with postreplication repair. Considerable progress has been made in the study of the relevant enzymes, particularly those from bacteria. In this workshop we focus on the recombination enzyme RecA protein. What structural changes take place in the protein and in DNA during repair. How does homologous pairing take place. How is ATP hydrolysis coupled to the stand exchange reaction and the formation of heteroduplx DNA. Turning to another enzyme needed for certain kinds of bacterial recombination, we will ask whether the purified recB protein and recC protein complement each other and are sufficient for exonuclease V activity. In higher cells, we would like to know whether sister exchanges, which occur in bacteria after uv irradiation, are also seen in animal cells

  12. Isolation and characterization of cellulolytic Bacillus licheniformis ...

    African Journals Online (AJOL)

    Eight cellulose degrading bacteria were isolated from compost and were identified as Bacillus licheniformis by 16S rRNA sequencing. Among the eight isolates, Bacillus licheniformis B4, B7 and B8 showed the highest cellulase activity. B. licheniformis B4 and B8 showed the maximum cellulase activity during the stationary ...

  13. ORF Alignment: NC_005945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_005945 gi|49185498 >1kwfA 3 362 53 435 1e-84 ... ref|YP_019314.1| chitosanase [Bac...illus anthracis str. 'Ames Ancestor'] ... ref|NP_845032.1| chitosanase [Bacillus anthracis str. ... ... ... Ames] ref|YP_028750.1| chitosanase [Bacillus anthracis ... str. Sterne] ref|NP_656550.1| Glyco_... ... gb|AAP26518.1| chitosanase [Bacillus anthracis str. ... Ames] gb|AAT31789.1| chitosanase [...Bacillus anthracis ... str. 'Ames Ancestor'] gb|AAT54801.1| chitosanase ... [Bacillus anthraci

  14. ORF Alignment: NC_003995 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003995 gi|21400565 >1kwfA 3 362 53 435 1e-84 ... ref|YP_019314.1| chitosanase [Bac...illus anthracis str. 'Ames Ancestor'] ... ref|NP_845032.1| chitosanase [Bacillus anthracis str. ... ... ... Ames] ref|YP_028750.1| chitosanase [Bacillus anthracis ... str. Sterne] ref|NP_656550.1| Glyco_... ... gb|AAP26518.1| chitosanase [Bacillus anthracis str. ... Ames] gb|AAT31789.1| chitosanase [...Bacillus anthracis ... str. 'Ames Ancestor'] gb|AAT54801.1| chitosanase ... [Bacillus anthraci

  15. ORF Alignment: NC_003997 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003997 gi|30262655 >1kwfA 3 362 53 435 1e-84 ... ref|YP_019314.1| chitosanase [Bac...illus anthracis str. 'Ames Ancestor'] ... ref|NP_845032.1| chitosanase [Bacillus anthracis str. ... ... ... Ames] ref|YP_028750.1| chitosanase [Bacillus anthracis ... str. Sterne] ref|NP_656550.1| Glyco_... ... gb|AAP26518.1| chitosanase [Bacillus anthracis str. ... Ames] gb|AAT31789.1| chitosanase [...Bacillus anthracis ... str. 'Ames Ancestor'] gb|AAT54801.1| chitosanase ... [Bacillus anthraci

  16. ORF Alignment: NC_007530 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_007530 gi|47527965 >1kwfA 3 362 53 435 1e-84 ... ref|YP_019314.1| chitosanase [Bac...illus anthracis str. 'Ames Ancestor'] ... ref|NP_845032.1| chitosanase [Bacillus anthracis str. ... ... ... Ames] ref|YP_028750.1| chitosanase [Bacillus anthracis ... str. Sterne] ref|NP_656550.1| Glyco_... ... gb|AAP26518.1| chitosanase [Bacillus anthracis str. ... Ames] gb|AAT31789.1| chitosanase [...Bacillus anthracis ... str. 'Ames Ancestor'] gb|AAT54801.1| chitosanase ... [Bacillus anthraci

  17. Getting the big beast to work--systems biotechnology of Bacillus megaterium for novel high-value proteins.

    Science.gov (United States)

    Korneli, Claudia; David, Florian; Biedendieck, Rebekka; Jahn, Dieter; Wittmann, Christoph

    2013-01-20

    The high industrial relevance of the soil bacterium Bacillus megaterium as host for recombinant proteins is driving systems-wide analyses of its metabolic and regulatory networks. The present review highlights novel systems biology tools available to unravel the various cellular components on the level of metabolic and regulatory networks. These provide a rational platform for systems metabolic engineering of B. megaterium. In line, a number of interesting studies have particularly focused on studying recombinant B. megaterium in its industrial bioprocess environment thus integrating systems metabolic engineering with systems biotechnology and providing the full picture toward optimal processes. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. [Characteristics of Bacillus cereus dissociants].

    Science.gov (United States)

    Doroshenko, E V; Loĭko, N G; Il'inskaia, O N; Kolpakov, A I; Gornova, I B; Klimanova, E V; El'-Registan, G I

    2001-01-01

    The autoregulation of the phenotypic (populational) variability of the Bacillus cereus strain 504 was studied. The isolated colonial morphotypes of this bacterium were found to differ in their growth characteristics and the synthesis of extracellular proteases. The phenotypic variabilities of vegetative proliferating cells and those germinated from endospores and cystlike refractory cells were different. Bacterial variants also differed in the production of the d1 and d2 factors (the autoinducers of dormancy and autolysis, respectively) and sensitivity to them. The possible role of these factors in the dissociation of microorganisms is discussed.

  19. Parton recombination model

    International Nuclear Information System (INIS)

    Hwa, R.C.

    1978-08-01

    Low P/sub T/ meson production in hadronic collisions is described in the framework of the parton model. The recombination of quark and antiquark is suggested as the dominant mechanism in the large x region. Phenomenological evidences for the mechanism are given. The application to meson initiated reactions yields the quark distribution in mesons. 21 references

  20. The resistance of Bacillus atrophaeus spores to the bactericidal activity of peracetic acid is influenced by both the nature of the solid substrates and the mode of contamination.

    Science.gov (United States)

    Grand, I; Bellon-Fontaine, M-N; Herry, J-M; Hilaire, D; Moriconi, F-X; Naïtali, M

    2010-11-01

    To evaluate the impact of the mode of contamination in relation with the nature of solid substrates on the resistance of spores of Bacillus atrophaeus -selected as surrogates of Bacillus anthracis- to a disinfectant, peracetic acid. Six materials confronted in urban and military environments were selected for their different structural and physicochemical properties. In parallel, two modes of contamination were examined, i.e. deposition and immersion. Deposition was used to simulate contamination by an aerosol and immersion by an extended contact with liquids. A pronounced difference in the biocontamination levels and spatial organization of spores was observed depending on the mode of contamination and the nature of the solid substrate considered, with consequences on decontamination. Contamination by immersion led to lower efficiency of peracetic acid decontamination than contamination by deposition. Infiltration of spores into porous materials after immersion is one reason. In contrast, the deposition mode aggregates cells at the surface of materials, explaining the similar disinfecting behaviour of porous and nonporous substrates when considering this inoculation route. The inoculation route was shown to be as influential a parameter as material characteristics (porosity and wettability) for decontamination efficacy. These results provide comparative information for the decontamination of B. atrophaeus spores in function of the mode of contamination and the nature of solid substrates. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology. No claim to French government works.

  1. MHC Class II and Non-MHC Class II Genes Differentially Influence Humoral Immunity to Bacillus anthracis Lethal Factor and Protective Antigen

    OpenAIRE

    Garman, Lori; Dumas, Eric K.; Kurella, Sridevi; Hunt, Jonathan J.; Crowe, Sherry R.; Nguyen, Melissa L.; Cox, Philip M.; James, Judith A.; Farris, A. Darise

    2012-01-01

    Anthrax Lethal Toxin consists of Protective Antigen (PA) and Lethal Factor (LF), and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class I...

  2. MHC Class II and Non-MHC Class II Genes Differentially Influence Humoral Immunity to Bacillus anthracis Lethal Factor and Protective Antigen

    Directory of Open Access Journals (Sweden)

    Judith A. James

    2012-12-01

    Full Text Available Anthrax Lethal Toxin consists of Protective Antigen (PA and Lethal Factor (LF, and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class II versus non-MHC class II genes in the humoral response to PA and LF immunization using three immunized strains of inbred mice: A/J (H-2k at the MHC class II locus, B6 (H-2b, and B6.H2k (H-2k. IgG antibody titers to LF were controlled primarily by the MHC class II locus, whereas IgG titers to PA were strongly influenced by the non-MHC class II genetic background. Conversely, the humoral fine specificity of reactivity to LF appeared to be controlled primarily through non-MHC class II genes, while the specificity of reactivity to PA was more dependent on MHC class II. Common epitopes, reactive in all strains, occurred in both LF and PA responses. These results demonstrate that MHC class II differentially influences humoral immune responses to LF and PA.

  3. Dataset - Evaluation of Standardized Sample Collection, Packaging, and Decontamination Procedures to Assess Cross-Contamination Potential during Bacillus anthracis Incident Response Operations

    Data.gov (United States)

    U.S. Environmental Protection Agency — Spore recovery data during sample packaging decontamination tests. This dataset is associated with the following publication: Calfee, W., J. Tufts, K. Meyer, K....

  4. Decontamination Efficacy of Three Commercial Off-the-Shelf Sporicidal Agents on Medium-Sized Panels Contaminated with Surrogates of Bacillus anthracis

    Science.gov (United States)

    2012-04-01

    Pressure-treated (PT) wood lumber All the panels were made with 7/16 in. thick 4 ft2 oriented strand board ( OSB ) as a backing material (Home Depot...No. 2B finish 20 gauge) stainless steel sheets glued to the OSB backing board with construction adhesive to form a single 4 ft2 panel. PT lumber...PT lumber was secured to the OSB with a single VA in. exterior screw (Home Depot [Cat. No. 131-537]) at each end of the board. The brick panels were

  5. Characterization of Potential Antimicrobial Targets in Bacillus spp. II. Branched-Chain Aminotransferase and Methionine Regeneration in B. cereus and B. anthracis

    National Research Council Canada - National Science Library

    Berger, B

    2002-01-01

    .... Four putative family III aminotransferases, two with homology to branched-chain amino acid aminotransferases and two with homology to D- amino acid aminotransferases, were cloned from B. cereus...

  6. Compilation of colony forming unit data for Bacillus anthracis and B. atrophaeus before and after exposure to various fogging treatments using peracetic acid or hydrogen peroxide

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data set contains CFU data for positive controls and test coupons for each test, for each material, and for each microorganism used. Also included are efficacy data...

  7. Effect of Ultrasonic Waves on the Heat Resistance of Bacillus cereus and Bacillus licheniformis Spores

    Science.gov (United States)

    Burgos, J.; Ordóñez, J. A.; Sala, F.

    1972-01-01

    Heat resistance of Bacillus cereus and Bacillus licheniformis spores in quarter-strength Ringer solution decreases markedly after ultrasonic treatments which are unable to kill a significant proportion of the spore population. This effect does not seem to be caused by a loss of Ca2+ or dipicolinic acid. The use of ultrasonics to eliminate vegetative cells or to break aggregates in Bacillus spore suspensions to be used subsequently in heat resistance experiments appears to be unadvisable. PMID:4627969

  8. Site directed recombination

    Science.gov (United States)

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  9. Nonradiative recombination in semiconductors

    CERN Document Server

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  10. Recombination epoch revisited

    International Nuclear Information System (INIS)

    Krolik, J.H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons. 18 references

  11. Dielectronic recombination theory

    International Nuclear Information System (INIS)

    LaGattuta, K.J.

    1991-01-01

    A theory now in wide use for the calculation of dielectronic recombination cross sections (σ DR ) and rate coefficients (α DR ) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of σ DR have been described by Fano and by Seaton. We will not consider those theories here. Calculations of α DR have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of σ DR . While the measurements of σ DR for δn ≠ 0 excitations have tended to agree very well with calculations, the case of δn = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain

  12. Current research efforts with Bacillus thuringiensis

    Science.gov (United States)

    Normand R. Dubois

    1991-01-01

    The bioassay of 260 strains of Bacillus thuringiensis (Bt) and 70 commercial preparations show that regression coefficient estimates may be as critical as LC5O estimates when evaluating them for future consideration.

  13. Antimicrobial effect of lactobacillus and bacillus derived ...

    African Journals Online (AJOL)

    This study focused on the screening, production, extraction of biosurfactants from Lactobacillus and Bacillus bacteria and their antimicrobial properties against causal microorganisms of food borne infections (food borne pathogens). The biosurfactants were investigated for potential antimicrobial activity using disk diffusion.

  14. Bacillus and biopolymer: Prospects and challenges

    Directory of Open Access Journals (Sweden)

    Swati Mohapatra

    2017-12-01

    Full Text Available The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.

  15. Molecular detection of TasA gene in endophytic Bacillus species ...

    African Journals Online (AJOL)

    Molecular detection of TasA gene in endophytic Bacillus species and characterization of the gene in Bacillus amyloliquefaciens. ... African Journal of Biotechnology ... in Bacillus amyloliquefaciens PEBA20 and 7 strains of Bacillus subtilis, ...

  16. Genetic determination of the radioprotective effect of cysteamine on γ-irradiated Bacillus subtilis cells

    International Nuclear Information System (INIS)

    Kalinin, V.L.; Oskolkova, O.B.; Stepanova, I.M.

    1979-01-01

    A study was made of a lethal effect of 60 Co-γ-rays on Bacillus subtilis cells: a wild type strain and recombination-deficient mutants rec A, rec B, rec D, rec F, rec K, rec L and rec O exposed in the absence and in the presence of cysteamine (H -2 M). It was established that the protective efficiency of cysteamine for repair- and recombination-deficient mutants is significantly lower than that for the wild type (DMF 2.2-3.0). The most deficient in sensitivity to the protective action of cysteamine are rec B mutants (DMF 0.8-1.2). These data suggest that in B. subtilis, like in E. coli, the radioprotective effect of cysteamine is genetically determined and depends on the activity of repair systems

  17. Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidusd-arabinose isomerase

    International Nuclear Information System (INIS)

    Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2008-01-01

    Recombinant B. pallidusd-arabinose isomerase was crystallized and diffraction data were collected to 2.3 Å resolution. d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidusd-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-altrose. Recombinant B. pallidusd-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution

  18. Larvicidal activity of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus.

    Science.gov (United States)

    DE Lara, Ana Paula DE Souza Stori; Lorenzon, Lucas Bigolin; Vianna, Ana Muñoz; Santos, Francisco Denis Souza; Pinto, Luciano Silva; Aires Berne, Maria Elisabeth; Leite, Fábio Pereira Leivas

    2016-10-01

    Effective control of gastrointestinal parasites is necessary in sheep production. The development of anthelmintics resistance is causing the available chemically based anthelmintics to become less effective. Biological control strategies present an alternative to this problem. In the current study, we tested the larvicidal effects of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus larvae. Bacterial suspensions [2 × 108 colony-forming units (CFU) g-1 of the feces] of B. thuringiensis var. israelensis and recombinant Escherichia coli expressing Cry11Aa toxin were added to naturally H. contortus egg-contaminated feces. The larvae were quantified, and significant reductions of 62 and 81% (P var. israelensis and recombinant E. coli expressing Cry11Aa toxin were then orally administered to lambs naturally infected with H. contortus. Twelve hours after administration, feces were collected and submitted to coprocultures. Significant larvae reductions (P var. israelensis is a promising new class of biological anthelmintics for treating sheep against H. contortus.

  19. Response of Bacillus cereus ATCC 14579 to challenges with sublethal concentrations of enterocin AS-48

    Directory of Open Access Journals (Sweden)

    Gálvez Antonio

    2009-10-01

    Full Text Available Abstract Background Enterocin AS-48 is produced by Enterococcus faecalis S48 to compete with other bacteria in their environment. Due to its activity against various Gram positive and some Gram negative bacteria it has clear potential for use as a food preservative. Here, we studied the effect of enterocin AS-48 challenges on vegetative cells of Bacillus cereus ATCC 14579 by use of transcriptome analysis. Results Of the 5200 genes analysed, expression of 24 genes was found to change significantly after a 30 min treatment with a subinhibitory bacteriocin concentration of 0.5 μg/ml. Most of up-regulated genes encode membrane-associated or secreted proteins with putative transmembrane segments or signal sequences, respectively. One operon involved in arginine metabolism was significantly downregulated. The BC4206-BC4207 operon was found to be the most upregulated target in our experiments. BC4206 codes for a PadR type transcriptional regulator, while BC4207 codes for a hypothetical membrane protein. The operon structure and genes are conserved in B. cereus and B. thuringiensis species, but are not present in B. anthracis and B. subtilis. Using real-time qPCR, we show that these genes are upregulated when we treated the cells with AS-48, but not upon nisin treatment. Upon overexpression of BC4207 in B. cereus, we observed an increased resistance against AS-48. Expression of BC4207 in B. subtilis 168, which lacks this operon also showed increased resistance against AS-48. Conclusion BC4207 membrane protein is involved in the resistance mechanism of B. cereus cells against AS-48.

  20. Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae

    Directory of Open Access Journals (Sweden)

    Eivind B. Drejer

    2018-05-01

    Full Text Available Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B. methanolicus, B. coagulans, B. smithii, B. licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production.

  1. Effect of deoxyribonucleic acid replication inhibitors on bacterial recombination

    International Nuclear Information System (INIS)

    Canosi, U.; Siccardi, A.G.; Falaschi, A.; Mazza, G.

    1976-01-01

    Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction

  2. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  3. Cell Physiology and Protein Secretion of Bacillus licheniformis Compared to Bacillus subtilis

    NARCIS (Netherlands)

    Voigt, Birgit; Antelmann, Haike; Albrecht, Dirk; Ehrenreich, Armin; Maurer, Karl-Heinz; Evers, Stefan; Gottschalk, Gerhard; van Dijl, Jan Maarten; Schweder, Thomas; Hecker, Michael

    2009-01-01

    The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can

  4. Recombinant Innovation and Endogenous Transitions

    OpenAIRE

    Koen Frenken; Luis R. Izquierdo; Paolo Zeppini

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create “short-cuts” which reduce switching costs allowing agents to escape a technological lock-in. As a result, recombinant innovations speed up technological progress allowing transitions that are impossible with only branching ...

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

    Science.gov (United States)

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

    2015-03-01

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

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

    KAUST Repository

    Li, Yongxin

    2015-03-24

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

  7. Expression of recombinant Antibodies

    Directory of Open Access Journals (Sweden)

    André eFrenzel

    2013-07-01

    Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  8. On the relict recombination lines

    International Nuclear Information System (INIS)

    Bershtejn, I.N.; Bernshtejn, D.N.; Dubrovich, V.K.

    1977-01-01

    Accurate numerical calculation of intensities and profiles of hydrogen recombination lines of cosmological origin is made. Relie radiation distortions stipulated by recombination quantum release at the irrevocable recombination are investigated. Mean number calculation is given for guantums educing for one irrevocably-lost electron. The account is taken of the educed quantums interraction with matter. The main quantum-matter interrraction mechanisms are considered: electronic blow broadening; free-free, free-bound, bound-bound absorptions Recombination dynamics is investigated depending on hydrogen density and total density of all the matter kinds in the Universe

  9. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp. plantarum and 'Bacillus oryzicola' are later heterotypic synonyms of Bacillus velezensis based on phylogenomics.

    Science.gov (United States)

    Dunlap, Christopher A; Kim, Soo-Jin; Kwon, Soon-Wo; Rooney, Alejandro P

    2016-03-01

    Bacillus velezensis was previously reported to be a later heterotypic synonym of Bacillus amyloliquefaciens , based primarily on DNA-DNA relatedness values. We have sequenced a draft genome of B. velezensis NRRL B-41580 T . Comparative genomics and DNA-DNA relatedness calculations show that it is not a synonym of B. amyloliquefaciens. It was instead synonymous with Bacillus methylotrophicus. ' Bacillus oryzicola ' is a recently described species that was isolated as an endophyte of rice ( Oryza sativa ). The strain was demonstrated to have plant-pathogen antagonist activity in greenhouse assays, and the 16S rRNA gene was reported to have 99.7 % sequence similarity with Bacillus siamensis and B. methylotrophicus , which are both known for their plant pathogen antagonism. To better understand the phylogenetics of these closely related strains, we sequenced the genome of ' B . oryzicola ' KACC 18228. Comparative genomic analysis showed only minor differences between this strain and the genomes of B. velezensis NRRL B-41580 T , B. methylotrophicus KACC 13015 T and Bacillus amyloliquefaciens subsp. plantarum FZB42 T . The pairwise in silico DNA-DNA hybridization values calculated in comparisons between the strains were all greater than 84 %, which is well above the standard species threshold of 70 %. The results of morphological, physiological, chemotaxonomic and phylogenetic analyses indicate that the strains share phenotype and genotype coherence. Therefore, we propose that B. methylotrophicus KACC 13015 T , B. amyloliquefaciens subsp. plantarum FZB42 T , and ' B. oryzicola' KACC 18228 should be reclassified as later heterotypic synonyms of B. velezensis NRRL B-41580 T , since the valid publication date of B. velezensis precedes the other three strains.

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

    Science.gov (United States)

    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.

  11. Expression of Bacillus thuringiensis serovar. israelensis toxins in Asticcacaulis excentricus to control dipteran larvae of vectors of diseases

    Directory of Open Access Journals (Sweden)

    Óscar Enrique Guevara

    2004-01-01

    Full Text Available Bacillus thuringiensis cry genes encode for a diverse group of crystal-forming proteins that exhibit insecticidal activity towards dipteran, lepidopteran and coleopteran larvae. The effectiveness of insecticides based on mosquito larvicidal B. thuringiensis strains can be enhanced by using aquatic prosthecated bacteria as alternative hosts, since they do not sink, cytoplasmic located toxins are protected f rom UV radiation and, most importantly, mosquito larvae feed on them. An Asticcacaulis excentricus reference strain was transformed with the cry1 1Aa gene from Bacillus thuringiensis serovar. israelensis. Western blot and electrophoresis were used to test recombinant protein expression; Western blot revealed a 72 kDa protein corresponding to B. thuringiensis serovar. israelensis Cry1 1 Aa. These aquatic bacte­rias toxicity achieved 50% mortality at 23 ng/mL concentration in f irst instar Culex quinquefasciatus larvae. Other bioassays indicated that recombinant A. excentricus is toxic against Aedes aegyptiand Anopheles albimanus first instar larvae. Buoyancy tests demonstrated the advantage of A. excentricus over B. thuringiensis. Key words: Asticcacaulis excentricus, Bacillus thuringiensis, prosthecated bacteria, dengue, malaria.

  12. Feather wastes digestion by new isolated strains Bacillus sp. in ...

    African Journals Online (AJOL)

    Feather wastes digestion by new isolated strains Bacillus sp. in Morocco. ... The most efficient isolated strain selected was compared with Bacillus subtilis ATCC 6633. Results showed ... African Journal of Biotechnology Vol.3(1) 2004: 67-70 ...

  13. Production of amylolytic enzymes by bacillus spp

    Energy Technology Data Exchange (ETDEWEB)

    Dawood, Elham Shareif [Department of Botany, Faculty of Science, University of Khartoum, Khartoum (Sudan)

    1997-12-01

    Sixty six bacteria and twenty fungi were isolated from various sources. These varied from rotten fruites to local drinks and soil samples from different parts of Sudan. On the basis of index of amylolytic activity, forty one bacteria and twelve fungi were found to hydrolyse strach. The best ten strach hydrolysing isolates were identified all as bacilli (Bacillus licheniformis SUD-K{sub 1}, SUD-K{sub 2}, SUD-K{sub 4}, SUD-O, SUD-SRW, SUD-BRW, SUD-By, Bacillus subtilis SUD-K{sub 3}, and Bacillus circulans SUD-D and SUD-K{sub 7}). Their amylase productivity was studied with respect to temperature and time. Amylolytic activity was measured by spectrophotometer, the highest activity was produced in around 24 hours of growth in all; six of which gave the highest amylase activity at 50 deg C and the rest at 45C. Based on the thermal production six isolates were chosen for further investigation. These were Bacillus licheniformis SUD-K{sub 1}, SUD-K{sub 2}, SUD-K{sub 4}, SUD-O, Bacillus subtilis SUD-K{sub 3} and Bacillus circulans SUD-K{sub 7}. The inclusion of strach and Mg{sup ++} ions in the culture medium gave the highest enzyme yield. The Ph 9.0 was found to be the optimum for amylase production for all isolates except Bacillus subtilis SUD-K{sub 3} which had an optimum at pH 7.0. Three isolates (Bacillus licheniformis SUD-K{sub 1}, SUD-K{sub 4} and SUD-O recorded highestamylase production in a medium supplemented with peptone while the rest (Bacillus licheniformis SUD-K{sub 2}, Bacillus subtilis SUD-K{sub 3} and Bacillus circulans SUD-K{sub 7}) gave highest amylase productivity in a medium supplemented with malt extract. Four isolates (Bacillus licheniformis SUD-K{sub 1} and Bacillus subtilis SUD-K{sub 3} gave maximum amylase production in a medium containing 0.5% soluble strach while the rest (gave maximum amylase production at 2%. Soluble strach was found to be best substrate among the different carbon sources tested. The maximum temperature for amylase activity

  14. Production of amylolytic enzymes by bacillus spp

    International Nuclear Information System (INIS)

    Dawood, Elham Shareif

    1997-12-01

    Sixty six bacteria and twenty fungi were isolated from various sources. These varied from rotten fruites to local drinks and soil samples from different parts of Sudan. On the basis of index of amylolytic activity, forty one bacteria and twelve fungi were found to hydrolyse strach. The best ten strach hydrolysing isolates were identified all as bacilli (Bacillus licheniformis SUD-K 1 , SUD-K 2 , SUD-K 4 , SUD-O, SUD-SRW, SUD-BRW, SUD-By, Bacillus subtilis SUD-K 3 , and Bacillus circulans SUD-D and SUD-K 7 ). Their amylase productivity was studied with respect to temperature and time. Amylolytic activity was measured by spectrophotometer, the highest activity was produced in around 24 hours of growth in all; six of which gave the highest amylase activity at 50 deg C and the rest at 45C. Based on the thermal production six isolates were chosen for further investigation. These were Bacillus licheniformis SUD-K 1 , SUD-K 2 , SUD-K 4 , SUD-O, Bacillus subtilis SUD-K 3 and Bacillus circulans SUD-K 7 . The inclusion of strach and Mg ++ ions in the culture medium gave the highest enzyme yield. The Ph 9.0 was found to be the optimum for amylase production for all isolates except Bacillus subtilis SUD-K 3 which had an optimum at pH 7.0. Three isolates (Bacillus licheniformis SUD-K 1 , SUD-K 4 and SUD-O recorded highestamylase production in a medium supplemented with peptone while the rest (Bacillus licheniformis SUD-K 2 , Bacillus subtilis SUD-K 3 and Bacillus circulans SUD-K 7 ) gave highest amylase productivity in a medium supplemented with malt extract. Four isolates (Bacillus licheniformis SUD-K 1 and Bacillus subtilis SUD-K 3 gave maximum amylase production in a medium containing 0.5% soluble strach while the rest (gave maximum amylase production at 2%. Soluble strach was found to be best substrate among the different carbon sources tested. The maximum temperature for amylase activity ranged from 60-70 deg C and 1% strach concentration was optimum for all isolates

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

    Science.gov (United States)

    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.

  16. Dissociative recombination of dications

    International Nuclear Information System (INIS)

    Seiersen, K.; Heber, O.; Jensen, M.J.; Safvan, C.P.; Andersen, L. H.

    2003-01-01

    Dissociative recombination (DR) of doubly-charged positive ions has been studied at the heavy ion storage ring ASTRID. Low-energy electrons were scattered on the dication of the N 2 molecule, and the absolute cross section was measured in the energy range of 10 -4 -50 eV. From the measured cross section, a thermal rate coefficient of 5.8x10 -7 cm 3 s -1 at 300 K was extracted. Furthermore, we present new results on the CO 2+ DR rate, and a summary and comparison of measured DR rate coefficients for both the singly and doubly-charged ions of CO, CO 2 , and N 2 is presented

  17. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as w...

  18. Hadron Correlations and Parton Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, R.J. [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)]. E-mail: rjfries@comp.tamu.edu

    2007-02-15

    Parton recombination has been found to be an extremely useful model to understand hadron production at the Relativistic Heavy Ion Collider. It is particularly important to explore its connections with hard processes. This article reviews some of the aspects of the quark recombination model and places particular emphasis on hadron correlations.

  19. Heat activation and stability of amylases from Bacillus species | Ajayi ...

    African Journals Online (AJOL)

    Leitch and Collier sporulating Bacillus medium was used to isolate some strains of Bacillus species from soil, wastewater and food sources in Ibadan, Oyo State, Nigeria, by heat activation method. Heat treatment at 80oC allowed the growth of sporulating Bacillus species, in the culture sample source without other bacteria ...

  20. Auger recombination in sodium iodide

    Science.gov (United States)

    McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

    2014-03-01

    Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

  1. Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

    National Research Council Canada - National Science Library

    Fan, Maomian; Roper, Shelly; Andrews, Carrie; Allman, Amity; Bruno, John; Kiel, Jonathan

    2008-01-01

    ...). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin...

  2. Preliminary report for analysis of genome wide mutations from four ciprofloxacin resistant B. anthracis Sterne isolates generated by Illumina, 454 sequencing and microarrays for DHS

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, Crystal [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vergez, Lisa [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Hinckley, Aubree [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Thissen, James [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Gardner, Shea [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McLoughlin, Kevin [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jackson, Paul [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Ellingson, Sally [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hauser, Loren [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Brettin, Tom [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Fofanov, Viacheslav [Eureka Genomics, Hercules, CA (United States); Koshinsky, Heather [Eureka Genomics, Hercules, CA (United States); Fofanov, Yuriy [Univ. of Houston, TX (United States)

    2011-06-21

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. As the result of a different DHS project, we have selected for and isolated a large number of ciprofloxacin resistant B. anthracis Sterne isolates. These isolates vary in the concentrations of ciprofloxacin that they can tolerate, suggesting multiple mutations in the samples. In collaboration with University of Houston, Eureka Genomics and Oak Ridge National Laboratory, we analyzed the ciprofloxacin resistant B. anthracis Sterne isolates by microarray hybridization, Illumina and Roche 454 sequencing to understand the error rates and sensitivity of the different methods. The report provides an assessment of the results and a complete set of all protocols used and all data generated along with information to interpret the protocols and data sets.

  3. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis Form an "Operational Group B. amyloliquefaciens" within the B. subtilis Species Complex.

    Science.gov (United States)

    Fan, Ben; Blom, Jochen; Klenk, Hans-Peter; Borriss, Rainer

    2017-01-01

    The plant growth promoting model bacterium FZB42 T was proposed as the type strain of Bacillus amyloliquefaciens subsp. plantarum (Borriss et al., 2011), but has been recently recognized as being synonymous to Bacillus velezensis due to phylogenomic analysis (Dunlap C. et al., 2016). However, until now, majority of publications consider plant-associated close relatives of FZB42 still as " B. amyloliquefaciens ." Here, we reinvestigated the taxonomic status of FZB42 and related strains in its context to the free-living soil bacterium DSM7 T , the type strain of B. amyloliquefaciens . We identified 66 bacterial genomes from the NCBI data bank with high similarity to DSM7 T . Dendrograms based on complete rpoB nucleotide sequences and on core genome sequences, respectively, clustered into a clade consisting of three tightly linked branches: (1) B. amyloliquefaciens , (2) Bacillus siamensis , and (3) a conspecific group containing the type strains of B. velezensis, Bacillus methylotrophicus , and B. amyloliquefaciens subsp. plantarum . The three monophyletic clades shared a common mutation rate of 0.01 substitutions per nucleotide position, but were distantly related to Bacillus subtilis (0.1 substitutions per nucleotide position). The tight relatedness of the three clusters was corroborated by TETRA, dDDH, ANI, and AAI analysis of the core genomes, but dDDH and ANI values were found slightly below species level thresholds when B. amyloliquefaciens DSM7 T genome sequence was used as query sequence. Due to these results, we propose that the B. amyloliquefaciens clade should be considered as a taxonomic unit above of species level, designated here as "operational group B. amyloliquefaciens " consisting of the soil borne B. amyloliquefaciens , and plant associated B. siamensis and B. velezensis , whose members are closely related and allow identifying changes on the genomic level due to developing the plant-associated life-style.

  4. The population and evolutionary dynamics of homologous gene recombination in bacterial populations.

    Directory of Open Access Journals (Sweden)

    Bruce R Levin

    2009-08-01

    Full Text Available In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination -- broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT -- plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1 the contribution of HGR to the rate of adaptive evolution in these populations and (2 the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1 HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2 once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent

  5. Antibacterial potential components of Bacillus species and ...

    African Journals Online (AJOL)

    Honey is a sweet viscous liquid produced by honey bee, Apis mellifera from the nectar of plants. Honey is a natural product that has been used from ancient times till now as food and for medicinal purpose. This study was carried out to determine the mode of action of Bacillus species and antibiotics residues in branded and ...

  6. Preliminary investigations reveal that Bacillus thuringiensis δ ...

    African Journals Online (AJOL)

    The imminent introduction of transgenic crops into Kenya requires a rigorous assessment of the potential risks involved. This study focused on the possible effect of Bacillus thuringiensisδ-endotoxin [CryIA(c)] on arbuscular mycorrhizal fungi (AMF) associated with sorghum. In green house experiments, sorghum seedlings ...

  7. Antimicrobials of Bacillus species: mining and engineering

    NARCIS (Netherlands)

    Zhao, Xin

    2016-01-01

    Bacillus sp. have been successfully used to suppress various bacterial and fungal pathogens. Due to the wide availability of whole genome sequence data and the development of genome mining tools, novel antimicrobials are being discovered and updated,;not only bacteriocins, but also NRPs and PKs. A

  8. Molecular characterization of Lepidopteran specific Bacillus ...

    African Journals Online (AJOL)

    Guest

    2013-05-15

    May 15, 2013 ... Bacillus thuringiensis (Bt) strains pathogenic to Lepidopteran insects and native to hilly zone soils of. Karnataka (India) were explored. 19 strains were isolated from the soils and identified by morphological and microscopic characters. Toxicity level of the Bt isolates was tested by treating third Instar larvae ...

  9. The Regulatory RNAs of Bacillus subtilis

    NARCIS (Netherlands)

    Mars, Ruben

    2014-01-01

    In vrijwel alle organismen wordt RNA aangemaakt dat niet codeert voor eiwit, maar een regulerende functie heeft. Dit proefschrift beschrijft de identificatie van ~1600 nieuwe potentiële regulatie-RNAs in de bodembacterie Bacillus subtilis die veel voor biotechnologische toepassingen ingezet wordt.

  10. Bacillus thuringiensis and its application in agriculture

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... Key words: Bacillus thuringiensis, endotoxins, crop plants. INTRODUCTION ..... of resistance in the pest and unfavorable interactions with beneficial .... with slower resistance evolution in North Carolina compared to .... level of 0.18% cross pollination in the experimental rice lines. .... Ecology and Safety.

  11. The Cell Wall of Bacillus subtilis

    NARCIS (Netherlands)

    Scheffers, Dirk-Jan; Graumann, Peter

    2012-01-01

    The cell wall of Bacillus subtilis is a rigid structure on the outside of the cell that forms the first barrier between the bacterium and the environment, and at the same time maintains cell shape and withstands the pressure generated by the cell’s turgor. In this chapter, the chemical composition

  12. Type I signal peptidases of Bacillus subtilis

    NARCIS (Netherlands)

    Tjalsma, Harold; Bolhuis, Albert; Bron, Sierd; Jongbloed, Jan; Meijer, Wilfried J.J.; Noback, Michiel; van Roosmalen, Maarten; Venema, Gerhardus; van Dijl, Jan Maarten; Hopsu Havu, VK; Jarvinen, M; Kirschke, H

    1997-01-01

    Bacillus subtilis contains at least three chromosomally-encoded type I signal peptidases (SPases; SipS, SipT, and SipU), which remove signal peptides from secretory proteins. In addition, certain B. subtilis (natto) strains contain plasmid-encoded type I SPases (SipP). The known type I SPases from

  13. Oxygen-hydrogen recombination system

    International Nuclear Information System (INIS)

    Sato, Shuichiro; Takejima, Masaki.

    1981-01-01

    Purpose: To avoid reduction in the performance of catalyst used for an oxygen-hydrogen recombiner in the off gas processing system of a nuclear reactor. Constitution: A thermometer is provided for the detection of temperature in an oxygen-hydrogen recombiner. A cooling pipe is provided in the recombiner and cooling medium is introduced externally. The cooling medium may be water or air. In accordance with the detection value from the thermometer, ON-OFF control is carried out for a valve to control the flow rate of the cooling medium thereby rendering the temperature in the recombiner to a predetermined value. This can prevent the catalyst from being exposed to high temperature and avoid the reduction in the performance of the catalyst. (Ikeda, J.)

  14. Controlled Release from Recombinant Polymers

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  15. Hydrogen recombiner development at AECL

    International Nuclear Information System (INIS)

    Dewit, W.A.; Koroll, G.W.; Loesel Sitar, J.; Graham, W.R.C.

    1997-01-01

    Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial

  16. Review of Parton Recombination Models

    International Nuclear Information System (INIS)

    Bass, Steffen A

    2006-01-01

    Parton recombination models have been very successful in explaining data taken at RHIC on hadron spectra and emission patterns in Au+Au collisions at transverse momenta above 2 GeV/c, which have exhibited features which could not be understood in the framework of basic perturbative QCD. In this article I will review the current status on recombination models and outline which future challenges need to be addressed by this class of models

  17. Recombinant snake venom prothrombin activators

    OpenAIRE

    L?vgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  18. DNA SEQUENCE SIMILARITY REQUIREMENTS FOR INTERSPECIFIC RECOMBINATION IN BACILLUS. (R825348)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

    CSIR Research Space (South Africa)

    Visser, Daniel F

    2010-03-01

    Full Text Available expression. Batch fermentations A 1.5 l InFors HT batch fermentor (Labfors, Switzerland) containing 1 l of GMO 20 medium was inoculated with a 50 ml inoculum (overnight culture of E. coli JM109 [pMSPNP] in LB medium). The composition of the GMO 20...

  20. Delayed recombination and cosmic parameters

    International Nuclear Information System (INIS)

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-01-01

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n s , and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z * =1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: ε α i <0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  1. Utilization of corn starch as sustrate for ß-Amylase by Bacillus SPP

    African Journals Online (AJOL)

    Corn starch was used as substrate for ß -amylase production from ten(10) amylolytic species of the genus Bacillus isolated locally from soil, waste water and food sources. Ten bacillus strains was made up of two strains each of Bacillus macerans, Bacillus licheniformis and Bacillus circulans. Also included are B. coagulans, ...

  2. L-Glutamic acid production by Bacillus spp. isolated from vegetable ...

    African Journals Online (AJOL)

    Ogiri” (fermented vegetable proteins) in Nigeria. The isolates were identified as Bacillus subtilis (6), (27.3%), Bacillus pumilus (5), (22.7%), Bacillus licheniformis (5), (27.3%) and Bacillus polymyxa (6), (22.7%). Four species of the Bacillus isolates ...

  3. ORF Alignment: NC_003995 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003995 gi|21402596 >1m65A 4 232 339 570 1e-51 ... ref|YP_021441.1| php domain prot...ein [Bacillus anthracis str. 'Ames Ancestor'] ... ref|NP_846998.1| PHP domain protein [Bacillus anthr...acis ... str. Ames] ref|YP_030695.1| PHP domain protein [Bacillus ... anthracis str. Sterne] r.... A2012] ... gb|AAP28484.1| PHP domain protein [Bacillus anthracis ... str. Ames] gb|AAT33916.1| PHP... domain protein [Bacillus ... anthracis str. 'Ames Ancestor'] gb|AAT56746.1| PHP ... dom

  4. ORF Alignment: NC_005945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_005945 gi|49187443 >1m65A 4 232 339 570 1e-51 ... ref|YP_021441.1| php domain prot...ein [Bacillus anthracis str. 'Ames Ancestor'] ... ref|NP_846998.1| PHP domain protein [Bacillus anthr...acis ... str. Ames] ref|YP_030695.1| PHP domain protein [Bacillus ... anthracis str. Sterne] r.... A2012] ... gb|AAP28484.1| PHP domain protein [Bacillus anthracis ... str. Ames] gb|AAT33916.1| PHP... domain protein [Bacillus ... anthracis str. 'Ames Ancestor'] gb|AAT56746.1| PHP ... dom

  5. ORF Alignment: NC_003997 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003997 gi|30264621 >1m65A 4 232 339 570 1e-51 ... ref|YP_021441.1| php domain prot...ein [Bacillus anthracis str. 'Ames Ancestor'] ... ref|NP_846998.1| PHP domain protein [Bacillus anthr...acis ... str. Ames] ref|YP_030695.1| PHP domain protein [Bacillus ... anthracis str. Sterne] r.... A2012] ... gb|AAP28484.1| PHP domain protein [Bacillus anthracis ... str. Ames] gb|AAT33916.1| PHP... domain protein [Bacillus ... anthracis str. 'Ames Ancestor'] gb|AAT56746.1| PHP ... dom

  6. Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis

    International Nuclear Information System (INIS)

    Weinrauch, Y.; Dubnau, D.

    1987-01-01

    Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2 H and 15 N, in the presence of 32 Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases

  7. A Cumulative Spore Killing Approach: Synergistic Sporicidal Activity of Dilute Peracetic Acid and Ethanol at Low pH Against Clostridium difficile and Bacillus subtilis Spores.

    Science.gov (United States)

    Nerandzic, Michelle M; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J

    2016-01-01

    Background.  Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5-2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods.  We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results.  Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200-2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions.  Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin.

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

    Directory of Open Access Journals (Sweden)

    shila khajavi shojaei

    2016-06-01

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

  9. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    Energy Technology Data Exchange (ETDEWEB)

    Moriya, Takashi J., E-mail: takashi.moriya@ipmu.jp [Kavli Institute for the Physics and Mathematics of the Universe, Todai Institutes for Advanced Study, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8583 (Japan)

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  10. Meiotic recombination in human oocytes.

    Directory of Open Access Journals (Sweden)

    Edith Y Cheng

    2009-09-01

    Full Text Available Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1 in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

  11. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  12. PRODUCTION OF FIBRINOLYTIC ENZYME (NATTOKINASE) FROM BACILLUS SP.

    OpenAIRE

    Padma Singh, Rekha Negi*, Vani Sharma, Alka Rani, Pallavi and Richa Prasad

    2018-01-01

    During present study Nattokinase which is a novel fibrinolytic enzyme was produced by Bacillus sp. To screen and extract nattokinase enzyme from Bacillus sp. were isolated from soil of different agricultural field by serial dilution method. Out of 10 isolate, one strain i.e. B3 produced nattokinase on screening medium. B3 was identified by biochemical characterization. The caseinolytic activity of Nattokinase was 0.526 U/ml and the selected isolate Bacillus sp. could produce active nattokinas...

  13. Protection of Bacillus pumilus Spores by Catalases

    OpenAIRE

    Checinska, Aleksandra; Burbank, Malcolm; Paszczynski, Andrzej J.

    2012-01-01

    Bacillus pumilus SAFR-032, isolated at spacecraft assembly facilities of the National Aeronautics and Space Administration Jet Propulsion Laboratory, is difficult to kill by the sterilization method of choice, which uses liquid or vapor hydrogen peroxide. We identified two manganese catalases, YjqC and BPUM_1305, in spore protein extracts of several B. pumilus strains by using PAGE and mass spectrometric analyses. While the BPUM_1305 catalase was present in six of the B. pumilus strains teste...

  14. Electric hydrogen recombiner special tests

    International Nuclear Information System (INIS)

    Wilson, J.F.

    1975-12-01

    Westinghouse has produced an electric hydrogen recombiner to control hydrogen levels in reactor containments following a postulated loss-of-coolant accident. The recombiner underwent extensive testing for NRC qualification (see WCAP 7709-L and Supplements 1, 2, 3, 4). As a result, WCAP 7709-L and Supplements 1, 2, 3, and 4 have been accepted by the NRC for reference in applications not committed to IEEE-323-1974. Supplement 5 and the next supplement will demonstrate conformance to IEEE-323-1974. This supplement describes additional tests, beyond those necessary to qualify the system, which will be referenced in supplement 6. Each test has demonstrated a considerable margin of safety over required performance. Concurrently, the test results increased the fund of technical information on the electric hydrogen recombiner

  15. Bioaccumulation of copper, zinc, cadmium and lead by Bacillus sp., Bacillus cereus, Bacillus sphaericus and Bacillus subtilis Bioacumulação de cobre, zinco, cádmio e chumbo por Bacillus sp., Bacillus cereus, Bacillus sphaericus e Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Antonio Carlos Augusto da Costa

    2001-03-01

    Full Text Available This work presents some results on the use of microbes from the genus Bacillus for uptake of cadmium, zinc, copper and lead ions. Maximum copper bioaccumulations were 5.6 mol/g biomass for B. sphaericus, 5.9 mol/g biomass for B. cereus and B. subtilis, and 6.4 mol/g biomass for Bacillus sp. Maximum zinc bioaccumulations were 4.3 mol/g biomass for B. sphaericus, 4.6 mol/g biomass for B. cereus, 4.8 mol/g biomass for Bacillus sp. and 5.0 mol/g biomass for B. subtilis. Maximum cadmium bioaccumulations were 8.0 mol/g biomass for B. cereus, 9.5 mol/g biomass for B. subtilis, 10.8 mol/g biomass for Bacillus sp. and 11.8 mol/g biomass for B. sphaericus. Maximum lead biomaccumulations were 0.7 mol/g biomass for B. sphaericus, 1.1 mol/g biomass for B. cereus, 1.4 mol/g biomass for Bacillus sp. and 1.8 mol/g biomass for B. subtilis. The different Bacillus strains tested presented distinct uptake capacities, and the best results were obtained for B. subtilis and B. cereus.Este trabalho apresenta resultados de acumulação dos íons metálicos cádmio, zinco, cobre e chumbo por bactérias do gênero Bacillus. A bioacumulação máxima de cobre foi 5,6 mol/g biomassa para B. sphaericus, 5,9 mol/g biomassa para B. cereus e B. subtilis, e 6,4 mol/g biomassa para Bacillus sp.. A bioacumulação máxima de zinco foi 4,3 mol/g biomassa para B. sphaericus, 4,6 mol/g biomassa para B. cereus, 4,8 mol/g biomassa para Bacillus sp. e 5,0 mol/g biomassa para B. subtilis. A bioacumulação máxima de cádmio foi 8,0 mol/g biomassa para B. cereus, 9,5 mol/g biomassa para B. subtilis, 10,8 mol/g biomassa para Bacillus sp. e 11,8 mol/g biomassa para B. sphaericus. A bioacumulação máxima de chumbo foi 0,7 mol/g biomassa para B. sphaericus, 1,1 mol/g biomassa para B. cereus, 1,4 mol/g biomassa para Bacillus sp. e 1,8 mol/g biomassa para B. subtilis. As distintas linhagens de Bacillus testadas apresentaram variáveis capacidades de carregamento de íons metálicos, sendo os

  16. Isolation of bacillus thuringiensis from different samples from Mansehra District

    International Nuclear Information System (INIS)

    Younis, F.; Lodhi, A.F.; Raza, G.

    2009-01-01

    The insecticidal activity of Bacillus thuringiensis has made it very interesting for the control of a variety of agricultural pests and human disease vectors. The present study is an attempt to explore the potential and diversity. of Bacillus thuringiensis. from the local environment for the control of cotton spotted bollworm (Earias sp.), a major pest of cotton. Two hundred and ninety eight samples of soil, grain dust, wild animal dung, birds dropping, decaying leaves and dead insects were collected from different ecological environments of Mansehra District yielding 438 Bacillus thuringiensis isolates that produce parasporal crystalline inclusions. In this study the soil samples were found to be the richest source for Bacillus thuringiensis. (author)

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Indu Khatri

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne

    1987-01-01

    The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...... in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up...... to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA....

  2. Study of the solubility of a modified Bacillus licheniformis alpha-amylase around the isoelectric point

    DEFF Research Database (Denmark)

    Faber, Cornilius; Hobley, Timothy John; Mollerup, Jørgen

    2007-01-01

    The solubility of a modified recombinant Bacillus licheniformis alpha-amylase (mBLA) has been studied by batch crystallization. A semi-pure preparation was chosen containing five isoforms with pI values from 6 to 7.3 (weighted average of 6.6). Small amounts (... sodium sulfate at all pH values and increased with 0.5 mol.L-1 sodium thiocyanate at pH 7 and pH 8. The effect of anions on alpha-amylase solubility followed the Hofmeister series, and only weak evidence of reversal was seen below the isoelectric point. Cations had little effect on solubility. The sign...... and magnitude of the alpha-amylase zeta potential was determined in the presence and absence of 0.1 mol.L-1 salt. Qualitatively, zeta potential correctly predicted the different salts influence on mBLA solubility....

  3. Surface display of Clonorchis sinensis enolase on Bacillus subtilis spores potentializes an oral vaccine candidate.

    Science.gov (United States)

    Wang, Xiaoyun; Chen, Wenjun; Tian, Yanli; Mao, Qiang; Lv, Xiaoli; Shang, Mei; Li, Xuerong; Yu, Xinbing; Huang, Yan

    2014-03-10

    Clonorchis sinensis (C. sinensis) infections remain the common public health problem in freshwater fish consumption areas. New effective prevention strategies are still the urgent challenges to control this kind of foodborne infectious disease. The biochemical importance and biological relevance render C. sinensis enolase (Csenolase) as a potential vaccine candidate. In the present study, we constructed Escherichia coli/Bacillus subtilis shuttle genetic engineering system and investigated the potential of Csenolase as an oral vaccine candidate for C. sinensis prevention in different immunization routes. Our results showed that, compared with control groups, both recombinant Csenolase protein and nucleic acid could induce a mixed IgG1/IgG2a immune response when administrated subcutaneously (Psinensis infection. Csenolase derived oral vaccine conferred worm reduction rate and egg reduction rate at 60.07% (Psinensis prevention. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Ÿztürk, Sibel; Ÿalık, Pınar; Ÿzdamar, Tunçer H

    2016-04-01

    Bacillus subtilis is a highly promising production system for various biomolecules. This review begins with the algorithm of fed-batch operations (FBOs) and then illustrates the approaches to design the initial production medium and/or feed stream. Additionally, the feeding strategies developed with or without feedback control for fed-batch B. subtilis fermentations were compiled with a special emphasis on recombinant protein (r-protein) production. For biomolecule production by wild-type B. subtilis, due to the different intracellular production patterns, no consensus exists on the FBO strategy that gives the maximum productivity, whereas for r-protein production appropriate feeding strategies vary depending on the promoter used. Thus, we conclude that the B. subtilis community is still seeking an approved strong promoter and generalized FBO strategies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2

    Directory of Open Access Journals (Sweden)

    Tadahiko Kajiwara

    2010-09-01

    Full Text Available The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2. The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9–11 and temperature range of 45–60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8 and pNP-laurate (C12 and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

  6. An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

    Science.gov (United States)

    Xu, X-X; Zhang, Y-Q; Freed, S; Yu, J; Gao, Y-F; Wang, S; Ouyang, L-N; Ju, W-Y; Jin, F-L

    2016-12-01

    Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

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

    International Nuclear Information System (INIS)

    Van, C.K.; Kuzin, Yu.Yu.; Kozlovskii, Yu.E.; Prozorov, A.A.

    1986-01-01

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

  8. Production and recombination of gluons

    International Nuclear Information System (INIS)

    Temiraliev, A.T.

    2006-01-01

    Full text: Nonlinear Markov process of parton production has been considered. The Kolmogorov equation is applied for the evolution equation based on the approximation of independent gluons production in every decay act. We introduced a 'crossing' parameter and used the combination relations to obtain nonlinear recombination equation for the evolution of gluon structure function. (author)

  9. Recombinator of hydrogen and oxygen

    International Nuclear Information System (INIS)

    Stejskal, J.; Klein, O.; Scholtz, G.; Schmidt, P.; Olaussson, A.

    1976-01-01

    Improvements are proposed for the well known reactors for the catalytic recombination of hydrogen and oxygen, which should permit this being used in contiuous operation in nuclear reactors (BWRs). The improvements concern the geometric arrangement of gas-inlet and -outlet pipes, the inclination of the axis of the catalyst container and the introduction of remote operation. (UWI) [de

  10. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  11. Recombination in hepatitis C virus.

    Science.gov (United States)

    González-Candelas, Fernando; López-Labrador, F Xavier; Bracho, María Alma

    2011-10-01

    Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus.

  12. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

    Science.gov (United States)

    Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

    2017-01-01

    In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Live recombinant BHV/BRSV vaccine

    NARCIS (Netherlands)

    Keil, G.M.; Rijsewijk, F.A.M.

    1998-01-01

    The present invention refers to synthetic Bovine Respiratory Syncytium virus genes. Also the invention relates to live attenuated Bovine Herpesvirus recombinants carrying such synthetic genes. Furthermore, the invention relates to vaccines based on these live attenuated recombinants, for the

  14. Hadron production at RHIC: recombination of quarks

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    We discuss quark recombination applied to the hadronization of a quark gluon plasma. It has been shown that the quark recombination model can explain essential features of hadron production measured in high energy heavy ion collisions.

  15. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Affinity purification of recombinant human plasminogen activator from ... Screening antibody was performed using rhPA milk in an ELISA-elution assay. ... useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

  16. Graded Recombination Layers for Multijunction Photovoltaics

    KAUST Repository

    Koleilat, Ghada I.; Wang, Xihua; Sargent, Edward H.

    2012-01-01

    it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron

  17. Recombinant innovation and endogenous technological transitions

    NARCIS (Netherlands)

    Frenken, K.; Izquierdo, L.R.; Zeppini, P.

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create "short-cuts" which reduce

  18. Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus.

    Science.gov (United States)

    Lee, Nam Keun; Yeo, In-Cheol; Park, Joung Whan; Kang, Byung-Sun; Hahm, Young Tae

    2010-09-01

    In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 degrees C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5mug/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254nm. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Enzyme activities and antibiotic susceptibility of colonial variants of Bacillus subtilis and Bacillus licheniformis.

    OpenAIRE

    Carlisle, G E; Falkinham, J O

    1989-01-01

    A nonmucoid colonial variant of a mucoid Bacillus subtilis strain produced less amylase activity and a transparent colonial variant of a B. licheniformis strain produced less protease activity compared with their parents. Antibiotic susceptibility patterns of the colonial variants differed, and increased resistance to beta-lactam antibiotics was correlated with increased production of extracellular beta-lactamase.

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

    Science.gov (United States)

    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.

  1. Role of fatty acids in Bacillus environmental adaptation

    Directory of Open Access Journals (Sweden)

    Sara Esther Diomande

    2015-08-01

    Full Text Available The large bacterial genus genus Bacillus is widely distributed in the environment and is able to colonize highly diverse niches. Some Bacillus species harbour pathogenic characteristics. The fatty acid (FA composition is among the essential criteria used to define Bacillus species. Some elements of the FA pattern composition are common to Bacillus species, whereas others are specific and can be categorized in relation to the ecological niches of the species. Bacillus species are able to modify their FA patterns to adapt to a wide range of environmental changes, including changes in the growth medium, temperature, food processing conditions, and pH. Like many other Gram-positive bacteria, Bacillus strains display a well-defined FA synthesis II system that is equilibrated with a FA degradation pathway and regulated to efficiently respond to the needs of the cell. Like endogenous FAs, exogenous FAs may positively or negatively affect the survival of Bacillus vegetative cells and the spore germination ability in a given environment. Some of these exogenous FAs may provide a powerful strategy for preserving food against contamination by the Bacillus pathogenic strains responsible for foodborne illness.

  2. Evaluation of antifungal activity from Bacillus strains against ...

    African Journals Online (AJOL)

    In this study, 30 bacterial strains isolated from marine biofilms were screened for their antifungal activity against Rhizoctonia solani by dual culture assay. Two bacterial strains, Bacillus subtilis and Bacillus cereus, showed a clear antagonism against R. solani on potato dextrose agar (PDA) medium. The antagonistic activity ...

  3. Increasing the alkaline protease activity of Bacillus cereus and ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... cereus and Bacillus polymyxa simultaneously with the start of sporulation phase as a ... microbial forms to inactivation by chemical or physical agents. .... alkaline pH, 9, 10 and 11 and the pH of the culture media was optimized with .... incubation temperature for alkaline protease production by Bacillus ...

  4. Optimizing Bacillus circulans Xue-113168 for biofertilizer production ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-12-28

    Dec 28, 2016 ... In this study, Bacillus circulans Xue-113168 biofertilizer was produced through solid state fermentation ... organic matter, NPK content from 8.83 to 16.16 kg hm2, and reduced chemical ... dependent on the nutritional components. ...... shell fish chitin wastes for the production of Bacillus subtilis W-118.

  5. Real-Time PCR Diagnostics for Detecting and Identifying Potential Bioweapons

    Science.gov (United States)

    2003-11-18

    pestis Bacillus cereus Salmonella enteritidis Yersinia pestis Bacillus thurigiensis Serratia odorifera Yersinia pestis Bacillus coagulans Shigella...10fg NTC 100pg-opt 10pg-opt 1pg-opt 100fg-opt 10fg-opt NTC-opt USAMRIID Specificity Organism Organism Organism Acineobacter baumanni Bacillus subtilis...var niger Staphylococcus saprophyticus Bacillus anthracis BA0068 Bacillus bronchiseptica Staphylococcus epidermidis Bacillus anthracis Clostridium

  6. Population inversion in recombining hydrogen plasma

    International Nuclear Information System (INIS)

    Furukane, Utaro; Yokota, Toshiaki; Oda, Toshiatsu.

    1978-11-01

    The collisional-radiative model is applied to a recombining hydrogen plasma in order to investigate the plasma condition in which the population inversion between the energy levels of hydrogen can be generated. The population inversion is expected in a plasma where the three body recombination has a large contribution to the recombining processes and the effective recombination rate is beyond a certain value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

  7. Regulation of homologous recombination in eukaryotes

    OpenAIRE

    Heyer, Wolf-Dietrich; Ehmsen, Kirk T.; Liu, Jie

    2010-01-01

    Homologous recombination is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage including DNA double-stranded breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and ...

  8. The effect of a single recombination event

    DEFF Research Database (Denmark)

    Schierup, Mikkel Heide; Jensen, Thomas Mailund; Wiuf, Carsten

    We investigate the variance in how visible a single recombination event is in a SNP data set as a function of the type of recombination event and its age. Data is simulated under the coalescent with recombination and inference is by the popular composite likelihood methods. The major determinant...

  9. Effect of Bacillus subtilis on Granite Weathering: A Laboratory Experiment

    Science.gov (United States)

    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.

  10. Ecology and genomics of Bacillus subtilis.

    Science.gov (United States)

    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.

  11. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  12. Effect of garlic solution to Bacillus sp. removal

    Science.gov (United States)

    Zainol, N.; Rahim, S. R.

    2018-04-01

    Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacillus sp. was used as biofilm model in this study. The purpose of this study is to determine the effect of Garlic solution in term of ratio of water and Garlic solution (W/G) and ratio of Garlic solution to Bacillus sp. (GS/B) on Bacillus sp removal. Garlic solution was used to remove Bacillus sp. In this study, Garlic solution was prepared by crushing the garlic and mixed it with water. the Garlic solution was added into Bacillus sp. mixture and mixed well. The mixture then was spread on nutrient agar. The Bacillus sp. weight on agar plate was measured by using dry weight measurement method. In this study, initially Garlic solution volume and Garlic solution concentration were studied using one factor at time (OFAT). Later two-level-factorial analysis was done to determine the most contributing factor in Bacillus sp. removal. Design Expert software (Version 7) was used to construct experimental table where all the factors were randomized. Bacilus sp removal was ranging between 42.13% to 99.6%. The analysis of the results showed that at W/G of 1:1, Bacillus sp. removal increased when more Garlic solution was added to Bacillus sp. Effect of Garlic solution to Bacillus sp. will be understood which in turn may be beneficial for the industrial purpose.

  13. Recombination Catalysts for Hypersonic Fuels

    Science.gov (United States)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  14. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    Nakai, Sayaka; Machida, Isamu; Tsuji, Satsuki

    1985-01-01

    Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G 2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

  15. Construction of a highly efficient Bacillus subtilis 168 whole-cell biocatalyst and its application in the production of L-ornithine.

    Science.gov (United States)

    Wang, Meizhou; Xu, Meijuan; Rao, Zhiming; Yang, Taowei; Zhang, Xian

    2015-11-01

    L-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of L-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 °C, respectively. In addition, the recombinant arginase exhibited a strong Mn(2+) preference. When using whole-cell biocatalyst-based bioconversion, a hyper L-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for L-ornithine production in industry.

  16. Interface recombination influence on carrier transport

    International Nuclear Information System (INIS)

    Konin, A

    2013-01-01

    A theory of interface recombination in the semiconductor–semiconductor junction is developed. The interface recombination rate dependence on the nonequilibrium carrier densities is derived on the basis of a model in which the interface recombination occurs through the mechanism of trapping. The general relation between the interface recombination parameters at small carrier density deviation from the equilibrium ones is obtained. The validity of this relation is proved considering the generation of the Hall electric field in the extrinsic semiconductor sample. The anomalous Hall electromotive force in a weak magnetic field was investigated and interpreted by means of a new interface recombination model. The experimental data corroborate the developed theory. (paper)

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

    Directory of Open Access Journals (Sweden)

    Li Li

    Full Text Available ABSTRACT 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 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. 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.

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

    Science.gov (United States)

    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.

  19. An azoreductase, aerobic NADH-dependent flavoprotein discovered from Bacillus sp.: functional expression and enzymatic characterization.

    Science.gov (United States)

    Ooi, Toshihiko; Shibata, Takeshi; Sato, Reiko; Ohno, Hiroaki; Kinoshita, Shinichi; Thuoc, Tran Linh; Taguchi, Seiichi

    2007-05-01

    The gene coding for an azoreductase, designated as an azrA, was cloned by polymerase chain reaction amplification from the genomic DNA of Bacillus sp. strain B29 isolated from soil. The azrA encoded a protein of 208 amino acids with calculated molecular mass of 22,766 Da. The enzyme was heterologously expressed in Escherichia coli with a strong band of 23 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified recombinant AzrA was a homodimer with a native molecular mass of 48 kDa containing two molecules of flavin mononucleotide (FMN; oxidized). This activity was oxygen insensitive and was nicotinamide adenine dinucleotide (reduced form; NADH) dependent. Recombinant AzrA exhibited a broad pH stability between 6 and 10 with a temperature optimum of 60-80 degrees C. The enzyme cleaved the model azo compound of methyl red [MR, 4'-(dimethylamino)-azobenzene-2-carboxylic acid] into 2-aminobenzoic acid and N, N'-dimethyl-p-phenylenediamine by ping-pong mechanism. The enzyme was not only able to decolorize MR but also able to decolorize sulfonated azo dyes such as Orange I and Acid Red 88.

  20. DNA repair and the evolution of transformation in Bacillus subtilis. 3. Sex with damaged DNA

    International Nuclear Information System (INIS)

    Hoelzer, M.A.; Michod, R.E.

    1991-01-01

    Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination. The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome. Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged. In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells. In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA. The authors document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA. These results suggest that sites of transformation are often damaged sites in the recipient cell's genome

  1. Recombinant Cyclophilins Lack Nuclease Activity

    OpenAIRE

    Manteca, Angel; Sanchez, Jesus

    2004-01-01

    Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

  2. Workshop on Radio Recombination Lines

    CERN Document Server

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

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

    Directory of Open Access Journals (Sweden)

    Ratu SAFITRI

    2015-10-01

    Full Text Available This study was conducted in order to determine the ability of bacterial consortium: Bacillus coagulans, Bacilus licheniformis, Bacillus pumilus, Bacillus subtilis, Nitrosomonas sp., and Pseudomonas putida in bioremediation of wastewater origin Cisirung WWTP. This study uses an experimental method completely randomized design (CRD, which consists of two treatment factors (8x8 factorial design. The first factor is a consortium of bacteria (K, consisting of 8 level factors (k1, k2, k3, k4, k5, k6, k7, and k8. The second factor is the time (T, consisting of a 7 level factors (t0, t1, t2, t3, t4, t5, t6, and t7. Test parameters consist of BOD (Biochemical Oxygen Demand, COD (Chemical Oxygen Demand, TSS (Total Suspended Solid, Ammonia and Population of Microbes during bioremediation. Data were analyzed by ANOVA, followed by Duncan test. The results of this study showed that the consortium of Bacillus pumilus, Bacillus subtilis, Bacillus coagulans, Nitrosomonas sp., and Pseudomonas putida with inoculum concentration of 5% (k6 is a consortium of the most effective in reducing BOD 71.93%, 64.30% COD, TSS 94.85%, and 88.58% of ammonia.

  4. Potential of Bacillus spp produces siderophores insuppressing thewilt disease of banana plants

    Science.gov (United States)

    Kesaulya, H.; Hasinu, J. V.; Tuhumury, G. NC

    2018-01-01

    In nature, different types of siderophore such as hydroxymate, catecholets and carboxylate, are produced by different bacteria. Bacillus spp were isolated from potato rhizospheric soil can produce siderophore of both catecholets and salicylate type with different concentrations. Various strains of Bacillus spp were tested for pathogen inhibition capability in a dual culture manner. The test results showed the ability of inhibition of pathogen isolated from banana wilt disease. From the result tested were found Bacillus niabensis Strain PT-32-1, Bacillus subtilis Strain SWI16b, Bacillus subtilis Strain HPC21, Bacillus mojavensis Strain JCEN3, and Bacillus subtilis Strain HPC24 showed different capabilities in suppressing pathogen.

  5. Consequences of recombination on traditional phylogenetic analysis

    DEFF Research Database (Denmark)

    Schierup, M H; Hein, J

    2000-01-01

    We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mt......DNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination....... With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may...

  6. Fast Neutron Radiation Effects on Bacillus Subtili

    International Nuclear Information System (INIS)

    Chen Xiaoming; Zhang Jianguo; Chu Shijin; Ren Zhenglong; Zheng Chun; Yang Chengde; Tan Bisheng

    2009-01-01

    To examine the sterilizing effect and mechanism of neutron radiation, Bacillus subtilis var. niger. strain (ATCC 9372) spores were irradiated with the fast neutron from the Chinese fast burst reactor II(CFBR-II). The plate-count results indicated that the D 10 value was 384.6 Gy with a neutron radiation dose rate of 7.4 Gy/min. The rudimental catalase activity of the spores declined obviously with the increase in the radiation dose. Meanwhile, under the scanning electron microscope, no visible influence of the neutron radiation on the spore configuration was detected even if the dose was increased to 4 kGy. The content and distribution of DNA double-strand breaks induced by neutron radiation at different doses were measured and quantified by pulsed-field gel electrophoresis (PFGE). Further analysis of the DNA release percentage (PR), the DNA breakage level (L), and the average molecular weight, indicated that DNA fragments were obviously distributed around the 5 kb regions at different radiation doses, which suggests that some points in the DNA molecule were sensitive to neutron radiation. Both PR and L varied regularly to some extent with the increase in radiation dose. Thus neutron radiation has a high sterilization power, and can induce falling enzyme activity and DNA breakage in Bacillus subtilis spores

  7. Bacillus beijingensis sp. nov. and Bacillus ginsengi sp. nov., isolated from ginseng root.

    Science.gov (United States)

    Qiu, Fubin; Zhang, Xiaoxia; Liu, Lin; Sun, Lei; Schumann, Peter; Song, Wei

    2009-04-01

    Four alkaligenous, moderately halotolerant strains, designated ge09, ge10(T), ge14(T) and ge15, were isolated from the internal tissue of ginseng root and their taxonomic positions were investigated by using a polyphasic approach. Cells of the four strains were Gram-positive-staining, non-motile, short rods. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains ge09 and ge10(T) formed one cluster and strains ge14(T) and ge15 formed another separate cluster within the genus Bacillus. 16S rRNA gene sequence similarities with type strains of other Bacillus species were less than 97 %. Levels of DNA-DNA relatedness among the four strains showed that strains ge09 and ge10(T) and strains ge14(T) and ge15 belonged to two separate species; the mean level of DNA-DNA relatedness between ge10(T) and ge14(T) was only 28.7 %. Their phenotypic and physiological properties supported the view that the two strains represent two different novel species of the genus Bacillus. The DNA G+C contents of strains ge10(T) and ge14(T) were 49.9 and 49.6 mol%, respectively. Strains ge10(T) and ge14(T) showed the peptidoglycan type A4alpha l-Lys-d-Glu. The lipids present in strains ge10(T) and ge14(T) were diphosphatidylglycerol, phosphatidylglycerol, a minor amount of phosphatidylcholine and two unknown phospholipids. Their predominant respiratory quinone was MK-7. The fatty acid profiles of the four novel strains contained large quantities of branched and saturated fatty acids. The predominant cellular fatty acids were iso-C(15 : 0) (42.5 %), anteiso-C(15 : 0) (22.2 %), anteiso-C(17 : 0) (7.3 %) and C(16 : 1)omega7c alcohol (5.7 %) in ge10(T) and iso-C(15 : 0) (50.7 %) and anteiso-C(15 : 0) (20.1 %) in ge14(T). On the basis of their phenotypic properties and phylogenetic distinctiveness, two novel species of the genus Bacillus are proposed, Bacillus beijingensis sp. nov. (type strain ge10(T) =DSM 19037(T) =CGMCC 1.6762(T)) and Bacillus ginsengi sp. nov. (type strain ge14

  8. Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots.

    Science.gov (United States)

    Stukenbrock, Eva H; Dutheil, Julien Y

    2018-03-01

    Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. Copyright © 2018 Stukenbrock and Dutheil.

  9. Heavy metals and their radionuclides uptake by Bacillus Licheniformis

    International Nuclear Information System (INIS)

    Ramadan, A.A.; Ahmed, M.M.; Abo-state, M.A.M.; Sarhan, M.; Faroqe, M.

    2007-01-01

    Bacillus licheniformis is a gram positive spore forming bacterium. Different concentrations of cobalt affected the ability of Co uptake and growth of Bacillus licheniformis. As the concentration increased, both the uptake and growth were decreased. Maximum Co uptake was found at ph 7.0, while for growth was ph 8.0. The optimum temperature for uptake and growth was 40 degree C and 20% inoculum size represents the maximum cobalt uptake by Bacillus licheniformis. Also, maximum uptake was recorded after 72 hours, incubation period. As the concentration of cesium was increased till 400 mg/l, the uptake was also increased. The optimum cesium uptake and growth was at ph 8.0. The optimum growth was at 45 degree C while Cs uptake was found at 35 degree C and 15% inoculum size represented the maximum Cs uptake. After 72 hour incubation period, maximum Cs uptake was recorded. Generally, Bacillus licheniformis removed more than 80% of Co and 50% of Cs from the broth medium. Addition of clay to Bacillus licheniformis increased both Co or Cs uptake. Bacillus licheniformis was gamma resistant and 10 KGy reduced the viability by 5.3 log cycles. The irradiated and non-irradiated cultures can grow on 500 or 700 mg Co or Cs. Bacillus licheniformis removed 99.32% of the Co radionuclides and 99.28% of Cs radionuclides

  10. Antifungal activity of indigenous Bacillus spp. isolated from soil

    Directory of Open Access Journals (Sweden)

    Bjelić Dragana Đ.

    2017-01-01

    Full Text Available Biocontrol using plant growth-promoting rhizobacteria (PGPR represents an alternative approach to disease management, since PGPR are known to promote growth and reduce diseases in various crops. Among the different PGPR, members of the genus Bacillus are prefered for most biotechnological uses due to their capability to form extremely resistant spores and produce a wide variety of metabolites with antimicrobial activity. The objective of this research was to identify antagonistic bacteria for management of the plant diseases. Eleven isolates of Bacillus spp. were obtained from the soil samples collected from different localities in the Province of Vojvodina. The antifungal activity of bacterial isolates against five fungal species was examined using a dual plate assay. Bacillus isolates exhibited the highest antifungal activity against Fusarium proliferatum, Fusarium oxysporum f. sp. cepae and Alternaria padwickii, while they had the least antagonistic effect on Fusarium verticillioides and Fusarium graminearum. Molecular identification showed that effective bacterial isolates were identified as Bacillus safensis (B2, Bacillus pumilus (B3, B11, Bacillus subtilis (B5, B7 and Bacillus megaterium (B8, B9. The highest antagonistic activity was exhibited by isolates B5 (from 39% to 62% reduction in fungal growth and B7 (from 40% to 71% reduction in fungal growth. These isolates of B. subtilis could be used as potential biocontrol agents of plant diseases. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR-31073

  11. Screening of Bacillus Species with Potentials of Antibiotics Production

    OpenAIRE

    Faruk Adamu KUTA; Lohya NIMZING; Priscilla Yahemba ORKA’A

    2009-01-01

    Sixteen soil samples were collected from different refuse dump sites in Minna, the capital Niger State, and analysed for the presence of Bacillus species. Physical-chemical analysis of the soil samples revealed the followings: PH value 6.89-8.47; moisture content 1.58 – 21.21% and temperature 27-28ºC. Using both pour plate and streak method of inoculation, total bacterial count in the soil samples ranged from 3.8×104 cfu/g 16.0×104 cfu/g. The identified Bacillus species included: Bacillus cer...

  12. Reparation and Immunomodulating Properties of Bacillus sp. Metabolites from Permafrost.

    Science.gov (United States)

    Kalenova, L F; Melnikov, V P; Besedin, I M; Bazhin, A S; Gabdulin, M A; Kolyvanova, S S

    2017-09-01

    An ointment containing metabolites of Bacillus sp. microorganisms isolated from permafrost samples was applied onto the skin wound of BALB/c mice. Metabolites isolated during culturing of Bacillus sp. at 37°C produced a potent therapeutic effect and promoted wound epithelialization by 30% in comparison with the control (ointment base) and by 20% in comparison with Solcoseryl. Treatment with Bacillus sp. metabolites stimulated predominantly humoral immunity, reduced the time of wound contraction and the volume of scar tissue, and promoted complete hair recovery. These metabolites can be considered as modulators of the wound process with predominance of regeneration mechanisms.

  13. Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis

    International Nuclear Information System (INIS)

    Marrero, R.; Yasbin, R.E.

    1988-01-01

    By use of the Bacillus subtilis bacteriophage cloning vehicle Phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages Phi 105Rec Phi1 (3.85-kilobase insert) and Phi 105Rec Phi4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE + strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage Phi105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either Phi 105Rec Phi 1 or Phi 105RecPhi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages Phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages Phi 105RecPhi 1 and Phi 105Rec Phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA + gene product antibodies. Collectively, these data demonstrate that the recE + gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination

  14. Characteristics and Application of a Novel Species of Bacillus: Bacillus velezensis.

    Science.gov (United States)

    Ye, Miao; Tang, Xiangfang; Yang, Ru; Zhang, Hongfu; Li, Fangshu; Tao, Fangzheng; Li, Fei; Wang, Zaigui

    2018-03-16

    Bacillus velezensis has been investigated and applied more and more widely recently because it can inhibit fungi and bacteria and become a potential biocontrol agent. In order to provide more clear and comprehensive understanding of B. velezensis for researchers, we collected the recent relevant articles systematically and reviewed the discovery and taxonomy, secondary metabolites, characteristics and application, gene function, and molecular research of B. velezensis. This review will give some direction to the research and application of this strain for the future.

  15. Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food

    DEFF Research Database (Denmark)

    Rosenquist, Hanne; Ørum-Smidt, Lasse; Andersen, Sigrid R

    2005-01-01

    Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains....../or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis....

  16. Characterization of an L-arabinose isomerase from Bacillus coagulans NL01 and its application for D-tagatose production.

    Science.gov (United States)

    Mei, Wending; Wang, Lu; Zang, Ying; Zheng, Zhaojuan; Ouyang, Jia

    2016-06-30

    L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed. The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h). In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular

  17. Improving Mycobacterium bovis Bacillus Calmette-Guèrin as a Vaccine Delivery Vector for Viral Antigens by Incorporation of Glycolipid Activators of NKT Cells

    OpenAIRE

    Venkataswamy, Manjunatha M.; Ng, Tony W.; Kharkwal, Shalu S.; Carreño, Leandro J.; Johnson, Alison J.; Kunnath-Velayudhan, Shajo; Liu, Zheng; Bittman, Robert; Jervis, Peter J.; Cox, Liam R.; Besra, Gurdyal S.; Wen, Xiangshu; Yuan, Weiming; Tsuji, Moriya; Li, Xiangming

    2014-01-01

    Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approac...

  18. Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

    International Nuclear Information System (INIS)

    Fraenkel, A.H.M.

    1974-01-01

    Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

  19. Vaccine platform recombinant measles virus.

    Science.gov (United States)

    Mühlebach, Michael D

    2017-10-01

    The classic development of vaccines is lengthy, tedious, and may not necessarily be successful as demonstrated by the case of HIV. This is especially a problem for emerging pathogens that are newly introduced into the human population and carry the inherent risk of pandemic spread in a naïve population. For such situations, a considerable number of different platform technologies are under development. These are also under development for pathogens, where directly derived vaccines are regarded as too complicated or even dangerous due to the induction of inefficient or unwanted immune responses causing considerable side-effects as for dengue virus. Among platform technologies are plasmid-based DNA vaccines, RNA replicons, single-round infectious vector particles, or replicating vaccine-based vectors encoding (a) critical antigen(s) of the target pathogens. Among the latter, recombinant measles viruses derived from vaccine strains have been tested. Measles vaccines are among the most effective and safest life-attenuated vaccines known. Therefore, the development of Schwarz-, Moraten-, or AIK-C-strain derived recombinant vaccines against a wide range of mostly viral, but also bacterial pathogens was quite straightforward. These vaccines generally induce powerful humoral and cellular immune responses in appropriate animal models, i.e., transgenic mice or non-human primates. Also in the recent first clinical phase I trial, the results have been quite encouraging. The trial indicated the expected safety and efficacy also in human patients, interestingly independent from the level of prevalent anti-measles immunity before the trial. Thereby, recombinant measles vaccines expressing additional antigens are a promising platform for future vaccines.

  20. CRMAGE: CRISPR Optimized MAGE Recombineering

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli...... that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red...