Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases

Pomerantsev, Andrei P.; McCall, Rita M.; Chahoud, Margaret; Hepler, N.; Fattah, Rasem J.; Leppla, Stephen H.

doi:10.1371/journal.pone.0183346

Cited by 17 publications

(19 citation statements)

References 22 publications

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“…B . anthracis Ames BH500 strain 11 with the genotype pXO1-, pXO2-, Spo0A- (GBAA_4394), NprB- (0599), TasA- (1298), Cam- (1290), InhA1- (1295), InhA2- (0672), MmpZ- (3159), CysP1- (1995), VpR- (4584), NprC- (2183), S41- (5414) was used in this study. Genetic maps for both plasmids are presented in the Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, expressing this protein in a modified, nonpathogenic, B. anthracis host may offer an attractive strategy. Here we employed the B. anthracis strain BH500, which is asporogenic, lacks both virulence plasmids, and is deleted for ten extracellular proteases 11 .…”

Section: Introductionmentioning

confidence: 99%

“…The differences seen in essential pathways required for protein expression including: central carbon metabolism, amino acid biosynthesis, transcription, translation, folding and secretion, were evaluated to identify plausible bottlenecks. The genes identified provide targets for genetic engineering to increase the effectiveness of B. anthracis strains as production hosts 11 .…”

Section: Introductionmentioning

confidence: 99%

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Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Sharma

Leppla

Pomerantsev

et al. 2018

Sci Rep

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Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. The present study examines the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a control strain carrying an empty plasmid. The strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was conducted. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase σ54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Also, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases, which was associated with decreased specific growth rates. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Sharma

Leppla

Pomerantsev

et al. 2018

Sci Rep

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“…B. anthracis Ames BH480 strain 25 (with the genotype pXO1 -, pXO2 -, Spo0A -(GBAA_4394), NprB -(GBAA_0599), TasA -(GBAA_1298), Cam -(GBAA_1290), InhA1 -(GBAA_1295), InhA2 -(GBAA_0672), MmpZ -(GBAA_3159), CysP1 -(GBAA_1995), VpR -(GBAA_4584), and the pYS5 plasmid containing original pagA promoter from pXO1, including 162 bp of B. anthracis DNA upstream of the PA start codon used in this study were obtained from Institute of Genomics & Integrative Biology, Delhi. The pYS plasmid was transformed into damand dcm -E. coli SCS110 strain.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Production and Purification of Protective Antigen of Bacillus anthracis and Development of a Sandwich ELISA for its Detection

et al. 2020

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Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purified and used for the development of a sandwich ELISA for detection of PA. The detection sensitivity of ELISA was found to be 3.9 ng/ mL PA.

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“…Recombinant B. anthracis PA 83 WT was produced as described in (51). F427A mutant of PA (PA 83 F427A) was created by Quick-Change mutagenesis of the pYS5 expression plasmid.…”

Section: Chemicalsmentioning

confidence: 99%

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel

Abolfath

Kolberg

Karginov

et al. 2019

Biophysical Journal

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Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA 63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA 63 more effectively. To understand the physical basis of PA 63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the highresolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-b-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-a-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-b-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-g-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA 63 variants and PA 63 F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the ''f-clamp.'' We show that the cationic blockers interact with PA 63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-p, Coulomb, and salt-concentration-independent p-p or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the f-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA 63 F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the f-clamp. We also show that seven-and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA 63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.

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Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases

Cited by 17 publications

References 22 publications

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Production and Purification of Protective Antigen of Bacillus anthracis and Development of a Sandwich ELISA for its Detection

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel

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