Polyaminated, acetylated and stop codon readthrough of recombinant Francisella tularensis universal stress protein in Escherichia coli.

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses.

Mass Spectrometry of Putrescine, Spermidine, and Spermine Covalently Attached to Universal Stress Protein and Bovine Albumin.

Bacterial and mammalian cells are rich in putrescine, spermidine, and spermine. Polyamines are required for optimum fitness, but the biological function of these small aliphatic compounds has only been partially revealed. Known functions of polyamines include interaction with nucleic acids that alters gene expression and with proteins that modulate activity.

Arginine Catabolism and Polyamine Biosynthesis Pathway Disparities Within Subpopulations.

is a highly infectious zoonotic pathogen with as few as 10 organisms causing tularemia, a disease that is fatal if untreated. Although subspecies (type A) and subspecies (type B) share over 99.5% average nucleotide identity, notable differences exist in genomic organization and pathogenicity.

Differences in Blood-derived Type B Strains from Clinical Cases of Tularemia.

can cause the zoonotic disease tularemia and is partitioned into subspecies due to differences in chromosomal organization and virulence. The subspecies (type B) is generally considered more clonal than the other subpopulations with moderate virulence compared to the hypervirulent A.I clade.

Differentiation of Francisella tularensis Subspecies and Subtypes.

The highly infectious and zoonotic pathogen is the etiologic agent of tularemia, a potentially fatal disease if untreated. Despite the high average nucleotide identity, which is >99.2% for the virulent subspecies and >98% for all four subspecies, including the opportunistic microbe subsp.

Oligomerization of bacterially expressed H1N1 recombinant hemagglutinin contributes to protection against viral challenge.

Vaccination is the most effective intervention to prevent influenza and control the spread of the virus. Alternatives are needed to the traditional egg-based vaccine strategy for a more rapid response to new outbreaks. Two different hemagglutinin (HA) fragments (rHA1 and rHA1) derived from influenza A virus subtype H1N1 were expressed in Escherichia coli and characterized by immunoblot, gel filtration, hemagglutination, and competitive binding assays.

Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile.

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized.

Development of potential broad spectrum antimicrobials using C2-symmetric 9-fluorenone alkyl amine.

DNA-dependent RNA primase is essential for de novo primer synthesis during DNA replication in all living organisms. Bacterial DnaG primase is an attractive target for inhibition because it is essential, low in copy number and structurally distinct from eukaryotic and archaeal primases. DnaG primase is sensitive to known inhibitors including suramin and doxorubicin.

Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay.

The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.

Reclassification of Wolbachia persica as Francisella persica comb. nov. and emended description of the family Francisellaceae.

The taxonomic status of the bacterium Wolbachia persica is described, and based on the evidence presented, transfer of this species to the genus Francisella as Francisella persica comb. nov. is proposed.

Discovery of bicyclic inhibitors against menaquinone biosynthesis.

Introduction: Menaquinone is used for transporting electrons and is essential for the aerobic and anaerobic respiratory systems of all pathogens and prokaryotes. Many Gram-positive bacteria use only menaquinone in the electron transport system. Thus, menaquinone biosynthesis is a potential target for the development of inhibitors against bacteria including drug-resistant pathogens.

Francisella tularensis Subtype A.II Genomic Plasticity in Comparison with Subtype A.I.

Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F.

Francisella tularensis bacteria associated with feline tularemia in the United States.

Tularemia in the United States was examined by reviewing 106 Francisella tularensis isolates, mostly from Nebraska, collected during 1998-2012: 48% of Nebraska cases were cat-associated; 7/8 human cases were caused by subtype A.I. A vaccine is needed to reduce feline-associated tularemia, and cat owners should protect against bites/scratches and limit their pet's outdoor access.

Polyproline promotes tetramerization of recombinant human butyrylcholinesterase.

Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers and monomers that are cleared from the circulation within minutes.

Functional interplay of DnaE polymerase, DnaG primase and DnaC helicase within a ternary complex, and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis.

Bacillus subtilis has two replicative DNA polymerases. PolC is a processive high-fidelity replicative polymerase, while the error-prone DnaEBs extends RNA primers before hand-off to PolC at the lagging strand. We show that DnaEBs interacts with the replicative helicase DnaC and primase DnaG in a ternary complex.

Application of chromosomal DNA and protein targeting for the identification of Yersinia pestis.

Purpose: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis.

Experimental Design: Computational analysis was used to discover chromosomal targets unique to Y. pestis.

Francisella tularensis molecular typing using differential insertion sequence amplification.

Tularemia is a potentially fatal disease that is caused by the highly infectious and zoonotic pathogen Francisella tularensis. Despite the monomorphic nature of sequenced F. tularensis genomes, there is a significant degree of plasticity in the organization of genetic elements.

Class-specific restrictions define primase interactions with DNA template and replicative helicase.

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases.

Genetic analysis of the Staphylococcus epidermidis macromolecular synthesis operon: Serp1129 is an ATP binding protein and sigA transcription is regulated by both sigma(A)- and sigma(B)-dependent promoters.

Background: The highly conserved macromolecular synthesis operon (MMSO) contains both dnaG (primase) and sigA (primary sigma factor). However, in previously evaluated gram-positive species, the MMSO is divergent upstream of dnaG. The MMSO of Bacillus subtilis contains three open reading frames (ORFs) that are differentially regulated by multiple promoters.

Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines.

The placement of the extreme thermophile Aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. We investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically DnaG primase. Although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for A.

Staphylococcus aureus primase has higher initiation specificity, interacts with single-stranded DNA stronger, but is less stimulated by its helicase than Escherichia coli primase.

The study of primases from model organisms such as Escherichia coli, phage T7 and phage T4 has demonstrated the essential nature of primase function, which is to generate de novo RNA polymers to prime DNA polymerase. However, little is known about the function of primases from other eubacteria. Their overall low primary sequence homology may result in functional differences.

Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome.

The bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC, and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase.

Myricetin inhibits Escherichia coli DnaB helicase but not primase.

Primase and DnaB helicase play central roles during DNA replication initiation and elongation. Both enzymes are drug targets because they are essential, persistent among bacterial genomes, and have different sequences than their eukaryotic equivalents. Myricetin is a ubiquitous natural product in plants that is known to inhibit a variety of DNA polymerases, RNA polymerases, reverse transcriptases, and telomerases in addition being able to inhibit kinases and helicases.

Domain swapping reveals that the C- and N-terminal domains of DnaG and DnaB, respectively, are functional homologues.

The bacterial primase (DnaG)-helicase (DnaB) interaction is mediated by the C-terminal domain of DnaG (p16) and a linker that joins the N- and C-terminal domains (p17 and p33 respectively) of DnaB. The crystal and nuclear magnetic resonance structures of p16 from Escherichia coli and Bacillus stearothermophilus DnaG proteins revealed a unique structural homology with p17, despite the lack of amino acid sequence similarity. The functional significance of this is not clear.

Bovine serum amyloid A3 gene structure and promoter analysis: induced transcriptional expression by bacterial components and the hormone prolactin.

Regulation of the bovine Saa3 promoter in response to signaling molecules associated with lactation or bacterial infection was assessed using a luciferase reporter system. Although the liver is the primary site for the production of acute phase proteins, typically serum amyloid A1 (SAA1) and serum amyloid A2 (SAA2), analysis of the differential expression of serum amyloid A3 (SAA3) by mammary epithelial cells is limited. Gram-negative bacterial lipopolysaccharide (LPS) and the Gram-positive bacterial lipoteichoic acid (LTA) substantially upregulated transcriptional expression driven by the Saa3 promoter in bovine mammary epithelial cells by 18.