Shiga-Toxin-Producing Strains of O104:H4 and a Strain of O157:H7, Which Can Cause Human Hemolytic Uremic Syndrome, Differ in Biofilm Formation in the Presence of CO and in Their Ability to Grow in a Novel Cell Culture Medium.

One pathogen that commonly causes gastrointestinal illnesses from the consumption of contaminated food is O157:H7. In 2011 in Germany, however, there was a prominent outbreak of bloody diarrhea with a high incidence of hemolytic uremic syndrome (HUS) caused by an atypical, more virulent O104:H4 strain. To facilitate the identification of this lesser-known, atypical O104:H4 strain, we wanted to identify phenotypic differences between it and a strain of O157:H7 in different media and culture conditions.

Development, Phenotypic Characterization and Genomic Analysis of a Panel for Tularemia Vaccine Testing.

is one of several biothreat agents for which a licensed vaccine is needed to protect against this pathogen. To aid in the development of a vaccine protective against pneumonic tularemia, we generated and characterized a panel of isolates that can be used as challenge strains to assess vaccine efficacy. Our panel consists of both historical and contemporary isolates derived from clinical and environmental sources, including human, tick, and rabbit isolates.

A novel selective medium for the isolation of Burkholderia mallei from equine specimens.

Background: Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations (e.g. horses, donkeys, and mules).

Avirulent Bacillus anthracis Strain with Molecular Assay Targets as Surrogate for Irradiation-Inactivated Virulent Spores.

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B.

Using a color-coded ambigraphic nucleic acid notation to visualize conserved palindromic motifs within and across genomes.

Background: Ambiscript is a graphically-designed nucleic acid notation that uses symbol symmetries to support sequence complementation, highlight biologically-relevant palindromes, and facilitate the analysis of consensus sequences. Although the original Ambiscript notation was designed to easily represent consensus sequences for multiple sequence alignments, the notation's black-on-white ambiguity characters are unable to reflect the statistical distribution of nucleotides found at each position. We now propose a color-augmented ambigraphic notation to encode the frequency of positional polymorphisms in these consensus sequences.

Rapid countermeasure discovery against Francisella tularensis based on a metabolic network reconstruction.

In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention.

Disrupting the luxS quorum sensing gene does not significantly affect Bacillus anthracis virulence in mice or guinea pigs.

Many bacterial species use secreted quorum-sensing autoinducer molecules to regulate cell density- and growth phase-dependent gene expression, including virulence factor production, as sufficient environmental autoinducer concentrations are achieved. Bacillus anthracis, the causative agent of anthrax, contains a functional autoinducer (AI-2) system, which appears to regulate virulence gene expression. To determine if the AI-2 system is necessary for disease, we constructed a LuxS AI-2 synthase-deficient mutant in the virulent Ames strain of B.

Biolog phenotype microarrays.

Phenotype microarrays nicely complement traditional genomic, transcriptomic, and proteomic analysis by offering opportunities for researchers to ground microbial systems analysis and modeling in a broad yet quantitative assessment of the organism's physiological response to different metabolites and environments. Biolog phenotype assays achieve this by coupling tetrazolium dyes with minimally defined nutrients to measure the impact of hundreds of carbon, nitrogen, phosphorous, and sulfur sources on redox reactions that result from compound-induced effects on the electron transport chain. Over the years, we have used Biolog's reproducible and highly sensitive assays to distinguish closely related bacterial isolates, to understand their metabolic differences, and to model their metabolic behavior using flux balance analysis.

Pressure cycling technology in systems biology.

Systems biologists frequently seek to integrate complex data sets of diverse analytes into a comprehensive picture of an organism's biological state under defined environmental conditions. Although one would prefer to collect these data from the same sample, technical limitations with traditional sample preparation methods often commit the investigator to extracting one type of analyte at the expense of losing all others. Often, volume further constrains the range of experiments that can be collected from a single sample.

CpG oligodeoxyribonucleotides protect mice from Burkholderia pseudomallei but not Francisella tularensis Schu S4 aerosols.

Studies have shown that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis live vaccine strain (LVS), when administered before parenteral challenge. Given the potential to develop CpG ODN as a pre-treatment for multiple bacterial biological warfare agents, we examined survival, histopathology, and cytokine data from CpG ODN-treated C57BL/6 mice to determine whether previously-reported protection extended to aerosolized B. pseudomallei 1026b and highly virulent F.

A novel brain heart infusion broth supports the study of common Francisella tularensis serotypes.

Francisella tularensis Schu S4, LVS and U112 have become model organisms for the study of Francisella pathogenesis, and represent a cross section of the different F. tularensis subspecies. Both Schu S4 and LVS are fastidious organisms, requiring medium fortified with supplements and nutrients for enhanced growth.

Exogenous Yersinia pestis quorum sensing molecules N-octanoyl-homoserine lactone and N-(3-oxooctanoyl)-homoserine lactone regulate the LcrV virulence factor.

LcrV is a key Yersinia pestis antigen, immune regulator, and component of the type III secretion system (T3SS). Researchers have shown that N-acyl-homoserine lactones (AHLs) can down-regulate the expression of the LcrV homolog, PcrV, in Pseudomonas aeruginosa. Using ELISA, western blot, DNA microarray analysis, and real time PCR we demonstrate that the addition of AHL molecules N-octanoyl-homoserine lactone (C8) or N-(3-oxooctanoyl)-homoserine lactone (oxo-C8) to Y.

Simplicity, function, and legibility in an enhanced ambigraphic nucleic acid notation.

We previously showed that an ambigraphic nucleic acid notation, based on symmetrical lowercase Roman characters, permits users to complement DNA by physically rotating the sequence text 180 degrees . This article describes an enhanced ambigraphic notation, which uses concept-related symbol design, rather than the arbitrary set of symbols that constitute the Roman alphabet, to logically encode the four DNA bases and 11 ambiguity characters. As ambigrams, the symbols continue to permit the rapid derivation of complementary sequences and visualization of palindromic DNA.

An artificially evolved albumin binding module facilitates chemical shift epitope mapping of GA domain interactions with phylogenetically diverse albumins.

Protein G-related albumin-binding (GA) modules occur on the surface of numerous Gram-positive bacterial pathogens and their presence may promote bacterial growth and virulence in mammalian hosts. We recently used phage display selection to evolve a GA domain, PSD-1 (phage selected domain-1), which tightly bound phylogenetically diverse albumins. With respect to PSD-1's broad albumin binding specificity, it remained unclear how the evolved binding epitope compared to those of naturally occurring GA domains and whether PSD-1's binding mode was the same for different albumins.

Structure, dynamics, and stability variation in bacterial albumin binding modules: implications for species specificity.

Protein G-related albumin-binding (GA) modules are frequently expressed on the surfaces of bacterial cells. The limited amino acid sequence variation among GA modules results in structural and functional differences with possible implications for bacterial pathogenesis and host specificity. In particular, the streptococcal G148-GA3 and F.

The practical and pedagogical advantages of an ambigraphic nucleic acid notation.

The universally applied IUPAC notation for nucleic acids was adopted primarily to facilitate the mental association of G, A, T, C, and the related ambiguity characters with the bases they represent. However it is possible to create a notation that offers greater support for the basic manipulations and analyses to which genetic sequences frequently are subjected. By designing a nucleic acid notation around ambigrams, it is possible to simplify the frequently applied process of reverse complementation and aid the visualization of palindromes.

Using offset recombinant polymerase chain reaction to identify functional determinants in a common family of bacterial albumin binding domains.

The 46 amino acid GA albumin binding module is a putative virulence factor that has been identified in 16 domains from four bacterial species. Aside from their possible effects on pathogenicity and host specificity, the natural genotypic and phenotypic variations that exist among members of this module offer unique opportunities for researchers to identify and explore functional determinants within the well-defined sequence space. We used a recently developed in vitro recombination technique, known as offset recombinant PCR, to shuffle seven homologues that encode a broad range of natural GA polymorphisms.

G148-GA3: a streptococcal virulence module with atypical thermodynamics of folding optimally binds human serum albumin at physiological temperatures.

The third albumin binding domain of streptococcal protein G strain 148 (G148-GA3) belongs to a novel class of prokaryotic albumin binding modules that is thought to support virulence in several bacterial species. Here, we characterize G148-GA3 folding and albumin binding by using differential scanning calorimetry and isothermal titration calorimetry to obtain the most complete set of thermodynamic state functions for any member of this medically significant module. When buffered at pH 7.

Directed evolution of highly homologous proteins with different folds by phage display: implications for the protein folding code.

To better understand how amino acid sequences specify unique tertiary folds, we have used random mutagenesis and phage display selection to evolve proteins with a high degree of sequence identity but different tertiary structures (homologous heteromorphs). The starting proteins in this evolutionary process were the IgG binding domains of streptococcal protein G (G(B)) and staphylococcal protein A (A(B)). These nonhomologous domains are similar in size and function but have different folds.

Offset recombinant PCR: a simple but effective method for shuffling compact heterologous domains.

DNA shuffling and other in vitro recombination strategies have proven highly effective at generating complex libraries for mutagenesis studies. While most recombination techniques employ DNA polymerases in part of a multi-step process, few seek to exploit the natural recombinogenic tendencies and exponential amplification rates of PCR. Here, we characterize a simple but effective method for using standard PCR to promote high recombination frequencies among compact heterologous domains by locating the domains near one end of the template.