Probabilistic Limit of Detection for Ricin Identification Using a Shotgun Proteomics Assay.

Robust and highly specific methods for the detection of the protein toxin ricin are of interest to the law enforcement community. In previous studies, methods based on liquid chromatography-tandem mass spectrometry shotgun proteomics have been proposed. The successful implementation of this approach relies on specific data evaluation criteria addressing (1) the quality of the mass spectrometric data, (2) the confidence of peptide identifications (peptide-spectrum matches), and (3) the number and sequence specificity of peptides detected. We present such data evaluation criteria and use a novel approach to establish the limit of detection for this ricin assay. Specifically, we use logistic regression to determine the probability of detection for individual ricin peptides at different concentrations. We then apply basic rules from probability theory, combining these individual peptide probabilities into an overall assay limit of detection. This procedure yields an assay limit of detection for ricin at 42.5 ng on column or 21.25 ng/µL for a 2-µL injection. We also show that, despite the conventional wisdom that detergents are deleterious to mass spectrometric analyses, the presence of Tween-20 did not prevent detection of ricin peptides, and indeed assays performed in buffers that included Tween-20 gave better results than assays performed using other buffer formulations with or without detergent removal.

Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency in Bacillus anthracis Sterne.

Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, the in vitro contributions of petrobactin to sporulation as well as growth imply that petrobactin may be required for B. anthracis transmission via the spore during natural infections, in addition to its key known functions during active anthrax infections.IMPORTANCEBacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.

Genome Sequence of the Attenuated Carbosap Vaccine Strain of Bacillus anthracis.

The Bacillus anthracis Carbosap genome, which includes the pXO1 and pXO2 plasmids, has been shown to encode the major B. anthracis virulence factors, yet this strain's attenuation has not yet been explained. Here we report the draft genome sequence of this strain, and a comparison to fully virulent B. anthracis.