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Optimization of methods for ruling out Bacillus anthracis leads to increased yields, faster turnaround times, and a lighter workload. We used 72 environmental non-B. anthracis bacilli to validate methods for [...]

PcrA is an essential helicase in gram-positive bacteria, and a gene encoding this helicase has been identified in all such organisms whose genomes have been sequenced so far. The precise role of PcrA that makes it essential for cell growth is not known; however, PcrA does not appear to be necessary for chromosome replication. The pcrA gene was identified in the genome of Bacillus anthracis on the basis of its sequence homology to the corresponding genes of Bacillus subtilis and Staphylococcus aureus, with which it shares 76 and 72% similarity, respectively. The pcrA gene of B. anthracis was isolated by PCR amplification and cloning into Escherichia coli. The PcrA protein was overexpressed with a Hi[s.sub.6] fusion at its amino-terminal end. The purified His-PcrA protein showed ATPase activity that was stimulated in the presence of single-stranded (ss) DNA (ssDNA). Interestingly, PcrA showed robust 3'[right arrow]5' as well as 5'[right arrow]3' helicase activities, with substrates containing a duplex region and a 3' or 5' ss poly(dT) tail. PcrA also efficiently unwound oligonucleotides containing a duplex region and a 5' or 3' ss tail with the potential to form a secondary structure. DNA binding experiments showed that PcrA bound much more efficiently to oligonucleotides containing a duplex region and a 5' or 3' ss tail with a potential to form a secondary structure than to those with ssDNAs or duplex DNAs with ss poly(dT) tails. Our results suggest that specialized DNA structures and/or sequences represent natural substrates of PcrA in biochemical processes that are essential for the growth and survival of gram-positive organisms, including B. anthracis.

Both the protective antigen (PA) and the poly([gamma]-D-glutamic acid) capsule ([gamma]DPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-[gamma]DPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic [gamma]DPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis [gamma]DPGA, Bacillus pumilus [gamma]DLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the D or L configuration with active groups at the N or C termini, were bound at 5-32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-[gamma]DPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-[gamma]DPGA were elicited by decamers of [gamma]DPGA at 10-20 mol per protein bound to the N- or C-terminal end. High IgG anti-[gamma]DPGA levels were elicited by two injections of 2.5 [micro]g of [gamma]DPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies, rPA was the most effective carrier. Anti-[gamma]DPGA induced opsonophagocytic killing of B. anthracis tox-, cap+. [gamma]DPGA conjugates may enhance the protection conferred by PA alone. [gamma]DPGA-rPA conjugates induced both anti-PA and anti-[gamma]DPGA.

After the intentional release of Bacillus anthracis through the U.S. Postal Service in the fall of 2001, many environments were contaminated with B. anthracis spores, and frequent inquiries were made [...]

The bacterium Bacillus anthracis causes the death of macrophages, which may allow it to avoid detection by the innate immune system. We found that B. anthracis lethal factor (LF) selectively induces apoptosis of activated macrophages by cleaving the amino-terminal extension of mitogen-activated protein kinase (MAPK) kinases (MKKs) that activate p38 MAPKs. Because macrophages that are deficient in transcription factor nuclear factor κB (NF-κB) are also sensitive to activation-induced death and p38 is required for expression of certain NF-κB target genes, p38 is probably essential for synergistic induction of those NF-κB target genes that prevent apoptosis of activated macrophages. This dismantling of the p38 MAPK module represents a strategy used by B. anthracis to paralyze host innate immunity., Bacillus anthracis, the causative agent of anthrax, has gained notoriety as a potential biowarfare and bioterrorism agent. During inhalation anthrax, the most lethal form of the disease, B. anthracis spores [...]

To the Editor: Forensic and epidemiologic investigation of the 2001 bioterrorism-associated anthrax attacks used multiple-locus variable-number tandem-repeat analysis (MLVA) to identify the attack strain as Ames (1). Strain identity was [...]

A study was conducted to determine the gamma irradiation dose for inactivating virulent Bacillus anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. The results suggested that the clinical and public health laboratories which test specimens for Bacillus anthracis could potentially integrate gamma irradiation into sample processing protocols without compromising the sensitivity of the B. anthtracis assays.

A pulsed field gel electrophoresis method is developed for discriminating Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Bacillus anthracis is distinguished from its closest relatives by using the PFGE profiles from NotI digestion.

Multiple-locus (MLVA) and other molecular typing methods were used to investigate Bacillus anthracis diversity in Georgia and to characterize vaccine strains used in former Soviet Union. The MLVA-based data establishes genetic diversity of Bacillus anthracis in Southern Caucasus.

A microbiological sampling strategy was undertaken to find heavy contamination within 642 separate plastic bags containing the mail delivered to the office of a United States senator, which was found to be intentionally filled with dried Bacillus anthracis spores. Results demonstrate that there was successful integration of microbiological methodology with law enforcement and hazmat objectives, and procedures and also highlight some of the basic deficiencies in bioterrorism response at the time described.

A study is presented on the effect of ionic strength and voltage on the anthrax toxin complex, which consists of three different molecules, protective antigen (PA), lethal factor (LF), and edema factor (EF). The results suggest that PA(sub 63) heptamers contain a high-affinity binding site for LF inside domain I or the channel vestibule, while the binding is ionic-strength-dependent.

Research describes the synthesis of peptide ligands specific to Bacillus anthracis spores and to spores from a phylogeneticlly similar variant of B. anthacis. The petide sequences are ATYPLPIR abd SLLPGLP, respectively. Data indicate that the peptides are highly specific without any false-positive binding.

The Bacillus collagen-like protein (BclA) possesses an internal highly polymorphic collagen-like region (CLR) in Bacillus anthracis. Data indicate that BclA is a structural component of the filaments that cover the outer surface of the exosporium. Furthermore, the variation in the length of these hair-like filaments is due to the variation in the size of CLR as shown by gene exchange between strains of B. anthracis.

Bacterial spore radiation inactivation analysis shows that under identical conditions of preparation and assaying, spores from Bacillus anthacis and two strains of B. subtilis exhibit essentially identical inactivation kinetics, suggesting that the UV dose effective for B. subtilis strains can be use as a biodosimetry model for B. anthracis.

The carbon nanotubes represent a unique displaying scaffold for multivalent monosaccharide ligands that bind effectively with Bacillus anthracis spores in the presence of a divalent cation. The binding results in substantial aggregation of the spores and corresponding CFC reduction.

----- Bacillus anthracis is an aerobic, Gram-positive, spore-forming, nonmotile Bacillus species. ----- Its spores germinate when they are in an environment rich in amino acids, nucleosides, and glucose, such as [...]

Bacillus anthracis is distinguished from its more benign relations among the bacilli by the possession of plasmids containing virulence genes. The virulence genes are transcribed in response to elevated environmental [...]