Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.

The three separate proteins that make up anthrax toxin-protective antigen (PA), edema factor (EF), and lethal factor (LF)-act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF). PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation ofEF or LF into the cytosol. PA can be separated, after mild trypsinolysis, into two fragments, PA6s (65 kDa) and PA" (20 kDa). We demonstrate that trypsin-cleaved PA is capable of forming cationselective channels in planar phospholipid bilayer membranes and that this activity is confined to the PAO1 fragment; PA20, LF, and EF are devoid of channel-forming activity. These PA65 channels exhibit pH-dependent and voltage-dependent activity-a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins.The pathogenesis ofBacillus anthracis, the causative agent of anthrax, depends on two important virulence factors: an antiphagocytic poly(D-glutamic acid) capsule and "anthrax toxin." The latter term refers collectively to three proteins encoded by plasmid pXO1 (1): edema factor (EF; 89 kDa), lethal factor (LF; 83 kDa), and protective antigen (PA; 85 kDa), the last so named because of its use in vaccines to generate antibodies which protect against anthrax infection. These three toxin components have no known biological effect when administered individually to experimental animals, but they act in binary combinations to produce two distinct reactions. Intradermal coinjection of PA and EF (a combination termed "edema toxin") produces edema, while coinjection of PA and LF (a combination termed "lethal toxin") causes death in susceptible animals. EF has been shown to be a calmodulin-dependent adenylate cyclase (2), whereas the molecular action of LF remains unknown. EF must penetrate to the cytosolic compartment to contact calmodulin and substrate ATP, and it is generally assumed that LF also penetrates to the cytosol, where it is believed to inactivate an essential cellular component. Although the mechanism(s) by which EF and LF are translocated to the cytoplasm is poorly understood, PA clearly plays a central role. Leppla and coworkers (4, 5) have proposed a mechanism of toxicity in which (i) PA binds to a receptor on the cell surface; (ii) a small N-terminal piece of PA (PA20; 20 kDa) is removed proteolytically, leaving the larger C-terminal piece (PA65; 65 kDa) bound to the receptor; (iii) EF or LF binds to PA65; (iv) the receptor-PA-EF or receptor-PA-LF complex undergoes receptor-mediated endocytosis; and finally, (v) EF or LF (within an endocytic or derivative vesicle) undergoes membrane translocation and is released into the cytosol.Recent studies on LF (6) suggest that toxicity requires passage through an acidic vesicle, a step reminiscent of the pathway leading to intoxication by diphtheria, tetanus, and botulinum toxins. Indeed, the entire sequence of events is quite analogous...

The pag gene of Bacillus anthracis, located on plasmid pXO1 (185 kb), encodes protective antigen, a component of the anthrax lethal and edema toxins. Synthesis of protective antigen is enhanced during growth of the organism with elevated levels of CO2. The CO2 effect is at the level of transcription, and pXO1-encoded regulatory factors have been implicated in control of pag expression. We used a Tn917-LTV3 insertion mutant of B. anthracis in which the wild-type pag gene on pXO1 was replaced with a pag-lacZ transcriptional fusion to monitor pag promoter activity. Expression of the pag-lacZ fusion is induced five- to eightfold during growth in 5% CO2 compared with growth in air. Growth in 20% CO2 increases transcription up to 19-fold. By monitoring pag-lacZ expression in atmospheres with different O2 and CO2 concentrations, we demonstrated definitively that the CO2 effect is specific and not simply a result of increased anaerobiosis. The results of 5' end mapping of pag transcripts indicate multiple sites of transcript initiation. We have determined two major apparent start sites, designated P1 and P2, located at positions -58 and -26 relative to the translation initiation codon, respectively. Analysis of total RNA from late-log-phase cells shows comparable initiation from P1 and P2 in wild-type strains grown in aerobic conditions. However, initiation from P1 is increased approximately 10-fold in cultures grown with an elevated level (5%) of CO2. We have identified a locus on pXO1, more than 13 kb upstream from the pag gene, which enhances pag transcription. When added in trans, this locus increases the level of transcripts with 5' ends mapping to P1 but has no effect on the level of transcripts with 5' ends mapping to P2. The CO2 effect on P1 is observed only in the presence of the activator locus.

This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections. Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mφ). During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death. Experiments focus on the early events occurring during the first few hours of the B. anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes. We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mφ and replicate within the cytoplasm of these cells. Release from the Mφ occurs 4–6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals. Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mφ, whereas the capsule plasmid pXO2 is not. The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required. This suggests that Mφ release of anthrax bacilli is atxA regulated. The putative ‘escape’ genes may be located on the chromosome and/or on pXO1.

The “Bacillus cereus group” includes several Bacillus species with closely related phylogeny. The most well-studied members of the group, Bacillus anthracis, B. cereus, and B. thuringiensis are known for their pathogenic potential. Here we present the historical rationale for speciation and discuss shared and unique features of these bacteria. Aspects of cell morphology and physiology, and genome sequence similarity and gene synteny support close evolutionary relationships for these three species. For many strains, distinct differences in virulence factor synthesis provide facile means for species assignment. B. anthracis is the causative agent of anthrax. Some B. cereus strains are commonly recognized as food poisoning agents, but strains can also cause localized wound and eye infections as well as systemic disease. Certain B. thuringiensis strains are entomopathogens and have been commercialized for use as biopesticides, while some strains have been reported to cause infection in immunocompromised individuals. In this chapter we compare and contrast B. anthracis, B. cereus, and B. thuringiensis, including ecology, cell structure and development, virulence attributes, gene regulation and genetic exchange systems, and experimental models of disease.

Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen). Expression of the toxin genes by B. anthracis is enhanced during growth under elevated levels of CO2. This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a transactivator of anthrax toxin synthesis. Here we show that transcription of atxA does not appear to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new efficient method for gene replacement in B. anthracis, we constructed an atxA-null mutant in which the atxA-coding sequence on pXO1 is replaced with an omega km-2 cassette. Transcription of all three toxin genes is decreased in the absence of atxA. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5' ends mapping to P1 are decreased in the atxA-null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the P1 site is sufficient for atxA-mediated activation of this transcript. The cya and lef genes each have one apparent start site for transcription. Transcripts with 5' ends mapping to these sites are not detected in the atxA-null mutant. The atxA-null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atxA-null mutant-infected mice. These data suggest that the atxA gene product also regulates toxin gene expression during infection.

Virulent and certain avirulent strains of BaciUus anthracis harbor a plasmid, designated pXO2, which is involved in the synthesis of capsules. Two classes of rough, noncapsulated (Cap-) variants were isolated from the capsule-producing (Cap') Pasteur vaccine strains ATCC 6602 and ATCC 4229. One class was cured of pXO2, and the other class still carried it. Reversion to Cap' was demonstrable only in rough variants which had retained pXO2. Proof that pXO2 is involved in capsule synthesis came from experiments in which the plasmid was transferred by CP-51-mediated transduction and by a mating system in which plasmid transfer is mediated by a BaciUus thuringiensis fertility plasmid, pX012. Cells of Bacillus cereus and a previously noncapsulated (pXO2-) strain of B. anthracis produced capsules after the acquisition of pXO2. R. A. Packer Colorado Virulent, Cap' Tox+ pXO1, pXO2 A. McChesney NH Virulent, Cap' Tox+ pXO1, pXO2 USAMRII)d Vollum 1B Virulent, Cap' Tox+ pXO1, pXO2 USAMRIID Vollum lB-1 Avirulent, Cap+ Tox-pXO2 This study Vollum 1B VNR-1 Avirulent, Cap-Tox+ pXO0 This study Weybridge (Sterne) Avirulent, Cap-Tox+ pXOl MREe Weybridge UM44 Ind-pXOl UVf of Weybridge Weybridge A Colony variant of Weybridge pXOl C. B. Thorne Weybridge A UM23C1 Ura-, cured of pXO1 None C. B. Thorne Weybridge A UM23C1 tdlO Ura-Cap' pXO2 This study 4229 (Pasteur)

Bacillus anthracis, the agent of anthrax, produces a poly-Dglutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD 50 and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.