Theresa M. Koehler

The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria

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.

Early Bacillus anthracis–macrophage interactions: intracellular survival and escape

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.

Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax

Bacillus anthracis, the agent of anthrax, produces a poly‐D‐glutamic 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 LD50 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.

Sequence, assembly and analysis of pX01 and pX02.

Bacillus anthracis plasmids pX01 and pX02, harboured by the Sterne and Pasteur strains, respectively, have been sequenced by random ‘shotgun’ cloning and high throughout sequence analysis. These sequences have been assembled (Sequencher) to generate a circulate pX01 plasmid containing 181 656 bp and a single linear (gapped) pX02 contig containing at least 93·479 bp. Initial annotation suggests that the two plasmids combined contain at least 200 potential open reading frames (ORFs) with < 40% having significant similarity to sequences registered in open databases. Collectively, only 118 566 bp of the pX01 DNA (65%) represent predicted coding regions. This value is similar to published gene densities for other plasmids and is indicative of the larger intergenic spaces in plasmids vs those found in the chromosomes of the parental microbes (85–93% gene density). A 70 kbp region including the toxin genes (cya, lef and pag) is distinct from the remainder of the pX01 sequence: (1) it has a lower gene density (58 vs 70%) than the remaining 111 kbp; (2) it contains all but one of the co‐regulated transcriptional fusions identified by transposon mutagenesis ( Hoffmaster & Koehler 1997 ) and (3) it contains a significantly higher proportion of positive BLAST scores (62 vs 20%) for putative ORFs. These data suggest different origins for the two regions of pX01.

The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence

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 trans‐activator of anthrax toxin synthesis. Here we show that transcription of atx A 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 atx A‐null mutant in which the atx A‐coding sequence on pXO1 is replaced with an ωkm‐2 cassette. Transcription of all three toxin genes is decreased in the absence of atx A. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5′ ends mapping to P1 are decreased in the atx A‐null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the Pi site is sufficient for atx A‐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 afxA‐null mutant. The atx A‐null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atx A‐null mutant‐infected mice. These data suggest that the atx A gene product also regulates toxin gene expression during infection.

Control of Anthrax Toxin Gene Expression by the Transition State Regulator abrB

Bacillus anthracis produces the anthrax toxin proteins protective antigen (PA), lethal factor (LF), and edema factor (EF) in a growth phase-dependent manner when cultured in liquid medium. Expression of the toxin genes pagA, lef, and cya peaks in late log phase, and steady-state levels of the toxin proteins are highest during the transition into stationary phase. Here we show that an apparent transition state regulator negatively regulates toxin gene expression. We identified two orthologues of the B. subtilis transition state regulator abrB in the B. anthracis genome: one on the chromosome and one on the 182-kb virulence plasmid pXO1. The orthologue located on the chromosome is predicted to encode a 94-amino-acid protein that is 85% identical to B. subtilis AbrB. The hypothetical protein encoded on pXO1 is 41% identical to B. subtilis AbrB but missing 27 amino acid residues from the amino terminus compared to the B. subtilis protein. Deletion of the pXO1-encoded abrB orthologue did not affect toxin gene expression under the conditions tested. However, a B. anthracis mutant in which the chromosomal abrB gene was deleted expressed pagA earlier and at a higher level than the parent strain. Expression of a transcriptional pagA-lacZ fusion in the abrB mutant was increased up to 20-fold during early exponential growth compared to the parent strain and peaked in mid-exponential rather than late exponential phase. In contrast to the strong effect of abrB on pagA expression, lef-lacZ and cya-lacZ expression during early-log-phase growth was increased only two- to threefold in the abrB null mutant. Western hybridization analysis showed increased PA, LF, and EF synthesis by the mutant. As is true in B. subtilis, the B. anthracis abrB gene is negatively regulated by spo0A. Our findings tie anthrax toxin gene expression to the complex network of postexponential phase adaptive responses that have been well studied in B. subtilis.

Bacillus anthracis Genetics and Virulence Gene Regulation

The Bacillus anthracis genome consists of an approximately 5.3-Mb chromosome and two plasmids, pXO1 (182 kb) and pXO2 (96 kb). Genetic analysis has focused primarily on the structural genes for the anthrax toxin proteins, pagA, lef, and cya, the biosynthetic genes for capsule synthesis, capB, capC, and capA, and a gene associated with depolymerization of capsule, dep. The three toxin genes are located at distinct loci on pXO1, while the cap and dep genes are arranged in an apparent operon on pXO2. Additional genes that may play a role in B. anthracis virulence include the germination operon gerX and the general stress transcription factor sigB. Host-related signals affecting transcription of the toxin and capsule genes include temperature (37 degrees C) and bicarbonate/CO2. The B. anthracis plasmids carry two regulatory genes that share little sequence similarity with regulators in other bacteria. The pXO1-encoded gene atxA positively controls expression of the toxin and capsule genes, and has been implicated in control of other genes of unknown function. atxA mutants are avirulent in mice, and mice infected with atxA-null strains show a decreased immunological response to the toxin proteins. The pXO2-encoded regulator, acpA, shares sequence similarity with atxA. Yet acpA function appears to be restricted to positive control of capsule gene expression. The chromosomal gene abrB, a homologue of a well-characterized B. subtilis transition state regulator, controls growth phase-specific transcription of the toxin genes. Genetic manipulation of B. anthracis can be achieved by using natural means of DNA transfer and by electroporation of recombinant DNAs into B. anthracis. Genetic exchange can occur between B. anthracis strains and between B. anthracis and closely-related species. Although pXO1 and pXO2 are not self-transmissible, these plasmids and others can be transferred by conjugative plasmids originating in B. thuringiensis. Generalized transducing phage that permit inter-species transfer of chromosomal and plasmid DNA have also been described.

Global Effects of Virulence Gene Regulators in a Bacillus anthracis Strain with Both Virulence Plasmids

ABSTRACT Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxA and acpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1+ pXO2+ parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA-regulated genes were affected synergistically in an atxA acpA mutant. Yet overall, acpA appears to be a minor regulator with fewer targets than atxA. In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpA expression was positively affected by atxA, although atxA-activated capsule gene transcription is not acpA dependent. The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues. Given the global effect of atxA on gene expression in B. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated.

Bacillus anthracis multiplication, persistence, and genetic exchange in the rhizosphere of grass plants

ABSTRACT Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Coinoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species.

Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin

ABSTRACT We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B. anthracis in rich media in vitro. To further support the alo gene product being a hemolysin, recombinant B. anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at ∼30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.