Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.     

Comparison of Minisatellite Polymorphisms in the Bacillus cereus Complex: a Simple Assay for Large-Scale Screening and Identification of Strains Most Closely Related to Bacillus anthracis

Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.     

Bacillus anthracis physiology and genetics

Bacillus anthracis is a member of the Bacillus cereus group species (also known as the “group 1 bacilli”), a collection of Gram-positive spore-forming soil bacteria that are non-fastidious facultative anaerobes with very similar growth characteristics and natural genetic exchange systems. Despite their close physiology and genetics, the B. cereus group species exhibit certain species-specific phenotypes, some of which are related to pathogenicity. B. anthracis is the etiologic agent of anthrax. Vegetative cells of B. anthracis produce anthrax toxin proteins and a poly-d-glutamic acid capsule during infection of mammalian hosts and when cultured in conditions considered to mimic the host environment. The genes associated with toxin and capsule synthesis are located on the B. anthracis plasmids, pXO1 and pXO2, respectively. Although plasmid content is considered a defining feature of the species, pXO1- and pXO2-like plasmids have been identified in strains that more closely resemble other members of the B. cereus group. The developmental nature of B. anthracis and its pathogenic (mammalian host) and environmental (soil) lifestyles of make it an interesting model for study of niche-specific bacterial gene expression and physiology.     

φ20, A Temperate Bacteriophage Isolated from Bacillus anthracis Exists as a Plasmidial Prophage

This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage φ20 isolated from B. anthracis Sterne 34F₂ (pXO1⁺ pXO2⁻) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The φ20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.     

Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis

The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.     

Novel Giant Siphovirus from Bacillus anthracis Features Unusual Genome Characteristics

Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.     

不同品系小鼠对炭疽芽胞杆菌弱毒株芽胞的敏感性研究

通过比较四种品系小鼠对炭疽芽胞杆菌（Bacillus anthracis）（简称炭疽杆菌）弱毒株芽胞的敏感性，确定炭疽杆菌弱毒株芽胞攻毒合适的动物模型。采用炭疽杆菌弱毒株A16Q1（pXO1-、pXO2+）和A16PI2（pXO1+、pXO2-）的芽胞对四种品系小鼠（DBA/2、KM、ICR和BALB/c）进行腹腔攻毒，记录小鼠死亡时间，计算LD₅₀、绘制存活曲线并统计分析。运用较敏感的KM小鼠研究不同canSNP基因型毒素缺陷株（含pXO2拷贝数不同）芽胞的毒力差异。利用更为敏感的DBA/2小鼠评价S-层蛋白BA3338对荚膜缺陷株芽胞毒力的影响。结果表明，在四种品系小鼠中，毒素缺陷株芽胞的毒力均高于荚膜缺陷株芽胞的毒力。DBA/2小鼠对炭疽杆菌弱毒株芽胞的剂量依赖关系最好，最为敏感，其次是KM小鼠，而ICR小鼠和BALB/c小鼠对炭疽杆菌弱毒株芽胞不敏感。确定了DBA/2小鼠和KM小鼠在炭疽杆菌弱毒株芽胞研究中的适用性。使用KM小鼠评价了不同canSNP基因型炭疽杆菌芽胞的毒力差异，结果表明，不同canSNP基因型炭疽杆菌由于所含pXO2质粒拷贝数的差异导致芽胞的毒力不同。使用DBA/2小鼠评价了S-层蛋白BA3338缺失对炭疽杆菌芽胞毒力的影响，表明BA3338基因的缺失导致炭疽杆菌芽胞毒力降低。     

Cell wall carbohydrate compositions of strains from the Bacillus cereus group of species correlate with phylogenetic relatedness

Members of the Bacillus cereus group contain cell wall carbohydrates that vary in their glycosyl compositions. Recent multilocus sequence typing (MLST) refined the relatedness of B. cereus group members by separating them into clades and lineages. Based on MLST, we selected several B. anthracis, B. cereus, and B. thuringiensis strains and compared their cell wall carbohydrates. The cell walls of different B. anthracis strains (clade 1/Anthracis) were composed of glucose (Glc), galactose (Gal), N-acetyl mannosamine (ManNAc), and N-acetylglucosamine (GlcNAc). In contrast, the cell walls from clade 2 strains (B. cereus type strain ATCC 14579 and B. thuringiensis strains) lacked Gal and contained N-acetylgalactosamine (GalNAc). The B. cereus clade 1 strains had cell walls that were similar in composition to B. anthracis in that they all contained Gal. However, the cell walls from some clade 1 strains also contained GalNAc, which was not present in B. anthracis cell walls. Three recently identified clade 1 strains of B. cereus that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241, had cell wall compositions that closely resembled those of the B. anthracis strains. It was also observed that B. anthracis strains cell wall glycosyl compositions differed from one another in a plasmid-dependent manner. When plasmid pXO2 was absent, the ManNAc/Gal ratio decreased, while the Glc/Gal ratio increased. Also, deletion of atxA, a global regulatory gene, from a pXO2⁻ strain resulted in cell walls with an even greater level of Glc.     

The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”.     

The genome and variation of Bacillus anthracis

The Bacillus anthracis genome reflects its close genetic ties to Bacillus cereus and Bacillus thuringiensis but has been shaped by its own unique biology and evolutionary forces. The genome is comprised of a chromosome and two large virulence plasmids, pXO1 and pXO2. The chromosome is mostly co-linear among B. anthracis strains and even with the closest near neighbor strains. An exception to this pattern has been observed in a large inversion in an attenuated strain suggesting that chromosome co-linearity is important to the natural biology of this pathogen. In general, there are few polymorphic nucleotides among B. anthracis strains reflecting the short evolutionary time since its derivation from a B. cereus-like ancestor. The exceptions to this lack of diversity are the variable number tandem repeat (VNTR) loci that exist in genic and non genic regions of the chromosome and both plasmids. Their variation is associated with high mutability that is driven by rapid insertion and deletion of the repeats within an array. A notable example is found in the vrrC locus which is homologous to known DNA translocase genes from other bacteria.     

Identification of Three Noncontiguous Regions on Bacillus anthracis Plasmid pXO1 That Are Important for Its Maintenance

Bacillus anthracis pXO1 minireplicon (MR) plasmid consisting of open reading frames (ORFs) GBAA_pXO1_0020 to GBAA_pXO1_0023 is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (having 181,677 bp and 217 ORFs) is extremely stable under the same growth conditions. Two genetic tools developed for DNA manipulation in B. anthracis (Cre-loxP and Flp-FRT systems) were used to identify pXO1 regions important for plasmid stability. We localized a large segment of pXO1 that enables stable plasmid maintenance during vegetative growth. Further genetic analysis identified three genes that are necessary for pXO1 maintenance: amsP (GBAA_pXO1_0069), minP (GBAA_pXO1_0082), and sojP (GBAA_pXO1_0084). Analysis of conserved domains in the corresponding proteins indicated that only AmsP (activator of maintenance system of pXO1) is predicted to bind DNA, due to its strong helix-turn-helix domain. Two conserved domains were found in the MinP protein (Min protein from pXO1): an N-terminal domain having some similarity to the B. anthracis septum site-determining protein MinD and a C-terminal domain that resembles a baculovirus single-stranded-DNA-binding protein. The SojP protein (Soj from pXO1) contains putative Walker box motifs and belongs to the ParA family of ATPases. No sequences encoding other components of type I plasmid partition systems, namely, cis-acting centromere parS and its binding ParB protein, were identified within the pXO1 genome. A model describing the role of the MinP protein in pXO1 distribution between daughter cells is proposed.     

Identification of a Ligand on the Wip1 Bacteriophage Highly Specific for a Receptor on Bacillus anthracis

Tectiviridae is a family of tailless bacteriophages with Gram-negative and Gram-positive hosts. The family model PRD1 and its close relatives all infect a broad range of enterobacteria by recognizing a plasmid-encoded conjugal transfer complex as a receptor. In contrast, tectiviruses with Gram-positive hosts are highly specific to only a few hosts within the same bacterial species. The cellular determinants that account for the observed specificity remain unknown. Here we present the genome sequence of Wip1, a tectivirus that infects the pathogen Bacillus anthracis. The Wip1 genome is related to other tectiviruses with Gram-positive hosts, notably, AP50, but displays some interesting differences in its genome organization. We identified Wip1 candidate genes for the viral spike complex, the structure located at the capsid vertices and involved in host receptor binding. Phage adsorption and inhibition tests were combined with immunofluorescence microscopy to show that the Wip1 gene product p23 is a receptor binding protein. His-p23 also formed a stable complex with p24, a Wip1 protein of unknown function, suggesting that the latter is involved with p23 in host cell recognition. The narrow host range of phage Wip1 and the identification of p23 as a receptor binding protein offer a new range of suitable tools for the rapid identification of B. anthracis.     

Investigating the genome diversity of B. cereus and evolutionary aspects of B. anthracis emergence

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax—a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a “species” DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.     

The Bacillus anthracis Protein MprF Is Required for Synthesis of Lysylphosphatidylglycerols and for Resistance to Cationic Antimicrobial Peptides

During inhalational anthrax, Bacillus anthracis survives and replicates in alveolar macrophages, followed by rapid invasion into the host's bloodstream, where it multiplies to cause heavy bacteremia. B. anthracis must therefore defend itself from host immune functions encountered during both the intracellular and the extracellular stages of anthrax infection. In both of these niches, cationic antimicrobial peptides are an essential component of the host's innate immune response that targets B. anthracis. However, the genetic determinants of B. anthracis contributing to resistance to these peptides are largely unknown. Here we generated Tn917 transposon mutants in the ΔANR strain (pXO1⁻ pXO2⁻) of B. anthracis and screened them for altered protamine susceptibility. A protamine-sensitive mutant identified carried the transposon inserted in the BA1486 gene encoding a putative membrane protein homologous to MprF known in several gram-positive pathogens. A mutant strain with the BAS1375 gene (the orthologue of BA1486) deleted in the Sterne 34F2 strain (pXO1⁺ pXO2⁻) of B. anthracis exhibited hypersusceptibility not only to protamine but also to α-helical cathelicidin LL-37 and β-sheet defensin human neutrophil peptide 1 compared to the wild-type Sterne strain. Analysis of membrane lipids using isotopic labeling demonstrated that the BAS1375 deletion mutant is unable to synthesize lysinylated phosphatidylglycerols, and this defect is rescued by genetic complementation. Further, we determined the structures of these lysylphosphatidylglycerols by using various mass spectrometric analyses. These results demonstrate that in B. anthracis a functional MprF is required for the biosynthesis of lysylphosphatidylglycerols, which is critical for resistance to cationic antimicrobial peptides.     

Toxin-Independent Virulence of Bacillus anthracis in Rabbits

The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation.     

Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A,spo0B,and spo0F in Phage AP50c Infection of Bacillus anthracis

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.     

Virulence plasmid stability in environmentally occurring <Emphasis Type="Italic">Bacillus anthracis</Emphasis> from North East Turkey

The Bacillus anthracis virulence plasmid pXO2, which encodes for a polypeptide capsule, can be lost during long term laboratory storage. To determine if pXO2 is lost in nature we screened B. anthracis isolates obtained from B. anthracis spores from contaminated animal burial sites in Turkey for their ability to express a capsule upon primary culture. A total of 672 B. anthracis colonies were examined of which ten produced a mixed mucoid (capsule +ve)/non-mucoid (capsule ?ve) phenotype and a further one colony yielded non-mucoid colonies upon repeated culture. Screening by PCR using pXO2 specific primers revealed that seven of these isolates had eliminated the plasmid. Of the four colonies which were positive by PCR, one regained the ability to express a capsule upon repeated culture suggesting that the defect was reversible. This is an important observation as capsule expression is a principal marker of virulence and in the absence of PCR serves as a key diagnostic marker. The results of this preliminary study suggest that pXO2 is lost in nature and that further studies are need to determine the mechanisms by which this occurs.     

A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.