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Recent observations have shed light on some of the endogenous iron-acquisition mechanisms of members of the Bacillus cereus sensu lato group. In particular, pathogens in the B. cereus group use siderophores with both unique chemical structures and biological roles. This review will focus on recent discoveries in siderophore biosynthesis and biology in this group, which contains numerous human pathogens, most notably the causative agent of anthrax, Bacillus anthracis. DOI 10.1099/mic.0.039404-0

Capsule depolymerase (CapD) is a [gamma]-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence. DOI 10.1099/mic.0.035857-0

PagR is a transcriptional repressor in Bacillus anthracis that controls the chromosomal S-layer genes eag and sap, and downregulates the protective antigen pagA gene by direct binding to their promoter regions. The PagR protein sequence is similar to those of members of the ArsR repressor family involved in the repression of arsenate-resistance genes in numerous bacteria. The crystal structure of PagR was solved using multi-wavelength anomalous diffraction (MAD) techniques and was refined with 1.8 [Angstrom] resolution diffraction data. The PagR molecules form dimers, as observed in all SmtB/ArsR repressor family proteins. In the crystal lattice four PagR dimers pack together to form an inactive octamer. Model-building studies suggest that the dimer binds to a DNA duplex with a bend of around 40[degrees]. DOI 10.1099/mic.0.033548-0

Modern taxonomy, diagnostics, and forensics of bacteria benefit from technologies that provide data for genome-based classification and identification of strains; however, full genome sequencing is still costly, lengthy, and labor intensive. Therefore, other methods are needed to estimate genomic relatedness among strains in an economical and timely manner. Although DNA--DNA hybridization and techniques based on genome fingerprinting or sequencing selected genes like 16S rDNA, gyrB, or rpoB are frequently used as phylogenetic markers, analyses of complete genome sequences showed that global measures of genome relatedness, such as the average genome conservation of shared genes, can provide better strain resolution and give phylogenies congruent with relatedness revealed by traditional phylogenetic markers. Bacterial genomes are characterized by a high gene density; therefore, we investigated the integration of mass spectrometry-based proteomic techniques with statistical methods for phylogenomic classification of bacterial strains. For this purpose, we used a set of well characterized Bacillus cereus group strains isolated from poisoned food to describe a method that relies on liquid chromatography-electrospray ionization-tandem mass spectrometry of tryptic peptides derived from whole cell digests. Peptides were identified and matched to a prototype database (DB) of reference bacteria with fully sequenced genomes to obtain their phylogenetic profiles. These profiles were processed for predicting genomic similarities with DB bacteria estimated by fractions of shared peptides (FSPs). FSPs served as descriptors for each food isolate and were jointly analyzed using hierarchical cluster analysis methods for revealing relatedness among investigated strains. The results showed that phylogenomic classification of tested food isolates was in consonance with results from established genomic methods, thus validating our findings. In conclusion, the proposed approach could be used as an alternative method for predicting relatedness among microbial genomes of B. cereus group members and potentially may circumvent the need for whole genome sequencing for phylogenomic typing of strains. 10.1021/ac9015648

All Bacillus spores are encased in macromolecular shells. One of these is a proteinacious shell called the coat that, in Bacillus subtilis, provides critical protective functions. The Bacillus anthracis spore is the infectious particle for the disease anthrax. Therefore, the coat is of particular interest because it may provide essential protective functions required for the appearance of anthrax. Here, we analyse a protein component of the spore outer layers that was previously designated BxpA. Our data indicate that a significant amount of BxpA is located below the spore coat and associated with the cortex. By SDS-PAGE, BxpA migrates as a 9 kDa species when extracted from Sterne strain spores, and as 11 and 14 kDa species from Ames strain spores, even though it has predicted masses of 27 and 29 kDa, respectively, in these two strains. We investigated the possibility that BxpA is subject to post-translational processing as previously suggested. In B. subtilis, a subset of coat proteins is proteolysed or cross-linked by the spore proteins YabG or Tgl, respectively. To investigate the possibility that similar processing occurs in B. anthracis, we generated mutations in the yabG or tgl genes in the Sterne and Ames strains and analysed the consequences for BxpA assembly by SDS-PAGE. We found that in a tgl mutant of B. anthracis, the apparent mass of BxpA increased. This is consistent with the possibility that Tgl directs the cross-linking of BxpA into a form that normally does not enter the gel. Unexpectedly, the apparent mass of BxpA also increased in a yabG mutant, suggesting a relatively complex role for proteolysis in spore protein maturation in B. anthracis. These data reveal a previously unobserved event in spore protein maturation in B. anthracis. We speculate that proteolysis and cross-linking are ubiquitous spore assembly mechanisms throughout the genus Bacillus. DOI 10.1099/mic.0.033407-0

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation. doi: 10.1128/JB.01050-09

The susceptibility of most Bacillus anthracis strains to [beta]-lactam antibiotics is intriguing considering that the closely related species Bacillus cereus and Bacillus thuringiensis typically produce [beta]-lactamases and the B. anthracis genome harbors two [beta]-lactamase genes, bla1 and bla2. We show that [beta]-lactamase activity associated with B. anthracis is affected by two genes, sigP (BA2502) and rsiP (BA2503), predicted to encode an extracyto-plasmic function sigma factor and an anti-sigma factor, respectively. Deletion of the sigP-rsiP locus abolished [beta]-lactamase activity in a naturally occurring penicillin-resistant strain and had no effect on [beta]-lactamase activity in a prototypical penicillin-susceptible strain. Complementation with sigP and rsiP from the penicillin-resistant strain, but not with sigP and rsiP from the penicillin-susceptible strain, conferred constitutive [beta]-lactamase activity in both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsiP in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsiP homologues are required for inducible penicillin resistance in these species. Expression of the B. cereus or B. thuringiensis sigP and rsiP genes in a B. anthracis sigP-rsiP-null mutant confers inducible production of [beta]-lactamase activity, suggesting that while B. anthracis contains the genes necessary for sensing [beta]-lactam antibiotics, the B. anthracis sigP and rsiP gene products are not sufficient for bla induction. doi: 10.1128/JB.00691-09

Industrial anthrax, also known as woolsorter's disease, was a serious threat in the 19th and early 20th centuries when the wool industry was flourishing. The causal agent, Bacillus anthracis, was [...]

The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJl or SleB. Compared to the wild type, germinating spores without CwlJl suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CWlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and lunH (similar to an inosine--uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB--GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB--GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB--GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. lunH--GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB--GFP and lunH--GFP resides in the region between the coat and the exosporium, called the interspace.

Proteins of the WXG100 family represent the protolypieal substrates of bacterial type VII secretion systems that typically encompass 100 residues, lack canonical signal peptides, and form helix-turn-helix hairpin structures with WXG positioned in the turn element. Bacillus anthracis encodes six WXG100 proteins, herein referred to as EsxB, EsxL, EsxP, EsxQ, EsxV, and EsxW. With the exception of EsxB, B. anthracis proteins harbor C-terminal extensions that are appended to canonical WXG domains. When cultured in liquid broth, B. anthracis secretes two substrates, EsxB and EsxW, into the extracellular environment. EsxB is required for the stability and secretion of EsxW; however, EsxW is dispensable for EsxB secretion. In agreement with the hypothesis that EsxB binding to substrates promotes recognition and secretion by the type VII pathway, EsxB is reported to interact with EsxB and EsxW. Unlike deletions in mycobacterial EsxB, deletion of five N- or C-terminal residues does not affect the ability of mutant B. anthracis EsxB to travel the type VII pathway and initiate secretion of EsxW. Translational fusion of ubiquitin to the N or C terminus of EsxB also had no effect, while ubiquitin insertion into the center turn abrogated secretion. Anthrax-infected guinea pigs mounted humoral immune responses to EsxB, EsxP, and EsxW, which suggests that B. anthracis activates the type VII secretion pathway during infection.

The AtxA virulence regulator of Bacillus anthracis is required for toxin and capsule gene expression. AtxA is a phosphotransferase system regulatory domain-containing protein whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. Here we report that transcription of the atxA gene occurs from two independent promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M. Mock, and T. M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose transcription start sites are separated by 650 bp. Both promoters have -10 and -35 consensus sequences compatible with recognition by [[sigma].sup.A]. containing RNA polymerase, and neither promoter depends on the sporulation sigma factor SigH. The dual promoter activity and the extended untranslated mRNA suggest that as-yet-unknown regulatory mechanisms may act on this region to influence the level of AtxA in the cell.

In Bacillus anthracis, the novel type III pantothenate kinase ([PanK.sub.Ba]; encoded by coaX) catalyzes the first committed step in coenzyme A biosynthesis. We have demonstrated by analyzing the growth characteristics of a conditional coaX mutant that [PanK.sub.Ba] is an essential enzyme, thus contributing to its validation as a new antimicrobial target.

The current model for virulence gene regulation in Bacillus anthracis involves several trans-acting factors, the most important of which appears to be the anthrax toxin activator encoded by the atxA gene. AtxA is a positive regulator of the toxin genes pagA, cya and lef, and of a number of other plasmid- and chromosome-encoded genes. The AtxA protein (56 kDa) possesses a predicted winged-helix DNA-binding domain and phosphotransferase system-regulated domains, but the mechanism for positive regulation of AtxA target genes is not known. Sequence similarities in the promoter regions of AtxA-regulated genes are not apparent, and recombinant AtxA binds DNA with a high affinity in a non-specific manner. We hypothesized that the toxin genes possess common structural features or cis-acting elements that are required for positive regulation. We employed deletion analyses to determine the minimal sequences required for atxA-mediated toxin gene expression. In silico modelling and in vitro experiments using double-stranded DNA corresponding to the toxin gene promoter regions indicated significant curvature associated with these regions. These findings suggest that the structural topology of the DNA plays an important role in the control of anthrax toxin gene expression.

Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that found in other species. During germination, B. anthracis released peptidoglycan fragments into the surrounding medium more quickly than some other species. A major lytic enzymatic activity was a glucosaminidase, probably YaaH, that cleaved between N- acetylglucosamine and muramic-[delta]-lactam. An epimerase activity previously proposed to function on spore peptidoglycan was not detected, and it is proposed that glucosaminidase products were previously misidentified as epimerase products. Spore cortex lyric enzymes and their regulators are attractive targets for development of germination inhibitors to kill spores and for development of activators to cause loss of resistance properties for decontamination of spore release sites.

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 [...]

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time is investigated. Peroxygen biocides prove to be useful for Bacillus anthracis spore inactivation when food residue is present.

The essential Bacillus anthracis nrdE gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the nrdE pre-mRNA is spliced and that the homing endonuclease cleaves an intronless nrdE gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3' extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly, nrdE genes from several other Bacillaceae were also susceptible to cleavage, with those of Bacillus cereus, Staphylococcus epidermidis (nrdE1), B. anthracis, and Bacillus thuringiensis serovar konkukian being better substrates than those of Bacillus subtilis, Bacillus lichenformis, and S. epidermidis (nrdE2). On the other hand, nrdE genes from Lactococcus lactis, Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium ammoniagenes were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase nrdE gene is a frequent target for self-splicing IVSs. A comparison of nrdE genes from seven gram-positive low-G+C bacteria, two bacteriophages, and Nocardia farcinica showed five different insertion sites for self-splicing 1VSs within the coding region of the nrdE gene.

Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because L-glutamate stereoisomerization to D-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of L-glutamate and D-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of L-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features.

The study demonstrates the importance of the two-component system Bacillus anthracis respiratory response A and B (BrrA-BrrB). The system is found to act as an extremely efficient virulence factor regulator of B. anthracis toxin genes without affecting the aerobic growth.

A report is presented on cloning, functional expression and characterization of three new N-acetyltransferases (NATs) genes from the pathogen Bacillus anthracis. The three NAT homologues exhibit distinct structural and enzymatic properties.

Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.

The biochemical characterization of AsbC locus in Bacillus anthracis, the first reported 3,4-dihydroxybenzoic acid-AMP-ligase and a key component in the biosynthesis of DHB-spermidine (DHB-SP), the first isolable intermediate in petrobactin biosynthesis, are described. AsbC and its homologues are unique compared to other adenylation domains at key binding domain residues, thereby defining a new A-domain family.

A transposon site hybridization (TraSH) assay was developed for functional analysis of the Bacillus anthracis genome using a mini-Tnl0 transposon which permitted analysis of 82% of this pathogen's genes. The system, used to identify genes required for generation of infectious anthrax spores, spore germination, and optimal growth on rich medium, was predictive of the contributions of two conserved hypothetical genes for the phenotypes examined.

The initiation of sporulation in Bacillus species is controlled by the phosphorelay signal transduction system. Multiple regulatory elements act on the phosphorelay to modulate the level of protein phosphorylation in response to cellular, environmental, and metabolic signals. In Bacillus anthracis nine possible histidine sensor kinases can positively activate the system, while two response regulator aspartyl phosphate phosphatases of the Rap family negatively impact the pathway by dephosphorylating the SpoOF intermediate response regulator. In this study, we have characterized the B. anthracis members of the Spo0E family of phosphatases that specifically dephosphorylate the Spo0A response regulator of the phosphorelay and master regulator of sporulation. The products of four genes were able to promote the dephosphorylation of Spo0A~P in vitro. The overexpression of two of these B. anthracis Spo0E-like proteins from a multicopy vector consistently resulted in a sporulation.deficient phenotype. A third gene was found to be not transcribed in vivo. A fourth gene encoded a prematurely truncated protein due to a base pair deletion that nevertheless was subject to translational frameshift repair in an Escherichia coli protein expression system. A fifth Spo0E-like protein has been structurally and functionally characterized as a phosphatase of Spo0A~P by R. N. Grenha et al. (J. Biol. Chem. 281:37993-38003, 2006). We propose that these proteins may contribute to maintain B. anthracis in the transition phase of growth during an active infection and therefore contribute to the virulence of this organism.

An extended bioinformatic analysis has described the genome-based link for the absence of GHS biosynthesis, the presence of the type III pantothenate kinase (PanK) and the presence of the coenzyme A-disulfide reductase (CoADR)-like enzymes in a number of bacteria and spirochetes. The results have indicated that the type III PanK in the spore-forming Bacillus anthracis plays a vital role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.

We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for routS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rig phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N.methyl-N'-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C [right arrow] T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rift phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific.

The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the ash operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus (asbABCDEF) were constructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis when strains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew to a very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis ash mutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenous petrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl citrate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxybenzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This work sets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomal peptide synthetase-independent biosynthetic system.

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals, such as elevated C[O.sub.2], and the trans-acting positive regulator AtxA. In addition to these requirements, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. During the late exponential phase of growth, when AbrB levels begin to decrease, toxin synthesis increases. Here we report that toxin gene expression also requires the presence of sigH, a gene encoding the RNA polymerase sigma factor associated with development in Bacillus subtilis. In the well-studied B. subtilis system, [[sigma].sup.H] is required for sporulation and other post-exponential-phase processes and is part of a feedback control pathway for abrB expression. Our data indicate that a Bacillus anthracis sigH-null mutant is asporogenous and toxin deficient. Yet the sigma factor is required for toxin gene expression in a manner that is independent of the pathway leading to post-exponential-phase gene expression. [[sigma].sup.H] positively controls atxA in an AbrB-independent manner. These findings, combined with previous observations, suggest that the steady-state level of atxA expression is critical for optimal toxin gene transcription. We propose a model whereby, under toxin-inducing growth conditions, control of toxin gene expression is fine-tuned by the independent effects of [[sigma].sup.H] and AbrB on the expression of atxA.

A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 [mm.sup.2]. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 [+ or -] 31 (n = 11), 2742 [+ or -] 38 (n = 3), 3053 [+ or -] 19 (n = 2), 2777 [+ or -] 26 (n = 2), 2953 [+ or -] 24 (n = 2), and 3105 [+ or -] 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 [min.sup.-1]. A hindrance factor (6) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x [10.sup.5] BT + BC spores/mL, suggesting that [alpha] is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ~272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ~270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXOl-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Piasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

Systemic anthrax, caused by inhalation or ingestion of Bacillus anthracis spores, is characterized by rapid microbial growth stages that require iron. Tightly bound and highly regulated in a mammalian host, iron is scarce during an infection. To scavenge iron from its environment, B. anthracis synthesizes by independent pathways two small molecules, the siderophores bacillibactin (BB) and petrobactin (PB). Despite the great efficiency of BB at chelating iron, PB may be the only siderophore necessary to ensure full virulence of the pathogen. In the present work, we show that BB is specifically bound by siderocalin, a recently discovered innate immune protein that is part of an antibacterial iron-depletion defense. In contrast, neither PB nor its ferric complex is bound by siderocalin. Although BB incorporates the common 2,3-dihydroxybenzoyl iron-chelating subunit, PB is novel in that it incorporates the very unusual 3,4-dihydroxybenzoyl chelating subunit. This structural variation results in a large change in the shape of both the iron complex and the free siderophore that precludes siderocalin binding, a stealthy evasion of the immune system. Our results indicate that the blockade of bacterial siderophore-mediated iron acquisition by siderocalin is not restricted to enteric pathogenic organisms and may be a general defense mechanism against several different bacterial species. Significantly, to evade this innate immune response, B. anthracis produces PB, which plays a key role in virulence of the organism. This analysis argues for antianthrax strategies targeting siderophore synthesis and uptake. bacillibactin | Bacillus anthracis | petrobactin | siderocalin

The effectiveness of a commercial autoclave for treating simulated building decontamination residue (BDR) is evaluated. The results have shown that the packing density and material type of the BDR in the autoclave have a major impact on the effectiveness of the decontamination process.

The Bacillus anthracis chromosome encodes four unique, putative superoxide dismutase (sod) genes. During exponential growth and sporulation, sodA1, sodA2, and sodC are transcribed constitutively throughout the growth cycle as individual genes. In contrast, the transcription of sod15 occurs mainly during late exponential and sporulation phases as part of a four-gene operon that may be involved in spore formation. Vegetative cell and spore lysates of wild-type Sterne and superoxide dismutase deletion ([DELTA]sod) mutants show detectable SOD activity for SODA1 and SODA2, and protein analysis suggests that these two proteins form active homodimers and heterodimers. A comparison of the growth of parental versus [DELTA]sod mutants under various chemical oxidative stresses indicates that [DELTA]sodA1 mutants are particularly sensitive to endogenously produced superoxide, whereas [DELTA]sodA2, [DELTA]sod15, and [DELTA]sodC mutants remain as resistant to this stress as the parental strain. In addition, in mouse survival assays, [DELTA]sod15 and [DELTA]sodA1 were responsible for less end-point death, but the level of decreased virulence does not fall within a statistically significant range. Collectively, these data show that sodA1 acts as a major protectant from intracellular superoxide stress, that sod15 is transcribed as part of an operon that may play a role in cell morphology, and that sodA2 and sodC may have minor roles that are not apparent in the conditions tested here.

Prevention of inhalational anthrax after Bacillus anthracis spore exposure requires a prolonged course of antibiotic prophylaxis. In response to the 2001 anthrax attack in the United States, [approximately equal to] 10,000 people were offered 60 days of antibiotic prophylaxis to prevent inhalational anthrax, but adherence to this regimen was poor. We sought to determine whether a short course of antibiotic prophylaxis after exposure could protect non-human primates from a high-dose spore challenge if vaccination was combined with antibiotics. Two groups of 10 rhesus macaques were exposed to [approximately equal to] 11,600 LDs0 of spores by aerosol. Both groups were given ciprofloxacin by orogastric tube twice daily for 14 days, beginning 1-2 h after exposure. One group also received three doses of the licensed human anthrax vaccine (anthrax vaccine adsorbed) after exposure. In the ciprofloxacin-only group, four of nine monkeys (44%) survived the challenge. In contrast, all 10 monkeys that received 14 days of antibiotic plus anthrax vaccine adsorbed survived (P = 0.011). Thus postexposure vaccination enhanced the protection afforded by 14 days of antibiotic prophylaxis alone and completely protected animals against inhalational anthrax. These data provide evidence that postexposure vaccination can shorten the duration of antibiotic prophylaxis required to protect against inhalational anthrax and may impact public health management of a bioterrorism event. Bacillus anthracis | treatment | vaccine

High-throughput microscale platforms have transformed modern analytical investigations. Traditional microarray analyses involve a comparative approach, with two samples, a known control and an unknown sample, hybridized side-by-side and then contrasted for genetic differences. The samples are labeled with separate dyes and hybridized together, providing a differential expression pattern based on the reporter intensities. In contrast, the fiber-optic microarray platform described herein is analyzed with a microscope, thereby enabling the use of virtually any reporter, including quantum dots. The instrumentation takes advantage of the narrow emission bands characteristic of quantum dots to perform multiplexed detection of Bacillus anthracis. Advancing beyond the standard red/green microarray experiment, a panel of eight reporters were linked to eight B. anthracis samples and simultaneously analyzed in a microarray format. The ability to employ an assortment of reporters, along with the capacity to simultaneously hybridize eight samples confers an unprecedented flexibility to array-based analyses, providing a 4-fold increase in throughput over standard two-color assays.

In the environment, the gram-positive bacterium Bacillus anthracis persists as a metabolically dormant endospore. Upon inoculation into the host the endospores germinate and outgrow into vegetative bacilli able to cause disease. The dramatic morphogenic changes to the bacterium during germination and outgrowth are numerous and include major rearrangement of and modifications to the bacterial surface. Such modifications occur during a time in the B. anthracis infectious cycle when the bacterium must guard against a multitude of innate immune mediators. The dltABCD locus of B. anthracis encodes a cell wall D-alanine esterification system that is initiated by transcriptional activation during endospore outgrowth. The level of transcription from the dltABCD operon determined B. anthracis resistance to cationic antibacterial peptides during vegetative growth and cationic peptide, enzymatic, and cellular mediators of innate immunity during outgrowth. Mutation of dltABCD was also attenuating in a mouse model of infection. We propose that the dltABCD locus is important for protection of endosporeforming bacteria from environmental assault during outgrowth and that such protection may be critical during the establishment phase of anthrax.

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60[degrees] rotational angle of [C.sub.[alpha]]([C.sub.[beta]]-[C.sub.1])-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens. anthrax | electron paramagnetic resonance | hydroxyurea | N-methyl hydroxylamine | bacterial growth inhibition

Bacillus anthracis begins its infectious cycle as a metabolically dormant cell type, the endospore. Upon entry into a host, endospores rapidly differentiate into vegetative bacilli through the process of germination, thus initiating anthrax. Elucidation of the signals that trigger germination and the receptors that recognize them is critical to understanding the pathogenesis of B. anthracis. Individual mutants deficient in each of the seven putative germinant receptor-encoding loci were constructed via temperature-dependent, plasmid insertion mutagenesis and used to correlate these receptors with known germinant molecules. These analyses showed that the GerK and GerL receptors are jointly required for the alanine germination pathway and also are individually required for recognition of either proline and methionine (GerK) or serine and valine (GerL) as cogerminants in combination with inosine. The germinant specificity of GerS was refined from a previous study in a nonisogenic background since it was required only for germination in response to aromatic amino acid cogerminants. The gerA and gerY loci were found to be dispensable for recognition of all known germinant molecules. In addition, we show that the promoter of each putative germinant receptor operon, except that of the gerA locus, is active during sporulation. A current model of B. anthracis endospore germination is presented.

Bacillus anthracis secretes three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. Here we demonstrate an approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular weight compound that prevents LF and EF entry into cells. Guided by the sevenfold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of sevenfold symmetry, [beta]-cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3-aminopropylthio)-[beta]-cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations (in 0.1 M KCI). The compound protected RAW 264.7 mouse macrophages from cytotoxicity of anthrax lethal toxin (= PA + LF). More importantly, it completely protected the highly susceptible Fischer F344 rats from lethal toxin. We anticipate that this approach will serve as the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax. infectious diseases | membrane transport | modified cyclodextrins

Pyrosequencing technology is a sequencing method that screens DNA nucleotide incorporation in real time. A set of coupled enzymatic reactions, together with bioluminescence, detects incorporated nucleotides in the form of [...]

Anthrax is a disease caused by infection with spores from the bacteria Bacillus anthracis. These spores enter the body, where they germinate into bacteria and secrete a tripartite toxin that causes local edema and, in systemic infections, death. Recent studies identified the cellular receptor for anthrax toxin (ATR), a type I membrane protein. ATR is one of the splice variants of the tumor endothelial marker 8 (TEM8) gene. ATR and TEM8 are identical throughout their extracellular and transmembrane sequence, and both proteins function as receptors for the toxin. ATR/TEM8 function and expression have been associated with development of the vascular system and with tumor angiogenesis. TEM8 is selectively upregulated in endothelial cells during blood vessel formation and tumor-igenesis. However, selective expression of TEM8 in endothelial cells contradicts the presumably ubiquitous expression of the receptor. To resolve this controversial issue, we evaluated the distribution of ATR/TEM8 in a variety of tissues. For this purpose, we generated and characterized a novel anti-ATR/TEM8 polyclonal antibody. Here, we show that this novel antibody recognizes all three ATR/TEM8 isoforms, which are widely and differentially expressed in various tissue types. We found that ATR/TEM8 expression is not only associated with tumor endothelial cells, as previously described. Indeed, ATR/TEM8 is highly and selectively expressed in the epithelial cells lining those organs that constitute the anthrax toxin's sites of entry, i.e., the lung, the skin, and the intestine. In fact, we show that ATR/TEM8 is highly expressed in the respiratory epithelium of the bronchi of the lung and is particularly abundant in the ciliated epithelial cells coating the bronchi. Furthermore, immunostaining of skin biopsies revealed that ATR/TEM8 is highly expressed in the keratinocytes of the epidermis. Finally, we show that the epithelial cells lining the small intestine strongly express ATR/TEM8 isoforms. This is the first demonstration that the ATR/TEM8 protein is highly expressed in epithelial cells, which represent the primary location for bacterial invasion. These results suggest that the ATR/TEM8 expression pattern that we describe here is highly relevant for understanding the pathogenesis of anthrax infection. anthrax; epithelia; lung; skin; intestine; toxin entry; receptor; bacterial pathogenesis

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3- methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl) acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (11 inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) [approximately equal to] 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit 'point of no return' model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax. Bacillus anthracis | hydroxamate

Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B. anthracis, showing that it lies closer to a newly described 'C' branch than to either of two previously described 'A' or 'B' branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.

Four swab materials were evaluated for their efficiency in recovery of Bacillus anthracis spores from steel coupons. Cotton, macrofoam, polyester, and rayon swabs were used to sample coupons inoculated with [...]

The lack of identified exposures in 2 of the 11 cases of bioterrorism-related inhalation anthrax in 2001 raised uncertainty about the infectious dose and transmission of Bacillus anthracis. We used [...]

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.

A major Bacillus anthracis spore coat protein of 13.4 kDa, designated Cot[alpha], was found only in the Bacillus cereus group. A stable ca. 30-kDa dimer of this protein was also present in spore coat extracts. Cot[alpha], which is encoded by a monocistronic gene, was first detected late in sporulation, consistent with a [[sigma].sup.K]-regulated gene. On the basis of immunogold labeling, the protein is in the outer spore coat and absent from the exosporium. In addition, disruption of the gene encoding Cot[alpha] resulted in spores lacking a dark-staining outer spore coat in thin-section electron micrographs. The mutant spores were stable upon heating or storage, germinated at the same rate as the wild type, and were resistant to lysozyme. They were, however, more sensitive than the wild type to phenol, chloroform, and hypochlorite but more resistant to diethylpyrocarbonate. In all cases, resistance or sensitivity to these reagents was restored by introducing a clone of the cot[alpha] gene into the mutant. Since Cot[alpha] is an abundant outer spore coat protein of the B. cereus group with a prominent role in spore resistance and sensitivity, it is a promising target for the inactivation of B. anthracis spores.