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The bacterial spore cortex is critical for spore stability and dormancy and must be hydrolyzed by germination-specific lytic enzymes (GSLEs), which allows complete germination and vegetative cell outgrowth. We created in-frame deletions of three genes that encode GSLEs that have been shown to be active in Bacillus anthracis germination: sleB, cwlJ1, and cwlJ2. Phenotypic analysis of individual null mutations showed that the removal of any one of these genes was not sufficient to disrupt spore germination in nutrient-rich media. This finding indicates that these genes have partially redundant functions. Double and triple deletions of these genes resulted in more significant defects. Although a small subset of [DELTA]sleB [DELTA]cwlJ1 spores germinate with wild-type kinetics, for the overall population there is a 3-order-of-magnitude decrease in the colony-forming efficiency compared with wild-type spores. [DELTA]sleB [DELTA]cwlJ1 [DELTA]cwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both [DELTA]sleB [DELTA]cwlJ1 and [DELTA]sleB [DELTA]cwlJ1 [DELTA]cwlJ2 spores are significantly attenuated, but are not completely devoid of virulence, in a mouse model of inhalation anthrax. Although unable to germinate in standard nutrient-rich media, spores lacking SleB, CwlJ1, and CwlJ2 are able to germinate in whole blood and serum in vitro, which may explain the persistent low levels of virulence observed in mouse infections. This work contributes to our understanding of GSLE activation and function during germination. This information may result in identification of useful therapeutic targets for the disease anthrax, as well as provide insights into ways to induce the breakdown of the protective cortex layer, facilitating easier decontamination of resistant spores.

Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell. In contrast, the unusual 3,4-DHBA component of petrobactin renders the siderocalin system incapable of obstructing bacterial iron uptake. Although recent genetic and biochemical studies have revealed selected early steps in petrobactin biosynthesis, the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the B. anthracis siderophore biosynthetic (asb) operon, asbF (BA1986), has remained unclear. In this study we demonstrate that 3,4-DHBA is produced through conversion of the common bacterial metabolite 3-dehydroshikimate (3-DHS) by AsbF--a 3-DHS dehydratase. Elucidation of the cocrystal structure of AsbF with 3,4-DHBA, in conjunction with a series of biochemical studies, supports a mechanism in which an enolate intermediate is formed through the action of this 3-DHS dehydratase metalloenzyme. Structural and functional parallels are evident between AsbF and other enzymes within the xylose isomerase TIM-barrel family. Overall, these data indicate that microbial species shown to possess homologs of AsbF may, like B. anthracis, also rely on production of the unique 3,4-DHBA metabolite to achieve full viability in the environment or virulence within the host. AsbF structure | dehydratase | siderophore | virulence factor

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.

Microarray analyses were conducted to evaluate the paraquat-induced global transcriptional response of Bacillus anthracis Sterne (34[F.sub.2]) to varying levels of endogenous superoxide stress. Data revealed that the transcription of genes putatively involved in metal/ion transport, bacillibactin siderophore biosynthesis, the glyoxalase pathway, and oxidoreductase activity was perturbed most significantly. A B. anthracis mutant lacking the superoxide dismutase gene sodA1 ([DELTA]sodA1) had transcriptional responses to paraquat similar to, but notably larger than, those of the isogenic parental strain. A small, unique set of genes was found to be differentially expressed in the [DELTA]sodA1 mutant relative to the parental strain during growth in rich broth independently of induced oxidative stress. The bacillibactin siderophore biosynthetic genes were notably overexpressed in Sterne and [DELTA]sodA1 cells after treatment with paraquat. The bacillibactin siderophore itself was isolated from the supernatants and lysates of cells grown in iron-depleted medium and was detected at lower levels after treatment with paraquat. This suggests that, while transcriptional regulation of these genes is sensitive to changes in the redox environment, additional levels of posttranscriptional control may exist for bacillibactin biosynthesis, or the enzymatic siderophore pipeline may be compromised by intracellular superoxide stress or damage. The [DELTA]sodA1 mutant showed slower growth in a chelated iron-limiting medium but not in a metal-depleted medium, suggesting a connection between the intracellular redox state and iron/metal ion acquisition in B. anthracis. A double mutant lacking both the sodA1 and sodA2 genes ([DELTA]sodA1 [DELTA]sodA2) was attenuated for growth in manganese-depleted medium, suggesting a slight level of redundancy between sodA1 and sodA2, and a role for the sod genes in manganese homeostasis.

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.

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.

The life cycle of Bacillus anthracis includes both vegetative and endospore morphologies which alternate based on nutrient availability, and there is considerable evidence indicating that the ability of this organism to cause anthrax depends on its ability to progress through this life cycle in a regulated manner. Here we report the use of a custom B. anthracis GeneChip in defining the gene expression patterns that occur throughout the entire life cycle in vitro. Nearly 5,000 genes were expressed in five distinct waves of transcription as the bacteria progressed from germination through sporulation, and we identified a specific set of functions represented within each wave. We also used these data to define the temporal expression of the spore proteome, and in doing so we have demonstrated that much of the spore's protein content is not synthesized de novo during sporulation but rather is packaged from preexisting stocks. We explored several potential mechanisms by which the cell could control which proteins are packaged into the developing spore, and our analyses were most consistent with a model in which B. anthracis regulates the composition of the spore proteome based on protein stability. This study is by far the most comprehensive survey yet of the B. anthracis life cycle and serves as a useful resource in defining the growth-phase-dependent expression patterns of each gene. Additionally, the data and accompanying bioinformatics analyses suggest a model for sporulation that has broad implications for B. anthracis biology and offer new possibilities for microbial forensics and detection.

Author(s): Timothy D. Read (corresponding author) [1, 2]; Scott N. Peterson [1, 3]; Nicolas Tourasse [4, 7]; Les W. Baillie [1, 2, 5]; Ian T. Paulsen [1, 6]; Karen E. [...]

Research has been conducted on the amino acid and purine ribonuclease combinations responsible for the endospore germination of Bacillus anthracis (Delta)Sterne. Results indicate that this germination requires multipartite signals and that gerS-encoded proteins are the aromatic-responsive germination receptors.

Doctors must become familiar with anthrax because it could be used as a biological weapon. Anthrax is a bacterial infection that can cause death within days and is almost 100% fatal. It is transmitted by spores, which can be inhaled. The bacteria produces a toxin called endotoxin, which is the agent that actually kills the patient. A complete description of the disease, its signs and symptoms, diagnosis and treatment is included.

Research was conducted to examine the hypothesis that in vivo-induced genes, which are coordinately induced by low Mg2+ and low pH, also demonstrated coordinate induction upon entry into cultured murine macrophages and cultured human epithelial cells. The investigation of ivi expression patterns demonstrates the inherent versatility of pathogens to express a given gene at different host sites and its ability to modify their expression.

In vivo expression technology (IVET) has been used to identify > 100 Salmonella typhimurium genes that are specifically expressed during infection of BALB/c mice and/or murine cultured macrophages. Induction of these genes is shown to be required for survival in the animal under conditions of the IVET selection. One class of in vivo induced (ivi) genes, iviVI-A and iviVI-B, constitute an operon that resides in a region of the Salmonella genome with low G+C content and presumably has been acquired by horizontal transfer. These ivi genes encode predicted proteins that are similar to adhesins and invasins from prokaryotic and eukaryotic pathogens (Escherichia coli [tia], Plasmodium falciparum [PfEMP1]) and have coopted the PhoPQ regulatory circuitry of Salmonella virulence genes. Examination of the in vivo induction profile indicates (i) many ivi genes encode regulatory functions (e.g., phoPQ and pmrAB) that serve to enhance the sensitivity and amplitude of virulence gene expression (e.g., spvB); (ii) the biochemical function of many metabolic genes may not represent their sole contribution to virulence; (iii) the host ecology can be inferred from the biochemical functions of ivi genes; and (iv) nutrient limitation plays a dual signaling role in pathogenesis: to induce metabolic functions that complement host nutritional deficiencies and to induce virulence functions required for immediate survival and spread to subsequent host sites.

A transcriptional-fusion factor pIVET8 contains lacZY genes and chloroamphenicol acetyltransferase and enables a selection of bacterial genes in host tissues. The principle of this method, which makes use of the in vivo expression technology (IVET), is antibiotic resistance. This technique enables an understanding of the resistance mechanisms of Salmonella typhimurium and the nature of the genes involved. The identification of these genes has implications on antimicrobial drug and vaccine development.

Mice depleted of macrophages by silica injections become resistant to the toxin of Bacillus anthracis. This toxin causes lysis of macrophages in vitro but has no effect on other cells. Macrophages regulate the action of lethal toxin in vivo. Systemic shock and death resulting from the effect of cytokines produced by macrophages have to be induced by the anthrax toxin.

A Bayesian hidden Markov model that incorporates comparative genomic data with traditional predictors such as intergenic distances was constructed to explore the possibility of using phylogenetic information to aid in operon prediction. The findings show that the constructed algorithm is capable of highly sensitive and accurate operon prediction in a wide range of bacterial genomes and that these predictions could lead to the rapid discovery of new functional relationships among genes.

Research has been conducted on bacterial endospores. Results suggest that the addition of exogenous adenosine and L-alanine is required for Bacillus anthracis in vivo germination.