An improved genetic tool suitable for routine markerless allelic exchange in Bacillus anthracis has been constructed. Its utility was demonstrated by the introduction of insertions, deletions, and missense mutations on the chromosome and plasmid pXO1 of the Sterne strain of B. anthracis.Bacillus anthracis, a gram-positive, spore-forming bacterium, is the causative agent of anthrax. Full virulence requires the production of a toxin and the formation of a protective capsule. The primary genes involved in the production of these two virulence factors are contained within two large nonessential plasmids, pXO1 and pXO2 (13). Distributed elsewhere on the two plasmids and on the chromosome are genes involved in regulating the expression of anthrax toxin, the capsule, and a number of other genes potentially involved in virulence (2, 6, 11). The entire genomic sequence of the Ames strain of B. anthracis has recently been determined, enabling the identification of interesting, potentially virulence-related genes by reverse genetics (19).The ideal mutation to initially introduce in such "genomemining" studies is an in-frame deletion of the candidate gene. Such a mutation avoids problems with polarity and other effects on the expression of surrounding genes, which accompany either insertion or less-precise deletion mutations and which can complicate interpretation. In addition, the ability to readily introduce missense mutations, which enables a determination of the effects of single-amino-acid substitutions, is necessary for more-sophisticated genetic analyses of structure-function relationships. Both of these types of desirable mutations are "markerless" in that they are not necessarily associated with a phenotype that can be selected or screened for during genetic manipulation (e.g., antibiotic resistance in the case of an insertion mutation). For B. anthracis, markerless gene replacements for some loci have been reported (3), but the methods by which these mutations have been isolated can be time and labor-intensive. This is due to the lack of a counterselection scheme, in which, by selecting for the loss of a plasmid vector, one can select for the second of two successive crossovers between such a vector and the chromosome in order to achieve gene replacement.An alternative to counterselection schemes involves the use of the intron-encoded homing restriction enzyme I-SceI. The ability of this enzyme, which recognizes an 18-bp sequence, to cleave an introduced site that is essentially unique in a genome has been exploited in the promotion of homologous recombination in organisms as diverse as bacteria, Drosophila, and other higher eukaryotes (4,20,22). In one case, the use of I-SceI in promoting allelic exchange in Escherichia coli has been reported (17). In such a scheme, the integration of a suicide plasmid by a cloned region of homology containing the desired genetic change results in one of the two crossovers required to effect allelic exchange. To promote the second, the synthesis of the I-SceI enzyme results in cl...

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 asb 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 asb 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 interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.Bacillus anthracis, the causative agent of anthrax, has come under increased scrutiny in recent years because of its potential role as a bioweapon (35). In the environment, B. anthracis exists primarily as a metabolically dormant endospore, and in this morphology the bacterium is both highly infectious and resistant to a wide range of harsh conditions (42). When the spores are inhaled, they reach the alveolar spaces of the lung, where they are efficiently taken up by resident phagocytes (5, 9, 48). It is commonly believed that the host cells then migrate across the alveolocapillary barrier, transporting the intracellular bacteria into the lymphatic system (26,40). During this time, the bacteria germinate, transforming from spores into vegetative bacilli, which begin to replicate within the phagocytes (15, 50). Eventually, the bacteria kill the phagocytes and escape into the extracellular environment, and the resulting sepsis ultimately leads to death of the host (23,24,43,52,57).Since the progression of anthrax is typically quite rapid once the systemic phase of the infection begins (16, 24), successful intervention depends on early diagnosis and treatment. Given this fact, it is particularly important from a therapeutic standpoint that the early events in anthrax are well understood. Most of these events occur within the context of the h...

Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the ⌬bclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of ⌬bclA and wild-type spores. The ⌬bclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.Bacillus anthracis is a gram-positive, spore-forming bacillus that can cause anthrax (15). The spore is the form of the organism found in its natural habitat, the soil, and is also the infectious form for herbivores, the typical vertebrate host for the bacterium, and humans (15). The B. anthracis spore is covered by a loose balloon-like membranous structure called the exosporium (8). BclA (for bacillus collagen-like protein of anthracis) was first described by Sylvestre et al. (23), who constructed an insertional bclA mutant and compared it to its wild-type parent. These investigators and, subsequently, others found that BclA is a glycoprotein and a major component of the hair-like projections that cover the exosporium (16,22,23,25). BclA is also an immunodominant marker on the outside of the spore (22). The finding that BclA does not play a significant role in the virulence of a Sterne-like strain for mice was first reported by Sylvestre et al. (23). Sterne strains contain pXO1 but not pX02 and are attenuated in humans and many other animals except certain mouse strains (26). In support of the findings of Sylvestre and colleagues, Bozue and coworkers recently constructed a bclA mutant of the fully virulent B. anthracis Ames strain and showed that the absence of BclA had no impact on the lethality of that strain for guinea pigs or mice (5). Whether BclA, the substance on the spore with which the host cells probably first interact, plays a more subtle role in B. anthracis patho...

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 S iderophore production in pathogenic bacteria has gained considerable attention because of its crucial function in essential iron uptake by many microbes and the relevance of siderophore-associated proteins as molecular markers of various infectious agents (1). In Bacillus anthracis, the causative agent of anthrax, 2 siderophores, petrobactin and bacillibactin (Fig. 1A), play a significant role during iron acquisition (2-4), but only petrobactin is absolutely essential for full virulence within a mammalian host (5). This siderophore was initially discovered from the Gram-negative marine bacterium Marinobacter hydrocarbonoclasticus, whose genome bears a biosynthetic gene cluster homologous to the B. anthracis asb operon (2). Recent genetic and chemical analysis suggests that petrobactin biosynthesis may also be a prerequisite for virulence in related Bacillus species (6). These studies highlight the importance of elucidating the mechanisms of siderophore production in pathogenic microbes as a target for abrogating infection by organisms like B. anthracis, a rapidly virulent microbe with proven potential as a bioterrorism agent. Based on these factors, we have initiated studies to investigate key biosynthetic enzymes for petrobactin assembly in efforts to establish new antimicrobial targets t...

Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (wbbO) from Klebsiella pneumoniae 43816. Analysis of the mutant lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the absence of O antigen. In vitro, there were no detectable differences between wild-type K. pneumoniae and the O-antigendeficient mutant in regard to avid binding by murine complement C3 or resistance to serum-or whole-bloodmediated killing. Nevertheless, the 72-h 50% lethal dose of the wild-type strain was 30-fold greater than that of the mutant (2 ؋ 10 3 versus 6 ؋ 10 4 CFU) after intratracheal injection in ICR strain mice. Despite being less lethal, the mutant organism exhibited comparable intrapulmonary proliferation at 24 h compared to the level of the wild type. Whole-lung chemokine expression (CCL3 and CXCL2) and bronchoalveolar inflammatory cell content were also similar between the two infections. However, whereas the wild-type organism produced bacteremia within 24 h of infection in every instance, bacteremia was not seen in mutant-infected mice. These results suggest that during murine pneumonia caused by K. pneumoniae, O antigen contributes to lethality by increasing the propensity for bacteremia and not by significantly changing the early course of intrapulmonary infection.

Lack of available iron is one of many environmental challenges that a bacterium encounters during infection and adaptation to iron starvation is important for the pathogen to efficiently replicate within the host. Here we define the transcriptional response of B. anthracis Sterne (34F2) to iron depleted conditions. Genome-wide transcript analysis showed that B. anthracis undergoes considerable changes in gene expression during growth in iron-depleted media, including the regulation of known and candidate virulence factors. Two genes encoding putative internalin proteins were chosen for further study. Deletion of either gene (GBAA0552 or GBAA1340) resulted in attenuation in a murine model of infection. This attenuation was amplified in a double mutant strain. These data define the transcriptional changes induced during growth in low iron conditions and illustrate the potential of this dataset in the identification of putative virulence determinants for future study.

About 10% of the nalidixic acid-resistant (Nal r ) mutants in a transposition-induced library exhibited a growth factor requirement as the result of cysH, icdA, metE, or purB mutation. Resistance in all of these mutants required a functional AcrAB-TolC efflux pump, but the EmrAB-TolC pump played no obvious role. Transcription of acrAB was increased in each type of Nal r mutant. In the icdA and purB mutants, each of the known signaling pathways appeared to be used in activating the AcrAB-TolC pump. The metabolites that accumulate upstream of the blocks caused by the mutations are hypothesized to increase the levels of the AcrAB-TolC pump, thereby removing nalidixic acid from the organism.Quinolone antibiotics constitute one of the most widely used classes of antibacterial agent. Although the original quinolone, nalidixic acid (Nal) (Fig. 1), was effective primarily against gram-negative bacteria, fluoroquinolone derivatives are potent against a wide variety of gram-negative and gram-positive bacteria. The primary targets of the quinolones are DNA topoisomerase II (DNA gyrase) and topoisomerase IV (10,19,37).Because of the therapeutic importance of these antibiotics, the appearance of bacterial mutants resistant to quinolones is of considerable concern. Many such mutants in a variety of bacteria have been characterized. These fall into two broad classes: those altered in the target topoisomerases and those failing to accumulate the drug to levels seen in the wild type. No mutant resistance stemming from the breakdown or modification of quinolones has been described.Resistant mutants of Escherichia coli and of a wide variety of other gram-negative and gram-positive bacteria selected in the laboratory or isolated from clinical settings have been shown to be altered in one or more of the genes encoding components of the two topoisomerases (gyrA, gyrB, parC, and parE in E. coli) (34). These mutations produce resistance of various levels, and further mutations often increase the resistance (18).Quinolone-resistant mutants containing mutations in genes other than those for the topoisomerases are also obtained frequently when only a low level of drug is used in selection or when a secondary mutation increases resistance beyond that of the primary mutation. Where the basis for such mutation has been determined, it has usually resulted from the activation of an efflux pump (31, 32) that removes the quinolone during or after entry so it does not reach a toxic level. In E. coli, activation of either the AcrAB-TolC pump (4, 9, 28) or the EmrABTolC pump (26) provides resistance. AcrAB-TolC appears to be the main pump providing intrinsic resistance to low levels of many toxic compounds in nature (38). Three signaling pathways controlling the expression of the acrAB genes and the formation of the AcrAB-TolC pump are known; these involve the MarAB, SoxRS, and RobA proteins (5,11,15,33,39).Thus, there are several potential ways of generating low-level resistance to quinolones through the activation of efflux pumps.Screening mutant...