(p)ppGpp is required for virulence of Shigella flexneri.

Infection by the enteric pathogen Shigella flexneri requires transit through the gastrointestinal tract and invasion of and replication within the cells of the host colonic epithelium. This process exposes the pathogen to a range of diverse microenvironments. Furthermore, the unique composition and physical environment of the eukaryotic cell cytosol represents a stressful environment for S. flexneri, and extensive physiological adaptations are needed for the bacterium to thrive. In this work, we show that disrupting synthesis of the stringent response alarmone (p)ppGpp in S. flexneri diminished expression of key virulence genes, including ipaA, ipaB, ipaC, and icsA, and it reduced bacterial invasion and intercellular spread. Deletion of the (p)ppGpp synthase gene relA alone had no effect on S. flexneri virulence, but disruption of both relA and the (p)ppGpp synthase/hydrolase gene spoT resulted in loss of (p)ppGpp synthesis and virulence. While the relA spoT deletion mutant was able to invade a cultured human epithelial cell monolayer, albeit at reduced levels, it was unable to maintain the infection and spread to adjacent cells, as indicated by loss of plaque formation. Complementation with spoT on a plasmid vector restored plaque formation. Thus, SpoT alone is sufficient to provide the necessary level of (p)ppGpp for virulence. These results indicate that (p)ppGpp is required for S. flexneri virulence and adaptation to the intracellular environment, adding to the repertoire of signaling pathways that affect Shigella pathogenesis.

Antibody responses of variable lymphocyte receptors in the lamprey.

Lamprey and hagfish, the living representatives of jawless vertebrates, use genomic leucine-rich-repeat cassettes for the combinatorial assembly of diverse antigen receptor genes encoding variable lymphocyte receptors of two types: VLRA and VLRB. We describe here the VLRB-bearing lineage of lymphocytes in sea lamprey. These cells responded to repetitive carbohydrate or protein determinants on bacteria or mammalian cells with lymphoblastoid transformation, proliferation and differentiation into plasmacytes that secreted multimeric antigen-specific VLRB antibodies. Lacking a thymus and the ability to respond to soluble protein antigens, lampreys seem to have evolved a B cell-like system for adaptive humoral responses.

X-ray crystal structure of a reiterative transcription complex reveals an atypical RNA extension pathway.

Reiterative transcription is a noncanonical form of RNA synthesis in which a nucleotide specified by a single base in the DNA template is repetitively added to the nascent transcript. Here we determined the crystal structure of an RNA polymerase, the bacterial enzyme from Thermus thermophilus, engaged in reiterative transcription during transcription initiation at a promoter resembling the pyrG promoter of Bacillus subtilis The structure reveals that the reiterative transcript detours from the dedicated RNA exit channel and extends toward the main channel of the enzyme, thereby allowing RNA extension without displacement of the promoter recognition σ-factor. Nascent transcripts containing reiteratively added G residues are eventually extended by nonreiterative transcription, revealing an atypical pathway for the formation of a transcription elongation complex.

Activities of gyrase and topoisomerase IV on positively supercoiled DNA.

Although bacterial gyrase and topoisomerase IV have critical interactions with positively supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and topoisomerase IV to relax and cleave positively supercoiled DNA were analyzed. Gyrase removed positive supercoils ∼10-fold more rapidly and more processively than it introduced negative supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positive supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positively supercoiled substrates ∼3-fold faster than negatively supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positively supercoiled (compared with negatively supercoiled) DNA, whereas topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than topoisomerase IV to safely remove positive supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positive supercoils, rather than induction of negative supercoils.

Interactions between Quinolones and Bacillus anthracis Gyrase and the Basis of Drug Resistance.

Gyrase appears to be the primary cellular target for quinolone antibacterials in multiple pathogenic bacteria, including Bacillus anthracis, the causative agent of anthrax. Given the significance of this type II topoisomerase as a drug target, it is critical to understand how quinolones interact with gyrase and how specific mutations lead to resistance. However, these important issues have yet to be addressed for a canonical gyrase. Therefore, we utilized a mechanistic approach to characterize interactions of quinolones with wild-type B. anthracis gyrase and enzymes containing the most common quinolone resistance mutations. Results indicate that clinically relevant quinolones interact with the enzyme through a water-metal ion bridge in which a noncatalytic divalent metal ion is chelated by the C3/C4 keto acid of the drug. In contrast to other bacterial type II topoisomerases that have been examined, the bridge is anchored to gyrase primarily through a single residue (Ser85). Substitution of groups at the quinolone C7 and C8 positions generated drugs that were less dependent on the water-metal ion bridge and overcame resistance. Thus, by analyzing the interactions of drugs with type II topoisomerases from individual bacteria, it may be possible to identify specific quinolone derivatives that can overcome target-mediated resistance in important pathogenic species.

Activity of quinolone CP-115,955 against bacterial and human type II topoisomerases is mediated by different interactions.

CP-115,955 is a quinolone with a 4-hydroxyphenyl at C7 that displays high activity against both bacterial and human type II topoisomerases. To determine the basis for quinolone cross-reactivity between bacterial and human enzymes, the activity of CP-115,955 and a series of related quinolones and quinazolinediones against Bacillus anthracis topoisomerase IV and human topoisomerase IIα was analyzed. Results indicate that the activity of CP-115,955 against the bacterial and human enzymes is mediated by different interactions. On the basis of the decreased activity of quinazolinediones against wild-type and resistant mutant topoisomerase IV and the low activity of quinolones against resistant mutant enzymes, it appears that the primary interaction of CP-115,955 with the bacterial system is mediated through the C3/C4 keto acid and the water-metal ion bridge. In contrast, the drug interacts with the human enzyme primarily through the C7 4-hydroxyphenyl ring and has no requirement for a substituent at C8 in order to attain high activity. Despite the fact that the human type II enzyme is unable to utilize the water-metal ion bridge, quinolones in the CP-115,955 series display higher activity against topoisomerase IIα in vitro and in cultured human cells than the corresponding quinazolinediones. Thus, quinolones may be a viable platform for the development of novel drugs with anticancer potential.

Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance.

Although quinolones are the most commonly prescribed antibacterials, their use is threatened by an increasing prevalence of resistance. The most common causes of quinolone resistance are mutations of a specific serine or acidic residue in the A subunit of gyrase or topoisomerase IV. These amino acids are proposed to serve as a critical enzyme-quinolone interaction site by anchoring a water-metal ion bridge that coordinates drug binding. To probe the role of the proposed water-metal ion bridge, we characterized wild-type, GrlA(E85K), GrlA(S81F/E85K), GrlA(E85A), GrlA(S81F/E85A) and GrlA(S81F) Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg(2+) concentration required to produce maximal quinolone-induced DNA cleavage and restricted the divalent metal ions that could support quinolone activity. Individual mutation of Ser81 or Glu85 partially disrupted bridge function, whereas simultaneous mutation of both residues abrogated protein-quinolone interactions. Results provide functional evidence for the existence of the water-metal ion bridge, confirm that the serine and glutamic acid residues anchor the bridge, demonstrate that the bridge is the primary conduit for interactions between clinically relevant quinolones and topoisomerase IV and provide a likely mechanism for the most common causes of quinolone resistance.

Transcription start site sequence and spacing between the -10 region and the start site affect reiterative transcription-mediated regulation of gene expression in Escherichia coli.

Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3' end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli, the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3' end of a nascent transcript during transcription initiation. Immediately following the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promoter sequences. One of the operons regulated (in part) by UTP-sensitive reiterative transcription in E. coli is the carAB operon, which encodes the first enzyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucleotide sequence at and near the transcription start site and spacing between the start site and -10 region of the promoter on reiterative transcription and gene regulation. Our results indicate that these variables are important determinants in establishing the extent of reiterative transcription, levels of productive transcription, and range of gene regulation.

Cryo-EM analysis of the organization of BclA and BxpB in the Bacillus anthracis exosporium.

Bacillus anthracis and other pathogenic Bacillus species form spores that are surrounded by an exosporium, a balloon-like layer that acts as the outer permeability barrier of the spore and contributes to spore survival and virulence. The exosporium consists of a hair-like nap and a paracrystalline basal layer. The filaments of the nap are comprised of trimers of the collagen-like glycoprotein BclA, while the basal layer contains approximately 20 different proteins. One of these proteins, BxpB, forms tight complexes with BclA and is required for attachment of essentially all BclA filaments to the basal layer. Another basal layer protein, ExsB, is required for the stable attachment of the exosporium to the spore. To determine the organization of BclA and BxpB within the exosporium, we used cryo-electron microscopy, cryo-sectioning and crystallographic analysis of negatively stained exosporium fragments to compare wildtype spores and mutant spores lacking BclA, BxpB or ExsB (ΔbclA, ΔbxpB and ΔexsB spores, respectively). The trimeric BclA filaments are attached to basal layer surface protrusions that appear to be trimers of BxpB. The protrusions interact with a crystalline layer of hexagonal subunits formed by other basal layer proteins. Although ΔbxpB spores retain the hexagonal subunits, the basal layer is not organized with crystalline order and lacks basal layer protrusions and most BclA filaments, indicating a central role for BxpB in exosporium organization.

Bacillus anthracis GrlAV96A topoisomerase IV, a quinolone resistance mutation that does not affect the water-metal ion bridge.

The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis topoisomerase IV on quinolone activity. The results indicate that this mutation causes an ∼ 3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.

Microbicidal power of alpha radiation in sterilizing germinating Bacillus anthracis spores.

Radioimmunotherapy (RIT) takes advantage of the specificity and affinity of the antigen-antibody interaction to deliver microbicidal radioactive nuclides to a site of infection. In this study, we investigated the microbicidal properties of an alpha particle-emitting 213Bi-labeled monoclonal antibody (MAb), EA2-1 (213Bi-EA2-1), that binds to the immunodominant antigen on Bacillus anthracis spores. Our results showed that dormant spores were resistant to 213Bi-EA2-1. Significant spore killing was observed following treatment with EA2-1 labeled with 300 µCi 213Bi; however, this effect was not dependent on the MAb. In contrast, when spores were germinating, 213Bi-EA2-1 mediated MAb-specific killing in a dose-dependent manner. Dormant spores are very resistant to RIT, and RIT should focus on targeting vegetative cells and germinating spores.

Crystal structure and induced stability of trimeric BxpB: implications for the assembly of BxpB-BclA complexes in the exosporium of Bacillus anthracis.

The outermost exosporium layer of Bacillus anthracis spores, the causative agents of anthrax, is comprised of a basal layer and an external hair-like nap. The nap includes filaments composed of trimers of the collagen-like glycoprotein BclA. Essentially all BclA trimers are attached to the spore in a process in which part of the 38-residue amino-terminal domain (NTD) of BclA forms an extremely stable interaction with the basal layer protein BxpB. Evidence indicates that the BclA-BxpB interaction is direct and requires trimeric BxpB. To further investigate the nature of the BclA-BxpB interaction, we determined the crystal structure of BxpB. The structure was trimeric with each monomer consisting of 11 ß strands with connecting loops. The structure did not include apparently disordered amino acids 1-19, which contain the only two cysteine residues of the 167-residue BxpB. The orientation of the structure reveals regions of BxpB that could be involved in interacting with the BclA NTD and with adjacent cysteine-rich proteins in the basal layer. Furthermore, the BxpB structure closely resembles that of the 134-residue carboxyl-terminal domain of BclA, which forms trimers that are highly resistant to heat and detergent. We demonstrated that BxpB trimers do not share this resistance. However, when BxpB trimers are mixed with a peptide containing residues 20-38 of BclA, they form a complex that is as stable as BclA-BxpB complexes extracted from spores. Together, our results provide new insights into the mechanism of BclA-BxpB attachment and incorporation into the exosporium. IMPORTANCE The B. anthracis exosporium plays major roles in spore survival and infectivity, but the complex mechanism of its assembly is poorly understood. Key steps in this process are the stable attachment of collagen-like BclA filaments to the major basal layer structural protein BxpB and the insertion of BxpB into an underlying basal layer scaffold. The goal of this study is to further elucidate these interactions thereby advancing our understanding of exosporium assembly, a process shared by many spore-forming bacteria including important human pathogens.

Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance.

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlA(S81F) and GrlA(S81Y) quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg(2+) bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg(2+) concentrations to support DNA cleavage by GrlA(S81F) topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the enzyme-quinolone-DNA ternary complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg(2+) bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically relevant quinolone resistance mutations in bacterial type II topoisomerases.

CD14-Mac-1 interactions in Bacillus anthracis spore internalization by macrophages.

Anthrax, a potentially lethal disease of animals and humans, is caused by the Gram-positive spore-forming bacterium Bacillus anthracis. The outermost exosporium layer of B. anthracis spores contains an external hair-like nap formed by the glycoprotein BclA. Recognition of BclA by the integrin Mac-1 promotes spore uptake by professional phagocytes, resulting in the carriage of spores to sites of spore germination and bacterial growth in distant lymphoid organs. We show that CD14 binds to rhamnose residues of BclA and acts as a coreceptor for spore binding by Mac-1. In this process, CD14 induces signals involving TLR2 and PI3k that promote inside-out activation of Mac-1, thereby enhancing spore internalization by macrophages. As observed with mice lacking Mac-1, CD14(-/-) mice are also more resistant than wild-type mice to infection by B. anthracis spores. Additionally, after B. anthracis spore challenge of CD14(-/-) mice, interference with the CD14-mediated signaling pathways results in increased mortality. Our results show that the binding and uptake of B. anthracis spores by phagocytic cells is a dynamic process and involves multiple receptors and signaling pathways.

ExsB, an unusually highly phosphorylated protein required for the stable attachment of the exosporium of Bacillus anthracis.

The outermost layer of the Bacillus anthracis spore, the exosporium, is composed of a paracrystalline basal layer and an external hair-like nap. The nap is formed from a single collagen-like glycoprotein, while the basal layer contains many different proteins, including a 186-amino acid protein called ExsB. In this study, we discovered that ExsB is unusually highly phosphorylated, with at least 14 of its 19 threonine residues modified. The phosphorylated threonines are included in seven contiguous approximately 12-residue imperfect repeats, which presumably contain kinase recognition sequences. We demonstrated that a B. anthracis DeltaexsB mutant unable to synthesize ExsB produced spores with an exosporium that was readily sloughed, indicating that ExsB was required for stable exosporium attachment. This unstable exosporium also lacked the enzyme alanine racemase, which is normally tightly associated with the exosporium. Additionally, purified DeltaexsB spores lacking a visible exosporium were devoid of most exosporium proteins but, surprisingly, retained the putative exosporium proteins BxpC and CotB-1. Finally, we showed that transcription of the exsB gene occurred only during the late stages of sporulation, and we used an active and phosphorylated ExsB-EGFP fusion protein to monitor ExsB localization to wild-type and DeltabxpB mutant exosporia.

The integrin Mac-1 (CR3) mediates internalization and directs Bacillus anthracis spores into professional phagocytes.

Anthrax, a disease caused by Bacillus anthracis, affects animals and humans. Because the inert spore is the infectious form of the organism that first contacts the potential host, the interaction between the host and spore exosporium is vital to the initiation of disease. Here, we demonstrate that the integrin Mac-1 is essential for the recognition of the major exosporium protein BclA by phagocytic cells. Expression of Mac-1, but not p150/95, in CHO cells markedly enhanced infection with Sterne strain of B. anthracis spores (WT spores). Conversely, CD11b(-/-) macrophages demonstrated a significant decrease in spore uptake when compared with macrophages from normal C57BL/6 mice. However, when CD11b(-/-) macrophages were infected with DeltabclA spores, spore ingestion was no different from their C57BL/6 counterparts. DeltabclA spores were also efficiently internalized by all CHO cell lines tested, independently of Mac-1 expression. Taken together, these results show that there is an alternative Mac-1-independent pathway involved in spore uptake that is unmasked only in the absence of BclA. Survival studies, using C57BL/6 and CD11b(-/-) mice, revealed that CD11b(-/-) mice are more resistant to infection with WT but not DeltabclA spores. Our experiments also show that DeltabclA spores are more virulent than WT spores in C57BL/6 and A/J mice. Overall, our data indicate that the Mac-1/BclA interaction may play a major role in B. anthracis pathogenesis by promoting spore uptake by professional phagocytes and subsequent access to a favorable niche for transport, germination, and outgrowth in lymphoid tissues.

Structure and specificity of lamprey monoclonal antibodies.

Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores. The recombinant VLR-B antibodies possess 8-10 uniform subunits that collectively bind antigen with high avidity. Sequence analysis, mutagenesis, and modeling studies show that antigen binding involves residues in the beta-sheets lining the VLR-B concave surface. EM visualization reveals tetrameric and pentameric molecules having a central core and highly flexible pairs of stalk-region "arms" with antigen-binding "hands." Remarkable antigen-binding specificity, avidity, and stability predict that these unusual LRR-based monoclonal antibodies will find many biomedical uses.

Sequence motifs and proteolytic cleavage of the collagen-like glycoprotein BclA required for its attachment to the exosporium of Bacillus anthracis.

Bacillus anthracis spores are enclosed by an exosporium comprised of a basal layer and an external hair-like nap. The filaments of the nap are composed of trimers of the collagen-like glycoprotein BclA. The attachment of essentially all BclA trimers to the exosporium requires the basal layer protein BxpB, and both proteins are included in stable high-molecular-mass exosporium complexes. BclA contains a proteolytically processed 38-residue amino-terminal domain (NTD) that is essential for basal-layer attachment. In this report, we identify three NTD submotifs (SM1a, SM1b, and SM2, located within residues 21 to 33) that are important for BclA attachment and demonstrate that residue A20, the amino-terminal residue of processed BclA, is not required for attachment. We show that the shortest NTD of BclA-or of a recombinant protein-sufficient for high-level basal-layer attachment is a 10-residue motif consisting of an initiating methionine, an apparently arbitrary second residue, SM1a or SM1b, and SM2. We also demonstrate that cleavage of the BclA NTD is necessary for efficient attachment to the basal layer and that the site of cleavage is somewhat flexible, at least in certain mutant NTDs. Finally, we propose a mechanism for BclA attachment and discuss the possibility that analogous mechanisms are involved in the attachment of many different collagen-like proteins of B. anthracis and closely related Bacillus species.

Characterization of the enzymes encoded by the anthrose biosynthetic operon of Bacillus anthracis.

Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-ß-L-rhamnose α-1,3-L-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit ß-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.

The spore-specific alanine racemase of Bacillus anthracis and its role in suppressing germination during spore development.

Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinant l-alanine to the germination inhibitor d-alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of the alr gene, which encodes Alr, is restricted to sporulating cells and that the bulk of alr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped two alr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Deltaalr mutant of B. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Deltaalr spores germinate more efficiently in the presence of l-alanine, presumably because of their inability to convert exogenous l-alanine to d-alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Deltaalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produce D-alanine during the late stages of sporulation to suppress premature germination of the developing spore.