The protective antigen component of the anthrax toxin is essential for the delivery of the enzymatic components of the toxin into host target cells. The toxin genes and other virulence genes of B. anthracis are regulated by multiple trans -acting regulators that respond to a variety of host-related signals.

Bacillus anthracis, the anthrax agent, exhibits robust proliferation in diverse niches of mammalian hosts. The metabolic attributes of B. anthracis that permit rapid growth in multiple mammalian tissues have not been established. We posit that branched-chain amino acid (BCAA) (isoleucine, leucine, and valine) metabolism is key to B. anthracis pathogenesis. Increasing evidence indicates the relationships between B. anthracis virulence and the expression of BCAA-related genes. The expression of some BCAA-related genes is altered during culture in bovine blood in vitro, and the bacterium exhibits valine auxotrophy in a blood serum mimic medium. Transcriptome analyses have revealed that the virulence regulator AtxA, which positively affects the expression of the anthrax toxin and capsule genes, negatively regulates genes predicted to be associated with BCAA biosynthesis and transport. Here, we show that B. anthracis growth in defined medium is severely restricted in the absence of exogenous BCAAs, indicating that BCAA transport is required for optimal growth in vitro. We demonstrate functional redundancy among multiple BrnQ-type BCAA transporters. Three transporters are associated with isoleucine and valine transport, and the deletion of one, BrnQ3, attenuates virulence in a murine model for anthrax. Interestingly, an ilvD-null mutant lacking dihydroxy acid dehydratase, an enzyme essential for BCAA synthesis, exhibits unperturbed growth when cultured in medium containing BCAAs but is highly attenuated in the murine model. Finally, our data show that BCAAs enhance AtxA activity in a dose-dependent manner, suggesting a model in which BCAAs serve as a signal for virulence gene expression. IMPORTANCE Infection with B. anthracis can result in systemic disease with large numbers of the bacterium in multiple tissues. We found that branched-chain amino acid (BCAA) synthesis is insufficient for the robust growth of B. anthracis; access to BCAAs is necessary for the proliferation of the pathogen during culture and during infection in a murine model for anthrax. B. anthracis produces an unusually large repertoire of BCAA-related transporters. We identified three isoleucine/valine transporters with partial functional redundancy during culture. The deletion of one of these transporters, BrnQ3, resulted in attenuated virulence. Interestingly, a BCAA biosynthesis mutant grew well in medium containing BCAAs but, like BrnQ3, was attenuated for virulence. These results suggest that BCAAs are limiting in multiple niches during infection and further our understanding of the nutritional requirements of this important pathogen.

Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named "XrrA and XrrB" (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post-translational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr- EI- mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.

The Bacillus cereus group includes several Bacillus species with closely related phylogeny. The most well-studied members of the group, B. anthracis, B. cereus, and B. thuringiensis, are known for their pathogenic potential. Here, we present the historical rationale for speciation and discuss shared and unique features of these bacteria. Aspects of cell morphology and physiology, and genome sequence similarity and gene synteny support close evolutionary relationships for these three species. For many strains, distinct differences in virulence factor synthesis provide facile means for species assignment. B. anthracis is the causative agent of anthrax. Some B. cereus strains are commonly recognized as food poisoning agents, but strains can also cause localized wound and eye infections as well as systemic disease. Certain B. thuringiensis strains are entomopathogens and have been commercialized for use as biopesticides, while some strains have been reported to cause infection in immunocompromised individuals. In this article we compare and contrast B. anthracis, B. cereus, and B. thuringiensis, including ecology, cell structure and development, virulence attributes, gene regulation and genetic exchange systems, and experimental models of disease.

Chapter 55 The Bacillus cereus Group: Bacillus Species with Pathogenic Potential Monika Ehling-Schulz, Monika Ehling-SchulzSearch for more papers by this authorDidier Lereclus, Didier LereclusSearch for more papers by this authorTheresa M. Koehler, Theresa M. KoehlerSearch for more papers by this author Monika Ehling-Schulz, Monika Ehling-SchulzSearch for more papers by this authorDidier Lereclus, Didier LereclusSearch for more papers by this authorTheresa M. Koehler, Theresa M. KoehlerSearch for more papers by this author Book Editor(s):Vincent A. Fischetti, Vincent A. Fischetti The Rockefeller UniversitySearch for more papers by this authorRichard P. Novick, Richard P. Novick Skirball Institute for Molecular Medicine, NYU Medical CenterSearch for more papers by this authorJoseph J. Ferretti, Joseph J. Ferretti Department of Microbiology & Immunology, University of Oklahoma Health Science CenterSearch for more papers by this authorDaniel A. Portnoy, Daniel A. Portnoy Department of Molecular and Cellular Microbiology, University of California, BerkeleySearch for more papers by this authorMiriam Braunstein, Miriam Braunstein Department of Microbiology and Immunology, University of North Carolina, Chapel HillSearch for more papers by this authorJulian I. Rood, Julian I. Rood Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash UniversitySearch for more papers by this author First published: 01 October 2019 https://doi.org/10.1128/9781683670131.ch55 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary The microorganisms constituting the Bacillus cereus group are Gram-positive low-GC-content bacteria belonging to the phylum Firmicutes. The group of spore-forming, aerobic, facultative anaerobic, rod-shaped bacteria comprises at least eight closely related species: B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. cytotoxicus, and B. toyonensis (1). With the exception of B. cytotoxicus, which is the most divergent of the group, with a chromosome of 4.085 Mb (2), the genomes of the B. cereus group species are highly conserved, with sizes of 5.2- to 5.9-Mb and very similar 16S rRNA gene sequences. Gram-Positive Pathogens, Third Edition RelatedInformation

Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075, an atxA-regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 "skiA" for "sporulation kinase inhibitor." Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.

SummaryBacillus anthracis produces three regulators, AtxA, AcpA and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed ‘PCVRs’ (Phosphoenolpyruvate‐dependent phosphotransferase regulation Domain‐Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA level or activity result in capBCADE transcription in strains lacking acpA and acpB. We used RNA‐Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Plasmid and chromosome‐borne genes were PCVR controlled, with AtxA, AcpA and AcpB having a ≥ 4‐fold effect on transcript levels of 145, 130 and 49 genes respectively. Several genes were coregulated by two or three PCVRs. We determined that AcpA and AcpB form homomultimers, as shown previously for AtxA, and we detected AtxA‐AcpA heteromultimers. In co‐expression experiments, AcpA activity was reduced by increased levels of AtxA. Our data show that the PCVRs have specific and overlapping activity and that PCVR stoichiometry and potential heteromultimerization can influence target gene expression.

To survive adverse conditions, some bacterial species are capable of developing into a cell type, the "spore," which exhibits minimal metabolic activity and remains viable in the presence of multiple environmental challenges. For some pathogenic bacteria, this developmental state serves as a means of survival during transmission from one host to another. Spores are the highly infectious form of these bacteria. Upon entrance into a host, specific signals facilitate germination into metabolically active replicating organisms, resulting in disease pathogenesis. In this article, we will review spore structure and function in well-studied pathogens of two genera, Bacillus and Clostridium, focusing on Bacillus anthracis and Clostridium difficile, and explore current data regarding the lifestyles of these bacteria outside the host and transmission from one host to another.

SummaryAtxA is a critical transcriptional regulator of plasmid‐encoded virulence genes in Bacillus anthracis. Bacillus cereus G9241, which caused an anthrax‐like infection, has two virulence plasmids, pBCXO1 and pBC210, that each harbor toxin genes and a capsule locus. G9241 also produces two orthologs of AtxA: AtxA1, encoded on pBCXO1, and AtxA2, encoded on pBC210. The amino acid sequence of AtxA1 is identical to that of AtxA from B. anthracis, while the sequences of AtxA1 and AtxA2 are 79% identical and 91% similar to one another. We found by qRT‐PCR that AtxA1 and AtxA2 function as positive regulators of toxin (AtxA1) and capsule operon (both) transcription in G9241 and that a ΔatxA1 mutant produced lower levels of the anthrax toxins and no hyaluronic acid capsule. Deletion of atxA1 or atxA2 decreased the virulence of spores administered intranasally or subcutaneously to C57BL/6 mice but not to A/J mice, and deletion of both genes rendered spores avirulent in A/J mice. In addition, unlike AtxA1, AtxA2 did not form stable homomultimers in vitro, although AtxA1 and AtxA2 formed heterodimers. Our data show that AtxA1 is the primary regulator of G9241 virulence factor expression and that AtxA1 and AtxA2 are both required for full virulence.

The best-studied members of the Bacillus cereus group, B. anthracis, B. thuringiensis, and B. cereus sensu stricto, are pathogens with common and unique features that facilitate their ability to cause disease. As the etiological agent of anthrax, B. anthracis is the most renowned member of the B. cereus group. Inhalation or ingestion of B. anthracis spores can result in a lethal hemorrhagic septicemia. Anthrax toxin represents an interesting variation on the classic A-B toxin model: one binding/translocating B component, protective antigen (PA), and two enzymatic A components, edema factor (EF) and lethal factor (LF). Opportunistic infections caused by B. thuringiensis and B. cereus sensu stricto are relatively uncommon, but they can have serious consequences whether local or systemic. The chromosomes of B. anthracis, B. thuringiensis, and B. cereus sensu stricto reveal striking sequence similarity and gene synteny, but virulence-associated plasmid content can allow facile discrimination of the three species. A large number of virulence factors have been established for the pathogenic B. cereus group species. Anthrax toxin is the best-studied and arguably the most important virulence factor produced by B. anthracis. The entomopathogenesis of B. thuringiensis is dependent upon the production of characteristic insecticidal parasporal crystals called cryotoxins (Cry) and cytolysins (Cyt). B. anthracis, B. thuringiensis, and B. cereus senso stricto secrete pore-forming toxins of the cholesterol-dependent cytolysin (CDC) family.

In this article we investigate the incorporation of sulfur into Cu(In,Ga)Se2 thin films by controlled diffusion. For this purpose, the conventional stacked elemental layer process has been extended by a subsequent sulfur diffusion step. The sulfur gradient is desirable for achieving a graded band gap of the absorber layer which allows to optimize the absorption properties. The sulfur incorporation can be influenced by the sulfurization temperature, the sulfur supply (partial pressure) and the temperature of the Cu(In,Ga)Se2 pre-treatment. The presence of binary selenides and small grains enhances the speed of sulfur incorporation. A combination of Raman spectroscopy and in-situ as well as ex-situ X-ray diffraction has been used in order to obtain detailed information about the diffusion process. The sulfur incorporation occurs at sulfurization temperatures in the range of 575°C in step 1. By decreasing the selenization temperature in step 2 the amount of sulfur incorporation is enhanced. Furthermore an additional defect spinel phase CuIn5S8 is obtained when sulfur is supplied in excess.

SummaryThe Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA‐binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system‐regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein–protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA‐binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high‐resolution full‐length structure of a PRD‐containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

ABSTRACT Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed “nutritional immunity.” Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis , the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCE The mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood—hemoglobin and serum albumin—can satiate the amino acid requirement for Bacillus anthracis , the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for bacterial pathogens.

Objective: Abdominal aortic vascular graft infection often involves several different organisms. Antibiotic polymethyl methacrylate (PMMA) beads may be effective in controlling infection after debridement, but bacterial species identification and antibiotic susceptibility are often not available at the time of operation, generating a need for a broad-spectrum drug combination for empirical use. We sought to determine an effective antibiotic in PMMA beads for use in abdominal vascular graft infection.Methods: PMMA beads were impregnated with combinations of antibiotics, consisting of daptomycin, tobramycin, and meropenem. Antibiotics were selected on the basis of activity spectrum and heat stability. Beads were placed on separate agar plates with vancomycin-resistant Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus. Antibiotic inhibition was recorded by use of a modified agar-based disk-diffusion method.Results: Daptomycin alone was not active against K. pneumoniae (average = 0 mm). Tobramycin alone was not active against vancomycin-resistant E. faecalis, K. pneumoniae, or methicillin-resistant S. aureus. Tobramycin and daptomycin in combination had moderate broad-spectrum activity with 8- to 14-mm mean inhibition halos. Meropenem showed strong activity against all tested organisms with > 15-mm mean inhibition halos. The addition of daptomycin to meropenem provided improved coverage of gram-positive organisms. The presence of tobramycin reduced the efficacy of meropenem.Conclusions: Antibiotic PMMA beads containing 10% meropenem with 2.5% daptomycin had excellent in vitro activity against typical bacterial species associated with abdominal vascular graft infections. The addition of antibiotic beads may be a useful adjunct in managing such cases. Further studies are required to determine efficacy in clinical practice.

The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself.

The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis. We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wildtype and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA - mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA - mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5' and 3' deletion analysis and site-directed mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A+T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between -13 and +36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax.

Summary AtxA, a unique regulatory protein of unknown molecular function, positively controls expression of the major virulence genes of Bacillus anthracis . The 475 amino acid sequence of AtxA reveals DNA binding motifs and regions similar to proteins associated with the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). We used strains producing native and functional epitope‐tagged AtxA proteins to examine protein–protein interactions in cell lysates and in solutions of purified protein. Co‐affinity purification, non‐denaturing polyacrylamide gel electrophoresis and bis(maleimido)hexane (BMH) cross‐linking experiments revealed AtxA homo‐multimers. Dimers were the most abundant species. BMH cross‐links available cysteines within 13 Å. To localize interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions were tested for multimerization and cross‐linking. All mutants multimerized, but one mutation, C402S, prevented cross‐linking. Thus, BMH uses C402 to make the inter‐molecular bond between AtxA proteins, but C402 is not required for protein–protein interaction. C402 is in a region bearing amino acid similarity to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein oligomerization. Finally, cultures grown with elevated CO 2 /bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO 2 /bicarbonate, suggesting that this host‐associated signal enhances AtxA function by shifting the dimer/monomer equilibrium towards the dimeric state.

ABSTRACTBacillus anthracisshares many regulatory loci with the nonpathogenicBacillusspeciesBacillus subtilis. One such locus issinIR, which inB. subtiliscontrols sporulation, biofilm formation, motility, and competency. AsB. anthracisis not known to be motile, to be naturally competent, or to readily form biofilms, we hypothesized that theB. anthracis sinIRregulon is distinct from that ofB. subtilis. A genome-wide expression microarray analysis ofB. anthracisparental andsinRmutant strains indicated limited convergence of theB. anthracisandB. subtilisSinR regulons. TheB. anthracisregulon includes homologues of someB. subtilisSinR-regulated genes, including the signal peptidase genesipWnear thesinIRlocus and the sporulation genespoIIE. TheB. anthracisSinR protein also negatively regulates transcription of genes adjacent to thesinIRlocus that are unique to theBacillus cereusgroup species. These includecalYandinhA1, structural genes for the metalloproteases camelysin and immune inhibitor A1 (InhA1), which have been suggested to be associated with virulence inB. cereusandB. anthracis, respectively. Electrophoretic mobility shift assays revealed direct binding ofB. anthracisSinR to promoter DNA from strongly regulated genes, such ascalYandsipW, but not to the weakly regulatedinhA1gene. Assessment of camelysin and InhA1 levels in culture supernates fromsinR-,inhA1-, andcalY-null mutants showed that the concentration of InhA1 in the culture supernatant is inversely proportional to the concentration of camelysin. Our data are consistent with a model in which InhA1 protease levels are controlled at the transcriptional level by SinR and at the posttranslational level by camelysin.