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Streptococcus pyogenes , also known as group A Streptococcus , causes a wide variety of diseases, leading to 517,000 deaths yearly. The two-component CovRS system, which responds to MgCl 2 and the antimicrobial peptide LL-37, controls the expression of 15% of the genome.

Membranes contain lipids that are composed of fatty acids (FA) and a polar head. Membrane homeostasis is crucial for optimal bacterial growth and interaction with the environment. Bacteria synthesize their FAs via the FASII pathway. Gram-positive bacteria can incorporate exogenous FAs which need to be phosphorylated to become substrate of the lipid biosynthetic pathway. In many species including staphylococci, streptococci and enterococci, this phosphorylation is carried out by the Fak complex, which is composed of two subunits, FakA and FakB. FakA is the kinase. FakB proteins are members of the DegV family, proteins known to bind FAs. Two or three FakB types have been identified depending on the bacterial species and characterized by their affinity for saturated and/or unsaturated FAs. Some species such as Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possess an uncharacterized additional DegV protein. We identify here this DegV member as a fourth FakB protein, named FakB4. The fakB4 gene is co-regulated with FASII genes suggesting an interaction with endogenous fatty acids. fakB4 deletion has no impact on membrane phospholipid composition nor on the percentage of other major lipids. However, the fakB4 mutant strain produced more lipids and more extracellular membrane vesicles than the wild-type strain. This suggests that FakB4 is involved in endogenous FA binding and controls FA storage or catabolism resulting in a limitation of extracellular FA release via membrane vesicles.

Group A Streptococcus (GAS), a Gram-positive human-specific pathogen, yields 517,000 deaths annually worldwide, including 163,000 due to invasive infections and among them puerperal fever. Before efficient prophylactic measures were introduced, the mortality rate for mothers during childbirth was approximately 10%; puerperal fever still accounts for over 75,000 maternal deaths annually. Yet, little is known regarding the factors and mechanisms of GAS invasion and establishment in postpartum infection. We characterized the early steps of infection in an ex vivo infection model of the human decidua, the puerperal fever portal of entry. Coordinate analysis of GAS behavior and the immune response led us to demonstrate that (a) GAS growth was stimulated by tissue products; (b) GAS invaded tissue and killed approximately 50% of host cells within 2 hours, and these processes required SpeB protease and streptolysin O (SLO) activities, respectively; and (c) GAS impaired the tissue immune response. Immune impairment occurred both at the RNA level, with only partial induction of the innate immune response, and protein level, in an SLO- and SpeB-dependent manner. Our study indicates that efficient GAS invasion of the decidua and the restricted host immune response favored its propensity to develop rapid invasive infections in a gynecological-obstetrical context.

International audience; An outbreak of nosocomial infections due to Streptococcus pyogenes (Group A Streptococcus; GAS) occurred in a post-surgery oncology unit and concerned more than 60 patients and lasted 20 months despite enhanced infection control and prophylaxis measures. All GAS strains were characterized (emm genotype, toxin gene profile and pulse-field gel electrophoresis subtype). Selected strains were sequenced and phylogenetic relationship established. Capacity to form biofilm and interaction with human pulmonary epithelial cells and macrophages were determined. Twenty-six GAS strains responsible for invasive infections (II) and 57 for non-II or colonization were isolated from patients (n = 66) or healthcare workers (n = 13). Seventy strains shared the same molecular markers and 69 the same PFGE pattern; 56 were sequenced. They all belonged to the emerging emm89 clade 3; all but 1 were clonal. Whole genome sequencing identified 43 genetic profiles with sporadic mutations in regulatory genes and acquired mutations in 2 structural genes. Except for two regulatory gene mutants, all strains tested had the same biofilm formation capacity and displayed similar adherence and invasion of pulmonary epithelial cells and phagocytosis and survival in human macrophages. This large outbreak of GAS infection in a post-surgery oncology unit, a setting that contains highly susceptible patients, arose from a strain of the emergent emm89 clade. No relationship between punctual or acquired mutations, invasive status, and strain phenotypic characteristics was found. Noteworthy, the phenotypic characteristics of this clone account for its emergence and its remarkable capacity to elicit outbreaks.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall–anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28(Nt), R28 promotes bacterial adhesion to both endometrial–epithelial and endometrial–stromal cells. R28(Nt) was further subdivided into two domains, and we found that both are involved in cell binding. R28(Nt) and both subdomains interacted directly with the laminin-binding α3β1, α6β1, and α6β4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28(Nt) also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype.

Context Emm1-type group A Streptococcus (GAS), or Streptococcus pyogenes, is mostly responsible for invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The recommended treatment of severe invasive GAS infections is a combination of clindamycin and penicillin. Until 2012, almost all emm1 isolates were susceptible to clindamycin. Objectives We aimed to identify the phenotypic and genotypic characteristics of emm1 GAS clone resistant to clindamycin. Methods GAS strains were characterized by emm sequence typing, detection of genes encoding pyrogenic exotoxins or superantigens. Cluster analysis was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic susceptibility was assessed using disk diffusion and resistance genes were detected by PCR. Results A total of 1321 GAS invasive isolates were analyzed between January 2011 and December 2012. The overall number of invasive isolates resistant to clindamycin was 52 (3.9%); seven of them were emm1 isolates. All isolates had the same genomic markers: macrolide resistance due to the presence of the erm(B) gene, emm subtype 1.0, the same toxin or superantigen profile, PFGE pattern and sequence type. Conclusion This is the first description of highly virulent GAS emm1 isolates resistant to clindamycin in France. This article strengthens the need for monitoring the epidemiology of invasive GAS strains as they could lead to changes in treatment guidelines.

International audience; Streptococcus agalactiae (group B streptococcus (GBS)) is the leading cause of invasive infections among newborns in industrialized countries, with two described syndromes: early-onset disease (EOD) and late-onset disease (LOD). Since the introduction in many countries of intrapartum antibioprophylaxis (IAP), the incidence of EOD has dramatically decreased, whereas that of LOD remains unchanged. We describe the clinical and bacteriological characteristics of 438 GBS neonatal invasive infections notified to the French National Reference Centre for Streptococci in France from 2007 to 2012. Clinical data were retrieved from hospitalization reports or questionnaires. Capsular type, assignment to the hypervirulent clonal complex (CC)17 and antibiotic susceptibility profiles were determined. One hundred and seventy-four (39.7%) and 264 (60.3%) isolates were responsible for EOD, including death in utero, and LOD, respectively. EOD was associated with bacteraemia (n = 103, 61%) and LOD with meningitis (n = 145, 55%). EOD was mainly due to capsular polysaccharide (CPS) III isolates (n = 99, 57%) and CPS Ia isolates (n = 40, 23%), and CPS III isolates were responsible for 80% (n = 211) of LOD cases. CC17 accounted for 80% (n = 121) of CPS III isolates responsible for meningitis (n = 151; total cases of meningitis, 188). Bad outcome risk factors were low gestational age and low birthweight. LOD represents almost 60% of cases of neonatal GBS disease in France and other countries in which IAP has been implemented. This observation reinforces the need to develop new prevention strategies targeting CC17, which is predominant in GBS neonatal infections.

The Group B Streptococcus (GBS) 'hypervirulent' ST-17 clone is strongly associated with invasive neonatal meningitis. Comparative genome analyses revealed that the serine-rich repeat (Srr) glycoprotein Srr2 is a cell wall-anchored protein specific for ST-17 strains, the non-ST-17 isolates expressing Srr1. Here, we unravel the binding capacity of GBS Srr proteins to relevant components of the host fibrinolysis pathway. We demonstrate that: (i) Srr2 binds plasminogen and plasmin whereas Srr1 does not; (ii) the ability of ST-17 strains to bind fibrinogen reflects a high level surface display of Srr2 combined with a higher affinity of Srr2 than Srr1 to bind this ligand; and (iii) Srr2 binding to host plasma proteins results in the formation of bacterial aggregates that are efficiently endocytosed by phagocytes. Importantly, we show that Srr2 increased bacterial survival to phagocytic killing and bacterial persistence in a murine model of meningitis. We conclude that Srr2 is a multifaceted adhesin used by the ST-17 clone to hijack ligands of the host coagulation system, thereby contributing to bacterial dissemination and invasiveness, and ultimately to meningitis.

Sixty-three cases of Streptococcus pyogenes meningitis in adults were studied. Three predominant emm types were associated with meningitis: emm1 (44%), emm3 (11%), and emm6 (11%). Streptococcal toxic shock syndrome and mortality rates were 40% and 38%, respectively.

Resume Le streptocoque du groupe A (SGA) est un germe humain responsable d’un large eventail de manifestations cliniques. Les infections invasives a SGA sont en recrudescence depuis le milieu des annees 1980. Afin d’etudier l’epidemiologie des infections invasives a SGA en France, nous avons caracterise toutes les souches invasives recues au Centre national de reference des streptocoques (CNR-Strep) entre 2007 et 2011 et analyse les questionnaires de renseignements cliniques accompagnant les souches. Pour chaque souche ont ete determines le genotype emm , les genes de superantigenes et la sensibilite aux antibiotiques. Sur les 2 603 souches invasives non redondantes de SGA etudiees, 65,1 % ( n = 1 695) provenaient d’hemoculture. Un syndrome de choc toxique streptococcique (SCTS) etait decrit dans 16,4 % ( n = 428) des cas, notamment en cas de dermohypodermite necrosante (DHN), d’infections pleuropulmonaires ou osteo-articulaires ( p ≤ 0,001). Le taux de mortalite s’elevait a 10,6 %. Au total, 102 genotypes emm differents ont ete identifies. Trois genotypes predominaient, atteignant pres de 60 % des souches : emm 1 (26,7 %), emm 28 (16,4 %), et emm 89 (12,8 %). La preponderance de chaque genotype emm etait variable selon les annees et l’âge des patients. Chez les emm 1 (36,8 %), emm 12 (12,9 %) et emm 4 (9,5 %). La distribution des genes de superantigenes ( Spe A, Spe C et Ssa ) etait restreinte a certains genotypes emm . Entre 2007 et 2011, le taux de resistance aux macrolides des SGA a diminue de 7,8 a 5,5 %. Les souches emm 1 restent les plus repandues, en particulier lors des manifestations cliniques les plus severes (SCTS, DHN).

International audience; Purpose: Bacillus causes a sight-threating infection of the posterior segment of the eye. The robust intraocular inflammatory response in this disease is likely activated via host innate receptor interactions with components of the Bacillus cell envelope. S-layer proteins (SLPs) of some Gram-positive pathogens contribute to the pathogenesis of certain infections. The potential contributions of SLPs in eye infection pathogenesis have not been considered. Here, we explored the role of a Bacillus SLP (SlpA) in endophthalmitis pathogenesis.Methods: The phenotypes and infectivity of wild-type (WT) and S-layer deficient (ΔslpA) Bacillus thuringiensis were compared. Experimental endophthalmitis was induced in C57BL/6J mice by intravitreally injecting 100-CFU WT or ΔslpA B. thuringiensis. Infected eyes were analyzed by bacterial counts, retinal function analysis, histology, and inflammatory cell influx. SLP-induced inflammation was also analyzed in vitro. Muller cells (MIO-M1) were treated with purified SLP. Nuclear factor-κB (NF-κB) DNA binding was measured by ELISA and expression of proinflammatory mediators from Muller cells was measured by RT-qPCR.Results: Tested phenotypes of WT and ΔslpA B. thuringiensis were similar, with the exception of absence of the S-layer in the ΔslpA mutant. Intraocular growth of WT and ΔslpA B. thuringiensis was also similar. However, eyes infected with the ΔslpA mutant had significantly reduced inflammatory cell influx, less inflammatory damage to the eyes, and significant retention of retinal function compared with WT-infected eyes. SLP was also a potent stimulator of the NF-κB pathway and induced the expression of proinflammatory mediators (IL6, TNFα, CCL2, and CXCL-1) in human retinal Muller cells.Conclusions: Taken together, our results suggest that SlpA contributes to the pathogenesis of Bacillus endophthalmitis, potentially by triggering innate inflammatory pathways in the retina.

Summary Peptidoglycan deacetylases (PGNG-dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG-dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG-dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG-dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S-layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.

The Bacillus cereus group of bacteria is a group of closely related species that are of medical and economic relevance, including B. anthracis , B. cereus , and B. thuringiensis . Bacteria from the Bacillus cereus group encode three large, highly conserved genes of unknown function (named crdA , crdB , and crdC ) that are composed of 16 to 35 copies of a repeated domain of 132 amino acids at the protein level. Bioinformatic analysis revealed that there is a phylogenetic bias in the genomic distribution of these genes and that strains harboring all three large genes mainly belong to cluster III of the B. cereus group phylogenetic tree. The evolutionary history of the three large genes implicates gain, loss, duplication, internal deletion, and lateral transfer. Furthermore, we show that the transcription of previously identified antisense open reading frames in crdB is simultaneously regulated with its host gene throughout the life cycle in vitro , with the highest expression being at the onset of sporulation. In B. anthracis , different combinations of double- and triple-knockout mutants of the three large genes displayed slower and less efficient sporulation processes than the parental strain. Altogether, the functional studies suggest an involvement of these three large genes in the sporulation process.

Capsule and toxin are the major virulence factors of Bacillus anthracis. The B. anthracis pleiotropic regulator CodY activates toxin gene expression by post-translationally regulating the accumulation of the global regulator AtxA. However, the role of CodY on B. anthracis capsulation and virulence of encapsulated strains has been unknown. The role of CodY in B. anthracis virulence was studied in mouse and guinea pig models. Spore outgrowth and dissemination of the vegetative cells was followed in mice by bioluminescent imaging. We also determined the state of capsulation and the iron requirement for growth of the codY mutant. In all models tested, the codY mutant strain was strongly attenuated compared to the wild-type strain and, in mice, also compared to the atxA strain. The disruption of codY did not affect either ex vivo or in vivo capsulation, whereas atxA deletion affected ex vivo capsulation only. The disruption of codY led to a delayed initiation of dissemination but similar kinetics of subsequent spread of the bacilli. The codY mutant cannot grow on heme iron as sole iron source, whereas the parental and complemented strains can. The lack of CodY-mediated transcription weakens virulence by controlling iron acquisition and synthesis of toxin, but without modifying capsulation.

The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database (http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing (Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.

Fifteen years ago, AtxA was isolated as a toxin gene activator and five years later it was shown to be a Bacillus anthracis master regulator. AtxA controls the expression of more than a hundred genes belonging to all genetic elements, the chromosome and both virulence plasmids, including those encoding the major virulence factors. AtxA can activate or repress gene expression. The mechanism by which AtxA exerts its control is unknown; it is indirect on some genes but may be direct on others. The expression of many AtxA-controlled genes is induced by the presence of bicarbonate/CO(2). AtxA links the metabolic state and virulence gene expression.

Bacillus anthracis is a Gram positive organism possessing a complex parietal structure. An S-layer, a bi-dimensional crystalline layer, and a peptidic capsule surround the thick peptidoglycan of bacilli harvested during infection. A review of the current literature indicates that elements from each of these three structures, as well as membrane components, have been studied. So-called cell-wall secondary polymers, be they attached to the cell-wall or to the membrane play important functions, either per se or because they permit the anchoring of proteins. Some surface proteins, whichever compartment they are attached to, play, as had been hypothesized, key roles in virulence. Others, of yet unknown function, are nevertheless expressed in vivo. This review will focus on well-studied polymers or proteins and indicate, when appropriate, the mechanisms by which they are targeted to their respective locations.

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The σ A-promoted transcription of spo0A was found to be crucial for cereulide production, while the σ H-driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

International audience; The Gram-positive, spore-forming pathogen Bacillus anthracis is the aetiological agent of anthrax. Its main virulence factors are two toxins and an anti-phagocytic capsule. When B. anthracis is grown in laboratory culture, the highest expression of the anthrax toxin genes occurs during entry into stationary phase, suggesting that nutrient limitation is an environmental cue which induces toxin production. A common bacterial response to starvation is the so-called stringent response, in which the hyperphosphorylated guanosine nucleotide (p)ppGpp is the effector molecule. In Escherichia coli, Bacillus subtilis and other bacteria, accumulation of this molecule leads to down-regulation of stable RNA synthesis and upregulation of the expression of genes involved in survival under nutrient-poor conditions. This study focuses on the stringent response of B. anthracis. We show that in B. anthracis the relA gene is responsible for the synthesis of (p)ppGpp and the stringent down-regulation of stable RNA synthesis upon starvation for the essential amino acids isoleucine, leucine and valine. The deletion of relA did not affect the expression of the virulence gene pagA or virulence in a mouse model of infection. In contrast, spore counts upon growth and sporulation in a defined medium were approximately 10,000-fold lower for the relA deletion mutant than for the parental strain. The contribution of the stringent response to efficient sporulation of B. anthracis is notable, as this suggests that the stringent response may contribute to the persistence of B. anthracis in the natural environment.

Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria carry two plasmids, pXO1, and pXO2, encoding toxins and capsule synthetic-enzymes (capB, C, A, dep), respectively. The PXO1 plasmid strongly enhances capsule formation. This influence was studied by analysing the expression of a capB-lacZ fusion in various backgrounds. The beta-galactosidase activities were similar in a delta atxA strain and a pXO1 cured strain. Moreover, the capB-lacZ expression level could be restored, in a pXO1 cured strain, by addition of atxA in trans. Thus, we conclude that the pX01 influence on capsule synthesis is mediated by AtxA, the pXO1-encoded trans-activator of the toxin gene expression.

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. It belongs to theBacillus cereusgroup, which also containsBacillus cereusandBacillus thuringiensis. MostB. anthracisstrains are sensitive to phage γ, but mostB. cereusandB. thuringiensisstrains are resistant to the lytic action of phage γ. Here, we report the identification of a protein involved in the bacterial receptor for the γ phage, which we term GamR (Gamma phagereceptor). It is an LPXTG protein (BA3367, BAS3121) and is anchored by the sortase A. AB. anthracissortase A mutant is not as sensitive as the parental strain nor as the sortase B and sortase C mutants, whereas the GamR mutant is resistant to the lytic action of the phage. Electron microscopy reveals the binding of the phage to the surface of the parental strain and its absence from the GamR mutant. SpontaneousB. anthracismutants resistant to the phage harbor mutations in the gene encoding the GamR protein. AB. cereusstrain that is sensitive to the phage possesses a protein similar (89% identity) to GamR.B. thuringiensis97-27, a strain which, by sequence analysis, is predicted to harbor a GamR-like protein, is resistant to the phage but nevertheless displays phage binding.

Several examples of bacterial surface-structure anchoring have been described, but they do not include polyglutamate capsule. Bacillus anthracis capsule, which is composed only of poly-gamma- d-glutamate, is one of the two major virulence factors of the bacterium. We analysed its anchoring. We report that the polyglutamate is anchored directly to the peptidoglycan and that the bond is covalent. We constructed a capD mutant strain, capD being the fourth gene of the capsule biosynthetic operon. The mutant bacilli are surrounded by polyglutamate material that is not covalently anchored. Thus, CapD is required for the covalent anchoring of polyglutamate to the peptidoglycan. Sequence similarities suggest that CapD is a gamma-glutamyltranspeptidase. Furthermore, CapD is cleaved at the gamma-glutamyltranspeptidase consensus cleavage site, and the two subunits remain associated, as necessary for gamma-glutamyltranspeptidase activity. Other Gram-positive gamma-glutamyltranspeptidases are secreted, but CapD is located at the Bacillus surface, associated both with the membrane and the peptidoglycan. Polyglutamate is hydrolysed by CapD indicating that it is a CapD substrate. We suggest that CapD catalyses the capsule anchoring reaction. Interestingly, the CapD(-) strain is far less virulent than the parental strain.

Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis are three closely related species belonging to the B. cereus group (20, 25, 48). These bacteria are gram-positive spore-forming microorganisms, the spores of which can be isolated from many sources, including insects, soils, dust, and foods (10, 11, 22). B. anthracis, the etiologic agent of anthrax, produces a capsule and toxins encoded by genes carried by the pXO1 and pXO2 plasmids (for a review see reference 35). The expression of these genes is under the control of the key regulator AtxA and the minor regulators AcpA and PagR (16, 24, 36). B. thuringiensis is an entomopathogenic bacterium that produces specific insecticidal toxins encoded by plasmid genes (45). These toxins, the Cry and Cyt proteins, are synthesized when the bacterium enters the stationary phase or the sporulation phase (2). B. cereus is an opportunistic pathogen that is responsible for food poisoning, pneumonia, and endophthalmitis, possibly due to production of an emetic toxin, enterotoxins, and degradative enzymes (3, 8, 15, 26, 34). B. cereus and B. thuringiensis generally produce extracellular proteins that are potentially involved in virulence. Transcription of the genes encoding these proteins is under control of the pleiotropic activator PlcR (1, 30, 38). These putative virulence factors include phospholipases (phosphatidylinositol-specific phospholipase C [PI-PLC], phosphatidylcholine-specific phospholipase C [PC-PLC], sphingomyelinase), cholesterol-dependent hemolysins (cereolysin Clo in B. cereus and thuringiolysin Tlo in B. thuringiensis), proteases (ColB, InhA, Sfp, NprB, NprP2), a cytotoxin (CytK), and hemolytic (Hbl) and nonhemolytic (Nhe) enterotoxins. Disruption of the plcR gene in B. cereus strain ATCC 14579 and B. thuringiensis strain 407 Cry− drastically reduces the hemolytic (Hly−) and cytotoxic properties (43) of these strains, decreases their motility, and attenuates the symptoms of endophthalmitis (9). Two-dimensional (2D) gel electrophoresis has shown that most of the proteins secreted by wild-type B. cereus ATTC 14579 (including phospholipases, hemolysins, enterotoxins, and proteases) are not secreted by the ΔplcR mutant strain (13). Opp, an oligopeptide permease, is necessary for plcR expression, suggesting that uptake of a small peptide is required to activate PlcR-regulated gene expression (14). PlcR activation requires a small peptide, designated PapR, which acts as a cell-cell signaling peptide. Agaisse and collaborators (1) showed that papR is a PlcR-regulated gene located 70 bp downstream from plcR and encoding a 48-amino-acid peptide. This peptide is secreted by the cell and then reimported via Opp, presumably as a pentapeptide. Intracellular PapR is required for binding of PlcR to its DNA targets and thus is required to trigger expression of the PlcR regulon (46). The importance of the PlcR regulon in the B. cereus group was recently emphasized by the publication of the genome sequences of B. cereus ATCC 14579 and B. anthracis Ames (21, 42). More than 100 genes putatively belong to the PlcR regulon on the basis of the presence of a PlcR DNA-binding sequence upstream from these genes in B. cereus ATCC 14579. Most of these genes are also present in B. anthracis. However, B. anthracis does not produce the PlcR-regulated extracellular proteins even though the corresponding genes are present. Most B. cereus and B. thuringiensis strains are hemolytic on sheep blood agar plates, whereas B. anthracis is not hemolytic. This characteristic is commonly used to distinguish between B. anthracis and B. cereus or B. thuringiensis, although it is not considered to be a decisive criterion (29). B. anthracis Sterne does not produce PlcR-regulated proteins due to a nonsense mutation in the plcR gene (1). Mignot and collaborators (33) showed that this mutation in the plcR gene of B. anthracis might result from incompatibility between the PlcR and AtxA regulons. Indeed, B. anthracis is unable to sporulate when it is complemented with a functional plcR gene that restores expression of the PlcR regulon. Point mutations in regulators or in major virulence genes could be one of the mechanisms responsible for the genetic shaping and divergence of closely related populations (5). For example, there is genetic variability among different Clostridium difficile clinical isolates due to deletions in the tcdC gene, which is the putative transcriptional repressor of the A and B toxins and thus affects the toxicity of strains (47). Thus, a point mutation in a key regulator gene, in association with acquisition of mobile genetic elements harboring virulence factors (transposons, plasmids, bacteriophages), may result in distinctive differences between closely related species that may lead to a specialization, like multiplication in mammals for B. anthracis (for a review see reference 18, 19). In this study, we first aimed to determine whether, because of their monomorphism, distinct B. anthracis strains had the same plcR mutation. We then sought B. thuringiensis and B. cereus strains that had lost phenotypic characteristics like hemolysis and lecithinase activity. We wanted to find out whether inactivation of a pleiotropic regulator, such as PlcR, was restricted to B. anthracis or if it was widespread in other species belonging to the B. cereus group.

Summary Transcription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal believed to reflect the host environment. A 180 kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation, pagR and atxA; the latter encodes a major trans-activator. It has long been known that pXO1 genes have major effects on the physiology of B. anthracis, probably through regulatory cross-talk between plasmid and chromosomal genes. Accordingly, we found that the chromosomal S-layer genes, sap and eag, are regulated by pXO1 genes so that only eag is significantly expressed in the presence of CO2. This effect results from the product of pagR acting as the most downstream element of a signalling cascade initiated by AtxA. In vitro evidence showed that PagR is a transcription factor that controls the S-layer genes by direct binding on their promoter regions. This work provides evidence that AtxA is a master regulator that co-ordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements.

Ribotyping of various Bacillus strains with one restriction enzyme (AccI) revealed significant similarity between Bacillus anthracis strains, Bacillus thuringiensis and Bacillus cereus strains, which are all members of the Bacillus cereus group. A further ribotyping study of 10 virulent and 8 attenuated B. anthracis strains, using 4 endonucleases and both 23S and 16S probes independently, was performed. The discrimination index D of Hunter and Gaston showed that the best combination for future large-scale ribotyping studies would be either the combination of AccI and 23S, or that of EcoRI and 16S. Depending on the B. anthracis strain analyzed 10 or 11 rRNA operons were found. In all cases, many strains were grouped into 2 to 3 patterns. Attenuated strains, including a laboratory-cured strain, yielded aberrant patterns.

Summary Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are members of the Bacillus cereus group. These bacteria express virulence in diverse ways in mammals and insects. The pathogenic properties of B. cereus and B. thuringiensis in mammals results largely from the secretion of non-specific toxins, including haemolysins, the production of which depends upon a pleiotropic activator PlcR. In B. anthracis, PlcR is inactive because of a nonsense mutation in the plcR gene. This suggests that the phenotypic differences between B. anthracis on the one hand and B. thuringiensis and B. cereus on the other could result at least partly from loss of the PlcR regulon. We expressed a functional PlcR in B. anthracis. This resulted in the transcriptional activation of genes weakly expressed in the absence of PlcR. The transcriptional activation correlated with the induction of enzymatic activities and toxins including haemolysins. The toxicity of a B. anthracis PlcR 1 strain was assayed in the mouse subcutaneous and nasal models of infection. It was no greater than that of the parental strain, suggesting that the PlcR regulon has no influence on B. anthracis virulence. The PlcR regulon had dramatic effects on the sporulation of a B. anthracis strain containing the virulence plasmid pXO1. This resulted from incompatible interactions with the major AtxA-controlled virulence regulon. We propose that the PlcR-controlled regulon in B. anthracis has been counterselected on account of its disadvantageous effects.

▪ Abstract Bacillus anthracis was shown to be the etiological agent of anthrax by R. Koch and L. Pasteur at the end of the nineteenth century. The concepts on which medical microbiology are based arose from their work on this bacterium. The link between plasmids and major virulence factors of B. anthracis was not discovered until the 1980s. The three toxin components are organized in two A-B type toxins, and the bacilli are covered by an antiphagocytic polyglutamic capsule. Structure-function analysis of the toxins indicated that the common B-domain binds to a ubiquitous cell receptor and forms a heptamer after proteolytic activation. One enzyme moiety is an adenylate cyclase and the other is a Zn2+metalloprotease, which is able to cleave MAPKKs. The capsule covers an S-layer sequentially composed of two distinct proteins. Knowledge of the toxins facilitates the design of safer veterinary vaccines. Spore-structure analysis could contribute to the improvement of human nonliving vaccines. The phylogeny of B. anthracis within the Bacillus cereus group is also reviewed.

Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus, in some B. thuringiensis strains and in B. anthracis, an S-layer has been described. We investigated how the S-layer is distributed in B. cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non-B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis. Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis, except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.

Despite its possible role in virulence, there has been little molecular characterization of members of the S-layer protein family of the Bacillus cereus group. It is hypothesized that the components of the S-layers are likely to display similar anchoring structures in Bacillus thuringiensis and Bacillus anthracis. Based on inferred sequence similarities, a DNA fragment encoding the cell-wall-anchoring domain of an S-layer component of Bac. thuringiensis subsp. galleriae NRRL 4045 was isolated. The complete gene was identified and sequenced. An ORF of 2463 nt was identified, which was predicted to encode a protein of 821 aa, SlpA. The mature SlpA protein (792 residues) carries three S-layer homology (SLH) motifs towards its amino terminus, each about 50 aa long. These motifs were sufficient to bind Bac. thuringiensis and Bac. anthracis cell walls in vitro by interacting with peptidoglycan-associated polymers, confirming a common wall-anchoring mechanism. The slpA null-allele mutant was constructed and shown to possess no other abundant surface protein. It is proposed that the method described in this paper could be applied to the identification and deletion of any Bac. cereus S-layer gene and is of great value for the molecular and functional characterization of these genes.

The operon encoding the general stress transcription factor ς B and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium Bacillus anthracis by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the Bacillus subtilis sigB operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the B. subtilis sigB operon. Therefore, the B. anthracis sigB operon contains three genes rather than eight as in B. subtilis . The B. anthracis operon is preceded by a ς B -like promoter sequence, the expression of which depends on an intact ς B transcription factor in B. subtilis . It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on B. subtilis ς B . We found that in B. anthracis , both these promoters were induced during the stationary phase and induction required an intact sigB gene. The sigB operon was induced by heat shock. Mutants from which sigB was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenic sigB mutant strain was also less virulent than the parental strain in the mouse model. B. anthracis ς B may therefore be a minor virulence factor.

Bacillus anthracis , the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinant B. anthracis strain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.

Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S-layer). Their corresponding genes have been cloned, shown to be clustered on the chromosome and sequenced. EA1 and Sap each possess three 'S-layer homology' motifs. Single and double disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of both the non-capsulated and capsulated bacilli. When present, the capsule is exterior to, and completely covers, the S-layer proteins, which form an array beneath it. Nevertheless, the presence of these proteins is not required for normal capsulation of the bacilli. Thus both structures are compatible, and yet neither is required for the correct formation of the other. Bacillus anthracis has, therefore, a very complex cell wall organization for a gram-positive bacterium.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Bacillus anthracis produces two toxins composed of three proteins. Genetic tools were constructed to study the regulation of toxin synthesis. They included transcriptional fusions with various reporter genes, in replicative and integrative vectors. The reporter gene xylE, encoding catechol 2,3-dioxygenase, may be valuable for screening of strong promoters, as expression of the gene can be visualized directly and the studies of regulation in B. anthracis. Therefore, transcriptional fusions between a lacZ reporter gene and the toxin genes were constructed. Experiments with a multicopy plasmid in trans suggested that the transcriptional activator(s) of the toxin genes were not titrated. B. anthracis strains, which contain pXO1 carrying multiple copies of fusions, were analysed. Expression of the reporter gene was proportional to the fusion copy number. Indeed, single integration of a suicide plasmid can be distinguished from multiple integration according to the level of resistance to an appropriate antibiotic. Finally, recombination in B. anthracis was found to be very efficient (approximately 10(-2) recombinants per transconjugant cell.

Bacillus anthracis is the causative agent of anthrax that is characterized by septicemia and toxemia. Many vaccine strategies were described to counteract anthrax infection. In contrast with veterinary live vaccines, currently human vaccines are acellular with the protective antigen, a toxin component, as the main constituent. However, in animal models this vaccine is less efficient than the live vaccine. In this study, we analyzed the protection afforded by a single extractable surface element. The poly-γ-d-glutamate capsule is covalently linked to the peptidoglycan. A preparation of peptidoglycan-linked poly-γ-d-glutamate (GluPG) was tested for its immunogenicity and its protective effect. GluPG injection, in mice, elicited the production of specific antibodies directed against poly-glutamate and partially protected the animals against lethal challenges with a non-toxinogenic strain. When combined to protective antigen, GluPG immunization conferred full protection against cutaneous anthrax induced with a fully virulent strain.

Group A Streptococcus (GAS) infections present high morbidity and mortality rates and consequently remain a significant health problem. The emm3 isolates induce more severe pathologies than all others. In this study, we tested, on a collection of invasive and non-invasive emm3 clinical isolates, whether in that genotype the invasive status of the strains affects the innate immune response. We show that phagocytosis is dependent on the invasiveness of the isolates. Interestingly, all emm3 isolates compromise macrophage integrity, already noticeable 1 h after infection. Inflammatory modulators (IL-6, TNF-α and IFN-β) are nevertheless detected during at least 6 h post-infection. This is a likely consequence of the macrophages not being all infected. The efficient and rapid induction of macrophage death could explain the virulence of the emm3 strains.

The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N-terminal two-thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I. The suggested active-site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.

While outbreaks of animal anthrax zoonoses still regularly occur in France, little is known about the epidemiology links between them. We have used the eight-locus multilocus variable-number tandem repeat analysis typing technique against a collection of 50 Bacillus anthracis isolates from France. There were eight distinct genotypes belonging to two dissimilar genetic clusters. Regional strain patterns were observed, with the B2 genotypes prevalent in southern France and the A1a genotypes found only in northern France.

International audience; Bacillus anthracis toxin gene expression requires AtxA, a virulence regulator that also activates capsule gene transcription and controls expression of more than a hundred genes. Here we report that atxA mRNA is 2.7-kb-long and ends, after a 500 nt-long 3' untranslated region, with a stem loop structure followed by a run of U's. The presence of this structure stabilizes atxA mRNA and is necessary for AtxA maximal accumulation, full expression of the PA toxin gene, pagA and optimal PA accumulation. This structure displays terminator activity independently of its orientation when cloned between an inducible promoter and a reporter gene. The 3.6-kb-long DNA fragment carrying both AtxA promoters and the terminator is sufficient for full expression of pagA in the presence of bicarbonate. No pXO1-encoded element other than the DNA fragment encompassing the 2.7 kb atxA transcript and the pagA promoter is required for bicarbonate induction of pagA transcription.

Poly-gamma-glutamate has been described in many Gram-positive organisms. When anchored to the surface, it is a capsule and as such a virulence factor. Based on sequence similarities, few Gram-negative organisms have been suggested to synthesize poly-gamma-glutamate. For the first time, a Gram-negative bacterium, Fusobacterium nucleatum, is shown to produce and secrete poly-gamma-glutamate. Putative poly-gamma-glutamate-synthesizing genes from Gram-negative organisms have been compared with their Gram-positive homologs by in silico analysis, i.e., gene sequence and phylogenetic analysis. Clusters of three instead of four genes were highlighted by our screen. The products of the first two genes display similarity with their Gram-positive equivalents, yet the sequences from the Gram-negative organisms can be distinguished from those of the Gram-positives. Interestingly, the sequence of the predicted product of the third gene is conserved among Gram-negative bacteria but displays no similarity to that of either the third or fourth gene of the Gram-positive operons. It is suggested that, like for Gram-positive bacteria, poly-gamma-glutamate has a role in virulence for pathogens and one in survival for other Gram-negative bacteria.

Bacillus anthracis is the etiological agent of anthrax. In the 1980s, a link was discovered between plasmids and major virulence factors of B. anthracis. Indeed, the three toxin components are encoded by pXO1, a 181-kb plasmid, and the poly-glutamate capsule biosynthetic operon is carried by pXO2, a 97-kb plas-mid. The functions encoded by a few other genes were described after screening or selecting for specific phenotypes, such as regulation of virulence factor synthesis. Despite a renewal of interest in B. anthracis at the end of the twentieth century, which prompted further research and led to complete sequencing of both plas-mids in 1999, only few genes have been fully characterized. These include genes involved in replication, sporulation, and germination. Yet, 40% of the open reading frames (ORFs) are of unknown function, and for most of the others a function has only been predicted in silico. B. anthracis is, on a genetic basis, a Bacillus cereus. B. cereus strains also harbor megaplasmids, some sharing core sequences with pXO1 and pXO2. These plasmids also encode virulence factors or specific environmental adaptive pathways.

The aconitase (citB) gene of Bacillus subtilis is repressed during growth in a medium that contains a rapidly metabolizable carbon source and a source of 2-ketoglutarate. It is derepressed when either of these nutrient sources becomes limiting. Repression by rapidly metabolizable carbon sources was shown previously to depend at least in part on a DNA sequence located 67 to 84 base pairs upstream of the start point of citB transcription. In the present work, this region and surrounding DNA were mutagenized to identify more precisely the target for carbon catabolite repression. Mutations in a symmetric sequence located between positions -73 and -59 led to constitutive transcription from the citB promoter in media that normally provoke catabolite repression. By gel mobility shift assays, it was shown that at least one protein in extracts of B. subtilis binds to the symmetric sequence and that DNA of constitutive mutants binds to this protein much less effectively. A second sequence located near position -45 was also implicated in this regulation. A second form of regulation of citB was also investigated. This gene is known to be derepressed when cells are induced to sporulate by exhaustion of a nutrient broth medium or limitation of guanine nucleotide synthesis. The mutations that led to constitutivity with respect to the carbon source had no effect on citB expression in nutrient broth medium, indicating that control by catabolite repression and control by components of nutrient broth (presumably amino acids) act by different mechanisms.

Expression of the aconitase (citB) gene of Bacillus subtilis is subject to catabolite repression in cells grown in minimal media. In nutrient broth medium, citB expression is low in growing cells but is induced when cells enter sporulation. A 600-base-pair DNA fragment that extends from positions -400 through +200, relative to the transcription start site, was shown to include all of the cis-acting sequences necessary for catabolite repression and sporulation-associated regulation. This was demonstrated by fusing this DNA fragment to the Escherichia coli lacZ gene, integrating the fusion in the amyE locus of the B. subtilis chromosome, and measuring the regulation of expression of beta-galactosidase. By creating a series of deletions from either end of the 600-base-pair fragment, it was possible to define a target for catabolite repression; at least part of this target lies within the sequence between positions -84 and -68. DNA fragments that included positions -84 through +36, when carried on high-copy plasmids, caused derepression of aconitase synthesis, as if a negative regulator were being titrated. The same plasmids caused derepression of citrate synthase activity as well. Deletion of the sequence between positions -84 and -67 abolished this titration effect for both enzymes. Mutations that altered the target for catabolite repression also affected the inducibility of citB at the onset of sporulation, at least when sporulation was induced by the addition of decoyinine, an inhibitor of guanine nucleotide synthesis. When sporulation was induced by exhaustion of nutrient broth, there was no detectable difference in expression of citB-lacZ fusions whether or not they had the citB sequence from positions -84 to -67, suggesting that the mechanisms of regulation of citB in minimal medium and nutrient broth are different.

The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that catalyze the N-acetylation of arylamines and their N-hydroxylated metabolites. These enzymes play a key role in detoxication of numerous drugs and xenobiotics. We report here the cloning, functional expression, and characterization of three new NAT genes (termed banatA, banatB, and banatC) from the pathogen Bacillus anthracis. The sequences of the corresponding proteins are approximately 30% identical with those of characterized eukaryotic and prokaryotic NAT enzymes, and the proteins were recognized by an anti-NAT antibody. The three genes were endogenously expressed in B. anthracis, and NAT activity was found in cell extracts. The three NAT homologues exhibited distinct structural and enzymatic properties, some of which have not previously been observed with other NAT enzymes. Recombinant BanatC displayed strong NAT activity toward several prototypic NAT substrates, including the sulfonamide antibiotic sulfamethoxazole (SMX). As opposed to BanatC, BanatB also had acetyl-CoA (AcCoA) and p-nitrophenyl acetate (PNPA) hydrolysis activity in the absence of arylamine substrates, indicating that it may act as an AcCoA hydrolase. BanatA was devoid of NAT or AcCoA/PNPA hydrolysis activities, suggesting that it may be a new bacterial NAT-like protein with unknown function. Expression of BanatC in Escherichia coli afforded higher-than-normal resistance to SMX in the recombinant bacteria, whereas an inactive mutant of the enzyme did not. These data indicate that BanatC could contribute to the resistance of B. anthracis to SMX.

Several examples of bacterial surface-structure anchoring have been described, but they do not include polyglutamate capsule. Bacillus anthracis capsule, which is composed only of poly-gamma- d-glutamate, is one of the two major virulence factors of the bacterium. We analysed its anchoring. We report that the polyglutamate is anchored directly to the peptidoglycan and that the bond is covalent. We constructed a capD mutant strain, capD being the fourth gene of the capsule biosynthetic operon. The mutant bacilli are surrounded by polyglutamate material that is not covalently anchored. Thus, CapD is required for the covalent anchoring of polyglutamate to the peptidoglycan. Sequence similarities suggest that CapD is a gamma-glutamyltranspeptidase. Furthermore, CapD is cleaved at the gamma-glutamyltranspeptidase consensus cleavage site, and the two subunits remain associated, as necessary for gamma-glutamyltranspeptidase activity. Other Gram-positive gamma-glutamyltranspeptidases are secreted, but CapD is located at the Bacillus surface, associated both with the membrane and the peptidoglycan. Polyglutamate is hydrolysed by CapD indicating that it is a CapD substrate. We suggest that CapD catalyses the capsule anchoring reaction. Interestingly, the CapD(-) strain is far less virulent than the parental strain.

Bacillus anthracis, the aetiological agent of anthrax, synthesizes two surface-layer (S-layer) proteins. S-layers are two-dimensional crystalline arrays that completely cover bacteria. In rich medium, the B. anthracis S-layer consists of Sap during the exponential growth phase. Sap is a modular protein composed of an SLH (S-layer homology)-anchoring domain followed by a putative crystallization domain (Sapc). A projection map of the two-dimensional Sap array has been established on deflated bacteria. In this work, the authors used two approaches to investigate whether Sapc is the crystallization domain. The purified Sapc polypeptide (604 aa) was sufficient to form a crystalline structure, as illustrated by electron microscopy. Consistent with this result, the entire Sapc domain promoted auto-interaction in a bacterial two-hybrid screen developed for the present study. The screen was derived from a system that takes advantage of the Bordetella pertussis cyclase subdomain structure to enable one to identify peptides that interact. A screening strategy was then employed to study Sapc subdomains that mediate interaction. A random library, derived from the Sapc domain, was constructed and screened. The selected polypeptides interacting with the complete Sapc were all larger (155 aa and above) than the mean size of the randomly cloned peptides (approx. 60 residues). This result suggests that, in contrast with observations for other interactions studied with this two-hybrid system, large fragments were required to ensure efficient interaction. It was noteworthy that only one polypeptide, which spanned aa 148–358, was able to interact with less than the complete Sapc, in fact, with itself.

Transcription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal mimicking the host environment. A 182-kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation of transcription, namely atxA and, pagR, the second gene of the pag operon. AtxA has major effects on the physiology of B. anthracis. It coordinates the transcription activation of the toxin genes with that of the capsule biosynthetic enzyme operon, located on the second virulence plasmid, pXO2. In rich medium, B. anthracis synthesises alternatively two S-layer proteins (Sap and EA1). An exponential phase "Sap-layer" is subsequently replaced by a stationary phase "EA1-layer". S-layer gene transcription is controlled by alternative sigma factors and by Sap acting as a transcriptional repressor of eag. Furthermore, in vitro in presence of CO2 and in vivo, AtxA is part of the sap and eag regulatory network. Only eag is significantly expressed in these conditions and this is due to AtxA activating eag and repressing sap transcription. PagR, and not AtxA itself, is the direct effector of this regulation by binding to sap and eag promoter regions. Therefore, PagR mediates the effect of AtxA on eag and sap and is the most downstream element of a signalling cascade initiated by AtxA. Taken together, these results indicate that the B. anthracis transcriptional regulator AtxA is controlling the synthesis of the three toxin components and of the surface elements (capsule and S-layer). Thus, AtxA is a master regulator that coordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements.

Transcription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal believed to reflect the host environment. A 180 kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation, pagR and atxA; the latter encodes a major trans-activator. It has long been known that pXO1 genes have major effects on the physiology of B. anthracis, probably through regulatory cross-talk between plasmid and chromosomal genes. Accordingly, we found that the chromosomal S-layer genes, sap and eag, are regulated by pXO1 genes so that only eag is significantly expressed in the presence of CO2. This effect results from the product of pagR acting as the most downstream element of a signalling cascade initiated by AtxA. In vitro evidence showed that PagR is a transcription factor that controls the S-layer genes by direct binding on their promoter regions. This work provides evidence that AtxA is a master regulator that co-ordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements.