In figure 3, what do the letters C, S, P, and m indicate in panel A?I need help finding the answer in the article link to article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106848/ The Capsule and S-Layer: Two Independent and YetCompatible Macromolecular Structures in Bacillus anthracisSTÉPHANE MESNAGE,¹ EVELYNE TOSI-COUTURE,² PIERRE GOUNON,² MICHÈLE MOCK,¹AND AGNES FOUET¹*Toxines et Pathogénie Bactériennes (CNRS URA 1858)¹ and Station Centrale de Microscopie Electronique, ²Institut Pasteur, Paris, FranceReceived 5 September 1997/Accepted 22 October 1997Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulentbacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, arecomponents of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has previouslyonly been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesizedunder conditions where the poly-y-D-glutamic acid capsule is present. Structural and immunological analysesshow that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of singleand double S-layer protein mutants shows that the presence of these proteins is not required for normalcapsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in thepresence of the capsule. Thus, both structures are compatible, and yet neither is required for the correctformation of the other.Bacillus anthracis, a gram-positive spore-forming bacterium,is the causative agent of anthrax. This disease, to which manyanimals, including humans, are susceptible, involves toxemiaand septicemia. In the mammalian host, B. anthracis bacillisynthesize two toxins (lethal and edema toxins) (31) and acapsule (18) encoded by two large plasmids, pX01 and pXO2,respectively (12, 21). The capsule is composed of poly-y-D-glutamic acid and has antiphagocytic properties (13, 31, 37).Although unusual, a similar capsule is also found on Bacilluslicheniformis bacilli (9). In the absence of pXO2 or the inducerbicarbonate, the cell does not produce a capsule and the cellwall appears layered. These layers are composed of fragmentsdisplaying a highly patterned ultrastructure (10, 16). This typeof cell surface is now referred to as the surface layer (S-layer).S-layers are present on the surfaces of many archaea andbacteria (for reviews, see references 29 and 30). Most areformed by noncovalent, entropy-driven assembly of a single(glyco)protein protomer on the bacterial surface, giving rise toproteinaceous paracrystalline layers. Generally, a single S-layer is present, constituting 5 to 10% of total cell protein. Itssynthesis is thus presumably energy consuming for the bacte-rium. Numerous bacteria have S-layers, suggesting that theyplay important roles in the interaction between the cell and itsenvironment. Various functions have been proposed for S-layers, including shape maintenance and molecular sieving,and they can serve in phage fixation. The S-layer may be avirulence factor, protecting pathogenic bacteria against com-plement killing, facilitating binding of bacteria to host mole-cules, or enhancing their ability to associate with macrophages(for reviews, see references 27 and 29).Some bacteria, such as cyanobacteria or Azotobacter spp.,possess both a capsule and an S-layer; however, to our knowl-edge, their structural relationships have not been analyzedthrough simultaneous genetic and cytologic studies. Both ofthese features have been independently described for the sur-* Corresponding author. Mailing address: Toxines et PathogénieBactériennes, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex15, France. Phone: 33 1 45 68 86 54. Fax: 33 1 45 68 89 54. E-mail:afouet@pasteur.fr.52face of the pathogenic bacterium B. anthracis. The componentsof the B. anthracis S-layer are two abundant surface proteins,EA1 and Sap (6, 20). Previous analyses of the B. anthracisS-layer used plasmid-cured strains; consequently, the interac-tion, if any, between the capsule and the S-layer could not bestudied. Temporal or environmental regulation could be suchthat only one or the other structure is ever present at the cellsurface. However, we show that S-layer proteins are synthe-sized under conditions where the bacilli are capsulated. Wedetermined the localizations of capsule and S-layer compo-nents and analyzed whether the S-layer is necessary for propercapsulation. Finally, the assembly of the S-layer proteins in atwo-dimensional crystal was examined in the presence of thecapsule.MATERIALS AND METHODSPlasmids, bacterial strains, mating experiments, and culture conditions. Theplasmids used to disrupt sap (encoding Sap), eag (encoding EA1), and bothgenes, i.e., PEA1207, pSAL322, and pSAL303, respectively, were described pre-viously (6, 20) and are listed in Table 1. The construction of B. anthracis CAF10,a pXO2 transductant of plasmidless strain 9131, and its regulation of capsulesynthesis have already been reported (8). Escherichia coli JM83 harboringpRK24 was used for mating experiments (34, 35). Allelic exchange was carriedout as previously described (26) with the spectinomycin resistance cassette as aselectable marker (24). sap, eag, and both genes were disrupted in CAF10 byheterogramic conjugation, giving CBA91, CSM91, and CSM11, respectively (Ta-ble 1). E. coli cells were grown in Luria broth on agar plates (22). Capsulesynthesis was induced by growing B. anthracis cells in brain heart infusion me-dium (Difco Laboratories) in the presence of 0.6% sodium bicarbonate or onCAP plates (28) in a 5% CO₂ atmosphere for electron microscopy. Antibioticswere used at the following concentrations: kanamycin, 40 µg/ml for E. coli;erythromycin, 5 µg/ml for B. anthracis; and spectinomycin, 60 µg/ml for both E.coli and B. anthracis.you wProtein analysis. To test the in vivo expression of EA1 and Sap, the synthesisof antibodies was assayed. The rationale of this experiment is that antibodies areproduced only if the antigen is synthesized in vivo. Seven Swiss mice wereinjected with 106 spores of strain CAF10 and sacrificed after 30 days. Their serawere pooled. The gel loading samples were obtained as follows. B. anthracis cellswere harvested at anan optical density at 600 nm of =2. Pellets were washed in 25mM Tris-HCl (pH 8.0), sonicated until complete clarification, and resuspendedin Laemmli buffer (19). Samples (3 μg ofof pellet protein and 20 μl of trichloro-acetic acid-precipitated supernatant protein) were loaded on sodium dodecylsulfate-10% polyacrylamide gels. Separated proteins were transferred to nitro-cellulose sheets by use of the Bio-Rad Trans-Blot system. The sera were used at1/200 dilution. Western blots were developed with the ECL Western blottinganalysis system (Amersham), with a 1/10,000 dilution of the second antibody. 54ABMESNAGE ET AL.FIG. 2. Immunolabeling of S-layer proteins in capsulated CAF10 bacteria.Thin sections were incubated with anti-EA1 (A) or anti-Sap (B) antibodies andthen with 10-nm gold-conjugated anti-rabbit antibodies. The capsule is visible inthese micrographs but was only weakly stained with the protocol used. Bar, 200nm.ABGSm(20). We therefore used strain CAF10, a pXO2 transductant ofstrain 9131 (8), to address the question.J. BACTERIOL.CAF10 was grown in the presence of bicarbonate to inducecapsule synthesis, stained with India ink, and examined byphase-contrast microscopy. The vast majority of the bacteriawere capsulated (Fig. 1A). The presence of EA1 or Sap in thepellets or the supernatants was verified by Western blot anal-yses and suggested that, in vitro in a given population, thepoly-y-D-glutamate capsule, EA1, and Sap could be simulta-neously present (data not shown).These prior experiments showed that a population of cellscould synthesize both capsule and S-layer proteins but couldnot definitively prove that a single cell possessed both at thesame time. To confirm that individual bacterial cells harboredthese three components simultaneously, we analyzed capsu-lated CAF10 bacteria by immunoelectron microscopy (Fig. 2Aand B and Fig. 3A). Optimized capsule visualization (Fig. 3)and immunolabeling (Fig. 2) are not compatible. However,despite these technical limitations, the capsulevisible inthin sections where EA1 (Fig. 2A) and Sap (Fig. 2B) werehighlighted by the corresponding antibodies. This result indi-cated that on the surface of CAF10 bacilli grown in the pres-ence of bicarbonate, EA1, Sap, and the capsule were presentsimultaneously. Moreover, the sites of antibody binding indi-cated that the corresponding antigens were localized under thecapsule (Fig. 2A and B). In thin sections (Fig. 3A), EA1 andSap were found between the capsule and the peptidoglycan. Acomplementary immunoelectron microscopy approach wasused to examine whole-mount cells incubated with specificantibodies. In noncapsulated strains, i.e., 9131 (20) and non-induced CAF10 bacilli (Fig. 4A and C), the anti-Sap and anti-EA1 antibodies covered the entire bacterial surface, whereasno antibody was detected on the cell surface of CAF10 grownCDFIG. 3. Capsule visualization in CAF10 and its various S-layer gene deletion derivatives. (A) CAF10 (EA1+ Sap*). (B) CBA91 (EA1+). (C) CSM91 (Sap+). (D)CSM11. Thin sections of cultures grown in capsule synthesis-inducing conditions were treated during fixation and embedding to highlight the peptidoglycan (p), theS-layer (s), and the fibrillar capsule (c) (see Materials and Methods). The cytoplasmic membrane is also indicated (m). The cell surface components (p, s, c, and m)are indicated in panel A. Note that the S-layer is absent from the bacterium in panel D. Bar, 250 nm.

This description is about the cellular structure of the bacterium bacillus anthracic. This is the…

Compatible Macromolecular Structures in Bacillus anthr STÉPHANE MESNAGE,¹ EVELYNE TOSI-COUTURE,2 PIERRE GOUNON,2 MICHÈLE MO AND AGNÈS FOUET¹* Toxines et Pathogénie Bactériennes (CNRS URA 1858)¹ and Station Centrale de Microscopie Electron Institut Pasteur, Paris, France Received 5 September 1997/Accepted 22 October 1997 Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully acilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antig omponents of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has pr nly been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synt nder conditions where the poly-y-D-glutamic acid capsule is present. Structural and immunological how that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis nd double S-layer protein mutants shows that the presence of these proteins is not required for apsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, eve resence of the capsule. Thus, both structures are compatible, and yet neither is required for the ormation of the other. anthracis, a gram-positive spore-forming bacterium, ative agent of anthrax. This disease, to which many ncluding humans, are susceptible, involves toxemia emia. In the mammalian host, B. anthracis bacilli two toxins (lethal and edema toxins) (31) and a 3) encoded by two large plasmids, pX01 and pXO2, y (12, 21). The capsule is composed of poly-y-D- cid and has antiphagocytic properties (13, 31, 37). unusual, a similar capsule is also found on Bacillus is bacilli (9). In the absence of pXO2 or the inducer e, the cell does not produce a capsule and the cell face of the pathogenic bacterium B. anthracis. of the B. anthracis S-layer are two abundant EA1 and Sap (6, 20). Previous analyses of S-layer used plasmid-cured strains; conseque tion, if any, between the capsule and the S-l studied. Temporal or environmental regulati that only one or the other structure is ever p surface. However, we show that S-layer pro sized under conditions where the bacilli are determined the localizations of capsule and nents and analyzed whether the S-layer is nec