prior to this study, why did researchers never notice that B. anthracis can make both an S-layer and a capsule simultaneously?I need help finding the answer in the article and explan in the shortest way pleasei linked the url for the full article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106848/ Capsule observation. The aspect and homogeneity of capsulation werechecked by India ink exclusion (4).Electron microscopy. (i) Thin sections. Cells were fixed with 2% formaldehyde(made freshly from paraformaldehyde) and 2.5% glutaraldehyde in 0.1 M caco-dylate buffer (pH 7.2) containing 5 mM CaCl₂ (14, 17). After being washed, thecells were postfixed for 2 h with 2% OsO4 in the same buffer. The pelleted.bacteria were embedded in 2% low-melting-point agar (type IX; Sigma) (36).The samples were then treated for 16 h with 0.5% uranyl acetate in water. Afterextensive washing, small blocks were dehydrated with alcohol and embedded inSpurr's medium (Ladd Inc.) (32). Thin sections were stained conventionally andobserved with a Philips CM12 electron microscope.(ii) Immunocytochemistry with thin sections. B. anthracis cells were fixed with2% formaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate-buffered saline(14 mM Na₂HPO4, 7 mM NaH₂PO4, 150 mM NaCl) (PBS) (pH 7.4) for 1 h,rinsed in the same buffer, and embedded in 2% low-melting-point agar (36).Small blocks containing bacteria were embedded in Lowicryl HM20 (Poly-sciences Ltd.) at -50°C following the progressively lower temperatures protocolof Carlemalm et al. (3) as described by Newman and Hobot (25). Thin sectionswere collected onto Formvar-carbon-coated nickel grids and incubated succes-sively at room temperature with the following solutions: PBS-50 mM NH4Cl for10 min; PBS-1% bovine serum albumin (BSA) -1% normal goat serum-0.1%Tween 20 for 10 min; specific anti-EA1 or anti-Sap antibodies diluted 1/50 inPBS-1% BSA-1% normal goat serum-0.1% Tween 20 for 1 h; PBS-0.1% BSAthree times for 5 min each time; goat immunoglobulin G (heavy and light chains)anti-rabbit immunoglobulin-gold conjugate diluted 1/20 in PBS-0.01% gelatin0854 MESNAGE ET AL.ABfor 1 h; PBS three times for 5 min each time; PBS-1% glutaraldehyde for 5 min;and five times with water. The thin sections were then stained by incubation with2% uranyl acetate in water for 35 min and then in lead tartrate for 2 min (23).(iii) Immunocytochemistry with whole-mount cells. Immunocytochemistrywith whole-mount cells was carried out as previously described (20).(iv) Negative staining experiments. B. anthracis cells were resuspended in a1/10 volume of 25 mM Tris-HCl (pH 8.0)-10 mM MgCl₂ with 0.25 or 0.5%glutaraldehyde for EA1 or Sap, respectively, in the presence of approximately 30μl of 425- to 600-μm glass beads (Sigma) and disrupted by vortexing for 30 s. Thistreatment disintegrated the capsule. Negative staining was performed as previ-ously described (20). Micrographs were recorded with a Philips CM12 electronmicroscope under low-dose (17 electrons/Å/s) transmission electron microscopyconditions.ABFIG. 1. Homogeneity of the capsulation state of B. anthracis cells. Cultures of CAF10 (A) or of its derivative, CSM11, with deletions of both S-layer genes (B), grownin capsule synthesis-inducing conditions were incubated in the presence of India ink. The capsule appears as a bright halo surrounding the cells under the lightmicroscope. Magnification, X1,600.RESULTSCosynthesis and respective localization of the capsule andthe S-layer components. All reported data on the B. anthracisS-layer is from noncapsulated strains (6, 7, 10, 16, 20). Wetherefore investigated whether the capsule and the S-layercomponents, EA1 and Sap, could all be simultaneouslypresent. The genes for EA1 and Sap are chromosomal andhave been well characterized for the plasmid-free strain 9131J. BACTERIOL.(20). We therefore used strain CAF10, a pXO2 transductant ofstrain 9131 (8), to address the question.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 capsule was visible 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- 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.ACSmJ. BACTERIOL.(20). We therefore used strain CAF10, a pXO2 transductant ofstrain 9131 (8), to address the question.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 capsule was visible 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 grownс это

Bacillus anthracis is a gram-positive bacteria. It is a virulent and capsulated bacteria. It causes…

Answered: prior to this study, why did…

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