One of the early results shows that the post-centrifugation pellet of encapsulated cells also contains EA1 and/or Sap. Why is this not proof that Bacillus anthracis cells have both an S-layer and a capsule simultaneously?I need help finding the answer in the article and explain in short answer link to article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106848/ 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.ABGmJ. 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 grownDFIG. 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. ABFIG. 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.ABCmTERIOL.(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 grownDFIG. 3. Capsule visualization in CAF10 and its various S-layer gene deletion derivatives. (A) CAF10 (EA1+ Sap*). (B) CBA91 (EA1¹). (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)

The presence of EA1 and/or Sap in the post-centrifugation pellet of encapsulated cells does not…

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Chapter1: The Human Body: An Orientation

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