When was bacillus megaterium discovered

Immuno-reactions in all panels were performed with mRFP-recognizing anti-His antibody conjugated with horseradish peroxidase. To analyze whether adsorbed mRFP molecules were tightly bound to the spore surface, post-adsorption reaction spores were washed twice with PBS buffer at pH 3.

As shown in Figure 1C , and supported by densitometric analysis of the dot blot Supplementary Table 2 , the washes at pH 3. Therefore, results presented in Figure 1 suggest that mRFP was efficiently adsorbed and tightly bound to B. To assess whether spore-adsorbed mRFP molecules retained their fluorescence properties, we performed a fluorescence microscopy analysis.

As shown in Figure 2 , post-adsorption reaction spores were associated with a strong fluorescence signal visible around the entire spore surface. Fluorescence microscopy analysis of B. The same microscopy field was observed by phase contrast and fluorescence microscopy. The merge panel is reported. The exposure time was ms. PV is a QM Bcured strain lacking all seven plasmids and, as a consequence, totally lacking the exosporium Manetsberger et al.

In parallel, we also used spores of B. To compare the adsorption efficiency of spores of the B. After the adsorption reactions spores were collected by centrifugation, proteins extracted by SDS-DTT treatment and analyzed by western blotting with mRFP-recognizing anti-His antibody. In an attempt to quantify these apparent differences, unbound mRFP from the adsorption reactions was serially diluted and analyzed by dot blotting Figure 3B.

Monomeric Red Fluorescent Protein adsorption to spores of B. Spores in the pellet fractions were used to extract surface proteins that were subsequently analyzed by western blot A , while the supernatants were serially diluted and analyzed by dot blot B.

Serial dilutions of purified mRFP were used as standards. Immuno-reactions in both panels were performed with anti-His antibody conjugated with horseradish peroxidase.

The adsorption efficiency of spores of the three strains was also analyzed by fluorescence microscopy Figure 4A and Supplementary Figure S1. Microscopy images were analyzed by ImageJ software v1. The analysis of 80 spores of each strain indicated an average fluorescence value per pixel of Efficiency of adsorption of mRFP to spores of B. Exposure times are indicated. Phase contrast and red fluorescence overlays are shown merge panel. B Box plots displaying the fluorescence intensity of eighty different spores of each strain.

The line dividing the box indicates the median value for each strain. P value is less than 0. In addition, results of Figure 4 indicated that B. After the reactions spores were collected by centrifugation and the supernatants containing unbound mRFP were serially diluted and analyzed by dot blotting Figure 5A. Figure 5B displays the results of a densitometric analyses of the dot blot?? Quantitative assessment of mRFP adsorption to B. The reaction mixtures were subsequently subject to centrifugation and the supernatants serially diluted and analyzed by dot blot A.

Error bars show the standard errors of the mean from three experiments and the P value never above 0. An immuno-fluorescence microscopy approach was employed to assess whether adsorbed mRFP molecules were exposed on the surface of B. Based on the results presented in Figure 6 , we hypothesized that mRFP molecules infiltrate through the exosporium and localizes in the inter-coat space between the outer coat and the exosporium, i.

This hypothesis implies that if the exosporium is lacking then all mRFP should be available to the antibody. When the exosporium was not present PV adsorbed mRFP was available to the antibody all around the spore and a complete green ring was formed, supporting the hypothesis that mRFP is internal to the exosporium in QM B spores. Immunofluorescence analysis of mRFP-adsorbed B. The same microscopy field for each reaction is reported together with the merge panel.

The exposure time was ms for all images. Immunofluorescence of mRFP adsorbed to B. For each field phase contrast and immunofluorescence microscopy are shown. While QM B spores used in the experiments of Figure 7 showed a complete red fluorescent ring as in Figure 2 , PV spores showed a very weak red fluorescent signal. With PV spores a red signal was only observed using long exposure times at the fluorescence microscope Figure 4.

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Jason Lee for help with testing the A5 medium, and Dr. Iain Hay for advice and experimental assistance. The authors are also grateful to the Manawatu Microscopy and Imaging Centre for technical assistance in electron microscopy. The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Bioline Reagents Ltd.

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