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Facilitator QuestionsOnce engulfed, how many membranes surround the forespore? Indicate the origin of each membrane. FM4-64 is a red fluorescent dye that binds to, but cannot cross, cellular membranes (i.e. it is non-membrane permeable). In contrast, MitoTracker Green is a green fluorescent dye that binds to and crosses lipid bilayers (i.e. membrane permeable). Once inserted into a membrane, both dyes are able to freely diffuse in the lateral plane of the membrane. Explain how you could use these two dyes to distinguish sporangia that are in the process of forespore engulfment versus those have completed forespore engulfment. How could localization of the anti-anti-?F factor E to the asymmetric septum cause preferential activation of ?F in the forespore? The DNA translocase that pumps the forespore chromsome into the forespore is also present during vegetative growth of B. subtilis. Why might that be? Given the availability of fusions of the gene for the Green Fluorescent Protein to promoters under the control of ?F, how could you test the proposition that only a portion of the chromosome is initially present in the forespore after asymmetric division? (Clue: ?F is active immediately following asymmetric septation.) Gene regulation during B. subtilis sporulation has often been described as “criss-cross” regulation. Why?
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