Question

Spo0A is part of the Spo0A phosphorelay and two-component
regulatory system that
coordinates sporulation of Bacillus subtilis.
a. Spo0A initiates sporulation when B. subtilis senses high cell density. Draw a
schematic to illustrate the key steps.
b. What mutations could you engineer to block activation but not expression of
Spo0A?
c. What mutations could you engineer to generate a strain that initiates sporulation at
a cell density higher than normal? Lower?

Answer 1

a. Steps of phosphorelay initiating sporulation

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b. The regulatory function of the anti-anti-sigmaF SpoIIAA can act as inhibitor of Spo0A activation. This effect does not require sigmaF activity, and can be abolished by expression of the phosphorelay-independent form Spo0A-Sad67. This suggests SpoIIAA directly interferes with Spo0A approximately P generation. IPTG-directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB can block Spo0A. The active form of the inhibitor is SpoIIAA and not SpoIIAA-P.

c. A new strain can be generated by placing  inducible Ptet promoter. Ptet regulatory elements can be placed upstream of the master regulator of sporulation, spo0A. This can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). This strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. TetR, represses both Ptet and PtetR. This causes a negative feedback loop to the system allowing an increased level of control.