σK of Clostridium acetobutylicum Is the First Known Sporulation-Specific Sigma Factor with Two Developmentally Separated Roles, One Early and One Late in Sporulation

Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specificsigma factors, sigma(H) (early), sigma(F), sigma(E), sigma(G), and sigma(K) (late). Here we show that the Clostridium acetobutylicum sigma(K) acts both early, prior to Spo0A expression, and late, past sigma(G) activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (Delta sigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the Delta sigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and sigma(G) proteins were not detectable by Western analysis, while sigma(F) protein levels were significantly reduced in the Delta sigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the Delta sigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that sigma(K) is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the Delta sigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, sigma(F), sigma(E), and sigma(G), but not sporulation, which was blocked past the sigma(G) stage of development, thus demonstrating that sigma(K) is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.