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-specific sigma factors,H (early),F,E,G, andK (late). Here we show that the Clostridium acetobutyli-cumK acts both early, prior to Spo0A expression, and late, pastG activation, thus departing from the B. subtilismodel. The C. acetobutylicum sigK deletion (sigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electronmicroscopy demonstrated that thesigKmu-tant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A andG proteins were not detectable byWestern analysis, whileF protein levels were significantly reduced in thesigKmutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in thesigKmutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show thatK is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in thesigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A,F,E, andG, but not sporulation, which was blocked past theG stage of development, thus demonstrating thatK 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. Clostridium acetobutylicum is a Gram-positive obligate anaer-obe that is capable of forming environmentally resistant en