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

Abstract

e 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 , and K (late). Here we show that the Clostridium acetobutylicum K acts both early, prior to Spo0A expression, and late, past G activation, thus departing from the B. subtilis model. 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 electron microscopy demonstrated that the ⌬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 G proteins were not detectable by Western analysis, while F protein levels were significantly reduced in the ⌬sigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ⌬sigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that K is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ⌬sigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, F , E , and G , but not sporulation, which was blocked past the G stage of development, thus demonstrating that 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.

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

“…Surprisingly, disruption of sigK in C. difficile did not lead to the complete loss of sporulation, as 10 3 heat-resistant spores/ml were detected at 72 h (25,94). This finding is in stark contrast to the sigK mutants generated in B. subtilis, C. perfringens, C. botulinum, and C. acetobutylicum, all of which resulted in an asporogenous phenotype (37,63,64,66). TEM image analysis revealed that the C. difficile sigK mutant produced forespores with what appears to be a cortex layer, but no coat layer was observed (94) (Fig.…”

“…TEM image analysis (Fig. 5) showed that these cells proceeded to a more advanced stage of spore morphogenesis than did cells of the C. acetobutylicum sigG disruption mutant (26) and appeared to be releasing an immature or severely underdeveloped forespore (66). These findings demonstrate that K plays a critical role in late sporulation as well as in very early sporulation prior to Spo0A activation, as supported by the nonsporulating phenotype of the ⌬sigK(p94Spo0A) strain.…”

“…Deletion of the sigK gene in C. acetobutylicum (66,67) abolished the mutant's (named ⌬sigK) ability to express Spo0A, as evidenced by the lack of Spo0A protein in Western blots, and hence, the ability to sporulate and produce solvents was also abolished. Moreover, protein and/or transcript levels of F , E , and G were all diminished.…”

“…A third phenotype relates to the mechanism by which, as discussed above, K activity becomes available for very, very early exponential growth of C. acetobutylicum to make Spo0A expression possible (66). We argued that epigenetic inheritance may play a role here (66).…”

“…However, spore assays and TEM analysis revealed that the ⌬sigK(p94Spo0A) strain (Fig. 5) was still unable to successfully complete the sporulation process (66). TEM image analysis (Fig.…”