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In order to better characterize the initial stages of sporulation past Spo0A activation and the associated solventogenesis in the important industrial and model organismClostridium acetobutylicum , thespoIIE gene was successfully disrupted and its expression was silenced. By silencingspoIIE , sporulation was blocked prior to asymmetric division, and no mature spores or any distinguishable morphogenetic changes developed. Upon plasmid-based complementation ofspoIIE , sporulation was restored, although the number of spores formed was below that of the plasmid control strain. To investigate the impact of silencingspoIIE on the regulation of sporulation, transcript levels ofsigF ,sigE , andsigG were examined by semiquantitative reverse transcription-PCR, and the corresponding σF , σE , and σG protein levels were determined by Western analysis. Expression ofsigF was significantly reduced in the inactivation strain, and this resulted in very low σF protein levels. Expression ofsigE was barely detected, and nosigG transcript was detected at all; consequently, no σE or σG proteins were detected. These data suggest an autostimulatory role for σF inC. acetobutylicum , in contrast to the model organism for endospore formation,Bacillus subtilis , and confirm that high-level expression of σF is required for expression of σE and σG . Unlike the σF and σE inactivation strains, the SpoIIE inactivation strain did not exhibit inoculum-dependent solvent formation and produced good levels of solvents from both exponential- and stationary-phase inocula. Thus, we concluded that SpoIIE does not control solvent formation.

journal of bacteriology

SpoIIE Is Necessary for Asymmetric Division, Sporulation, and Expression of σ F , σ E , and σ G but Does Not Control Solvent Production in Clostridium acetobutylicum ATCC 824