Molekularbiologische Untersuchungen zur Regulation der Sporulation in Clostridium acetobutylicum

The DNA-binding protein Spo0A is the master regulator for initiation of sporulation in Bacillus subtilis, which is also the case in Clostridium acetobutylicum. Radioactive primer extension experiments revealed two different transcription start poits for spo0A in C. acetobutylicum. Like in B. subtilis the activity of the two start points are depending on the growth phase. The signal S1 allowed to identify a clear sA-dependent -10- and -35-sequence. A sH-dependent -10- and -35-promoter sequence can be postulated from the other signal S3. But in C. acetobutylicum there does not exist the known promoter switching from B. subtilis. The second transcription start point is active only awhile in the stationary phase. It seems that the transcription of spo0A from this start point allows the organism to increase the amount of spo0A transcript momentarily over a critical level, so sporulation can be initiated in due time. Upstream of the clostridial spo0A gene two imperfect palindromic sequences were identified. Closer inspections showed an overlapping of this sequences with potential binding sites of the repressor protein AbrB. Binding of AbrB at the promoter region of spo0A could mean an additional control and very precise regulation of spo0A expression before initiation of sporulation. As a member of the response regulator family of transcription factors Spo0A must be activated by phosphorylation, which is mediated by a phosphorelay consisting of multiple orphan histidin kinases and two phosphorelay proteins, Spo0F and Spo0B in B. subtilis. Clostridia do not contain components of the so-called phosphorelay, with the exception of the transcription factor Spo0A. Overexpression of potential Spo0A phosphorylating orphan histidine kinases of C. acetobutylicum was not successful with the exception of the kinase encoded by CAC0437. Furthermore, the ClosTron® Gene Knockout System was used as an alternative method to identify the kinase responsible for Spo0A phosphorylation