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Function of the ATP-dependent Metalloprotease FtsH during sporulation in Bacillus subtilis
(2008)
- The analysis of the function of the ftsH gene of Bacillus subtilis started about ten years ago. It was shown at that time that an ftsH knockout was viable, but exhibited a pleiotropic phenotype. Cells are sensitive to salt and heat shock, exhibit filamentous growth, are difficult to be transformed and are almost unable to sporulate. Despite the severe phenotype caused by the absence of the ftsH gene, the precise functions of this protein remained unclear. This PhD thesis presents data to elucidate the function of ftsH during sporulation. Furthermore, it describes the construction of a cold-inducible expression system. The major finding of this thesis is that the FtsH protease interferes with the synthesis and/or phosphorylation of Spo0A, the master regulator during initiation of sporulation called phase 0. In the ftsH knockout, the amount of Spo0A is greatly reduced, and the small amounts present are inactive. When the wild-type spo0A allele was replaced by an IPTG-inducible allele coding for mutant Spo0A protein being fully active in the absence of phosphorylation (Spo0A-Sad67), spores were formed at a normal rate in an ftsH knockout. Again, this result indicates that FtsH is clearly involved in the formation of active Spo0A and that this protease is only essential during stage 0 of sporulation. To become active, Spo0A needs to be phosphorylated by the multi-component system called phosphorelay. Since no active Spo0A is present in an ftsH knockout, it was hypothesized that FtsH has to degrade one or more negative regulator(s) either preventing the phosphorylation of Spo0A or/and being involving in its rapid dephosphorylation. The further analysis focused on four antagonists of the phosphorelay, three Rap phosphatases being involved in the dephosphorylation of Spo0F~P, and Spo0E which targets Spo0A~P. When a null allele in any one of them was combined with the ftsH knockout, the wild-type amount of Spo0A was restored only in the case of the ftsH spo0E knockout and the sporulation frequency was increased by two to three orders of magnitude in all double knockouts, but remained below 1%. Since overexpression of Spo0E reduces the sporulation frequency and removal of the gene from the genome has an opposite effect, a direct interaction between FtsH and Spo0E was envisaged. In vitro proteolysis assays with purified GST-FtsH and GST-Spo0E showed that Spo0E is indeed a target of FtsH. In contrast, the two homologs of Spo0E, YisI and YnzD, remained stable upon incubation with FtsH. Since all three proteins are distinguished by a C-terminal extension of about 25 amino acids present in Spo0E, but not in the two other phosphatases, these additional amino acids could serve as a target for FtsH. When two mutant versions of Spo0E, Spo0E94 and Spo0E11, with truncated C-terminal ends were analyzed, they turned out to be stable in the presence of FtsH. When the C-terminal 25 amino acids was transferred to YnzD, this fusion protein became unstable when incubated with FtsH. In conclusion, the C-terminal end of Spo0E confers instability to this enzyme. Since a spo0E knockout in a wild-type background does not result in a sporulation frequency close to 100% and a combination of a spo0E and an ftsH knockout raises the sporulation frequency only close to 1%, it can be concluded that there are additional targets for FtsH interfering with the synthesis of active Spo0A. Moreover, it is likely that FtsH also exerts a function late during sporulation. It could be shown that SpoVM, a small peptide essential for spore morphogenesis, inhibits the proteolytic activity of the B. subtilis FtsH protease in vitro. It can be inferred that SpoVM also inhibits activity of FtsH during sporulation, and in the absence of SpoVM, FtsH will degrade at least one protein essential for successful completion of sporulation. When the intracellular proteomes of spoVM+ and spoVM- cells were compared, a total of 83 proteins were identified being either completely absent or present in reduced amounts in the absence of the peptide. Analysis of the expression of the spoVM gene revealed that cells started to synthesize the spoVM transcript at stage 2 while the SpoVM peptide accumulated at stage 4. The 5ยด untranslated region of the spoVM transcript has been identified to act as a negative regulator of its own transcription or translation. Furthermore, a cold-inducible expression system has been constructed allowing intra- and extracellular production of recombinant proteins. This expression system makes use of a two-component signal transduction system, which senses changes in the fluidity of the cytoplasmic membrane.
