- Trap-Mutante (1) (remove)
- Identification of substrate proteins of FtsH during sporulation of Bacillus subtilis (2012)
- FtsH is an ATP- and Zn2+-dependent metalloprotease anchored in the cytoplasmic membrane by two transmembrane segments. It is the unique membrane-bound AAA-protease in bacteria that performs a variety of regulatory functions. In B. subtilis, an ftsH knockout exhibits a pleiotropic phenotype such as filamentous growth, sensitivity towards heat, osmotic shock and cells are unable to sporulate. Recently, it has been shown that ftsH knockout cells fail to entry sporulation stage II due to lack of a sufficient amount of Spo0A~P and the first substrate of FtsH identified in B. subtilis is the Spo0E phosphatase, a negative regulator that dephosphorylates Spo0A~P. However, the sporulation frequency in a spo0E ftsH double mutant strain was only partly restored, we hypothesized that FtsH might degrade additional substrate proteins involved in sporulation. To identify these proteins, two different strategies were applied. By using the 2D gel technique, the proteomes of an ftsH wild-type strain was compared with an ftsH null mutant. Several proteins were identified to be either up- or down-regulated in the absence of FtsH. One of them up-regulated about 4-fold was identified as Spo0M. Since ftsH did not interfere with transcription of spo0M, an in vitro proteolysis assay was established using purified components. It was shown that Spo0M was degraded by FtsH in an ATP- and time-dependent way. In the second strategy, an ftsHtrap mutant was constructed and tested for loss of its proteolytic activity. Protease trap mutants are still able to bind substrate proteins, but are unable to degrade them. By using FtsHtrap fused to a GST-tag, YwnF, a membrane protein, was trapped and identified as a substrate of FtsH by mass spectrometry. However, further experiments will be required to confirm YwnF as a target of FtsH. The last part of this thesis was focused on the eag gene, which forms a bicistronic operon with Spo0E. Construction and analysis of an eag insertion mutant exhibited a slight increase in the sporulation frequency and in the amount of Spo0A. A transcriptional fusion between the promoter of the spo0E-eag operon and the lacZ reporter gene revealed an increase in the beta-galactosidase activity from t0 when the cells were grown in sporulation medium. Since the Eag protein may be an integral membrane protein, it may bind excess Spo0E thereby preventing it from dephosphorylating Spo0A~P. Alternatively, Eag may bind Spo0E and present it as a modulator to FtsH for degradation.