The Spo0E phosphatase of Bacillus subtilis is a substrate of the FtsH metalloprotease.

In the absence of the ATP-dependent metalloprotease FtsH, the sporulation frequency of Bacillus subtilis cells is reduced by several orders of magnitude. This indicates that FtsH has to degrade or to regulate the steady-state level of one or more proteins that interfere with successful sporulation. Here, we show that the amount of the master regulator protein Spo0A is reduced in an ftsH knockout and the small amounts of Spo0A protein present are inactive. Phosphorylation of Spo0A occurs through a phosphorelay. Four negative regulators have been identified here which directly interfere with the phosphorelay through ftsH, namely the phosphatases RapA, RapB, RapE and Spo0E. If a null allele in any one of them was combined with an ftsH knockout, the sporulation frequency was increased by two to three orders of magnitude, but remained below 1 %. When purified Spo0E was incubated with FtsH, partial degradation of the phosphatase was observed. In contrast, two mutant versions of Spo0E with truncated C-termini remained stable. Transfer of the C-terminal 25 aa of Spo0E to a shorter homologue of Spo0E, YnzD, which is not a substrate of FtsH, conferred instability. When a mutant Spo0A was produced that was active in the absence of phosphorylation, spores were formed at a normal rate in an ftsH knockout, indicating that ftsH is needed only during phase 0.