The SpoOE 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 SpoOA is reduced in an ftsH knockout and the small amounts of SpoOA protein present are inactive. Phosphorylation of SpoOA 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 SpoOE. 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 SpoOE was incubated with FtsH, partial degradation of the phosphatase was observed. In contrast, two mutant versions of SpoOE with truncated C-termini remained stable. Transfer of the C-terminal 25 aa of SpoOE to a shorter homologue of SpoOE, YnzD, which is not a substrate of FtsH, conferred instability. When a mutant SpoOA 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 O.