Expression and localization of SpoIISA toxin during the life cycle of Bacillus subtilis

The previously identified spoIIS locus encodes a toxin–antitoxin system in Bacillus subtilis. It comprises two genes, spoIISA encoding a toxin and spoIISB encoding an antitoxin, which lies adjacent to each other on the chromosome. Each of the spoIIS coding sequences is preceded by a promoter region and the two genes together constitute an operon. The function of SpoIISA is unknown, although it has been shown that the absence of SpoIISB or loss of its function leads to a block in sporulation at stage II. The cytoplasmic membrane has been proposed as the target of the SpoIISA toxin. Heterologously expressed SpoIISA–SpoIISB was shown to be functional in Escherichia coli, where again the cytoplasmic membrane was the most probable target for SpoIISA toxicity. Here we analyzed the effects of SpoIISA production during vegetative growth of B. subtilis and during sporulation by following the levels of SpoIISA. SpoIISA levels increase at the point of entry into stationary phase of cell cultures grown in sporulation-inducing medium. However, SpoIISA expression appears to be unrelated to the sporulation process, since it is independent of the major early sporulation-specific transcription factor, Spo0A. We also investigated SpoIISA localization within the cell. We confirmed the predicted localization of SpoIISA at the B. subtilis cytoplasmic membrane. In addition, we observed localization of SpoIISA in higher level structures in a cell-wall-dependent manner.