Regulation Mechanism of Initiation of Sporulation in Bacillus subtilis

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A Bacillus subtilis gene (ispU) encodes a 277-amino-acid-protein whose COOH-half showed significant similarity to those of RsbR and RsbS, both of which were shown to regulate the σィイD1BィエD1 activity. ispU was expressed before the onset of sporulation. An ispU-null mutation (ΔispU) caused a 30-min delay in the expression of σィイD1HィエD1-dependent genes, such as kinA, spoVG, and the Ps promoter of spo0A. Corresponding delay in appearance of heat-resistant spores was also observed in the ΔispU mutant. On the other hand the ΔispU mutation had no effect on either the expression of spo0H encoding σィイD1HィエD1 or the accumulation of σィイD1HィエD1 at the onset of sporulation, indicating possible involvement of ISpU in modulating σィイD1HィエD1 activity at a posttranslational level.There were six other genes paralogous to ispU within the B. subtilis genome, among which only the deletion of yojH produced the same phenotype as that of ΔispU mutation on kinA expression. Double mutation of ispU and yojH caused a further reduction of kinA expression, indicating that IspU and YojH have a same function in the σィイD1HィエD1-activation pathway.When we purified IspU from crude extracts of B. subtilis using a CNBr-activated Sepharose column coupled with an anti-IspU antibody, co-purification of two proteins of 30 kDa and 32 kDa was observed. NHィイD22ィエD2-terminal amino acid sequencing revealed that these proteins were YojH and RsbR, respectively- These proteins were not purified when the crude extract of ΔispU mutant was used. This result indicates that IspU is interacting with YojH and RsbR in vivo.