A Conserved Cysteine Residue of Bacillus subtilis SpoIIIJ Is Important for Endospore Development

During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σG coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation.在枯草芽孢杆菌（Bacillus subtilis）的芽孢形成过程中，晚期前芽孢特异性σ因子σG的活性启动，与母细胞完成对前芽孢的吞噬恰好同步。在此阶段，前芽孢转变为游离原生质体，被母细胞细胞质包裹，并由源自不对称分裂隔膜的两层膜与母细胞分隔开来。与外界培养基隔绝的前芽孢，其后续的基因表达依赖于由母细胞和前芽孢共同合成的蛋白质组装而成的SpoIIIA-SpoIIQ分泌系统。属于YidC/Oxa1/Alb3家族的膜蛋白插入酶SpoIIIJ，参与SpoIIIA-SpoIIQ复合物的组装。本研究发现，SpoIIIJ以单体与二聚体的混合形式存在，二者通过二硫键实现稳定。研究表明，SpoIIIJ的跨膜区段2（TM2）内的Cys134残基，对维持该蛋白的二聚体形式具有关键作用。采用半胱氨酸反应性试剂对Cys134进行标记仅能在严苛条件下完成，这提示膜内的SpoIIIJ单体之间至少部分通过TM2发生紧密结合。将Cys134替换为丙氨酸（Ala）会导致单体形式的积累，并降低SpoIIIJ在活体内的功能。综上，活体内SpoIIIJ的活性似乎依赖于二聚体的形成。