Protein-protein interaction network supporting growth and differentiation of Bacillus subtilis

1)我们对基因组测序鉴定的基因新构建了约300个突变体，完成了国际突变体构建项目，并报道了271个基因是枯草芽孢杆菌在LB培养基中37℃生长所必需的。2)在功能未知的必需基因中，我们发现yacA编码一种负责赖苷形成的rna修饰酶，yneS和plsX对磷脂生物合成的第一步至关重要。此外，GTP必需结合蛋白之一YlqF已被证明参与了50S核糖体亚基组装的最后一步。3)我们尝试构建了57个核糖体蛋白基因的敲除突变体，发现22个是生长所必需的。4)我们系统分析了双敲除副同源基因对的合成致死性，发现polA-yacP双突变体是由于冈崎片段的引物RNA去除缺陷而致死性的。5)结合蛋白质组学、转录组学和遗传分析，除先前报道的119个基因外，我们新鉴定出175个在产孢过程中特异性表达的基因，并确定了与这些基因表达相关的sigma因子。我们还分析了大约90个孢子蛋白的GFP融合物的定位，发现了至少10种不同的定位模式。6)我们尝试通过酵母双杂交分析和体内蛋白复合物的质谱分析来分析蛋白质-蛋白质相互作用网络，并发现了一些有趣的相互作用；调控ECF sigma因子活性的抗sigma蛋白，调控能力发育的ComK的Rap蛋白，参与调控磷传递诱导产孢起始的YheIH蛋白，与FtsZ相互作用参与细胞分裂的YlqF蛋白，与DnaA和DnaA相互作用调控基因组复制起始的YabA蛋白。

1) We newly constructed about 300 mutants of the genes identified by genome sequencing, to complete the international mutant construction project, and reported that 271 genes are essential for the B. subtilis growth at 37℃ in LB medium.2) Among essential genes without known function, we have found that yacA encodes an RNA-modifying enzyme responsible for lysidine formation and yneS, together with plsX, is essential for the first step of phospholipids biosynthesis. In addition, one of the essential GTP binding proteins, YlqF, has been demonstrated to participate in the last step of 50S ribosome subunit assembly.3) We have attempted to construct knock out mutant of each of 57 ribosome protein genes, and found that 22 are dispensable for the growth.4) We have systematically analyzed the synthetic lethality of double knockout of paralogous gene pairs, and found that the polA-yacP double mutant is lethal due to the defect in removal of primer RNA of Okazaki fragments.5) By combination of proteome, transcriptome and genetic analysis, in addition to 119 genes previously reported, we have newly identified 175 genes that are expressed specifically during sporulation, and determined sigma factors responsible for their expression. We have also analyzed the localization of GFP fusions of about 90 spore proteins, and found at least 10 different the localization patterns.6) We have attempted the analysis of protein-protein interaction network by using yeast two-hybrid analysis and mass spectroscopic analysis of in vivo protein complex, and identified several interesting interactions; anti-sigma proteins regulating activities of ECF sigma factors, Rap proteins regulating ComK responsible for competence development, YheIH involved in regulation of phosphor-relay inducing initiation of sporulation, YlqF interacting with FtsZ and involved in cell division, and YabA interacting with DnaA and DnaA and regulating initiation of genome replication.