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

1)我们新构建了约300个基因的突变体，以完成国际突变体构建计划，并报道了271个基因对枯草杆菌在Lb培养基中37℃的生长是必需的。2)在未知功能的必需基因中，我们发现yacA编码一种负责赖氨酸形成的rna修饰酶，ynes与plsX一起对于磷脂生物合成的第一步是必不可少的。3)我们尝试构建了57个核糖体蛋白基因的敲除突变体，发现其中22个对生长是必不可少的。4)我们系统地分析了平行基因对的双敲除的合成致死性，发现Pola-yacP双突变体是由于Okazaki片段的引物RNA去除缺陷所致。5)结合蛋白质组、转录组和遗传分析，我们新鉴定了在孢子萌发过程中特异表达的175个基因并确定了影响其表达的西格玛因子。我们还分析了大约90个孢子蛋白的GFP融合定位，发现了至少10种不同的定位模式。6)我们尝试利用酵母双杂交分析和体内蛋白质复合体的质谱分析来分析蛋白质-蛋白质相互作用网络，发现了几个有趣的相互作用：抗sigma蛋白调节ECF sigma因子的活性，Rap蛋白调节ComK的活性负责能力的发展，YheIH参与调节磷酸继电器诱导孢子形成的启动，YlqF与FtsZ相互作用并参与细胞分裂，Yaba与Dna A和Dna A相互作用并调节基因组复制的开始。

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.