Cellular homeostasis pathways in bacteria

 描述(由申请人提供)：细菌中的细胞平衡途径维持细胞平衡对于平衡细胞生长至关重要。然而，令人惊讶的是，人们对促进动态平衡的过程之间的联系知之甚少。许多以转录为中心的研究已经阐明了其中的一些联系，特别是对特定压力的反应，但对动态平衡的其他贡献仍然是间接的。在这里，我们将重点放在细菌的两个未被研究的特征上，以了解它们对细胞内稳态的贡献。第一, 我们正在梳理细胞包膜隔间中的过程，这些过程将其复杂的功能相互关联，并与细胞质协调。其次，我们正在探索细胞如何控制蛋白质的丰度，以应对不同的条件和细胞分化。由于蛋白质生产构成了细菌细胞消耗的大部分能量，因此蛋白质生产可以根据需要进行改变是至关重要的。我们的生物学研究 是由基因组规模的技术提供动力的。我们使用化学基因组学识别新的包膜途径，也使用我们目前正在开发的方法进行池遗传相互作用分析。该方法基于CRISPRi(CRISPR/Cas9干扰)技术进行的双击倒。我们正在使用测量蛋白质丰度的全球技术(核糖体图谱+mRNAseq)来确定如何控制蛋白质丰度，同时我们正在开发一种新的方法来测量基因组规模的mRNA衰变。在每种情况下，我们都使用我们生成的数据集作为起点，对有趣的发现进行详细的机械分析。重要的是，因为支持我们研究的方法很容易在细菌中移植，我们现在正在研究大肠杆菌和枯草杆菌的包膜，从而能够进行跨革兰氏阳性/革兰氏阴性的进化比较。我们还在积极努力，将这些方法移植到与医学和环境相关的非模式生物。最后，由于我们的高质量数据集符合为机械研究提供可靠切入点所必需的标准，因此它们是科学界研究多细胞过程的重要资源。

 DESCRIPTION (provided by applicant): Cellular Homeostasis Pathways in Bacteria Maintaining cellular homeostasis is critical for balanced cell growth. Yet remarkably little is known about the connections between processes that contribute to homeostasis. A number of transcription-focused studies have elucidated some of this wiring, especially for responses to specific stresses, but other contributions to homeostasis remain oblique. Here we focus on two understudied features of bacteria to understand their contributions to cellular homeostasis. First, we are teasing apart processes in the envelope compartment of the cell that interlock its complex functions, and coordinate with the cytoplasm. Second, we are exploring how the cell controls protein abundance both in response to differential conditions, and in response to cell differentiation. As protein production constitutes the majority of the energy expended by the bacterial cell, it is critical that protein production can change as needed. Our biological studies are powered by genome-scale technologies. We identify novel envelope pathways using chemical genomics, and also with our method, currently in development, for pooled genetic interaction analysis. This method is based on double knockdowns made with CRISPRi (CRISPR/Cas9 interference) technology. We are determining how protein abundance is controlled using global technologies to measure protein abundance (ribosome profiling + mRNAseq) coupled with a new method we are developing to measure mRNA decay at genome scale. In each case, we use the datasets we generate as a starting point for detailed mechanistic analysis of interesting findings. Importantly, because the methods powering our research are readily portable across bacteria, we are now studying the envelope both in E. coli and in B. subtilis, enabling evolutionary comparison across the Gram-positive/Gram- negative divide. We are also actively working to port these methods to medically and environmentally relevant non-model organisms. Finally, as our high quality datasets meet the standard necessary for providing a reliable entry point for mechanistic studies, they are an essential resource for the study of multiple cell processes by the scientific community.