A genomic and structural study of FtsZ constriction in cell division

描述（由申请人提供）：FtsZ（丝状温敏Z）是细菌细胞分裂所必需的蛋白质。它组装把细胞夹成两半的z形环。我对FtsZ函数的两个不同方面感兴趣。1. FtsZ是脂质体在体外产生收缩力所必需的唯一蛋白。来自外来细菌的FtsZ和一些突变的FtsZ也能引起大肠杆菌的细胞分裂。然而，测试的某些形式的FtsZ需要获得未知的基因组突变来抑制有缺陷的FtsZ表型并允许分裂。这些抑制菌株的突变目前尚不清楚，但可能是参与细胞分裂的其他途径的功能丧失突变。该项目旨在利用高通量测序技术对13个抑制菌株的全基因组进行重测序，以绘制抑制突变，并进一步确定细菌细胞分裂中感兴趣的基因。2. 通过x射线晶体学可以看到，FtsZ蛋白的c端（Ct）尾部区域缺乏明确的结构，这表明它主要是作为连接FtsZ球形结构域与FtsA结合位点和膜的间隔物。然而，一些在Ct尾部有少量20aa插入的FtsZ突变体不能发挥细胞分裂的功能。本项目旨在研究Ct尾部功能在体内和体外的作用。Ct尾部将在FtsZ球形区域外进行表达和纯化，用于核磁共振结构测定。不同形式的FtsZ将使用不同的Ct尾部序列和长度来表达，并评估该蛋白在体外脂质体中产生收缩力的能力，以及在体内对细胞分裂的作用。

DESCRIPTION (provided by applicant): FtsZ (filamentous temperature sensitive Z) is an essential protein for bacterial cell division. It assembles the Z-ring that pinches the cell in two. I am interested in two different aspects of FtsZ function. 1. FtsZ is the only protein necessary for constriction force generation in liposomes in vitro. FtsZ from foreign bacteria and some mutant FtsZ were also able to cause cell division in E. coli. However, some forms of FtsZ tested required the acquisition of unknown genomic mutations to suppress the defective FtsZ phenotype and allow division. The mutations in these suppressor strains are currently unknown, but are likely loss-of- function mutations in other pathways involved in cell division. This project aims to utilize high-throughput sequencing technology to resequence the entire genome of each of the 13 suppressor strains to map the suppressor mutation and identify further genes of interest in bacterial cell division. 2. The FtsZ protein has a C-terminal (Ct) tail region that lacks a lacks a well-defined structure as visualized through X-ray crystallography, indicating that it serves primarily as a spacer to link the FtsZ globular domain to the FtsA binding site and the membrane. However, some FtsZ mutants with small 20aa insertions in the Ct tail are incapable of functioning for cell division. This project aims to examine the role of the Ct tail function in vivo and in vitro. The Ct tail will be expressed and purified apart from the FtsZ globular domain for NMR structure determination. Varied forms of FtsZ will be expressed using alternative Ct tail sequences and length, and the ability of the protein to produce a constriction force in liposomes in vitro, and to function for cell division in vivo will be assessed. Public Health Relevance: FtsZ is a required protein for cell division in most bacteria. A detailed understanding of FtsZ structure and function would increase the possibility and efficacy of targeting the protein pharmacologically to inhibit bacterial division, thus creating potential new antibiotics.