A genomic and structural study of FtsZ constriction in cell division

描述(申请人提供)：FtsZ(丝状温度敏感Z)是细菌细胞分裂所必需的蛋白质。它组装Z形环，将细胞挤压成两半。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紧缩力所必需的唯一蛋白质。外源细菌的FtsZ和一些突变体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，以抑制有缺陷的FtsZ表型并允许分裂。这些抑制株中的突变目前尚不清楚，但很可能是参与细胞分裂的其他途径中的功能丧失突变。该项目旨在利用高通量测序技术对13株抑制子菌株的整个基因组进行重新测序，以绘制抑制子突变图谱，并进一步确定细菌细胞分裂中感兴趣的基因。2.FtsZ蛋白有一个C-末端(Ct)尾部区域，缺乏X射线结晶学显示的明确结构，表明它主要作为一个间隔区将FtsZ球状结构域与FTSA结合位点和膜连接起来。然而，一些在Ct尾部插入20aa小片段的FtsZ突变体不能发挥细胞分裂的功能。本项目旨在研究Ct Tail功能在体内和体外的作用。CT尾部将在FtsZ球状结构域之外进行表达和纯化，以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，并将评估该蛋白质在体外在脂质体中产生紧缩力的能力，以及在体内对细胞分裂的功能。 公共卫生相关性：FtsZ是大多数细菌细胞分裂所必需的蛋白质。对FtsZ结构和功能的详细了解将增加药物靶向蛋白质以抑制细菌分裂的可能性和有效性，从而创造潜在的新抗生素。

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.