Genetic and Biochemical Analysis of a Peptide Export System

本实验旨在阐明细菌体内多肽和蛋白质输出的机制。具体来说，将要使用的系统是Microcin B17 （MccB17）的出口，这是一种由大肠杆菌产生的肽抗生素。mcb17本身是mcbA的产物，mcbB到mcbG这6个基因对于mcb17的产生和免疫是必需的。抗生素作用于敏感的肠道细菌细胞，阻断DNA复制。mcbE和mcbF基因产物可能介导mcb17的输出；mcbE和mcbF及其外基因抑制子的突变将描述mcb17输出机制中这些组成部分的作用、相互作用和功能域。细胞内和细胞外的MccB17将通过生化和细胞分离研究以及电子显微镜来表征。MccB17分子中阻止其输出的突变将识别运输的功能域；允许改变肽输出的这些突变的基因外抑制因子将识别输出肽与输出所需蛋白质之间的相互作用。斯克维尔斯基博士通过“女性研究机会发起奖”确立了自己作为独立研究者的地位。这项工作很有趣，因为在大肠杆菌中，向细胞外输出蛋白质是很罕见的。在这些研究中获得的信息将有许多可能的实际应用于细菌克隆基因产品的商业生产

The objective of the experiments proposed is to elucidate mechanisms of peptide and protein export in bacteria. Specifically, the system to be used is the export of Microcin B17 (MccB17), a peptide antibiotic produced by E. coli. Six genes, mcbB through mcbG, are necessary for production of and immunity to MccB17, itself the product of mcbA. The antibiotic acts on sensitive enteric bacterial cells to block DNA replication. The mcbE and mcbF gene products appear to mediate export of MccB17; mutations in mcbE and mcbF and extragenic suppressors thereof will delineate the roles, interactions, and functional domains of these components of the MccB17 export machinery. Intracellular and extracellular MccB17 will be characterized by biochemical and cell fractionation studies and by electron microscopy. Mutations in the MccB17 molecule that prevent its export will identify functional domains for transport; extragenic suppressors of these mutations that allow export of the altered peptide will identify interactions between the exported peptide and the proteins necessary for its export.%%% Dr. Skvirsky is establishing herself as an independent investigator through this Research Opportunities for Women Research Initiation award. The work promises to be interesting because export of proteins to the exterior of the cell is rare in the bacterium E.coli. The information obtained in these studies will have numerous possible practical applications to the commercial production of products of cloned genes in bacteria.***//