Structure and Genetic Control of Colicines

结肠菌素的结构和遗传控制

• 批准号: 6510292
• 负责人: DONALD R HELINSKI
• 金额: $ 42.91万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-02-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510292
• 关键词: DNA binding protein; DNA replication; Escherichia coli; Pseudomonas; bacterial genetics; bacterial proteins; colicines; fluorescent in situ hybridization; green fluorescent proteins; helicase; intracellular transport; plasmids; protein binding; protein localization; protein transport

The research in this proposal is concerned with genetic and biochemical mechanisms responsible for the initiation of replication and partitioning of the broad-host-range plasmid RK2 in Escherichia coli and distantly related Gram-negative bacteria. Plasmid RK2 specifies resistance to the antibiotics ampicillin, tetracycline and kanamycin, and will replicate and is stably maintained in a wide range of Gram-negative bacteria. Mechanisms of plasmid replication initiation and segregation to daughter cells will be investigated in E. coli, Pseudomonas putida and Pseudomonas aeruginosa using biochemical, genetic and cytological techniques. RK2 encodes a replication initiation protein (TrfA) and a replication origin that has as its main features 17 base pair repeats (iterons) that are bound by the TrfA protein, four DnaA boxes, and an A+T rich sequence that contains four 13-mer sequences. The plasmid also contains two regions, including the par operons, which are involved in stable maintenance. A major thrust of the proposed research is understanding the unique properties of this plasmid that account for its ability to initiate its replication and faithfully partition itself during cell division in a wide range of bacteria. To this end, the activities of the key host proteins DnaA and DnaB of E. coli, P. aeruginosa, and P. putida along with the plasmid specific initiation protein in the initiation of replication of RK2 and narrow-host-range plasmids P1 and F will be determined. In addition, the activities of these various host proteins (along with the DnaC protein of E. coli) at the chromosomal replication origins of E. coli and the two Pseudomonas strains will be compared. Both FISH and GFP-tagging techniques will be used to localize the RK2 plasmid in wild-type and mutant E. coli strains and in bacteria distantly related to E. coli. GFP-tagging of RK2 and of specific replication proteins will be used for time-lapse analysis of the dynamic movement of this plasmid and replication proteins during cell growth and division. These various studies should contribute to our understanding of the fundamental processes of initiation of DNA replication, DNA segregation, and the dissemination of antibiotic resistance in bacteria.

本提案中的研究涉及负责广泛宿主范围质粒 RK2 在大肠杆菌和远缘革兰氏阴性细菌中复制和分配的遗传和生化机制。质粒 RK2 对抗生素氨苄青霉素、四环素和卡那霉素具有抗性，并且可以在多种革兰氏阴性细菌中复制并稳定维持。 将利用生化、遗传和细胞学技术在大肠杆菌、恶臭假单胞菌和铜绿假单胞菌中研究质粒复制起始和分离到子细胞的机制。 RK2 编码复制起始蛋白 (TrfA) 和复制起点，其主要特征是由 TrfA 蛋白结合的 17 个碱基对重复序列（迭代子）、四个 DnaA 盒以及包含四个 13 聚体序列的富含 A+T 的序列。 该质粒还包含两个区域，包括参与稳定维持的 par 操纵子。 拟议研究的一个主要推动力是了解该质粒的独特特性，这些特性解释了它在多种细菌的细胞分裂过程中启动复制并忠实地分割自身的能力。 为此，将测定大肠杆菌、铜绿假单胞菌和恶臭假单胞菌的关键宿主蛋白 DnaA 和 DnaB 以及 RK2 和窄宿主范围质粒 P1 和 F 复制起始中的质粒特异性起始蛋白的活性。 此外，还将比较大肠杆菌和两种假单胞菌菌株染色体复制起点处的这些不同宿主蛋白（以及大肠杆菌的 DnaC 蛋白）的活性。 FISH 和 GFP 标记技术将用于在野生型和突变型大肠杆菌菌株以及与大肠杆菌远缘的细菌中定位 RK2 质粒。 RK2 和特定复制蛋白的 GFP 标记将用于在细胞生长和分裂过程中对该质粒和复制蛋白的动态运动进行延时分析。 这些不同的研究应该有助于我们理解细菌中 DNA 复制起始、DNA 分离和抗生素耐药性传播的基本过程。