Structure and Genetic Control of Colicines

本提案中的研究涉及负责大肠杆菌和远亲革兰氏阴性菌中广宿主范围质粒RK 2的复制和分配启动的遗传和生化机制。质粒RK 2指定对抗生素氨苄青霉素、四环素和卡那霉素的抗性，并且将在广泛的革兰氏阴性细菌中复制并稳定维持。 质粒复制起始和分离到子细胞的机制将在E.大肠杆菌、恶臭假单胞菌和铜绿假单胞菌。 RK 2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（iteron）、4个DnaA盒和含有4个13-mer序列的富含A+T的序列。 质粒还含有两个区域，包括par操纵子，其参与稳定维持。 拟议研究的一个主要目标是了解这种质粒的独特性质，这些性质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自我分裂的能力。 为此，本文对大肠杆菌的关键宿主蛋白DnaA和DnaB的活性进行了研究。大肠杆菌、铜绿假单胞菌和恶臭假单胞菌沿着质粒特异性起始蛋白在RK 2和窄宿主范围质粒P1和F复制起始中的作用。 此外，这些不同的宿主蛋白（沿着E. coli）的染色体复制起点。大肠杆菌和两株假单胞菌进行比较。 FISH和GFP标记技术将用于在野生型和突变型E.大肠杆菌菌株和与大肠杆菌有远亲关系的细菌。杆菌 RK 2和特定复制蛋白的GFP标记将用于在细胞生长和分裂期间该质粒和复制蛋白的动态运动的延时分析。 这些不同的研究应该有助于我们理解DNA复制，DNA分离和细菌中抗生素耐药性传播的基本过程。

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.