Structure and Genetic Control of Colicines

这项建议中的研究涉及负责广泛宿主范围的质粒RK2在大肠杆菌和远亲关系的革兰氏阴性细菌中复制和分割的遗传和生化机制。RK2对氨苄西林、四环素和卡那霉素等抗生素产生抗药性，并能在广泛的革兰氏阴性菌中复制并稳定保持。利用生化、遗传学和细胞学技术，对大肠杆菌、恶臭假单胞菌和铜绿假单胞菌的复制起始和分离到子代细胞的机制进行了研究。RK2编码一个复制起始蛋白(TrfA)和一个复制起始点，其主要特征是由TrfA蛋白结合的17个碱基对重复(迭代子)，4个Dna A盒，以及包含4个13聚体序列的A+T丰富序列。该质粒还包含两个区域，包括参与稳定维持的PAR操纵子。拟议的研究的一个主要目的是了解这种质粒的独特性质，这些性质解释了它在广泛的细菌的细胞分裂过程中启动复制和忠实地分割自己的能力。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Dna A和Dna B以及质粒特异性起始蛋白在启动RK2和窄宿主质粒P1和F复制中的活性。此外，这些不同的宿主蛋白(连同大肠杆菌的Dna C蛋白)在大肠杆菌和两株假单胞菌的染色体复制起始处的活性将被比较。FISH和GFP标签技术将被用来在野生型和突变型大肠杆菌菌株以及与大肠杆菌有远缘关系的细菌中定位RK2质粒。RK2和特定复制蛋白的GFP标记将用于对该质粒和复制蛋白在细胞生长和分裂过程中的动态运动进行时移分析。这些不同的研究应该有助于我们理解DNA复制的启动、DNA分离和抗生素耐药性在细菌中传播的基本过程。

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.