Structure and Genetic Control of Colicines

本提案的研究涉及大肠杆菌和远亲革兰氏阴性菌中广泛宿主质粒RK2的复制和分裂启动的遗传和生化机制。质粒RK2指定了对抗生素氨苄西林、四环素和卡那霉素的耐药性，并将在广泛的革兰氏阴性细菌中复制并稳定维持。利用生物化学、遗传学和细胞学技术研究大肠杆菌、恶臭假单胞菌和铜绿假单胞菌质粒复制起始和分离到子细胞的机制。RK2编码一个复制起始蛋白（TrfA）和一个复制起始蛋白，其主要特征是由TrfA蛋白结合的17个碱基对重复序列（iterons）、4个dna盒和一个包含4个13-mer序列的a +T富序列。质粒还包含两个区域，包括par操纵子，这两个区域参与稳定的维持。这项研究的主要目的是了解这种质粒的独特性质，这种性质解释了它在各种细菌的细胞分裂过程中启动复制和忠实地分裂自己的能力。为此，我们将测定大肠杆菌、铜绿假单胞菌和恶臭假单胞菌的关键宿主蛋白DnaA和DnaB以及质粒特异性起始蛋白在RK2和窄宿主范围质粒P1和F的复制起始中的活性。此外，还将比较这些不同宿主蛋白（以及大肠杆菌的DnaC蛋白）在大肠杆菌和两种假单胞菌染色体复制起点的活性。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亲的细菌中定位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时序分析。这些不同的研究应该有助于我们理解DNA复制起始、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.