This project will provide a detailed understanding of the mechanism of plasmid DNA transfer during bacterial conjugation. Bacterial mating is a basic means of genetic exchange that has been described in both gram-positive and gram-negative organisms. In gram-negative bacteria, conjugation systems are encoded by plasmids, and mediate the transfer of plasmid and chromosomal DNA from donor to recipient cell. Bacterial conjugation is significant in clinical medicine because plasmids specify both antibiotic resistance and important virulence traits, and conjugation is the major mechanism for the spread of these plasmids in pathogenic gram-negative bacteria. The broad host range conjugative plasmid RK2 will be used as a model system to study plasmid DNA transfer. The RK2 conjugation system differs from the previously studied F factor of E. coli in several fundamental properties, including host range, sex pili, and mating physiology. We have identified and sequenced the origin of transfer (oriT) of RK2, and have isolated the RK2 relaxation complex which nicks at oriT and initiates the transfer process. The major structural feature of the 112 base pair oriT sequence is a 19 base pair inverted repeat, which may represent the DNA recognition site for the transfer proteins.
The specific aims of this proposal are: 1) to determine the minimal DNA sequence required for a functional origin of transfer, 2) to determine the structural requirements for oriT and the role of the inverted repeats, and 3) to characterize the transfer proteins which interact with oriT. These studies will provide basic information on the protein-DNA interactions during plasmid transfer. In addition, the project will involve two practical applications of broad interest to bacterial geneticists: 1) construction of improved broad host range cloning vectors based on RK2, and 2) the development of a general system for RK2-mediated transfer of the bacterial chromosome. These applications will facilitate genetic manipulations in many bacteria which currently lack a convenient genetic system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028924-06
Application #
3276289
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-09-01
Project End
1986-08-31
Budget Start
1985-09-01
Budget End
1986-08-31
Support Year
6
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Cole, S P; Guiney, D G (1995) Site-specific mutations in the traI relaxase and upstream region of plasmid RP4. Plasmid 34:236-9
Cole, S P; Lanka, E; Guiney, D G (1993) Site-directed mutations in the relaxase operon of RP4. J Bacteriol 175:4911-6
Waters, V L; Guiney, D G (1993) Processes at the nick region link conjugation, T-DNA transfer and rolling circle replication. Mol Microbiol 9:1123-30
Waters, V L; Strack, B; Pansegrau, W et al. (1992) Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity. J Bacteriol 174:6666-73
Waters, V L; Hirata, K H; Pansegrau, W et al. (1991) Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids. Proc Natl Acad Sci U S A 88:1456-60
Yakobson, E; Deiss, C; Hirata, K et al. (1990) Initiation of DNA synthesis in the transfer origin region of RK2 by the plasmid-encoded primase: detection using defective M13 phage. Plasmid 23:80-4
Guiney, D G; Deiss, C; Simnad, V et al. (1989) Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes. J Bacteriol 171:4100-3
Beninger, P R; Chikami, G; Tanabe, K et al. (1988) Physical and genetic mapping of the Salmonella dublin virulence plasmid pSDL2. Relationship to plasmids from other Salmonella strains. J Clin Invest 81:1341-7
Fierer, J; Chikami, G; Hatlen, L et al. (1988) Active immunization with LD842, a plasmid-cured strain of Salmonella dublin, protects mice against group D and group B Salmonella infection. J Infect Dis 158:460-3
Guiney, D G; Deiss, C; Simnad, V (1988) Location of the relaxation complex nick site within the minimal origin of transfer region of RK2. Plasmid 20:259-65

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