The long term goal of this project is to elucidate the mechanism of broad host range conjugation mediated by the IncP plasmids of gram negative bacteria. The P plasmids carry antibiotic resistance genes and are capable of transferring DNA to most gram negative bacteria as well as gram positives and yeast. Recent evidence also suggests a relationship between IncP conjugation and Agrobacterium mediated DNA transfer to plants. Study of IncP conjugation will enhance the understanding of gene exchange mechanisms and facilitate use of P plasmids as genetic tools in a wide variety of medically important bacteria. One broad goal of this current proposal is to define the structural interactions between essential transfer proteins and binding sites within the origin of transfer that lead to initiation of the transfer process. A 250 basepair(bp) sequence is required for oriT function, and two plasmid specific proteins, TraJ and TraI, are required for the site specific nicking that initiates transfer at oriT.
Specific aims for this project are to use site directed mutagenesis to define the role of the 19 bp inverted repeat and the sequence adjacent to the nick site, and to correlate these structural changes with protein-DNA interactions between the essential TraJ and TraI proteins of the nicking complex and their binding sites in oriT. A second broad goal is to define transfer genes and gene products responsible for the broad host range property.
Specific aims are to analyze the role of the two DNA primase proteins in host range by constructing clones that produce one or the other exclusively, and using these to complement a primase deletion mutant in transfer to a variety of gram negative bacteria, to determine the roles in conjugation of the two genes that are encoded by different reading frames in traI.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028924-12
Application #
2175318
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-09-01
Project End
1994-12-31
Budget Start
1993-01-01
Budget End
1994-12-31
Support Year
12
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Internal Medicine/Medicine
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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