The research in this proposal deals with the characterization of genetic and biochemical mechanisms responsible for the regulation of replication and for stable maintenance of the broad-host-range plasmid RK2 in bacteria. This plasmid specifies resistance to the antibiotics tetracycline, ampicillin and kanamycin and is stably maintained in the extrachromosomal state in a wide range of Gram-negative bacteria. Two components of this plasmid that are absolutely required for its replication are the trfA gene that encodes two replication initiation proteins (44 kDa and 33 kDa), the smaller of which is the result of an internal translational start in the same open reading frame, and a replication origin sequence that contains as its main feature eight 17 bp repeats (iterons) in groups of five and three. The 44 kDa and 33 kDa proteins are essentially equivalent in activity in the bacterium Escherichia coli and both proteins specifically bind to the iterons at the RK2 replication origin. The major thrust of this proposal is to examine the nature of the interactions between the TrfA replication protein(s) and the sequence at the replication origin that are responsible for the regulation of initiation of replication and the ability of this antibiotic resistance plasmid to be maintained in a wide range of bacteria. These studies also will be concerned with analyses of the interactions between the TrfA protein/origin sequence complex and specific host replication proteins isolated from several Gram-negative bacteria. The analyses will be carried out in vivo and in vitro and will involve both wild-type and mutant TrfA proteins, including defective and copy-up mutants. An attempt will be made to identify regions of the TrfA protein responsible for its various interactions with the replication origin and the host replication proteins. Finally, a region of the RK2 plasmid that appears to encode a broad-host-range plasmid partitioning function will be examined in order to understand the mechanism(s) responsible for the partitioning of a plasmid to daughter cells upon cell division. In addition to providing information on the regulation of initiation of replication and the stable maintenance of an extrachromosomal element in bacteria, a practical fallout of this basic study will be the construction of high-copy number and stably maintained plasmid vectors for gene cloning in a wide range of Gram-negative bacteria of medical and agricultural importance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI007194-27
Application #
3124351
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-02-01
Project End
1996-05-31
Budget Start
1992-06-01
Budget End
1993-05-31
Support Year
27
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Verheust, Celine; Helinski, Donald R (2007) The incC korB region of RK2 repositions a mini-RK2 replicon in Escherichia coli. Plasmid 58:195-204
Konieczny, I; Helinski, D R (1997) The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone. Proc Natl Acad Sci U S A 94:14378-82
Cereghino, J L; Helinski, D R; Toukdarian, A E (1994) Isolation and characterization of DNA-binding mutants of a plasmid replication initiation protein utilizing an in vivo binding assay. Plasmid 31:89-99
Roberts, R C; Strom, A R; Helinski, D R (1994) The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss. J Mol Biol 237:35-51
Perri, S; Helinski, D R (1993) DNA sequence requirements for interaction of the RK2 replication initiation protein with plasmid origin repeats. J Biol Chem 268:3662-9
Cereghino, J L; Helinski, D R (1993) Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria. J Biol Chem 268:24926-32
Fang, F C; Durland, R H; Helinski, D R (1993) Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria. Gene 133:1-8
Roberts, R C; Spangler, C; Helinski, D R (1993) Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2. J Biol Chem 268:27109-17
Lin, J; Helinski, D R (1992) Analysis of mutations in trfA, the replication initiation gene of the broad-host-range plasmid RK2. J Bacteriol 174:4110-9
Greener, A; Lehman, S M; Helinski, D R (1992) Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria. Genetics 130:27-36

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