Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI007194-37
Application #
6510292
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Korpela, Jukka K
Project Start
1978-02-01
Project End
2006-04-30
Budget Start
2002-05-01
Budget End
2003-04-30
Support Year
37
Fiscal Year
2002
Total Cost
$429,100
Indirect Cost

  Institution

Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

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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