Research efforts in the lab are focused on the genetic and biochemical basis of stable maintenance of plasmids in bacteria. Particular interest is placed on the biochemical interactions between the replication initiation protein (Rep) and the origin of replication (oriV) of the broad-host range, antibiotic resistance, plasmid RK2, which is capable of replication and stable maintenance in virtually all Gram-negative bacteria. A number of mutants of the Rep protein have been isolated and purified to determine various levels of interaction between the initiation protein and the replication origin including DNA binding, replication control, and interaction with bacterial host proteins. Bacterial replication initiation proteins DnaA and DnaB have been isolated from Pseudomonas aeruginosa and Pseudomonas putida to compare at the in vitro level the mechanism of replication initiation of plasmid RK2 in these bacteria with events taking place in E. coli. These comparisons are yielding critical information on the molecular basis of the broad host-range properties of RK2 and on DNA replication initiation in bacteria other than E. coli. To complement these biochemical and molecular genetic studies, cell biology techniques are being used to determine the location and dynamic movement of plasmids in E. coli and other Gram-negative bacteria during growth and cell division.
Initiation of DNA replication in an iteron-containing plasmid. In this model the various proteins indicated are host specified, except for the plasmid-encoded Rep protein. Pogliano, J., Ho, T.Q., Zhong, Z. and Helinski, D.R. (2001). Multicopy plasmids are clustered and localized in Escherichia coli. Proc. Natl. Acad. Sci. USA, 98: 4486-4491. Caspi, R., Pacek, M., Consiglieri, G., Helinski, D.R., Toukdarian, A. and Konieczny, I. (2001). A broad host range replicon with different requirements for replication initiation in three bacterial species. EMBO J., 20:3262-3271. Zhong, Z., Toukdarian, A., Helinski, D., Knauf, V., Sykes, S., Wilkinson, J.E., O'Bryne, C., Shea, T., DeLoughery, C. and Caspi, R. (2001). Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate. Appl. Environ. Microbiol., 67:5771-5779. Jiang, Y., Pogliano, J., Helinski,
D.R. and Konieczny, I. (2002) ParE toxin encoded by the broad-host-range
plasmid RK2 is an inhibitor of Escherichia coli gyrase.
Molecular Microbiol., 44:971-979.
Donald Helinski earned his Ph.D. at Case Western Reserve University and held a postdoctoral fellowship at Stanford University. He serves on the editorial boards of Plasmid and Genetic Engineering. Professor Helinski is a member of the National Academy of Sciences, the American Academy of Arts and Sciences, the European Molecular Biology Organization and the American Academy of Microbiology. |
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