Abstract

The objectives of the studies outlined in this proposal are (1) to determine the mechanisms of plasmid stabilization by the kil loci, (2) to reveal the functions of the twelve gene products expressed by the kil loci, and (3) to understand the host-specificity of these systems. In addition, the incC and korB genes of the korA operon will be studied for their possible involvement in active partition of RK2. For each system, the essential genes and sites will be identified and characterized in detail by genetic analysis. The experiments will determine whether stabilization occurs by active partition, plasmid addiction, multimer resolution, or by novel mechanisms. A variety of genetic approaches (yeast two hybrid assays, suppressor mutants, toxicity-resistant mutants) and biochemical approaches (affinity chromatography, coimmunoprecipitation) will be used to detect interactions among plasmid proteins and between plasmid and host proteins. Each system displays unique properties that will be exploited for analysis.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029085-21
Application #
6385408
Study Section
Special Emphasis Panel (ZRG5-MBC-2 (04))
Program Officer
Rhoades, Marcus M
Project Start
1981-04-01
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2003-03-31
Support Year
21
Fiscal Year
2001
Total Cost
$561,436
Indirect Cost

  Institution

Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
167204994
City
New York
State
NY
Country
United States
Zip Code
10032

  Publications

Siddique, Azeem; Figurski, David H (2012) Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids. Plasmid 68:93-104
Siddique, Azeem; Figurski, David H (2002) The active partition gene incC of IncP plasmids is required for stable maintenance in a broad range of hosts. J Bacteriol 184:1788-93
Kachlany, Scott C; Fine, Daniel H; Figurski, David H (2002) Purification of secreted leukotoxin (LtxA) from Actinobacillus actinomycetemcomitans. Protein Expr Purif 25:465-71
Wilson, James W; Figurski, David H (2002) Host-specific incompatibility by 9-bp direct repeats indicates a role in the maintenance of broad-host-range plasmid RK2. Plasmid 47:216-23
Bhattacharyya, A; Figurski, D H (2001) A small protein-protein interaction domain common to KlcB and global regulators KorA and TrbA of promiscuous IncP plasmids. J Mol Biol 310:51-67
Rosche, T M; Siddique, A; Larsen, M H et al. (2000) Incompatibility protein IncC and global regulator KorB interact in active partition of promiscuous plasmid RK2. J Bacteriol 182:6014-26
Wilson, J W; Sia, E A; Figurski, D H (1997) The kilE locus of promiscuous IncP alpha plasmid RK2 is required for stable maintenance in Pseudomonas aeruginosa. J Bacteriol 179:2339-47
Jovanovic, O S; Figurski, D H (1997) A potential new gene (tccA) on IncP plasmid RK2 and transposon Tn1721: relationship of its product to the TrwC relaxase/helicase of IncW plasmid R388. Plasmid 38:220-3
Sia, E A; Kuehner, D M; Figurski, D H (1996) Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required. J Bacteriol 178:1457-64
Sia, E A; Roberts, R C; Easter, C et al. (1995) Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. J Bacteriol 177:2789-97

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