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Journal of Bacteriology, November 2000, p. 6014-6026, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2

Thomas M. Rosche,dagger Azeem Siddique, Michelle H. Larsen,Dagger and David H. Figurski*

Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Received 10 April 2000/Accepted 5 August 2000

Replication of the broad-host-range, IncPalpha plasmid RK2 requires two plasmid loci: trfA, the replication initiator gene, and oriV, the origin of replication. While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts. The product of the incC gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC has the properties expected of a component of an active partition system. The smaller polypeptide product of incC (IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2. This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for incC-mediated incompatibility is a KorB-binding site (OB). We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins. Elevated expression of the incC and korB genes individually has no obvious effect on Escherichia coli cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing incC, korB, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in an incC-dependent manner. Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2.

* Corresponding author. Mailing address: Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 W. 168th St., New York, NY 10032. Phone: (212) 305-3425. Fax: (212) 305-1468. E-mail: figurski{at}cuccfa.ccc.columbia.edu.

dagger Present address: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Dagger Present address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.

Journal of Bacteriology, November 2000, p. 6014-6026, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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