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

Rosche, Thomas M.; Siddique, Azeem; Larsen, Michelle H.; Figurski, David H.

doi:10.1128/jb.182.21.6014-6026.2000

Cited by 37 publications

(36 citation statements)

References 96 publications

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“…5). However, the RK2 par region used, O B1 incC korB, contained only one of three KorB binding sites present in RK2 par, and this site, O B1 , had previously been shown not to be as effective as O B3 in stabilizing RK2 derivatives (39,48).…”

Section: Discussionmentioning

confidence: 99%

Localization of the Naturally Occurring Plasmid ColE1 at the Cell Pole

2007

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The naturally occurring plasmid ColE1 was found to localize as a cluster in one or both of the cell poles of Escherichia coli. In addition to the polar localization of ColE1 in most cells, movement of the plasmid to the midcell position was observed in time-lapse studies. ColE1 could be displaced from its polar location by the p15A replicon, pBAD33, but not by plasmid RK2. The displacement of ColE1 by pBAD33 resulted in an almost random positioning of ColE1 foci in the cell and also in a loss of segregational stability, as evidenced by the large number of cells carrying pBAD33 with no visible ColE1 focus and as confirmed by ColE1 stability studies. The addition of the active partitioning systems of the F plasmid (sopABC) or RK2 (O B1 incC korB) resulted in movement of the ColE1 replicon from the cell pole to within the nucleoid region. This repositioning did not result in destabilization but did result in an increase in the number of plasmid foci, most likely due to partial declustering. These results are consistent with the importance of par regions to the localization of plasmids to specific regions of the cell and demonstrate both localization and dynamic movement for a naturally occurring plasmid that does not encode a replication initiation protein or a partitioning system that is required for plasmid stability.

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Section: Discussionmentioning

confidence: 99%

Localization of the Naturally Occurring Plasmid ColE1 at the Cell Pole

2007

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“…Gels were either prepared for Western blot analysis or stained overnight with 0.5% Coomassie blue and destained in 50% methanol-10% acetic acid for 1 to 2 h. Detection of TadA-T7 was carried out using an anti-T7 monoclonal antibody (Novagen). Western blot analysis was performed as described by Rosche et al (55).…”

Section: Methodsmentioning

confidence: 99%

Nonspecific Adherence and Fibril Biogenesis by Actinobacillus actinomycetemcomitans : TadA Protein Is an ATPase

et al. 2001

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Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfatepolyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.

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“…RP4 is a 60 kbp IncP-a plasmid that contains a partitioning module comprising the ParA-like ATPase IncC, a site-specific DNA-binding ParB-like protein, KorB, and DNA sequences to which KorB bind (Rosche et al 2000; (Delbruck et al 2002). Yeast two-hybrid studies have implicated the C terminus in dimerization of full length KorB (Lukaszewicz et al 2002).…”

Section: Structural Insights From the Broad-host-range Plasmid Rp4mentioning

confidence: 99%

Towards understanding the molecular basis of bacterial DNA segregation

Leonard

Møller‐Jensen

Löwe

2005

Phil. Trans. R. Soc. B

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Bacteria ensure the fidelity of genetic inheritance by the coordinated control of chromosome segregation and cell division. Here, we review the molecules and mechanisms that govern the correct subcellular positioning and rapid separation of newly replicated chromosomes and plasmids towards the cell poles and, significantly, the emergence of mitotic-like machineries capable of segregating plasmid DNA. We further describe surprising similarities between proteins involved in DNA partitioning (ParA/ParB) and control of cell division (MinD/MinE), suggesting a mechanism for intracellular positioning common to the two processes. Finally, we discuss the role that the bacterial cytoskeleton plays in DNA partitioning and the missing link between prokaryotes and eukaryotes that is bacterial mechano-chemical motor proteins.

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

Cited by 37 publications

References 96 publications

Localization of the Naturally Occurring Plasmid ColE1 at the Cell Pole

Localization of the Naturally Occurring Plasmid ColE1 at the Cell Pole

Nonspecific Adherence and Fibril Biogenesis by Actinobacillus actinomycetemcomitans : TadA Protein Is an ATPase

Towards understanding the molecular basis of bacterial DNA segregation

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