Multicopy Plasmids are Clustered and Localized in Escherichia coli

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 8; pp. 4486 - 4491
• Main Authors: Pogliano, Joe, Ho, Thanh Quoc, Zhong, Zhenping, Helinski, Donald R.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 10-04-2001; National Acad Sciences
• Subjects: Bacteria; Bacterial Proteins - genetics; Base Sequence; Biological Sciences; Cell cycle; Cell growth; Cell membranes; Cellular biology; Chromosomes; Daughter cells; DNA; DNA Primers; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins; Fluorescence in situ hybridization; Geodetic position; Green Fluorescent Proteins; Lac Repressors; Luminescent Proteins - genetics; Microscopy; multicopy plasmids; plasmid pUC19; plasmid RK2; Plasmids; Repressor Proteins - genetics; Subcellular Fractions - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.081075798

We localized the multicopy plasmid RK2 in Escherichia coli and found that the number of fluorescent foci observed in each cell was substantially less than the copy number of the plasmid, suggesting that many copies of RK2 are grouped into a few multiplasmid clusters. In minimal glucose media, the majority of cells had one or two foci, with a single focus localized near midcell, and two foci near the 1/4 and 3/4 cell positions. The number of foci per cell increased with cell length and with growth rate, and decreased upon entering stationary phase, suggesting a coordination of RK2 replication or segregation with the bacterial cell cycle. Time-lapse microscopy demonstrated that partitioning of RK2 foci is achieved by the splitting of a single focus into two or three smaller foci, which are capable of separating with rapid kinetics. A derivative of the high-copy-number plasmid pUC19 containing the lacO array was also localized by tagging with GFP-Lacl. Whereas many of the cells contained numerous, randomly diffusing foci, most cells exhibited one or two plasmid clusters located at midcell or the cell quarter positions. Our results suggest a model in which multicopy plasmids are not always randomly diffusing throughout the cell as previously thought, but can be replicated and partitioned in clusters targeted to specific locations.