Localization of the naturally occurring plasmid ColE1 at the cell pole

Abstract

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.

FIG. 1.

Localization of the ColE1 derivative pAS2 in E. coli. Cells of E. coli MGTRY(pAS2) with one (A), two (B and C), or three (D and E) foci or after treatment with cephalexin (F) are shown. In all panels, the nucleoid has been stained with DAPI. The bars in panels A and F correspond to 1 μm. Cells shown in panels B to D are at the same magnification as those in panel A.

FIG. 2.

Location of pAS2 foci in E. coli in the presence or absence of a second replicon. The position of foci in cells of MGTRY(pAS2) (A and B), MGTRY(pAS2)(pBAD33) (C and D), or MGTRY(pAS2)(RK2) (E and F) was determined as a fraction of cell length. Each point represents a single focus. The position of pAS2 in those cells with only a single focus (•) is shown in panels A, C, and E. The position of pAS2 foci in cells with two foci is shown in panels B, D, and F (▴, first focus; □, second focus).

FIG. 3.

Time-lapse photography of single cells of MGTRY(pAS2), showing movement of polar foci (A) or cell division in the absence of focus duplication (B). The time after transfer of the culture to an agarose pad for microscopic observation is noted in the top left corner of each picture. The bars correspond to 1 μm.

FIG. 4.

Localization of ColE1 in the presence of a second plasmid. Plasmid pAS2 was localized in E. coli MGTRY in the presence of plasmids pBAD33 (A) or RK2 (B) or in E. coli MG1655 in the presence of plasmid pCV234 (C). In panel A, cells were visualized for pAS2 and the nucleoid was stained with DAPI. In panels B and C, only foci are shown. The bar in each panel corresponds to 1 μm.

FIG. 5.

Influence of a second replicon on stability of ColE1 derivatives in E. coli. The stabilities of ColE1 derivative pAS2 in MG1655 (□) (A), of pAS4 (pAS2 + sopABC) in MG1655 (□) (B), and of pAS6 (pAS2 + O_B1 incC korB) in MGTRY (□) (C) were determined as the percentage of cells maintaining Gm resistance in the absence of selection, as described in Materials and Methods. Stability was also determined for pAS2 (A) and pAS4 (B) in the presence of pBAD33 (○) or RK2 (▵) and for pAS6 in the presence of pBAD33 (C) (○). Results shown are the averages and standard deviations from two or three experiments.

FIG. 6.

Localization of ColE1 derivatives carrying heterologous partition systems. (A to C) MGTRY(pAS4) (A, foci only; B, DAPI staining of nucleoid; C, merge of A and B); (D to F) MGTRY(pAS6) (D, foci only; E, DAPI staining of nucleoid; F, merge of D and E). The bar in panel A corresponds to 1 μm.