Plasmid DNA supercoiling and gyrase activity in Escherichia coli wild-type and rpoS stationary-phase cells

Abstract

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

FIG. 1.

Agarose gel electrophoresis separation of reporter plasmid pMS01 isolated from MC4110 stationary-phase cells. Plasmid DNA topoisomers were isolated and separated through 1% agarose gels containing chloroquine at 12 μg/ml as described in the text. Migration was from top to bottom. (A) Distribution of plasmid topoisomers as a function of growth phase. (B) Plasmid supercoiling of 18-h stationary-phase cells after dilution into fresh medium. SC, supercoiled plasmids; EP, exponential phase; SP, stationary phase; R, time (in minutes) after the addition of fresh medium to 18-h stationary-phase cultures.

FIG. 2.

Expression of transcriptional λΦ(gyrA-lacZ) or λΦ(gyrB-lacZ) fusions along the growth curve and in 18-h stationary-phase cells after transfer to fresh medium. MC4100 derivatives bearing the transcriptional fusions were grown in LB-MOPS medium. Similar results were obtained in three independent experiments. Solid symbols, cell growth (at OD₅₅₀); open symbols, β-galactosidase levels expressed in Miller units as described in the text. (A and B) Expression of the transcriptional gyrA-lacZ (A) and gyrB-lacZ (B) fusions along the growth curve. (C and D) Expression of the transcriptional gyrA-lacZ (C) and gyrB-lacZ (D) fusions during the recovery of growth induced in stationary-phase cells by the addition of fresh medium.

FIG. 3.

Immunoblot analyses of extracts from exponential and stationary-phase MC4100 cells with anti-gyrA and anti-gyrB monoclonal antibodies. Total protein samples were separated by electrophoresis through 7.5% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes as decribed in the text. Similar results were obtained in three independent experiments. EP, exponential phase; SP, stationary phase.

FIG. 4.

RpoS influences the level of relaxation of plasmids isolated from stationary-phase cells and the capacity of these cells to supercoil plasmids after dilution into fresh medium. Plasmids were isolated, and topoisomers were separated as described for Fig. 1. Abbreviations are as defined for Fig. 1. wt, wild type.