Role of chaperones and ATP synthase in DNA gyrase reactivation in Escherichia coli stationary-phase cells after nutrient addition

Abstract

Escherichia coli stationary-phase (SP) cells contain relaxed DNA molecules and recover DNA supercoiling once nutrients become available. In these cells, the reactivation of DNA gyrase, which is a DNA topoisomerase type IIA enzyme, is responsible for the recovery of DNA supercoiling. The results presented in this study show that DNA gyrase reactivation does not require cellular chaperones or polyphosphate. Glucose addition to SP cells induced a slow recovery of DNA supercoiling, whereas resveratrol, which is an inhibitor of ATP synthase, inhibited the enzyme reactivation. These results suggest that DNA gyrase, which is an ATP-dependent enzyme, remains soluble in SP cells, and that its reactivation occurs primarily due to a rapid increase in the cellular ATP concentration.

Keywords: ATP synthase; DNA gyrase; Escherichia coli; Stationary phase.

Figure 1

DNA gyrase reactivation in stationary-phase cells with low levels of the main chaperones. Cells were grown in LB-MOPS medium at 30°C. Strains used included the following: a) BB7222 (wild type) and BB7224 (ΔrpoH), and b) C600 (wild type) and CAG9310 groEL140 bearing the reporter plasmid pMS01. Strain BB7224 expresses very low levels of the main cellular chaperones, except for GroE, while CAG9310 carries the temperature-sensitive groEL140 mutation. To induce the recovery of the DNA SC level in stationary-phase cells, cell cultures were diluted in pre-warmed LB-MOPS medium. a: 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, and 3) stationary-phase cells diluted 1:10 in LB-MOPS at 30°C and incubated for 5 min. b: 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, 3) stationary-phase cells diluted 1:30 in LB-MOPS at 30°C and incubated for 5 min, and 4) stationary-phase cells diluted 1:30 in LB-MOPS at 43°C and incubated for 5 min. Before dilution in LP-MOPS at 43°C, SP cell cultures were incubated for 15 min at 43°C. Plasmid topoisomers were isolated and separated on 1% agarose gels containing 10 μg/mL chloroquine. Migration proceeded from top to bottom. Topoisomers more supercoiled migrated more rapidly in the gel. Similar results were obtained in at least three independent experiments.

Figure 2

DNA gyrase reactivation in stationary-phase cells with low levels of polyphosphate, polyP. Cells grown in LB-MOPS media at 37°C. The strains used were BW25113 (wt) and BW25113 Δppk, bearing the reporter plasmid pMS01. To induce the recovery of the DNA SC level in stationary-phase cells, cell cultures were diluted 1:10 in pre-warmed LB-MOPS medium. 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, 3) stationary-phase cells diluted 1:10 in LB-MOPS and incubated for 30 sec, and 4) stationary-phase cells diluted 1:10 in LB-MOPS and incubated for 1 min. Plasmid topoisomers were isolated and separated as described in Figure 1. Similar results were obtained in at least three independent experiments.

Figure 3

Effect of glucose as carbon source on DNA gyrase reactivation in MC4100 stationary-phase cells. Cells were grown in LB-MOPS at 37°C. To induce the recovery of the DNA SC level in stationary-phase cells, cell cultures were diluted 1:10 in pre-warmed LB-MOPS or 1:30 in a 0.86% NaCl solution with or without 0.4% glucose. 1) Exponentially growing cells; 2) 48 hr stationary-phase cells; 3) and 4) stationary-phase cells diluted in LB-MOPS and incubated for 3 or 5 min, respectively; 5) and 6) stationary-phase cells diluted in 0.86% NaCl and incubated for 3 or 5 min, respectively; 7) and 8) stationary-phase cells diluted in 0.86% NaCl-0.4% glucose and incubated for 3 or 5 min, respectively. Plasmid topoisomers were isolated and separated as described in Figure 1. Similar results were obtained in at least three independent experiments.

Figure 4

Effect of ATPase inhibitors resveratrol (RVT), piceatannol (PCT) or sodium azide on DNA gyrase reactivation in MC4100 stationary-phase cells. Cells were grown at 37°C in LB-MOPS medium. To induce the recovery of the DNA SC level, cell cultures were diluted 1:10 in pre-warmed LB-MOPS medium with or without the inhibitor. a: 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, 3) stationary-phase cells diluted in LB-MOPS media and incubated 1 min, 4), 5) and 6) stationary-cells diluted in LB-MOPS with RVT 400 μM, 1.2 mM, or 2.0 mM, respectively. The diluted cultures were incubated 1 min. b: 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, 3) stationary-phase cells diluted in LB-MOPS and incubated 1 min, 4), 5) and 6) stationary-phase cells diluted in LB-MOPS with PCT 100 μM, 200 μM or 300 μM, respectively. The diluted cultures were incubated 1 min. c: 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, 3) stationary-phase cells diluted in LB-MOPS and incubated 10 min, 4) stationary-phase cells diluted in LB-MOPS-sodium azide 3 mM and incubated 10 min, 5) stationary-phase cells diluted in LB-MOPS-sodium azide 5 mM and incubated 5 min, 6) stationary-phase cells diluted in LB-MOPS-sodium azide 5 mM and incubated 10 min. Plasmid topoisomers were isolated and separated as described in Figure 1. Similar results were obtained in at least three independent experiments.