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

Gutiérrez-Estrada A, Ramírez-Santos J, Gómez-Eichelmann Mdel C - Springerplus (2014)

Bottom Line: In these cells, the reactivation of DNA gyrase, which is a DNA topoisomerase type IIA enzyme, is responsible for the recovery of DNA supercoiling.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Institute of Biomedical Research, National Autonomous University of México, P.O. Box 70228, México City, 04510 México.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig4: 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.

Mentions: To determine the effect of RVT, PCT, and sodium azide on the recovery of DNA gyrase activity, the DNA SC level was determined in SP cells grown in LB-MOPS and in SP cells diluted 1:10 in LB-MOPS with or without the ATP synthase inhibitor. DMSO or DMSO-RVT, PCT or sodium azide added 1 hr before diluting the SP cells, did not modify the plasmid topoisomer distribution in these cells. The recovery of the DNA SC level was complete after 1 min incubation of SP cells diluted in LB-MOPS; whereas the recovery of SP cells diluted in LB-MOPS with 400 μM RVT was partial, this recovery was completely inhibited by 1.2 mM RVT and 2.0 mM RVT(Figure 4a). PCT 100 μM induces an inhibitory effect similar to 400 μM RVT in vitro or in cell cultures (Dadi et al. 2009). However, 100 μM, 300 μM or 500 μM PCT did not inhibit DNA SC recovery (Figure 4b). The effects of RVT and of PCT in cell cultures were determined after 20 hr of growth in LB medium (Dadi et al. 2009), whereas, in this study, the effect on DNA SC recovery was determined 1 min after polyphenol addition to SP cells. The different inhibition levels observed with RVT and with PCT most likely occur due to higher cell membrane permeability for RVT than for PCT. The RVT molecule has three hydroxyl groups compared with four in PCT. Sodium azide (1 and 3 mM), which is a potent inhibitor of ATP synthase which inhibits ATP hydrolysis, but not ATP synthesis (Gledhill et al. 2007; Zharova and Vinogradov 2004), did not inhibit DNA gyrase recovery (Figure 4c).Figure 4


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

Gutiérrez-Estrada A, Ramírez-Santos J, Gómez-Eichelmann Mdel C - Springerplus (2014)

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.
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Fig4: 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.
Mentions: To determine the effect of RVT, PCT, and sodium azide on the recovery of DNA gyrase activity, the DNA SC level was determined in SP cells grown in LB-MOPS and in SP cells diluted 1:10 in LB-MOPS with or without the ATP synthase inhibitor. DMSO or DMSO-RVT, PCT or sodium azide added 1 hr before diluting the SP cells, did not modify the plasmid topoisomer distribution in these cells. The recovery of the DNA SC level was complete after 1 min incubation of SP cells diluted in LB-MOPS; whereas the recovery of SP cells diluted in LB-MOPS with 400 μM RVT was partial, this recovery was completely inhibited by 1.2 mM RVT and 2.0 mM RVT(Figure 4a). PCT 100 μM induces an inhibitory effect similar to 400 μM RVT in vitro or in cell cultures (Dadi et al. 2009). However, 100 μM, 300 μM or 500 μM PCT did not inhibit DNA SC recovery (Figure 4b). The effects of RVT and of PCT in cell cultures were determined after 20 hr of growth in LB medium (Dadi et al. 2009), whereas, in this study, the effect on DNA SC recovery was determined 1 min after polyphenol addition to SP cells. The different inhibition levels observed with RVT and with PCT most likely occur due to higher cell membrane permeability for RVT than for PCT. The RVT molecule has three hydroxyl groups compared with four in PCT. Sodium azide (1 and 3 mM), which is a potent inhibitor of ATP synthase which inhibits ATP hydrolysis, but not ATP synthesis (Gledhill et al. 2007; Zharova and Vinogradov 2004), did not inhibit DNA gyrase recovery (Figure 4c).Figure 4

Bottom Line: In these cells, the reactivation of DNA gyrase, which is a DNA topoisomerase type IIA enzyme, is responsible for the recovery of DNA supercoiling.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Institute of Biomedical Research, National Autonomous University of México, P.O. Box 70228, México City, 04510 México.

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.

No MeSH data available.


Related in: MedlinePlus