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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

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.
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Fig1: 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.

Mentions: The SC level, which is a reporter of DNA gyrase activity, of the pMS01 plasmid was determined in growing and SP cells, as well as in SP cells after nutrient addition. BB7222, BB7224, C600 and CAG9310 cells were grown at 30°C in LB-MOPS medium. To determine DNA gyrase reactivation, wt and rpoH SP cells were diluted 1:10 in LB-MOPS medium at 30°C (Figure 1a); on the other hand, wt and groEL140 cells were diluted 1:30 in LB-MOPS at 30°C and 43°C (Figure 1b). The 1:30 dilution favored a fast temperature increment of the LB-MOPS medium. DNA gyrase reactivation in SP cells grown at 37°C is observed 30 sec-1 min after addition of nutrients (Reyes-Domínguez et al. 2003). This reactivation in wt cells grown at 30°C is observed after 3–5 min.Figure 1


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230433&req=5

Fig1: 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.
Mentions: The SC level, which is a reporter of DNA gyrase activity, of the pMS01 plasmid was determined in growing and SP cells, as well as in SP cells after nutrient addition. BB7222, BB7224, C600 and CAG9310 cells were grown at 30°C in LB-MOPS medium. To determine DNA gyrase reactivation, wt and rpoH SP cells were diluted 1:10 in LB-MOPS medium at 30°C (Figure 1a); on the other hand, wt and groEL140 cells were diluted 1:30 in LB-MOPS at 30°C and 43°C (Figure 1b). The 1:30 dilution favored a fast temperature increment of the LB-MOPS medium. DNA gyrase reactivation in SP cells grown at 37°C is observed 30 sec-1 min after addition of nutrients (Reyes-Domínguez et al. 2003). This reactivation in wt cells grown at 30°C is observed after 3–5 min.Figure 1

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