2026-05-17T05:43:40Zhttps://repisalud.isciii.es/rest/oai/requestoai:repisalud.isciii.es:20.500.12105/160922025-02-27T17:52:16Zcom_20.500.12105_2052com_20.500.12105_2051col_20.500.12105_19609col_20.500.12105_25179
Repisalud
author
García-López, Míriam
author
Megías, Diego
author
Ferrandiz-Avellano, Maria-Jose
author
de la Campa, Adela G
funder
Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)
funder
Ministerio de Ciencia e Innovación (España)
funder
Agencia Estatal de Investigación (España)
2023-05-18T09:11:19Z
2023-05-18T09:11:19Z
2023-01
Front Microbiol. 2023 Jan 11;13:1094692.
10.3389/fmicb.2022.1094692
1664-302X
Frontiers in microbiology
36713152
http://hdl.handle.net/20.500.12105/16092
Two enzymes are responsible for maintaining supercoiling in the human pathogen Streptococcus pneumoniae, gyrase (GyrA2GyrB2) and topoisomerase I. To attain diverse levels of topoisomerase I (TopoI, encoded by topA), two isogenic strains derived from wild-type strain R6 were constructed: PZn topA, carrying an ectopic topA copy under the control of the ZnSO4-regulated PZn promoter and its derivative ΔtopAPZn topA, which carries a topA deletion at its native chromosomal location. We estimated the number of TopoI and GyrA molecules per cell by using Western-blot and CFUs counting, and correlated these values with supercoiling levels. Supercoiling was estimated in two ways. We used classical 2D-agarose gel electrophoresis of plasmid topoisomers to determine supercoiling density (σ) and we measured compaction of nucleoids using for the first time super-resolution confocal microscopy. Notably, we observed a good correlation between both supercoiling calculations. In R6, with σ = -0.057, the average number of GyrA molecules per cell (2,184) was higher than that of TopoI (1,432), being the GyrA:TopoI proportion of 1:0.65. In ΔtopAPZn topA, the number of TopoI molecules depended, as expected, on ZnSO4 concentration in the culture media, being the proportions of GyrA:TopoI molecules in 75, 150, and 300 μM ZnSO4 of 1:0.43, 1:0.47, and 1:0.63, respectively, which allowed normal supercoiling and growth. However, in the absence of ZnSO4, a higher GyrA:TopoI ratio (1:0.09) caused hyper-supercoiling (σ = -0.086) and lethality. Likewise, growth of ΔtopAPZn topA in the absence of ZnSO4 was restored when gyrase was inhibited with novobiocin, coincidentally with the resolution of hyper-supercoiling (σ change from -0.080 to -0.068). Given that TopoI is a monomer and two molecules of GyrA are present in the gyrase heterotetramer, the gyrase:TopoI enzymes proportion would be 1:1.30 (wild type R6) or of 1:1.26-0.86 (ΔtopAPZn topA under viable conditions). Higher proportions, such as 1:0.18 observed in ΔtopAPZn topA in the absence of ZnSO4 yielded to hyper-supercoiling and lethality. These results support a role of the equilibrium between gyrase and TopoI activities in supercoiling maintenance, nucleoid compaction, and viability. Our results shed new light on the mechanism of action of topoisomerase-targeting antibiotics, paving the way for the use of combination therapies.
eng
DNA gyrase
DNA supercoiling
DNA topoisomerase I
Regulation of supercoiling
Supercoiling homeostasis
The balance between gyrase and topoisomerase I activities determines levels of supercoiling, nucleoid compaction, and viability in bacteria
research article
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BalanceBetweenGyraseTopoisomeraseI_2023.pdf
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text/plain
BalanceBetweenGyraseTopoisomeraseI_2023.pdf.txt