The balance between gyrase and topoisomerase I activities determines levels of supercoiling, nucleoid compaction, and viability in bacteria.

Two enzymes are responsible for maintaining supercoiling in the human pathogen , gyrase (GyrAGyrB) and topoisomerase I. To attain diverse levels of topoisomerase I (TopoI, encoded by ), two isogenic strains derived from wild-type strain R6 were constructed: P , carrying an ectopic copy under the control of the ZnSO-regulated P promoter and its derivative ΔP , which carries a 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 ΔP , the number of TopoI molecules depended, as expected, on ZnSO concentration in the culture media, being the proportions of GyrA:TopoI molecules in 75, 150, and 300 μM ZnSO of 1:0.43, 1:0.47, and 1:0.63, respectively, which allowed normal supercoiling and growth. However, in the absence of ZnSO, a higher GyrA:TopoI ratio (1:0.09) caused hyper-supercoiling (σ = -0.086) and lethality. Likewise, growth of ΔP in the absence of ZnSO 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 (ΔP under viable conditions). Higher proportions, such as 1:0.18 observed in ΔP in the absence of ZnSO 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.