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 Streptococcus pneumoniae, gyrase (GyrA(2)GyrB(2)) 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: P(Zn)topA, carrying an ectopic topA copy under the control of the ZnSO(4)-regulated P(Zn) promoter and its derivative ΔtopAP(Zn)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 ΔtopAP(Zn)topA, the number of TopoI molecules depended, as expected, on ZnSO(4) concentration in the culture media, being the proportions of GyrA:TopoI molecules in 75, 150, and 300 μM ZnSO(4) of 1:0.43, 1:0.47, and 1:0.63, respectively, which allowed normal supercoiling and growth. However, in the absence of ZnSO(4), a higher GyrA:TopoI ratio (1:0.09) caused hyper-supercoiling (σ = −0.086) and lethality. Likewise, growth of ΔtopAP(Zn)topA in the absence of ZnSO(4) 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 (ΔtopAP(Zn)topA under viable conditions). Higher proportions, such as 1:0.18 observed in ΔtopAP(Zn)topA in the absence of ZnSO(4) 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.