StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible P-Zn promoter (strain Delta staRP(Zn)staR) or of the maltose-inducible P-Mal promoter (strain Delta staRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of Delta staRP(Zn)staR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (sigma) in vivo, which was higher in the absence of StaR (sigma = -0.049) than when StaR was overproduced (sigma = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

Automated summary

In this study, a new topoisomerase I regulator protein (StaR) was characterized in Streptococcus pneumoniae. It was found that StaR directly affects novobiocin susceptibility and needs to be maintained within a narrow range.