| Autor: |
de Vasconcelos Junior AA; Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain., Tirado-Vélez JM; Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain., Martín-Galiano AJ; Unidades Centrales Científico-Técnicas, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain., Megias D; Unidad de Microscopía Confocal, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain., Ferrándiz MJ; Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain., Hernández P; Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain., Amblar M; Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain., de la Campa AG; Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain.; Presidencia, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain. |
| Abstrakt: |
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 ZnSO 4 -inducible P Zn promoter (strain Δ staR P Zn staR ) or of the maltose-inducible P Mal promoter (strain Δ staR pLS1ROM staR) . 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 Δ staR P Zn staR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -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. |
