Single-cell analysis of StkP activity reveals the temporal dynamics of DivIVA phosphorylation in Streptococcus pneumoniae

ABSTRACT Serine/threonine phosphorylation has emerged as a central regulatory mechanism of the bacterial cell cycle. However, the dynamics and temporal selectivity of protein phosphorylation through the cell cycle remain unamenable to existing analytical tools. Although fluorescent phosphorylation biosensors are widely available in single eukaryotic cells, such tools have yet to be adapted for bacteria. To address this gap, we developed an in vivo fluorescent biosensor specifically designed to monitor serine/threonine phosphorylation in single bacterial cells. Specifically, this sensor reports protein phosphorylation mediated by the serine/threonine kinase StkP, the main regulator of cell division in Streptococcus pneumoniae . We showed that StkP activity is constitutive throughout the cell cycle. However, we observed that StkP-mediated protein phosphorylation is heterogeneous from cell to cell in early stages of the cell cycle. Importantly, our results revealed that the cell division protein DivIVA is found phosphorylated at the cell poles, suggesting an additional regulatory layer controlling its function at this location. Collectively, this work provides original insights into the temporal dynamics of StkP activity and the regulation of the pneumococcal cell cycle by phosphorylation. IMPORTANCE How bacterial serine/threonine phosphorylation is dynamically regulated in living single cells remains inaccessible, largely because efficient reporters required to monitor phosphorylation in vivo are lacking. In this study, we designed a fluorescent biosensor approach that enables direct visualization of serine/threonine phosphorylation dynamics in single bacterial cells. Using this approach, we revealed that the activity of StkP, the serine/threonine kinase that regulates cell division in Streptococcus pneumoniae , is constitutive throughout the cell cycle, yet StkP-mediated protein phosphorylation is unexpectedly heterogeneous across individual cells in the early stages of division. Importantly, our reporter also showed that the phosphorylation of the key cell division protein DivIVA is highly dynamic. Together, these findings provide the conceptual basis to improve our understanding of the role of phosphorylation in pneumococcal cell division and pave the way for investigating phosphorylation dynamics in other bacterial species.