Molecular dissection of the chromosome partitioning protein RocS and regulation by phosphorylation

Chromosome segregation in bacteria is a critical process ensuring that each daughter cell receives an accurate copy of the genetic material during cell division. Active segregation factors, such as the ParABS system or SMC complexes, are usually essential for this process, but they are surprisingly dispensable in Streptococcus pneumoniae. Rather, chromosome segregation in S. pneumoniae relies on the protein Regulator of Chromosome Segregation (RocS), although the molecular mechanisms involved remain elusive. By combining genetics, in vivo imaging, and biochemical approaches, we dissected the molecular features of RocS involved in chromosome segregation. We investigated the respective functions of the three RocS domains, specifically the C-terminal amphipathic helix (AH), the N-terminal DNA-binding domain (DBD), and the coiled-coil domain (CCD) separating the AH and the DBD. Notably, we found that a single AH is not sufficient for membrane binding and that RocS requires prior oligomerization to interact with the membrane. We further demonstrated that this self-interaction was driven by the N-terminal part of the CCD. On the other hand, we revealed that the C-terminal part of the CCD corresponds to a domain of unknown function (DUF 536) and is defined by three conserved glutamines, which play a crucial role in RocS-mediated chromosome segregation. Finally, we showed that the DBD is phosphorylated by the unique serine-threonine kinase of S. pneumoniae StkP and that mimicking this phosphorylation abrogated RocS binding to DNA. Overall, this study offers new insights into chromosome segregation in Streptococci and paves the way for a deeper understanding of RocS-like proteins in other bacteria.IMPORTANCEBacteria have evolved a variety of mechanisms to properly segregate their genetic material during cell division. In this study, we performed a molecular dissection of the chromosome partitioning protein Regulator of Chromosome Segregation (RocS), a pillar element of chromosome segregation in S. pneumoniae that is also generally conserved in the Streptococcaceae family. Our systematic investigation sheds light on the molecular features required for successful pneumococcal chromosome segregation and the regulation of RocS by phosphorylation. In addition, our study also revealed that RocS shares functional domains with the Par protein, involved in an atypical plasmid segregation system. Therefore, we expect that our findings may serve to extend our understanding of RocS and RocS-like proteins while broadening the repertoire of partitioning systems used in bacteria.