ABSTRACTParB proteins bind centromere-like DNA sequences called parSsites and are involved in plasmid and chromosome segregation in bacteria. We previously showed that the opportunistic human pathogen Streptococcus pneumoniaecontains four parSsequences located close to the origin of replication which are bound by ParB. Using chromatin immunoprecipitation (ChIP), we found here that ParB spreads out from one of these parSsites, parS(−1.6°), for more than 5 kb and occupies the nearby comCDEoperon, which drives competence development. Competence allows S. pneumoniaeto take up DNA from its environment, thereby mediating horizontal gene transfer, and is also employed as a general stress response. Mutating parS(−1.6°) or deleting parBresulted in transcriptional up-regulation of comCDEand ssbB(a gene belonging to the competence regulon), demonstrating that ParB acts as a repressor of competence. However, genome-wide transcription analysis showed that ParB is not a global transcriptional regulator. Different factors, such as the composition of the growth medium and antibiotic-induced stress, can trigger the sensitive switch driving competence. This work shows that the ParB-parSchromosome segregation machinery also influences this developmental process.IMPORTANCEStreptococcus pneumoniae(pneumococcus) is an important human pathogen responsible for more than a million deaths each year. Like all other organisms, S. pneumoniaemust be able to segregate its chromosomes properly. Not only is understanding the molecular mechanisms underlying chromosome segregation in S. pneumoniaetherefore of fundamental importance, but also, this knowledge might offer new leads for ways to target this pathogen. Here, we identified a link between the pneumococcal chromosome segregation system and the competence-developmental system. Competence allows S. pneumoniaeto take up and integrate exogenous DNA in its chromosome. This process plays a crucial role in successful adaptation to—and escape from—host defenses, antibiotic treatments, and vaccination strategies. We show that the chromosome segregation protein ParB acts as a repressor of competence. To the best of our knowledge, this is the first example of a ParB protein controlling bacterial competence.