Suppression and synthetic-lethal genetic relationships of gpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin-binding protein interactions in Streptococcus pneumoniae D39

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side-wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that divIVA mutations are not epistatic to gpsB division-protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of gpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. gpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, gpsB mutations are synthetically lethal with pbp1a, but not pbp2a or pbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP-PBP2x.