Suppression and Synthetic-Lethal Genetic Relationships of ΔgpsB Mutations\ud Indicate That GpsB Mediates Protein Phosphorylation and Penicillin-Binding\ud Protein Interactions in Streptococcus pneumoniae D39

摘要

GpsB regulatory protein and StkP protein kinase have been proposed as molecular\udswitches that balance septal and peripheral (side-wall like) peptidoglycan (PG)\udsynthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this\udswitching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB\uddivision-protein mutations in progenitor D39 and related genetic backgrounds; nor is\udGpsB required for StkP localization or FDAA labeling at septal division rings. However,\udwe confirm that reduction of GpsB amount leads to decreased protein phosphorylation\udby StkP and report that the essentiality of ΔgpsB mutations is suppressed by\udinactivation of PhpP protein phosphatase, which concomitantly restores protein\udphosphorylation levels. ΔgpsB mutations are also suppressed by other classes of\udmutations, including one that eliminates protein phosphorylation and may alter division.\udMoreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or\udΔpbp1b, mutations, suggesting GpsB activation of PBP2a activity. Consistent with this\udresult, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a,\udPBP2b, and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents\udPBP2x migration to septal centers. These results support a model in which GpsB\udnegatively regulates peripheral PG synthesis by PBP2b and positively