Interaction of Penicillin-Binding Protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae†R6 morphogenesis.

Bacterial cell growth and division require the co-ordinated action of peptidoglycan biosynthetic enzymes and cell morphogenesis proteins. However, the regulatory mechanisms that allow generating proper bacterial shape and thus preserving cell integrity remain largely uncharacterized, especially in ovococci. Recently, the conserved eukaryotic-like Ser/Thr protein kinase of Streptococcus pneumoniae (StkP) was demonstrated to play a major role in cell shape and division. Here, we investigate the molecular mechanisms underlying the regulatory function(s) of StkP and show that it involves one of the essential actors of septal peptidoglycan synthesis, Penicillin-Binding Protein 2x (PBP2x). We demonstrate that StkP and PBP2x interact directly and are present in the same membrane-associated complex in S. pneumoniae. We further show that they both display a late-division localization pattern at the division site and that the positioning of PBP2x depends on the presence of the extracellular PASTA domains of StkP. We demonstrate that StkP and PBP2x interaction is mediated by their extracellular regions and that the complex formation is inhibited in vitro in the presence of cell wall fragments. These data suggest that the role of StkP in cell division is modulated by an interaction with PBP2x.

Nascent teichoic acids insertion into the cell wall directs the localization and activity of the major pneumococcal autolysin LytA.

The bacterial cell wall is in part composed of the peptidoglycan (PG) layer that maintains the cell shape and sustains the basic cellular processes of growth and division. The cell wall of Gram-positive bacteria also carries teichoic acids (TAs). In this work, we investigated how TAs contribute to the structuration of the PG network through the modulation of PG hydrolytic enzymes in the context of the Gram-positive Streptococcus pneumoniae bacterium. Pneumococcal TAs are decorated by phosphorylcholine residues which serve as anchors for the Choline-Binding Proteins, some of them acting as PG hydrolases, like the major autolysin LytA. Their binding is non covalent and reversible, a property that allows easy manipulation of the system. In this work, we show that the release of LytA occurs independently from its amidase activity. Furthermore, LytA fused to GFP was expressed in pneumococcal cells and showed different localization patterns according to the growth phase. Importantly, we demonstrate that TAs modulate the enzymatic activity of LytA since a low level of TAs present at the cell surface triggers LytA sensitivity in growing pneumococcal cells. We previously developed a method to label nascent TAs in live cells revealing that the insertion of TAs into the cell wall occurs at the mid-cell. In conclusion, we demonstrate that nascent TAs inserted in the cell wall at the division site are the specific receptors of LytA, tuning in this way the positioning of LytA at the appropriate place at the cell surface.