Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division

Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.尽管历经多年深入研究，学界对于协调细菌细胞生长与分裂的调控网络仍有诸多未解之处。肺炎链球菌（Streptococcus pneumoniae）如何形成其标志性的椭球形细胞形态，其机制在很大程度上仍不明确。本研究针对细胞分裂旁系同源蛋白DivIVA与GpsB，以及丝氨酸/苏氨酸激酶StkP之间的相互作用展开分析。我们观察到，divIVA基因的缺失会阻碍细胞伸长，导致细胞变短并趋于圆形。与之相反，GpsB的缺失会抑制细胞分裂，并触发细胞异常伸长。值得注意的是，ΔgpsB突变体的伸长细胞呈现出螺旋状的FtsZ分布模式，而非正常的Z环结构，同时伴随DivIVA与肽聚糖（peptidoglycan, PG）合成相关蛋白的螺旋状分布。尤为关键的是，divIVA的缺失能够抑制ΔgpsB细胞的伸长表型。上述数据表明，DivIVA可促进细胞伸长，而GpsB则对此过程起到拮抗作用。蛋白质相互作用分析显示，GpsB与DivIVA均不直接结合FtsZ，而是通过细胞分裂蛋白EzrA与FtsZ产生间接相互作用；此外，GpsB可直接与DivIVA结合。上述结果与“DivIVA与GpsB作为分子开关，协调外周与隔膜肽聚糖合成，并通过EzrA将这些过程与Z环相连”的结论相符。对转肽酶PBP2x、PBP2b以及脂质翻转酶FtsW、RodA在ΔgpsB细胞中的共定位分析进一步证实，存在一个大型的单一肽聚糖组装复合体。最后，本研究证实GpsB对于StkP的隔膜定位与激酶活性不可或缺，因此也参与了StkP介导的DivIVA磷酸化过程。综上，我们提出StkP/DivIVA/GpsB三者构成的调控模块，可精细调控负责肺炎链球菌椭球形细胞形态的两种肽聚糖合成模式——外周合成与隔膜合成。