Evolution of paralogous multicomponent systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria

Many bacteria decorate flagellin with sialic acid-like sugars such as pseudaminic acid (Pse) by O-glycosylation on serine or threonine residues. Evidence for sufficiency of sialylation by a conserved flagellin glycosyltransferase (fGT) system is lacking, presumably because of (a) missing component(s). Here, we reconstituted two Maf-type fGTs from the Gram-negative bacterium Shewanella oneidensis MR-1 in a heterologous host producing a Pse donor sugar. While Maf-1 is sufficient for flagellin glycosylation, Maf-2 reconstitution requires a newly identified, cis-encoded and conserved specificity factor GlfM, predicted to form a four-helix bundle. While GlfM binds Maf-2 to form a ternary complex with flagellin, the C-terminal tetratricopeptide repeat (TPR) domain of Maf-1 confers flagellin acceptor and O-glycosylation specificity at preferred serine residues. GlfM from Gram-negative and Gram-positive bacteria are functional, providing evidence for convergent evolution of specialized flagellin modification systems with acceptor serine selectivity, while also shaping the evolution of bacterial tripartite and bipartite O-glycosylation systems.

许多细菌通过对丝氨酸或苏氨酸残基进行O-糖基化修饰，将假单胞菌酸（pseudaminic acid, Pse）这类唾液酸类糖修饰于鞭毛蛋白之上。目前尚缺乏保守鞭毛蛋白糖基转移酶（fGT）系统可介导唾液酸化修饰的充分证据，推测其原因在于存在缺失的功能组分。本研究在能够产生Pse供体糖的异源宿主中，重构了来自革兰氏阴性菌希瓦氏菌MR-1的两种Maf型fGT。其中Maf-1即可独立完成鞭毛蛋白的糖基化修饰，而Maf-2的重构则需要新发现的顺式编码保守特异性因子GlfM，该因子被预测可形成四螺旋束结构。研究发现，GlfM可与Maf-2结合，与鞭毛蛋白形成三元复合物；而Maf-1的C端四肽重复（TPR）结构域则可赋予鞭毛蛋白受体特异性，并在偏好性丝氨酸残基处实现O-糖基化修饰。来自革兰氏阴性菌与革兰氏阳性菌的GlfM均具有功能活性，这为具备受体丝氨酸选择性的特化鞭毛蛋白修饰系统的趋同进化提供了证据，同时也阐明了细菌三组分与双组分O-糖基化系统的演化历程。