Functional dissection of the three N-terminal general secretory pathway domains and the Walker motifs of the traffic ATPase PilF from Thermus thermophilus
Functional dissection of the three N-terminal general secretory pathway domains and the Walker motifs of the traffic ATPase PilF from Thermus thermophilus
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DOI：
10.1007/s00792-018-1008-9
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发表时间：
2018-05-01
期刊：
影响因子：
2.9
通讯作者：
Averhoff, Beate
中科院分区：
文献类型：
--
作者：
Kruse, Kerstin;Salzer, Ralf;Averhoff, Beate
嗜热菌的交通ATPase PILF促进菌毛的组装和DNA的摄取。PILF不同于其他交通性ATPase的是一个三重的总分泌途径II，蛋白E，N-末端结构域(GSPIIABC)。我们通过分析一组PILF缺失衍生物和PILF突变体，研究了GSPII结构域、Walker A基序和催化谷氨酸在体内和体外的作用。在这里，我们报告了缺乏前两个或所有三个GSPII结构域的PILF变体不能形成稳定的六聚体，表明三重GSPII结构域在复合体形成和/或稳定性中的作用。一个PILF Delta GSPIIC突变体在菌毛组装过程中明显受损，这导致了GSPIIC结构域在菌毛组装中发挥重要作用的结论。有趣的是，PILF Delta GSPIIC突变体具有高度变形性。这表明GSPIIC对转化效率有很强的影响。PilFa匕首GSPIIA突变体表现出野生型抽动，但减少了毛发介导的抽动运动，表明GSPIIA在毛发动力学中发挥作用。此外，我们报告了具有ATP结合Walker A基序或催化谷氨酸残基缺陷的PILF突变体在连接和自然转化方面存在缺陷。这些发现表明，在PILF的自然转化和菌毛组装的双重功能中，ATP结合和水解酶都是必不可少的。
The traffic ATPase PilF of Thermus thermophilus powers pilus assembly as well as uptake of DNA. PilF differs from other traffic ATPases by a triplicated general secretory pathway II, protein E, N-terminal domain (GSPIIABC). We investigated the in vivo and in vitro roles of the GSPII domains, the Walker A motif and a catalytic glutamate by analyzing a set of PilF deletion derivatives and pilF mutants. Here, we report that PilF variants devoid of the first two or all three GSPII domains do not form stable hexamers indicating a role of the triplicated GSPII domain in complex formation and/or stability. A pilF Delta GSPIIC mutant was significantly impaired in piliation which leads to the conclusion that the GSPIIC domain plays a vital role in pilus assembly. Interestingly, the pilF Delta GSPIIC mutant was hypertransformable. This suggests that GSPIIC strongly affects transformation efficiency. A pilFa dagger GSPIIA mutant exhibited wild-type piliation but reduced pilus-mediated twitching motility, suggesting that GSPIIA plays a role in pilus dynamics. Furthermore, we report that pilF mutants with a defect in the ATP binding Walker A motif or in the catalytic glutamate residue are defective in piliation and natural transformation. These findings show that both, ATP binding and hydrolysis, are essential for the dual function of PilF in natural transformation and pilus assembly.