Vignon Guillaume, Köhler Rolf, Larquet Eric, Giroux Stéphanie, Prévost Marie-Christine, Roux Pascal, Pugsley Anthony P
Unité de Génétique Moléculaire (CNRS URA 2172), Institut Pasteur, Paris, France.
J Bacteriol. 2003 Jun;185(11):3416-28. doi: 10.1128/JB.185.11.3416-3428.2003.
The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).
革兰氏阴性菌的分泌装置或II型分泌机制包括几种IV型菌毛样蛋白(假菌毛蛋白),这些蛋白是分泌所绝对必需的。我们之前报道过,在琼脂平板上生长的、高水平表达产酸克雷伯菌支链淀粉酶(Pul)分泌基因的大肠杆菌K-12细胞表面,存在由假菌毛蛋白PulG组成的束状菌毛(N. Sauvonnet、G. Vignon、A. P. Pugsley和P. Gounon,《欧洲分子生物学组织杂志》19:2221 - 2228,2000年)。我们在此表明,PulG是纯化菌毛中唯一的假菌毛蛋白,并且这种现象并不局限于在大肠杆菌中重组的Pul分泌装置或PulG。例如,大肠杆菌内源性gsp分泌基因的高水平表达导致由假菌毛蛋白GspG组成的束状菌毛的产生,并且Pul分泌装置能够形成由来自其他细菌分泌系统的PulG样蛋白组成的菌毛。通过添加八种不同肽段使C末端延长得到的PulG衍生物也能组装成菌毛并在分泌中发挥作用。其中三个C末端肽段显示沿组装菌毛的全长暴露。因此,PulG的C末端可能代表免疫原性表位或其他肽序列插入的允许位点。这些PulG变体之一,其C末端带有六个组氨酸标签,形成了非极性、非束状的菌毛,这表明束状形成和极性定位与PulG在分泌中发挥功能的能力无关。我们提出,PulG菌毛是周质“假菌毛”的一种人为表现形式,并且周质内假菌毛的延伸和收缩循环推动支链淀粉酶通过外膜中的分泌通道。只有当主要假菌毛亚基(PulG)过量产生导致菌毛长度控制和收缩失调时,才会产生延伸到外膜之外的异常长的菌毛。