Data_Sheet_1_Agrobacterium tumefaciens Type IV and Type VI Secretion Systems Reside in Detergent-Resistant Membranes.pdf

Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen Agrobacterium tumefaciens. Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in A. tumefaciens.

细胞膜并非均质，而是划分为横向微区室，这些微区室被视为真核生物和原核生物中各种生理过程的生物化学反应中心。鉴于其特殊的脂质和蛋白质组成，其中一些微区室对非离子洗涤剂的处理具有抵抗力，并可被纯化成洗涤剂抵抗膜（DRMs）。在本研究中，我们报道了来自革兰氏阴性植物病原体根癌农杆菌的DRMs蛋白质组。通过无标记液相色谱-串联质谱技术，我们鉴定了在正常生长条件和模拟致病性生长条件下分离的DRMs中富集的蛋白质。如SPFH（ stomatin/prohibitin/flotillin/HflKC）蛋白家族中的HflK、HflC和Atu3772等显著的微区室标记蛋白，以及蛋白酶FtsH在所有条件下均高度富集于DRMs中。此外，参与细胞壁生物发生、运输和分泌，以及运动性和趋化性相关蛋白在DRMs中均有过度表达。最引人注目的是，我们发现与致病性相关的蛋白质，如VirA/VirG双组分系统，以及跨越膜的IV型和VI型分泌系统在DRMs中富集。对这两种分泌系统及其标记蛋白的细胞定位进行荧光显微镜观察，其结果与蛋白质组学方法的结果一致。这些发现表明，在根癌农杆菌中，致病性特征被微区室化，并分布于功能微区室之中。