Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei 106, Taiwan;
Mol Cell Proteomics. 2013 Oct;12(10):2701-13. doi: 10.1074/mcp.M113.029330. Epub 2013 Jul 4.
Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili-mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili-related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production.
嗜热栖热菌 HB27 是一种极端嗜热的真细菌，具有高频自然转化能力。因此，该生物常被用作研究热环境中 IV 型菌毛介导功能的模式生物，如游离 DNA 的摄取、黏附、扭动运动和生物膜形成。在这项研究中，采用鸟枪法和高精度质谱分析了嗜热栖热菌 HB27 的磷酸化蛋白质组。鉴定出了 93 个独特的磷酸肽，包括 53 个磷酸化蛋白中的 67 个体内磷酸化位点。Ser/Thr/Tyr 磷酸化位点的分布为 57%/36%/7%。磷酸化蛋白主要参与中心代谢途径和蛋白质/细胞包膜生物合成。根据这项分析，首次发现 IV 型菌毛相关成分 ATP 酶马达 PilF 在 Thr-368 和 Ser-372 上发生磷酸化。通过 PilF 的点突变，模拟磷酸化突变体 T368D 和 S372E 导致菌毛缺失和扭动运动缺失表型，而非磷酸化突变体 T368V 和 S372A 则显示菌毛和扭动运动。此外，模拟磷酸化突变体由于增加了胞外多糖的产生，表现出更高的生物膜形成能力和更高的初始附着率。总之，PilF 的磷酸化可能调节与胞外多糖产生相关的菌毛和生物膜形成。
