Phosphoproteomic analysis reveals the effects of PilF phosphorylation on type IV pilus and biofilm formation in Thermus thermophilus HB27
Date Issued
2013
Date
2013
Author(s)
Wu, Wan-Ling
Abstract
Protein phosphorylation has been established as an important post-translational modification in bacterial cells. Bacterial phosphoproteomics provides a better understanding of the regulatory networks in physiological processes. In this study, the shotgun approach was applied to analyze the phosphoproteome of the extremely thermophilic eubacterium T. thermophilus HB27. The genus Thermus isolated from its natural hot spring environment with a high frequency of natural competence often used as a thermophilic model to investigate the molecular basis of type IV pili (T4P)-mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. 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. Sequence alignment and comparison revealed that the identified phosphoproteins, phosphosites, and their flanked regions involved in central metabolic pathways were highly conserved. Interestingly, four phosphosugar mutases were phosphorylated on a conserved serine residue and also found in all studied prokaryotic phosphoproteomes. According to this analysis, the identified phosphoproteins such as chaperonin GroEL, DAG kinase, MurE, and PilF involved in the protein synthesis machinery and regulation of cell envelope biosynthesis were correlated with thermoadaptation. Moreover, the ATPase motor PilF, a T4P-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 the nonpiliated and nontwitching phenotypes, whereas non-phosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with higher initial attachment rate, caused by increasing exopolysaccharide production. Therefore, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production. In summary, this study of thermophilic bacteria phosphoproteomics indicated that phosphorylated thermostable proteins may play important roles in regulating many metabolic pathways and adaptation processes in a thermal environment.
Subjects
嗜熱性菌屬
磷酸化蛋白質體學
陰離子交換樹酯和二氧化鈦親和性磷酸胜肽萃取
質譜儀分析儀/第四型纖毛/ATP分解酶活性PilF蛋白質/抽扯性運動/生物膜生成/胞外多糖產生
Type
thesis
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