Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci
Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci
复制标题
DOI：
10.1111/j.1462-5822.2005.00674.x
复制
发表时间：
2006-06-01
影响因子：
3.4
通讯作者：
Ichinose, Y
中科院分区：
文献类型：
--
作者：
Taguchi, F;Takeuchi, K;Ichinose, Y
糖基化岛是糖基化所必需的遗传区域。紫丁香假单胞菌鞭毛蛋白糖基化岛。Tabaci 6605由三个ORF组成：ORF1、ORF2和ORF3。Orf1和ORF2编码糖基转移酶，它们的缺失突变体Delta orf1和Delta ORF2分别存在鞭毛蛋白糖基化缺陷或产生部分糖基化的鞭毛蛋白。用天冬氨酸N-肽酶消化野生型细菌的糖基化鞭毛蛋白和Delta orf1突变体的非糖基化鞭毛蛋白，并随后进行高效液相分析，发现了候选的糖基化氨基酸。通过产生定点Ser/Ala替换突变体，在143、164、176、183、193和201位确定了所有糖基化氨基酸残基。基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱仪(MS)分析表明，每个糖链大小约为540Da。在所有糖基化缺陷突变体保持游动能力的同时，Delta orf1、Delta ORF2和Ser/Ala替代突变体的聚集能力降低。所有糖基化突变体也被发现在附着在聚苯乙烯表面的能力和在烟草中引起疾病的能力方面受到损害。根据预测的鞭毛蛋白的三级结构，S176和S183可能位于鞭毛的最外表面。因此，这些丝氨酸的丙氨酸取代作用比其他丝氨酸更强。这些结果表明，紫丁香拟青霉鞭毛蛋白的糖基化。Tabaci 6605是细菌毒力所必需的。鞭毛蛋白的糖基化也可能掩盖了鞭毛蛋白分子的激发子功能。
A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.