Taguchi Fumiko, Takeuchi Kasumi, Katoh Etsuko, Murata Katsuyoshi, Suzuki Tomoko, Marutani Mizuri, Kawasaki Takayuki, Eguchi Minako, Katoh Shizue, Kaku Hanae, Yasuda Chihiro, Inagaki Yoshishige, Toyoda Kazuhiro, Shiraishi Tomonori, Ichinose Yuki
The Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka 1-1-1, Okayama 700-8530, Japan.
Cell Microbiol. 2006 Jun;8(6):923-38. doi: 10.1111/j.1462-5822.2005.00674.x.
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, Deltaorf1 and Deltaorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Deltaorf1 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 Deltaorf1, Deltaorf2 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.
糖基化岛是糖基化所需的遗传区域。丁香假单胞菌烟草致病变种6605中鞭毛蛋白的糖基化岛由三个开放阅读框组成：orf1、orf2和orf3。Orf1和orf2编码推定的糖基转移酶，它们的缺失突变体Deltaorf1和Deltaorf2分别表现出鞭毛蛋白糖基化缺陷或产生部分糖基化的鞭毛蛋白。使用天冬氨酸N肽酶消化野生型细菌的糖基化鞭毛蛋白和Deltaorf1突变体的非糖基化鞭毛蛋白，并进行后续的高效液相色谱分析，揭示了候选的糖基化氨基酸。通过产生定点丝氨酸/丙氨酸取代突变体，确定了所有糖基化氨基酸残基位于第143、164、176、183、193和201位。基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱（MS）分析表明，每个聚糖约为540 Da。虽然所有糖基化缺陷突变体都保留了游动能力，但Deltaorf1、Deltaorf2和丝氨酸/丙氨酸取代突变体的群体运动能力降低。还发现所有糖基化突变体在粘附聚苯乙烯表面的能力和在烟草中致病的能力方面都受损。基于鞭毛蛋白的预测三级结构，预计S176和S183位于鞭毛的最外表面。因此，这些丝氨酸被丙氨酸取代的效果比其他丝氨酸更强。这些结果表明，丁香假单胞菌烟草致病变种6605中鞭毛蛋白的糖基化是细菌致病力所必需的。鞭毛蛋白的糖基化也可能掩盖鞭毛蛋白分子的激发子功能。
