start-ver=1.4 cd-journal=joma no-vol=8 cd-vols= no-issue=6 article-no= start-page=923 end-page=938 dt-received= dt-revised= dt-accepted= dt-pub-year=2006 dt-pub=20066 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=TaguchiFumiko en-aut-sei=Taguchi en-aut-mei=Fumiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=TakeuchiKasumi en-aut-sei=Takeuchi en-aut-mei=Kasumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KatohEtsuko en-aut-sei=Katoh en-aut-mei=Etsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MurataKatsuyoshi en-aut-sei=Murata en-aut-mei=Katsuyoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=SuzukiTomoko en-aut-sei=Suzuki en-aut-mei=Tomoko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=MarutaniMizuri en-aut-sei=Marutani en-aut-mei=Mizuri kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=KawasakiTakayuki en-aut-sei=Kawasaki en-aut-mei=Takayuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=EguchiMinako en-aut-sei=Eguchi en-aut-mei=Minako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=KatohShizue en-aut-sei=Katoh en-aut-mei=Shizue kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=kakuHanae en-aut-sei=kaku en-aut-mei=Hanae kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=YasudaChihiro en-aut-sei=Yasuda en-aut-mei=Chihiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=InagakiYoshishige en-aut-sei=Inagaki en-aut-mei=Yoshishige kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=ToyodaKazuhiro en-aut-sei=Toyoda en-aut-mei=Kazuhiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=ShiraishiTomonori en-aut-sei=Shiraishi en-aut-mei=Tomonori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= en-aut-name=IchinoseYuki en-aut-sei=Ichinose en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=15 ORCID= affil-num=1 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=2 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=3 en-affil= kn-affil=National Institute of Agrobiological Sciences affil-num=4 en-affil= kn-affil=National Institute of Agrobiological Sciences affil-num=5 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=6 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=7 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=8 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=9 en-affil= kn-affil=National Institute of Agrobiological Sciences affil-num=10 en-affil= kn-affil=National Institute of Agrobiological Sciences affil-num=11 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=12 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=13 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=14 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University affil-num=15 en-affil= kn-affil=The Graduate School of Natural Science and Technology, Okayama University en-keyword=Gram-Negative bacteria kn-keyword=Gram-Negative bacteria en-keyword=Posttranslational modification kn-keyword=Posttranslational modification en-keyword=Protein Glycosylation kn-keyword=Protein Glycosylation en-keyword=Perception kn-keyword=Perception en-keyword=Aeruginosa kn-keyword=Aeruginosa en-keyword=Cells kn-keyword=Cells en-keyword=Specificity kn-keyword=Specificity en-keyword=Expression kn-keyword=Expression en-keyword=Plasmids kn-keyword=Plasmids en-keyword=Pathways kn-keyword=Pathways END