Post-translational Modification Of Flagellins And The Functional Difference Between Flagellin FlaA And FlaB In Shewanella Oneidensis

Most proteins must be undergone post-translational modification (PTM) before becoming biologically active. One of the mjor forms of PTMs is glycosylation, especially for flagellin. Glycosylation of flagellins has been well characterized in a number of pathogenic bacteria. However, the process has not been visited in S. oneidensis. S.oneidensis is a highly motile organism by virtue of a polar, glycosylated flagellum composed of flagellins FlaA and FlaB. Although these two flagellins share a high level of sequence identity (89%), they differ from each other in functionality substantially, with FlaB as the predominant flagellin. In this study, we investigated that PTM of flagellin FlaB, important aspects regarding flagellin function, and underlying mechanism that differes FlaA from FlaB functionally.A genetic system was constructed to allow overproduction of functional FlaB, which was essential for subsequent analyses. The functional flagellin FlaB was expressed, isolated, and analyzed with nanoLC-MS and-MS/MS. In combination with the cysteine mutational analysis, we proposed that the FlaB flagellin protein from S. oneidensis was modified at five serine residues with a series of novel O-linked post-translational modifications (PTMs) that differ from each other by14Da. These PTMs are composed in part of a274Da sugar residue, the remainder appears to be composed of a second residue whose mass varies by14Da depending on the PTM. In addition, a number of lysine residues are found to be methylated by FliB(SO4160), an analogue of the lysine methyltransferase of Salmonella enterica serovar Typhimurium.In spit of the high sequence conservation between FlaA and FlaB flagellins, with respect to function the major flagellin is FlaB andthe minor flagellin is FlaA. To unveil this difference, we analyzed their promoter, secretion efficiency by the direct-secretion sequence of type Ⅲ secretion system, the flagellin3D structure and the variable region. While some differences were observed in promoter activities and secretion efficiencies between FlaA and FlaB, they were not sufficient to explain the distinct function of these two flagellins. However, residue changes at a few sites were unexpectedly able to distinct FlaA and flaB from each other. The link area between the third α-helix(α3) and Loop3decides the major flagellin, the sites of T129and T134for FlaB are key amino acids. Loop4in Shewanella belong to conserved region in variable region. and this region of FlaB was scanned by alanine. The alanine replaced sites F148, Q149and V150almost lost cell motility, respectively. Those sites are important and irreplaceable. We scanned serine or threonine in the variable region of FlaB by alanine. The T179site almost lost cell motility. When the flagellum assembles the macromolecule, threonine hydroxy of T179site may form hydrogen bond with adjacent amino acid to stabilize the macromolecule.