flio regulation of flip in the formation of the salmonella enterica flagellumflio监管抛在沙门氏菌鞭毛的形成.pdfVIP

FliO Regulation of FliP in the Formation of the Salmonella enterica Flagellum 1 1 2 1 Clive S. Barker , Irina V. Meshcheryakova , Alla S. Kostyukova , Fadel A. Samatey * 1Trans-Membrane Trafficking Unit, Okinawa Institute of Science and Technology, Okinawa, Japan, 2 Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America Abstract The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the DfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N- terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO43–125 or FliO1–95 was able to rescue motility of the DfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO43–125, should contain beta-structure and alpha-helices. FliO43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and it