cross-complementation study of the flagellar type iii export apparatus membrane protein flhbcross-complementation鞭毛的研究类型iii flhb出口装置膜蛋白质.pdfVIP

Cross-Complementation Study of the Flagellar Type III Export Apparatus Membrane Protein FlhB Clive S. Barker, Fadel A. Samatey* Trans-membrane Trafficking Unit, Okinawa Institute of Science and Technology, Onna, Kunigami, Okinawa, Japan Abstract The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram- negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of- function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, whi