Genetic Analysis of Flaeellum Biogenesis in Caulobacter

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1) During the course of this grant we plan to : examine the expression of cloned flagellar genes in strains containing various fla mutations as a means of determining regulatory interactions among the flagellar genes. 2) Complete the characterization of strains containing mutations in newly identified flagellar genes and to determining how these new genes fit into the flagellar genes regulatory hierarchy. 3) Complete the construction of a physical map of the C. crescentus chromosome using pulsed field gel eletrophoresis (PFGE) and to determine the location of additional nutritional genes so that new mutations can be mapped rapidly and precisely using PEGE rather than the more tedious methods of classical genetics. 4) Confirm that f1bT encodes a repressor protein which regulates flagellin gene expression by examining the expression of reporter genes fused to flagellin gene promoters in strains containing various levels of f1bT expression, by gel retardation assays, and by the isolation of operator mutants which are insensitive to the f1bT repressor. 5) Examine the role of the gene involved in flagellin processing by isolating and characterizing unlinked suppressor mutations.//