A MEMBRANE-ASSOCIATED PROTEIN, FLIX, IS REQUIRED FOR AN EARLY STEP INCAULOBACTER FLAGELLAR ASSEMBLY

The ordered assembly of the Caulobacter crescentus flagellum is accomp lished in part through the organization of the flagellar structural ge nes in a regulatory hierarachy of four classes. Class LI genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene p roducts required for early events in flagellar assembly, we used the k nown phenotypes of class LI mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene, fliX. A fliX null mutant is nonmotile, lacks a fl agellum, and exhibits a marked cell division defect. Epistasis experim ents placed fliX within class II of the flagellar regulatory hierarchy , suggesting that FliX functions at an early stage in flagellar assemb ly. The fliX gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. Full expression of fliX was found to be dependent on ctr A, and DNase I footprinting analysis demonstra ted a direct interaction between CtrA and the fliX promoter. The fliX gene is located upstream and is divergently transcribed from the class III flagellar gene flgI, which encodes the basal body P-ring monomer. Analysis of the fliX-flgI intergenic region revealed an arrangement o f cis-acting elements similar to that of another set of Caulobacter cl ass II and class III flagellar genes, fliX-flgF, that is also divergen tly transcribed. In parallel with the FliL protein, FliX copurifies wi th the membrane fraction, and although its expression is cell cycle co ntrolled, the protein is present throughout the cell cycle.