ANALYSIS OF THE FLAGELLAR BASAL BODY OF CAULOBACTER CRESCENTUS
HAHNENBERGER, KAREN M.
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The bacterial flagellum is a complex structure composed of a transmembrane basal body, a hook, and a filament. In C. crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. To help define the order of assembly of the flagellar components and to identify the genes involved in the early steps of basal body construction, mutants defective in basal body formation have been analyzed.;The C. crescentus basal body consists of a central rod surrounded by five basal rings. Mutants in the flaD flaB flaC gene cluster were found to be unable to assemble a complete basal body. The flaDBCmotC region was cloned and the genes were localized by subcloning and complementation analysis. Complementation analysis of Tn 5 insertion mutants indicated the existence of at least four transcriptional units in the region and identified the presence of two new genes designated flbN and flbO. Mutants in flbN, flaB, flaC and flbO were unable to assemble any basal body structure and are likely to be involved in early steps of basal body formation. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook distal rings. The flaD gene may therefore encode the structural protein of the next basal body ring assembled.;DNA sequence analysis of the flaD gene indicates that it encodes a protein of 27Kd. The predicted amino acid sequence includes a hydrophobic leader sequence similar to that present in a basal ring protein from Salmonella. Sl nuclease protection experiments using RNA isolated from synchronized cells at various time points during the cell cycle demonstrate that the expression of the flaD gene is temporally regulated and it is expressed coincident with flagellum assembly.;All of the mutants in this cluster exhibited pleiotropic effects on the expression of other flagellar and chemotaxis functions, including the level of synthesis of flagellins, the hook protein, and hook protein precursor, and the level of chemotaxis methylation, permitting the placement of those genes within the regulatory hierarchy of flagellar gene expression.