Isolation of a Caulobacter gene cluster specifying flagellum production by using nonmotile Tn5 insertion mutants

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Abstract
C. crescentus assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. Of the 26 genes required for flagellum production at least 4, flaY, E, F and G, map together in a single cluster. DNA was isolated from this region of the chromosome by using a nonmotile mutant with a [transposon] Tn5 insertion into flaE. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into plasmid pBR325 and selected by transposon-encoded kanamycin resistance. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. At least 3 promotors and 3 major transcripts originated from the cloned gene cluster. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. Deletions extending rightward into this gene cluster eliminated 1 of the 2 flagellin proteins normally synthesized by C. crescentus. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. The leftward end of this cluster contains a region that may function in a regulatory capacity, whereas the rightward end may contain sequences overlapping a flagellin structural gene.