STRUCTURE AND FUNCTION OF A FLAGELLAR GENE CLUSTER (MOLECULAR CLONING, DIFFERENTIATION, BACTERIAL MOTILITY)

Abstract

Caulobacter crescentus assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. This developmental process represents an attractive system for the study of differential gene expression. Four of the 24 genes required for flagellum production--flaY,E,F, and G map together in a single chromosomal location. To study the regulation of flagellum biogenesis I have cloned this gene cluster. DNA was isolated from this region of the genome using a nonmotile mutant with a Tn5 insertion into flaE. The Tn5-flaE region was cloned into pBR325 using transposon-encoded kanamycin resistance. The resulting plasmid was used to recover the flaYE region from a wild type gene bank.;The clone isolated by this procedure was used to determine the location of insertions and extent of deletions in 9 fla mutants mapped genetically to this region. When cell extracts from these mutants were immunoprecipitated with antiflagellin antibody, mutations mapping in the leftward portion of the clone were shown to cause loss of flagellar assembly due, in part, to down-regulation of flagellin synthesis in the corresponding mutant. Rightward mutations resulted in a mutant with a qualitative change in flagellin synthesis, eliminating one or more of the three distinct flagellins normally detected in Caulobacter. This result suggests that a locus containing a flagellin structural gene has been disrupted. Sequencing data from another laboratory has demonstrated the presence of one of the three possible flagellin genes (Mr = 29,000) in this rightward region.;In vivo and in vitro analysis of this flagellar locus has shown that it encodes at least 5 transcripts. One of these RNAs, which hybridized with the 29K flagellin gene probe, was found to vary during the cell cycle, demonstrating that this transcript is regulated. The promotor for the 29K flagellin gene was identified using an in vitro run-off transcription assay and was found to function using either C. crescentus or E. coli RNA polymerase. Similarly, RNA polymerase isolated from cells at any stage in the cell cycle is able to utilize this promotor in vitro. However, this flagellin is not detected in E. coli cells which contain the gene on a plasmid, although abundant homologous mRNAs are detected by Northern blot hybridization. This result suggests that specific but as yet unidentified factors may control the expression of this gene in vivo. Finally, several fractions of the cell cycle specific C. crescentus RNA polymerase were found to contain a novel activity which resulted in the production of a previously unreported transcript from an E. coli plasmid vector. The significance of this last finding is not yet known.