Control of gene expression during development in Caulobacter crescentus

Abstract

A novel method has been developed to analyze chromosome replication in vivo using pulsed field gel electrophoresis.;Analyses of the hierarchical regulation of flagellar gene expression identified three new promoter probe insertion mutants that failed to form a flagellum. Molecular and genetic dissection of one of these insertion mutants, the basal body gene flbN, led to the discovery of a unique overlapping genetic architecture, subsequently found among several flagellar genes. The direction of transcription of the flbN gene was identified, and the precise locations of the 5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar} ends were mapped by both nuclease S1 and primer extension analyses. The cis-acting regulatory control regions responsible for positive and negative control of transcription were identified. The flbN gene encoded the basal body L-ring, and the gene promoter utilizes the consensus sigma 54 recognition sequence associated with several C. crescentus flagellar genes. Analyses also identified a sequence upstream of the promoter responsible for proper cell cycle activation of transcription and a trans-acting factor that binds to this site. This work has helped demonstrate that the ordered expression of flagellar genes reflects the order of assembly, and showed that the ordered synthesis of two contiguous genes within the basal body cluster, flaD and flbN, coincides with their positions relative to each other on the chromosome.;A positive regulator of many flagellar genes was cloned. This gene, flaS, is required early in the flagellar biogenesis pathway for the expression of genes that encode the structural components of the flagellum. The mutation in the flaS mutant strain SC508 is a deletion of approximately 100 base pairs within the coding region. The flaS gene is autoregulated as well as subject to negative control by other genes at the same level of the flagellar hierarchy. The flaS gene is among the earliest transcribed flagellar genes, and is therefore, required early in the assembly pathway before the synthesis of the basal body and hook genes. We have used these genetic analyses to construct a model of the flagellar hierarchy that includes both positive and negative regulation within a network of flagellar gene interactions. (Abstract shortened with permission of author.).