Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (Pvan) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (Pxyl). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

Temporally and spatially controlled master regulators drive the Caulobacter cell cycle by regulating the expression of >200 genes. Rapid clearance of the master regulator, CtrA, by the ClpXP protease is a critical event that enables the initiation of chromosome replication at specific times in the cell cycle. We show here that a previously unidentified single domain-response regulator, CpdR, when in the unphosphorylated state, binds to ClpXP and, thereby, causes its localization to the cell pole. We further show that ClpXP localization is required for CtrA proteolysis. When CpdR is phosphorylated, ClpXP is delocalized, and CtrA is not degraded. Both CtrA and CpdR are phosphorylated via the same CckA histidine kinase phospho-signaling pathway, providing a reinforcing mechanism that simultaneously activates CtrA and prevents its degradation by delocalizing the CpdR͞ClpXP complex. In swarmer cells, CpdR is in the phosphorylated state, thus preventing ClpXP localization and CtrA degradation. As swarmer cells differentiate into stalked cells (G1͞S transition), unphosphorylated CpdR accumulates and is localized to the stalked cell pole, where it enables ClpXP localization and CtrA proteolysis, allowing the initiation of DNA replication. Dynamic protease localization mediated by a phosphosignaling pathway is a novel mechanism to integrate spatial and temporal control of bacterial cell cycle progression.Caulobacter ͉ ClpXP ͉ phosphorylation ͉ proteolysis ͉ temporal control

Using 62 probe-level datasets obtained with a custom-designed Caulobacter crescentus microarray chip, we identify transcriptional start sites of 769 genes, 53 of which are transcribed from multiple start sites. Transcriptional start sites are identified by analyzing probe signal cross-correlation matrices created from probe pairs tiled every 5 bp upstream of the genes. Signals from probes binding the same message are correlated. The contribution of each promoter for genes transcribed from multiple promoters is identified. Knowing the transcription start site enables targeted searching for regulatory-protein binding motifs in the promoter regions of genes with similar expression patterns. We identified 27 motifs, 17 of which share no similarity to the characterized motifs of other C. crescentus transcriptional regulators. Using these motifs, we predict coregulated genes. We verified novel promoter motifs that regulate stress-response genes, including those responding to uranium challenge, a stress-response sigma factor and a stress-response noncoding RNA.

Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.