Dividing cells must coordinate cell cycle events to ensure genetic stability. Here we identify an essential two-component signal transduction protein that controls multiple events in the Caulobacter cell cycle, including cell division, stalk synthesis, and cell cycle-specific transcription. This protein, CtrA, is homologous to response regulator transcription factors and controls transcription from a group of cell cycle-regulated promoters critical for DNA replication, DNA methylation, and flagellar biogenesis. CtrA activity in the cell cycle is controlled both transcriptionally and by phosphorylation. As purified CtrA binds an essential DNA sequence motif found within its target promoters, we propose that CtrA acts in a phosphorelay signal transduction system to control bacterial cell cycle events directly at the transcriptional level.

one of the authors regrets that she inadvertently omitted references to the computer program and protein potential function that the authors used for their simulations of barnase cited above. The following sentence should have been the first sentence of the Methods section: Molecular dynamics simulations were performed with the program ENCAD (44) and the potential energy function of Levitt et al. (45).

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DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.

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In Caulobacter crescentus, the global response regulator CtrA controls chromosome replication and determines the fate of two different cell progenies. Previous studies proposed that CtrA represses replication by binding to five sites, designated [a–e], in the replication origin. We show that phosphorylated CtrA binds sites [a–e] with 35‐ to 100‐fold lower Kd values than unphosphorylated CtrA. CtrA phosphorylation stimulates two distinct modes of binding to the replication origin. Phosphorylation stimulates weak intrinsic protein–protein cooperation between half‐sites and does not stimulate CtrA–P binding unless protein–DNA contacts are made at both half‐sites. CtrA phosphorylation also stimulates cooperative binding between complete sites [a] and [b]. However, binding to each of the other CtrA‐binding sites [c], [d] and [e] is completely independent and suggests a modular organization of replication control by CtrA. We therefore propose a model where a phosphorelay targets separate biochemical activities inside the replication origin through both cooperative and independent CtrA‐binding sites.

Only one of the two chromosomes in the asymmetric Caulobacter predivisional cell initiates replication in the progeny cells. Transcription from a strong promoter within the origin occurs uniquely from the replication-competent chromosome at the stalked pole of the predivisional cell. This regulated promoter has an unusual sequence organization, and transcription from this promoter is essential for regulated {cell type-specific) replication. Our analysis defines a new class of bacterial origins and suggests a coupling between transcription and replication that is consistent with the phylogenetic relationship of Caulobacter to the ancestral mitochondrion.

SummaryThe Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori ) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNAbinding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control.