Faithful chromosome segregation is an essential component of cell division in all organisms. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. MreB selectively interacts, directly or indirectly, with origin-proximal regions of the chromosome, arguing that the origin-proximal region segregates via an MreB-dependent mechanism not used by the rest of the chromosome.

A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. The CtrA/GcrA regulatory circuit controls expression of polar differentiation factors and the timing of DNA replication. CtrA functions as a silencer of the replication origin and GcrA as an activator of components of the replisome and the segregation machinery.

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

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the Cl subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.Chromosomal DNA methylation is widespread in prokaryotes and eukaryotes and can affect critical processes such as DNA replication (1, 2), transcription (3-6), and repair of mutational lesions (7). We are examining the regulation and function of a DNA methyltransferase, CcrM*, found in Caulobacter crescentus, a bacterium that undergoes cellular differentiation during each cell cycle (9). Caulobacter chromosomal DNA exhibits cell cycle-dependent patterns of methylation. Constitutive expression of the ccrM gene, yielding chromosomes that are fully methylated throughout the cell cycle, results in an altered developmental program, indicating that variations in methylation state are of regulatory significance (10). Understanding the role of DNA methylation in growth and development has been an elusive goal in many systems. There is abundant evidence correlating the level of cytosine methylation of eukaryotic DNA with gene expression and/or differentiation states (3)(4)(5)(11)(12)(13), but only recently has the role of DNA methylation in eukaryotic organisms been addressed genetically. A deficiency in cytosine methylation results in embryonic lethality in mice (14); in contrast, mutations resulting in deficiencies in DNA methylation in Arabidopsis thaliana (15, 16) and Neurospora crassa (17) are not lethal but cause abnormalities in chromosome segregation behavior. In prokaryotes, the only "regulatory" DNA methyltransferase that has been extensively examined has been the Dam methyltransferase of Escherichia coli and related enterics. Dam methylation is important for temporal control of chromosomal replication (1, 2) and for directing mismatch repair (7) The ccrM (cell-cycle regulated methyltransferase) locus encodes a DNA methyltransferase (CcrM) responsible for N6 methylation of adenine in GANTC sequences (10). In Caulobacter, the single chromosome replicates just once during the cell cycle (19,20). We have shown previously that the remethylation of newly replicated (and thereby hemimethylated) GANTC sites is restricted to the predivisional cell (10), near completion of chromosome replication, and that transcription of the ccrM gene occurs during a similar time fram...

The transcriptional regulator CtrA controls several key cell-cycle events in Caulobacter crescentus, including the initiation of DNA replication, DNA methylation, cell division, and f lagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems. Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. We report here that ctrA expression is under the control of two promoters: a promoter (P1) that is active only in the early predivisional cell and a stronger promoter (P2) that is active in the late predivisional cell. Both promoters exhibit CtrA-mediated feedback regulation: the early P1 promoter is negatively controlled by CtrA, and the late P2 promoter is under positive feedback control. The CtrA protein footprints conserved binding sites within the P1 and P2 promoters. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events.