Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus.
Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle. (+info)
The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle.
In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. PfliQ is activated earlier than PccrM. Phosphorylated CtrA (CtrA approximately P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in PccrM significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrM expression. Our data indicate that differences in the affinity of CtrA approximately P for PfliQ and PccrM regulate, in part, the temporal expression of these genes. However, the timing of fliQ transcription but not of ccrM transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA approximately P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA approximately P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle. (+info)
Cell cycle-dependent polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division.
The master CtrA response regulator functions in Caulobacter to repress replication initiation in different phases of the cell cycle. Here, we identify an essential histidine kinase, CckA, that is responsible for CtrA activation by phosphorylation. Although CckA is present throughout the cell cycle, it moves to a cell pole in S phase, and upon cell division it disperses. Removal of the membrane-spanning region of CckA results in loss of polar localization and cell death. We propose that polar CckA functions to activate CtrA just after the initiation of DNA replication, thereby preventing premature reinitiations of chromosome replication. Thus, dynamic changes in cellular location of critical signal proteins provide a novel mechanism for the control of the prokaryote cell cycle. (+info)
Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease.
The region of the Caulobacter crescentus chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the C. crescentus ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The C. crescentus clpX gene was shown by complementation analysis to be functional in Escherichia coli. However, clpX from E. coli was not able to substitute for the essential nature of the clpX gene in C. crescentus. The clpP and clpX genes are separated on the C. crescentus chromosome by an open reading frame pointing in the opposite direction from the clp genes, and transcription of clpP and clpX was found to be uncoupled. clpP is transcribed as a monocistronic unit with a promoter (PP1) located immediately upstream of the 5' end of the gene and a terminator structure following its 3' end. PP1 is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for clpX (PX1, PX2, and PX3) were mapped in the clpP-clpX intergenic region. In contrast to PP1, the clpX promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the clpP and clpX promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in C. crescentus. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP. (+info)
Cell cycle expression and transcriptional regulation of DNA topoisomerase IV genes in caulobacter.
DNA replication and differentiation are closely coupled during the Caulobacter crescentus cell cycle. We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the parE and parC genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D. Ward and A. Newton, Mol. Microbiol. 26:897-910, 1997). We have examined the cell cycle regulation of parE and parC and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis. The regulation of parE and parC expression is not strictly coordinated, however. The rate of parE transcription increases ca. 20-fold during the G1-to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication. Transcription from the parC promoter, by contrast, is induced only two- to threefold during this cell cycle period. Steady-state ParE levels are also regulated, increasing ca. twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected. These results suggest that topo IV activity may be regulated primarily through parE expression. The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major Caulobacter sigma factor sigma73. We also present evidence that a conserved 8-mer sequence motif located in the spacers between the -10 and -35 elements of the parE and parC promoters is required for maximum levels of parE transcription, which raises the possibility that it may function as a positive regulatory element. The pattern of parE transcription and the parE and parC promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication. (+info)
Feedback control of a master bacterial cell-cycle regulator.
The transcriptional regulator CtrA controls several key cell-cycle events in Caulobacter crescentus, including the initiation of DNA replication, DNA methylation, cell division, and flagellar 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. (+info)
Regulation of podJ expression during the Caulobacter crescentus cell cycle.
The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of the Caulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJ results in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors. (+info)
Bacterial cells: The migrating kinase and the master regulator.
It is becoming clear that, as in eukaryotes, proteins in bacterial cells are targeted to specific cellular locations. The most recently discovered example is a remarkable histidine kinase that oscillates between polar and global distributions while temporally regulating transcription and DNA replication in Caulobacter. (+info)