Justine Collier

A DNA methylation ratchet governs progression through a bacterial cell cycle

The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G1 and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. The timing of DnaA accumulation was found to be regulated by the methylation state of the dnaA promoter, which in turn depends on the chromosomal position of dnaA near the origin of replication and restriction of CcrM synthesis to the end of the cell cycle. The dnaA gene is preferentially transcribed from a fully methylated promoter. DnaA initiates DNA replication and activates the transcription of the next cell-cycle regulator, GcrA. With the passage of the replication fork, the dnaA promoter becomes hemimethylated, and DnaA accumulation drops. GcrA then activates the transcription of the next cell-cycle regulator, CtrA, once the replication fork passes through the ctrA P1 promoter, generating two hemimethylated copies of ctrA. The ctrA gene is preferentially transcribed from a hemimethylated promoter. CtrA then activates the transcription of ccrM, to bring the newly replicated chromosome to the fully methylated state, promoting dnaA transcription and the start of a new cell cycle. We show that the cell-cycle timing of CcrM is critical for Caulobacter fitness. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust cell-cycle control.

High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization

Significance The bacterial cytoskeletal protein FtsZ, which forms a constricting “Z-ring” during cell division, is the major cytoskeletal protein involved in cell division in almost all prokaryotes, and is a key next-generation antibiotic target. However, the small size of the Z-ring, approximately 500 nm in diameter, makes it difficult to observe in vivo. We provide a quantitative nanoscale picture of Z-ring organization in live cells; these results improve our understanding of the structural and force-generating roles of FtsZ in bacterial cell division. To achieve this, we created an automated modality of superresolution fluorescence microscopy, allowing high-throughput live cell microscopy at nanoscale resolution; this technique should be broadly useful in prokaryotic and eukaryotic systems. We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of “Z-ring” organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.

DnaA couples DNA replication and the expression of two cell cycle master regulators

Cell cycle progression in Caulobacter is driven by the master transcriptional regulators CtrA and GcrA. The cellular levels of CtrA and GcrA are temporally and spatially out‐of‐phase during the cell cycle, with CtrA repressing gcrA transcription and GcrA activating ctrA transcription. Here, we show that DnaA, a protein required for the initiation of DNA replication, also functions as a transcriptional activator of gcrA, which in turn activates multiple genes, notably those involved in chromosome replication and segregation. The cellular concentration of DnaA is cell cycle‐controlled, peaking at the time of replication initiation and gcrA induction. Regulated proteolysis of GcrA contributes to the cell cycle variations in GcrA abundance. We propose that DnaA couples DNA replication initiation with the expression of the two oscillating regulators GcrA and CtrA and that the DnaA/GcrA/CtrA regulatory cascade drives the forward progression of the Caulobacter cell cycle.

Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein in Caulobacter

Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.

Epigenetic regulation of the bacterial cell cycle.

N(6)-methyl-adenines can serve as epigenetic signals for interactions between regulatory DNA sequences and regulatory proteins that control cellular functions, such as the initiation of chromosome replication or the expression of specific genes. Several of these genes encode master regulators of the bacterial cell cycle. DNA adenine methylation is mediated by Dam in gamma-proteobacteria and by CcrM in alpha-proteobacteria. A major difference between them is that CcrM is cell cycle regulated, while Dam is active throughout the cell cycle. In alpha-proteobacteria, GANTC sites can remain hemi-methylated for a significant period of the cell cycle, depending on their location on the chromosome. In gamma-proteobacteria, most GATC sites are only transiently hemi-methylated, except regulatory GATC sites that are protected from Dam methylation by specific DNA-binding proteins.

The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach

DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle

Significance Caulobacter crescentus, a bacterium with an inherent asymmetric cell division, uses dynamic changes in chromosome methylation state to synchronize chromosome replication with cell-cycle regulation. We identified the N6-methyladenine and 5-methylcytosine methylation state of every base pair at five times in the cell cycle to show that 4,515 GANTC sites, recognized by the CcrM methyltransferase, change from full- to hemimethylation upon passage of the replication fork. Significantly, 27 of the GANTC sites are protected from methylation at all times. We also identified four previously unknown methylation motifs and the cognate methyltransferase for two of these motifs. The ability to track the state of the methylome in exquisite temporal detail will be invaluable to investigations of microbial epigenetic regulation. The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N6-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.

Architecture and inherent robustness of a bacterial cell-cycle control system

A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

Regulation of chromosomal replication in Caulobacter crescentus.

The alpha-proteobacterium Caulobacter crescentus is characterized by its asymmetric cell division, which gives rise to a replicating stalked cell and a non-replicating swarmer cell. Thus, the initiation of chromosomal replication is tightly regulated, temporally and spatially, to ensure that it is coordinated with cell differentiation and cell cycle progression. Waves of DnaA and CtrA activities control when and where the initiation of DNA replication will take place in C. crescentus cells. The conserved DnaA protein initiates chromosomal replication by directly binding to sites within the chromosomal origin (Cori), ensuring that DNA replication starts once and only once per cell cycle. The CtrA response regulator represses the initiation of DNA replication in swarmer cells and in the swarmer compartment of pre-divisional cells, probably by competing with DnaA for binding to Cori. CtrA and DnaA are controlled by multiple redundant regulatory pathways that include DNA methylation-dependent transcriptional regulation, temporally regulated proteolysis and the targeting of regulators to specific locations within the cell. Besides being critical regulators of chromosomal replication, CtrA and DnaA are also master transcriptional regulators that control the expression of many genes, thus connecting DNA replication with other events of the C. crescentus cell cycle.

DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus

DNA methylation regulates many processes, including gene expression, by superimposing secondary information on DNA sequences. The conserved CcrM enzyme, which methylates adenines in GANTC sequences, is essential to the viability of several Alphaproteobacteria. In this study, we find that Caulobacter crescentus cells lacking the CcrM enzyme accumulate low levels of the two conserved FtsZ and MipZ proteins, leading to a severe defect in cell division. This defect can be compensated by the expression of the ftsZ gene from an inducible promoter or by spontaneous suppressor mutations that promote FtsZ accumulation. We show that CcrM promotes the transcription of the ftsZ and mipZ genes and that the ftsZ and mipZ promoter regions contain a conserved CGACTC motif that is critical to their activities and to their regulation by CcrM. In addition, our results suggest that the ftsZ promoter has the lowest activity when the CGACTC motif is non‐methylated, an intermediate activity when it is hemi‐methylated and the highest activity when it is fully methylated. The regulation of ftsZ expression by DNA methylation may explain why CcrM is essential in a subset of Alphaproteobacteria.