scholarly journals Two different cell-cycle processes determine the timing of cell division in Escherichia coli

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexandra Colin ◽  
Gabriele Micali ◽  
Louis Faure ◽  
Marco Cosentino Lagomarsino ◽  
Sven van Teeffelen
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Division ◽  
Single Cells ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Cell Width ◽  
Division Process ◽  
Balanced Contributions ◽  
Independent Process

Cells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

scholarly journals Two different cell-cycle processes determine the timing of cell division in Escherichia coli

2021 ◽  
Author(s):  
Alexandra Colin ◽  
Gabriele Micali ◽  
Louis Faure ◽  
Marco Cosentino Lagomarsino ◽  
Sven van Teeffelen
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Division ◽  
Single Cells ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Cell Width ◽  
Division Process ◽  
Balanced Contributions ◽  
Independent Process

AbstractCells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting 21 for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

scholarly journals Energy Starvation Induces a Cell Cycle Arrest in Escherichia coli by Triggering Degradation of the DnaA Initiator Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Godefroid Charbon ◽  
Belén Mendoza-Chamizo ◽  
Christopher Campion ◽  
Xiaobo Li ◽  
Peter Ruhdal Jensen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Growth Conditions ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Atp Depletion ◽  
Initiator Protein ◽  
Cycle Arrest ◽  
A Cell

During steady-state Escherichia coli growth, the amount and activity of the initiator protein, DnaA, controls chromosome replication tightly so that initiation only takes place once per origin in each cell cycle, regardless of growth conditions. However, little is known about the mechanisms involved during transitions from one environmental condition to another or during starvation stress. ATP depletion is one of the consequences of long-term carbon starvation. Here we show that DnaA is degraded in ATP-depleted cells. A chromosome replication initiation block is apparent in such cells as no new rounds of DNA replication are initiated while replication events that have already started proceed to completion.

scholarly journals Blocking, Bending, and Binding: Regulation of Initiation of Chromosome Replication During the Escherichia coli Cell Cycle by Transcriptional Modulators That Interact With Origin DNA

2021 ◽  
Vol 12 ◽  
Author(s):  
Julia E. Grimwade ◽  
Alan C. Leonard
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Chromosome Replication ◽  
Integration Host Factor ◽  
Replication Initiation ◽  
Critical Event ◽  
E Coli ◽  
Chromosome Duplication ◽  
Recognition Sites ◽  
Transcriptional Modulators

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin (oriC) DNA and recruit proteins needed to build new replication forks. Functional orisomes comprise the conserved initiator protein, DnaA, bound to a set of high and low affinity recognition sites in oriC. Orisomes must be assembled each cell cycle. In Escherichia coli, the organism in which orisome assembly has been most thoroughly examined, the process starts with DnaA binding to high affinity sites after chromosome duplication is initiated, and orisome assembly is completed immediately before the next initiation event, when DnaA interacts with oriC’s lower affinity sites, coincident with origin unwinding. A host of regulators, including several transcriptional modulators, targets low affinity DnaA-oriC interactions, exerting their effects by DNA bending, blocking access to recognition sites, and/or facilitating binding of DnaA to both DNA and itself. In this review, we focus on orisome assembly in E. coli. We identify three known transcriptional modulators, SeqA, Fis (factor for inversion stimulation), and IHF (integration host factor), that are not essential for initiation, but which interact directly with E. coli oriC to regulate orisome assembly and replication initiation timing. These regulators function by blocking sites (SeqA) and bending oriC DNA (Fis and IHF) to inhibit or facilitate cooperative low affinity DnaA binding. We also examine how the growth rate regulation of Fis levels might modulate IHF and DnaA binding to oriC under a variety of nutritional conditions. Combined, the regulatory mechanisms mediated by transcriptional modulators help ensure that at all growth rates, bacterial chromosome replication begins once, and only once, per cell cycle.

scholarly journals Single-cell data and correlation analysis support the independent double adder model in both Escherichia coli and Bacillus subtilis

2020 ◽  
Author(s):  
Guillaume Le Treut ◽  
Fangwei Si ◽  
Dongyang Li ◽  
Suckjoon Jun
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Correlation Analysis ◽  
Size Control ◽  
Replication Initiation ◽  
Biological Models ◽  
Value Analysis ◽  
E Coli

AbstractThe reference point for cell-size control in the cell cycle is a fundamental biological question. We previously reported that we were unable to reproduce the conclusions of Witz et al.’s eLife paper (Witz, van Nimwegen, and Julou 2019) entitled, “Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism”, despite extensive efforts. In this ‘replication double adder’ (RDA) model, both replication and division cycles are determined via replication initiation as the sole implementation point of size control. Witz et al. justified the RDA model using a type of correlation analysis (the “I-value analysis”) that they developed. By contrast, we previously showed that, in both Escherichia coli and Bacillus subtilis, replication initiation and cell division are determined by balanced biosynthesis of key cell cycle proteins (e.g., DnaA for initiation and FtsZ for cell division) and their accumulation to their respective threshold numbers, which Witz et al. coined the ‘independent double adder’ (IDA) model. The adder phenotype is a natural quantitative consequence of these mechanistic principles. In a recent bioRxiv response to our report, Witz and colleagues explicitly confirmed two important limitations of the I-value analysis: (1) it is only applicable to non-overlapping cell cycles, wherein E. coli is known to deviate from the adder principle, and (2) it is only applicable to select biological models and, for example, cannot evaluate the IDA model. These limitations of the I-value analysis were not explained in the original eLife paper and were overlooked during the review process. In this report, we show using data analysis, mathematical modeling, and experiments why the I-value analysis - in its current implementation - cannot compare different biological models. Furthermore, the RDA model is incompatible with the adder principle and is not broadly supported by experimental data. For completeness, we also provide a detailed point-by-point response to Witz et al.’s response (Witz, Julou, and van Nimwegen 2020) in the Supplemental Information.

scholarly journals A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy

2015 ◽  
Vol 184 ◽  
pp. 425-450 ◽  
Author(s):  
Jacek T. Mika ◽  
Aster Vanhecke ◽  
Peter Dedecker ◽  
Toon Swings ◽  
Jeroen Vangindertael ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Bacterial Genome ◽  
Genetically Engineered ◽  
Replication Initiation ◽  
Cell Protein ◽  
Experimental Conditions ◽  
Localization Microscopy ◽  
E Coli ◽  
Copy Numbers

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20–30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA–PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA–PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA–PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA–PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA–PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.

scholarly journals The Escherichia coli datA site promotes proper regulation of cell division

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 703-710 ◽  
Author(s):  
Morigen Morigen ◽  
Ingvild Flåtten ◽  
Kirsten Skarstad
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Binding Sites ◽  
Replication Initiation ◽  
Permissive Temperature ◽  
Initiator Protein ◽  
Dnaa Protein ◽  
Daughter Cells ◽  
Initiation Of Replication ◽  
Replication Initiator

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA. An excess of datA sites provided on plasmids has been found to lead to both a delay in initiation of replication and in cell division during exponential growth. Here we have investigated the effect of datA on the cell division block that occurs upon inhibition of replication initiation in a dnaC2 mutant. We found that this checkpoint mechanism was aided by the presence of datA. In cells where datA was deleted or an excess of DnaA was provided, cell division occurred in the absence of replication and anucleate cells were formed. This finding indicates that loss of datA and/or excess of DnaA protein promote cell division. This conclusion was supported by the finding that the lethality of the division-compromised mutants ftsZ84 and ftsI23 was suppressed by deletion of datA, at the lowest non-permissive temperature. We propose that the cell division block that occurs upon inhibition of DNA replication is, at least in part, due to a drop in the concentration of the ATP–DnaA protein.

scholarly journals Evidence for polarity of chromosome replication in F−strains ofEscherichia coli

1966 ◽  
Vol 8 (1) ◽  
pp. 119-124 ◽  
Author(s):  
W. D. Donachie ◽  
Millicent Masters
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Dna Replication ◽  
Cell Separation ◽  
Chromosome Replication ◽  
Ofescherichia Coli ◽  
Cell Cycles ◽  
Gene Replication ◽  
Serine Deaminase

The inducibility of three enzymes (β-galactosidase, tryptophanase and D-serine deaminase) has been measured at various times during the cell cycles of three strains ofEscherichia coli(K12 58–161 F−, B/r F–and 15T−). In each strain sharp increases in inducibility of these enzymes occurred at characteristic periods in each cell cycle. Such increases depend on DNA replication and therefore probably reflect synchronized gene replication. It is inferred that chromosome replication in these F−strains is sequential from a fixed origin.Infection with F′Lack+results in an extra period of increase in inducibiity of β-galactosidase in each cell cycle. It is concluded that the F′ episome replicates once in each cell cycle at a time soon after cell separation.

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

2009 ◽  
Vol 191 (18) ◽  
pp. 5706-5716 ◽  
Author(s):  
Justine Collier ◽  
Lucy Shapiro
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Regulatory Mechanism ◽  
Caulobacter Crescentus ◽  
Replication Initiation ◽  
Dnaa Protein ◽  
Additional Layer ◽  
Initiation Of Dna Replication ◽  
The Right ◽  
Feedback Regulatory Mechanism

ABSTRACT 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.

scholarly journals Changes in Cell Size and DNA Content inSulfolobus Cultures during Dilution and Temperature Shift Experiments

1999 ◽  
Vol 181 (18) ◽  
pp. 5669-5675 ◽  
Author(s):  
Karin Hjort ◽  
Rolf Bernander
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Stationary Phase ◽  
Growth Medium ◽  
Temperature Shift ◽  
Chromosome Replication ◽  
Cellular Growth ◽  
Inhibition Of Growth ◽  
Ice Water ◽  
Fresh Growth Medium

ABSTRACT Stationary-phase cultures of different hyperthermophilic species of the archaeal genus Sulfolobus were diluted into fresh growth medium and analyzed by flow cytometry and phase-fluorescence microscopy. After dilution, cellular growth started rapidly but no nucleoid partition, cell division, or chromosome replication took place until the cells had been increasing in size for several hours. Initiation of chromosome replication required that the cells first go through partition and cell division, revealing a strong interdependence between these key cell cycle events. The time points at which nucleoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When exponentially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing that the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79°C were shifted to room temperature or to ice-water baths, the cells were found to “freeze” in mid-growth. After a shift back to 79°C, growth, replication, and division rapidly resumed and the mode and kinetics of the resumption differed depending upon the nature and length of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.

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