Coordination between E. coli Cell Size and Cell Cycle Mediated by DnaA

Sixty years ago, bacterial cell size was found as an exponential function of growth rate. Fifty years ago, a more general relationship was proposed, in which the cell mass was equal to the initiation mass multiplied by the ratio of the total time of the C and D periods to the doubling time. This relationship has recently been experimentally confirmed by perturbing doubling time, C period, D period or the initiation mass. However, the underlying molecular mechanism remains unclear. Here, we developed a mechanistic and kinetic model to describe how the initiator protein DnaA mediates the initiation of DNA replication in E. coli. In the model, we introduced an initiation probability function involving competitive binding of DnaA-ATP (active) and DnaA-ADP (inactive) at replication origin to determine the initiation of replication. In addition, we considered RNAP availability, ppGpp inhibition, DnaA autorepression, DnaA titration by chromosomal sites, hydrolysis of DnaA-ATP along with DNA replication, reactivation of DnaA-ADP and established a kinetic description of these DnaA regulatory processes. We simulated DnaA kinetics and obtained a self-consistent cell size and a regular DnaA oscillation coordinated with the cell cycle at steady state. The relationship between the cell size obtained by the simulation and the growth rate, C period, D period or initiation mass reproduces the results of the experiment. This model also predicts how the number of DnaA and the initiation mass vary with the perturbation parameters (including those reflecting the mutation or interference of DnaA regulatory processes), which is comparable to experimental data. The results suggest that the regulatory mechanisms of DnaA level and activity are associated with the invariance of initiation mass and the cell size general relationship for matching frequencies of replication initiation and cell division. This study may provide clues for concerted control of cell size and cell cycle in synthetic biology.

Download Full-text

Instructive simulation of the bacterial cell division cycle

Microbiology ◽

10.1099/mic.0.049403-0 ◽

2011 ◽

Vol 157 (7) ◽

pp. 1876-1885 ◽

Cited By ~ 29

Author(s):

Arieh Zaritsky ◽

Ping Wang ◽

Norbert O. E. Vischer

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Cell Mass ◽

Time Lapse ◽

Replication Initiation ◽

Bacterial Cells ◽

Bacterial Cell Division ◽

Cycle Simulation ◽

New Ideas

The coupling between chromosome replication and cell division includes temporal and spatial elements. In bacteria, these have globally been resolved during the last 40 years, but their full details and action mechanisms are still under intensive study. The physiology of growth and the cell cycle are reviewed in the light of an established dogma that has formed a framework for development of new ideas, as exemplified here, using the Cell Cycle Simulation (CCSim) program. CCSim, described here in detail for the first time, employs four parameters related to time (replication, division and inter-division) and size (cell mass at replication initiation) that together are sufficient to describe bacterial cells under various conditions and states, which can be manipulated environmentally and genetically. Testing the predictions of CCSim by analysis of time-lapse micrographs of Escherichia coli during designed manipulations of the rate of DNA replication identified aspects of both coupling elements. Enhanced frequencies of cell division were observed following an interval of reduced DNA replication rate, consistent with the prediction of a minimum possible distance between successive replisomes (an eclipse). As a corollary, the notion that cell poles are not always inert was confirmed by observed placement of division planes at perpendicular planes in monstrous and cuboidal cells containing multiple, segregating nucleoids.

Download Full-text

Sinorhizobium meliloti, a Slow-Growing Bacterium, Exhibits Growth Rate Dependence of Cell Size under Nutrient Limitation

mSphere ◽

10.1128/msphere.00567-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 6

Author(s):

Xiongfeng Dai ◽

Zichu Shen ◽

Yiheng Wang ◽

Manlu Zhu

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Cell Size ◽

Sinorhizobium Meliloti ◽

Cell Cycle Progression ◽

Bacterial Species ◽

Progression Rate ◽

Content Type ◽

E Coli ◽

Slow Growing

ABSTRACTBacterial cells need to coordinate the cell cycle with biomass growth to maintain cell size homeostasis. For fast-growing bacterial species likeEscherichia coliandBacillus subtilis, it is well-known that cell size exhibits a strong dependence on the growth rate under different nutrient conditions (known as the nutrient growth law). However, cell size changes little with slow growth (doubling time of >90 min) forE. coli, posing the interesting question of whether slow-growing bacteria species also observe the nutrient growth law. Here, we quantitatively characterize the cell size and cell cycle parameter of a slow-growing bacterium,Sinorhizobium meliloti, at different nutrient conditions. We find thatS. melilotiexhibits a threefold change in its cell size when its doubling time varies from 2 h to 6 h. Moreover, the progression rate of its cell cycle is much longer than that ofE. coli, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate. Our study shows that the nutrient growth law holds robustly regardless of the growth capacity of the bacterial species, generalizing its applicability among the bacterial kingdom.IMPORTANCEThe dependence of cell size on growth rate is a fundamental principle in the field of bacterial cell size regulation. Previous studies of cell size regulation mainly focus on fast-growing bacterial species such asEscherichia coliandBacillussubtilis. We find here thatSinorhizobium meliloti, a slow-growing bacterium, exhibits a remarkable growth rate-dependent cell size pattern under nutrient limitation, generalizing the applicability of the empirical nutrient growth law of cell size. Moreover,S. melilotiexhibits a much slower speed of cell cycle progression thanE. colidoes, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate.

Download Full-text

Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism

10.1101/593590 ◽

2019 ◽

Author(s):

Guillaume Witz ◽

Erik van Nimwegen ◽

Thomas Julou

Keyword(s):

Cell Cycle ◽

Cell Size ◽

Cell Cycle Control ◽

Size Control ◽

Time Lapse ◽

Growth Conditions ◽

Replication Initiation ◽

Stochastic Simulations ◽

E Coli ◽

Wide Range

AbstractLiving cells proliferate by completing and coordinating two essential cycles, a division cycle that controls cell size, and a DNA replication cycle that controls the number of chromosomal copies in the cell. Despite lacking dedicated cell cycle control regulators such as cyclins in eukaryotes, bacteria such as E. coli manage to tightly coordinate those two cycles across a wide range of growth conditions, including situations where multiple nested rounds of replication progress simultaneously. Various cell cycle models have been proposed to explain this feat, but it has been impossible to validate them so far due to a lack of experimental tools for systematically testing their different predictions. Recently new insights have been gained on the division cycle through the study of the structure of fluctuations in growth, size, and division in individual cells. In particular, it was found that cell size appears to be controlled by an adder mechanism, i.e. the added volume between divisions is held approximately constant and fluctuates independently of growth rate and cell size at birth. However, how replication initiation is regulated and coupled to cell size control remains unclear, mainly due to scarcity of experimental measurements on replication initiation at the single-cell level. Here, we used time-lapse microscopy in combination with microfluidics to directly measure growth, division and replication in thousands of single E. coli cells growing in both slow and fast growth conditions. In order to compare different phenomenological models of the cell cycle, we introduce a statistical framework which assess their ability to capture the correlation structure observed in the experimental data. Using this in combination with stochastic simulations, our data indicate that, instead of thinking of the cell cycle as running from birth to division, the cell cycle is controlled by two adder mechanisms starting at the initiation of replication: the added volume since the last initiation event controls the timing of both the next division event and the next replication initiation event. Interestingly the double-adder mechanism identified in this study has recently been found to explain the more complex cell cycle of mycobacteria, suggesting shared control strategies across species.

Download Full-text

Bacillus subtilis Cell Cycle as Studied by Fluorescence Microscopy: Constancy of Cell Length at Initiation of DNA Replication and Evidence for Active Nucleoid Partitioning

Journal of Bacteriology ◽

10.1128/jb.180.3.547-555.1998 ◽

1998 ◽

Vol 180 (3) ◽

pp. 547-555 ◽

Cited By ~ 105

Author(s):

Michaela E. Sharpe ◽

Philippe M. Hauser ◽

Robert G. Sharpe ◽

Jeffery Errington

Keyword(s):

Cell Cycle ◽

Bacillus Subtilis ◽

Dna Replication ◽

Cell Cycle Progression ◽

Cell Mass ◽

Cell Length ◽

Cycle Progression ◽

Average Cell ◽

E Coli ◽

Initiation Of Dna Replication

ABSTRACT Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that ofE. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.

Download Full-text

Altered patterns of ribonucleic acid synthesis during the cell cycle: a mechanism compensating for variation in gene concentration

Journal of Cell Science ◽

10.1242/jcs.35.1.25 ◽

1979 ◽

Vol 35 (1) ◽

pp. 25-40

Author(s):

R.S. Fraser ◽

P. Nurse

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cell Size ◽

Ribosomal Rna ◽

Gene Dosage ◽

Cell Mass ◽

Small Cells ◽

Wild Type ◽

Protein Ratio ◽

The Mean

In the fission yeast Schizosaccharomyces pombe, a series of diploid mutants divides at smaller cell sizes than wild type. In these smaller strains, the mean gene concentration (defined by previous authors as the DNA to protein ratio) is higher than in wild type. Such an increase in gene concentration should also increase the concentration of those components such as messenger and ribosomal RNA, whose rate of synthesis is determined by gene dosage. We show that the mean concentrations of these 2 RNA species in the small cells are not increased, but are the same as in wild type. The small mutant cells are thus able to compensate for changes in gene concentration. This compensation is shown to operate through differences in the patterns of synthesis of RNA during the cell cycle. In all the strains of the diploid series, the rates of synthesis of messenger and ribosomal RNA double as steps once in each cell cycle. The timings of the steps in the cell cycle appear to be cell-size related, since the smaller the cell at division, the later are the steps in the cell cycle. In contrast, there is comparatively little variation in the timing of DNA replication in the cycles of cells of different sizes. We propose that after DNA replication, there is a delay before doubling in the rate of transcription. Such a cell mass-related delay is all that is required to compensate for increased gene concentration, and results in the same mean functional DNA concentration in all strains. This mechanism will maintain the same mean messenger and ribosomal RNA concentrations in cells dividing at different sizes. Ways in which the cell size-related control over transcription may operate are discussed.

Download Full-text

DNA Double Strand Break Repair in E. coli Perturbs Cell Division and Chromosome Dynamics

10.1101/802389 ◽

2019 ◽

Author(s):

M.A. White ◽

E. Darmon ◽

M.A. Lopez-Vernaza ◽

D.R.F. Leach

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Cell Size ◽

Strand Break ◽

Dsb Repair ◽

Average Cell Size ◽

Average Cell ◽

E Coli ◽

Dna Double Strand Break

AbstractTo prevent the transmission of damaged genomic material between generations, cells require a system for accommodating DNA repair within their cell cycles. We have previously shown that Escherichia coli cells subject to a single, repairable site-specific DNA double-strand break (DSB) per DNA replication cycle reach a new average cell length, with a negligible effect on population growth rate. We show here that this new cell size distribution is caused by a DSB repair-dependent delay in completion of cell division. This delay occurs despite unperturbed cell size regulated initiation of both chromosomal DNA replication and cell division. Furthermore, despite DSB repair altering the profile of DNA replication across the genome, the time required to complete chromosomal duplication is invariant. The delay in completion of cell division is accompanied by a DSB repair-dependent delay in individualization of sister nucleoids. We suggest that DSB repair events create inter-sister connections that persist until those chromosomes are separated by a closing septum.Author SummaryThe bacterium Escherichia coli has a remarkable cell cycle where overlapping rounds of DNA replication can occur in a single generation between cell birth and division. This implies a complex coordination network between growth, genome duplication and cell division to ensure that the right number of genomes are created and distributed to daughter cells at all growth rates. This network must be robust to a number of unpredictable challenges. One such challenge is broken DNA, something that in E. coli is estimated to occur in ~20% of cell division cycles. In this work we perturb the E. coli cell cycle by elevating the frequency of repairable DNA double-strand breaks to determine which parameters of the cell cycle are conserved and which are changed. Our results demonstrate that this perturbation does not alter the average cell size at initiation of DNA replication or initiation of cell division. Furthermore, it does not alter the time taken to replicate the genome or the generation time. However, it does delay the segregation of the DNA to daughter cells and the completion of cell division explaining the increase in average cell size observed previously.

Download Full-text

Regulation of DNA Replication Licensing and Re-Replication by Cdt1

International Journal of Molecular Sciences ◽

10.3390/ijms22105195 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5195

Author(s):

Hui Zhang

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Genome Stability ◽

E3 Ligase ◽

S Phase ◽

Replication Initiation ◽

Nuclear Antigen ◽

Repair Synthesis ◽

Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

Download Full-text

Evaluation of genomic sequence-based growth rate methods for synchronized Synechococcus cultures

Applied and Environmental Microbiology ◽

10.1128/aem.01743-21 ◽

2021 ◽

Author(s):

Julia Carroll ◽

Nicolas Van Oostende ◽

Bess B. Ward

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Dna Replication ◽

Growth Rates ◽

Genomic Sequence ◽

Niche Differentiation ◽

Trophic Levels ◽

Individual Species ◽

Cell Counts

Standard methods for calculating microbial growth rates (μ) through the use of proxies, such as in situ fluorescence, cell cycle, or cell counts, are critical for determining the magnitude of the role bacteria play in marine carbon (C) and nitrogen (N) cycles. Taxon-specific growth rates in mixed assemblages would be useful for attributing biogeochemical processes to individual species and understanding niche differentiation among related clades, such as found in Synechococcus and Prochlorococcus . We tested three novel DNA sequencing-based methods (iRep, bPTR, and GRiD) for evaluating growth of light synchronized Synechococcus cultures under different light intensities and temperatures. In vivo fluorescence and cell cycle analysis were used to obtain standard estimates of growth rate for comparison with the sequence-based methods (SBM). None of the SBM values were correlated with growth rates calculated by standard techniques despite the fact that all three SBM were correlated with percentage of cells in S phase (DNA replication) over the diel cycle. Inaccuracy in determining the time of maximum DNA replication is unlikely to account entirely for the absence of relationship between SBM and growth rate, but the fact that most microbes in the surface ocean exhibit some degree of diel cyclicity is a caution for application of these methods. SBM correlate with DNA replication but cannot be interpreted quantitatively in terms of growth rate. Importance Small but abundant, cyanobacterial strains such as the photosynthetic Synechococcus spp. are essential because they contribute significantly to primary productivity in the ocean. These bacteria generate oxygen and provide biologically-available carbon, which is essential for organisms at higher trophic levels. The small size and diversity of natural microbial assemblages means that taxon-specific activities (e.g., growth rate) are difficult to obtain in the field. It has been suggested that sequence-based methods (SBM) may be able to solve this problem. We find, however, that SBM can detect DNA replication and are correlated with phases of the cell cycle but cannot be interpreted in terms of absolute growth rate for Synechococcus cultures growing under a day-night cycle, like that experienced in the ocean.

Download Full-text

Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5767-5777.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5767-5777 ◽

Cited By ~ 54

Author(s):

Amit Vas ◽

Winnie Mok ◽

Janet Leatherwood

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Origin Recognition Complex ◽

Safety Net ◽

Replication Initiation ◽

Initiation Factor ◽

Phosphorylation Sites ◽

Cdc2 Kinase ◽

Origin Recognition ◽

Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

Download Full-text

Regulation of the start of DNA replication in Schizosaccharomyces pombe

Journal of Cell Science ◽

10.1242/jcs.112.6.939 ◽

1999 ◽

Vol 112 (6) ◽

pp. 939-946 ◽

Cited By ~ 2

Author(s):

C.R. Carlson ◽

B. Grallert ◽

T. Stokke ◽

E. Boye

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Growth Rate ◽

Schizosaccharomyces Pombe ◽

Growth Rates ◽

Cell Separation ◽

Cell Mass ◽

Nitrogen Sources ◽

S Phase ◽

G1 Phase

Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30–50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate.

Download Full-text