The Absence of (p)ppGpp Renders Initiation of Escherichia coli Chromosomal DNA Synthesis Independent of Growth Rates

ABSTRACT The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase. IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

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Differential Effects of Myeloperoxidase-Derived Oxidants on Escherichia coli DNA Replication

Infection and Immunity ◽

10.1128/iai.66.6.2655-2659.1998 ◽

1998 ◽

Vol 66 (6) ◽

pp. 2655-2659 ◽

Cited By ~ 12

Author(s):

Henry Rosen ◽

Bryce R. Michel ◽

Donald R. vanDevanter ◽

James P. Hughes

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Dna Synthesis ◽

Dna Sequences ◽

Regression Line ◽

Chromosomal Dna ◽

Chloride System ◽

E Coli ◽

Total Dna ◽

Cell Envelopes

ABSTRACT The microbicidal myeloperoxidase (MPO)-H2O2-chloride system strongly inhibitsEscherichia coli DNA synthesis. Also, cell envelopes from MPO-treated E. coli cells lose their ability to interact with hemimethylated DNA sequences of oriC, the chromosomal origin of replication, raising the prospect that suppression of DNA synthesis involves impairment of oriC-related functions (H. Rosen, et al. Proc. Natl. Acad. Sci. USA, 87:10048–10052, 1990). To evaluate whether origin-specific DNA sequences play a role in the MPO effect on E. coli DNA synthesis, plasmid DNA replication was compared to total (chromosomal) DNA replication for six plasmids with three distinct origins of replication. Plasmid pCM700 replication, replicating from oriC, was as sensitive to MPO-mediated inhibition as was total (chromosomal) DNA replication. A regression line describing this relationship had a slope of 0.90, and ther 2 was 0.89. In contrast, the replication activities of three of four non-oriC plasmids, pUC19, pACYC184, and pSC101, demonstrated significant early resistance to inhibition by MPO-derived oxidants. The exception to this resistance pattern was plasmid pSP102, which has an origin derived from P1 phage. pSP102 replication declined similarly to that of total DNA synthesis. The regression line for pSP102 replication versus total DNA synthesis had a slope of 0.95, and the r 2 was 0.92. The biochemical requirements for P1-mediated replication are strikingly similar to those for oriC-mediated replication. It is proposed that one of these requirements, common to oriC and the P1 origin but not critical to the replication of the other non-oriC plasmids, is an important target for MPO-mediated oxidations that mediate the initial decline in E. colichromosomal DNA synthesis.

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Replication of Bacteriophage M13. Mechanism of Single-Strand DNA Synthesis in an Escherichia coli Mutant Thermosensitive in Chromosomal DNA Replication

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1972.tb02111.x ◽

1972 ◽

Vol 30 (3) ◽

pp. 403-412 ◽

Cited By ~ 18

Author(s):

Walter L. Staudenbauer ◽

Peter Hans Hofschneider

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Dna Synthesis ◽

Single Strand ◽

Chromosomal Dna ◽

Coli Mutant ◽

Bacteriophage M13

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Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli

Biochemical Journal ◽

10.1042/bj20031255 ◽

2004 ◽

Vol 379 (3) ◽

pp. 553-562 ◽

Cited By ~ 17

Author(s):

Subhasis B. BISWAS ◽

Stephen FLOWERS ◽

Esther E. BISWAS-FISS

Keyword(s):

Escherichia Coli ◽

Quantitative Analysis ◽

Dna Replication ◽

Dna Binding ◽

Binding Affinity ◽

Replication Fork ◽

Dna Helicase ◽

Replication Initiation ◽

Chromosomal Dna ◽

Dnac Protein

In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC–DNA and DnaC–DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6•DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.

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DpiA Binding to the Replication Origin of Escherichia coli Plasmids and Chromosomes Destabilizes Plasmid Inheritance and Induces the Bacterial SOS Response

Journal of Bacteriology ◽

10.1128/jb.185.20.6025-6031.2003 ◽

2003 ◽

Vol 185 (20) ◽

pp. 6025-6031 ◽

Cited By ~ 21

Author(s):

Christine Miller ◽

Hanne Ingmer ◽

Line Elnif Thomsen ◽

Kirsten Skarstad ◽

Stanley N. Cohen

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Replication Origin ◽

Sos Response ◽

Dna Helicase ◽

Replication Initiation ◽

Chromosomal Dna ◽

E Coli ◽

Dna Replication Initiation ◽

Helicase Dnab

ABSTRACT The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size—both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB—both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids—reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.

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Timing of nuclear division cycle in Neurospora crassa

Journal of Cell Science ◽

10.1242/jcs.48.1.127 ◽

1981 ◽

Vol 48 (1) ◽

pp. 127-136

Author(s):

E. Martegani ◽

F. Tome ◽

F. Trezzi

Keyword(s):

Growth Rate ◽

Dna Replication ◽

Dna Synthesis ◽

Neurospora Crassa ◽

Exponential Growth ◽

Nuclear Division ◽

S Phase ◽

G1 Phase ◽

Rate Dependent ◽

Nutritional Conditions

The timing of the nuclear division cycle in Neurospora crassa was studied by inhibiting DNA synthesis with hydroxyurea added to exponentially growing cultures at concentrations that do not inhibit cytoplasmic growth. After the addition of hydroxyurea only the nuclei that have passed the S-phase divide, and therefore by counting the increase in the average number of nuclei per hypha, the stage in the cycle in which completion of S phase occurred was determined in different nutritional conditions of exponential growth. The stage at which DNA replication terminates is different in different conditions of exponential growth; however, when the length of G2+M phases is calculated a constancy of these phases is shown. Our data indicate that in N. crassa the coordination between cytoplasmic growth and the nuclear division cycle is achieved mainly through a growth-rate-dependent expansion of the G1 phase, according to a sizer-plus-timer model of control of the nuclear division cycle.

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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.

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Escherichia colipopulations adapt to complex, unpredictable fluctuations without any trade-offs across environments

10.1101/045369 ◽

2016 ◽

Author(s):

Shraddha Karve ◽

Devika Bhave ◽

Dhanashri Nevgi ◽

Sutirth Dey

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Growth Rates ◽

Complex Environments ◽

Multiple Stresses ◽

Trade Off ◽

Trade Offs ◽

Fluctuating Environments ◽

Laboratory Populations ◽

Lower Variation

AbstractIn nature, organisms are simultaneously exposed to multiple stresses (i.e. complex environments) that often fluctuate unpredictably. While both these factors have been studied in isolation, the interaction of the two remains poorly explored. To address this issue, we selected laboratory populations ofEscherichia coliunder complex (i.e. stressful combinations of pH, H2O2and NaCl) unpredictably fluctuating environments for ~900 generations. We compared the growth rates and the corresponding trade-off patterns of these populations to those that were selected under constant values of the component stresses (i.e. pH, H2O2and NaCl) for the same duration. The fluctuation-selected populations had greater mean growth rate and lower variation for growth rate over all the selection environments experienced. However, while the populations selected under constant stresses experienced severe tradeoffs in many of the environments other than those in which they were selected, the fluctuation-selected populations could by-pass the across-environment trade-offs completely. Interestingly, trade-offs were found between growth rates and carrying capacities. The results suggest that complexity and fluctuations can strongly affect the underlying trade-off structure in evolving populations.

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DNA replication and repair of Tilapia cells. II. Effects of temperature on DNA replication and ultraviolet repair in Tilapia ovary cells

Journal of Cell Science ◽

10.1242/jcs.89.2.263 ◽

1988 ◽

Vol 89 (2) ◽

pp. 263-272

Author(s):

J.D. Chen ◽

F.H. Yew

Keyword(s):

Molecular Weight ◽

Dna Replication ◽

Dna Synthesis ◽

Mammalian Cells ◽

Excision Repair ◽

Control Level ◽

High Molecular Weight Dna ◽

Wide Range ◽

Effects Of Temperature ◽

Fish Cells

TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15–34 degrees C). While most fish cells lack DNA excision repair and are hypersensitive to ultraviolet light (u.v.), Tilapia cells are more u.v.-resistant than mammalian cells. In this paper we report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 degrees C to the sublethal high temperature of 37 degrees C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8 h at 37 degrees C the rate of DNA synthesis overshot the 31 degrees C control level by 180%. When moved to low temperature (18 degrees C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. u.v. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3 h at 37, 31 and 25 degrees C, but not at 18 degrees C. Initiation of nascent DNA synthesis was blocked at 4 J m-2 in TO-2 cells compared with less than or equal to 1 J m-2 in mammalian cells. After 9 J m-2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate, and they could be chased into high molecular weight DNA with little delay. TO-2 cells showed low levels of u.v.-induced excision repair; but this was prominent compared with other fish cells. The u.v.-induced incision rate has been measured at various temperatures, and the activation energy of incision estimated to be 13 kcal mol-1 (1 cal approximately equal to 4.184 J).

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Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy

mBio ◽

10.1128/mbio.00143-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Yanyu Zhu ◽

Mainak Mustafi ◽

James C. Weisshaar

Keyword(s):

Escherichia Coli ◽

Fluorescence Microscopy ◽

Rna Polymerase ◽

Stationary Phase ◽

Exponential Growth ◽

Mean Square Displacement ◽

Quiescent State ◽

Chromosomal Dna ◽

Content Type ◽

E Coli

ABSTRACT In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is “compacted” or “supercompacted,” and there are suggestions that the cytoplasm is “glass-like.” Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli. Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

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