Copy‐number control of the <i>Escherichia coli</i> chromosome: a plasmidologist's view

Nordström, Kurt; Dasgupta, Santanu

doi:10.1038/sj.embor.7400681

Cited by 37 publications

(29 citation statements)

References 47 publications

(66 reference statements)

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“…3B). These values are in good agreement with the expected copy number of the cI locus under our experimental conditions (26). We used these measured copy numbers to delineate the transcriptional activity of individual gene copies.…”

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Measurement of gene regulation in individual cells reveals rapid switching between promoter states

Sepúlveda

Zhang

et al. 2016

Science

119

134

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In vivo mapping of transcription-factor binding to the transcriptional output of the regulated gene is hindered by probabilistic promoter occupancy, the presence of multiple gene copies, and cell-to-cell variability. We demonstrate how to overcome these obstacles in the lysogeny maintenance promoter of bacteriophage lambda, PRM. We simultaneously measured the concentration of the lambda repressor CI and the number of mRNAs from PRM in individual E. coli cells, and used a theoretical model to identify the stochastic activity corresponding to different CI binding configurations. We found that switching between promoter configurations is faster than mRNA lifetime, and that individual gene copies within the same cell act independently. The simultaneous quantification of transcription factor and promoter activity, followed by stochastic theoretical analysis, provides a tool that can be applied to other genetic circuits.

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“…The copy-number distribution of cI mRNA in a lysogen (Fig. 3A) represents the combined contribution from multiple copies of the P RM - cI gene in each cell (26). To identify the contribution of a single gene copy, we first examined how the cI gene copy number varies during the cell cycle.…”

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confidence: 99%

Measurement of gene regulation in individual cells reveals rapid switching between promoter states

Sepúlveda

Zhang

et al. 2016

Science

119

134

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“…However, it is uncertain whether plasmid DNA replicates during spheroplast growth. Several factors influence the synchronous regulation between chromosome and plasmid (Nordström and Dasgupta, 2006).In this study, we measured the chromosomal and plasmid DNA of E. coli spheroplasts by using real-time quantitative PCR, in order to elucidate the change in copy numbers of chromosomal and plasmid DNA during the growth of spheroplasts. …”

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confidence: 99%

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Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of <i>Escherichia coli</i>

Takahashi

Nishida

2015

J. Gen. Appl. Microbiol.

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None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.cillin (an inhibitor of peptidoglycan synthesis) was shown to generate giant protoplasts (Kuroda et al., 1998;Kusaka, 1967;Nakamura et al., 2011).Generally, DNA duplicates before cell division, and it is distributed to each cell during cell division. Most bacteria undergo symmetric binary fission. During the growth of spheroplasts, although cell division was not observed, DNA replication was, indeed, observed (Kuroda et al., 1998;Nakamura et al., 2011). However, it is uncertain whether plasmid DNA replicates during spheroplast growth. Several factors influence the synchronous regulation between chromosome and plasmid (Nordström and Dasgupta, 2006).In this study, we measured the chromosomal and plasmid DNA of E. coli spheroplasts by using real-time quantitative PCR, in order to elucidate the change in copy numbers of chromosomal and plasmid DNA during the growth of spheroplasts. Materials and MethodsPreparation of giant spheroplasts from E. coli. Giant spheroplasts were prepared using a modified version of the method previously described by Kusaka (1967) and the spheroplast incubation method reported by Kuroda et al. (1998). E. coli SCS1 (Stratagene) cells, which was transformed with the plasmid pHRP311 (14 kbp) with an RSF1010 replicon, were grown on marine broth agar (Difco) containing streptomycin under aerobic conditions. The harvested cells (0.003 g) were suspended in a buffer (1 mL) consisting of 0.1 M Tris-HCl (pH 7.6) and 0.3 M sucrose. Lysozyme (Wako) (200 µg/mL) was added to the cell suspension, and the suspension was incubated at 37°C for 15 min. After the suspension was divided into 2 aliquots (500 µL each), the cells were harvested (centrifugation Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of Escherichia coli (Received August 8, 2015; Accepted September 9, 2015) Sawako Takahashi and Hiromi Nishida* Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Japan We generated spheroplasts from Escherichia coli carrying a broad-host-range plasmid. In the presence of penicillin, the spheroplasts did not divide but grew and enlarged in marine broth, whereas, in the absence of penicillin, they elongated. We quantified cellular DNA at different time points by using real-time quantitative PCR. Both chromosomal and plasmid DNA had replicated during spheroplast growth not only in the absence but also in the presence of penicillin. Thus, plasmid DNA and chromosomal DNA replication might be regulated synchronously during the growth of spheroplasts. Biotechnology

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“…This too seems to be constant in a large variety of prokaryotic cells and is thought to be the minimum length of time required to accurately replicate a chromosome containing the minimum amount of information necessary to create a cell (Donachie 1968;Bipatnath et al 1998;Nordstrom and Dasgupta 2006). Primitive prokaryotes must have faced the challenge of coordinating this molecular constraint with the external environment.…”

Section: From Molecular Constraints To a Primordial Clock: The Path Fmentioning

confidence: 99%

Evolution of the Clock from Yeast to Man by Period-Doubling Folds in the Cellular Oscillator

Klevecz¹,

Li²

2007

Cold Spring Harbor Symposia on Quantitative Biology

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Copy‐number control of the Escherichia coli chromosome: a plasmidologist's view

Cited by 37 publications

References 47 publications

Measurement of gene regulation in individual cells reveals rapid switching between promoter states

Measurement of gene regulation in individual cells reveals rapid switching between promoter states

Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of <i>Escherichia coli</i>

Evolution of the Clock from Yeast to Man by Period-Doubling Folds in the Cellular Oscillator

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