Association of the Folded Chromosome with the Cell Envelope of
            <i>Escherichia coli</i>
            : Nature of the Membrane-Associated DNA

Drlica, Karl; Burgi, Elizabeth; Worcel, Abraham

doi:10.1128/jb.134.3.1108-1116.1978

Cited by 21 publications

(8 citation statements)

References 48 publications

(49 reference statements)

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“…However, current techniques for assaying chromosome supercoiling in vivo do not have the resolution to probe a small (∼250-bp) locus and its immediate proximal regions. Most assays of chromosome topology only provide information on the average supercoiling across the genome (27, 68, 69). Recent efforts to use psoralen, which preferentially binds negatively supercoiled DNA, with cross-linking and deep sequencing do not yet offer the sensitivity or resolution to probe the topological state of specific loci like oriC (70).…”

Section: Discussionmentioning

confidence: 99%

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

Kraemer

Sanderlin

Laub

2019

mBio

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The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factor dnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication, oriC. Active transcription normally introduces negative supercoils into oriC to help promote replication initiation, so the accumulation of ppGpp reduces initiation potential at oriC by reducing transcription. We find that maintaining transcription near oriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein gene seqA also relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils into oriC. IMPORTANCE To survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation in Escherichia coli for decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

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Section: Discussionmentioning

confidence: 99%

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

Kraemer

Sanderlin

Laub

2019

mBio

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“…For instance, fibril lengths of rapidly sedimenting associations of predominantly supercoiled plastid DNA attached to protein patches that can be prepared from controlled partial chloroplast lysates and are reminiscent to isolated bacterial nucleoids (e.g. Drlica et al 1978 ) can amount up to a dozen plastome equivalents (Herrmann and Possingham 1980 ; for membrane association of ptDNA see Herrmann and Kowallik 1970 , Kowallik and Haberkorn 1971 ).…”

Section: Introductionmentioning

confidence: 99%

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

et al. 2009

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Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10−2 pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.

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“…Specific restriction fragments from the origin region of Escherichia coli have been identified in the outer membrane fraction prepared after mechanically disrupting the bacteria, although DNA from other parts of the chromosome was also present in this fraction (14,15,26). In contrast, when membrane-bound nucleoids of E. coli were digested with restriction enzymes, the DNA remaining associated with the membrane appeared to be nonspecific (4).…”

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confidence: 99%

Identification of specific restriction fragments associated with a membrane subparticle from Bacillus subtilis

Sargent¹,

Bennett²,

Burdett³

1983

J Bacteriol

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When lysates of Bacillus subtilis were treated with restriction endonucleases EcoRI or HindlIl, almost all of the DNA was released from the major plasma membrane fraction that was sedimentable at low speed. However, a very small part of the released DNA, when centrifuged at high speed, appeared to be bound to small membrane fragments. On agarose gels, this material, prepared with either enzyme, contained only a small number of restriction fragments, and the DNA in the sample hybridized with 11 to 12 EcoRI or HindlIl fragments of chromosomal DNA. This DNA was used after nick-translation to screen Charon 4A clone banks for phages containing membrane-bound fragments. One of these was studied in detail. Only a part (about 5 kilobases) of the region present in this clone is important in binding the DNA to the membrane subparticle.

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Association of the Folded Chromosome with the Cell Envelope of Escherichia coli : Nature of the Membrane-Associated DNA

Cited by 21 publications

References 48 publications

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

Identification of specific restriction fragments associated with a membrane subparticle from Bacillus subtilis

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