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PubMed1994

The bacterial nucleoid revisited.

This review compares the results of different methods of investigating the morphology of nucleoids of bacteria grown under conditions favoring short generation times. We consider the evidence from fixed and stained specimens, from phase-contrast and fluorescence microscopy of growing bacteria, and from electron microscopy of whole as well as thinly sectioned ones. It is concluded that the nucleoid of growing cells is in a dynamic state: part of the chromatin is "pulled out" of the bulk of the nucleoid in order to be transcribed. This activity is performed by excrescences which extend far into the cytoplasm so as to reach the maximum of available ribosomes. Different means of fixation provide markedly different views of the texture of the DNA-containing plasm of the bulk of the nucleoid. Conventional chemical fixatives stabilize the cytoplasm of bacteria but not their protein-low chromatin. Uranyl acetate does cross-link the latter well but only if the cytoplasm has first been fixed conventionally. In the interval between the two fixations, the DNA arranges itself in liquid-crystalline form, supposedly because of loss of supercoiling. In stark contrast, cryofixation preserves bacterial chromatin in a finely granular form, believed to reflect its native strongly negatively supercoiled state. In dinoflagellates the DNA of their permanently visible chromosomes (also low in histone-like protein) is natively present as a liquid crystal. The arrangement of chromatin in Epulocystis fishelsoni, one of the largest known prokaryotes, is briefly described.

Animals↗

PubMed1999

Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli.

An image cytometric method for quantifying integrated fluorescence was developed to measure the relative DNA contents of bacterial nucleoids. Image analysis was performed with newly developed macros in combination with the program Object-Image, all downloadable from http://simon.bio.uva.nl/object-image.html. Four aspects of the method were investigated. (i) Good linearity was found over a ten-fold range of fluorescence intensity in a test with a calibration kit of fluorescent latex spheres. (ii) The accuracy of the method was tested with a narrowly distributed Escherichia coli population, which was obtained by growing cells into stationary phase. The width of the image cytometric distribution was approximately 6%, in good agreement with results obtained by flow cytometry. (iii) The error contribution of manual focusing could be kept below 2%, although a strong dependency between integrated fluorescence and focus position was observed. (iv) The results were verified with a flow cytometer, which gave similar distributions for the DNA contents per cell expressed in chromosome equivalents (4.8 fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effect on the total fluorescence of bacterial nucleoids. Experiments using nucleoids with the same amount of DNA in either a dispersed or a compact conformation showed no significant difference in integrated fluorescence, indicating that it is possible to determine the DNA content per nucleoid independently of the actual organization of the DNA.

Analysis of Variance↗

PubMed1995

Nucleoids of pea chloroplasts: microscopic and chemical characterization. Occurrence of histone-like proteins.

The chloroplast genome is highly condensed and packed into discrete structures called "nucleoids". Each pea chloroplast contains 16-20 spherical nucleoids randomly located in the matrix of organelle. Nucleoids are shown to contain DNA, RNA and proteins (1:0.62:2.3). Approximately 30 polypeptides (mol. masses 94 to 12 kD) have been found in nucleoids. Chloroplast DNA is associated with acidic as well as basic proteins, two of which are electrophoretically similar to histones H2A+H2B and H3 of pea cell nuclei. Amino acid composition of these proteins demonstrates high similarity with "HU" proteins of E. coli.

Cell Fractionation↗

PubMed2025

The small bacterial membrane protein YohP induces nucleoid condensation in E. coli and inhibits oligomerization of antimicrobial peptides.

Prokaryotic organisms execute multiple stress response mechanisms in order to cope with rapidly changing environments. Some mechanisms respond to specific cues, such as the OxyR-dependent response to hydrogen peroxide or the SOS-response that is induced upon DNA-damage. These specific responses complement general mechanisms that respond to multiple and diverse stressors. One example is nucleoid condensation, which is a rapid and effective mechanism for genome protection and observed in response to various stresses, including entry into stationary phase. Recently, the upregulation of small membrane proteins (SMPs) in response to stress was observed, but details on how this emerging class of proteins modulate the stress response is largely unknown. Here, we demonstrate that the production of two SMPs, YohP and YncL, cause nucleoid condensation in Escherichia coli. Nucleoid condensation is the result of YohP-/YncL-induced sublethal membrane depolarization, which induces the phage-shock response and leads to a reduction of global protein synthesis. YohP production also prevents the oligomerization of the antimicrobial peptide magainin-2 in the E. coli membrane and reduces the metabolic activity of E. coli cells. Thus, the synthesis of YohP and likely of other SMPs potentially protects bacterial cells against some unfavorable conditions by shifting them into a metabolically silent state.

YncL↗

PubMed1986

Proteins tightly bound to DNA and DNA-synthesizing activity of nucleoids from Escherichia coli.

Membrane-attached nucleoids were isolated from E. coli and separated from proteins by 2 M NaCl. Disintegration of such nucleoids by ultrasound and subsequent centrifugation resulted in the formation of two fractions: a sediment (fraction I) and a supernatant (fraction II). The protein:DNA ratio of fraction I was equal to 27 and was different from that to fraction II (2.6). More than 70% of the proteins not dissociating at 2 M NaCl and bound to DNA of both fractions were polypeptides with molecular weights (Mw) of 31,000-23,000 daltons (31-23 Kdal). After pulse labelling of the cells with [3H]-thymidine, the specific radioactivity of newly synthesized DNA associated with fraction I was shown to be considerably higher than that of fraction II. The analysis of DNA-synthesizing activities in fractions I and II showed that both nucleoid fractions contained DNA polymerase I. After dissolving the two fractions in 8 M urea - 0.15% sodium dodecylsulphate (SDS) they were chromatographed on hydroxyapatite. DNA-protein complexes were obtained that did not dissociate at 4 M guanidine X HCl - 5 M urea and 1% SDS. The main protein of the complexes was a 31 Kdal polypeptide tightly bound to DNA.

Bacterial Proteins↗

PubMed2006

Yme2p is a mediator of nucleoid structure and number in mitochondria of the yeast Saccharomyces cerevisiae.

A large number of gene products have been identified that either directly or indirectly alter the inheritance of mitochondrial DNA. In yeast, we have used a unique genetic screen based on the transfer of DNA from mitochondria to nucleus to identify nuclear-encoded gene products that are targeted to mitochondria and impact the stable inheritance of mitochondrial DNA. A specific allele of one of these genes, yme2-4, prevents even the low wild-type rate of mitochondrial DNA transfer to the nucleus and imparts significant temperature-sensitive and respiratory-growth defects. Intra- and extragenic suppressors of the yme2-4 growth phenotypes were isolated and analysis of these interacting genes reveals that both YME2 and its suppressors influence the structure and number of mitochondrial nucleoids. The yme2-4 allele decreases the average number of mtDNA nucleoids found in cells and the sensitivity of DNA in toluene-treated mitochondria to digestion by DNA exonuclease, effects reversed by intra- and extragenic suppressors. The extragenic suppressor, a missense allele of ILV5, encodes an enzyme of the branched-chain amino acid biosynthetic pathway that is also a component of mitochondrial nucleoids. A null allele of ILV5 suppresses transfer of mitochondrial DNA to the nucleus and displays synthetic interactions with yme2-4.

Alleles↗

PubMed2004

Phosphorylation by protein kinase CKII modulates the DNA-binding activity of a chloroplast nucleoid-associated protein.

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein-DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this localization is unknown. MFP1 is a DNA-binding, coiled-coil protein associated with the thylakoid membranes of mature chloroplasts. It is also a component of nucleoids, suggesting a function at the interface of the chloroplast genome and the photosynthetic membranes. Several thylakoid proteins are phosphorylated by a protein kinase CKII-like activity and the alpha subunit of a chloroplast-located CKII has recently been identified as a component of the chloroplast transcription complex. Here, we show evidence for the phosphorylation of MFP1 in purified chloroplasts from tobacco (Nicotiana tabacum L.). We demonstrate that the DNA-binding domain of MFP1 is a substrate for CKII and that phosphorylation by CKII inhibits DNA binding. Using site-directed mutagenesis, we identify a conserved twin CKII site in the DNA-binding domain that is required for the inhibition of DNA binding. Phosphorylation of MFP1 by chloroplast CKII as a possible means to modulate its DNA-binding activity is discussed.

Amino Acid Sequence↗

PubMed1989

Nucleoids, a subnuclear system capable of chain elongation.

Nucleoids, prepared by salt extraction of non-DNase-digested nuclei, have properties similar, but not identical, to those of nuclear matrices which are prepared by salt extraction of DNase-digested nuclei. Nuclear matrices retained less pulse-labelled DNA, slightly less bound DNA polymerase alpha and DNA primase, but had greater in vitro DNA synthesis and in vitro priming. Nucleoids contained larger (110 S) DNA chains than nuclear matrices (30 S). Each type of residual nuclear structure could synthesize 4.5 S Okazaki fragments. When extracted with increasing concentrations of salt, DNase-digested nucleo lost the ability for further elongation of the 4.5 S DNA intermediate after 0.1-0.2 M NaCl, whereas undigested nuclei retained this ability up to 0.9 M NaCl. Chain elongation to 28 S DNA chains could be restored to nucleoids, but not to nuclear matrices, by the addition of nuclear extracts.

Antibodies, Monoclonal↗

PubMedDate not supplied

Functional consequences of improved structural information on bacterial nucleoids.

Section of Escherichia coli cells, cryofixed, freeze-substituted into acetone and resin-embedded, show nucleoids with coralline shape. The excrescencies reach far into the cytoplasm. Membrane contact is no longer excluded. Comparison with phase contrast light microscopy shows that the fine excrescencies cannot be resolved and therefore lead "artificially" to a more confined aspect of the nucleoid. The packing density of the DNA in the nucleoids is like that of eukaryotic interphase nuclei and would thus allow diffusion in and out of even large macromolecules. The transcription has, however, been demonstrated to occur only at the periphery; it requires a very dynamic state of the chromatin. The chromatin fine structure is now more granular than fibrillar, as it was previously. The granular structure is compatible with--but there is no proof for--the existence of compactosomes, which would form as a consequence of unrestrained supercoiling.

Cell Nucleus↗

PubMed1991

Escherichia coli DNA-binding protein H-NS is localized in the nucleoid.

Electron microscope localization of the 15.4-kDa DNA-binding protein H-NS was carried out in Escherichia coli cells subjected to cryosubstitution followed by immuno-labelling with the protein A/gold technique. Three types of E. coli cells were used: (1) "normal" cells growing exponentially at 37 degrees C; (2) "cold-shocked" cells two hours after the shift from 37 degrees C to 10 degrees C; and (3) cells in which an expression vector had been induced to overproduce H-NS. The results clearly indicate that in all 3 cases, the vast majority of the molecules reacting with anti-H-NS antibodies are localized within the nucleoid and at the border between the nucleoid and the ribosome-rich cytoplasm, which supports the premise that H-NS is implicated in the condensation of the nucleoid.

Bacterial Proteins↗

PubMed2005

Restricted diffusion of DNA segments within the isolated Escherichia coli nucleoid.

To study the dynamics and organization of the DNA within isolated Escherichia coli nucleoids, we track the movement of a specific DNA region. Labeling of such a region is achieved using the Lac-O/Lac-I system. The Lac repressor-GFP fusion protein binds to the DNA section where tandem repeats of the Lac operator are inserted, which allows us to monitor the motion of the DNA. The movement of such a GFP spot is followed at 48 ms temporal resolution during 12s. The spots are found to diffuse within a confined space, so that the nucleoid appears to behave like a viscoelastic network. The distribution of the "particle" position in time can be fitted to a Gaussian function indicating that the motion of the particle is Brownian. An average self-diffusion constant Ds=0.12 microm(2) s-1 is derived via the time auto-correlation functions of the displacement and is compatible with the collective diffusion coefficient measured previously by dynamic light scattering. Restriction of a DNA sequence to a small region of the nucleoid is tentatively related to the existence of so-called supercoiling domains.

Chromosomes, Bacterial↗

PubMed2006

Shape and compaction of Escherichia coli nucleoids.

The genomic DNA in cells of Escherichia coli is localized in one or two compact, phase-like regions with characteristic shapes. Nucleoids undergo progressive changes in shape and compaction in the presence of drugs such as chloramphenicol or puromycin. Forces which influence nucleoid shape and compaction are reviewed, with particular emphasis on crowding effects of the cytoplasm and confinement effects of the cell envelope. Based in part on the theory of Kornyshev and Leikin for interaction between DNA duplexes, the folding of DNA caused by binding of DNA-associated proteins is suggested to antagonize DNA condensation and, thereby, increase access to DNA sequences. These views are incorporated into a working model for nucleoid organization.

Chromosomes, Bacterial↗

PubMed2005

SlmA, a nucleoid-associated, FtsZ binding protein required for blocking septal ring assembly over Chromosomes in E. coli.

Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.

Carrier Proteins↗

PubMed1993

Treatment with a polyamine analog alters DNA-matrix association in HeLa cell nuclei: a nucleoid halo assay.

The polyamine analog 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4) depletes polyamines and inhibits the growth of tumor cells in tissue culture. We treated HeLa cells in culture with BE-4-4-4 for different time periods to produce different degrees of polyamine depletion. The cells were lysed and dehistonized to obtain nucleoids containing DNA attached to the nuclear matrix. Titration of the nucleoids with propidium iodide caused an uncoiling of negatively supercoiled DNA, resulting in the formation of a halo surrounding the nucleoid periphery. The halo diameters in both the BE-4-4-4-treated cells and the untreated control cells were measured using a fluorescence image analysis system. As compared to the control cells, the BE-4-4-4-treated cells showed a 20-25% decrease in halo diameter, indicating that there was less relaxation of the negative supercoils in the nuclear DNA of the BE-4-4-4-treated cells than in the controls.

Cell Division↗

PubMed1999

Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival.

Upon sensing an impending saturation level of their population density, Escherichia coli cells enter into the stationary phase. We have identified structural and functional modulations of the nucleoid, the transcription apparatus and the translation machinery occurring during the transition from exponential growth to stationary phase. The major DNA-binding proteins, Fis, HU and Hfq, in the exponential-phase nucleoid are replaced by a single stationary-phase protein Dps, thereby compacting the nucleoid and ultimately leading to silencing of the DNA functions. The transcription apparatus is modified by replacing the major promoter recognition subunit, sigma70, with sigmaS. A stationary-phase protein, Rsd (Regulator of Sigma D), with the binding activity of sigma70 is involved in the efficient replacement of sigma and/or the storage of unused sigma70. Changes in cytoplasmic composition also differentially influence the activity of Esigma70 and EsigmaS holoenzymes. Together, these effects may result in the preferential transcription of stationary-phase specific genes. The translation machinery is also modulated in stationary phase, by the formation of translationally incompetent 100S ribosomes. A small stationary-phase protein, RMF (Ribosome Modulation Factor), is involved in the dimerization of 70S ribosome monomers.

Bacterial Proteins↗

PubMed1996

The effects on Escherichia coli of expression of the cloned bacteriophage T4 nucleoid disruption (ndd) gene.

Immediately after T4 bacteriophage infection, the Escherichia coli nucleoid undergoes rapid delocalization. The ndd gene of T4 is responsible for this nuclear disruption phenomenon. We have cloned two alleles of this gene and studied the effects of their expression on E. coli cells. We have shown that the Ndd protein (i) is able to reproduce the disruption of the nucleoid characteristic of T4 infection, (ii) is highly toxic and results in a logarithmic decrease in cell viability, and (iii) inhibits genomic DNA replication by blocking progression of replication forks. Induction of Ndd does not result in degradation of genomic DNA and does not significantly alter the general processes of transcription and translation during the entire period of exponential cell death. These results support the notion that the target of Ndd is some aspect of the nucleoid architecture.

Bacterial Proteins↗

PubMed1991

Membrane-bounded nucleoid in the eubacterium Gemmata obscuriglobus.

The freshwater budding eubacterium Gemmata obscuriglobus possesses a DNA-containing nuclear region that is bounded by two nuclear membranes. The membrane-bounded nature of the nucleoid in this bacterium was shown by thin sectioning of chemically fixed cells, thin sectioning of freeze-substituted cells, and freeze-fracture/freeze-etch. The fibrillar nucleoid was surrounded by electron-dense granules that were in turn enveloped by two nuclear membranes separated by an electron-transparent space. Immunogold labeling of thin sections of conventionally fixed cells with anti-double-stranded DNA antibody demonstrated double-stranded DNA associated with fibrillar material within the membrane boundary. The occurrence of a membrane-bounded nucleoid in a eubacterial prokaryote is a significant exception to the evidence supporting the prokaryote/eukaryote dichotomous classification of cell structure.

Journal Article↗

PubMed1999

Nucleoid proteins of pea chloroplasts: detection of a protein homologous to ribosomal protein.

Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction. Using RP-HPLC, the component composition of the basic proteins was studied. SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol. masses of components from 17 to 30 kDa. One polypeptide with mol. mass of 28 kDa (P28) was obtained by RP-HPLC. The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae. The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed.

Amino Acid Sequence↗