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PubMed1976

In vitro transcription of the tryptophan operon in isolated bacterial nucleoids.

In vitro transcription of the trp operon in isolated nucleoids from Escherichia coli was studied. RNA synthesis in this system occurred primarily as a continuation of transcription which had been initiated in vivo; little or no initiation of new RNA chains was observed. Transcription of the trp operon in nucleoids by endogenous RNA polymerase procedded efficiently and ceases sequentially in the order of the gene sequence within the operon. Under these conditions, no appreciable exonuccleolytic digestion of nascent 3H-RNA was found, though some endonucleolytic cleavage was generally seen. Little or no incorporation of 14C-leucine into polypeptides was observed, inspite of tha fact that considerable number of ribosomes and nascent RNA chains were found attached to the isolated nucleoids. The synthesis of trp mRNA continued in the presence of chloramphenicol or fusidic acid, or under conditions where the rebosomal translocation factor G was inactivated. From these and other kinetic studies of trp mRNA synthesis in nucleoids obtained from nonsense strong polar mutants of the trp operon, it was shown that transcription in nucleoids was not connected functionally with transloational processes and thus unable to exhibit polarity effected by a nonsense mutation or by general translational blockage. In studies employing nucleoids from nonsense strong polar mutants of the trp operon, it was demonstrated that RNA polymerase are scantily distributed over the region downstream from the nonsense mutation site of the operon, thereby supporting a notion that in vivo transcription is eventually terminated near the nonsense mutation.

DNA, Bacterial↗

PubMed1990

Changes in nucleoid viscosity following X-irradiation of rat thymic and splenic cells in vitro.

Unscheduled DNA synthesis (UDS) suggested a higher DNA repair capacity of X-irradiated rat thymic (T) cells when compared to splenic (S) cells (Tempel 1980). In the present investigations, damage and repair of DNA supercoiling was measured in T- and S-cells following X-irradiation in vitro by using the nucleoid sedimentation technique and a simplified low-shearing viscometric test. - X-irradiation resulted in a dose (0.6-19.2 Gy) - dependent reduction in sedimentation and viscosity of nucleoids. Within a post-irradiation period of 30-45 min after a challenge dose of 19.2 Gy, DNA repair was accompanied by an increase in nucleoid sedimentation and viscosity in T-cells by about 60 and 300, in S-cells by almost 40 and 100%, resp. The increase in nucleoid viscosity within a 30 min repair period could be reduced in a concentration-dependent manner by DNA polymerase - inhibitors and proteinase K. - The higher DNA repair capacity of T-cells as reflected by UDS is confirmed therefore by the nucleoid characteristics. Apart from this suggestion, measuring nucleoid viscosity may be considered as a sensitive, simple and rapid device to detect radiation-induced DNA supercoiling phenomena.

Animals↗

PubMed1981

The chromosome structure and the cell cycle of Caulobacter crescentus. Isolation and analysis of envelope-free nucleoids.

Envelope-free nucleoids were isolated from an asymmetrically dividing bacterium, Caulobacter crescentus. In the nucleoid fraction, most of the DNA and nascent RNA in the cell and about 2% of the total cellular proteins were recovered. The sedimentation coefficient of the nucleoid was constant (1260 S) during the G1 period of the swarmer cell cycle and increased to 1940 S during the S period. Since both replicating (S period) and non-replicating (G1 period) chromosomes had a similar superhelical concentration, the increase in the sedimentation coefficient was simply explained by duplication of the nucleoid structure. The duplicated nucleoid was shown to segregate prior to the cell division. The pulse-labeled proteins recovered in the nucleoid fraction contained several stage-specific species, most of which were detected at the beginning of S period.

Bacterial Proteins↗

PubMed1983

In vitro transcription from Bacillus subtilis nucleoids by homologous RNA polymerase.

The effect of DNA structural features on RNA synthesis was investigated. Purified Bacillus subtilis nucleoids templated from vegetative cells were transcribed by the homologous RNA polymerase using Hg-UTP as one of the nucleotide substrates. Low RNA polymerase/DNA ratios were used during transcription in order to avoid nonspecific initiations. The rate of synthesis of total RNA was 40% greater on nucleoid templates than on naked DNA. The proportion of asymmetric transcript synthesized on nucleoid templates (HvsL strand transcripts) was close to that observed in vivo, whereas with naked DNA this value was at least 3-times lower. The percentage of rRNA relative to the total RNA, synthesized in the in vitro system with the nucleoid template, approaches the rate of the in vivo transcription. The size of the RNA synthesized on nucleoids was large and heterogeneous while with naked DNA it was homogeneous and of about 6 S. Our results suggest that the supercoiled, folded nucleoids may retain some of the structural features responsible for the regulation of RNA synthesis in vivo.

Bacillus subtilis↗

PubMed2006

Transmission electron microscopy of the bacterial nucleoid.

Water-containing biological material cannot withstand the vacuum of the transmission electron microscope. The classical solution to this problem has been to dehydrate chemically fixed biological samples and then embed them in resin. During such treatment, the bacterial nucleoid is especially prone to aggregation, which affects its global shape and fine structure. Initial attempts to deal with aggregation by optimizing chemical fixation yielded contradictory results. Two decades ago, the situation improved with the introduction of freeze-substitution. This method is based on dehydration of unfixed cryo-immobilized samples at low temperature, which substantially reduces aggregation. As a result, the global shape of the nucleoid can be fairly well defined. Overall, in actively growing bacteria, the nucleoids are dispersed and "coralline" but become more confined when growth ceases. However, it is usually impossible to determine the molecular arrangement of DNA in the nucleoids of freeze-substituted bacteria because crystallization and the subsequent removal of water during substitution result in unavoidable distortions at the ultrastructural level. Recently, cryo-electron microscopy of vitreous sections has enabled the fully hydrated bacterial nucleoid to be studied close to the native state. Such studies have revealed aspects of bacterial nucleoid organization that are not preserved by freeze-substitution, including locally parallel or twisted bundles of DNA filaments, which are more frequently observed once bacterial growth has stopped, whereas in actively growing bacteria, the DNA is seen to be in a mostly disordered pattern.

Bacteria↗

PubMed1999

Delayed nucleoid segregation in Escherichia coli.

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.

Aztreonam↗

PubMed1988

Fluorescence microscopic studies on mitochondria and mitochondrial nucleoids in a wild-type strain and respiratory mutants of Candida albicans.

A wild-type strain and two respiratory mutants of Candida albicans were examined for mitochondria and mitochondrial nucleoids (mt-nucleoids) using the fluorescent dyes, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) and 4', 6-diamidino-2-phenylindole (DAPI). Rapidly growing cells of the wild type possessed one or a few giant branched mitochondria that were intensely stained with DASPMI. When a bud emerged, an end of the giant mitochondrion extended into the bud and the mitochondrion was divided and partitioned into mother and daughter cells by cytokinesis. Cell cycle-associated fragmentation or fusion of mitochondria were not demonstrated. The mutant KRD-8, that possesses cristate mitochondria but respires at a lower level, was shown to contain one or a few, less stainable giant mitochondria per cell. DASPMI failed to stain cells of the mutant KRD-19 which lacks cytochrome aa3 and cristate mitochondria. About eight and 10 mt-nucleoids were detected as discrete fluorescent spots in DAPI-stained cells of the wild type and KRD-8, respectively. KRD-19 cells also possessed mt-nucleoids, although the number of mt-nucleoids per cell seemed to be smaller than that of the wild type. In all the strains, mt-nucleoids existed discretely throughout the budding cycle, and the increase of their number per cell appeared to correlate with the cellular volume but not with nuclear division.

Candida albicans↗

PubMed1999

Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA.

Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.

Alleles↗

PubMed2002

Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids.

Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.

Algal Proteins↗

PubMed2005

Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome.

The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.

Cell Nucleus↗

PubMed1997

The bacterial nucleoid visualized by fluorescence microscopy of cells lysed within agarose: comparison of Escherichia coli and spirochetes of the genus Borrelia.

The nucleoids of Escherichia coli and the spirochetes Borrelia burgdorferi and Borrelia hermsii, agents of Lyme disease and relapsing fever, were examined by epifluorescence microscopy of bacterial cells embedded in agarose and lysed in situ with detergent and protease. The typical E. coli nucleoid was a rosette in which 20 to 50 long loops of DNA emanated from a dense node of DNA. The percentages of cells in a population having nucleoids with zero, one, two, and three nodes varied with growth rate and growth phase. The borrelia nucleoid, in contrast, was a loose network of DNA strands devoid of nodes. This nucleoid structure difference correlates with the unusual genome of Borrelia species, which consists primarily of linear replicons, including a 950-kb linear chromosome and linear plasmids. This method provides a simple, direct means to analyze the structure of the bacterial nucleoid.

Borrelia↗

PubMed2000

Analysis of nucleoid morphology during germination and outgrowth of spores of Bacillus species.

After a few minutes of germination, nucleoids in the great majority of spores of Bacillus subtilis and Bacillus megaterium were ring shaped. The major spore DNA binding proteins, the alpha/beta-type small, acid-soluble proteins (SASP), colocalized to these nucleoid rings early in spore germination, as did the B. megaterium homolog of the major B. subtilis chromosomal protein HBsu. The percentage of ring-shaped nucleoids was decreased in germinated spores with lower levels of alpha/beta-type SASP. As spore outgrowth proceeded, the ring-shaped nucleoids disappeared and the nucleoid became more compact. This change took place after degradation of most of the spores' pool of major alpha/beta-type SASP and was delayed when alpha/beta-type SASP degradation was delayed. Later in spore outgrowth, the shape of the nucleoid reverted to the diffuse lobular shape seen in growing cells.

Bacillus megaterium↗

PubMed2005

Fine structure of the Deinococcus radiodurans nucleoid revealed by cryoelectron microscopy of vitreous sections.

Transmission electron microscopy revealed that the nucleoid of the extremely radioresistant bacteria Deinococcus radiodurans may adopt an unusual ring shape. This led to the hypothesis that the tight toroidal package of the D. radiodurans genome might contribute to radioresistance by preventing diffusion of ends of double-stranded DNA breaks. The molecular arrangement of DNA in the nucleoid, which must be determined to test this hypothesis, is not discernible by conventional methods of electron microscopy. We have applied cryoelectron microscopy of vitreous sections and found that the DNA arrangement in D. radiodurans differs from toroidal spooling. Diffuse coralline nucleoids of exponentially growing D. radiodurans do not reveal any particular molecular order. Electron-dense granules are generally observed in the centers of nucleoids. In stationary-phase cells, the nucleoid segregates from cytoplasm and DNA filaments show locally parallel arrangements, with increasing aspects of cholesteric liquid crystalline phase upon prolonged starvation. The relevance of the observed nucleoid organization to the radiation resistance of D. radiodurans is discussed.

Cryoelectron Microscopy↗

PubMed1993

Detection of DNA in the nucleoids of chloroplasts and mitochondria in Euglena gracilis by immunoelectron microscopy.

DNA in the nucleoids of chloroplasts and of mitochondria in Euglena gracilis was detected with anti-DNA antibodies by immunoelectron microscopy. After treatment with the antibodies, DNA in these organelles combined with gold particles that had been coated with anti-IgM antibodies such that it was possible to trace the outlines of the nucleoids. Nucleoids in chloroplasts appeared to be composed of twisted threads 50-70 nm in diameter. The twisted threads were entangled to form thicker nodes of 100-200 nm diameter. Most nucleoids in mitochondria were spherical or ovoid, 70-130 nm in diameter. Nucleoids both in chloroplasts and in mitochondria contained cores with which DNA threads were in tight contact. The structure of the nucleoids was very different from those previously observed by conventional electron microscopy.

Animals↗

PubMed2001

Unique nucleoid structure during cell division of Thermococcus kodakaraensis KOD1.

The nucleoid structure and the partition in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 were observed by a combination of phase-contrast microscopy and fluorescence microscopy. The nucleoids occurred as rounded fluorescent foci centrally located in the cells and as differences in fluorescence intensity between exponential and stationary phases. The cellular space occupied by the nucleoid in the stationary phase was larger than that in the exponential phase. Various shapes of nucleoid in the exponential-phase cells were observed, indicating that nucleoid separation was processed under cell cycle control. The number of cells which showed distinctive division stages was counted and the proportions of dividing cells were determined. About half of the observed cells were in the replication stage. More than 40% of the counted cells possessed a fully replicated but not separated form of nucleoid. Only 8% of the total cells clearly showed visible constriction. These results suggested that the post-replication period before cell division was relatively as long as the eucaryal gap period (G2); however, the period of visible cell constriction was almost the same as that of the bacteria.

Journal Article↗

PubMed1991

Novobiocin treatment reverses radiation-induced alterations in higher-order DNA structure in L5178Y nucleoids.

We have studied the effect of novobiocin treatment on radiation-induced damage and its repair in higher-order DNA structure in two mouse leukemia cell lines differing in their radiosensitivity, L5178Y-R (LY-R) and L5178Y-S (LY-S). We used the fluorescent halo technique to measure alterations in the superhelical density and the topological constraints of DNA in LY-R and LY-S nucleoids. The results for untreated cells show that both cell lines reached maximal DNA unwinding at the same concentration of propidium iodide (PI), whereas LY-S nucleoids were less efficient in their ability to rewind their DNA. The loop size did not differ significantly between the cell lines. Incubation of LY-R and LY-S cells with novobiocin at a concentration which does not influence survival (0.1 mM for 45 min), but inhibits DNA synthesis in LY-R cells (by 28%) to a greater extent than in LY-S cells (by 10%), also causes more DNA unwinding in LY-R nucleoids than in LY-S nucleoids. However, a decreased superhelical density was observed in nucleoids from both cell lines. Novobiocin applied before, and present during, irradiation prevents radiation-induced alterations in DNA supercoiling more efficiently in LY-R than in LY-S cells. The presence of novobiocin during the repair period increased DNA rewinding to levels not significantly different from control values in nucleoids from both cell lines.

Animals↗

PubMed1992

Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy.

The Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter. An hns mutant yielding a protein (H-NS delta 12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained. Overproduction of wild-type (wt) H-NS, but not of H-NS delta 12, resulted in a drastic loss of cell viability. The molecular events and the morphological alterations eventually leading to cell death were investigated. A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time. Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids. The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical. These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.

Bacterial Outer Membrane Proteins↗

PubMed1990

The Escherichia coli minB mutation resembles gyrB in defective nucleoid segregation and decreased negative supercoiling of plasmids.

Nucleoid segregation in the Escherichia coli minB mutant and in cells that over-produce minB gene products appeared defective as measured from fluorescence micrographs. Electrophoretic resolution of topoisomers of plasmid isolates from the minB strain revealed a decreased level of negative supercoiling; in addition, multimerization was observed. Over-production of the minB gene product also resulted in a decreased level of negative supercoiling. This phenotype is typical of the gyrB(ts) mutant, which is known to be affected in chromosome decatenation and supercoiling. We propose that the minB mutation and over-production of the minB gene products cause a defect in nucleoid segregation, which may be related to the decrease in negative supercoiling. As in the gyrB(ts) mutant, retardation of nucleoid segregation is proposed to inhibit constriction initiation in the cell centre and to give rise to nucleoid-free cell poles. As a consequence, these cells divide between nucleoid and cell pole, resulting in minicell and (sometimes) in anucleate cell formation.

Bacterial Proteins↗