Fluorescence Microscopy of the Dynamics of Supercoiling,Folding, and Condensation of Bacterial Chromosomes,Induced by Acridine Orange

Abstract

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and super- coiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA and indicate that kinks are an important intermediate step in many of the transitions.     

Filaments from Ignicoccus hospitalis Show Diversity of Packing in Proteins Containing N-Terminal Type IV Pilin Helices

Bacterial motility is driven by the rotation of flagellar filaments that supercoil. The supercoiling involves the switching of coiled-coil protofilaments between two different states. In archaea, the flagellar filaments responsible for motility are formed by proteins with distinct homology in their N-terminal portion to bacterial Type IV pilins. The bacterial pilins have a single N-terminal hydrophobic α-helix, not the coiled coil found in flagellin. We have used electron cryo-microscopy to study the adhesion filaments from the archaeon Ignicoccus hospitalis. While I. hospitalis is non-motile, these filaments make transitions between rigid stretches and curved regions and appear morphologically similar to true archaeal flagellar filaments. A resolution of ~ 7.5 Å allows us to unambiguously build a model for the packing of these N-terminal α-helices, and this packing is different from several bacterial Type IV pili whose structure has been analyzed by electron microscopy and modeling. Our results show that the mechanism responsible for the supercoiling of bacterial flagellar filaments cannot apply to archaeal filaments.     

Partitioning of the Escherichia coli chromosome: superhelicity and condensation

Segregation in Escherichia coli, the process of separating the replicated chromosomes into daughter progeny cells, seems to start long before the duplication of the genome reaches completion. Soon after initiation in mid-cell region, the daughter oriCs rapidly move apart to fixed positions inside the cell (quarter length positions from each pole) and are anchored there by yet unknown mechanism(s). As replication proceeds, the rest of the chromosome is sequentially unwound and then refolded. At termination, the two sister chromosomes are unlinked by decatenation and separated by supercoiling and/or condensation. Muk and Seq proteins are involved in different stages of this replication-cum-partition process and thus can be categorized as important partition proteins along with topoisomerases. E. coli strains, lacking mukB or seqA functions, are defective in segregation and cell division. The nucleoids in these mutant strains exhibit altered condensation and superhelicity as can be demonstrated by sedimentation analysis and by fluorescence microscopy. As the supercoiling of an extrachromosomal element (a plasmid DNA) was also influenced by the mukB and seqA mutations we concluded that the MukB and SeqA proteins are possibly involved in maintaining the general supercoiling activity in the cell. The segregation of E. coli chromosome might therefore be predominantly driven by factors that operate by affecting the superhelicity and condensation of the nucleoid (MukB, SeqA, topoisomerases and additional unknown proteins). A picture thus emerges in which replication and partition are no longer compartmentalized into separable stages with clear gaps (S and M phases in eukaryotes) but are parallel processes that proceed concomitantly through a cell cycle continuum.     

Chromosomal domains of supercoiling in Salmonella typhimurium

The chromosomes of enteric bacteria are divided into about 50 independently supercoiled domains. It is not known whether the net level of DNA supercoiling is similar in each domain, or whether the domains are differentially supercoiled. We have addressed this question genetically, using a supercoiling-sensitive promoter to probe the relative levels of supercoiling at defined points around the Salmonella typhimurium chromosome. We conclude that, within the limits of resolution of this approach, the level of supercoiling does not differ significantly between chromosomal domains, and that each domain responds in a similar fashion to factors that perturb supercoiling. These findings have implications for the organization of the bacterial genome.     

Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum.

Light and electron microscope observations showed that the filamentous, segmented bacterium commonly found attached to the ileal epithelium of rats and mice undergoes a complex life cycle. Filaments comprising up to 90 segments were attached to the microvillous border of absorptive epithelial cells by a specialized terminal holdfast segment. Starting at the free end of the filament and progressing toward the attached end, undifferentiated segments were converted into reproductive or mother segments. Within each mother cell two new holdfast segments developed. As the holdfasts matured, their mother cells degenerated and released them into the intervillar space where they attached, grew, and divided to produce new segmented filaments. Alternately, in some filaments, newly formed but not yet released holdfasts were converted into endospores, which were released in the same manner as holdfasts, presumably to spread the bacterial colony to other members of the rodent population.     

Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA–DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.     

Novel, attached, sulfur-oxidizing bacteria at shallow hydrothermal vents possess vacuoles not involved in respiratory nitrate accumulation

Novel, vacuolate sulfur bacteria occur at shallow hydrothermal vents near White Point, Calif. There, these filaments are attached densely to diverse biotic and abiotic substrates and extend one to several centimeters into the surrounding environment, where they are alternately exposed to sulfidic and oxygenated seawater. Characterizations of native filaments collected from this location indicate that these filaments possess novel morphological and physiological properties compared to all other vacuolate bacteria characterized to date. Attached filaments, ranging in diameter from 4 to 100 microm or more, were composed of cylindrical cells, each containing a thin annulus of sulfur globule-filled cytoplasm surrounding a large central vacuole. A near-complete 16S rRNA gene sequence was obtained and confirmed by fluorescent in situ hybridization to be associated only with filaments having a diameter of 10 microm or more. Phylogenetic analysis indicates that these wider, attached filaments form within the gamma proteobacteria a monophyletic group that includes all previously described vacuolate sulfur bacteria (the genera Beggiatoa, Thioploca, and Thiomargarita) and no nonvacuolate genera. However, unlike for all previously described vacuolate bacteria, repeated measurements of cell lysates from samples collected over 2 years indicate that the attached White Point filaments do not store internal nitrate. It is possible that these vacuoles are involved in transient storage of oxygen or contribute to the relative buoyancy of these filaments.     

Interactions stabilizing DNA tertiary structure in the Escherichia coli chromosome investigated with ionizing radiation

The structure of the bacterial chromosome was investigated after introducing breaks in the DNA with gamma irradiation. It is demonstrated that irradiation of the chromosome in the cell prior to isolation results in partial unfolding of the isolated condensed DNA, while irradiation of the chromosome after it is released from the cell has no demonstrable effect on DNA folding. The results indicate that RNA/DNA interactions which stabilize DNA folds are unstable when breaks are introduced in the DNA prior to isolation of the chromosome. It is suggested that the supercoiled state of the DNA is required for the initial stabilization of some of the critical RNA/DNA interaction in the isolated nucleoid. However, some of these interactions are not affected by irradiation of the cells. Remnant supercoiling in partially relaxed chromosomes containing a limited number of DNA breaks has the same superhelical density as the unirradiated chromosome. This suggests that restraints on rotation of the packaged DNA are formed prior to the physical unwinding which occurs at the sites of the radiation induced DNA breaks. — Analysis of the in vitro irradiated chromosomes shows that there are 100+-30 domains of supercoiling per genome equivalent of DNA. The introduction of up to 50 double-strand breaks per nucleoid does not influence rotor speed effects of the sedimentation coefficient of the chromosome.     

Differential and dynamic localization of topoisomerases in Bacillus subtilis

Visualization of topoisomerases in live Bacillus subtilis cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcellular topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the B. subtilis nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.     

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."     

SMC proteins in bacteria: condensation motors for chromosome segregation?

SMC proteins are a ubiquitous protein family, present in almost all organisms so far analysed except for a few bacteria. They function in chromosome condensation, segregation, cohesion, and DNA recombination repair in eukaryotes, and can introduce positive writhe into DNA in vitro. SMC proteins and the structurally homologous MukB protein are unusual ATPases that form antiparallel dimers, with long coiled coil segments separating globular ends capable of binding DNA. Recently, SMC proteins have been shown to be essential for chromosome condensation, segregation and cell cycle progression in bacteria. Identification of a suppressor mutation for MukB in topoisomerase I in Escherichia coli suggests that SMC proteins are involved in negative DNA supercoiling in vivo, and by this means organize and compact chromosomes. A model is discussed in which bacterial SMC proteins act after an initial separation of replicated chromosome origins into the future daughter cell, separating sister chromatids by condensing replicated DNA strands within both cell halves. This would be analogous to a pulling of DNA strands into opposite cell halves by a condensation mechanism exerted at two specialised subregions in the cell.     

Micro-video study of moving bacterial flagellar filaments. I. Passive rotation by hydrodynamic force in vitro.

Moving images of reconstituted single bacterial flagellar filaments in a dark-field microscope were recorded by an ultrasensitive video camera, and then transferred to 16 mm cinefilm for quantitative analysis of the dynamic properties of the filaments.Flagellar filaments are found to attach to a glass surface at only one end (the H -end). When attached helical filaments were subjected to viscous flow of methylcellulose solution, they rotated as a result of the hydrodynamic torque generated. Occasionally, two filaments associated into a bundle and rotated coordinately in the viscous flow, even though each filament was separately attached to the glass surface. In addition, we have observed partly rotating filaments which consisted of two portions, the rotating portion being connected end-to-end to the non-rotating portion.The magnitude of the hydrodynamic torque depended on the rotational friction which was determined by the manner of attachment. Based on hydrodynamic calculations, values of ?5 × 10^?12 and ?1 × 10^?13 dyne cm were obtained for the average torque for rotating filaments on glass and partly rotating filaments, respectively, in viscous fluid at a flow rate of 15 μm/s.     

Incomplete chromatin condensation in enlarged rat myelocytic leukemia cells

The distinguishable morphologic features of nuclei of acute myelogenous leukemia cells with enlarged size and finely distributed nuclear chromatin indicate incomplete chromosome condensation that can be related to elevated gene expression. To confirm this, interphase chromosome structures were studied in exponentially growing rat myelomonocytic leukemia 1 cells isolated at the University of Debrecen (My1/De cells). This cell line was established from primary rat leukemia chemically induced by 7,12-dimethylbenz[a]anthracene treatment. The enlarged nuclei of My1/De cells allowed improved fluorescent visualization of chromosomal structures. Increased resolution revealed major interphase intermediates consisting of (1) veil-like chromatin, (2) chromatin ribbon, (3) chromatin funnel, (4) chromatin bodies, (5) elongated prechromosomes, (6) seal-ring, spiral shaped, and circular chromosomal subunits, (7) elongated, bent, u- and v-shaped prechromosomes, and (8) metaphase chromosomes. Results confirmed the existence of the chromatin funnel, the first visible interphase chromosome generated by the supercoiling of the chromatin ribbon. Other intermediates not seen previously included the spiral subunits that are involved in the chromonemic folding of metaphase chromosomes. The existence of spiral subunits favors the helical coil model of chromosome condensation. Incomplete chromatin condensation in leukemia cells throughout the cell cycle is an indication of euchromatization contributing to enhanced gene expression and is regarded as a leukemic factor.     

Spermiogenesis in an iceryine coccid,Steatococcus tuberculatus morrison

The spermatozoon of Steatococcus is a motile filament containing a core of two chromosomes arranged in tandem and surrounded by more than 80 microtubules in 2 1/2 concentric rings. Two sperm develop from each binucleate spermatid in the form of long papillae. From the zone corresponding to the pole of the previous division microtubules appear and lengthen, assembly apparently occurring at their proximal undifferentiated ends. As they extend, they presumably push out the cytoplasmic papilla and co-extend a nuclear papilla through bridges with the nuclear envelope. Chromatin, attached to the envelope, is thus carried into the papilla, the shorter chromosome in the lead. 100 Å chromatin filaments are reduced to 20 Å and aligned as they enter the papilla. The filaments transform into 100 Å tubular fibrils, presumably by supercoiling. These then pack hexagonally, aggregate further into packed axial filaments, and finally condense into a nearly solid core in the mature sperm. Completed papillae (sperm) detach from the spermatid leaving behind nuclei devoid of chromatin. Following cycles of spiralization and despiralization, the sperm are bundled into hexagonal packs of 32 in register by cyst wall cells. The latter form primary and secondary sheaths and lay down a matrix within the bundle. As originally reported by Hughes Schrader (1946), no evidence of centriole, acrosome, mitochondrial derivative or structure suggesting flagellar axoneme is found in either the developing papilla or the mature sperm. The microtubules determine the axis of the developing sperm; polarity is set by the direction of sperm motion and is homologous with most flagellate sperm in that the nuclear material is anterior and the microtubule initiating center is posterior. All of the functions attributed to microtubules are manifest in differentiation of this sperm: extension, support, translocation and motility.This paper is affectionately dedicated to Professor Sally Hughes-Schrader on the occasion of her seventy-fifth birthday, with warm appreciation of her friendship, her exemplary science, her keen criticism, her contagious enthusiasm, and for leading me to Steatococcus.     

Novel, Attached, Sulfur-Oxidizing Bacteria at Shallow Hydrothermal Vents Possess Vacuoles Not Involved in Respiratory Nitrate Accumulation

Novel, vacuolate sulfur bacteria occur at shallow hydrothermal vents near White Point, Calif. There, these filaments are attached densely to diverse biotic and abiotic substrates and extend one to several centimeters into the surrounding environment, where they are alternately exposed to sulfidic and oxygenated seawater. Characterizations of native filaments collected from this location indicate that these filaments possess novel morphological and physiological properties compared to all other vacuolate bacteria characterized to date. Attached filaments, ranging in diameter from 4 to 100 μm or more, were composed of cylindrical cells, each containing a thin annulus of sulfur globule-filled cytoplasm surrounding a large central vacuole. A near-complete 16S rRNA gene sequence was obtained and confirmed by fluorescent in situ hybridization to be associated only with filaments having a diameter of 10 μm or more. Phylogenetic analysis indicates that these wider, attached filaments form within the gamma proteobacteria a monophyletic group that includes all previously described vacuolate sulfur bacteria (the genera Beggiatoa, Thioploca, and Thiomargarita) and no nonvacuolate genera. However, unlike for all previously described vacuolate bacteria, repeated measurements of cell lysates from samples collected over 2 years indicate that the attached White Point filaments do not store internal nitrate. It is possible that these vacuoles are involved in transient storage of oxygen or contribute to the relative buoyancy of these filaments.     

Cell cycle-dependent phosphorylation, nuclear localization, and activation of human condensin

Condensin, one of the most abundant components of mitotic chromosomes, is a conserved protein complex composed of two structural maintenance of chromosomes (SMC) subunits (SMC2- and SMC4-type) and three non-SMC subunits, and it plays an essential role in mitotic chromosome condensation. Purified condensin reconfigures DNA structure using energy provided by ATP hydrolysis. To know the regulation of condensin in somatic cells, the expression level, subcellular localization, and phosphorylation status of human condensin were examined during the cell cycle. The levels of condensin subunits were almost constant throughout the cell cycle, and the three non-SMC subunits were phosphorylated at specific sites in mitosis and dephosphorylated upon the completion of mitosis. Subcellular fractionation studies revealed that a proportion of condensin was tightly bound to mitotic chromosomes and that this form was phosphorylated at specific sites. Condensin purified from mitotic cells had much stronger supercoiling activity than that purified from interphase cells. These results suggest that condensin functions in somatic cells are regulated by phosphorylation in two ways during the cell cycle; the phosphorylation of specific sites correlates with the chromosomal targeting of condensin, and its biochemical activity is stimulated by phosphorylation.     

Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC

Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo. In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.     

Looping Probabilities in Model Interphase Chromosomes

Fluorescence in-situ hybridization (FISH) and chromosome conformation capture (3C) are two powerful techniques for investigating the three-dimensional organization of the genome in interphase nuclei. The use of these techniques provides complementary information on average spatial distances (FISH) and contact probabilities (3C) for specific genomic sites. To infer the structure of the chromatin fiber or to distinguish functional interactions from random colocalization, it is useful to compare experimental data to predictions from statistical fiber models. The current estimates of the fiber stiffness derived from FISH and 3C differ by a factor of 5. They are based on the wormlike chain model and a heuristic modification of the Shimada-Yamakawa theory of looping for unkinkable, unconstrained, zero-diameter filaments. Here, we provide an extended theoretical and computational framework to explain the currently available experimental data for various species on the basis of a unique, minimal model of decondensing chromosomes: a kinkable, topologically constraint, semiflexible polymer with the (FISH) Kuhn length of l_K = 300 nm, 10 kinks per Mbp, and a contact distance of 45 nm. In particular: 1), we reconsider looping of finite-diameter filaments on the basis of an analytical approximation (novel, to our knowledge) of the wormlike chain radial density and show that unphysically large contact radii would be required to explain the 3C data based on the FISH estimate of the fiber stiffness; 2), we demonstrate that the observed interaction frequencies at short genomic lengths can be explained by the presence of a low concentration of curvature defects (kinks); and 3), we show that the most recent experimental 3C data for human chromosomes are in quantitative agreement with interaction frequencies extracted from our simulations of topologically confined model chromosomes.     

Nucleoid Condensation and Cell Division in Escherichia coli MX74T2 ts52 After Inhibition of Protein Synthesis

The reorganization of the bacterial nucleoid of an Escherichia coli mutant, MX74T2 ts52, was studied by electron microscopy after protein synthesis inhibition by using whole mounts of cell ghosts, ultrathin-sectioning, and freeze-etching. The bacterial nucleoid showed two morphological changes after chloramphenicol addition: deoxyribonucleic acid (DNA) localization and DNA condensation. DNA localization was observed 10 min after chloramphenicol addition; the DNA appeared as a compact, solid mass. DNA condensation was observed at 25 min; the nucleoid appeared as a cytoplasm-filled sphere, often opened at one end. Ribosomes were observed in the center. Giant nucleoids present in some mutant filaments showed fused, spherical nucleoids arranged linearly, suggesting that the tertiary structure of the nucleoid reflects the number of replicated genomes. Inhibitors which directly or indirectly blocked protein synthesis and caused DNA condensation were chloramphenicol, puromycin, amino acid starvation, rifampicin, or carbonyl cyanide m-chlorophenyl hydrazone. All inhibitors that caused cell division in the mutant also caused condensation, although some inhibitors caused condensation without cell division. Nucleoid condensation appears to be related to chromosome structure rather than to DNA segregation upon cell division.     

Efficient supercoiling of DNA by a single condensin complex as revealed by electron spectroscopic imaging

Condensin, a five-subunit protein complex essential for mitotic chromosome condensation from yeast to humans, introduces positive supercoils into DNA in an ATP-dependent manner in vitro. We report here the direct visualization of this supercoiling reaction by electron spectroscopic imaging. In the presence of ATP, a single condensin complex is capable of introducing two or more compensatory supercoils into the protein-free region of a closed circular DNA. Within the condensin-bound region, approximately 190 bp of DNA is organized into a compact structure with two distinct domains, indicative of the formation of two oriented gyres. The current results suggest that the action of condensin is more dynamic and more efficient than that postulated before, providing fundamental insight into the energy-dependent mechanism of higher order chromatin folding.