Nucleoid Organization Research Articles

Nucleoid organization in the radioresistant bacterium Deinococcus radiodurans.

Processes favoring the exceptional resistance to genotoxic stress of Deinococcus radiodurans are not yet completely characterized. It was postulated that its nucleoid and chromosome(s) organization could participate in the DNA double strand break repair process. Here, we investigated the organization of chromosome 1 by localization of three chromosomal loci including oriC, Ter and a locus located in its left arm. For this purpose, we used a ParB-parS system to visualize the position of the loci before and after exposure to γ-rays. By comparing the number of fluorescent foci with the number of copies of the studied loci present in the cells measured by quantitative polymerase chain reaction (qPCR), we demonstrated that the 4-10 copies of chromosome 1 per cell are dispersed within the nucleoid before irradiation, indicating that the chromosome copies are not prealigned. Chromosome segregation is progressive but not co-ordinated, allowing each locus to be paired with its sister during part of the cell cycle. After irradiation, the nucleoid organization is modified, involving a transient alignment of the loci in the late stage of DNA repair and a delay of segregation of the Ter locus. We discuss how these events can influence DNA double strand break repair.

Integrating small molecule signalling and H-NS antagonism in Vibrio cholerae, a bacterium with two chromosomes.

H-NS is a well-established silencer of virulence gene transcription in the human pathogen Vibrio cholerae. Biofilm formation aids V. cholerae in colonizing both its host and its external environments, and H-NS silences biofilm gene expression. Cyclic-di-guanosine monophosphate acts through the DNA binding proteins VpsR and VpsT to overcome H-NS-mediated repression of biofilm genes, driving a transition between a planktonic and a colonial/biofilm lifestyle. The H-NS binding pattern has now been charted on both chromosomes in V. cholerae, but whether or not this abundant DNA-binding-and-bridging protein plays any roles in nucleoid organization in this bacterium remains an open question.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction.

The annotated whole-genome sequence of Mycobacterium tuberculosis indicated that Rv1388 (Mtihf) likely encodes a putative 20 kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or organization of mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF-duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg170, Arg171, and Arg173, which might be involved in DNA binding, and a conserved proline (P150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methylmethanesulfonate could be complemented with the wild-type Mtihf, but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, bind with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes compaction of DNA into nucleoid-like or higher-order filamentous structures. We hence propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Transcriptional Repressor TrmBL2 from Thermococcus kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt- and DNA Supercoiling-dependent Manner

Architectural DNA proteins play important roles in the chromosomal DNA organization and global gene regulation in living cells. However, physiological functions of some DNA-binding proteins from archaea remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon Thermococcus kodakarensis. Although histones and Alba proteins have been previously characterized, the DNA binding properties of TrmBL2 and its interplay with the other major architectural proteins in the chromosomal DNA organization and gene transcription regulation remain largely unexplored. Here, we report single-DNA studies showing that at low ionic strength (<300 mM KCl), TrmBL2 binds to DNA largely in non-sequence-specific manner with positive cooperativity, resulting in formation of stiff nucleoprotein filamentous patches, whereas at high ionic strength (>300 mM KCl) TrmBL2 switches to more sequence-specific interaction, suggesting the presence of high affinity TrmBL2-filament nucleation sites. Furthermore, in vitro assays indicate the existence of DNA binding competition between TrmBL2 and archaeal histones B from T. kodakarensis, which can be strongly modulated by DNA supercoiling and ionic strength of surrounding solution. Overall, these results advance our understanding of TrmBL2 DNA binding properties and provide important insights into potential functions of architectural proteins in nucleoid organization and gene regulation in T. kodakarensis.

The nucleoid as a smart polymer.

The nucleoid as a smart polymer.

The interplay between nucleoid organization and transcription in archaeal genomes.

The archaeal genome is organized by either eukaryotic-like histone proteins or bacterial-like nucleoid-associated proteins. Recent studies have revealed novel insights into chromatin dynamics and their effect on gene expression in archaeal model organisms. In this Progress article, we discuss the interplay between chromatin proteins, such as histones and Alba, and components of the basal transcription machinery, as well as between chromatin structure and gene-specific transcription factors in archaea. Such an interplay suggests that chromatin might have a role in regulating gene expression on both a global and a gene-specific level. Moreover, several archaeal transcription factors combine a global gene regulatory role with an architectural role, thus contributing to chromatin organization and compaction, as well as gene expression. We describe the emerging principles underlying how these factors cooperate in nucleoid structuring and gene regulation.

TrmBL2 Protein from Thermococcus Kodakarensis Competes with Histones for DNA Binding and Forms Filamentous Nucleoprotein Complexes that Affect DNA Structural State

Architectural DNA proteins play important roles in the genome organization and maintenance of its functionality in living cells. During past years DNA binding properties of different architectural proteins from bacterial and eukaryotic cells were extensively investigated. However, physiological functions of some DNA-architectural proteins from archaeal cells still remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon T. kodakarensis. While histones and Alba proteins have been characterized in detail, the DNA-binding properties of TrmBL2 remain largely unexplored. Here, we report single-DNA studies showing that TrmBL2 binds to DNA with positive cooperativity, resulting in formation of rigid nucleoprotein filaments. Our results indicate that polymerization of such filaments on DNA reduces with increasing ionic strength of solution due to the electrostatic screening of TrmBL2 non-specific binding to DNA, which is nearly abolished at > 300 mM KCl. Yet, patches of DNA covered by TrmBL2 can be seen on AFM images even at large salt concentrations, suggesting presence of high affinity TrmBL2-filament nucleation DNA sequences. Further DNA footprint analysis revealed that TrmBL2 preferentially interacts with G/C-rich sequences, similar to archaeal histones, thus, suggesting existence of TrmBL2-histone binding competition to overlapping DNA segments. This observation is supported by competitive binding assay, showing that TrmBL2 and archaeal histones indeed mutually occlude each other for DNA binding. Finally, DNA twisting experiments showed that TrmBL2-filaments weaken formation of positively supercoiled plectonemes during DNA winding and promote DNA transition into negatively supercoiled Z/L-state during unwinding. Taken together, these results advance our understanding of TrmBL2 DNA-binding properties and provide important insights into its potential functions in nucleoid organization and gene regulation in hyperthermophilic euryarchaea cells.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Investigating the Interplay between Nucleoid-Associated Proteins, DNA Curvature, and CRISPR Elements Using Comparative Genomics

Many prokaryotic and eukaryotic genomes feature a characteristic periodic signal in distribution of short runs of A or T (A-tracts) phased with the DNA helical period of ∼10–11 bp. Such periodic spacing of A-tracts has been associated with intrinsic DNA curvature. In eukaryotes, this periodicity is a major component of the nucleosome positioning signal but its physiological role in prokaryotes is not clear. One hypothesis centers on possible role of intrinsic DNA bends in nucleoid compaction. We use comparative genomics to investigate possible relationship between the A-tract periodicity and nucleoid-associated proteins in prokaryotes. We found that genomes with DNA-bridging proteins tend to exhibit stronger A-tract periodicity, presumably indicative of more prevalent intrinsic DNA curvature. A weaker relationship was detected for nucleoid-associated proteins that do not form DNA bridges. We consider these results an indication that intrinsic DNA curvature acts collaboratively with DNA-bridging proteins in maintaining the compact structure of the nucleoid, and that previously observed differences among prokaryotic genomes in terms DNA curvature-related sequence periodicity may reflect differences in nucleoid organization. We subsequently investigated the relationship between A-tract periodicity and presence of CRISPR elements and we found that genomes with CRISPR tend to have stronger A-tract periodicity. This result is consistent with our earlier hypothesis that extensive A-tract periodicity could help protect the chromosome against integration of prophages, possibly due to its role in compaction of the nucleoid.

Bacterial nucleoid structure probed by active drag and resistive pulse sensing

Recent biophysical approaches have provided key insights into the enthalpic and entropic forces that compact the nucleoid in the cell. Our biophysical approach combines two complementary, non-invasive and label-free techniques: a precisely timed steerable optical trap and a high throughput microcapillary Coulter counter. We demonstrate the ability of the latter technique to probe the physical properties and size of many purified nucleoids, at the individual nucleoid level. The DNA-binding protein H-NS is central to the organization of the bacterial genome. Our results show that nucleoids purified from the Δhns strain in the stationary phase expand approximately five fold more than the form observed in WT bacteria. This compaction is consistent with the role played by H-NS in regulating the nucleoid structure and the significant organizational changes that occur as the cell adapts to the stationary phase. We also study the permeability to the flow of ions and find that in the experiment nucleoids behave as solid colloids.

Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging

The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

Role of RNA polymerase and transcription in the organization of the bacterial nucleoid.

One of the major structural differences that provides a taxonomic distinction between eukaryotic and prokaryotic organisms is that prokaryotes lack the nuclear envelope, with exceptions such as planctomycetes 1, and genomic organization mechanisms found in eukaryotes. Instead, the prokaryotic genome is partitioned into a somewhat amorphous region of the cell known as the nucleoid 2–4. In both prokaryotes and eukaryotes, the genome must be compacted to fit into its allotted space while maintaining a level of organization that allows efficient functionality. In eukaryotes, this is accomplished by well-defined histone proteins that form nucleosomal and chromatin structures and yet can be remodeled to allow for the decondensation critical for gene expression 5–9. Although the bacterial genome must also be densely compacted, more than one-thousand-fold, and also organized for optimum functionality (chromosome replication/segregation, recombination and transcription) 10–14, the mechanisms by which this is accomplished remain unclear. The lack of “beads-on-a-string” morphology indicates different and/or multiple roles for nucleoid-associated proteins. Since transcription is a major cellular function and involves protein-DNA interaction and DNA topological domain structures, the transcription machinery themselves may play a role in nucleoid organization. Conversely, the nucleoid structure and organization may influence gene expression in a manner analogous to chromatin decondensation in eukaryotes. The interactions involved in nucleoid structure and gene expression continue to be challenging issues in the post-genomic era. Escherichia coli is a preferred model system for prokaryotic research due to the extensive studies into its genetic, physiology, biochemistry, and molecular biology mechanisms 15. The study of nucleoid organization is intrinsically difficult mainly due to the small size of bacteria in comparison to the limits of optical resolution of conventional instrumentation. Until very recently, images of the nucleoid have not changed much over the last four decades and reveal no structural details 16–18. Consequently, most of the earlier studies were focused on the biochemical properties and morphologies of isolated nucleoids and electron microscopic images after fixation 3–4,19–24. However, any preparation or fixation procedure will potentially introduce different organizations of the nucleoids or even artifacts. For example, different fixation procedures produced different nucleoid shapes of electron microscopic images 3. Additionally, growing evidence indicates that the organization of the nucleoid in the cell is plastic and sensitive to changes in growth and stress. The challenge of the research is to capture the “true” organization of the nucleoid reflecting the dynamic states of the nucleoid in living cells. Extensive studies have primarily emphasized the “histone-like” proteins including FIS, HU, H-NS, and IHF for their architectural roles in bending, looping, bridging and compacting DNA 25–36. The first three and RNA polymerase (RNAP) are the major nucleoid-associated proteins (NAPs) isolated from exponentially growing cells. Compared to the “histone-like” proteins, the binding of RNAP to DNA is much stronger. A prevailing view is that bacterial “histone-like proteins” are primarily responsible for the nucleoid organization and compaction in growing cells. Recent advances in the cell biology of E. coli RNAP and the nucleoid have shown that the distribution of RNAP, which is coupled to cell growth, plays an important role in the nucleoid dynamic structure. Emerging evidence indicates that formation of the transcription foci centered at the putative nucleolus structure is critical in nucleoid remodeling and influencing global gene expression. As other recent comprehensive reviews have dealt with the roles of other factors in bacterial nucleoid organization 37–43, this review provides an alternative and/or complementary perspective to the traditional views on bacterial nucleoid compaction and expansion.

Atomic Force Microscopy Analysis of the Role of Major DNA-Binding Proteins in Organization of the Nucleoid in Escherichia coli

Bacterial genomic DNA is packed within the nucleoid of the cell along with various proteins and RNAs. We previously showed that the nucleoid in log phase cells consist of fibrous structures with diameters ranging from 30 to 80 nm, and that these structures, upon RNase A treatment, are converted into homogeneous thinner fibers with diameter of 10 nm. In this study, we investigated the role of major DNA-binding proteins in nucleoid organization by analyzing the nucleoid of mutant Escherichia coli strains lacking HU, IHF, H–NS, StpA, Fis, or Hfq using atomic force microscopy. Deletion of particular DNA-binding protein genes altered the nucleoid structure in different ways, but did not release the naked DNA even after the treatment with RNase A. This suggests that major DNA-binding proteins are involved in the formation of higher order structure once 10-nm fiber structure is built up from naked DNA.

Genome conformation capture reveals that the Escherichia coli chromosome is organized by replication and transcription

To fit within the confines of the cell, bacterial chromosomes are highly condensed into a structure called the nucleoid. Despite the high degree of compaction in the nucleoid, the genome remains accessible to essential biological processes, such as replication and transcription. Here, we present the first high-resolution chromosome conformation capture-based molecular analysis of the spatial organization of the Escherichia coli nucleoid during rapid growth in rich medium and following an induced amino acid starvation that promotes the stringent response. Our analyses identify the presence of origin and terminus domains in exponentially growing cells. Moreover, we observe an increased number of interactions within the origin domain and significant clustering of SeqA-binding sequences, suggesting a role for SeqA in clustering of newly replicated chromosomes. By contrast, ‘histone-like’ protein (i.e. Fis, IHF and H-NS) -binding sites did not cluster, and their role in global nucleoid organization does not manifest through the mediation of chromosomal contacts. Finally, genes that were downregulated after induction of the stringent response were spatially clustered, indicating that transcription in E. coli occurs at transcription foci.

Four-Dimensional Imaging of E. coli Nucleoid Organization and Dynamics in Living Cells

Visualization of living E. coli nucleoids, defined by HupA-mCherry, reveals a discrete, dynamic helical ellipsoid. Three basic features emerge. (1) Nucleoid density coalesces into longitudinal bundles, giving a stiff, low-DNA-density ellipsoid. (2) This ellipsoid is radially confined within the cell cylinder. Radial confinement gives helical shape and directs global nucleoid dynamics, including sister segregation. (3) Longitudinal density waves flux back and forth along the nucleoid, with 5%-10% of density shifting within 5 s, enhancing internal nucleoid mobility. Furthermore, sisters separate end-to-end in sequential discontinuous pulses, each elongating the nucleoid by 5%-15%. Pulses occur at 20 min intervals, at defined cell-cycle times. This progression includes sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intranucleoid mechanical stress. These effects could comprise a chromosome-based cell-cycle engine. Overall, the presented results suggest a general conceptual framework for bacterial nucleoid morphogenesis and dynamics.

The Escherichia coli SMC Complex, MukBEF, Shapes Nucleoid Organization Independently of DNA Replication

SMC (structural maintenance of chromosomes) complexes function ubiquitously in organizing and maintaining chromosomes. Functional fluorescent derivatives of the Escherichia coli SMC complex, MukBEF, form foci that associate with the replication origin region (ori). MukBEF impairment results in mispositioning of ori and other loci in steady-state cells. These observations led to an earlier proposal that MukBEF positions new replicated sister oris. We show here that MukBEF generates and maintains the cellular positioning of chromosome loci independently of DNA replication. Rapid impairment of MukBEF function by depleting a Muk component in the absence of DNA replication leads to loss of MukBEF foci as well as mispositioning of ori and other loci, while rapid Muk synthesis leads to rapid MukBEF focus formation but slow restoration of normal chromosomal locus positioning.

NuST: analysis of the interplay between nucleoid organization and gene expression

Different experimental results suggest the presence of an interplay between global transcriptional regulation and chromosome spatial organization in bacteria. The identification and clear visualization of spatial clusters of contiguous genes targeted by specific DNA-binding proteins or sensitive to nucleoid perturbations can elucidate links between nucleoid structure and gene expression patterns. Similarly, statistical analysis to assess correlations between results from independent experiments can provide the integrated analysis needed in this line of research. NuST (Nucleoid Survey tools), based on the Escherichia coli genome, gives the non-expert the possibility to analyze the aggregation of genes or loci sets along the genome coordinate, at different scales of observation. It is useful to discover correlations between different sources of data (e.g. expression, binding or genomic data) and genome organization. A user can use it on datasets in the form of gene lists coming from his/her own experiments or bioinformatic analyses, but also make use of the internal database, which collects data from many published studies. NuST is a web server (available at http://www.lgm.upmc.fr/nust/). The website is implemented in PHP, SQLite and Ajax, with all major browsers supported, while the core algorithms are optimized and implemented in C. NuST has an extensive help page and provides a direct visualization of results as well as different downloadable file formats. A template Perl code for automated access to the web server can be downloaded at http://www.lgm.upmc.fr/nust/downloads/, in order to allow the users to use NuST in systematic bioinformatic analyses.

Electron tomography of the nucleoid of Gemmata obscuriglobus reveals complex liquid crystalline cholesteric structure

The nucleoid of the planctomycete Gemmata obscuriglobus is unique within the Bacteria in being both highly condensed and enclosed by a double-membrane nuclear envelope, seemingly analogous to the nucleus of eukaryotes. Here we have applied electron tomography to study high-pressure frozen, cryosubstituted cells of G. obscuriglobus and found multiple nested orders of DNA organization within the condensed nucleoid structure. Detailed examination of the nucleoid revealed a series of nested arcs characteristic of liquid crystalline cholesteric DNA structure. The finest fibers were arranged in parallel concentrically in a double-twist organization. At the highest order of nucleoid organization, several of these structures come together to form the core of the G. obscuriglobus nucleoid. The complex structure of DNA within this nucleoid may have implications for understanding the evolutionary significance of compartmentalized planctomycete cells.

Nucleoid organization

Nucleoid organization

Conversion of Commensal Escherichia coli K-12 to an Invasive Form via Expression of a Mutant Histone-Like Protein

ABSTRACTThe HUαE38K, V42L mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397–16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUαE38K, V42L was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins BimEL and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUαE38K, V42L variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes.