The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin

The bacterial chromosomal DNA is folded into a compact structure called nucleoid. The shape and size of this 'body' is determined by a number of factors. Major players are DNA supercoiling, macromolecular crowding and architectural proteins, associated with the nucleoid, which are the topic of this MicroReview. Although many of these proteins were identified more than 25 years ago, the molecular mechanisms involved in the organization and compaction of DNA have only started to become clear in recent years. Many of these new insights can be attributed to the use of recently developed biophysical techniques.     

A Systematic In Vitro Study of Nucleoprotein Complexes Formed by Bacterial Nucleoid-Associated Proteins Revealing Novel Types of DNA Organization

Bacterial nucleoid is a dynamic entity that changes its three-dimensional shape and compaction depending on cellular physiology. While these changes are tightly associated with compositional alterations of abundant nucleoid-associated proteins implicated in reshaping the nucleoid, their cooperation in regular long-range DNA organization is poorly understood. In this study, we reconstitute a novel nucleoprotein structure in vitro, which is stabilized by cooperative effects of major bacterial DNA architectural proteins. While, individually, these proteins stabilize alternative DNA architectures consistent with either plectonemic or toroidal coiling of DNA, the combination of histone-like protein, histone-like nucleoid structuring protein, and integration host factor produces a conspicuous semiperiodic structure. By employing a bottom-up in vitro approach, we thus characterize a minimum set of bacterial proteins cooperating in organizing a regular DNA structure. Visualized structures suggest a mechanism for nucleation of topological transitions underlying the reshaping of DNA by bacterial nucleoid-associated proteins.     

Impact of Self-Association on the Architectural Properties of Bacterial Nucleoid Proteins

The chromosomal DNA of bacteria is folded into a compact body called the nucleoid, which is composed essentially of DNA (～80%), RNA (～10%), and a number of different proteins (～10%). These nucleoid proteins act as regulators of gene expression and influence the organization of the nucleoid by bridging, bending, or wrapping the DNA. These so-called architectural properties of nucleoid proteins are still poorly understood. For example, the reason why certain proteins compact the DNA coil in certain environments but make the DNA more rigid instead in other environments is the subject of ongoing debates. Here, we address the question of the impact of the self-association of nucleoid proteins on their architectural properties and try to determine whether differences in self-association are sufficient to induce large changes in the organization of the DNA coil. More specifically, we developed two coarse-grained models of proteins, which interact identically with the DNA but self-associate differently by forming either clusters or filaments in the absence of the DNA. We showed through Brownian dynamics simulations that self-association of the proteins dramatically increases their ability to shape the DNA coil. Moreover, we observed that cluster-forming proteins significantly compact the DNA coil (similar to the DNA-bridging mode of H-NS proteins), whereas filament-forming proteins significantly increase the stiffness of the DNA chain instead (similar to the DNA-stiffening mode of H-NS proteins). This work consequently suggests that the knowledge of the DNA-binding properties of the proteins is in itself not sufficient to understand their architectural properties. Rather, their self-association properties must also be investigated in detail because they might actually drive the formation of different DNA-protein complexes.     

Crenarchaeal chromatin proteins Cren7 and Sul7 compact DNA by inducing rigid bends

Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoid-associated proteins, which are structurally homologous, but not conserved at the sequence level. Co-crystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein–DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.     

Multiple restraints to the unfolding of spermidine nucleoids from Escherichia coli

Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at approximately 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged.     

Integration Host Factor of Mycobacterium tuberculosis,mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.     

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid

DNA binding proteins, supercoiling, macromolecular crowders, and transient DNA attachments to the cell membrane have all been implicated in the organization of the bacterial chromosome. However, it is unclear what role these factors play in compacting the bacterial DNA into a distinct organelle-like entity, the nucleoid. By analyzing the effects of osmotic shock and mechanical squeezing on Escherichia coli, we show that macromolecular crowders play a dominant role in the compaction of the DNA into the nucleoid. We find that a 30% increase in the crowder concentration from physiological levels leads to a three-fold decrease in the nucleoid's volume. The compaction is anisotropic, being higher along the long axes of the cell at low crowding levels. At higher crowding levels, the nucleoid becomes spherical, and its compressibility decreases significantly. Furthermore, we find that the compressibility of the nucleoid is not significantly affected by cell growth rates and by prior treatment with rifampicin. The latter results point out that in addition to poly ribosomes, soluble cytoplasmic proteins have a significant contribution in determining the size of the nucleoid. The contribution of poly ribosomes dominates at faster and soluble proteins at slower growth rates.     

拟核结合蛋白与细菌基因的表达调控

拟核结合蛋白是细菌遗传物质组织和基因表达调控的关键. 细菌基因组压缩为致密的拟核必需有拟核结合蛋白的支撑. 拟核结合蛋白、DNA超螺旋和大分子簇在拟核的结构形成中起到重要作用,其中拟核结合蛋白最重要.拟核结合蛋白还影响细菌DNA的复制、重组、转录和修复等多个重要生理过程.作为全局调控因子,拟核结合蛋白是调控细菌适应环境变化所需基因表达的关键. 本文总结拟核结合蛋白的结构、功能和调控,特别是其在致病与非致病分枝杆菌中的差别,为寻找新药物靶标提供线索.     

Unveiling the role of Dps in the organization of mycobacterial nucleoid

In order to preserve genetic information in stress conditions, bacterial DNA is organized into higher order nucleoid structure. In this paper, with the help of Atomic Force Microscopy, we show the different structural changes in mycobacterial nucleoid at different points of growth in the presence of different concentrations of glucose in the medium. We also observe that in Mycobacterium smegmatis, two different Dps proteins (Dps1 and Dps2) promote two types of nucleoid organizations. At the late stationary phase, under low glucose availability, Dps1 binds to DNA to form a very stable toroid structure. On the other hand, under the same condition, Dps2-DNA complex forms an incompletely condensed toroid and finally forms a further stable coral reef structure in the presence of RNA. This coral reef structure is stable in high concentration of bivalent ion like Mg(2+).     

The bacterial nucleoid: a highly organized and dynamic structure

Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.     

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.     

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.     

Application of Physical Methods to the Study of Serum Lipoprotein

Abstract

One of the primary characteristics distinguishing prokaryotic from eukaryotic cells is the absence of a nucleus with a clearly defined nuclear membrane. In prokaryotic cells the DNA is condensed into a structure called the nucleoid. This structure has also been referred to attimes as the nuclear body, prokaryotic nucleus, bacterial chromosome, folded genome, or folded bacterial chromosome. The nomenclature sometimes becomes confusing because unfolded bacterial DNA free of other components of the nucleoid has also been referred to as the bacterial chromosome. To avoid such confusion, it would be preferable to reserve the terms nucleoid or bacterial chromosome to describe the condensed prokaryotic DNA structures which have some features analogous to the eukaryotic metaphase chromosome and condensed interphase chromatin. If this convention is followed, the terms “folded chromosome” or “folded genome” become ambiguous because they could equally mean “folded nucleoid.” These latter terms will, therefore, be avoided throughout this article.     

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli.

Growing bacterial cells forming division septa have sites near the septa that are sensitive to EDTA shock. Cells treated with EDTA incorporate proteins and other molecules from the surrounding medium, probably via vesiclelike lesions at the septa that are induced by EDTA. The amount of protein taken up is proportional to the protein concentration in the permeabilization medium. Incorporated molecules equilibrate throughout the cytoplasm, and those with affinity for DNA bind to the nucleoid. Conditions that promote the viability of permeabilized cells and help to avoid otherwise irreversible effects of EDTA are defined. Procedures for selecting cells that have incorporated protein and for studying the distribution of the protein and its effects in growing-dividing cells are described. The procedure may have several applications to molecular and cellular biology; however, we describe here the localization in living cells of the histonelike protein HU. Fluorescence microscopy of cells containing different amounts of fluorescein-labeled HU (varied from approximately 10(3) to 10(5) molecules per cell) showed that the HU concentrates in the nucleoid and is uniformly distributed throughout this structure. Control experiments demonstrated that unlabeled interior parts of the nucleoid can be resolved when labeled proteins that do not bind DNA or enter the nucleoid are introduced into living cells. It was concluded that in vivo added HU binds primarily DNA and that there are no intrinsic restrictions on major regions of the nucleoid to which the added HU protein may bind.