Nucleoid

The nucleoid is an irregularly shaped region within the prokaryotic cell that contains all or most of the genetic material. The chromosome of a typical prokaryote is circular, and its length is very large compared to the cell dimensions, so it needs to be compacted in order to fit. In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a nuclear membrane. Instead, the nucleoid forms by condensation and functional arrangement with the help of chromosomal architectural proteins and RNA molecules as well as DNA supercoiling. The length of a genome widely varies and a cell may contain multiple copies of it.
There is not yet a high-resolution structure known of a bacterial nucleoid, however key features have been researched in Escherichia coli as a model organism. In E. coli, the chromosomal DNA is on average negatively supercoiled and folded into plectonemic loops, which are confined to different physical regions, and rarely diffuse into each other. These loops spatially organize into megabase-sized regions called macrodomains, within which DNA sites frequently interact, but between which interactions are rare. The condensed and spatially organized DNA forms a helical ellipsoid that is radially confined in the cell. The 3D structure of the DNA in the nucleoid appears to vary depending on conditions and is linked to gene expression so that the nucleoid architecture and gene transcription are tightly interdependent, influencing each other reciprocally.

Background

In many bacteria, the chromosome is a single covalently closed double-stranded DNA molecule that encodes the genetic information in a haploid form. The size of the DNA varies from 500,000 to several million base pairs encoding from 500 to several thousand genes depending on the organism. The chromosomal DNA is present in cells in a highly compact, organized form called the nucleoid, which is not encased by a nuclear membrane as in eukaryotic cells. The isolated nucleoid contains 80% DNA, 10% protein, and 10% RNA by weight.
The gram-negative bacterium Escherichia coli is a model system for nucleoid research into how chromosomal DNA becomes the nucleoid, the factors involved therein, what is known about its structure, and how some of the DNA structural aspects influence gene expression.
There are two essential aspects of nucleoid formation; condensation of a large DNA into a small cellular space and functional organization of DNA in a three-dimensional form. The haploid circular chromosome in E. coli consists of ~ 4.6 million bp. If DNA is relaxed in the B form, it would have a circumference of ~1.5 millimeters. However, a large DNA molecule such as the E. coli chromosomal DNA does not remain a straight rigid molecule in a suspension. Brownian motion will generate curvature and bends in DNA. The maximum length up to which a double-helical DNA remains straight by resisting the bending enforced by Brownian motion is ~50 nm or 150 bp, which is called the persistence length. Thus, pure DNA becomes substantially condensed without any additional factors; at thermal equilibrium, it assumes a random coil form. The random coil of E. coli chromosomal DNA would occupy a volume of ~ 523 μm³, calculated from the radius of gyration where a'' is the Kuhn length, and N is the number of Kuhn length segments in the DNA. Although DNA is already condensed in the random coil form, it still cannot assume the volume of the nucleoid which is less than a micron. Thus, the inherent property of DNA is not sufficient: additional factors must help condense DNA further on the order of ~10³. The second essential aspect of nucleoid formation is the functional arrangement of DNA. Chromosomal DNA is not only condensed but also functionally organized in a way that is compatible with DNA transaction processes such as replication, recombination, segregation, and transcription. Almost five decades of research beginning in 1971, has shown that the final form of the nucleoid arises from a hierarchical organization of DNA. At the smallest scale, nucleoid-associated DNA architectural proteins condense and organize DNA by bending, looping, bridging or wrapping DNA. At a larger scale, DNA forms plectonemic loops, a braided form of DNA induced by supercoiling. At the megabase scale, the plectonemic loops coalesce into six spatially organized domains, which are defined by more frequent physical interactions among DNA sites within the same macrodomain than between different macrodomains. Long- and short-range DNA-DNA connections formed within and between the macrodomains contribute to condensation and functional organization. Finally, the nucleoid is a helical ellipsoid with regions of highly condensed DNA at the longitudinal axis.

Condensation and organization

Nucleoid-associated proteins (NAPs)

In eukaryotes, genomic DNA is condensed in the form of a repeating array of DNA-protein particles called nucleosomes.
A nucleosome consists of ~146 bp of DNA wrapped around an octameric complex of the histone proteins. Although bacteria do not have histones, they possess a group of DNA binding proteins referred to as nucleoid-associated proteins that are functionally analogous to histones in a broad sense. NAPs are highly abundant and constitute a significant proportion of the protein component of nucleoid.
A distinctive characteristic of NAPs is their ability to bind DNA in both a specific and non-sequence specific manner. As a result, NAPs are dual function proteins. The specific binding of NAPs is mostly involved in gene-specific transcription, DNA replication, recombination, and repair. At the peak of their abundance, the number of molecules of many NAPs is several orders of magnitude higher than the number of specific binding sites in the genome. Therefore, it is reasoned that NAPs bind to the chromosomal DNA mostly in the non-sequence specific mode and it is this mode that is crucial for chromosome compaction. Non-sequence specific binding of a NAP may not be completely random; there could be low-sequence specificity and or structural specificity due to sequence-dependent DNA conformation or DNA conformation created by other NAPs.
Although molecular mechanisms of how NAPs condense DNA in vivo are not well understood, based on the extensive in vitro studies it appears that NAPs participate in chromosome compaction via the following mechanisms: NAPs induce and stabilize bends in DNA, thus aid in DNA condensation by reducing the persistence length. NAPs condense DNA by bridging, wrapping, and bunching that could occur between nearby DNA segments or distant DNA segments of the chromosome. Another mechanism by which NAPs participate in chromosome compaction is by constraining negative supercoils in DNA thus contributing to the topological organization of the chromosome.
There are at least 12 NAPs identified in E. coli, the most extensively studied of which are HU, IHF, H-NS, and Fis. Their abundance and DNA binding properties and effect on DNA condensation and organization are summarized in the tables below.

Protein	Molecular mass	Native functional unit	Abundance¹ in growth phase	Abundance¹ in stationary phase
HUα and HUβ	~ 9	Homo- and hetero-dimer	55,000	30,000
IHFα and IHFβ	~ 11	Heterodimer	12,000	55,000
H-NS	~ 15	Homodimer	20,000	15,000
Fis	~ 11	Homodimer	60,000	Undetectable
Dps	~ 19	Dodecamer	6,000	180,000

¹ Abundance data were taken from; The number in the parenthesis is micromolar concentration calculated using the following formula: x x 10³. Cell volume in liter was determined by assuming volume of the E. coli cell to be 2 μm³.

Protein	Binding motif	Specific DNA binding affinity¹	Random DNA binding affinity¹
HU	A structural motif defined by bends and kinks in DNA	7.5 x 10⁻⁹	4.0 x 10⁻⁷
IHF	WATCAANNNNTTR	1.5 x 10⁻⁹	1.7 x 10⁻⁶
H-NS	TCGATAAATT	10-15 x 10⁻⁹	6 x 10⁻⁸
Fis	GNTYAAAWTTTRANC	0.2-1.0 x 10⁻⁹	>8.0 x 10⁻⁶
Dps	ND	ND	1.65 x 10⁻⁷
MatP	GTGACRNYGTCAC	8.0 x 10⁻⁹	ND
MukBEF	ND	ND	ND

¹ Binding affinity refers to equilibrium dissociation constant in molar units. ND = not determined

HU

Histone-like protein from E. coli strain U93 is an evolutionarily conserved protein in bacteria. HU exists in E. coli as homo- and heterodimers of two subunits HUα and HUβ sharing 69% amino acid identity. Although it is referred to as a histone-like protein, close functional relatives of HU in eukaryotes are high-mobility group proteins, and not histones. HU is a non-sequence specific DNA binding protein. It binds with low-affinity to any linear DNA. However, it preferentially binds with high-affinity to a structurally distorted DNA. Examples of distorted DNA substrates include cruciform DNA, bulged DNA, dsDNA containing a single-stranded break such as nicks, gaps, or forks. Furthermore, HU specifically binds and stabilizes a protein-mediated DNA loop. In the structurally specific DNA binding mode, HU recognizes a common structural motif defined by bends or kinks created by distortion, whereas it binds to a linear DNA by locking the phosphate backbone. While the high-affinity structurally-specific binding is required for specialized functions of HU such as site-specific recombination, DNA repair, DNA replication initiation, and gene regulation, it appears that the low-affinity general binding is involved in DNA condensation. In chromatin-immunoprecipitation coupled with DNA sequencing, HU does not reveal any specific binding events. Instead, it displays a uniform binding across the genome presumably reflecting its mostly weak, non-sequence specific binding, thus masking the high-affinity binding in vivo.
In strains lacking HU, the nucleoid is "decondensed", consistent with a role of HU in DNA compaction. The following in vitro studies suggest possible mechanisms of how HU might condense and organize DNA in vivo. Not only HU stably binds to distorted DNA with bends, it induces flexible bends even in a linear DNA at less than 100 nM concentration. In contrast, HU shows the opposite architectural effect on DNA at higher physiologically relevant concentrations. It forms rigid nucleoprotein filaments causing the straitening of DNA and not the bending. The filaments can further form a DNA network expandable both laterally and medially because of the HU-HU multimerization triggered by the non-sequence-specific DNA binding.
How are these behaviors of HU relevant inside the cell? The formation of filaments requires high-density binding of HU on DNA, one HU dimer per 9-20 bp DNA. But there is only one HU dimer every ~150 bp of the chromosomal DNA based on the estimated abundance of 30,000 HU dimers per cell. This indicates that the flexible bends are more likely to occur in vivo. The flexible bending would cause condensation due to a reduction in the persistence length of DNA as shown by magnetic tweezers experiments, which allow studying condensation of a single DNA molecule by a DNA binding protein. However, because of the cooperativity, the rigid filaments and networks could form in some regions in the chromosome. The filament formation alone does not induce condensation, but DNA networking or bunching can substantially contribute to condensation by bringing distant or nearby chromosome segments together.