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Ultrastructural studies of the nucleoids of the pleomorphic forms of Chlamydia psittaci 6BC: a comparison with bacteria.

The nucleoids of the various pleomorphic forms of Chlamydia psittaci have been examined by direct observation of infected cells and by observations on isolated particles. The fixation and staining methods used were the same as those routinely used for the examination of bacteria to facilitate the comparison of chlamydial fine structure with that of bacteria. The nucleoids of reticulate bodies were composed of fine fibrils which extended throughout these particles. The nucleoids of intermediate bodies are characterized by an electron-dense mass with which the fibrous elements are associated in a structurally coherent manner. As condensation of the intermediate bodies proceeds, the electron-dense mass becomes eccentrically located and the fibers form a distinct radiating structure. Large elementary bodies have a few fibers associated with their condensed electron-dense nucleoids but the more condensed mature elementary bodies have a very discrete and homogeneous electron-dense nucleoid which is separated from the cytoplasmic elements of these particles by a very distinct electron-transparent space. These highly condensed elementary body nucleoids are usually ovoid, but may be elongated or irregular, and a small number of these structures react very strongly with ruthenium red. While the nucleoid structure of reticulate bodies resembles that of the bacterial cell, both the condensation process and the nucleoid morphologies which result from it in intermediate and elementary bodies have no parallels among the bacteria. Thus we conclude that major differences in nucleoid organization exist between the chlamydia and the bacteria.

Bacteria

The Absence of E. coli Nucleoid-Associated Protein FIS at Low Temperature Leads to an Adaptation Response That Causes a Shift Towards Genome Compaction in Small Rods.

In contrast to the rod shape at 37°C, the morphology of Escherichia coli cells at temperatures just above the minimum temperature of growth is small rods. A study was initiated to determine the requirement of nucleoid-associated protein FIS for growth and genome compaction in the small rods at low temperature. Growth and nucleoid staining analyses revealed that the fis null mutant displayed decreased growth and initially formed filaments containing decondensed nucleoids at 12°C, indicating that FIS facilitates production of small rods with condensed nucleoids at low temperature. However, characterized by biphasic growth at low temperature, the fis null mutant exhibited increased growth, cell division, and nucleoid condensation following an acclimation phase. Therefore, the absence of FIS with nucleoid decondensation leads to an adaptation mechanism, termed FIS Null Adaptation Response, that causes a shift towards nucleoid condensation resulting in genome compaction in small rods. Furthermore, overproduction of the HsIVU protease suppressed the cold-sensitive phenotypes of the fis null mutant indicating that degradation of a natural substrate of the protease alleviates the requirement of FIS at low temperature. In addition, null mutations of genes encoding natural substrates of HsIVU (exoribonuclease RNAse R, and cell division inhibitor SulA) were identified as extragenic suppressors of the fis null mutation.

Escherichia coli

The Lrs14 family of DNA-binding proteins as nucleoid-associated proteins in the Crenarchaeal order Sulfolobales.

Organization of archaeal chromatin combines bacterial, eukaryotic, and unique characteristics. Many archaeal lineages harbor a wide diversity of small and highly expressed nucleoid-associated proteins, which are involved in DNA structuring. In Sulfolobales, representing model organisms within the Crenarchaeota, Sul7d, Cren7, Sul10a, and Sul12a are well-characterized nucleoid-associated proteins. Here, we combine evidence that the Lrs14 family of DNA binders is part of the repertoire of nucleoid-associated proteins in Sulfolobales. Lrs14-encoding genes are widespread within genomes of different members of the Sulfolobales, typically encoded as four to nine homologs per genome. The Lrs14 proteins harbor a winged helix-turn-helix DNA-binding domain and are typified by a coiled-coil dimerization. They are characterized by distinct sequence- and structure-based features, including redox-sensitive motifs and residues targeted for posttranslational modification, allowing a further classification of the family into five conserved clusters. Lrs14-like proteins have unique DNA-organizing properties. By binding to the DNA nonsequence specifically and in a highly cooperative manner, with a slight preference for AT-rich promoter regions, they introduce DNA kinks and are able to affect transcription of adjacent transcription units either positively or negatively. Genes encoding Lrs14-type proteins display considerable differential expression themselves in response to various stress conditions, with certain homologs being specific to a particular stressor. Taken together, we postulate that members of the Lrs14 family can be considered nucleoid-associated proteins in Sulfolobales, combining a DNA-structuring role with a global gene expression role in response to stress conditions.

DNA-Binding Proteins

The small bacterial membrane protein YohP induces nucleoid condensation in E. coli and inhibits oligomerization of antimicrobial peptides.

Prokaryotic organisms execute multiple stress response mechanisms in order to cope with rapidly changing environments. Some mechanisms respond to specific cues, such as the OxyR-dependent response to hydrogen peroxide or the SOS-response that is induced upon DNA-damage. These specific responses complement general mechanisms that respond to multiple and diverse stressors. One example is nucleoid condensation, which is a rapid and effective mechanism for genome protection and observed in response to various stresses, including entry into stationary phase. Recently, the upregulation of small membrane proteins (SMPs) in response to stress was observed, but details on how this emerging class of proteins modulate the stress response is largely unknown. Here, we demonstrate that the production of two SMPs, YohP and YncL, cause nucleoid condensation in Escherichia coli. Nucleoid condensation is the result of YohP-/YncL-induced sublethal membrane depolarization, which induces the phage-shock response and leads to a reduction of global protein synthesis. YohP production also prevents the oligomerization of the antimicrobial peptide magainin-2 in the E. coli membrane and reduces the metabolic activity of E. coli cells. Thus, the synthesis of YohP and likely of other SMPs potentially protects bacterial cells against some unfavorable conditions by shifting them into a metabolically silent state.

YncL

Completed Bacillus subtilis nucleoid as a doublet structure.

When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-1, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)-less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were foud to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.

Bacillus subtilis

Caulobacter crescentus nucleoid: analysis of sedimentation behavior and protein composition during the cell cycle.

The envelope-associated nucleoid (EAN) of Caulobacter crescentus has been isolated during major developmental stages in the cell cycle. The sedimentation coefficient of the Caulobacter EAN changes as a function of development and is closely correlated with the periodicity of DNA synthesis in this bacterium. The contribution of proteins to the structure of the Caulobacter nucleoid has been analyzed by using polyacrylamide gel electrophoresis of proteins labeled both for short intervals and continuously throughout the cell cycle. The EAN proteins are derived primarily from membranes and are sequentially associated with the EAN during the cell cycle. Several distinct proteins are isolated with the EAN at specific stages of differentiation. A 26,000 Mr protein, which appears uniquely associated with the swarmer cell EANs, was identified and shown to segregate preferentially with the swarmer cell EAN at cell division.

Bacteria

Polyphosphate acts as an architectural regulator of carbon fixation and nucleoid structure in cyanobacteria.

Polyphosphate (polyP) is a conserved inorganic polymer traditionally viewed as a stress-induced phosphate and energy reserve. In cyanobacteria, however, polyP granules are constitutively present and are frequently observed in close proximity to carboxysomes, the bacterial microcompartments that mediate CO2 fixation. Here, we show that polyP functions as a spatially organized regulator of the photosynthetic cytoplasm in Synechococcus elongatus. PolyP granules localize to the nucleoid and are periodically arranged along the cell axis, independent of the McdAB carboxysome positioning system. Despite this independence, polyP and carboxysomes associate non-randomly, and this association is enhanced when active carboxysome positioning by the McdAB system is disrupted. Loss of polyP synthesis leads to nucleoid expansion, an increased number of smaller carboxysomes with high mobility, and severe defects in growth under ambient CO2. Perturbation of polyP turnover further reveals structural connections to both carboxysomes and thylakoid membranes. Together, these findings identify polyP as an architectural integrator that couples chromosome organization, metabolic compartmentalization, and photosynthetic fitness.IMPORTANCEPolyphosphate (polyP) is a ubiquitous storage polymer found across all three domains of life. In bacteria, polyP is involved in virulence, energy metabolism, DNA structure, and stress. Here, we investigate the physical and functional relationships between polyP and the carboxysome-a carbon-fixing organelle that encapsulates the most abundant enzyme on Earth and is responsible for a significant amount of atmospheric CO2 sequestration. We discover that polyP is important for proper carboxysome assembly and organization in the cell and is also required for robust cell growth under ambient CO2 conditions. As carboxysomes are one class of the broader group of bacterial microcompartments, these findings have broader implications for polyP in the function and organization of the bacterial cytoplasm.

Polyphosphates

Polyphosphate acts as an architectural regulator of carbon fixation and nucleoid structure in cyanobacteria.

Polyphosphate (polyP) is a conserved inorganic polymer traditionally viewed as a stress-induced phosphate and energy reserve. In cyanobacteria, however, polyP granules are constitutively present and frequently observed in proximity to carboxysomes, the bacterial microcompartments that mediate CO2 fixation. Here we show that polyP functions as a spatially organized regulator of the photosynthetic cytoplasm in Synechococcus elongatus. PolyP granules localize to the nucleoid and are periodically arranged along the cell axis, independently of the McdAB carboxysome positioning system. Despite this independence, polyP and carboxysomes associate non-randomly, and this association is enhanced when active carboxysome positioning by the McdAB system is disrupted. Loss of polyP synthesis leads to nucleoid expansion, an increased number of smaller carboxysomes with high mobility, and severe defects in growth under ambient CO2. Perturbation of polyP turnover further reveals structural connections to both carboxysomes and thylakoid membranes. Together, these findings identify polyP as an architectural integrator that couples chromosome organization, metabolic compartmentalization, and photosynthetic fitness.

Journal Article

Identification, characterization and classification of prokaryotic nucleoid-associated proteins.

Common throughout life is the need to compact and organize the genome. Possible mechanisms involved in this process include supercoiling, phase separation, charge neutralization, macromolecular crowding, and nucleoid-associated proteins (NAPs). NAPs are special in that they can organize the genome at multiple length scales, and thus are often considered as the architects of the genome. NAPs shape the genome by either bending DNA, wrapping DNA, bridging DNA, or forming nucleoprotein filaments on the DNA. In this mini-review, we discuss recent advancements of unique NAPs with differing architectural properties across the tree of life, including NAPs from bacteria, archaea, and viruses. To help the characterization of NAPs from the ever-increasing number of metagenomes, we recommend a set of cheap and simple in vitro biochemical assays that give unambiguous insights into the architectural properties of NAPs. Finally, we highlight and showcase the usefulness of AlphaFold in the characterization of novel NAPs.

Archaea

Post-translational modifications of the nucleoid protein H-NS: sites, mechanisms, and regulatory cues.

Histone-like nucleoid structuring protein H-NS plays a pivotal role in orchestrating bacterial chromatin and regulating horizontal gene transfer (HGT) elements. In response to environmental signals, H-NS undergoes dynamic post-translational modifications (PTMs) that resemble the epigenetic codes of eukaryotic histones. This review explores how environmental cues regulate PTMs at specific sites within distinct domains of H-NS, thereby modulating its oligomerization and DNA-binding capabilities to reprogram bacterial responses. Notably, HGT elements commonly encode counter-silencing factors, including PTM-modifying enzymes, that counteract H-NS repression. We propose that combinatorial PTM patterns on H-NS form the bacterial histone-like epigenetic code, regulating the expression of HGT elements. Collectively, these interactions establish a sophisticated network of silencing and counter-silencing mechanisms that drive bacterial genome evolution.

Protein Processing, Post-Translational

Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid.

Most, if not all, host RNA synthesis was shut off after infection of Escherichia coli strain B/5 with a bacteriophage T4 multiple mutant defective in the abilities to induce (i) unfolding of the host nucleoid (unf-), (ii) nuclear disruption (ndd-), and (iii) host DNA degradation (denA-, denB-). The shutoff of host RNA synthesis and turn-on of phage RNA synthesis were slower after infection of E. coli with unf- phage than after infection with unf+ phage. This delay in the switchover from host RNA synthesis to phage RNA synthesis in unf- infections did not result in a measurable delay in the onset of nuclear disruption, deoxyribonucleoside monophosphate kinase synthesis, or DNA synthesis. unf39 did not complement alc (allows late transcription on cytosine-containing DNA) mutants, supporting the proposal of Sirotkin et al. [Nature (London) 265:28-32, 1977] that alc and unf are possibly the same gene.

Cell Nucleus

Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.

Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.

Binding Sites

Use of the fluorochrome 4'6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA.

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.

Acetabularia

Membrane-bound mitochondrial DNA: isolation, transcription and protein composition.

Mitochondrial membrane-bound DNA complex from bovine heart mitochondria lysed in the presence of Triton X-100 was isolated by differential centrifugation. The yield of "nucleoid" is about 30 microgram protein/mg mitochondrial protein. It contains about 3-5 microgram DNA/mg protein and varying amounts of RNA. The heart mitochondrial nucleoid actively synthesizes RNA. The nucleoid fraction contains about sixteen different proteins as evidenced by urea-SDS gel electrophoresis and about twenty-one proteins as evidenced by acid-urea gel electrophoresis. It appears that the nucleoid is attached to the inner membrane since it does contain cytochromes.

Animals

TFAM organizes DNA into compact higher order structures.

TFAM (Transcription Factor A, Mitochondrial) is an essential human protein that plays two key roles in mitochondrial DNA (mtDNA) homeostasis. TFAM acts as a transcription factor that specifically binds to promoter regions, but it is also solely responsible for organizing mtDNA into nucleoids by nonspecifically covering the entire genome. Many studies have addressed TFAM in transcription regulation, but its role as a genome organizing entity is not well characterized. The current understanding of how TFAM compacts DNA into nucleoids is based on crystal structures of a TFAM monomer bound to short fragments of DNA (22-28 bp). However, this does not adequately reflect the biological role of TFAM in organizing the nucleoid where multiple TFAM molecules oligomerize on the 16.5 kb genome to form the nucleoid. Here, we present a biochemical and structural analysis of TFAM oligomerization on longer DNA. Our results show that TFAM compacts longer segments of DNA into higher order complexes that are homogenous yet exhibit continuous conformational dynamics.

Major classification: Biological sciences

Rok from B. subtilis: Bridging genome structure and transcription regulation.

Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors. In this review, we provide arguments supporting this view using the Gram-positive bacterium Bacillus subtilis as a model. Proteins BsSMC, HBsu and Rok all impact the structure of the B. subtilis chromosome. Particularly for Rok, there is compelling evidence that it combines its structural function with a role as global gene regulator. Many studies describe either function of Rok, but rarely both are addressed at the same time. Here, we review both sides of the coin and integrate them into one model. Rok forms unusually stable DNA-DNA bridges and this ability likely underlies its repressive effect on transcription by either preventing RNA polymerase from binding to DNA or trapping it inside DNA loops. Partner proteins are needed to change or relieve Rok-mediated gene repression. Lastly, we investigate which features characterize H-NS-like proteins, a family that, at present, lacks a clear definition.

Bacillus subtilis