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Dromedary camels can survive and reproduce in desert areas. The unique anatomical structure of the kidney enables the camel to prevent water loss. The present study aimed to investigate the ultrastructure of the peroxisomes in the normal kidney of the adult dromedary camel. Tissue samples were taken from the cortex and outer medulla of the kidney of eight camels. The samples were then processed for histological and ultrastructural investigations. The epithelial cells of the proximal tubules displayed peroxisomes with varying sizes and shapes. The peroxisomes were observed in either dispersed or clustered arrangement. Each peroxisome exhibited a homogenous matrix enveloped by a single membrane. Several peroxisomes exhibited one or more dark marginal plates that were always strongly associated with the smooth endoplasmic reticulum. The intensity of the peroxisomal matrix differed significantly, either within the same cell or across different cells. The intensity was light or dark, with a few peroxisomes presenting a similar intensity to that of the mitochondria. Some peroxisomes contained nucleoids within their matrix. The peroxisomes in the first and second sections of proximal convoluted tubules were scattered and primarily located in the region between the microvilli and the underlying mitochondria. The peroxisomes in the third region were abundant and frequently aggregated in clusters throughout the cytoplasm. In the fourth region, the number of peroxisomes was low. The proximal straight tubule had a limited quantity of peroxisomes. In conclusion, peroxisomes in the proximal tubule in kidney of normal dromedary camel were similar in shape and size to other mammals; however, heterogeneity exists as a result of differences in species‐specific peroxisomal proteins. Peroxisomes are suggested to be a major source of metabolic energy and act as hydrogen peroxide (H2O2) scavengers, resulting in the release of water and oxygen. [ABSTRACT FROM AUTHOR]

Mammalian mitochondrial DNA (mtDNA) is replicated and transcribed by phage-like DNA and RNA polymerases, and our understanding of these processes has progressed substantially over the last several decades. Molecular mechanisms have been elucidated by biochemistry and structural biology and essential in vivo roles established by cell biology and mouse genetics. Single molecules of mtDNA are packaged by mitochondrial transcription factor A into mitochondrial nucleoids, and their level of compaction influences the initiation of both replication and transcription. Mutations affecting the molecular machineries replicating and transcribing mtDNA are important causes of human mitochondrial disease, reflecting the critical role of the genome in oxidative phosphorylation system biogenesis. Mechanisms controlling mtDNA replication and transcription still need to be clarified, and future research in this area is likely to open novel therapeutic possibilities for treating mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The bacterial chromosome is both highly supercoiled and bound by an ensemble of proteins and RNA, causing the DNA to form a compact structure termed the nucleoid. The nucleoid serves to condense, protect, and control access to the bacterial chromosome through a variety of mechanisms that remain incompletely understood. The nucleoid is also a dynamic structure, able to change both in size and composition. The dynamic nature of the bacterial nucleoid is particularly apparent when studying the effects of various stresses on bacteria, which require cells to protect their DNA and alter patterns of transcription. Stresses can lead to large changes in the organization and composition of the nucleoid on timescales as short as a few minutes. Here, we summarize some of the recent advances in our understanding of how stress can alter the organization of bacterial chromosomes. [ABSTRACT FROM AUTHOR]

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10- fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation. [ABSTRACT FROM AUTHOR]

In the last years, plant organelles have emerged as central coordinators of responses to internal and external stimuli, which can induce stress. Mitochondria play a fundamental role as stress sensors being part of a complex communication network between the organelles and the nucleus. Among the different environmental stresses, salt stress poses a significant challenge and requires efficient signaling and protective mechanisms. By using the why2 T-DNA insertion mutant and a novel knock-out mutant prepared by CRISPR/Cas9–mediated genome editing, this study revealed that WHIRLY2 is crucial for protecting mitochondrial DNA (mtDNA) integrity during salt stress. Loss-of-function mutants show an enhanced sensitivity to salt stress. The disruption of WHIRLY2 causes the impairment of mtDNA repair that results in the accumulation of aberrant recombination products, coinciding with severe alterations in nucleoid integrity and overall mitochondria morphology besides a compromised redox-dependent response and misregulation of antioxidant enzymes. The results of this study revealed that WHIRLY2-mediated structural features in mitochondria (nucleoid compactness and cristae) are important for an effective response to salt stress. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

DNA supercoiling is central to many fundamental processes of living organisms. Its average level along the chromosome and over time reflects the dynamic equilibrium of opposite activities of topoisomerases, which are required to relax mechanical stresses that are inevitably produced during DNA replication and gene transcription. Supercoiling affects all scales of the spatio-temporal organization of bacterial DNA, from the base pair to the large scale chromosome conformation. Highlighted in vitro and in vivo in the 1960s and 1970s, respectively, the first Physical models were proposed concomitantly in order to predict the deformation properties of the double helix. About fifteen years later, polymer physics models demonstrated on larger scales the plectonemic nature and the tree-like organization of supercoiled DNA. Since then, many works have tried to establish a better understanding of the multiple structuring and physiological properties of bacterial DNA in thermodynamic equilibrium and far from equilibrium. The purpose of this essay is to address upcoming challenges by thoroughly exploring the relevance, predictive capacity, and limitations of current physical models, with a specific focus on structural properties beyond the scale of the double helix. We discuss more particularly the problem of DNA conformations, the interplay between DNA supercoiling with gene transcription and DNA replication, its role on nucleoid formation and, finally, the problem of scaling up models. Our primary objective is to foster increased collaboration between physicists and biologists. To achieve this, we have reduced the respective jargon to aminimum and we provide some explanatory background material for the two communities. [ABSTRACT FROM AUTHOR]

How to adapt to a changing environment is a fundamental, recurrent problem confronting cells. One solution is for cells to organize their constituents into a limited number of spatially extended, functionally relevant, macromolecular assemblies or hyperstructures, and then to segregate these hyperstructures asymmetrically into daughter cells. This asymmetric segregation becomes a particularly powerful way of generating a coherent phenotypic diversity when the segregation of certain hyperstructures is with only one of the parental DNA strands and when this pattern of segregation continues over successive generations. Candidate hyperstructures for such asymmetric segregation in prokaryotes include those containing the nucleoid-associated proteins (NAPs) and the topoisomerases. Another solution to the problem of creating a coherent phenotypic diversity is by creating a growth-environment-dependent gradient of supercoiling generated along the replication origin-to-terminus axis of the bacterial chromosome. This gradient is modulated by transcription, NAPs, and topoisomerases. Here, we focus primarily on two topoisomerases, TopoIV and DNA gyrase in Escherichia coli , on three of its NAPs (H-NS, HU, and IHF), and on the single-stranded binding protein, SSB. We propose that the combination of supercoiling-gradient-dependent and strand-segregation-dependent topoisomerase activities result in significant differences in the supercoiling of daughter chromosomes, and hence in the phenotypes of daughter cells. [ABSTRACT FROM AUTHOR]

Bacteria frequently store excess carbon in hydrophobic granules of polyhydroxybutyrate (PHB) that in some growth conditions can occupy most of the cytoplasmic space. Different types of proteins associate to the surface of the granules, mainly enzymes involved in the synthesis and utilization of the reserve polymer and a diverse group of proteins known as phasins. Phasins have different functions, among which are regulating the size and number of the granules, modulating the activity of the granule‐associated enzymes and helping in the distribution of the granules inside the cell. Caulobacter crescentus is an oligotrophic bacterium that shows several morphological and regulatory traits that allow it to grow in very nutrient‐diluted environments. Under these conditions, storage compounds should be particularly relevant for survival. In this work, we show an initial proteomic characterization of the PHB granules and describe a new type of phasin (PhaH) characterized by the presence of an N‐terminal hydrophobic helix followed by a helix‐hairpin‐helix (HhH) domain. The hydrophobic helix is required for maximal PHB accumulation and maintenance during the stationary phase while the HhH domain is involved in determining the size of the PHB granules and their distribution in the cell. [ABSTRACT FROM AUTHOR]

DNA supercoiling is central to many fundamental processes of living organisms. Its average level along the chromosome and over time reflects the dynamic equilibrium of opposite activities of topoisomerases, which are required to relax mechanical stresses that are inevitably produced during DNA replication and gene transcription. Supercoiling affects all scales of the spatio-temporal organization of bacterial DNA, from the base pair to the large scale chromosome conformation. Highlighted in vitro and in vivo in the 1960s and 1970s, respectively, the first physical models were proposed concomitantly in order to predict the deformation properties of the double helix. About fifteen years later, polymer physics models demonstrated on larger scales the plectonemic nature and the tree-like organization of supercoiled DNA. Since then, many works have tried to establish a better understanding of the multiple structuring and physiological properties of bacterial DNA in thermodynamic equilibrium and far from equilibrium. The purpose of this essay is to address upcoming challenges by thoroughly exploring the relevance, predictive capacity, and limitations of current physical models, with a specific focus on structural properties beyond the scale of the double helix. We discuss more particularly the problem of DNA conformations, the interplay between DNA supercoiling with gene transcription and DNA replication, its role on nucleoid formation and, finally, the problem of scaling up models. Our primary objective is to foster increased collaboration between physicists and biologists. To achieve this, we have reduced the respective jargon to a minimum and we provide some explanatory background material for the two communities.

At higher order levels chromatin is organized into loops, and this looping plays an important role in transcription regulation. In our previous works we investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) of nucleoids obtained from lysed cells. It was shown that there are three parts of DNA in nucleoids: DNA in rather small loops which migrate rapidly; DNA in the loops up to ~150 kb, the migration of which is retarded; and larger loops that cannot migrate. Here we applied, for the first time, the pulse-field electrophoresis in the comet assay. Our results show that the first rapid step during the usual comet assay can be attributed to loops on the nucleoid surface while the second slow component represents loops inside the nucleoid. [ABSTRACT FROM AUTHOR]

A recurring challenge in cell biology is to define the molecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis, and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development, with decreased chlorophyll accumulation, impaired thylakoid formation, and down-regulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species (ROS)-responsive genes were up-regulated in pgp1-2. However, ROS production was not enhanced in the mutant even under strong light, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered down-regulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor gene regulating chloroplast development, in pgp1-2 up-regulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in plastid gene expression and plant development. [ABSTRACT FROM AUTHOR]

Phase separation underlies the formation of biomolecular condensates. We hypothesize the cellular processes that occur within condensates shape their structural features. We use the example of transcription to discuss structure–function relationships in condensates. Various types of transcriptional condensates have been reported across the evolutionary spectrum in the cell nucleus as well as in mitochondrial and bacterial nucleoids. In vitro and in vivo observations suggest that transcriptional activity of condensates influences their supramolecular structure, which in turn affects their function. Condensate organization thus becomes driven by differences in miscibility among the DNA and proteins of the transcription machinery and the RNA transcripts they generate. These considerations are in line with the notion that cellular processes shape the structural properties of condensates, leading to a dynamic, mutual interplay between structure and function in the cell. [ABSTRACT FROM AUTHOR]

WHIRLIES are plant-specific proteins binding to DNA in plastids, mitochondria, and nucleus. They have been identified as significant components of nucleoids in the organelles where they regulate the structure of the nucleoids and diverse DNA-associated processes. WHIRLIES also fulfil roles in the nucleus by interacting with telomers and various transcription factors, among them members of the WRKY family. While most plants have two WHIRLY proteins, additional WHIRLY proteins evolved by gene duplication in some dicot families. All WHIRLY proteins share a conserved WHIRLY domain responsible for ssDNA binding. Structural analyses revealed that WHIRLY proteins form tetramers and higher-order complexes upon binding to DNA. An outstanding feature is the parallel localization of WHIRLY proteins in two or three cell compartments. Because they translocate from organelles to the nucleus, WHIRLY proteins are excellent candidates for transducing signals between organelles and nucleus to allow for coordinated activities of the different genomes. Developmental cues and environmental factors control the expression of WHIRLY genes. Mutants and plants with a reduced abundance of WHIRLY proteins gave insight into their multiple functionalities. In chloroplasts, a reduction of the WHIRLY level leads to changes in replication, transcription, RNA processing, and DNA repair. Furthermore, chloroplast development, ribosome formation, and photosynthesis are impaired in monocots. In mitochondria, a low level of WHIRLIES coincides with a reduced number of cristae and a low rate of respiration. The WHIRLY proteins are involved in the plants' resistance toward abiotic and biotic stress. Plants with low levels of WHIRLIES show reduced responsiveness toward diverse environmental factors, such as light and drought. Consequently, because such plants are impaired in acclimation, they accumulate reactive oxygen species under stress conditions. In contrast, several plant species overexpressing WHIRLIES were shown to have a higher resistance toward stress and pathogen attacks. By their multiple interactions with organelle proteins and nuclear transcription factors maybe a comma can be inserted here? and their participation in organelle–nucleus communication, WHIRLY proteins are proposed to serve plant development and stress resistance by coordinating processes at different levels. It is proposed that the multifunctionality of WHIRLY proteins is linked to the plasticity of land plants that develop and function in a continuously changing environment. [ABSTRACT FROM AUTHOR]

The architectural protein histone-like protein from Escherichia coli strain U93 (HU) is the most abundant bacterial DNA binding protein and highly conserved among bacteria and Apicomplexan parasites. It not only binds to double-stranded DNA (dsDNA) to maintain DNA stability but also, interacts with RNAs to regulate transcription and translation. Importantly, HU is essential to cell viability for many bacteria; hence, it is an important antibiotic target. Here, we report that Gp46 from bacteriophage SPO1 of Bacillus subtilis is an HU inhibitor whose expression prevents nucleoid segregation and causes filamentous morphology and growth defects in bacteria. We determined the solution structure of Gp46 and revealed a striking negatively charged surface. An NMR-derived structural model for the Gp46–HU complex shows that Gp46 occupies the DNA binding motif of the HU and therefore, occludes DNA binding, revealing a distinct strategy for HU inhibition. We identified the key residues responsible for the interaction that are conserved among HUs of bacteria and Apicomplexans, including clinically significant Mycobacterium tuberculosis, Acinetobacter baumannii, and Plasmodium falciparum, and confirm that Gp46 can also interact with these HUs. Our findings provide detailed insight into a mode of HU inhibition that provides a useful foundation for the development of antibacteria and antimalaria drugs. [ABSTRACT FROM AUTHOR]

WHIRLIES are plant-specific proteins binding to DNA in plastids, mitochondria, and nucleus. They have been identified as significant components of nucleoids in the organelles where they regulate the structure of the nucleoids and diverse DNA-associated processes. WHIRLIES also fulfil roles in the nucleus by interacting with telomers and various transcription factors, among them members of the WRKY family. While most plants have two WHIRLY proteins, additional WHIRLY proteins evolved by gene duplication in some dicot families. All WHIRLY proteins share a conserved WHIRLY domain responsible for ssDNA binding. Structural analyses revealed that WHIRLY proteins form tetramers and higher-order complexes upon binding to DNA. An outstanding feature is the parallel localization of WHIRLY proteins in two or three cell compartments. Because they translocate from organelles to the nucleus, WHIRLY proteins are excellent candidates for transducing signals between organelles and nucleus to allow for coordinated activities of the different genomes. Developmental cues and environmental factors control the expression of WHIRLY genes. Mutants and plants with a reduced abundance of WHIRLY proteins gave insight into their multiple functionalities. In chloroplasts, a reduction of the WHIRLY level leads to changes in replication, transcription, RNA processing, and DNA repair. Furthermore, chloroplast development, ribosome formation, and photosynthesis are impaired in monocots. In mitochondria, a low level of WHIRLIES coincides with a reduced number of cristae and a low rate of respiration. The WHIRLY proteins are involved in the plants’ resistance toward abiotic and biotic stress. Plants with low levels of WHIRLIES show reduced responsiveness toward diverse environmental factors, such as light and drought. Consequently, because such plants are impaired in acclimation, they accumulate reactive oxygen species under stress conditions. In contrast, several plant species overexpressing WHIRLIES were shown to have a higher resistance toward stress and pathogen attacks. By their multiple interactions with organelle proteins and nuclear transcription factors maybe a comma can be inserted here? and their participation in organelle–nucleus communication, WHIRLY proteins are proposed to serve plant development and stress resistance by coordinating processes at different levels. It is proposed that the multifunctionality of WHIRLY proteins is linked to the plasticity of land plants that develop and function in a continuously changing environment.

Summary: Mitochondria play critical roles in generating ATP through oxidative phosphorylation (OXPHOS) and produce both damaging and signaling reactive oxygen species (ROS). They have reduced genomes that encode essential subunits of the OXPHOS machinery. Mitochondrial Transcription tERmination Factor‐related (mTERF) proteins are involved in organelle gene expression, interacting with organellar DNA or RNA.We previously found that mutations in Arabidopsis thaliana mTERF18/SHOT1 enable plants to better tolerate heat and oxidative stresses, presumably due to low ROS production and reduced oxidative damage.Here we discover that shot1 mutants have greatly reduced OXPHOS complexes I and IV and reveal that suppressor of hot1‐4 1 (SHOT1) binds DNA and localizes to mitochondrial nucleoids, which are disrupted in shot1. Furthermore, three homologues of animal ATPase family AAA domain‐containing protein 3 (ATAD3), which is involved in mitochondrial nucleoid organization, were identified as SHOT1‐interacting proteins. Importantly, disrupting ATAD3 function disrupts nucleoids, reduces accumulation of complex I, and enhances heat tolerance, as is seen in shot1 mutants.Our data link nucleoid organization to OXPHOS biogenesis and suggest that the common defects in shot1 mutants and ATAD3‐disrupted plants lead to critical changes in mitochondrial metabolism and signaling that result in plant heat tolerance. [ABSTRACT FROM AUTHOR]

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA. [ABSTRACT FROM AUTHOR]

Despite the absence of a membrane-enclosed nucleus, the bacterial DNA is typically condensed into a compact body--the nucleoid. This compaction influences the localization and dynamics of many cellular processes including transcription, translation, and cell division. Here, we develop a model that takes into account steric interactions among the components of the Escherichia coli transcriptional-translational machinery (TTM) and out-of-equilibrium effects of messenger RNA (mRNA) transcription, translation, and degradation, to explain many observed features of the nucleoid. We show that steric effects, due to the different molecular shapes of the TTM components, are sufficient to drive equilibrium phase separation of the DNA, explaining the formation and size of the nucleoid. In addition, we show that the observed positioning of the nucleoid at midcell is due to the out-of-equilibrium process of mRNA synthesis and degradation: mRNAs apply a pressure on both sides of the nucleoid, localizing it to midcell. We demonstrate that, as the cell grows, the production of these mRNAs is responsible for the nucleoid splitting into two lobes and for their well-known positioning to 1/4 and 3/4 positions on the long cell axis. Finally, our model quantitatively accounts for the observed expansion of the nucleoid when the pool of cytoplasmic mRNAs is depleted. Overall, our study suggests that steric interactions and out-ofequilibrium effects of the TTM are key drivers of the internal spatial organization of bacterial cells. [ABSTRACT FROM AUTHOR]

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA.

Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone. An active part of phase separation, transcription zone enforces segregation of the centrally positioned information phase (the nucleoid) from the surrounding action phase (the riboid), where translation happens, protein accumulates, and metabolism occurs. I speculate that HU NAP mostly tiles up the nucleoid periphery--facilitating DNA mobility but also supporting transcription in the interphase. Besides extruding plectonemically supercoiled DNA mega-loops, condensins could compact them into solenoids of uniform rings, while HU could support rigidity and rotation of these DNA rings. The two-phase cytoplasm arrangement allows the bacterial cell to organize the central dogma activities, where (from the cell center to its periphery) DNA replicates and segregates, DNA is transcribed, nascent mRNA is handed over to ribosomes, mRNA is translated into proteins, and finally, the used mRNA is recycled into nucleotides at the inner membrane. The resulting information-action conveyor, with one activity naturally leading to the next one, explains the efficiency of prokaryotic cell design--even though its main intracellular transportation mode is free diffusion. [ABSTRACT FROM AUTHOR]

[Display omitted] • DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the Toxoplasma gondii apicoplast. • DNA Gyrase A and B and Prex are required for apicoplast genome replication and the growth of the parasite. • Apicoplast nucleoid division and segregation initiate at the beginning of the S phase and conclude during mitosis. • Replication and division of the apicoplast nucleoid is highly coordinated with nuclear genomic replication and mitosis. Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites' metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle. [ABSTRACT FROM AUTHOR]

Mitochondrial transcription termination factors (mTERFs) are highly conserved proteins in metazoans. Plants have many more mTERF proteins than animals. The functions and the underlying mechanisms of plants’ mTERFs remain largely unknown. In plants, mTERF family proteins are present in both mitochondria and plastids and are involved in gene expression in these organelles through different mechanisms. In this study, we screened Arabidopsis mutants with pigment-defective phenotypes and isolated a T-DNA insertion mutant exhibiting seedling-lethal and albino phenotypes [seedling lethal 1 (sl1)]. The SL1 gene encodes an mTERF protein localized in the chloroplast stroma. The sl1 mutant showed severe defects in chloroplast development, photosystem assembly, and the accumulation of photosynthetic proteins. Furthermore, the transcript levels of some plastid-encoded proteins were significantly reduced in the mutant, suggesting that SL1/mTERF3 may function in the chloroplast gene expression. Indeed, SL1/mTERF3 interacted with PAP12/PTAC7, PAP5/PTAC12, and PAP7/PTAC14 in the subgroup of DNA/RNA metabolism in the plastid-encoded RNA polymerase (PEP) complex. Taken together, the characterization of the plant chloroplast mTERF protein, SL1/mTERF3, that associated with PEP complex proteins provided new insights into RNA transcription in the chloroplast.

Mitochondrial transcription termination factors (mTERFs) are highly conserved proteins in metazoans. Plants have many more mTERF proteins than animals. The functions and the underlying mechanisms of plants' mTERFs remain largely unknown. In plants, mTERF family proteins are present in both mitochondria and plastids and are involved in gene expression in these organelles through different mechanisms. In this study, we screened Arabidopsis mutants with pigment-defective phenotypes and isolated a T-DNA insertion mutant exhibiting seedling-lethal and albino phenotypes [ seedling lethal 1 (sl1)]. The SL1 gene encodes an mTERF protein localized in the chloroplast stroma. The sl1 mutant showed severe defects in chloroplast development, photosystem assembly, and the accumulation of photosynthetic proteins. Furthermore, the transcript levels of some plastid-encoded proteins were significantly reduced in the mutant, suggesting that SL1/mTERF3 may function in the chloroplast gene expression. Indeed, SL1/mTERF3 interacted with PAP12/PTAC7, PAP5/PTAC12, and PAP7/PTAC14 in the subgroup of DNA/RNA metabolism in the plastid-encoded RNA polymerase (PEP) complex. Taken together, the characterization of the plant chloroplast mTERF protein, SL1/mTERF3, that associated with PEP complex proteins provided new insights into RNA transcription in the chloroplast. [ABSTRACT FROM AUTHOR]

Gemmata obscuriglobus, distinctive member of phylum planctomycetes possesses uncommon features which make it unique from other bacteria and has gained significant attention in the field of microbial evolution and ecology. Presence of cellular characteristics like intracellular compartmentalization, condensed-membrane bound nucleoid, endocytosis-like mechanism and sterol biosynthesis has made it the intermediate member of prokaryotes and eukaryotes. These features which were previously thought to be restricted for eukaryotes has raised the question about its taxonomical classification and made it the target organism for studying the evolution of eukaryotes. G. obscuriglobus shows resistance to heavy metals and can also produce antimicrobial peptides, which reveal potential applications of this organism. This review intends to highlight the cellular morphology and molecular characteristics. Additionally, the article also outlines the unique features which question its classification in detail for better understanding about this bacterium. [ABSTRACT FROM AUTHOR]

Mycoplasma gallisepticum (MG) is one of the smallest free-living and self-replicating organisms, it is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, MG has a limited variety of DNA-binding proteins and transcription factors. To investigate the dynamic changes of the proteomic profile of MG nucleoid, that may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation, a quantitative proteomic study was performed on MG nucleoids obtained from the cell culture in logarithmic and stationary phases of synchronous growth. MG cells were grown on a liquid medium with a 9 h starvation period. Nucleoids were obtained from the cell culture at the 26th and the 50th hour (logarithmic and stationary growth phases respectively) by sucrose density gradient centrifugation. LC-MS analysis was carried out on an Ultimate 3000 RSLCnano HPLC system connected to a Fusion Lumos mass spectrometer, controlled by XCalibur software (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). For comprehensive peptide library generation one sample from each biological replicate was run in DDA mode. Then, all the samples were run in a single LC-MS DIA run. Identification of DDA files and DIA quantitation was performed with MaxQuant and Skyline software, correspondingly. All raw data generated from IDA and DDA acquisitions are presented in the PRIDE database with identifier PXD019077.

Abstract WHIRLY2 is a single‐stranded DNA binding protein associated with mitochondrial nucleoids. In the why 2‐1 mutant of Arabidopsis thaliana, a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild‐type and why 2‐1 mutant cell cultures. In contrast to the development of the vegetative parts, seed germination is compromised in the why 2‐1 mutant. In line with that, the expression level of why 2 in seeds of wild‐type plants is higher than that of why 3, whereas in adult plant no difference is found. Intriguingly, in early stages of shoots development of the why 2‐1 mutant, although not in seeds, the expression level of why 3 is enhanced. These results suggest that WHIRLY3 is a potential candidate to compensate for the lack of WHIRLY2 in the why 2‐1 mutant. Such compensation is possible only if the two proteins are localized in the same organelle. Indeed, in organello protein transport experiments using intact mitochondria and chloroplasts revealed that WHIRLY3 can be dually targeted into both, chloroplasts and mitochondria. Together, these data indicate that the alterations of mitochondria nucleoids are tightly linked to alterations of mitochondria morphology and functionality. This is even more evident in those phases of plant life when mitochondrial activity is particularly high, such as seed germination. Moreover, our results indicate that the differential expression of why 2 and why 3 predetermines the functional replacement of WHIRLY2 by WHIRLY3, which is restricted though to the vegetative parts of the plant.

Every organism across the tree of life compacts and organizes its genome with architectural chromatin proteins. While eukaryotes and archaea express histone proteins, the organization of bacterial chromosomes is dependent on nucleoid-associated proteins. In Escherichia coli and other proteobacteria, the histone-like nucleoid structuring protein (H-NS) acts as a global genome organizer and gene regulator. Functional analogues of H-NS have been found in other bacterial species: MvaT in Pseudomonas species, Lsr2 in actinomycetes and Rok in Bacillus species. These proteins complement hns− phenotypes and have similar DNA-binding properties, despite their lack of sequence homology. In this review, we focus on the structural and functional characteristics of these four architectural proteins. They are able to bridge DNA duplexes, which is key to genome compaction, gene regulation and their response to changing conditions in the environment. Structurally the domain organization and charge distribution of these proteins are conserved, which we suggest is at the basis of their conserved environment responsive behaviour. These observations could be used to find and validate new members of this protein family and to predict their response to environmental changes.

Comparative structural/molecular biology by single-molecule analyses combined with single-cell dissection, mass spectroscopy, and biochemical reconstitution have been powerful tools for elucidating the mechanisms underlying genome DNA folding. All genomes in the three domains of life undergo stepwise folding from DNA to 30–40 nm fibers. Major protein players are histone (Eukarya and Archaea), Alba (Archaea), and HU (Bacteria) for fundamental structural units of the genome. In Euryarchaeota, a major archaeal phylum, either histone or HTa (the bacterial HU homolog) were found to wrap DNA. This finding divides archaea into two groups: those that use DNA-wrapping as the fundamental step in genome folding and those that do not. Archaeal transcription factor-like protein TrmBL2 has been suggested to be involved in genome folding and repression of horizontally acquired genes, similar to bacterial H-NS protein. Evolutionarily divergent SMC proteins contribute to the establishment of higher-order structures. Recent results are presented, including the use of Hi-C technology to reveal that archaeal SMC proteins are involved in higher-order genome folding, and the use of single-molecule tracking to reveal the detailed functions of bacterial and eukaryotic SMC proteins. Here, we highlight the similarities and differences in the DNA-folding mechanisms in the three domains of life.

Defects in chloroplast development are 'retrograde-signalled' to the nucleus, reducing synthesis of photosynthetic or related proteins. The Arabidopsis cue8 mutant manifests virescence, a slow-greening phenotype, and is defective at an early stage in plastid development. Greening cotyledons or early leaf cells of cue8 exhibit immature chloroplasts which fail to fill the available cellular space. Such chloroplasts show reduced expression of genes of photosynthetic function, dependent on the plastid-encoded polymerase (PEP), while the expression of genes of housekeeping function driven by the nucleusencoded polymerase (NEP) is elevated, a phenotype shared with mutants in plastid genetic functions. We attribute this phenotype to reduced expression of specific PEP-controlling sigma factors, elevated expression of RPOT (NEP) genes and maintained replication of plastid genomes (resulting in densely coalesced nucleoids in the mutant), i.e. it is due to an anterograde nucleus-to-chloroplast correction, analogous to retention of a juvenile plastid state. Mutants in plastid protein import components, particularly those involved in housekeeping protein import, also show this 'retro-anterograde' correction. Loss of CUE8 also causes changes in mRNA editing. The overall response has strong fitness value: loss of GUN1, an integrator of retrograde signalling, abolishes elements of it (albeit not others, including editing changes), causing bleaching and eventual seedling lethality upon cue8 gun1. This highlights the adaptive significance of virescence and retrograde signalling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

WHIRLY2 is a single‐stranded DNA binding protein associated with mitochondrial nucleoids. In the why 2‐1 mutant of Arabidopsis thaliana, a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild‐type and why 2‐1 mutant cell cultures. In contrast to the development of the vegetative parts, seed germination is compromised in the why 2‐1 mutant. In line with that, the expression level of why 2 in seeds of wild‐type plants is higher than that of why 3, whereas in adult plant no difference is found. Intriguingly, in early stages of shoots development of the why 2‐1 mutant, although not in seeds, the expression level of why 3 is enhanced. These results suggest that WHIRLY3 is a potential candidate to compensate for the lack of WHIRLY2 in the why 2‐1 mutant. Such compensation is possible only if the two proteins are localized in the same organelle. Indeed, in organello protein transport experiments using intact mitochondria and chloroplasts revealed that WHIRLY3 can be dually targeted into both, chloroplasts and mitochondria. Together, these data indicate that the alterations of mitochondria nucleoids are tightly linked to alterations of mitochondria morphology and functionality. This is even more evident in those phases of plant life when mitochondrial activity is particularly high, such as seed germination. Moreover, our results indicate that the differential expression of why 2 and why 3 predetermines the functional replacement of WHIRLY2 by WHIRLY3, which is restricted though to the vegetative parts of the plant. [ABSTRACT FROM AUTHOR]

The skin barrier function is mostly provided by the stratum corneum (SC), the uppermost layer of the epidermis. To noninvasively analyze the physiological properties of the skin barrier functionin vivo, it is important to determine the SC thickness. Confocal Raman microscopy (CRM) is widely used for this task. In the present in vivo study, a new method based on the determination of the DNA concentration profile using CRM is introduced for determining the SC thickness. The obtained SC thickness values are compared with those obtained using other CRM-based methods determining the water and lipid depth profiles. The obtained results show almost no significant differences in SC thickness for the utilized methods. Therefore, the results indicate that it is possible to calculate the SC thickness by using the DNA profile in the fingerprint region, which is comparable with the SC thickness calculated by the water depth profiles (ANOVA test p = 0.77) and the lipid depth profile (ANOVA test p = 0.74). This provides the possibility to measure the SC thickness by using the DNA profile, in case the water or lipid profile analyses are influenced by a topically applied formulation. The increase in DNA concentration in the superficial SC (0–2 µm) is related to the DNA presence in the microbiome of the skin, which was not present in the SC depth below 4 µm. [ABSTRACT FROM AUTHOR]

Abstract Background In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. Results We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. Conclusion Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.

In a review of the features of the bacterial cells are Corynebacterium structure: characterized by an upper layer, highly organized cell wall, cytoplasmic membrane, cytoplasm, nucleoid. Described in detail the structure of the upper layer containing pili (fimbriae), microcapsule surface proteins - PS-2, DIP1281, 67-72r protein (hemagglutinin), porins, sialidase (neuraminidase). These components are the ability to initiate a serial of Corynebacterium work with the host cell, followed by colonization. It submitted a detailed description of the structure and functions of cell wall structures - cord factor, which is a second barrier permeability; arabinogalactan, peptidoglycan, lipomannan and lipoarabinomannan. The structure and function of the cytoplasmic membrane as the main diffusion barrier cell cytoplasm and the genome of Corynebacterium. Presented different molecular genetic methods for the identification and differentiation of closely related species of Corynebacterium.

Background: Living organisms survive in constantly changing environmental conditions due to universal strategies of adaptation to various stresses based on structural, biochemical, and genetic rearrangements. One of adaptive strategies utilized in bacterial cells involves the protection of the nucleoid from unfavorable factors via binding of DNA to specific histone-like proteins. This strategy leads to the condensation of DNA in complexes with Dps (DNA-binding protein from starved cells), which has been discovered in starved for up to 48 hrs cells of the E. coli bacterium Methods: Electron microscopy and X-ray diffraction of synchrotron radiation studies were used to reveal distinct forms of nucleoid condensation in dormant E. coli cells that were starving for longer period – up to 7 months. Results: We have found and described not only previously detected forms of nucleoid condensation: quasi-nanocrystalline, quasi-liquid crystalline and spore-like, but also observed a new type of nucleoid condensation in dormant cells - folded nucleosome-like. Conclusion: According to the recognized concept of a bacterial population as a multicellular organism, their heterogeneity allows to respond flexibly to the environmental changes and to survive in stressful situations. Heterogeneity is the reason why we observed several types of nucleoid condensation in dormant E. coli cells. Heterogeneity of dormant cells increases the ability of the whole population to survive under various stress conditions. Results, observed here, shed a new light both on the phenomenon of nucleoid condensation in prokaryotic cells and on the general problem of developing a response to the stress.

Atomic force microscopy is a high-resolution imaging technique useful for observing the structures of biomolecular complexes. This approach provides a straightforward method to characterize the binding behavior of different chromatin architectural proteins and to analyze the increasingly complex structural units assembled on the DNA. The protocol describes the preparation, AFM imaging, and structural analysis of chromatin that is reconstituted in vitro using purified proteins and DNA. Here, we describe the successful application of the method on the chromatin architectural proteins of the archaeon Sulfolobus solfataricus., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The universal response of Escherichia coli to stress is enhancing the synthesis of specific histone-like Dps proteins that bind bacterial DNA. As a result, two-dimensional and three-dimensional crystalline arrays can be observed in the cytoplasm of starving bacteria. Conditions for obtaining in vitro co-crystals of DNA-Dps were selected, and their elemental composition was studied using analytical electron microscopy. It was found that Dps in the co-crystal retains its ferritin-like activity; that is, it can stimulate the oxidation of Fe2+ ions to Fe3+ and facilitate the accumulation of iron in the form of Fe2O3 in the inner cavity of the oligomer. [ABSTRACT FROM AUTHOR]

Decades of research have probed the interplay between chromatin (genomic DNA associated with proteins and RNAs) and transcription by RNA polymerase (RNAP) in all domains of life. In bacteria, chromatin is compacted into a membrane-free region known as the nucleoid that changes shape and composition depending on the bacterial state. Transcription plays a key role in both shaping the nucleoid and organizing it into domains. At the same time, chromatin impacts transcription by at least five distinct mechanisms: (i) occlusion of RNAP binding; (ii) roadblocking RNAP progression; (iii) constraining DNA topology; (iv) RNA-mediated interactions; and (v) macromolecular demixing and heterogeneity, which may generate phase-separated condensates. These mechanisms are not mutually exclusive and, in combination, mediate gene regulation. Here, we review the current understanding of these mechanisms with a focus on gene silencing by H-NS, transcription coordination by HU, and potential phase separation by Dps. The myriad questions about transcription of bacterial chromatin are increasingly answerable due to methodological advances, enabling a needed paradigm shift in the field of bacterial transcription to focus on regulation of genes in their native state. We can anticipate answers that will define how bacterial chromatin helps coordinate and dynamically regulate gene expression in changing environments. Unlabelled Image • Bacterial transcription and chromatin affect each other topologically and sterically • Bacterial chromatin can both silence (H-NS) and facilitate (HU, Fis) transcription • The complex structure and dynamic nature of bacterial chromatin complicate its study • Occlusion, roadblocking, topology, RNA & phase separation mediate chromatin effects • New methods allow a paradigm shift to study gene transcription in its native state [ABSTRACT FROM AUTHOR]

DNA replication in human mitochondria has been studied for several decades; however, its mechanism still remains unclear. During the last 15 years, many new experimental data on the mitochondrial replication have appeared, although extremely contradictory. Two asynchronous (strand displacement and RITOLS) and one synchronous (strand coupled) replication models have been proposed. In the asynchronous models, replication from the origin in the H-chain starts earlier, so that the replication of the two chains ends at different times. The synchronous model is more traditional and implies two replication forks with leading and lagging strands initiated at the same origin. For each of the three models, both confirming and contradicting experimental data exist. Most likely, there is no single model of mitochondrial replication. It is possible that the unique mitochondrial replication machinery that has originated as a results of endosymbiosis has an unexpected variety of replication strategies to maintain the mitochondrial genome. An unusual combination of enzymes of different origin (phage, bacterial, eukaryotic) and unique features of the mitochondrial genome (existance of heavy and light chains, insertions of ribonucleotides, a variety of origins) can allow replication through different mechanisms. In human mitochondria, asynchronous replication seems to dominate; however, synchronous replication is also possible under certain conditions. In the human heart mitochondria, circular mitochondrial DNA (mtDNA) molecules can rearrange in a network of rapidly replicating linear genomes, thereby suggesting possible existence of a wide range of replication mechanisms in the mitochondria. The review describes the main stages of mtDNA replication and enzymes involved in this process, as well as discusses the prospects of mitochondrial replication studies. [ABSTRACT FROM AUTHOR]

The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB’s ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.

HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.