RESUMEN

DNA-protein interactions occur in biological processes such as genome replication, gene transcription, DNA repair, and chromatin compaction and organization. Mapping the distribution of the DNA-bound proteins on the chromosome is essential for understanding their associated biological process. Chromatin immunoprecipitation (ChIP) involves the antibody-mediated enrichment of DNA fragments bound by a target protein and has become one of the most powerful techniques for exploring the distribution of proteins on the chromosome. By incorporating quantitative polymerase chain reaction (qPCR) downstream of the ChIP assay, ChIP-qPCR was developed to describe binding profiles of DNA-associated proteins at a candidate locus. In this chapter, we describe ChIP-qPCR. We provide a step-by-step protocol for the preparation of a ChIP library of a 3× FLAG-tagged protein in bacteria, describe how downstream qPCR experiments can be performed with the appropriate controls, and explain how the data is analyzed. This chapter provides reliable technical guidance for ChIP-qPCR studies in bacteria.

Asunto(s)

Inmunoprecipitación de Cromatina , Inmunoprecipitación de Cromatina/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Bacterias/genética , Bacterias/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo

RESUMEN

Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E. coli. These methods include construction of genomic and plasmidic transcriptional and translational lacZ reporter gene fusions to study regulation of promoters; insertion of promoter cassettes to drive transcription into a locus of interest in the genome, for example, an H-NS-bound locus; and construction of isogenic hns and stpA mutants and precautions in doing so.

Asunto(s)

Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli , Transcripción Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Genes Reporteros , Plásmidos/genética , ADN Bacteriano/genética

RESUMEN

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.

RESUMEN

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.

Asunto(s)

ARN Polimerasas Dirigidas por ADN , Proteínas de Escherichia coli , Escherichia coli , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Elongación de la Transcripción Genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética

RESUMEN

Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in the different environmental niches they occupy. H-NS, the histone-like nucleoid structuring protein, is the best-studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently discovered HAEs encoding for phage defense systems, we show that a lack of H-NS causes a robust increase in the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity. We also identified few sites that are affected by TsrA independently of H-NS, suggesting TsrA may act with diverse regulatory mechanisms. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently discovered HAEs. IMPORTANCE: New strains of the bacterial pathogen Vibrio cholerae, bearing novel horizontally acquired elements (HAEs), frequently emerge. HAEs provide beneficial traits to the bacterium, such as antibiotic resistance and defense against invading bacteriophages. Xenogeneic silencers are proteins that help bacteria harness new HAEs and silence those HAEs until they are needed. H-NS is the best-studied xenogeneic silencer; it is one of the nucleoid-associated proteins (NAPs) in gamma-proteobacteria and is responsible for the proper regulation of HAEs within the bacterial transcriptional network. We studied the effects of H-NS and other NAPs on the HAEs of a clinical isolate of V. cholerae. Importantly, we found that H-NS partners with a small and poorly characterized protein, TsrA, to help domesticate new HAEs involved in bacterial survival and in causing disease. A proper understanding of the regulatory state in emerging isolates of V. cholerae will provide improved therapies against new isolates of the pathogen.

Asunto(s)

Proteínas Bacterianas , Cólera , Regulación Bacteriana de la Expresión Génica , Vibrio cholerae , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Vibrio cholerae/metabolismo , Cólera/microbiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Transcripción Genética , Virulencia , Factores de Virulencia/genética , Transferencia de Gen Horizontal

RESUMEN

The extremophile Deinococcus radiodurans maintains a highly organized and condensed nucleoid as its default state, possibly contributing to its high tolerance to ionizing radiation (IR). Previous studies of the D. radiodurans nucleoid were limited by reliance on manual image annotation and qualitative metrics. Here, we introduce a high-throughput approach to quantify the geometric properties of cells and nucleoids using confocal microscopy, digital reconstructions of cells, and computational modeling. We utilize this novel approach to investigate the dynamic process of nucleoid condensation in response to IR stress. Our quantitative analysis reveals that at the population level, exposure to IR induced nucleoid compaction and decreased the size of D. radiodurans cells. Morphological analysis and clustering identified six distinct sub-populations across all tested experimental conditions. Results indicate that exposure to IR induced fractional redistributions of cells across sub-populations to exhibit morphologies associated with greater nucleoid condensation and decreased the abundance of sub-populations associated with cell division. Nucleoid-associated proteins (NAPs) may link nucleoid compaction and stress tolerance, but their roles in regulating compaction in D. radiodurans are unknown. Imaging of genomic mutants of known and suspected NAPs that contribute to nucleoid condensation found that deletion of nucleic acid-binding proteins, not previously described as NAPs, can remodel the nucleoid by driving condensation or decondensation in the absence of stress and that IR increased the abundance of these morphological states. Thus, our integrated analysis introduces a new methodology for studying environmental influences on bacterial nucleoids and provides an opportunity to further investigate potential regulators of nucleoid condensation.IMPORTANCEDeinococcus radiodurans, an extremophile known for its stress tolerance, constitutively maintains a highly condensed nucleoid. Qualitative studies have described nucleoid behavior under a variety of conditions. However, a lack of quantitative data regarding nucleoid organization and dynamics has limited our understanding of the regulatory mechanisms controlling nucleoid organization in D. radiodurans. Here, we introduce a quantitative approach that enables high-throughput quantitative measurements of subcellular spatial characteristics in bacterial cells. Applying this to wild-type or single-protein-deficient populations of D. radiodurans subjected to ionizing radiation, we identified significant stress-responsive changes in cell shape, nucleoid organization, and morphology. These findings highlight this methodology's adaptability and capacity for quantitatively analyzing the cellular response to stressors for screening cellular proteins involved in bacterial nucleoid organization.

Asunto(s)

Deinococcus , Radiación Ionizante , Deinococcus/efectos de la radiación , Deinococcus/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética

RESUMEN

Bacterial chromosomes are large molecules that need to be highly compacted to fit inside the cells. Chromosome compaction must facilitate and maintain key biological processes such as gene expression and DNA transactions (replication, recombination, repair, and segregation). Chromosome and chromatin 3D-organization in bacteria has been a puzzle for decades. Chromosome conformation capture coupled to deep sequencing (Hi-C) in combination with other "omics" approaches has allowed dissection of the structural layers that shape bacterial chromosome organization, from DNA topology to global chromosome architecture. Here we review the latest findings using Hi-C and discuss the main features of bacterial genome folding.

RESUMEN

In every bacterium, nucleoid-associated proteins (NAPs) play crucial roles in chromosome organization, replication, repair, gene expression, and other DNA transactions. Their central role in controlling the chromatin dynamics and transcription has been well-appreciated in several well-studied organisms. Here, we review the diversity, distribution, structure, and function of NAPs from the genus Mycobacterium. We highlight the progress made in our understanding of the effects of these proteins on various processes and in responding to environmental stimuli and stress of mycobacteria in their free-living as well as during distinctive intracellular lifestyles. We project them as potential drug targets and discuss future studies to bridge the information gap with NAPs from well-studied systems.

RESUMEN

Escherichia coli has been a vital model organism for studying chromosomal structure, thanks, in part, to its small and circular genome (4.6 million base pairs) and well-characterized biochemical pathways. Over the last several decades, we have made considerable progress in understanding the intricacies of the structure and subsequent function of the E. coli nucleoid. At the smallest scale, DNA, with no physical constraints, takes on a shape reminiscent of a randomly twisted cable, forming mostly random coils but partly affected by its stiffness. This ball-of-spaghetti-like shape forms a structure several times too large to fit into the cell. Once the physiological constraints of the cell are added, the DNA takes on overtwisted (negatively supercoiled) structures, which are shaped by an intricate interplay of many proteins carrying out essential biological processes. At shorter length scales (up to about 1 kb), nucleoid-associated proteins organize and condense the chromosome by inducing loops, bends, and forming bridges. Zooming out further and including cellular processes, topological domains are formed, which are flanked by supercoiling barriers. At the megabase-scale both large, highly self-interacting regions (macrodomains) and strong contacts between distant but co-regulated genes have been observed. At the largest scale, the nucleoid forms a helical ellipsoid. In this review, we will explore the history and recent advances that pave the way for a better understanding of E. coli chromosome organization and structure, discussing the cellular processes that drive changes in DNA shape, and what contributes to compaction and formation of dynamic structures, and in turn how bacterial chromatin affects key processes such as transcription and replication.

RESUMEN

MucR belongs to a large protein family whose members regulate the expression of virulence and symbiosis genes in α-proteobacteria species. This protein and its homologs were initially studied as classical transcriptional regulators mostly involved in repression of target genes by binding their promoters. Very recent studies have led to the classification of MucR as a new type of Histone-like Nucleoid Structuring (H-NS) protein. Thus this review is an effort to put together a complete and unifying story demonstrating how genetic and biochemical findings on MucR suggested that this protein is not a classical transcriptional regulator, but functions as a novel type of H-NS-like protein, which binds AT-rich regions of genomic DNA and regulates gene expression.

RESUMEN

Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.

Asunto(s)

Proteínas Bacterianas , Hemaglutininas , Mycobacterium tuberculosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN/química , ADN/metabolismo , Hemaglutininas/genética , Hemaglutininas/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Eliminación de Gen , Proteínas de Unión al ADN/genética , Dominios Proteicos/genética , Microscopía de Fuerza Atómica

RESUMEN

This review summarizes current knowledge about the mechanisms of timely binding and dissociation of two nucleoid proteins, IHF and Fis, which play fundamental roles in the initiation of chromosomal DNA replication in Escherichia coli. Replication is initiated from a unique replication origin called oriC and is tightly regulated so that it occurs only once per cell cycle. The timing of replication initiation at oriC is rigidly controlled by the timely binding of the initiator protein DnaA and IHF to oriC. The first part of this review presents up-to-date knowledge about the timely stabilization of oriC-IHF binding at oriC during replication initiation. Recent advances in our understanding of the genome-wide profile of cell cycle-coordinated IHF binding have revealed the oriC-specific stabilization of IHF binding by ATP-DnaA oligomers at oriC and by an initiation-specific IHF binding consensus sequence at oriC. The second part of this review summarizes the mechanism of the timely regulation of DnaA activity via the chromosomal loci DARS2 (DnaA-reactivating sequence 2) and datA. The timing of replication initiation at oriC is controlled predominantly by the phosphorylated form of the adenosine nucleotide bound to DnaA, i.e., ATP-DnaA, but not ADP-ADP, is competent for initiation. Before initiation, DARS2 increases the level of ATP-DnaA by stimulating the exchange of ADP for ATP on DnaA. This DARS2 function is activated by the site-specific and timely binding of both IHF and Fis within DARS2. After initiation, another chromosomal locus, datA, which inactivates ATP-DnaA by stimulating ATP hydrolysis, is activated by the timely binding of IHF. A recent study has shown that ATP-DnaA oligomers formed at DARS2-Fis binding sites competitively dissociate Fis via negative feedback, whereas IHF regulation at DARS2 and datA still remains to be investigated. This review summarizes the current knowledge about the specific role of IHF and Fis in the regulation of replication initiation and proposes a mechanism for the regulation of timely IHF binding and dissociation at DARS2 and datA.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Factores de Integración del Huésped/genética , Factores de Integración del Huésped/metabolismo , Origen de Réplica , Replicación del ADN , Ciclo Celular , Adenosina Trifosfato/metabolismo , ADN Bacteriano/genética , Factor Proteico para Inverción de Estimulación/genética , Factor Proteico para Inverción de Estimulación/metabolismo

RESUMEN

In cells that are exposed to terrestrial sunlight, the indole moiety in the side chain of tryptophan (Trp) can suffer photo/oxidative damage (POD) by reactive oxygen species (ROS) and/or ultraviolet light (UV-B). Trp is oxidized to produce N-formylkynurenine (NFK), a UV-A-responsive photosensitizer that further degenerates into photosensitizers capable of generating ROS through exposure to visible light. Thus, Trp-containing proteins function as both victims, and perpetrators, of POD if they are not rapidly replaced through protein turnover. The literature indicates that protein turnover and DNA repair occur poorly in chromosomal interiors. We contend, therefore, that basic chromosomal proteins (BCPs) that are enveloped by DNA should have evolved to lack Trp residues in their amino acid sequences, since these could otherwise function as 'Trojan horse-type' DNA-damaging agents. Our global analyses of protein sequences demonstrates that BCPs consistently lack Trp residues, although DNA-binding proteins in general do not display such a lack. We employ HU-B (a wild-type, Trp-lacking bacterial BCP) and HU-B F47W (a mutant, Trp-containing form of the same bacterial BCP) to demonstrate that the possession of Trp is deleterious to BCPs and associated chromosomal DNA. Basically, we show that UV-B and UV-A (a) cause no POD in HU-B, but cause extensive POD in HU-B F47W (in vitro), as well as (b) only nominal DNA damage in bacteria expressing HU-B, but extensive DNA damage in bacteria expressing F47W HU-B (in vivo). Our results suggest that Trp-lacking BCPs could have evolved to reduce scope for protein-facilitated, sunlight-mediated damage of DNA by UV-A and visible light, within chromosomal interiors that are poorly serviced by protein turnover and DNA repair machinery.

Asunto(s)

Proteínas Bacterianas , Cromosomas , Daño del ADN , Genoma , Histonas , Estrés Oxidativo , Luz Solar , Triptófano , Humanos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/efectos de la radiación , Cromosomas/química , Cromosomas/metabolismo , Cromosomas/efectos de la radiación , Cromosomas Bacterianos/química , Cromosomas Bacterianos/metabolismo , Cromosomas Bacterianos/efectos de la radiación , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Genoma/genética , Genoma/efectos de la radiación , Histonas/química , Histonas/metabolismo , Histonas/efectos de la radiación , Concentración de Iones de Hidrógeno , Etiquetado Corte-Fin in Situ , Factores de Integración del Huésped/química , Oxidación-Reducción/efectos de la radiación , Fenilalanina/genética , Fármacos Fotosensibilizantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/química , Triptófano/deficiencia , Triptófano/genética , Triptófano/metabolismo , Rayos Ultravioleta

RESUMEN

Structure and function of bacterial nucleoid is controlled by the nucleoid-associated proteins (NAP). In any phase of growth, various NAPs, acting sequentially, condense nucleoid and facilitate formation of its transcriptionally active structure. However, in the late stationary phase, only one of the NAPs, Dps protein, is strongly expressed, and DNA-protein crystals are formed that transform nucleoid into a static, transcriptionally inactive structure, effectively protected from the external influences. Discovery of crystal structures in living cells and association of this phenomenon with the bacterial resistance to antibiotics has aroused great interest in studying this phenomenon. The aim of this work is to obtain and compare structures of two related NAPs (HU and IHF), since they are the ones that accumulate in the cell at the late stationary stage of growth, which precedes formation of the protective DNA-Dps crystalline complex. For structural studies, two complementary methods were used in the work: small-angle X-ray scattering (SAXS) as the main method for studying structure of proteins in solution, and dynamic light scattering as a complementary one. To interpret the SAXS data, various approaches and computer programs were used (in particular, the evaluation of structural invariants, rigid body modeling and equilibrium mixture analysis in terms of the volume fractions of its components were applied), which made it possible to determine macromolecular characteristics and obtain reliable 3D structural models of various oligomeric forms of HU and IHF proteins with ~2 nm resolution typical for SAXS. It was shown that these proteins oligomerize in solution to varying degrees, and IHF is characterized by the presence of large oligomers consisting of initial dimers arranged in a chain. An analysis of the experimental and published data made it possible to hypothesize that just before the Dps expression, it is IHF that forms toroidal structures previously observed in vivo and prepares the platform for formation of DNA-Dps crystals. The results obtained are necessary for further investigation of the phenomenon of biocrystal formation in bacterial cells and finding ways to overcome resistance of various pathogens to external conditions.

Asunto(s)

Proteínas de Unión al ADN , Hidrodinámica , Proteínas de Unión al ADN/metabolismo , Dispersión del Ángulo Pequeño , ADN Bacteriano/metabolismo , Difracción de Rayos X , Proteínas Bacterianas/metabolismo , ADN

RESUMEN

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

Asunto(s)

ADN-Topoisomerasas de Tipo I , Streptococcus pneumoniae , Humanos , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Novobiocina/farmacología , ADN Bacteriano/genética , Girasa de ADN/genética , Girasa de ADN/metabolismo

RESUMEN

The bacterial chromosome, known as its nucleoid, is an amorphous assemblage of globular nucleoprotein domains. It exists in a state of phase separation from the cell's cytoplasm, as an irregularly-shaped, membrane-less, intracellular compartment. This state (the nature of which remains largely unknown) is maintained through bacterial generations ad infinitum. Here, we show that HU and Dps, two of the most abundant nucleoid-associated proteins (NAPs) of Escherichia coli, undergo spontaneous complex coacervation with different forms of DNA/RNA, both individually and in each other's presence, to cause accretion and compaction of DNA/RNA into liquid-liquid phase separated condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) biphasic heterotypic mixed condensates in which HU-B helps to lower the Csat of HU-A and also (b) multiphasic heterotypic condensates, with Dps, in which demixed domains display different contents of HU and Dps. We believe that these modes of complex coacervation that are seen in vitro can serve as models for the in vivo relationships among NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in demixed subdomains that are characterized by differing grades of phase separation. Our results clearly demonstrate some quantitative, and some qualitative, differences in the coacervating abilities of different NAPs with DNA, potentially explaining (i) why E. coli has two isoforms of HU, and (ii) why changes in the abundances of HU and Dps facilitate the lag, logarithmic, and stationary phases of E. coli growth.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa , ADN Bacteriano , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/ultraestructura , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Isoformas de Proteínas/metabolismo , ARN Bacteriano

RESUMEN

Implementation of computational tools in the identification of novel drug targets for Tuberculosis (TB) has been a promising area of research. TB has been a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) localized primarily on the lungs and it has been one of the most successful pathogen in the history of mankind. Extensively arising drug resistivity in TB has made it a global challenge and need for new drugs has become utmost important.The involvement of Nucleoid-Associated Proteins (NAPs) in maintaining the structure of the genomic material and regulating various cellular processes like transcription, DNA replication, repair and recombination makes significant, has opened a new arena to find the drugs targeting Mtb. The current study aims to identify potential inhibitors of NAPs through a computational approach. In the present work we worked on the eight NAPs of Mtb, namely, Lsr2, EspR, HupB, HNS, NapA, mIHF and NapM. The structural modelling and analysis of these NAPs were carried out. Moreover, molecular interaction were checked and binding energy was identified for 2500 FDA-approved drugs that were selected for antagonist analysis to choose novel inhibitors targeting NAPs of Mtb. Drugs including Amikacin, streptomycin, kanamycin, and isoniazid along with eight FDA-approved molecules that were found to be potential novel targets for these mycobacterial NAPs and have an impact on their functions. The potentiality of several anti-tubercular drugs as therapeutic agents identified through computational modelling and simulation unlocks a new gateway for accomplishing the goal to treat TB. Complete framework of the methodology employed in this study to predict inhibitors against mycobacterial NAPs.

RESUMEN

The hyperactive Tn5 transposase in the ATAC-seq method has been widely used to determine the open DNA regions and understand the overall epigenomic regulation in the chromatins of eukaryotic cells. Here, we describe POP-seq (Prokaryotic chromatin Openness Profiling sequencing), an adaptation of the ATAC-seq method, to interrogate changes in the openness of prokaryotic nucleoids.

Asunto(s)

Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ADN , Genoma Bacteriano

RESUMEN

The massive sequencing of transposon insertion mutant libraries (Tn-Seq) represents a commonly used method to determine essential genes in bacteria. Using a hypersaturated transposon mutant library consisting of 400,096 unique Tn insertions, 523 genes were classified as essential in Escherichia coli K-12 MG1655. This provided a useful genome-wide gene essentiality landscape for rapidly identifying 233 of 301 essential genes previously validated by a knockout study. However, there was a discrepancy in essential gene sets determined by conventional gene deletion methods and Tn-Seq, although different Tn-Seq studies reported different extents of discrepancy. We have elucidated two causes of this discrepancy. First, 68 essential genes not detected by Tn-Seq contain nonessential subgenic domains that are tolerant to transposon insertion, which leads to the false assignment of an essential gene as a nonessential or dispensable gene. These genes exhibited a high level of transposon insertion in their subgenic nonessential domains. In contrast, 290 genes were additionally categorized as essential by Tn-Seq, although their knockout mutants were available. The comparative analysis of Tn-Seq and high-resolution footprinting of nucleoid-associated proteins (NAPs) revealed that a protein-DNA interaction hinders transposon insertion. We identified 213 false-positive genes caused by NAP-genome interactions. These two limitations have to be considered when addressing essential bacterial genes using Tn-Seq. Furthermore, a comparative analysis of high-resolution Tn-Seq with other data sets is required for a more accurate determination of essential genes in bacteria. IMPORTANCE Transposon mutagenesis is an efficient way to explore gene essentiality of a bacterial genome. However, there was a discrepancy between the essential gene set determined by transposon mutagenesis and that determined using single-gene knockout strains. In this study, we generated a hypersaturated Escherichia coli transposon mutant library comprising approximately 400,000 different mutants. Determination of transposon insertion sites using next-generation sequencing provided a high-resolution essentiality landscape of the E. coli genome. We identified false negatives of essential gene discovery due to the permissive insertion of transposons in the C-terminal region. Comparisons between the transposon insertion landscape with binding profiles of DNA-binding proteins revealed interference of nucleoid-associated proteins to transposon insertion, generating false positives of essential gene discovery. Consideration of these findings is required to avoid the misinterpretation of transposon mutagenesis results.

Asunto(s)

Escherichia coli K12 , Escherichia coli , Escherichia coli/genética , Mutagénesis Insercional , Escherichia coli K12/genética , Elementos Transponibles de ADN/genética , Genoma Bacteriano

RESUMEN

DNA supercoiling and nucleoid-associated proteins (NAPs) are two of the factors that govern the architecture of the bacterial genome, influencing the expression of the genetic information that it contains. Alterations to DNA topology, and to the numbers and types of NAPs, have pleiotropic effects on gene expression, suggesting that modifications to the production patterns of DNA topoisomerases and/or NAPs are likely to result in marked impacts on bacterial physiology. Knockout mutations in the genes encoding these proteins (where the mutants remain viable) result in clear physiological effects. However, genetic modifications that involve rewiring, or repositioning, of topoisomerase or NAP genes produce much more subtle outcomes. These findings demonstrate that the high-level regulatory circuitry of bacteria is robust in the face of genomic rearrangements that, a priori, might be expected to produce significant changes in bacterial lifestyle. Examples from genomic rewiring experiments, performed chiefly with the Gram-negative model bacteria Escherichia coli K-12 and Salmonella enterica serovar Typhimurium, will be used to illustrate these features. The results show not only the ability of naturally occurring bacteria to tolerate regulatory rewiring but also indicate the limits within which experiments in synthetic biology may be designed.

Asunto(s)

Escherichia coli K12 , ADN-Topoisomerasas/metabolismo , Escherichia coli/genética , Salmonella typhimurium/genética