2024: A "nucleoid space" odyssey featuring H-NS.

The three-dimensional architecture of the bacterial chromosome is intertwined with genome processes such as transcription and replication. Conspicuously so, that the structure of the chromosome permits accurate prediction of active genome processes. Although appreciation of this interplay has developed rapidly in the past two decades, our understanding of this subject is still in its infancy, with research primarily focusing on how the process of transcription regulates and is regulated by chromosome structure.

Histones and histone variant families in prokaryotes.

Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences.

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

Common throughout life is the need to compact and organize the genome. Possible mechanisms involved in this process include supercoiling, phase separation, charge neutralization, macromolecular crowding, and nucleoid-associated proteins (NAPs). NAPs are special in that they can organize the genome at multiple length scales, and thus are often considered as the architects of the genome.

Unravelling the Biophysical Properties of Chromatin Proteins and DNA Using Acoustic Force Spectroscopy.

Acoustic force spectroscopy (AFS) is a single-molecule micromanipulation technique that uses sound waves to exert force on surface-tethered DNA molecules in a microfluidic chamber. As large numbers of individual protein-DNA complexes are tracked in parallel, AFS provides insight into the individual properties of such complexes as well as their population averages. In this chapter, we describe in detail how to perform AFS experiments specifically on bare DNA, protein-DNA complexes, and how to extract their (effective) persistence length and contour length from force-extension relations.

Quantitation of DNA Binding Affinity Using Tethered Particle Motion.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered particle motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to measure binding constants of proteins binding to DNA reliably, provided that they distort DNA.

Tethered Particle Motion Analysis of DNA-Binding Properties of Architectural Proteins.

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. Tethered particle motion (TPM) permits analysis of DNA conformation and detection of changes in conformation induced by such proteins at the single molecule level in vitro. As many individual protein-DNA complexes can be investigated in parallel, these experiments have high throughput.

Quantitative Determination of DNA Bridging Efficiency of Chromatin Proteins.

DNA looping is important for genome organization in all domains of life. The basis of DNA loop formation is the bridging of two separate DNA double helices. Detecting DNA bridge formation generally involves the use of complex single-molecule techniques (atomic force microscopy, magnetic or optical tweezers).

Microscale Thermophoresis Analysis of Chromatin Interactions.

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.

ChIP-qPCR of FLAG-Tagged Proteins in Bacteria.

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.

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

Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors.

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli.

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking.

MucR from : New Insights into Its DNA Targets and Its Ability to Oligomerize.

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS.

DNA-bridging by an archaeal histone variant via a unique tetramerisation interface.

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into 'hypernucleosome' particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood.

Three-dimensional chromosome re-modelling: The integral mechanism of transcription regulation in bacteria.

Nucleoid-associated proteins (NAPs) are architectural proteins of the bacterial chromosome and transcription factors that dynamically organise the chromosome and regulate gene expression in response to physicochemical environmental signals. While the architectural and regulatory functions of NAPs have been verified independently, the coupling between these functions in vivo has not been conclusively proven. Here we describe a model NAP - histone-like nucleoid structuring protein (H-NS) - as a coupled sensor-effector that directly regulates gene expression by chromatin re-modelling in response to physicochemical environmental signals.

Specific DNA binding of archaeal histones HMfA and HMfB.

In archaea, histones play a role in genome compaction and are involved in transcription regulation. Whereas archaeal histones bind DNA without sequence specificity, they bind preferentially to DNA containing repeats of alternating A/T and G/C motifs. These motifs are also present on the artificial sequence "Clone20," a high-affinity model sequence for binding of the histones from .

The B. subtilis Rok protein is an atypical H-NS-like protein irresponsive to physico-chemical cues.

Nucleoid-associated proteins (NAPs) play a central role in chromosome organization and environment-responsive transcription regulation. The Bacillus subtilis-encoded NAP Rok binds preferentially AT-rich regions of the genome, which often contain genes of foreign origin that are silenced by Rok binding. Additionally, Rok plays a role in chromosome architecture by binding in genomic clusters and promoting chromosomal loop formation.

Ruthenium-Locked Helical Chirality: A Barrier of Inversion and Formation of an Asymmetric Macrocycle.

Upon coordination to metal centers, tetradentate ligands based on the 6,6'-bis(2″-aminopyridyl)-2,2'-bipyridine (bapbpy) structure form helical chiral complexes due to the steric clash between the terminal pyridines of the ligand. For octahedral ruthenium(II) complexes, the two additional axial ligands bound to the metal center, when different, generate diastereotopic aromatic protons that can be distinguished by NMR. Based on these geometrical features, the inversion barrier of helical [Ru(L)(RR'SO)Cl] complexes, where L is a sterically hindered bapbpy derivative and RR'SO is a chiral or achiral sulfoxide ligand, was studied by variable-temperature H NMR.

Chromosome Conformation Capture in Bacteria and Archaea.

The three-dimensional structure of the chromosome is encoded within its sequence and regulates activities such as replication and transcription. This necessitates the study of the spatial organization of the chromosome in relation to the underlying sequence. Chromosome conformation capture (3C) techniques are proximity ligation-based approaches that simplify the three-dimensional architecture of the chromosome into a one-dimensional library of hybrid ligation junctions.

System-Wide Analysis of the GATC-Binding Nucleoid-Associated Protein Gbn and Its Impact on Development.

Bacterial chromosome structure is, to a great extent, organized by a diverse group of proteins collectively referred to as nucleoid-associated proteins (NAPs). Many NAPs have been well studied in , including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of NAPs and recognizes a consensus sequence consisting of GATC followed by (A/T)T.

Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity.

Horizontal gene transfer facilitates dissemination of favourable traits among bacteria. However, foreign DNA can also reduce host fitness: incoming sequences with a higher AT content than the host genome can misdirect transcription. Xenogeneic silencing proteins counteract this by modulating RNA polymerase binding.

Special Issue: Role of Bacterial Chromatin in Environmental Sensing, Adaptation and Evolution.

A typical bacterial cell is micron-sized and contains a genome several million base pairs in length [...

HI-NESS: a family of genetically encoded DNA labels based on a bacterial nucleoid-associated protein.

The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding.