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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-II(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-1 NMR The coalescence energies for the inversion of the helical chirality of [Ru(bapbpy)(DMSO)(Cl)]Cl and [Ru(bapbpy)(MTSO)(Cl)]Cl (where MTSO is (R)-methyl p-tolylsulfoxide) were found to be 43 and 44 kJ/mol, respectively. By contrast, in [Ru(biqbpy)(DMSO)(Cl)]Cl (biqbpy = 6,6'-bis(aminoquinolyl)-2,2'-bipyridine increased strain caused by the larger terminal quinoline groups resulted in a coalescence temperature higher than 376 K, which pointed to an absence of helical chirality inversion at room temperature. Further increasing the steric strain by introducing methoxy groups ortho to the nitrogen atoms of the terminal pyridyl groups in bapbpy resulted in the serendipitous discovery of a ring-closing reaction that took place upon trying to make [Ru(OMe-bapbpy)(DMSO)Cl](+) (OMe-bapbpy = 6,6'-bis(6-methoxy-aminopyridyl)2,2'-bipyridine). This reaction generated, in excellent yields, a chiral complex [Ru(L '')(DMSO)Cl]Cl, where L '' is an asymmetric tetrapyridyl macrocycle. This unexpected transformation appears to be specific to ruthenium(II) as macrocyclization did not occur upon coordination of the same ligand to palladium(II) or rhodium(III).

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT–DNA complex. Structural investigations suggest that gp4 acts as an ‘electrostatic zipper’ between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their ‘half-open’ conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

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. Based on this, Rok was proposed to be a functional homolog of E. coli H-NS. However, it is largely unclear how Rok binds DNA, how it represses transcription and whether Rok mediates environment-responsive gene regulation. Here, we investigated Rok's DNA binding properties and the effects of physico-chemical conditions thereon. We demonstrate that Rok is a DNA bridging protein similar to prototypical H-NS-like proteins. However, unlike these proteins, the DNA bridging ability of Rok is not affected by changes in physico-chemical conditions. The DNA binding properties of the Rok interaction partner sRok are affected by salt concentration. This suggests that in a minority of Bacillus strains Rok activity can be modulated by sRok, and thus respond indirectly to environmental stimuli. Despite several functional similarities, the absence of a direct response to physico-chemical changes establishes Rok as disparate member of the H-NS family.

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. Deciphering the information contained in these libraries resolves chromosome architecture in a sequence-specific manner. This chapter describes the preparation of 3C libraries for bacteria and archaea. It details how the three-dimensional architecture of local chromatin can be extracted from the 3C library using qPCR (3C-qPCR), and it summarizes the processing of 3C libraries for next-generation sequencing (3C-Seq) for a study of global chromosome organization.

Bacterial chromosome structure is organized by a diverse group of proteins collectively referred to as nucleoid-associated proteins (NAPs). Many NAPs have been well studied in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of Actinobacteria NAPs and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein was designated Gbn for GATC-binding NAP. Deletion of gbn led to alterations in development and antibiotic production in Streptomyces coelicolor. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2800 binding regions, encompassing some 3600 GATCWT motifs, which comprise 55% of all such motifs in the S. coelicolor genome. DNA binding of Gbn in vitro increased DNA stiffness but not compaction, suggesting a role in regulation rather than chromosome organization. Transcriptomics analysis showed that Gbn binding generally leads to reduced gene expression. The DNA binding profiles were nearly identical between vegetative and aerial growth. Exceptions are SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognises the rare leucine codon CUA, respectively, which nearly exclusively bound during vegetative growth. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes.IMPORTANCEA large part of the chemical space of bioactive natural products is derived from Actinobacteria. Many of the biosynthetic gene clusters for these compounds are cryptic, in others words, they are expressed in nature but not in the laboratory. Understanding the global regulatory networks that control gene expression is key to the development of approaches to activate this biosynthetic potential. Chromosome structure has a major impact on the control of gene expression. In bacteria, the organization of chromosome structure is mediated by a diverse group of proteins referred to collectively as nucleoid-associated proteins (NAPs), which play an important role in the control of gene expression, nucleoid structure and DNA repair. We here present the discovery of a novel and extremely pleiotropic NAP, which we refer to as Gbn. Gbn is a sporulation-specific protein that occurs only in the Actinobacteria and binds to GATC sequences, with a subtle but broad effect on global gene expression. The discovery of Gbn is a new step towards better understanding of how gene expression and chromosome structure is governed in antibiotic-producing streptomycetes.

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. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS — a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin.

Architectural DNA–binding proteins are involved in many important DNA transactions by virtue of their ability to change DNA conformation. Histone-like protein from E. coli strain U93, HU, is one of the most studied bacterial architectural DNA–binding proteins. Nevertheless, there is still a limited understanding of how the interactions between HU and DNA are affected by ionic conditions and the structure of DNA. Here, using optical tweezers in combination with fluorescent confocal imaging, we investigated how ionic conditions affect the interaction between HU and DNA. We directly visualized the binding and the diffusion of fluorescently labelled HU dimers on DNA. HU binds with high affinity and exhibits low mobility on the DNA in the absence of Mg2+; it moves 30-times faster and stays shorter on the DNA with 8 mM Mg2+ in solution. Additionally, we investigated the effect of DNA tension on HU–DNA complexes. On the one hand, our studies show that binding of HU enhances DNA helix stability. On the other hand, we note that the binding affinity of HU for DNA in the presence of Mg2+ increases at tensions above 50 pN, which we attribute to force-induced structural changes in the DNA. The observation that HU diffuses faster along DNA in presence of Mg2+ compared to without Mg2+ suggests that the free energy barrier for rotational diffusion along DNA is reduced, which can be interpreted in terms of reduced electrostatic interaction between HU and DNA, possibly coinciding with reduced DNA bending.

Bacteria use specific silencing proteins to prevent spurious transcription of horizontally acquired DNA. Here, Forrest et al. describe differences in silencing strategies between E. coli and Bacillus subtilis, driven by the respective specificities of the silencing protein and the RNA polymerase in each organism.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. In this work, we compare xenogeneic silencing strategies of two distantly related model organisms: Escherichia coli and Bacillus subtilis. In E. coli, silencing is mediated by the H-NS protein that binds extensively across horizontally acquired genes. This prevents spurious non-coding transcription, mostly intragenic in origin. By contrast, binding of the B. subtilis Rok protein is more targeted and mostly silences expression of functional mRNAs. The difference reflects contrasting transcriptional promiscuity in E. coli and B. subtilis, largely attributable to housekeeping RNA polymerase sigma factors. Thus, whilst RNA polymerase specificity is key to the xenogeneic silencing strategy of B. subtilis, transcriptional promiscuity must be overcome to silence horizontally acquired DNA in E. coli.

Bacterial xenogeneic silencers, the H-NS family proteins play central roles in genome organization and in the regulation of foreign genes acquired from phages or through horizontal gene transfer. It is thought that the repression of these genes is directly dependent on their DNA binding modes. H-NS family proteins bind along DNA, forming lateral protein filaments, in which the H-NS protomers adopt a "half-open" conformational state. Under specific environmental conditions and without dissociating from DNA, these proteins adopt an "open" conformational state which can bridge two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the gp4 protein, which binds the H-NS family protein MvaT of Pseudomonas aeruginosa. Binding of gp4 was suggested to inhibit the silencing of phage LUZ24 DNA by MvaT. However, the mechanism by which gp4 modulates MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations by NMR spectroscopy revealed that gp4 acts as an "electrostatic zipper" between the N-terminal and DNA binding domains of MvaT protomers and stabilizes a structure resembling their "half-open" conformation. Based on these observations, we propose that binding of gp4 promotes a half-open bridging-incompetent state of MvaT, resulting in relief of MvaT-mediated gene silencing and adverse effects on P. aeruginosa homeostasis and growth. Conformational switching of H-NS proteins driven by phage "early proteins" provides a novel anti-repression mechanism. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

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&ndash, 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.

The topology of biological polymers such as proteins and nucleic acids is an important aspect of their 3D structure. Recently, two applications of topology to molecular chains have emerged as important theoretical developments that are beginning to find utility in heteropolymer characterization and design: namely, circuit topology (CT) and knot theory. Here, we review the application of these two theories to protein, RNA, and DNA/genome structure, focusing on connections to conventional 3D structural information and relevance to function and highlighting recent experimental findings. We conclude with a discussion of recent applications to molecular origami and engineering.

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive.Here, we focus on the H-NS family protein MvaT from P. aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strengths on electrostatic interactions between the appositively charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

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.

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA–DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

The epigenome is a heritable layer of information not encoded in the DNA sequence of the genome, but in chemical modifications of DNA or histones. These chemical modifications, together with transcription factors, operate as spatiotemporal regulators of genome activity. Dissecting epigenome function requires controlled site-specific alteration of epigenetic information. Such control can be obtained using designed DNA-binding platforms associated with effector domains to function as targeted transcription factors or epigenetic modifiers. Here, we review the use of dCas9 as a novel and versatile tool for fundamental studies on epigenetic landscapes, chromatin structure and transcription regulation, and the potential of this approach in basic research in these fields.

Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes. Advances in sequencing- and imaging-based techniques for chromosome structure analysis have led to a mature understanding of bacterial chromosome structure and dynamics. In this Review, Dame, Rashid and Grainger discuss the hierarchical nature of bacterial chromosome structure and how it is influenced by diverse types of nucleoid-associated proteins. Furthermore, they describe roles for nucleoid-associated proteins and chromosome structure, including in gene expression, chromosome segregation and cell cycle regulation.

Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes.

Rok from Bacillus subtilis is an abundant DNA binding protein similar in function to H-NS-like proteins found in many proteobacteria. Rok binds across the genome with a preference for A/T rich DNA. Such DNA often contains genes of foreign origin that are silenced due to Rok binding. Rok also has been implied in global organization of the B. subtilis genome. However, how Rok binds to DNA and how it represses transcription is unclear. Also, it is unknown whether Rok-mediated gene repression can be induced or relieved following changes in physico-chemical conditions, as noted for H-NS-like proteins. Here we investigate the DNA binding properties of Rok and determine the effects of physico-chemical conditions on these properties. We demonstrate that Rok is a DNA bridging protein similar to H-NS like proteins from E. coli (H-NS), Pseudomonas sp. (MvaT) and Mycobacteria (Lsr2). Strikingly, unlike these proteins, the ability of Rok to bridge DNA is not affected by changes in physico-chemical conditions. Not being a direct sensor of such changes sets Rok apart from other H-NS like proteins. It implies the existence of other (protein-mediated) mechanisms to relieve Rok-mediated gene silencing in response to changes in environmental conditions.

Bacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how cells cope with stressful conditions and how this may lead to dramatic morphological changes. These changes may not only allow harmless cells to withstand environmental insults but can also benefit pathogenic bacteria by enabling them to escape from the immune system and the activity of antibiotics. A better understanding of stress-induced morphogenesis will help us to develop new approaches to combat such harmful pathogens.

The maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA, modulate its conformation and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of C. difficile have been characterized to date.Here, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10 kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G+C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.The present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.

This chapter explains the chromosome organization and dynamics in bacteria. It reviews some of the main discoveries and open questions on the bacterial chromosome that emerged from studies, through the experimental, theoretical and computational work performed around them. The chapter highlights research directions in this area where theoretical and experimental work performed on bacteria can lead the way to formulating new questions concerning eukaryotic chromosomes, and, vice versa, attempt to identify where studies performed on eukaryotes can inform the field of the bacterial chromosome. It explores the four different but inter-related areas of chromosome folding, deoxyribonucleic acid-organizing proteins, chromosome dynamics, and role of molecular crowding, presented in separate sections. Molecular dynamics simulations with simple but explicit descriptions of crowders are available, and allow, for example, the study of the role of size and polydispersity of crowding agents on chromosome compaction.

Coarse-grained models have long been considered indispensable tools in the investigation of biomolecular dynamics and assembly. However, the process of simulating such models is arduous because unconventional force fields and particle attributes are often needed, and some systems are not in thermal equilibrium. Although modern molecular dynamics programs are highly adaptable, software designed for preparing all-atom simulations typically makes restrictive assumptions about the nature of the particles and the forces acting on them. Consequently, the use of coarse-grained models has remained challenging. Moltemplate is a file format for storing coarse-grained molecular models and the forces that act on them, as well as a program that converts moltemplate files into input files for LAMMPS, a popular molecular dynamics engine. Moltemplate has broad scope and an emphasis on generality. It accommodates new kinds of forces as they are developed for LAMMPS, making moltemplate a popular tool with thousands of users in computational chemistry, materials science, and structural biology. To demonstrate its wide functionality, we provide examples of using moltemplate to prepare simulations of fluids using many-body forces, coarse-grained organic semiconductors, and the motor-driven supercoiling and condensation of an entire bacterial chromosome.

Bacterial genomes are functionally organized and compactly folded into a structure referred to as bacterial chromatin or the nucleoid. An important role in genome folding is attributed to Nucleoid-Associated Proteins, also referred to as bacterial chromatin proteins. Although a lot of molecular insight in the mechanisms of operation of these proteins has been generated in the test tube, knowledge on genome organization in the cellular context is still lagging behind severely. Here, we discuss important advances in the understanding of three-dimensional genome organization due to the application of Chromosome Conformation Capture and super-resolution microscopy techniques. We focus on bacterial chromatin proteins whose proposed role in genome organization is supported by these approaches. Moreover, we discuss recent insights into the interrelationship between genome organization and genome activity/stability in bacteria.

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). Although DNA bridging can be qualitatively described, quantification of DNA bridging and bridging dynamics using these techniques is challenging. Here, we describe a novel biochemical assay capable of not only detecting DNA bridge formation, but also allowing for quantification of DNA bridging efficiency and the effects of physico-chemical conditions on DNA bridge formation.

The spatial organization of genomes is based on their hierarchical compartmentalization in topological domains. There is growing evidence that bacterial genomes are organized into insulated domains similar to the Topologically Associating Domains (TADs) detected in eukaryotic cells. Chromosome conformation capture (3C) technologies are used to analyze in vivo DNA proximity based on ligation of distal DNA segments crossed-linked by bridging proteins. By combining 3C and high-throughput sequencing, the Hi-C method reveals genome-wide interactions within topological domains and global genome structure as a whole. This chapter provides detailed guidelines for the preparation of Hi-C sequencing libraries for bacteria.

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.

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 reliably measure binding constants of proteins binding to DNA, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This makes it possible to measure binding properties. We use the bacterial protein Integration Host Factor (IHF) as an example to show how specific binding to DNA can be measured. Moreover, we show a new intuitive quantitative approach to displaying data obtained via TPM.

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.

Post-translational modification (PTM) of histones has been investigated in eukaryotes for years, revealing its widespread occurrence and functional importance. Many PTMs affect chromatin folding and gene activity. Only recently the occurrence of such modifications has been recognized in bacteria. However, it is unclear whether PTM of the bacterial counterparts of eukaryotic histones, nucleoid-associated proteins (NAPs), bears a comparable significance. Here, we scrutinize proteome mass spectrometry data for PTMs of the four most abundantly present NAPs in Escherichia coli (H-NS, HU, IHF and FIS). This approach allowed us to identify a total of 101 unique PTMs in the 11 independent proteomic studies covered in this review. Combined with structural and genetic information on these proteins, we describe potential effects of these modifications (perturbed DNA-binding, structural integrity or interaction with other proteins) on their function.

Genomes are arranged in a confined space in the cell, the nucleoid or nucleus. This arrangement is hierarchical and dynamic, and follows DNA/chromatin-based transactions or environmental conditions. Describing the interplay between local genome structure and gene activity is a long-standing quest in biology. Here, we focus on systematic studies correlating bacterial genome folding and function. Parallels on organizational similarities with eukaryotes are drawn. The biological relevance of hierarchical units in bacterial genome folding and the causal relationship between genome folding and its activity is unclear. We discuss recent quantitative approaches to tackle these questions. Moreover, we sketch a perspective of experiments necessary to iteratively and systematically build, test and improve structure–function models of bacterial chromatin.

The spatial organization of genomes is based on their hierarchical compartmentalization in topological domains. There is growing evidence that bacterial genomes are organized into insulated domains similar to the Topologically Associating Domains (TADs) detected in eukaryotic cells. Chromosome conformation capture (3C) technologies are used to analyze in vivo DNA proximity based on ligation of distal DNA segments crossed-linked by bridging proteins. By combining 3C and high-throughput sequencing, the Hi-C method reveals genome-wide interactions within topological domains and global genome structure as a whole. This chapter provides detailed guidelines for the preparation of Hi-C sequencing libraries for bacteria.