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

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

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. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

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. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea.

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. We outline how H-NS-binding partners and post-translational modifications modulate the role of H-NS as a transcription factor by influencing its DNA structuring properties. We consolidate our ideas in models of how H-NS may regulate the expression of the proVWX and hlyCABD operons by chromatin re-modelling. The interplay between chromosome structure and gene expression may be a common - but, at present, under-appreciated - concept of transcription regulation in bacteria.

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 Methanothermus fervidus. Here, we investigate the binding of HMfA and HMfB to Clone20 DNA. We show that specific binding at low protein concentrations (<30 nM) yields a modest level of DNA compaction, attributed to tetrameric nucleosome formation, whereas nonspecific binding strongly compacts DNA. We also demonstrate that histones impaired in hypernucleosome formation are still able to recognize the Clone20 sequence. Histone tetramers indeed exhibit a higher binding affinity for Clone20 than nonspecific DNA. Our results indicate that a high-affinity DNA sequence does not act as a nucleation site, but is bound by a tetramer which we propose is geometrically different from the hypernucleosome. Such a mode of histone binding might permit sequence-driven modulation of hypernucleosome size. These findings might be extrapolated to histone variants that do not form hypernucleosomes. Versatile binding modes of histones could provide a platform for functional interplay between genome compaction and transcription.

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. 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.

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 [RuII(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 1H 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).

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. 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.

System-Wide Analysis of the GATC-Binding Nucleoid-Associated Protein Gbn and Its Impact on Streptomyces 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 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, which is expressed in particular during sporulation, 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 2,800 binding regions, encompassing around 3,600 GATCWT motifs. This amounts to 55% of all such sequences in the S. coelicolor genome. DNA binding of Gbn in vitro minimally changes DNA conformation, suggesting a modest role in chromosome organization only, in addition to a gene regulatory role. 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 recognizes 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. IMPORTANCE A 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 eukaryotes. In bacteria, the organization of chromosome structure is mediated by multiple factors, including macromolecular biophysics processes, biological processes, and, more importantly, a diverse group of proteins referred to collectively as nucleoid-associated proteins (NAPs). We here present the discovery of a novel and extremely pleiotropic NAP, which we refer to as Gbn. Gbn is an Actinobacteria-specific protein that binds to GATC sequences, with a subtle but broad effect on global gene expression, especially during the late developmental stage. The discovery of Gbn is a new step toward better understanding of how gene expression and chromosome structure are governed in antibiotic-producing streptomycetes.

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. 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 σ 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.

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. 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.

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 [...].

Novel anti-repression mechanism of H-NS proteins by a phage protein.

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.

Direct visualization of the effect of DNA structure and ionic conditions on HU-DNA interactions.

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.

Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps.

JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A's genome and physical size are approximately one-tenth those of the model bacterial organism Escherichia coli's, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome's configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes.

Moltemplate: A Tool for Coarse-Grained Modeling of Complex Biological Matter and Soft Condensed Matter Physics.

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.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB-DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU.

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.

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins.

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 Pseudomonas 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 strength on electrostatic interactions between the oppositely 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.

Chromosome organization in bacteria: mechanistic insights into genome structure and function.

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.