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.

Bacterial histone HBb from Bdellovibrio bacteriovorus compacts DNA by bending.

Histones are essential for genome compaction and transcription regulation in eukaryotes, where they assemble into octamers to form the nucleosome core. In contrast, archaeal histones assemble into dimers that form hypernucleosomes upon DNA binding. Although histone homologs have been identified in bacteria recently, their DNA-binding characteristics remain largely unexplored. Our study reveals that the bacterial histone HBb (Bd0055) is indispensable for the survival of Bdellovibrio bacteriovorus, suggesting critical roles in DNA organization and gene regulation. By determining crystal structures of free and DNA-bound HBb, we unveil its distinctive dimeric assembly, diverging from those of eukaryotic and archaeal histones, while also elucidating how it binds and bends DNA through interaction interfaces reminiscent of eukaryotic and archaeal histones. Building on this, by employing various biophysical and biochemical approaches, we further substantiated the ability of HBb to bind and compact DNA by bending in a sequence-independent manner. Finally, using DNA affinity purification and sequencing, we reveal that HBb binds along the entire genomic DNA of B. bacteriovorus without sequence specificity. These distinct DNA-binding properties of bacterial histones, showcasing remarkable similarities yet significant differences from their archaeal and eukaryotic counterparts, highlight the diverse roles histones play in DNA organization across all domains of life.

Histones, traditionally known for organizing and regulating DNA in eukaryotes and archaea, have recently been discovered in bacteria, opening up a new frontier in our understanding of genome organization across the domains of life. Our study investigates the largely unexplored DNA-binding properties of bacterial histones, focusing on HBb in Bdellovibrio bacteriovorus. We reveal that HBb is essential for bacterial survival and exhibits DNA-binding properties similar to archaeal and eukaryotic histones. However, unlike eukaryotic and archaeal histones, which wrap DNA, HBb bends DNA without sequence specificity. This work not only broadens our understanding of DNA organization across different life forms but also suggests that bacterial histones may have diverse roles in genome organization.

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.

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.

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.

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.

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.

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.

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).

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.

Structure and function of archaeal histones.

The genomes of all organisms throughout the tree of life are compacted and organized in chromatin by association of chromatin proteins. Eukaryotic genomes encode histones, which are assembled on the genome into octamers, yielding nucleosomes. Post-translational modifications of the histones, which occur mostly on their N-terminal tails, define the functional state of chromatin. Like eukaryotes, most archaeal genomes encode histones, which are believed to be involved in the compaction and organization of their genomes. Instead of discrete multimers, in vivo data suggest assembly of "nucleosomes" of variable size, consisting of multiples of dimers, which are able to induce repression of transcription. Based on these data and a model derived from X-ray crystallography, it was recently proposed that archaeal histones assemble on DNA into "endless" hypernucleosomes. In this review, we discuss the amino acid determinants of hypernucleosome formation and highlight differences with the canonical eukaryotic octamer. We identify archaeal histones differing from the consensus, which are expected to be unable to assemble into hypernucleosomes. Finally, we identify atypical archaeal histones with short N- or C-terminal extensions and C-terminal tails similar to the tails of eukaryotic histones, which are subject to post-translational modification. Based on the expected characteristics of these archaeal histones, we discuss possibilities of involvement of histones in archaeal transcription regulation.

CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3.

The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.

StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

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.

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.

dCas9: A Versatile Tool for Epigenome Editing.

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.

Post-translational modification of nucleoid-associated proteins: an extra layer of functional modulation in bacteria?

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.

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity.

DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.

DNA recognition by Escherichia coli CbpA protein requires a conserved arginine-minor-groove interaction.

Curved DNA binding protein A (CbpA) is a co-chaperone and nucleoid associated DNA binding protein conserved in most γ-proteobacteria. Best studied in Escherichia coli, CbpA accumulates to >2500 copies per cell during periods of starvation and forms aggregates with DNA. However, the molecular basis for DNA binding is unknown; CbpA lacks motifs found in other bacterial DNA binding proteins. Here, we have used a combination of genetics and biochemistry to elucidate the mechanism of DNA recognition by CbpA. We show that CbpA interacts with the DNA minor groove. This interaction requires a highly conserved arginine side chain. Substitution of this residue, R116, with alanine, specifically disrupts DNA binding by CbpA, and its homologues from other bacteria, whilst not affecting other CbpA activities. The intracellular distribution of CbpA alters dramatically when DNA binding is negated. Hence, we provide a direct link between DNA binding and the behaviour of CbpA in cells.

E. coli Fis protein insulates the cbpA gene from uncontrolled transcription.

The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual "insulation" mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.

Chromosomal macrodomains and associated proteins: implications for DNA organization and replication in gram negative bacteria.

The Escherichia coli chromosome is organized into four macrodomains, the function and organisation of which are poorly understood. In this review we focus on the MatP, SeqA, and SlmA proteins that have recently been identified as the first examples of factors with macrodomain-specific DNA-binding properties. In particular, we review the evidence that these factors contribute towards the control of chromosome replication and segregation by specifically targeting subregions of the genome and contributing towards their unique properties. Genome sequence analysis of multiple related bacteria, including pathogenic species, reveals that macrodomain-specific distribution of SeqA, SlmA, and MatP is conserved, suggesting common principles of chromosome organisation in these organisms. This discovery of proteins with macrodomain-specific binding properties hints that there are other proteins with similar specificity yet to be unveiled. We discuss the roles of the proteins identified to date as well as strategies that may be employed to discover new factors.