Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.     

The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: a molecular perspective

The bacterial genome is folded into a compact structure called the nucleoid. Considerable compaction of the DNA molecule is required in order to reduce its volume below that of the cell. Several mechanisms, such as molecular crowding and DNA supercoiling contribute to the compactness of the nucleoid. Besides these mechanisms, a number of architectural proteins associate with the chromosomal DNA and cause it to fold into a compact structure by bridging, bending or wrapping DNA. In this review, we provide an overview of the major nucleoid-associated proteins from a structural perspective and we discuss their possible roles in dynamically shaping the bacterial nucleoid.     

A comparative proteomic approach to better define Deinococcus nucleoid specificities

Compared to radiation-sensitive bacteria, the nucleoids of radiation-resistant Deinococcus species show a higher degree of compaction. Such a condensed nucleoid may contribute to the extreme radiation resistance of Deinococcus by limiting dispersion of radiation-induced DNA fragments. Architectural proteins may play a role in this high degree of nucleoid compaction, but comparative genomics revealed only a limited number of Deinococcus homologs of known nucleoid-associated proteins (NAPs) from other species such as Escherichia coli. A comparative proteomic approach was used to identify potentially novel proteins from isolated nucleoids of Deinococcus radiodurans and Deinococcus deserti. Proteins in nucleoid enriched fractions were identified and semi-quantified by shotgun proteomics. Based on normalized spectral counts, the histone-like DNA-binding protein HU appeared to be the most abundant among candidate NAPs from both micro-organisms. By immunofluorescence microscopy, D. radiodurans HU and both DNA gyrase subunits were shown to be distributed throughout the nucleoid structure and absent from the cytoplasm. Taken together, our results suggest that D. radiodurans and D. deserti bacteria contain a very low diversity of NAPs, with HU and DNA gyrase being the main proteins involved in the organization of the Deinococcus nucleoids.     

NapM,a new nucleoid‐associated protein,broadly regulates gene expression and affects mycobacterial resistance to anti‐tuberculosis drugs

Nucleoid‐associated proteins (NAPs) play important roles in the global organization of bacterial chromosomes. However, potential NAPs and their functions are barely characterized in mycobacteria. In this study, NapM, an alkaline protein, functions as a new NAP. NapM is conserved in all of the sequenced mycobacterial genomes, and can recognize DNA in a length‐dependent but sequence‐independent manner. It prefers AT‐rich DNA and binds to the major groove. NapM possesses a clear DNA‐bridging function, and can protect DNA from DNase I digestion. NapM globally regulates the expression of more than 150 genes and the resistance of Mycobacterium smegmatis to two anti‐tuberculosis drugs, namely, rifampicin and ethambutol. An ABC transporter operon was found to be specifically responsible for the napM‐dependent ethambutol resistance of M. smegmatis. NapM also presents a similar regulation of anti‐tuberculosis drug resistance in M. tuberculosis. These results suggest that NapM is a new member of the mycobacterial NAP family. Our findings expand the range of identified NAPs and improve the understanding on the relationship between NAPs with antibiotic resistance in mycobacteria.     

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.     

拟核结合蛋白与细菌基因的表达调控

拟核结合蛋白是细菌遗传物质组织和基因表达调控的关键. 细菌基因组压缩为致密的拟核必需有拟核结合蛋白的支撑. 拟核结合蛋白、DNA超螺旋和大分子簇在拟核的结构形成中起到重要作用,其中拟核结合蛋白最重要.拟核结合蛋白还影响细菌DNA的复制、重组、转录和修复等多个重要生理过程.作为全局调控因子,拟核结合蛋白是调控细菌适应环境变化所需基因表达的关键. 本文总结拟核结合蛋白的结构、功能和调控,特别是其在致病与非致病分枝杆菌中的差别,为寻找新药物靶标提供线索.     

A Systematic In Vitro Study of Nucleoprotein Complexes Formed by Bacterial Nucleoid-Associated Proteins Revealing Novel Types of DNA Organization

Bacterial nucleoid is a dynamic entity that changes its three-dimensional shape and compaction depending on cellular physiology. While these changes are tightly associated with compositional alterations of abundant nucleoid-associated proteins implicated in reshaping the nucleoid, their cooperation in regular long-range DNA organization is poorly understood. In this study, we reconstitute a novel nucleoprotein structure in vitro, which is stabilized by cooperative effects of major bacterial DNA architectural proteins. While, individually, these proteins stabilize alternative DNA architectures consistent with either plectonemic or toroidal coiling of DNA, the combination of histone-like protein, histone-like nucleoid structuring protein, and integration host factor produces a conspicuous semiperiodic structure. By employing a bottom-up in vitro approach, we thus characterize a minimum set of bacterial proteins cooperating in organizing a regular DNA structure. Visualized structures suggest a mechanism for nucleation of topological transitions underlying the reshaping of DNA by bacterial nucleoid-associated proteins.     

Enhanced conformational flexibility of the histone-like (HU) protein from Mycoplasma gallisepticum

The histone-like (HU) protein is one of the major nucleoid-associated proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This small positively charged dimeric protein binds DNA in a non-sequence specific manner promoting DNA super-structures. The majority of HU proteins are highly conserved among bacteria; however, HU protein from Mycoplasma gallisepticum (HUMgal) has multiple amino acid substitutions in the most conserved regions, which are believed to contribute to its specificity to DNA targets unusual for canonical HU proteins. In this work, we studied the structural dynamic properties of the HUMgal dimer by NMR spectroscopy and MD simulations. The obtained all-atom model displays compliance with the NMR data and confirms the heterogeneous backbone flexibility of HUMgal. We found that HUMgal, being folded into a dimeric conformation typical for HU proteins, has a labile α-helical body with protruded β-stranded arms forming DNA-binding domain that are highly flexible in the absence of DNA. The amino acid substitutions in conserved regions of the protein are likely to affect the conformational lability of the HUMgal dimer that can be responsible for complex functional behavior of HUMgal in vivo, e.g. facilitating its spatial adaptation to non-canonical DNA-targets.     

Direct regulation of topoisomerase activity by a nucleoid-associated protein

The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.     

DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins

The volume of a typical Eschericia coli nucleoid is roughly 10⁴ times smaller than the volume of a freely coiling linear DNA molecule with the same length as the E. coli genome. We review the main forces that have been suggested to contribute to this compaction factor: macromolecular crowding (that “pushes” the DNA together), DNA charge neutralization by various polycationic species (that “glues” the DNA together), and finally, DNA deformations due to DNA supercoiling and nucleoid proteins. The direct contributions of DNA supercoiling and nucleoid proteins to the total compaction factor are probably small. Instead, we argue that the formation of the bacterial nucleoid can be described as a consequence of the influence of macromolecular crowding on thick, supercoiled protein-DNA fibers, that have been partly charge neutralized by small multivalent cations.     

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.     

The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin

The bacterial chromosomal DNA is folded into a compact structure called nucleoid. The shape and size of this 'body' is determined by a number of factors. Major players are DNA supercoiling, macromolecular crowding and architectural proteins, associated with the nucleoid, which are the topic of this MicroReview. Although many of these proteins were identified more than 25 years ago, the molecular mechanisms involved in the organization and compaction of DNA have only started to become clear in recent years. Many of these new insights can be attributed to the use of recently developed biophysical techniques.     

DNA damage induces nucleoid compaction via the Mre11‐Rad50 complex in the archaeon Haloferax volcanii

In prokaryotes the genome is organized in a dynamic structure called the nucleoid, which is embedded in the cytoplasm. We show here that in the archaeon Haloferax volcanii, compaction and reorganization of the nucleoid is induced by stresses that damage the genome or interfere with its replication. The fraction of cells exhibiting nucleoid compaction was proportional to the dose of the DNA damaging agent, and results obtained in cells defective for nucleotide excision repair suggest that breakage of DNA strands triggers reorganization of the nucleoid. We observed that compaction depends on the Mre11‐Rad50 complex, suggesting a link to DNA double‐strand break repair. However, compaction was observed in a radA mutant, indicating that the role of Mre11‐Rad50 in nucleoid reorganisation is independent of homologous recombination. We therefore propose that nucleoid compaction is part of a DNA damage response that accelerates cell recovery by helping DNA repair proteins to locate their targets, and facilitating the search for intact DNA sequences during homologous recombination.     

A-tract clusters may facilitate DNA packaging in bacterial nucleoid

Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be a propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816–3820]. To further elaborate this concept, we analyzed positioning of A-tracts (the sequence motifs introducing the most pronounced DNA curvature) in the Escherichia coli genome. First, we observed that the A-tracts are over-represented and distributed ‘quasi-regularly’ throughout the genome, including both the coding and intergenic sequences. Second, there is a 10–12 bp periodicity in the A-tract positioning indicating that the A-tracts are phased with respect to the DNA helical repeat. Third, the phased A-tracts are organized in ~100 bp long clusters. The latter feature was revealed with the help of a novel approach based on the Fourier series expansion of the A-tract distance autocorrelation function. Since the A-tracts introduce local bends of the DNA duplex and these bends accumulate when properly phased, the observed clusters would facilitate DNA looping. Also, such clusters may serve as binding sites for the nucleoid-associated proteins that have affinities for curved DNA (such as HU, H-NS, Hfq and CbpA). Therefore, we suggest that the ~100 bp long clusters of the phased A-tracts constitute the ‘structural code’ for DNA compaction by providing the long-range intrinsic curvature and increasing stability of the DNA complexes with architectural proteins.     

Streptomyces IHF uses multiple interfaces to bind DNA

BackgroundNucleoid associated proteins (NAPs) are essential for chromosome condensation in bacterial cells. Despite being a diverse group, NAPs share two common traits: they are small, oligomeric proteins and their oligomeric state is critical for DNA condensation. Streptomyces coelicolor IHF (sIHF) is an actinobacterial-specific nucleoid-associated protein that despite its name, shares neither sequence nor structural homology with the well-characterized Escherichia coli IHF. Like E. coli IHF, sIHF is needed for efficient nucleoid condensation, morphological development and antibiotic production in S. coelicolor.MethodsUsing a combination of crystallography, small-angle X-ray scattering, electron microscopy and structure-guided functional assays, we characterized how sIHF binds and remodels DNA.ResultsThe structure of sIHF bound to DNA revealed two DNA-binding elements on opposite surfaces of the helix bundle. Using structure-guided functional assays, we identified an additional surface that drives DNA binding in solution. Binding by each element is necessary for both normal development and antibiotic production in vivo, while in vitro, they act collectively to restrain negative supercoils.ConclusionsThe cleft defined by the N-terminal and the helix bundle of sIHF drives DNA binding, but the two additional surfaces identified on the crystal structure are necessary to stabilize binding, remodel DNA and maintain wild-type levels of antibiotic production. We propose a model describing how the multiple DNA-binding elements enable oligomerization-independent nucleoid condensation.General significanceThis work provides a new dimension to the mechanistic repertoire ascribed to bacterial NAPs and highlights the power of combining structural biology techniques to study sequence unspecific protein-DNA interactions.