Histone-like Nucleoid Structuring Protein Research Articles

Role of the Histone-Like Nucleoid Structuring Protein (H-NS) in the Regulation of Virulence Factor Expression and Stress Response in Vibrio vulnificus.

Temperature is one of the important parameters regulating the expression of virulence factors in bacteria. The global regulator, a histone-like nucleoid structuring protein (H-NS), is known to play a crucial role in this regulation. In the present study, we first clarified the role of H-NS in the temperature-dependent regulation of virulence factor production in Vibrio vulnificus, including that of the cytolytic toxin (V. vulnificus hemolysin: VVH) and the proteolytic enzyme (V. vulnificus protease: VVP). The expression of hns itself was subjected to temperature regulation, where hns was expressed more at 26 ℃ than at 37 ℃. VVH production and the expression of its gene vvhA were increased by disruption of the hns gene. H-NS appeared to affect the vvhA expression by the well-documented transcriptional silencing mechanism. On the other hand, hns disruption resulted in the reduction of VVP production and the expression of its gene vvpE. H-NS was suggested to positively regulate vvpE expression through the increase in the level of the rpoS mRNA. Moreover, H-NS was found to contribute to the survival of V. vulnificus in stressful environments. When compared to the wild type strain, the hns mutant exhibited reduced survival rates when subjected to acidic pH, hyperosmotic and oxidative stress.

Modeling the Binding of H-NS to DNA

Bacterial chromosomal DNA is organized within a structure called the nucleoid, which is distinctly different from the rest of the cytoplasm. Bacteria have a number of nucleoid-associated proteins that influence the organization of the nucleoid by bending, wrapping or bridging DNA. The Histone-like Nucleoid Structuring protein H-NS can bridge DNA by binding to two separate DNA duplexes, or shield the DNA by binding to distant sites on the same duplex, depending on external conditions. H-NS occurs in Gram-negative enterobacteria and silences genes involved in bacterial virulence and antibiotic resistance.

Bacterial nucleoid-associated protein uncouples transcription levels from transcription timing.

ABSTRACTThe histone-like nucleoid-structuring (H-NS) protein binds to horizontally acquired genes in the bacterium Salmonella enterica serovar Typhimurium, silencing their expression. We now report that overcoming the silencing effects of H-NS imposes a delay in the expression of genes activated by the transcriptional regulator PhoP. We determine that PhoP-activated genes ancestral to Salmonella are expressed before those acquired horizontally. This expression timing reflects the in vivo occupancy of the corresponding promoters by the PhoP protein. These results are surprising because some of these horizontally acquired genes reached higher mRNA levels than ancestral genes expressed earlier and were transcribed from promoters harboring PhoP-binding sites with higher in vitro affinity for the PhoP protein. Our findings challenge the often-made assumption that for genes coregulated by a given transcription factor, early genes are transcribed to higher mRNA levels than those transcribed at later times. Moreover, they provide a singular example of how gene ancestry can impact expression timing.

Equilibration of complexes of DNA and H-NS proteins on charged surfaces: A coarse-grained model point of view

The Histone-like Nucleoid Structuring protein (H-NS) is a nucleoid-associated protein, which is involved in both gene regulation and DNA compaction. Although it is a key player in genome organization by forming bridges between DNA duplexes, the precise structure of complexes of DNA and H-NS proteins is still not well understood. In particular, it is not clear whether the structure of DNA/H-NS complexes in the living cell is similar to that of complexes deposited on mica surfaces, which may be observed by AFM microscopy. A coarse-grained model, which helps getting more insight into this question, is described and analyzed in the present paper. This model is able of describing both the bridging of bacterial DNA by H-NS in the bulk and the deposition and equilibration of the complex on a charged surface. Simulations performed with the model reveal that a slight attraction between DNA and the charged surface is sufficient to let DNA/H-NS complexes reorganize from 3D coils to planar plasmids bridged by H-NS proteins similar to those observed by AFM microscopy. They furthermore highlight the antagonistic effects of the interactions between DNA and the surface. Indeed, increasing these interactions slows down the equilibration of naked plasmids on the surface but, on the other hand, enables a faster equilibration of DNA/H-NS complexes. Based on the distribution of the lifetimes of H-NS bridges and the time evolution of the number of trans-binding protein dimers during equilibration of the complexes on the surface, it is argued that the decrease of the equilibration time of the complex upon increase of the interaction strength between DNA and the surface is ascribable to the associated decrease of the probability to form new bridges between DNA and the proteins.

H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1

The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity.

Protein expression and transcription profiles of three strains of Aeromonas salmonicida ssp. salmonicida under normal and iron-limited culture conditions.

BackgroundAeromonas salmonicida is an important fish pathogen that produces a wide and varied array of virulence factors. Here we used iron deprivation by addition of the chelator 2’2-dipyridyl to induce the expression of several such virulence factors in three isolates of Aeromonas salmonicida (one avirulent and two virulent). By using SDS-PAGE followed by mass spectrometry, we identified proteins that appeared differentially expressed under these conditions. The differential transcription of the identified gene products were subsequently measured by reverse transcription quantitative real-time PCR (RT-qPCR).ResultsOur initial screening using SDS-PAGE identified five proteins that appeared differentially expressed in virulent and avirulent isolates or, within the same isolates, between bacteria cultivated under iron-rich or iron-deprived conditions. The transcription of the genes coding for these proteins were subsequently quantified by RT-qPCR. Results of this analysis demonstrated that the gene coding for alkyl hydroperoxide reductase (AhpC), a protein involved in oxidative stress response, was transcribed at a higher rate in the virulent strain as compared to the avirulent strain. Additionally, it was observed that addition of an iron chelator to the culture medium lead to a reduction of the transcription levels of the regulatory histone-like nucleoid structuring protein (H-NS). This was consistent in all three isolates. On the other hand, the transcription levels of the virulence array protein (VapA) and the protein ATP-synthetase F (ATPF) displayed only limited changes, despite being the dominant component of a protein fraction that displayed changes during the preliminary SDS-PAGE screening. This was true regardless of the culture conditions and of the isolates considered. Finally, transcription of the enzyme enolase was upregulated in the iron-deprived broths in all isolates.ConclusionsWe identified several genes differentially expressed under culture conditions known to lead to the overexpression of virulence factors. In addition, we identified alkyl hydroperoxide as being overexpressed in the virulent isolates compared to the avirulent isolates. The results from this study will contribute to enhance our understanding of the virulence of A. salmonicida and may suggest new directions for further research.

Locus of Enterocyte Effacement-encoded Regulator (Ler) of Pathogenic Escherichia coli Competes Off Histone-like Nucleoid-structuring Protein (H-NS) through Noncooperative DNA Binding

The locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) functions to activate transcription of virulence genes silenced by the histone-like nucleoid-structuring protein (H-NS). Despite its important role in the bacterial gene regulation, the binding mode of Ler to DNA and its mechanism in alleviating genes repressed by H-NS are largely unknown. In this study, we use magnetic tweezers to demonstrate that Ler binds extended DNA through a largely noncooperative process, which results in DNA stiffening and DNA folding depending on protein concentration. We also show that Ler can replace prebound H-NS on DNA over a range of potassium and magnesium concentrations. Our findings reveal the DNA binding properties of Ler and shed light to further understand the anti-silencing activity of Ler.

Identification of high-specificity H-NS binding site in LEE5 promoter of enteropathogenic Esherichia coli (EPEC)

Histone-like nucleoid structuring protein (H-NS) is a small but abundant protein present in enteric bacteria and is involved in compaction of the DNA and regulation of the transcription. Recent reports have suggested that H-NS binds to a specific AT rich DNA sequence than to intrinsically curved DNA in sequence independent manner. We detected two high-specificity H-NS binding sites in LEE5 promoter of EPEC centered at -110 and -138, which were close to the proposed consensus H-NS binding motif. To identify H-NS binding sequence in LEE5 promoter, we took a random mutagenesis approach and found the mutations at around -138 were specifically defective in the regulation by H-NS. It was concluded that H-NS exerts maximum repression via the specific sequence at around -138 and subsequently contacts a subunit of RNAP through oligomerization.

Widespread suppression of intragenic transcription initiation by H-NS

Widespread intragenic transcription initiation has been observed in many species. Here we show that the Escherichia coli ehxCABD operon contains numerous intragenic promoters in both sense and antisense orientations. Transcription from these promoters is silenced by the histone-like nucleoid structuring (H-NS) protein. On a genome-wide scale, we show that 46% of H-NS-suppressed transcripts in E. coli are intragenic in origin. Furthermore, many intergenic promoters repressed by H-NS are for noncoding RNAs (ncRNAs). Thus, a major overlooked function of H-NS is to prevent transcription of spurious RNA. Our data provide a molecular description for the toxicity of horizontally acquired DNA and explain how this is counteracted by H-NS.

The Force Dependent Protein-DNA Structure of H-NS

Horizontal gene transfer (HGT) is a major driver of bacterial evolution, allowing for rapid development of deleterious functions - such as virulence or antibiotic resistance. However, expressing foreign DNA without selection is at best a waste of resources and at worst fatal to the bacterium. Bacteria have, therefore, developed mechanisms to selectively silence foreign genes. For instance, the histone-like nucleoid-structuring protein (H-NS) silences foreign genes that are recognized by their AT content. It has previously been shown that the binding of H-NS to AT-rich DNA dramatically changes the persistence length of the DNA. However, we discovered that the persistence length measured under no load, via tethered particle motion (TPM), and under increasing H-NS concentrations was much less sensitive to concentration than when stretched with optical tweezers. Our results indicate a previously unknown protein-DNA structure which breaks down under an applied force.

Probing the Unbinding Kinetics of DNA-H-NS-DNA Protein Complexes by a High-Speed and High-Throughput Single-Molecule Pulling Assay using Atomic Force Microscopy

We demonstrate an atomic force microscopy (AFM) pulling assay to measure the unbinding kinetics of DNA-protein interactions. Here we use this technique to investigate the interactions between DNA and the abundant, non-specific nucleoid-associated protein H-NS (Histone-like Nucleoid-Structuring protein). In particular, we probe the unbinding kinetics of the DNA-H-NS-DNA bridges under the influence of one environmental factor, Mg2+-ions. In this experiment, biotinylated DNA molecules are attached on an avidin-coated mica surface. Then thiolated DNA molecules and H-NS proteins are added to the sample solution to form DNA-H-NS-DNA complexes. Individual DNA-protein complexes are then pulled by a gold-coated AFM tip. By measuring the force-distance curve, we gain information on the strength with which H-NS forms bridges between DNA molecules. Here, we probe both single DNA-H-NS-DNA complexes and sample many complexes by fast scanning of the AFM tip over the sample's surface and performing approach/pulling cycles for each position. This experimental approach leads to high-throughput measurements with single-molecule resolution and is widely applicable to other DNA-bridging proteins and receptor-ligand interactions.

A Single Molecule Study of Gene Silencing by HNS Protein DNA Interactions

Histone-like nucleoid structuring (H-NS) proteins are an important prokaryotic protein responsible for organizing and compacting chromosomal DNA. Recently, it was found that H-NS can identify foreign genes acquired by horizontal gene transfer (HGT), based on AT content, and selectively silence these genes. Single-molecule studies have shown that H-NS binding can affect DNA rigidity and sometimes loop the DNA. However, most of these studies are done with λ DNA, which is neither part of the bacterial genome nor a sequence commonly acquired by HGT. We have been using optical tweezers to study gene silencing by H-NS on a variety of Salmonella genes whose interaction with H-NS is well characterized in vivo. We have also been studying how the presence of various cofactors of H-NS, such as Hha, cooperatively affect these genes. These experiments greatly improve our understanding of the biophysical mechanism behind how H-NS protein selectively interacts with and silences foreign DNA.

Molecular Engineering of a Fluorescent Bioprobe for Sensitive and Selective Detection of Amphibole Asbestos

Fluorescence microscopy-based affinity assay could enable highly sensitive and selective detection of airborne asbestos, an inorganic environmental pollutant that can cause mesothelioma and lung cancer. We have selected an Escherichia coli histone-like nucleoid structuring protein, H-NS, as a promising candidate for an amphibole asbestos bioprobe. H-NS has high affinity to amphibole asbestos, but also binds to an increasingly common asbestos substitute, wollastonite. To develop a highly specific Bioprobe for amphibole asbestos, we first identified a specific but low-affinity amosite-binding sequence by slicing H-NS into several fragments. Second, we constructed a streptavidin tetramer complex displaying four amosite-binding fragments, resulting in the 250-fold increase in the probe affinity as compared to the single fragment. The tetramer probe had sufficient affinity and specificity for detecting all the five types of asbestos in the amphibole group, and could be used to distinguish them from wollastonite. In order to clarify the binding mechanism and identify the amino acid residues contributing to the probe’s affinity to amosite fibers, we constructed a number of shorter and substituted peptides. We found that the probable binding mechanism is electrostatic interaction, with positively charged side chains of lysine residues being primarily responsible for the probe’s affinity to asbestos.

H-NS can facilitate specific DNA-binding by RNA polymerase in AT-rich gene regulatory regions.

Extremely AT-rich DNA sequences present a challenging template for specific recognition by RNA polymerase. In bacteria, this is because the promoter −10 hexamer, the major DNA element recognised by RNA polymerase, is itself AT-rich. We show that Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions. Thus, at the Escherichia coli ehxCABD operon, RNA polymerase is unable to distinguish between the promoter −10 element and similar overlapping sequences. This problem is resolved in native nucleoprotein because the overlapping sequences are masked by H-NS. Our work provides mechanistic insight into nucleoprotein structure and its effect on protein-DNA interactions in prokaryotic cells.

Protein oligomers studied by solid‐state NMR – the case of the full‐length nucleoid‐associated protein histone‐like nucleoid structuring protein

Members of the histone-like nucleoid structuring protein (H-NS) family play roles both as architectural proteins and as modulators of gene expression in Gram-negative bacteria. The H-NS protein participates in modulatory processes that respond to environmental changes in osmolarity, pH, or temperature. H-NS oligomerization is essential for its activity. Structural models of different truncated forms are available. However, high-resolution structural details of full-length H-NS and its DNA-bound state have largely remained elusive. We report on progress in characterizing the biologically active H-NS oligomers with solid-state NMR. We compared uniformly ((13)C,(15)N)-labeled ssNMR preparations of the isolated N-terminal region (H-NS 1-47) and full-length H-NS (H-NS 1-137). In both cases, we obtained ssNMR spectra of good quality and characteristic of well-folded proteins. Analysis of the results of 2D and 3D (13)C-(13)C and (15)N-(13)C correlation experiments conducted at high magnetic field led to assignments of residues located in different topological regions of the free full-length H-NS. These findings confirm that the structure of the N-terminal dimerization domain is conserved in the oligomeric full-length protein. Small changes in the dimerization interface suggested by localized chemical shift variations between solution and solid-state spectra may be relevant for DNA recoginition.

H-NS Plays a Role in Expression of Acinetobacter baumannii Virulence Features

Acinetobacter baumannii has become a major problem in the clinical setting with the prevalence of infections caused by multidrug-resistant strains on the increase. Nevertheless, only a limited number of molecular mechanisms involved in the success of A. baumannii as a human pathogen have been described. In this study, we examined the virulence features of a hypermotile derivative of A. baumannii strain ATCC 17978, which was found to display enhanced adherence to human pneumocytes and elevated levels of lethality toward Caenorhabditis elegans nematodes. Analysis of cellular lipids revealed modifications to the fatty acid composition, providing a possible explanation for the observed changes in hydrophobicity and subsequent alteration in adherence and motility. Comparison of the genome sequences of the hypermotile variant and parental strain revealed that an insertion sequence had disrupted an hns-like gene in the variant. This gene encodes a homologue of the histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes seen in the autotransporter Ata, a type VI secretion system, and a type I pilus cluster. Interestingly, isolation and analysis of a second independent hypermotile ATCC 17978 variant revealed a mutation to a residue within the DNA binding region of H-NS. Taken together, these mutants indicate that the phenotypic and transcriptomic differences seen are due to loss of regulatory control effected by H-NS.

Pack it up, Pack it in: Unraveling H-NS Mediated Genome Packaging

Pack it up, Pack it in: Unraveling H-NS Mediated Genome Packaging

A Model of H-NS Mediated Compaction of Bacterial DNA

The histone-like nucleoid structuring protein (H-NS) is a nucleoid-associated protein, which is involved in both gene regulation and DNA compaction. H-NS can bind to DNA in two different ways: in trans, by binding to two separate DNA duplexes, or in cis, by binding to different sites on the same duplex. Based on scanning force microscopy imaging and optical trap-driven unzipping assays, it has recently been suggested that DNA compaction may result from the antagonistic effects of H-NS binding to DNA in trans and cis configurations. To get more insight into the compaction mechanism, we constructed a coarse-grained model description of the compaction of bacterial DNA by H-NS. These simulations highlight the fact that DNA compaction indeed results from the subtle equilibrium between several competing factors, which include the deformation dynamics of the plasmid and the several binding modes of protein dimers to DNA, i.e., dangling configurations, cis- and trans-binding. In particular, the degree of compaction is extremely sensitive to the difference in binding energies of the cis and trans configurations. Our simulations also point out that the conformations of the DNA-protein complexes are significantly different in bulk and in planar conditions, suggesting that conformations observed on mica surfaces may differ significantly from those that prevail in living cells.

The H-NS protein silences thepypregulatory network ofYersinia enterocoliticaand is involved in controlling biofilm formation

Horizontal gene transfer plays an important role in bacterial evolution. DNA acquired by horizontal gene transfer has to be incorporated into existing regulatory networks. The histone-like nucleoid structuring protein H-NS acts as a silencer of horizontally acquired genes to avoid potential damage. However, specific regulators can overcome H-NS repression, resulting in the integration of newly acquired genes into existing regulatory networks. Here, we analyzed the influence of H-NS on the transcription of the Yersinia enterocolitica hreP gene and its regulators pypA, pypB, and pypC by establishing a dominant-negative H-NS version. Using transcriptional fusions and electrophoretic mobility shift assays, we show that H-NS silences hreP, pypA, pypB, and pypC by direct interactions. While the H-NS antagonist RovA activates pypC, it has no effect on pypA and pypB. Furthermore, H-NS affects biofilm formation in Y.enterocolitica.

Rho and NusG suppress pervasive antisense transcription in Escherichia coli

Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.