Histone-like Nucleoid Structuring Research Articles

Nanoscale reorganizations of histone-like nucleoid structuring proteins in Escherichia coli are caused by silver nanoparticles

Silver nanoparticles (AgNPs) and ions (Ag+) have recently gained broad attention due to their antimicrobial effects against bacteria and other microbes. In this work, we demonstrate the use of super-resolution fluorescence microscopy for investigating and quantifying the antimicrobial effect of AgNPs at the molecular level. We found that subjecting Escherichia coli (E. coli) bacteria to AgNPs led to nanoscale reorganization of histone-like nucleoid structuring (H-NS) proteins, an essential nucleoid associated protein in bacteria. We observed that H-NS proteins formed denser and larger clusters at the center of the bacteria after exposure to AgNPs. We quantified the spatial reorganizations of H-NS proteins by examining the changes of various spatial parameters, including the inter-molecular distances and molecular densities. Clustering analysis based on Voronoi-tessellation were also performed to characterize the change of H-NS proteins’ clustering behavior. We found that AgNP-treatment led to an increase in the fraction of H-NS proteins forming clusters. Similar effects were observed for bacteria exposed to Ag+ ions, suggesting that the release of Ag+ ions plays an important role in the toxicity of AgNPs. On the other hand, we observed that AgNPs with two surface coatings showed difference in the nanoscale reorganization of H-NS proteins, indicating that particle-specific effects also contribute to the antimicrobial activities of AgNPs. Our results suggested that H-NS proteins were significantly affected by AgNPs and Ag+ ions, which has been overlooked previously. In addition, we examined the dynamic motion of AgNPs that were attached to the surface of bacteria. We expect that the current methodology can be readily applied to broadly and quantitatively study the spatial reorganization of biological macromolecules at the scale of nanometers caused by metal nanoparticles, which are expected to shed new light on the antimicrobial mechanism of metal nanoparticles.

Threonine Phosphorylation Fine-Tunes the Regulatory Activity of Histone-Like Nucleoid Structuring Protein in Salmonella Transcription.

Histone-like nucleoid structuring protein (H-NS) in enterobacteria plays an important role in facilitating chromosome organization and functions as a crucial transcriptional regulator for global gene regulation. Here, we presented an observation that H-NS of Salmonella enterica serovar Typhimurium could undergo protein phosphorylation at threonine 13 residue (T13). Analysis of the H-NS wild-type protein and its T13E phosphomimetic substitute suggested that T13 phosphorylation lead to alterations of H-NS structure, thus reducing its dimerization to weaken its DNA binding affinity. Proteomic analysis revealed that H-NS phosphorylation exerts regulatory effects on a wide range of genetic loci including the PhoP/PhoQ-regulated genes. In this study, we investigated an effect of T13 phosphorylation of H-NS that rendered transcription upregulation of the PhoP/PhoQ-activated genes. A lower promoter binding of the T13 phosphorylated H-NS protein was correlated with a stronger interaction of the PhoP protein, i.e., a transcription activator and also a competitor of H-NS, to the PhoP/PhoQ-dependent promoters. Unlike depletion of H-NS which dramatically activated the PhoP/PhoQ-dependent transcription even in a PhoP/PhoQ-repressing condition, mimicking of H-NS phosphorylation caused a moderate upregulation. Wild-type H-NS protein produced heterogeneously could rescue the phenotype of T13E mutant and fully restored the PhoP/PhoQ-dependent transcription enhanced by T13 phosphorylation of H-NS to wild-type levels. Therefore, our findings uncover a strategy in S. typhimurium to fine-tune the regulatory activity of H-NS through specific protein phosphorylation and highlight a regulatory mechanism for the PhoP/PhoQ-dependent transcription via this post-translational modification.

Predicting the mechanism and rate of H-NS binding to AT-rich DNA.

Bacteria contain several nucleoid-associated proteins that organize their genomic DNA into the nucleoid by bending, wrapping or bridging DNA. The Histone-like Nucleoid Structuring protein H-NS found in many Gram-negative bacteria is a DNA bridging protein and can structure DNA by binding to two separate DNA duplexes or to adjacent sites on the same duplex, depending on external conditions. Several nucleotide sequences have been identified to which H-NS binds with high affinity, indicating H-NS prefers AT-rich DNA. To date, highly detailed structural information of the H-NS DNA complex remains elusive. Molecular simulation can complement experiments by modelling structures and their time evolution in atomistic detail. In this paper we report an exploration of the different binding modes of H-NS to a high affinity nucleotide sequence and an estimate of the associated rate constant. By means of molecular dynamics simulations, we identified three types of binding for H-NS to AT-rich DNA. To further sample the transitions between these binding modes, we performed Replica Exchange Transition Interface Sampling, providing predictions of the mechanism and rate constant of H-NS binding to DNA. H-NS interacts with the DNA through a conserved QGR motif, aided by a conserved arginine at position 93. The QGR motif interacts first with phosphate groups, followed by the formation of hydrogen bonds between acceptors in the DNA minor groove and the sidechains of either Q112 or R114. After R114 inserts into the minor groove, the rest of the QGR motif follows. Full insertion of the QGR motif in the minor groove is stable over several tens of nanoseconds, and involves hydrogen bonds between the bases and both backbone and sidechains of the QGR motif. The rate constant for the process of H-NS binding to AT-rich DNA resulting in full insertion of the QGR motif is in the order of 106 M−1s−1, which is rate limiting compared to the non-specific association of H-NS to the DNA backbone at a rate of 108 M−1s−1.

Properties and directionality of intragenic promoters in E. coli

The histone-like nucleoid structuring (H-NS) protein targets and binds AT-rich DNA. Oligomerisation of H-NS along AT-rich genes represses transcription from spurious intragenic promoters within the coding sequences of AT-rich genes. In this work, we sought to better understand why promoters occur so frequently within AT-rich DNA. To do this, we compared the properties of (i) canonical promoters (ii) promoters within H-NS bound genes and (iii) promoters generated by random combinations of nucleotides. We have identified and analysed many active intragenic promoters distributed within AT-rich genes in the E. coli genome. We show that these promoters, and promoters from randomly generated AT-rich DNA, differ from canonical promoters in several ways. In particular, spurious promoters are often dependent on AT-tracts upstream of the promoter −10 element. These AT-tracts play a key role by altering DNA curvature and facilitating interactions between the promoter and RNA polymerase sigma factor. Promoter regions inside genes are also often bidirectional.

Identifying the region responsible for Brucella abortus MucR higher-order oligomer formation and examining its role in gene regulation

MucR is a member of the Ros/MucR family of prokaryotic zinc-finger proteins found in the α-proteobacteria which regulate the expression of genes required for the successful pathogenic and symbiotic interactions of these bacteria with the eukaryotic hosts. The structure and function of their distinctive zinc-finger domain has been well-studied, but only recently the quaternary structure of the full length proteins was investigated demonstrating their ability to form higher-order oligomers. The aim of this study was to identify the region of MucR involved in higher-order oligomer formation by analysing deletion and point mutants of this protein by Light Scattering, and to determine the role that MucR oligomerization plays in the regulatory function of this protein. Here we demonstrate that a conserved hydrophobic region at the N-terminus of MucR is responsible for higher-order oligomer formation and that MucR oligomerization is essential for its regulatory function in Brucella. All these features of MucR are shared by the histone-like nucleoid structuring protein, (H-NS), leading us to propose that the prokaryotic zinc-finger proteins in the MucR/Ros family control gene expression employing a mechanism similar to that used by the H-NS proteins, rather than working as classical transcriptional regulators.

Klebsiella pneumoniae Type VI Secretion System Contributes to Bacterial Competition, Cell Invasion, Type-1 Fimbriae Expression, and In Vivo Colonization.

BackgroundWe previously isolated a Klebsiella pneumoniae strain, NTUH-K2044, from a community-acquired pyogenic liver abscess (PLA) patient. Analysis of the NTUH-K2044 genome revealed that this strain harbors 2 putative type VI secretion system (T6SS)-encoding gene clusters.MethodsThe distribution of T6SS genes in the PLA and intestinal-colonizing K pneumoniae clinical isolates was examined. icmF1-, icmF2-, icmF1/icmF2-, and hcp-deficient K pneumoniae strains were constructed using an unmarked deletion method. The roles of T6SSs in antibacterial activity, type-1 fimbriae expression, cell adhesion, and invasion and intestinal colonization were determined.ResultsThe prevalence of T6SSs is higher in the PLA strains than in the intestinal-colonizing strains (37 of 42 vs 54 of 130). Deletion of icmF1/icmF2 and hcp genes significantly reduced interbacterial and intrabacterial killing. Strain deleted for icmF1 and icmF2 exhibited decreased transcriptional expression of type-1 fimbriae and reduced adherence to and invasion of human colorectal epithelial cells and was attenuated for in vivo competition to enable colonization of the host gut. Finally, Hcp expression in K pneumoniae was silenced by the histone-like nucleoid structuring protein via direct binding.ConclusionsThese results provide new insights into T6SS-mediated bacterial competition and attachment in K pneumoniae and could facilitate the prevention of K pneumoniae infection.

Metagenomic Analysis of Microbial Community Compositions and Cold-Responsive Stress Genes in Selected Antarctic Lacustrine and Soil Ecosystems.

This study describes microbial community compositions, and various cold-responsive stress genes, encompassing cold-induced proteins (CIPs) and cold-associated general stress-responsive proteins (CASPs) in selected Antarctic lake water, sediment, and soil metagenomes. Overall, Proteobacteria and Bacteroidetes were the major taxa in all metagenomes. Prochlorococcus and Thiomicrospira were highly abundant in waters, while Myxococcus, Anaeromyxobacter, Haliangium, and Gloeobacter were dominant in the soil and lake sediment metagenomes. Among CIPs, genes necessary for DNA replication, translation initiation, and transcription termination were highly abundant in all metagenomes. However, genes for fatty acid desaturase (FAD) and trehalose synthase (TS) were common in the soil and lake sediment metagenomes. Interestingly, the Lake Untersee water and sediment metagenome samples contained histone-like nucleoid structuring protein (H-NS) and all genes for CIPs. As for the CASPs, high abundances of a wide range of genes for cryo- and osmo-protectants (glutamate, glycine, choline, and betaine) were identified in all metagenomes. However, genes for exopolysaccharide biosynthesis were dominant in Lake Untersee water, sediment, and other soil metagenomes. The results from this study indicate that although diverse microbial communities are present in various metagenomes, they share common cold-responsive stress genes necessary for their survival and sustenance in the extreme Antarctic conditions.

A putative curved DNA region upstream of rcsA in Escherichia coli plays a key role in transcriptional regulation by H‐NS

It is well established that in Escherichia coli, the histone‐like nucleoid structuring (H‐NS) protein also functions as negative regulator of rcsA transcription. However, the exact mode of regulation of rcsA transcription by H‐NS has not been studied extensively. Here, we report the multicopy effect of dominant‐negative hns alleles on the transcription of rcsA based on expression of cps‐lac transcriptional fusion in ∆lon, ∆lon rpoB12, ∆lon rpoB77 and lon + strains. Our results indicate that H‐NS defective in recognizing curved DNA fails to repress rcsA transcription significantly, while nonoligomeric H‐NS molecules still retain the repressor activity to an appreciable extent. Together with bioinformatics analysis, our study envisages a critical role for the putative curved DNA region present upstream of rcsA promoter in the transcriptional regulation of rcsA by H‐NS.

Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model.

A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.

Waking the neighbours: disruption of H-NS repression by overlapping transcription.

The histone-like nucleoid structuring (H-NS) protein and its analogues bind large stretches of horizontally acquired AT-rich DNA in a broad range of bacterial species. Binding by H-NS silences the promoters within such DNA that would otherwise deplete the cellular pool of RNA polymerase. Selective de-repression can occur when sequence-specific DNA-binding proteins locally disrupt H-NS function; this mechanism is important for the regulation of many virulence genes. In this issue of Molecular Microbiology, Rangarajan and Schnetz show that when transcription from a neighbouring region invades an H-NS-bound locus, it can disrupt local H-NS repression. Moreover, they show that de-repression occurs in a dose-dependent manner, and they demonstrate a natural example of this in Escherichia coli. This finding has important implications for H-NS function and its impact on genome evolution.

Insights into transcriptional silencing and anti-silencing in Shigella flexneri: a detailed molecular analysis of the icsP virulence locus.

Transcriptional silencing and anti-silencing mechanisms modulate bacterial physiology and virulence in many human pathogens. In Shigella species, many virulence plasmid genes are silenced by the histone-like nucleoid structuring protein H-NS and anti-silenced by the virulence gene regulator VirB. Despite the key role that these regulatory proteins play in Shigella virulence, their mechanisms of transcriptional control remain poorly understood. Here, we characterize the regulatory elements and their relative spacing requirements needed for the transcriptional silencing and anti-silencing of icsP, a locus that requires remotely located regulatory elements for both types of transcriptional control. Our findings highlight the flexibility of the regulatory elements' positions with respect to each other, and yet, a molecular roadblock docked between the VirB binding site and the upstream H-NS binding region abolishes transcriptional anti-silencing by VirB, providing insight into transcriptional anti-silencing. Our study also raises the need to re-evaluate the currently proposed VirB binding site. Models of transcriptional silencing and anti-silencing at this genetic locus are presented, and the implications for understanding these regulatory mechanisms in bacteria are discussed.

New Insights into the Antimicrobial Mechanism of Silver Ions Revealed by Super-Resolution Fluorescence Microscopy

Silver compounds have been found to show antimicrobial activities against bacteria, making them a promising alternative to antibiotics. However, although exciting progresses have been made, the molecular mechanism of the antimicrobial effect of silver ions (Ag+) remains a subject of debate. In this work, we tested a hypothesis that was suggested by TEM imaging of bacteria – Ag+ causes DNA condensation – using super-resolution fluorescence microscopy in combination with biochemical assays. By examining, in vitro, the size of bacterial genomic DNA in the absence and presence of Ag+, we found that exposure to Ag+ does not directly result in DNA condensation. Instead, spatial reorganization of histone-like nucleoid structuring (H-NS) protein was observed in vivo using super-resolution fluorescence microscopy, which is expected to cause DNA compaction and condensation. It was also found that the expression of H-NS was enhanced in the presence of Ag+. In addition, we varied the treatment time and concentration of Ag+, from which we obtained the quantitative dose- and time-dependence. Our data showed that the previous hypothesis is incorrect, or at least incomplete. Our results suggest a new mechanism for Ag+'s antimicrobial activities.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

While structuring of the bacterial nucleoid by nucleoid-associated proteins (NAPs) is critical for proper chromosomal organization and compaction, DNA-dependent RNA polymerase (RNAP) must frequently interact with and overcome the barriers these NAPs impose upon transcription. One particular NAP in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS, that binds to DNA and forms nucleoprotein filaments. To specifically investigate the effect that H-NS filaments have on RNAP elongation, we developed an in vitro transcription assay to assess transcript elongation by RNAP when transcribing DNA bound by an H-NS filament. In this method, initiation and elongation by RNAP are uncoupled by initiating transcription in the presence of three rNTPs to halt elongation just downstream of the promoter. Before elongation is restarted, an H-NS filament is formed so that elongation occurs on an H-NS nucleoprotein filament template. We also describe visualization and analysis of the transcription products from the nucleoprotein template which provides insight into how H-NS and RNAP interact. This method is a starting point to determine effects of NAPs on RNAP elongation in a variety of conditions.

Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) measures the physical association between a protein and DNA in the cell. In combination with next-generation sequencing, the technique enables the identification of DNA targets for the corresponding protein across an entire genome. Here we describe the immunoprecipitation of Vibrio cholerae DNA bound to the histone-like nucleoid structuring protein (H-NS) tagged with the Flag epitope. The quality of the DNA obtained in this protocol is suitable for next-generation sequencing. The procedure described herein can be readily adapted to other bacteria and DNA-binding proteins.

Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.

The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF) was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU) caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.

Probing the mechanical stability of bridged DNA-H-NS protein complexes by single-molecule AFM pulling

Atomic force microscopy (AFM) has proven to be a powerful tool for the study of DNA-protein interactions due to its ability to image single molecules at the nanoscale. However, the use of AFM in force spectroscopy to study DNA-protein interactions has been limited. Here we developed a high throughput, AFM based, pulling assay to measure the strength and kinetics of protein bridging of DNA molecules. As a model system, we investigated the interactions between DNA and the Histone-like Nucleoid-Structuring protein (H-NS). We confirmed that H-NS both changes DNA rigidity and forms bridges between DNA molecules. This straightforward methodology provides a high-throughput approach with single-molecule resolution which is widely applicable to study cross-substrate interactions such as DNA-bridging proteins.

An H-NS Family Protein, Sfh, Regulates Acid Resistance by Inhibition of Glutamate Decarboxylase Expression in Shigella flexneri 2457T.

The glutamate-dependent acid-resistance system is the most effective acid tolerance pathway in Shigella, allowing survival in extremely acidic environments. However, the regulation of this system in Shigella remains elusive. In the current study, we identified significant differences in the levels of glutamate decarboxylase between three Shigella flexneri strains with different levels of acid resistance using blue native-polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF)/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the degree of acid resistance and the levels of GadA/B were significantly lower in strain 2457T compared with two other S. flexneri strains. It has been reported that plasmid pSf-R27 is expressed in strain 2457T but not in the other 142 sequenced S. flexneri isolates. pSf-R27 encodes protein Sfh, which belongs to a family of histone-like nucleoid-structuring (H-NS) proteins that participate in the transcriptional control of glutamate-dependent acid resistance, implicating pSf-R27 in the lower acid resistance of strain 2457T. Transformation of pSf-R27 or sfh alone into strain 301 resulted in decreased expression of GadA/B in the recombinant strains. Thus, we confirmed that H-NS family protein Sfh, bound to the gadA/B regulatory region and regulates the expression of glutamate decarboxylase at the transcriptional level. We also examined the acid tolerance of the wild-type and recombinant strains using flow cytometry and determined that the acid tolerance of S. flexneri is closely related to the expression of GadA/B. These findings further our understanding of the acid tolerance of S. flexneri, especially via the glutamate-dependent pathway.

In vitro studies of DNA condensation by bridging protein in a crowding environment

The macromolecules of the bacterial cell occupy 20–40% of the total cytosol volume, and crowded environments have long been known to compact and stabilize DNA. Nevertheless, investigations on DNA-protein binding are generally performed in the absence of crowding, which may yield an incomplete understanding of how nucleoid-assembling proteins work. A family of such proteins, abundant in Gram-negative bacteria, is the histone-like nucleoid structuring proteins (H-NS). Herein, the synergistic role of macromolecular crowding (mimicked using polyethylene glycol, PEG) and H-NS was investigated using fluorescence correlation spectroscopy (FCS) and enzyme protection assays. We show that crowding enhances the binding of H-NS to the AT-rich tracks of the DNA, where it preferentially binds to, protecting these tracks towards enzyme digestion, inducing some DNA condensation, and inhibiting the biological function of DNA. We further suggest that the looping of DNA chains, induced by H-NS, contributes to the synergistic effect of DNA-binding protein and crowding agents, on DNA condensation.

Characterisation of the regulatory function of the H-NS protein in the benthic bacterium Shewanella piezotolerans WP3 under cold conditions.

The histone-like nucleoid structuring (H-NS) protein is conserved in Gram-negative bacteria and plays important roles as a multifunctional bacterial modulator. However, its roles in deep-sea microorganisms warrant investigation. In this study, the relationship between temperature and the regulatory function of H-NS was characterised in the benthic bacterium Shewanella piezotolerans WP3. Comparative microarray analysis of the hns knockout mutant and wild-type strain identified a total of 264 differentially expressed genes at 4°C, which is significantly less than that at 20°C. Notably, H-NS was proved to be involved in the cold-mediated induction of lateral flagellar gene transcription. We further showed that the binding affinity of H-NS is higher at 4°C than 20°C, thus explaining the significant difference in the expression pattern at different temperatures.

Regulation systems of protease and hemolysin production in Vibrio vulnificus.

Vibrio vulnificus, a gram-negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema-forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone-like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.