Bacterial Nucleoid-associated Proteins Research Articles

Genetic Approaches to Study the Interplay Between Transcription and Nucleoid-Associated Proteins in Escherichia coli.

Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E. coli. These methods include construction of genomic and plasmidic transcriptional and translational lacZ reporter gene fusions to study regulation of promoters; insertion of promoter cassettes to drive transcription into a locus of interest in the genome, for example, an H-NS-bound locus; and construction of isogenic hns and stpA mutants and precautions in doing so.

Peptides designed from a bacteriophage capsid protein function as synthetic transcription repressors

The bacteriophage capsid protein, Psu (polarity suppression), inhibits the bacterial transcription terminator, Rho. In an effort to find nontraditional antibacterial agents, we previously designed peptides from the Psu C terminus that function as inhibitors of Rho. Here, we demonstrated that these peptides have positive surface-charge densities, and they downregulate many genes in Escherichia coli. We hypothesized that these peptides could bind to nucleic acids and repress gene expression. One of these peptides, peptide 33, represses invitro transcription from the T7A1 and Plac promoters efficiently by blocking the access of RNA polymerase to the promoter, a mode of transcription repression akin to many bacterial repressors. Invivo, expressions of the peptides reduce the total RNA level as well as transcription from Plac and Posm promoters significantly. However, they are less efficient in repressing transcription from the rRNA promoters with a very high turnover of RNA polymerase. The peptide 33 binds to both single and dsDNA as well as to RNA with dissociation constants ranging from 1 to 5μM exhibiting preferences for the single-stranded DNA and RNAs. These interactions are salt-resistant and not sequence-specific. Interactions with dsDNA are entropy-driven, while it is enthalpy-driven for the ssDNA. This mode of interaction with nucleic acids is similar to many nonspecific ssDNA-binding proteins. Expression of peptide 33 induces cell elongation and impaired cell division, possibly due to the dislodging of the DNA-binding proteins. Overall, we surmised that these synthetic transcription repressors would function like bacterial nucleoid-associated proteins.

The bacterial nucleoid-associated proteins, HU and Dps, condense DNA into context-dependent biphasic or multiphasic complex coacervates

The bacterial chromosome, known as its nucleoid, is an amorphous assemblage of globular nucleoprotein domains. It exists in a state of phase separation from the cell's cytoplasm, as an irregularly-shaped, membrane-less, intracellular compartment. This state (the nature of which remains largely unknown) is maintained through bacterial generations ad infinitum. Here, we show that HU, and Dps, two of the most abundant nucleoid-associated proteins (NAPs) of Escherichia coli, undergo spontaneous complex coacervation with different forms of DNA/RNA, both individually and in each other's presence, to cause accretion and compaction of DNA/RNA into liquid-liquid phase separated (LLPS) condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) bi-phasic heterotypic mixed condensates in which HU-B helps to lower the Csat of HU-A; and also (b) multi-phasic heterotypic condensates, with Dps, in which de-mixed domains display different contents of HU and Dps. We believe that these modes of complex coacervation that are seen in vitro can serve as models for the in vivo relationships amongst NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in de-mixed sub-domains that are characterized by differing grades of phase separation. Our results clearly demonstrate some quantitative, and some qualitative, differences in the coacervating abilities of different NAPs with DNA, potentially explaining (i) why E. coli has two isoforms of HU, and (ii) why changes in the abundances of HU and Dps facilitate the lag, logarithmic and stationary phases of E. coli growth.

Probing DNA-Amyloid Interaction and Gel Formation by Active Magnetic Wire Microrheology.

Recent studies have shown that bacterial nucleoid-associated proteins (NAPs) can bind to DNA and result in altered structural organization and bridging interactions. Under spontaneous self-assembly, NAPs may also form anisotropic amyloid fibers, whose effects are still more significant on DNA dynamics. To test this hypothesis, microrheology experiments on dispersions of DNA associated with the amyloid terminal domain (CTR) of the bacterial protein Hfq were performed using magnetic rotational spectroscopy (MRS). In this chapter, we survey this microrheology technique based on the remote actuation of magnetic wires embedded in a sample. MRS is interesting as it is easy to implement and does not require complex procedures regarding data treatment. Pertaining to the interaction between DNA and amyloid fibers, it is found that DNA and Hfq-CTR protein dispersions behave like a gel, an outcome that suggests the formation of a network of amyloid fibers cross-linked with the DNA strands. In contrast, the pristine DNA and Hfq-CTR dispersions behave as purely viscous liquids. To broaden the scope of the MRS technique, we include theoretical predictions for the rotation of magnetic wires regarding the generic behaviors of basic rheological models from continuum mechanics, and we list the complex fluids studied by this technique over the past 10years.

Specialization of the Reiterated Copies of the Heterodimeric Integration Host Factor Genes in Geobacter sulfurreducens.

Integration host factor (IHF) is a widely distributed small heterodimeric protein member of the bacterial Nucleoid-Associated Proteins (NAPs), implicated in multiple DNA regulatory processes. IHF recognizes a specific DNA sequence and induces a large bend of the nucleic acid. IHF function has been mainly linked with the regulation of RpoN-dependent promoters, where IHF commonly recognizes a DNA sequence between the enhancer-binding region and the promoter, facilitating a close contact between the upstream bound activator and the promoter bound, RNA polymerase. In most proteobacteria, the genes encoding IHF subunits (ihfA and ihfB) are found in a single copy. However, in some Deltaproteobacteria, like Geobacter sulfurreducens, those genes are duplicated. To date, the functionality of IHF reiterated encoding genes is unknown. In this work, we achieved the functional characterization of the ihfA-1, ihfA-2, ihfB-1, and ihfB-2 from G. sulfurreducens. Unlike the ΔihfA-2 or ΔihfB-1 strains, single gene deletion in ihfA-1 or ihfB-2, provokes an impairment in fumarate and Fe(III) citrate reduction. Accordingly, sqRT-PCR experiments showed that ihfA-1 and ihfB-2 were expressed at higher levels than ihfA-2 and ihfB-1. In addition, RNA-Seq analysis of the ΔihfA-1 and ΔihfB-2 strains revealed a total of 89 and 122 differentially expressed genes, respectively. Furthermore, transcriptional changes in 25 genes were shared in both mutant strains. Among these genes, we confirmed the upregulation of the pilA-repressor, GSU1771, and downregulation of the triheme-cytochrome (pgcA) and the aconitate hydratase (acnA) genes by RT-qPCR. EMSA experiments also demonstrated the direct binding of IHF to the upstream promoter regions of GSU1771, pgcA and acnA. PilA changes in ΔihfA-1 and ΔihfB-2 strains were also verified by immunoblotting. Additionally, heme-staining of subcellular fractions in ΔihfA-1 and ΔihfB-2 strains revealed a remarkable deficit of c-type cytochromes. Overall, our data indicate that at least during fumarate and Fe(III) citrate reduction, the functional IHF regulator is likely assembled by the products of ihfA-1 and ihfB-2. Also, a role of IHF controlling expression of multiple genes (other than RpoN-dependent) affects G. sulfurreducens physiology and extracellular electron transfer.

Crystal structure of histone-like protein from Streptococcus mutans refined to 1.9 Å resolution.

Nucleoid-associated proteins (NAPs) in prokaryotes play an important architectural role in DNA bending, supercoiling and DNA compaction. In addition to architectural roles, some NAPs also play regulatory roles in DNA replication and repair, and act as global transcriptional regulators in many bacteria. Bacteria encode multiple NAPs and some of them are even essential for survival. Streptococcus mutans, a dental pathogen, encodes one such essential NAP called histone-like protein (HLP). Here, the three-dimensional structure of S. mutans HLP has been determined to 1.9 Å resolution. The HLP structure is a dimer and shares a high degree of similarity with other bacterial NAPs, including HU. Since HLPs are essential for the survival of pathogenic streptococci, this structure determination is potentially beneficial for future drug development against these pathogens.

Structural Insights into the Regulation of Foreign Genes in Salmonella by the Hha/H-NS Complex

Hha facilitates H-NS-mediated silencing of foreign genes in bacteria. Two Hha monomers bind opposing faces of the H-NS N-terminal dimerization domain. Hha binds the dimerization domain of H-NS and may contact DNA via positively charged surface residues. The structure of Hha and H-NS in complex provides a mechanistic model of how Hha may affect gene regulation. The bacterial nucleoid-associated proteins Hha and H-NS jointly repress horizontally acquired genes in Salmonella, including essential virulence loci encoded within Salmonella pathogenicity islands. Hha is known to interact with the N-terminal dimerization domain of H-NS; however, the manner in which this interaction enhances transcriptional silencing is not understood. To further understand this process, we solved the x-ray crystal structure of Hha in complex with the N-terminal dimerization domain of H-NS (H-NS(1-46)) to 3.2 Å resolution. Two monomers of Hha bind to symmetrical sites on either side of the H-NS(1-46) dimer. Disruption of the Hha/H-NS interaction by the H-NS site-specific mutation I11A results in increased expression of the Hha/H-NS co-regulated gene hilA without affecting the expression levels of proV, a target gene repressed by H-NS in an Hha-independent fashion. Examination of the structure revealed a cluster of conserved basic amino acids that protrude from the surface of Hha on the opposite side of the Hha/H-NS(1-46) interface. Hha mutants with a diminished positively charged surface maintain the ability to interact with H-NS but can no longer regulate hilA. Increased expression of the hilA locus did not correspond to significant depletion of H-NS at the promoter region in chromatin immunoprecipitation assays. However, in vitro, we find Hha improves H-NS binding to target DNA fragments. Taken together, our results show for the first time how Hha and H-NS interact to direct transcriptional repression and reveal that a positively charged surface of Hha enhances the silencing activity of H-NS nucleoprotein filaments.

Mechanism of DNA organization by Mycobacterium tuberculosis protein Lsr2

Bacterial nucleoid-associated proteins, such as H-NS-like proteins in Enterobacteriaceae, are abundant DNA-binding proteins that function in chromosomal DNA organization and gene transcription regulation. The Mycobacterium tuberculosis Lsr2 protein has been proposed to be the first identified H-NS analogue in Gram-positive bacteria based on its capability to complement numerous in vivo functions of H-NS. Here, we report that Lsr2 cooperatively binds to DNA forming a rigid Lsr2 nucleoprotein complex that restricts DNA accessibility, similar to H-NS. On large DNA, the rigid Lsr2 nucleoprotein complexes can mediate DNA condensation into highly compact DNA conformations. In addition, the responses of Lsr2 nucleoprotein complex to environmental factors (salt concentration, temperature and pH) were studied over physiological ranges. These results provide mechanistic insights into how Lsr2 may mediate its gene silencing, genomic DNA protection and organization functions in vivo. Finally, our results strongly support that Lsr2 is an H-NS-like protein in Gram-positive bacteria from a structural perspective.

Evolution of genome base composition and genome size in bacteria

Evolution of genome base composition and genome size in bacteria

Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins

AbstractBacteria that cause chronic and/or recurrent diseases often rely on a biofilm lifestyle. The foundation of the biofilm structure is the extracellular polymeric substance (EPS) that acts as a barrier to both effectors of the immune system and antimicrobial agents. Recent work has highlighted extracellular DNA (eDNA) as a key component common to many pathogenic biofilms. Here, we show that the DNABII family of proteins, well known for their strong structural influences on intracellular DNA, was also critical for the integrity of the EPS matrix of biofilms that contain eDNA. In fact, antisera derived against a purified Escherichia coli DNABII family member rapidly disrupts the biofilm EPS formed by multiple human pathogens in vitro. In addition, when a member of this family of proteins was used as an immunogen in an animal model in which the bacteria had already formed a robust biofilm at the site of infection, the resultant targeted immune response strongly ameliorated this biofilm disease in vivo. Finally, this methodology to debulk the biofilm of EPS was shown to work synergistically with otherwise ineffective traditional anti-microbial approaches in vitro. We discuss the prospects for targeting DNABII family members as a potential universal strategy for treating biofilm diseases.

Distribution of Genes Encoding Nucleoid-Associated Protein Homologs in Plasmids

Bacterial nucleoid-associated proteins (NAPs) form nucleoprotein complexes and influence the expression of genes. Recent studies have shown that some plasmids carry genes encoding NAP homologs, which play important roles in transcriptional regulation networks between plasmids and host chromosomes. In this study, we determined the distributions of the well-known NAPs Fis, H-NS, HU, IHF, and Lrp and the newly found NAPs MvaT and NdpA among the whole-sequenced 1382 plasmids found in Gram-negative bacteria. Comparisons between NAP distributions and plasmid features (size, G+C content, and putative transferability) were also performed. We found that larger plasmids frequently have NAP gene homologs. Plasmids with H-NS gene homologs had less G+C content. It should be noted that plasmids with the NAP gene homolog also carried the relaxase gene involved in the conjugative transfer of plasmids more frequently than did those without the NAP gene homolog, implying that plasmid-encoded NAP homologs positively contribute to transmissible plasmids.

Bacterial nucleoid-associated proteins, nucleoid structure and gene expression

Emerging models of the bacterial nucleoid show that nucleoid-associated proteins (NAPs) and transcription contribute in combination to the dynamic nature of nucleoid structure. NAPs and other DNA-binding proteins that display gene-silencing and anti-silencing activities are emerging as key antagonistic regulators of nucleoid structure. Furthermore, it is becoming clear that the boundary between NAPs and conventional transcriptional regulators is quite blurred and that NAPs facilitate the evolution of novel gene regulatory circuits. Here, NAP biology is considered from the standpoints of both gene regulation and nucleoid structure.

Lsr2 is a nucleoid-associated protein that targets AT-rich sequences and virulence genes inMycobacterium tuberculosis

Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a unique nucleoid-associated protein that binds AT-rich regions of the genome, including genomic islands acquired by horizontal gene transfer and regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM and PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain-swapping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA-binding domain. Nuclear magnetic resonance analysis of the DNA-binding domain of Lsr2 and its interaction with DNA reveals a unique structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve a function similar to the Enterobacteriaceae H-NS, likely coordinating global gene regulation and virulence in this group of medically important bacteria.

Horizontally acquired homologues of the nucleoid‐associated protein H‐NS: implications for gene regulation

H-NS is one of the most intensively studied members of the family of bacterial nucleoid-associated proteins. It is a DNA-binding protein with a preference for A+T-rich DNA sequences, and it represses the transcription of hundreds of genes in Gram-negative bacteria, including pathogens. In most cases where the issue has been investigated, the repressive activity of H-NS is opposed by the intervention of an antagonistically acting DNA-binding protein, a remodelling of local DNA structure, or a combination of these two. H-NS activity can also be modulated by protein-protein interaction with members of the Hha/YdgT protein family, molecules that share partial amino acid sequence similarity to the oligomerization domain of H-NS. Of particular interest is the ability of H-NS to interact with the full-length paralogue StpA or full-length orthologues that have been acquired by horizontal DNA transfer. In this issue of Molecular Microbiology, Müller et al. describe the H-NS orthologue Hfp and present evidence that in bacteria that acquire Hfp the range of activities of H-NS is modified with important implications for the physiology of the bacterium.

DNA bridging and antibridging: a role for bacterial nucleoid-associated proteins in regulating the expression of laterally acquired genes

Horizontal DNA transfer plays a major role in the evolution of bacteria. It allows them to acquire new traits rapidly and these may confer fitness advantages as the bacteria compete with others in the environment. Historically, the mechanisms of horizontal DNA transfer, chiefly conjugation, transformation and transduction, have received a great deal of attention. Less attention has been focused on the regulatory problems that may accompany the acquisition of new genes by lateral routes. How are these genes integrated into the existing regulatory circuits of the cell? Does a process of 'plug-and-play' operate, or are the new genes silenced pending the evolution of regulatory mechanisms that make their expression not only safe but also beneficial to both the gene and its new host? Recent research shows that bacterial nucleoid-associated proteins such as H-NS, HU and Fis are important contributors to the processes of regulatory integration that accompany horizontal gene transfer. A key emerging theme is the antagonism that exists between the DNA-protein-DNA bridging activity of the H-NS repressor and the DNA-bending and DNA-wrapping activities of regulatory proteins that oppose H-NS.

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.

New Roles for Key Residues in Helices H1 and H2 of the Escherichia coli H-NS N-terminal Domain: H-NS Dimer Stabilization and Hha Binding

Bacterial nucleoid-associated proteins H-NS and Hha modulate gene expression in response to environmental factors. The N-terminal domain of H-NS is involved in homomeric and heteromeric protein-protein interactions. Homomeric interaction leads to the formation of dimers and higher oligomers. Heteromeric interactions with Hha-like proteins modify the modulatory properties of H-NS. In this study, we have used NMR and mutagenesis of the N-terminal domain of H-NS to identify the Hha-binding region around helices H1 and H2 of H-NS. Two conserved arginine residues, R12 and R15, located in the same side and in adjacent turns of helix H2 are shown to be involved in two different protein-protein interactions: R12 is essential for Hha binding and does not affect H-NS dimer formation, and R15 does not affect Hha binding but is essential for the proper folding of H-NS dimers. Our results demonstrate a close structural connection between Hha-H-NS interactions and H-NS dimerization that may be involved in a possible mechanism for the modulation of the H-NS regulatory activity by Hha.

The Putative Orf4 of Broad-Host-Range Conjugative Plasmid R446 Could Be Related to the H-NS Family of Bacterial Nucleoid-Associated Proteins

Theimldeterminant of IncM plasmid R446 was described initially as a short fragment of DNA causing (when cloned in multicopy plasmids) insensitivity to pilus-dependent bacteriophage lysis in bacteria harboring coresident IncM plasmids. We have performed a computational analysis of theimldeterminant of IncM plasmid R446 and found that a potential polypeptide (Orf4) shows similarity to the H-NS family of nucleoid-associated modulators of gene expression. The predicted protein has a molecular weight of 16.3 kDa and an isoelectric point of 5.50. Orf4 is adjacent and upstream of Orf5, a potential polypeptide related to the Hha/YmoA/RmoA family of proteins which modulate bacterial virulence expression (Hha and YmoA) and R100 transfer (RmoA) in response to environmental conditions.