Heat-stable Nucleoid Structuring Research Articles

Advancing evolution: Bacteria break down gene silencer to express horizontally acquired genes.

Horizontal gene transfer advances bacterial evolution. To benefit from horizontally acquired genes, enteric bacteria must overcome silencing caused when the widespread heat-stable nucleoid structuring (H-NS) protein binds to AT-rich horizontally acquired genes. This ability had previously been ascribed to both anti-silencing proteins outcompeting H-NS for binding to AT-rich DNA and RNA polymerase initiating transcription from alternative promoters. However, we now know that pathogenic Salmonella enterica serovar Typhimurium and commensal Escherichia coli break down H-NS when this silencer is not bound to DNA. Curiously, both species use the same protease - Lon - to destroy H-NS in distinct environments. Anti-silencing proteins promote the expression of horizontally acquired genes without binding to them by displacing H-NS from AT-rich DNA, thus leaving H-NS susceptible to proteolysis and decreasing H-NS amounts overall. Conserved amino acid sequences in the Lon protease and H-NS cleavage site suggest that diverse bacteria degrade H-NS to exploit horizontally acquired genes.

Degradation of gene silencer is essential for expression of foreign genes and bacterial colonization of the mammalian gut

Horizontal gene transfer drives bacterial evolution. To confer new properties, horizontally acquired genes must overcome gene silencing by nucleoid-associated proteins, such as the heat-stable nucleoid structuring (H-NS) protein. Enteric bacteria possess proteins that displace H-NS from foreign genes, form nonfunctional oligomers with H-NS, and degrade H-NS, raising the question of whether any of these mechanisms play a role in overcoming foreign gene silencing invivo. To answer this question, we mutagenized the hns gene and identified a variant specifying an H-NS protein that binds foreign DNA and silences expression of the corresponding genes, like wild-type H-NS, but resists degradation by the Lon protease. Critically, Escherichia coli expressing this variant alone fails to produce curli, which are encoded by foreign genes and required for biofilm formation, and fails to colonize the murine gut. Our findings establish that H-NS proteolysis is a general mechanism of derepressing foreign genes and essential for colonization of mammalian hosts.

Horizontally acquired regulatory gene activates ancestral regulatory system to promote Salmonella virulence.

Horizontally acquired genes are typically regulated by ancestral regulators. This regulation enables expression of horizontally acquired genes to be coordinated with that of preexisting genes. Here, we report a singular example of the opposite regulation: a horizontally acquired gene that controls an ancestral regulator, thereby promoting bacterial virulence. We establish that the horizontally acquired regulatory gene ssrB is necessary to activate the ancestral regulatory system PhoP/PhoQ of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mildly acidic pH, which S. Typhimurium experiences inside macrophages. SsrB promotes phoP transcription by binding upstream of the phoP promoter. SsrB also increases ugtL transcription by binding to the ugtL promoter region, where it overcomes gene silencing by the heat-stable nucleoid structuring protein H-NS, enhancing virulence. The largely non-pathogenic species S. bongori failed to activate PhoP/PhoQ in mildly acidic pH because it lacks both the ssrB gene and the SsrB binding site in the target promoter. Low Mg2+ activated PhoP/PhoQ in both S. bongori and ssrB-lacking S. Typhimurium, indicating that the SsrB requirement for PhoP/PhoQ activation is signal-dependent. By controlling the ancestral genome, horizontally acquired genes are responsible for more crucial abilities, including virulence, than currently thought.

Regulation of R1 Plasmid Transfer by H-NS, ArcA, TraJ, and DNA Sequence Elements.

In conjugative elements such as integrating conjugative elements (ICEs) or conjugative plasmids (CPs) transcription of DNA transfer genes is a prerequisite for cells to become transfer competent, i.e., capable of delivering plasmid DNA via bacterial conjugation into new host bacteria. In the large family of F-like plasmids belonging to the MobF12A group, transcription of DNA transfer genes is tightly controlled and dependent on the activation of a single promoter, designated PY. Plasmid encoded TraJ and chromosomally encoded ArcA proteins are known activators, whereas the nucleoid associated protein heat-stable nucleoid structuring (H-NS) silences the PY promoter. To better understand the role of these proteins in PY promoter activation, we performed in vitro DNA binding studies using purified H-NS, ArcA, and TraJR1 (TraJ encoded by the conjugative resistance plasmid R1). All proteins could bind to R1PY DNA with high affinities; however, only ArcA was found to be highly sequence specific. DNase I footprinting studies revealed three H-NS binding sites, confirmed the binding site for ArcA, and suggested that TraJ contacts a dyad symmetry DNA sequence located between −51 and −38 in the R1PY promoter region. Moreover, TraJR1 and ArcA supplied together changed the H-NS specific protection pattern suggesting that these proteins are able to replace H-NS from R1PY regions proximal to the transcription start site. Our findings were corroborated by PY-lacZ reporter fusions with a series of site specific R1PY promoter mutations. Sequential changes of some critical DNA bases in the TraJ binding site (jbs) from plasmid R1 to plasmid F led to a remarkable specificity switch: The PY promoter became activatable by F encoded TraJ whereas TraJR1 lost its activation function. The R1PY mutagenesis approach also confirmed the requirement for the host-encoded response-regulator ArcA and indicated that the sequence context, especially in the −35 region is critical for PY regulation and function.

Salmonella expresses foreign genes during infection by degrading their silencer

The heat-stable nucleoid structuring (H-NS, also referred to as histone-like nucleoid structuring) protein silences transcription of foreign genes in a variety of Gram-negative bacterial species. To take advantage of the products encoded in foreign genes, bacteria must overcome the silencing effects of H-NS. Because H-NS amounts are believed to remain constant, overcoming gene silencing has largely been ascribed to proteins that outcompete H-NS for binding to AT-rich foreign DNA. However, we report here that the facultative intracellular pathogen Salmonella enterica serovar Typhimurium decreases H-NS amounts 16-fold when inside macrophages. This decrease requires both the protease Lon and the DNA-binding virulence regulator PhoP. The decrease in H-NS abundance reduces H-NS binding to foreign DNA, allowing transcription of foreign genes, including those required for intramacrophage survival. The purified Lon protease degraded free H-NS but not DNA-bound H-NS. By displacing H-NS from DNA, the PhoP protein promoted H-NS proteolysis, thereby de-repressing foreign genes-even those whose regulatory sequences are not bound by PhoP. The uncovered mechanism enables a pathogen to express foreign virulence genes during infection without the need to evolve binding sites for antisilencing proteins at each foreign gene.

Growth Phase-Dependent Chromosome Condensation and Heat-Stable Nucleoid-Structuring Protein Redistribution in Escherichia coli under Osmotic Stress.

The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in Escherichia coli, including many involved in adaptation to osmotic stress. We have applied superresolved microscopy to quantify the intracellular and spatial reorganization of H-NS in response to a rapid osmotic shift. We found that H-NS showed growth phase-dependent relocalization in response to hyperosmotic shock. In stationary phase, H-NS detached from a tightly compacted bacterial chromosome and was excluded from the nucleoid volume over an extended period of time. This behavior was absent during rapid growth but was induced by exposing the osmotically stressed culture to a DNA gyrase inhibitor, coumermycin. This chromosomal compaction/H-NS exclusion phenomenon occurred in the presence of either potassium or sodium ions and was independent of the presence of stress-responsive sigma factor σS and of the H-NS paralog StpA.IMPORTANCE The heat-stable nucleoid-structuring (H-NS) protein coordinates the expression of over 200 genes in E. coli, with a large number involved in both bacterial virulence and drug resistance. We report on the novel observation of a dynamic compaction of the bacterial chromosome in response to exposure to high levels of salt. This stress response results in the detachment of H-NS proteins and their subsequent expulsion to the periphery of the cells. We found that this behavior is related to mechanical properties of the bacterial chromosome, in particular, to how tightly twisted and coiled is the chromosomal DNA. This behavior might act as a biomechanical response to stress that coordinates the expression of genes involved in adapting bacteria to a salty environment.

A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity.

Heat-stable nucleoid structuring protein (H-NS) plays a crucial role in gene silencing within prokaryotic cells and is important in pathogenesis. It was reported that H-NS silences nearly 5% of the genome, yet the molecular mechanism of silencing is not well understood. Here, we employed a highly-sensitive single-molecule counting approach, and measured the dissociation constant (KD) of H-NS binding to single DNA binding sites. Charged residues in the linker domain of H-NS provided the most significant contribution to DNA binding affinity. Although H-NS was reported to prefer A/T-rich DNA (a feature of pathogenicity islands) over G/C-rich DNA, the dissociation constants obtained from such sites were nearly identical. Using a hairpin unzipping assay, we were able to uncouple non-specific DNA binding steps from nucleation site binding and subsequent polymerization. We propose a model in which H-NS initially engages with non-specific DNA via reasonably high affinity (∼60 nM KD) electrostatic interactions with basic residues in the linker domain. This initial contact enables H-NS to search along the DNA for specific nucleation sites that drive subsequent polymerization and gene silencing.

Spatial Distribution of H-NS in E. coli under Environmental Stress

The heat-stable nucleoid structuring (H-NS) protein is an abundant DNA-binding protein in gram-negative bacteria. H-NS impacts both DNA condensation and globally regulates gene expression by binding to AT-rich domains of DNA. H-NS is known to play a key role in the adaptation of bacteria to thermal and osmotic shifts in the environment, although the mechanism is still poorly understood. In this study, we probed the spatial redistribution of H-NS in E. coli via super-resolved microscopy in response to osmotic stress and cold-shock at two phases of bacterial growth: exponential and stationary phase. While we saw little effect under conditions of cold-shock, we observed that in early stationary phase, under osmotic stress, H-NS markedly alters its intracellular organization, detaching from a tightly condensed chromosome, and moving toward the periphery of the cell. This response by H-NS to osmotic shock, notably, is not observed in exponential phase despite a similar condensation of the chromosomal DNA. To understand this observation we have been exploring the response of H-NS to other key regulators of the osmotic stress response.

H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production of Escherichia coli Stx2 Phage Lysogen

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli (E. coli) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δhns mutant (MG1655Δhns) of the E. coli K-12 strain MG1655 was obtained. The Δhns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δhns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.

Xenogeneic Silencing and Its Impact on Bacterial Genomes.

The H-NS (heat-stable nucleoid structuring) protein affects both nucleoid compaction and global gene regulation. H-NS appears to act primarily as a silencer of AT-rich genetic material acquired by horizontal gene transfer. As such, it is key in the regulation of most genes involved in virulence and in adaptation to new environmental niches. Here we review recent progress in understanding the biochemistry of H-NS and how xenogeneic silencing affects bacterial evolution. We highlight the strengths and weaknesses of some of the models proposed in H-NS-mediated nucleoprotein complex formation. Based on recent single-molecule studies, we also propose a novel mode of DNA compaction by H-NS termed intrabridging to explain over two decades of observations of the H-NS molecule.

Phage Encoded H-NS: A Potential Achilles Heel in the Bacterial Defence System

The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race.

H‐NS‐mediated repression of CRISPR‐based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO

The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

A divalent switch drives H-NS/DNA-binding conformations between stiffening and bridging modes

Heat-stable nucleoid structuring protein (H-NS) is an abundant prokaryotic protein that plays important roles in organizing chromosomal DNA and gene silencing. Two controversial binding modes were identified. H-NS binding stimulating DNA bridging has become the accepted mechanism, whereas H-NS binding causing DNA stiffening has been largely ignored. Here, we report that both modes exist, and that changes in divalent cations drive a switch between them. The stiffening form is present under physiological conditions, and directly responds to pH and temperature in vitro. Our findings have broad implications and require a reinterpretation of the mechanism by which H-NS regulates genes.

Biophysical Studies of H-NS Binding to DNA

Heat-stable nucleoid structuring protein (H-NS) is an abundant prokaryotic protein that organizes chromosomal DNA and plays an important role in gene silencing. Here we report that H-NS can have two distinct binding modes that can be switched from one to another by changes in divalent ion concentrations. Further, we show that the switching does not require dissociation of H-NS from the DNA. Our finding resolves an important controversy in the field in which mutually exclusive observations of H-NS/DNA interaction were reported. In single-molecule manipulation experiments, one binding mode leads to stiffening of DNA backbone, while the other leads to DNA folding. AFM imaging showed that the stiffening is caused by polymerization of H-NS along DNA starting from a few nucleation sites, while the folding is caused by formation of large DNA hairpins. The transition from stiffening to bridging occurs when the magnesium or calcium concentration is around or above 5 mM, suggesting that both binding modes are likely present at physiological conditions. Furthermore, the susceptibility of the two binding modes to physiological stimuli (pH and temperature), which are known modulators of H-NS activity, are discussed.

Protein mediated bridging motifs: A key mechanism in biopolymer organization

The protein-mediated bridging motif is ubiquitous and essential for shaping cellular structures in all organisms. In this paper, we dissect the bridging motif for a model system: the Heat-stable Nucleoid-Structuring protein (H-NS). We present data from two complementary single-molecule assays that probe the H-NS-DNA interaction: a dynamic optical-trap-driven unzipping assay and an equilibrium H-NS-mediated DNA looping assay. To quantitatively analyze and compare these assays, we develop a general theoretical framework for describing the bridging motif. The interplay between these experiments and our theoretical model not only infers the effective interaction free energy, the bridging conformation and the duplex-duplex spacing, but also reveals a second, unresolved, cis-binding motif that challenges our current understanding of the role of bridging proteins in chromatin structure. The theoretical framework we have developed is generally applicable to the description of protein-mediated bridging motifs in chromatin and cytoskeletal organization.

H-NS: a universal regulator for a dynamic genome.

The effect of the bacterial heat-stable nucleoid-structuring (H-NS) protein on gene expression is overwhelmingly negative and extends throughout the genome, pointing to an almost universal role for this nucleoid-associated protein as a transcriptional repressor. Its ability to exert widespread effects on gene expression probably reflects the fact that it binds to curved DNA, which is commonly found at promoters. H-NS and related proteins can engage in both homologous and heterologous protein–protein interactions. Recent data show that the genes that encode H-NS-like proteins can be carried on mobile genetic elements. This raises the possibility that horizontal gene transfer expands the repertoire of protein–protein interactions that nucleoid-associated proteins can engage in, with potentially profound consequences for the global gene-expression profile of the cell.