Explore the vast range of titles available.

Histone-like nucleoid structuring (H-NS) and H-NS-like proteins serve as global gene silencers and work with antagonistic transcriptional activators (counter-silencers) to properly coordinate the expression of virulence genes in pathogenic bacteria. In Brucella , MucR has been proposed as a novel H-NS-like gene silencer, but direct experimental evidence is lacking. Here, we show that MucR serves as an H-NS-like silencer of the Brucella abortus genes encoding the polar autotransporter adhesins BtaE and BmaC, the c-di-GMP-specific phosphodiesterase BpdB, and the quorum-sensing regulator BabR. We also demonstrate that the MarR-type transcriptional activator MdrA can displace MucR from the btaE promoter, supporting the existence of MucR counter-silencers in Brucella . Moreover, our chromatin immunoprecipitation (ChIP)-seq analysis identified 546 MucR enrichment peaks along the genome, including in the promoters of the genes encoding the Type IV secretion machinery and effectors and the quorum-sensing regulator VjbR. Importantly, MucR ChIP-seq peaks overlap with the previously described binding sites for the transcriptional activators VjbR, BvrR, and CtrA suggesting that these regulators serve as MucR counter-silencers and work in concert with MucR to coordinate virulence gene expression in Brucella . In addition, using chromosome conformation capture (Hi-C), we show that like H-NS in Escherichia coli , MucR alters the global structure of the Brucella nucleoid. Finally, a copy of the E. coli hns rescues the distinctive growth defect and elevated btaE expression of a B. abortus mucR mutant. Together, these findings solidify the role of MucR as a novel type of H-NS-like protein and suggest that MucR’s gene-silencing properties play a key role in virulence in Brucella . Histone-like nucleoid structuring (H-NS) and H-NS-like proteins coordinate host-associated behaviors in many pathogenic bacteria, often through forming silencer/counter-silencer pairs with signal-responsive transcriptional activators to tightly control gene expression. Brucella and related bacteria do not encode H-NS or homologs of known H-NS-like proteins, and it is unclear if they have other proteins that perform analogous functions during pathogenesis. In this work, we provide compelling evidence for the role of MucR as a novel H-NS-like protein in Brucella . We show that MucR possesses many of the known functions attributed to H-NS and H-NS-like proteins, including the formation of silencer/counter-silencer pairs to control virulence gene expression and global structuring of the nucleoid. These results uncover a new role for MucR as a nucleoid structuring protein and support the importance of temporal control of gene expression in Brucella and related bacteria.

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 in vivo. 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.

Cyclic di-GMP (c-di-GMP) is a second messenger that transduces extracellular stimuli into cellular responses and regulates various biological processes in bacteria. H-NS is a global regulatory protein that represses expression of many genes, but how H-NS activity is modulated by environmental signals remains largely unclear. Here, we show that high intracellular c-di-GMP levels, induced by environmental cues, relieve H-NS-mediated transcriptional silencing in Salmonella enterica serovar Typhimurium. We find that c-di-GMP binds to the H-NS protein to inhibit its binding to DNA, thus derepressing genes silenced by H-NS. However, c-di-GMP is unable to displace H-NS from DNA. In addition, a K107A mutation in H-NS abolishes response to c-di-GMP but leaves its DNA binding activity unaffected in vivo. Our results thus suggest a mechanism by which H-NS acts as an environment-sensing regulator in Gram-negative bacteria. H-NS is a global regulatory protein that represses expression of many genes in bacteria. Here, Li et al. show that a second messenger, cyclic di-GMP, binds to H-NS and inhibits its binding to DNA, thus relieving H-NS-mediated transcriptional silencing.

Abstract Background We 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. Methods The 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. Results The 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. Conclusions These results provide new insights into T6SS-mediated bacterial competition and attachment in K pneumoniae and could facilitate the prevention of K pneumoniae infection.

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference. We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner. The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster--in our case the first acquired spacer--and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.

In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS–silenced Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 5′ rapid amplification of complementary DNA ends sequencing (5’ RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS–bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD’s ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.

Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.

Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria and is believed to be a crucial player in bacterial chromatin organization via its DNA-bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically, it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the DNA-bridging activity of H-NS. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA-binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA-bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a ‘closed’ and an ‘open’, bridging competent, conformation driven by environmental cues and interaction partners. The genetic information every cell needs to work properly is encoded in molecules of DNA that are much longer than the cell itself. A key challenge in biology is to understand how DNA is organized to fit inside each cell, whilst still providing access to the information that it contains. Since the way DNA is organized can influence which genes are active, rearranging DNA plays an important role in controlling how cells behave. In Escherichia coli and many other bacteria, a protein called H-NS contributes to DNA reorganization by forming or rupturing loops in the DNA in response to changes in temperature, the levels of salt and other aspects of the cell’s surroundings. In controlling loop formation, it dictates whether specific genes are switched on or off. However, it remains unclear how H-NS detects the environmental changes. To address this question, van der Valk et al. used biochemical techniques to study the activity of H-NS from E. coli under different environmental conditions. The experiments show that changes in the environment cause structural changes to H-NS, altering its ability to form DNA loops. A previously unnoticed region of the protein acts as a switch to control these structural changes, and ultimately affects which genes are active in the cell. These findings shed new light on how bacteria organize their DNA and the strategies they have developed to adapt to different environments. The new protein region identified in H-NS may also be present in similar proteins found in other organisms. In the future, this knowledge may ultimately help to develop new antibiotic drugs that target H-NS proteins in bacteria.

Ros/MucR is a widespread family of bacterial zinc-finger-containing proteins that integrate multiple functions, such as symbiosis, virulence, transcription regulation, motility, production of surface components, and various other physiological processes in cells. This regulatory protein family is conserved in bacteria and is characterized by its zinc-finger motif, which has been proposed as the ancestral domain from which the eukaryotic C2H2 zinc-finger structure has evolved. The first prokaryotic zinc-finger domain found in the transcription regulator Ros was identified in Agrobacterium tumefaciens. In the past decades, a large body of evidence revealed Ros/MucR as pleiotropic transcriptional regulators that mainly act as repressors through oligomerization and binding to AT-rich target promoters. The N-terminal domain and the zinc-finger-bearing C-terminal region of these regulatory proteins are engaged in oligomerization and DNA binding, respectively. These properties of the Ros/MucR proteins are similar to those of xenogeneic silencers, such as H-NS, MvaT, and Lsr2, which are mainly found in other lineages. In fact, a novel functional model recently proposed for this protein family suggests that they act as H-NS-‘like’ gene silencers. The prokaryotic zinc-finger domain exhibits interesting structural and functional features that are different from that of its eukaryotic counterpart (a βββα topology), as it folds in a significantly larger zinc-binding globular domain (a βββαα topology). Phylogenetic analysis of Ros/MucR homologs suggests an ancestral origin of this type of protein in α-Proteobacteria. Furthermore, multiple duplications and lateral gene transfer events contributing to the diversity and phyletic distribution of these regulatory proteins were found in bacterial genomes.