Nucleoprotein filament formation is the structural basis for bacterial protein H-NS gene silencing

Lim, C.Y.H.; Lee, Sin Yi; Kenney, Linda J.; Yan, Jie

doi:10.1038/srep00509

Cited by 93 publications

(111 citation statements)

References 35 publications

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“…DNase footprinting on the clpV promoter further showed that H-NS binds to different regions, including the Ϫ35 box, and then spreads on downstream and upstream fragments. This observation is consistent with the two-step mechanism of H-NS silencing in which H-NS first binds to high-affinity sites that nucleate cooperative binding, leading to H-NS polymerization onto adjacent DNA regions (54,55,65,73). H-NSdependent silencing of the S. Typhimurium T6SS gene cluster is not an isolated case as previous studies showed that the Acinetobacter baumannii, Pseudomonas aeruginosa HSI-2 and HSI-3, Pseudomonas Putida, and Vibrio parahaemolyticus T6SS gene clusters as well as the Edwardsiella tarda T6SS evpP-evpO genes are repressed by H-NS-like proteins (80)(81)(82)(83)(84).…”

Section: Discussionsupporting

confidence: 73%

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H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

et al. 2015

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The secretion of bacterial toxin proteins is achieved by dedicated machineries called secretion systems. The type VI secretion system (T6SS) is a widespread versatile machine used for the delivery of protein toxins to both prokaryotic and eukaryotic cells. In Salmonella enterica serovar Typhimurium, the expression of the T6SS genes is activated during macrophage or mouse infection. Here, we show that the T6SS gene cluster is silenced by the histone-like nucleoid structuring H-NS protein using a combination of reporter fusions, electrophoretic mobility shift assays, DNase footprinting, and fluorescence microscopy. We further demonstrate that derepression of the S. Typhimurium T6SS genes induces T6SS-dependent intoxication of competing bacteria. Our results suggest that relieving T6SS H-NS silencing may be used as a sense-and-kill mechanism that will help S. Typhimurium to homogenize and synchronize the microbial population to gain efficiency during infection. During the course of infection, bacteria produce and secrete bacterial toxins. Secretion of these toxin proteins is achieved by dedicated, specialized machineries called secretion systems. The type VI secretion system (T6SS) is required for the virulence of several Gram-negative pathogens. In Vibrio cholerae, the T6SS translocates VgrG1, a toxin that carries a domain responsible for actin cross-linking in eukaryotic cells (1-4). However, the role of the T6SS is not limited to virulence toward eukaryotes; an increasing number of reports demonstrate that the T6SS is also involved in interbacterial intoxication (5-10). With bacteriocins and contact-dependent inhibition (CDI), the T6SS is therefore involved in shaping bacterial communities (11-14) by delivering antibacterial toxins, including murein hydrolases, DNases, and phospholipases, directly into the target recipient bacterial cell (5, 6, 15; for recent reviews see references 14, 16 and 17). The attacker cell is protected from these toxins by the coproduction of cognate proteins that confer immunity (15, 18). The T6SS therefore confers a growth advantage to bacteria in mixed cultures and has been suggested to be important in environmental niches where bacterial competition for nutrients is critical for survival or for the uptake of DNA for transformation and acquisition of new traits (14, 19). However, while the role of the T6SS in interbacterial competition has been evidenced and characterized under laboratory conditions, its role in shaping bacterial communities of the microbiota is not yet clearly elucidated.At the molecular level, the T6SS is assembled from 13 proteins, called core components, that form a transenvelope apparatus anchoring a cytoplasmic tubular structure to the membrane (20-24). Based on structural homologies with bacteriophage tail components, this tubular structure has been proposed to be constituted of an inner tube assembled by stacked hexameric rings of the Hcp protein, resembling the tail tube of bacteriophages, tipped by the VgrG protein (21,(25)(26)(27)). The current model descr...

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Section: Discussionsupporting

confidence: 73%

“…This observation suggests that H-NS spreads from an initial, high-affinity binding site used as a nucleation center to occupy an extensive DNA region. These characteristics of H-NS have already been studied in detail in previous work and have been proposed to be responsible for gene silencing (54,65,73).…”

Section: Resultsmentioning

confidence: 96%

H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

et al. 2015

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“…This region has a very high AT content (69.3%) with respect to that of the entire S. Typhimurium genome (47.8%), consistent with the fact that H-NS binds to and acts on AT-rich sequences (42,43). H-NS represses gene expression by forming nucleoprotein complexes, which trap or exclude the RNA polymerase at or from promoters, mainly by two mechanisms (45,46,(54)(55)(56)(57). By the first mechanism, H-NS binds to two sites located further apart, upstream and downstream of the promoter, and then induces a DNA-H-NS-DNA bridge that forms a repressor nucleoprotein stem-loop.…”

Section: Discussionmentioning

confidence: 82%

HilD Induces Expression of Salmonella Pathogenicity Island 2 Genes by Displacing the Global Negative Regulator H-NS from ssrAB

et al. 2014

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Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) have essential roles in the pathogenesis of Salmonella enterica. Previously, we reported transcriptional cross talk between SPI-1 and SPI-2 when the SPI-1 regulator HilD induces expression of the SsrA/B two-component system, the central positive regulator of SPI-2, during the growth of Salmonella to late stationary phase in LB rich medium. Here, we further define the mechanism of the HilD-mediated expression of ssrAB. Expression analysis of cat transcriptional fusions containing different regions of ssrAB revealed the presence of negative regulatory sequences located downstream of the ssrAB promoter. In the absence of these negative cis elements, ssrAB was expressed in a HilD-independent manner and was no longer repressed by the global regulator H-NS. Consistently, when the activity of H-NS was inactivated, the expression of ssrAB also became independent of HilD. Furthermore, electrophoretic mobility shift assays showed that both HilD and H-NS bind to the ssrAB region containing the repressing sequences. Moreover, HilD was able to displace H-NS bound to this region, whereas H-NS did not displace HilD. Our results support a model indicating that HilD displaces H-NS from a region downstream of the promoter of ssrAB by binding to sites overlapping or close to those sites bound by H-NS, which leads to the expression of ssrAB. Although the role of HilD as an antagonist of H-NS has been reported before for other genes, this is the first study showing that HilD is able to effectively displace H-NS from the promoter of one of its target genes.

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“…H-NS is a small (ϳ136-aminoacid) protein that preferentially binds as a dimer to curved double-stranded DNA, although it can also induce bending of noncurved DNA upon binding. H-NS is thought to repress gene expression by oligomerizing along the DNA from its primary nucleation sites or by bridging adjacent DNA segments, thus blocking RNA polymerase access to promoter sequences or preventing the formation of an active open complex (41,42). It has also been demonstrated that H-NS silences genes that are thought to have been acquired by horizontal transfer events, thus preventing the fitness cost for the receptor cell while newly acquired genes (including virulence factors) are integrated into and controlled by preexisting regulatory networks (reviewed in references 43 and 44).…”

Section: Discussionmentioning

confidence: 99%

Identification and Regulation of a Novel Citrobacter rodentium Gut Colonization Fimbria (Gcf)

et al. 2015

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The Gram-negative enteric bacterium Citrobacter rodentium is a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. For C. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novel C. rodentium colonization factor, called gut colonization fimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. A gcfA mutant shows a severe colonization defect within the first 10 days of infection. The gcf promoter is not active in C. rodentium under several in vitro growth conditions; however, it is readily expressed in a C. rodentium ⌬hns1 mutant lacking the closest ortholog of the Escherichia coli histone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region of gcf, further supporting its direct role as a repressor of the gcf promoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. The gcf operon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures. IMPORTANCEFimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection by C. rodentium in mice, despite being poorly expressed under in vitro growth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection. The natural mouse pathogen Citrobacter rodentium and the human pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) represent a family of enteric pathogenic bacteria that adhere to the host intestinal epithelial cells and inject effector proteins through a specialized type III secretion system (T3SS), subverting or hijacking several cell functions and leading to the formation of a distinctive histopathology known as the attaching and effacing (A/E) lesion (1, 2). These three pathogens produce similar lesions in the intestinal epithelium of their respective hosts, which are characterized by the intimate atta...

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Nucleoprotein filament formation is the structural basis for bacterial protein H-NS gene silencing

Cited by 93 publications

References 35 publications

H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

HilD Induces Expression of Salmonella Pathogenicity Island 2 Genes by Displacing the Global Negative Regulator H-NS from ssrAB

Identification and Regulation of a Novel Citrobacter rodentium Gut Colonization Fimbria (Gcf)

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