Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein.
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ABSTRACT:
SUBMITTER: Duysak T
PROVIDER: S-EPMC8346002 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Burkholderia cepacia complex bacteria comprise opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream of the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. This insertion sequence (IS) element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pilus synthesis, type VI secretion, and chromosome-associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line and reduced surface hydrophobicity and forms smaller cellular aggregates but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of the Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes. IMPORTANCE Members of the histone-like nucleoid structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, fulfilling a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein is one out of eight orthologs present in the B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits.
| S-EPMC8231446 | biostudies-literature
Project description:The cholera disease bacterium V. cholerae, can adopt planktonic or biofilm lifestyles depending on the intracellular concentration of the second messenger cyclic diguanylic acid (c-di-GMP). Biofilm formation protects Vibrios from stressful conditions and facilitates disease transmission by enhancing infectivity. The histone-like nucleoid structuring protein (H-NS) is a global regulator of genes associated with pathogenicity and responses to environmental stresses. H-NS represses the transcription of genes vpsT, vpsA and vpsL, which are required for the biosynthesis of the biofilm exopolysacchide matrix. Here we demonstrate that the c-di-GMP-binding protein VpsT disrupts H-NS nucleoprotein complexes at the vpsA and vpsL promoters and that this effect is enhanced by c-di-GMP. We used ChIP coupled with Next Generation Sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to identify additional loci repressed by H-NS affecting biofilm formation. This study showed that H-NS directly represses the transcription of genes encoding proteins present in the biofilm matrix such as the rbmA-F cluster, hemolysin and chitinase. Similar to vpsA and vpsL, the promoter region of vpsU, rbmA and rbmF exhibited overlapping H-NS and VpsT binding motifs. Deletion of vpsT increased H-NS occupancy at the vpsU, vpsA, vpsL, rbmA and rbmF promoters. Conversely, artificially increasing the c-di-GMP pool diminished H-NS occupancy at the above promoters. Deletion of vpsT did not affect H-NS occupancy at its own promoter. However, deletion of genes encoding the regulators AphA and VpsR significantly increased H-NS occupancy at the vpsT promoter. In sum, our study shows that c-di-GMP enhances biofilm formation by acting through VpsT to activate an H-NS anti-repression cascade. The Binding profile of V. cholerae H-NS to the genome was determined by ChIP followed by Next Generation Sequencing (ChIP-Seq) using the Illumina HiSeq2000 platform. V. cholerae C7258 cells expressing H-NS-FLAG fusion protein from the hns transcription and translation signals were collected from LB cultures grown to mid-exponential phase (OD600 0.5). An anti-FLAG Immunoprecipitation (IP) and an Input samples were used for the analysis.
2015-05-13 | E-GEOD-64249 | biostudies-arrayexpress
Project description:The cholera disease bacterium V. cholerae, can adopt planktonic or biofilm lifestyles depending on the intracellular concentration of the second messenger cyclic diguanylic acid (c-di-GMP). Biofilm formation protects Vibrios from stressful conditions and facilitates disease transmission by enhancing infectivity. The histone-like nucleoid structuring protein (H-NS) is a global regulator of genes associated with pathogenicity and responses to environmental stresses. H-NS represses the transcription of genes vpsT, vpsA and vpsL, which are required for the biosynthesis of the biofilm exopolysacchide matrix. Here we demonstrate that the c-di-GMP-binding protein VpsT disrupts H-NS nucleoprotein complexes at the vpsA and vpsL promoters and that this effect is enhanced by c-di-GMP. We used ChIP coupled with Next Generation Sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to identify additional loci repressed by H-NS affecting biofilm formation. This study showed that H-NS directly represses the transcription of genes encoding proteins present in the biofilm matrix such as the rbmA-F cluster, hemolysin and chitinase. Similar to vpsA and vpsL, the promoter region of vpsU, rbmA and rbmF exhibited overlapping H-NS and VpsT binding motifs. Deletion of vpsT increased H-NS occupancy at the vpsU, vpsA, vpsL, rbmA and rbmF promoters. Conversely, artificially increasing the c-di-GMP pool diminished H-NS occupancy at the above promoters. Deletion of vpsT did not affect H-NS occupancy at its own promoter. However, deletion of genes encoding the regulators AphA and VpsR significantly increased H-NS occupancy at the vpsT promoter. In sum, our study shows that c-di-GMP enhances biofilm formation by acting through VpsT to activate an H-NS anti-repression cascade.
2015-05-13 | GSE64249 | GEO
Project description:In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes involved in pathogenesis and stress response. In addition, H-NS inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance. Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression. An increased expression of genes associated with β-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH. The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains. Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.
| S-EPMC8446060 | biostudies-literature
Project description:Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites. H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli, but their direct effects on transcriptional pausing and termination are untested. In this study, we report that bridged H-NS filaments strongly increase pausing by E. coli RNA polymerase at a subset of pause sites with high potential for backtracking. Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action. By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy, we established that bridged filaments surround paused complexes. Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites. Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria.
| S-EPMC4337669 | biostudies-literature
Project description:The data described in this article pertain to the genome-wide transcription profiling of a Vibrio cholerae mutant lacking the histone-like nucleoid structuring protein (H-NS) and the mapping of the H-NS chromosome binding sites [1, 2]. H-NS is a nucleoid-associated protein with two interrelated functions: organization of the bacterial nucleoid and transcriptional silencing [3]. Both functions require DNA binding and protein oligomerization [4, 5]. H-NS commonly silences the expression of virulence factors acquired by lateral gene transfer [6]. The highly pleiotropic nature of hns mutants in V. cholerae indicates that H-NS impacts a broad range of cellular processes such as virulence, stress response, surface attachment, biofilm development, motility and chemotaxis. We used a V. cholerae strain harboring a deletion of hns and a strain expressing H-NS tagged at the C-terminus with the FLAG epitope to generate datasets representing the hns transcriptome and DNA binding profile under laboratory conditions (LB medium, 37°C). The datasets are publicly available at the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE62785 and GSE64249.
| S-EPMC4470426 | biostudies-literature
Project description: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.
| S-EPMC6616471 | biostudies-literature
Project description:Production of the Zn-metalloprotease hemagglutinin (HA)/protease by Vibrio cholerae has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor sigma(S) (RpoS), encoded by rpoS. The histone-like nucleoid structuring protein (H-NS) has been shown to repress rpoS expression in Escherichia coli. In V. cholerae strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a Deltahns mutant. The Deltahns mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of hns resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in Deltahns, DeltarpoS, and Deltahns DeltarpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in DeltarpoS and DeltarpoS Deltahns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The Deltahns mutant exhibited a lower degree of motility and lower levels of expression of flaA, flaC, cheR-2, and motX mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, Deltahns, DeltarpoS, and Deltahns DeltarpoS strains revealed that deletion of rpoS had a more severe negative effect on their expression. Interestingly, deletion of hns in the rpoS background resulted in higher expression levels of flaA, flaC, and motX, suggesting that H-NS represses the expression of these genes in the absence of sigma(S). Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.
| S-EPMC2576668 | biostudies-literature
Project description:The emergence of plasmid-mediated tigecycline resistance gene tet(X4) poses a challenging threat to public health. Based on the analysis of tet(X4)-positive plasmids in the NCBI database, we found that the IncX1-type plasmid is one of the most common vectors for spreading tet(X4) gene, but the mechanisms by which these plasmids adapt to host bacteria and maintain the persistence of antibiotic resistance genes (ARGs) remain unclear. Herein, we investigated the underlying mechanisms of how host bacteria modulate the fitness cost of IncX1 plasmids carrying tet(X4) gene. Interestingly, we found that the tet(X4)-bearing IncX1 plasmids encoding H-NS protein imposed low or no fitness cost in Escherichia coli and Klebsiella pneumoniae; instead, they partially promoted the virulence and biofilm formation in host bacteria. Regression analysis revealed that the expression of hns gene in plasmids was positively linked to the relative fitness of host bacteria. Furthermore, when pCE2::hns was introduced, the fitness of tet(X4)-positive IncX1 plasmid pRF55-1 without hns gene was significantly improved, indicating that hns mediates the improvement of fitness. Finally, we showed that the expression of hns gene is negatively correlated with the expression of tet(X4) gene, suggesting that the regulatory effect of H-NS on adaptability may be attributed to its inhibitory effect on the expression of ARGs. Together, our findings suggest the important role of plasmid-encoded H-NS protein in modulating the fitness of tet(X4)-bearing IncX1 plasmids, which shed new insight into the dissemination of tet(X4) gene in a biological environment.
| S-EPMC8632487 | biostudies-literature
Project description:The antimicrobial activity and mechanism of silver ions (Ag+) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag+ ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag+ ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag+ ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag+ ions: Ag+-induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria.IMPORTANCE As so-called "superbug" bacteria resistant to commonly prescribed antibiotics have become a global threat to public health in recent years, noble metals, such as silver, in various forms have been attracting broad attention due to their antimicrobial activities. However, most of the studies in the existing literature have relied on the traditional bioassays for studying the antimicrobial mechanism of silver; in addition, temporal resolution is largely missing for understanding the effects of silver on the molecular dynamics inside bacteria. Here, we report our study of the antimicrobial effect of silver ions at the nanoscale on the diffusive dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy. This work highlights the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria.
| S-EPMC7054089 | biostudies-literature