Source: https://www.omicsdi.org/dataset/arrayexpress-repository/E-GEOD-64249
Timestamp: 2019-04-18 11:14:11+00:00

Document:
ABSTRACT: 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.
Project description:Using a combination of ChIP-seq and RNA-seq we show that ToxT regulates expression of the small RNA TarB. TarB modulates the expression level of the transcriptioanl repressor VC0177. VC0177 controls expression of several genes including VC0179. The function of the Vibrio 7th pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing ChIP-seq and RNA-seq to map the regulon of the master virulence regulator ToxT, we identify a TCP island encoded small RNA that reduces the expression of a previously unrecognized VSP-1 encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and down-regulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae and implicates its occurrence in 7th pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide.
Project description:We report the genomic regions enriched in Histone Deacetylase 3 (HDAC3) in mouse livers. We also report the change of HDAC3 occupancy upon DAD mutations in NCOR and SMRT. HDAC3 enriched genomic regions in WT and NS-DADm mice livers using Illumina GAIIx.
Project description:Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used two shotgun proteomics approaches, a label-free one to identify proteins present at two weeks of pellicle formation, and isobaric labeling to assess the differences in the proteome occurring among pellicles formed by three strains of M. bovis BCG: the parental strain (WT), a previously reported knock out in BCG1419c (∆PDE) gene and a new knock out in BCG1416c (∆DGC) gene, in order to unravel the role played by genes involved in the metabolism c-di-GMP during pellicle mode of growth. Our findings strongly suggest that in M. bovis BCG, c-di-GMP may positively correlate with enhanced tolerance to nitrosative stress and negatively to cell wall permeability, therefore suggesting hypothesis relevant for adaption to tolerance to several antibiotics.
Project description:This work characterizes a P.aeruginosa strain with low levels of c-di-GMP resultant from the overexpression of the phosphodiesterase PA2133. Phenotypic characterization showed that biofilm formation, adhesion to epitheliel cells, and motility are severely impaired in this strain. We applied both, DIGE / MALDI-TOF/TOF and shotgun LC/MS/MS quantitative proteomic analysis to exoproteome and enriched membrane protein fractions. Results from both complementary strategies were in agreement and revealed motility, type III secretion, and chemotaxis to be the main affected processes by PA2133 overexpression. These results are the first to show that flagella synthesis, twitching motility, and chemotaxis are negatively regulated by low c-di-GMP levels in P.aeruginosa and thus confront our current undertsandings of c-di-GMP signalling.
Project description:In Staphylococcus aureus, the role of the GGDEF domain containing protein GdpS remains poorly understood. Previous studies reported that gdpS mutant strains had decreased biofilm formation due to changes in icaADBC expression that were independent of cyclic-di-GMP levels. We deleted gdpS in three unrelated S. aureus isolates, and analyzed the resultant mutants for alterations in biofilm formation, metabolism and transcription. Dynamic imaging during biofilm development showed that GdpS inhibited early biofilm formation in only two out of the three strains examined, without affecting bacterial survival. However, quantification of biofilm formation using crystal violet staining revealed that inactivation of gdpS affected biofilm formation in all three studied strains. Extraction of metabolites from S. aureus cells confirmed the absence of cyclic-di-GMP, suggesting that biofilm formation in this species differs from that in other Gram-positive organisms. In addition, targeted mutagenesis demonstrated that the GGDEF domain was not required for GdpS activity. Transcriptomic analysis revealed that the vast majority of GGDEF-regulated genes were involved in virulence, metabolism, cell wall biogenesis and eDNA release. Finally, expression of lrgAB or deletion of cidABC in a strain lacking gdpS confirmed the role of GdpS on regulation of eDNA production that occurred without an increase in cell autolysis. In summary, S. aureus GdpS contributes to cell-to-cell interactions during early biofilm formation by influencing expression of lrgAB and cidABC mediated eDNA release. We conclude that GdpS acts as a negative regulator of eDNA release. Three strains UAMS-1, SA113 and SA564 were used in this study to compare wt with gdpS mutant after 5 hours of growth in static conditions (biofilm formation).
Project description:Vibrio parahaemolyticus scr genes modulate expression of gene sets pertinent to swarming and biofilm formation. They do so by affecting the level of the second messenger c-di-GMP. Here we explore the extent of this regulation by comparing the transcriptomes of a scrABC mutant and its wild-type parental strain. The scope of transcriptional effects modulated by c-di-GMP includes ~100 genes that are positively and negatively regulated. An elevated cellular level of c-di-GMP represses the surface sensing regulon including the genes encoding the lateral flagellar and type three secretion systems while inducing expression of genes encoding cell surface molecules and capsular polysaccharide. Expression of a few transcriptional regulators was also affected, and here we describe the role of one, CpsQ. CpsQ is one of four V. parahaemolyticus homologs in the CsgD/VpsT family of regulators, members of which have been implicated in c-di-GMP signaling. Mutations in cpsQ, like defects in another previously identified capsule regulator, cpsR, suppress the sticky phenotype of scr mutants. By using a combination of mutant and reporter analyses in Vibrio and E. coli, CpsQ is shown to be the direct positive regulator of cpsA transcription and its cpsA-activating ability is found to be responsive to the cellular level of c-di-GMP. Unexpectedly, we find that a low level of this nucleotide diminishes the stability of CpsQ. The molecular interplay in this signaling circuit is further defined by demonstrating that CpsQ is epistatic to CpsS, a negative regulator of capsule. CpsR activates cpsQ, and CpsQ can also regulate its own transcription. Overall design: Wildtype Vibrio parahaemolyticus (LM5674) and scrABC mutant (LM6567) were grown on rich mediim agar plates and gene expression profiles were compared.
Project description:Vibrio parahaemolyticus scr genes modulate expression of gene sets pertinent to swarming and biofilm formation. They do so by affecting the level of the second messenger c-di-GMP. Here we explore the extent of this regulation by comparing the transcriptomes of a scrABC mutant and its wild-type parental strain. The scope of transcriptional effects modulated by c-di-GMP includes ~100 genes that are positively and negatively regulated. An elevated cellular level of c-di-GMP represses the surface sensing regulon including the genes encoding the lateral flagellar and type three secretion systems while inducing expression of genes encoding cell surface molecules and capsular polysaccharide. Expression of a few transcriptional regulators was also affected, and here we describe the role of one, CpsQ. CpsQ is one of four V. parahaemolyticus homologs in the CsgD/VpsT family of regulators, members of which have been implicated in c-di-GMP signaling. Mutations in cpsQ, like defects in another previously identified capsule regulator, cpsR, suppress the sticky phenotype of scr mutants. By using a combination of mutant and reporter analyses in Vibrio and E. coli, CpsQ is shown to be the direct positive regulator of cpsA transcription and its cpsA-activating ability is found to be responsive to the cellular level of c-di-GMP. Unexpectedly, we find that a low level of this nucleotide diminishes the stability of CpsQ. The molecular interplay in this signaling circuit is further defined by demonstrating that CpsQ is epistatic to CpsS, a negative regulator of capsule. CpsR activates cpsQ, and CpsQ can also regulate its own transcription. Wildtype Vibrio parahaemolyticus (LM5674) and scrABC mutant (LM6567) were grown on rich mediim agar plates and gene expression profiles were compared.
Project description:TICAM1 knockout and wild-type (TICAM1 knockout MEFs with restored TICAM1 expression) MEFs were treated by c-di-GMP or DMSO for 4 hours. Total RNA was analyzed via Illumina Mouse Ref-8 V2. Changes in gene induction, especially of interferon-stimulated genes, between c-di-GMP and DMSO treated cells were examined. Total RNA from c-di-GMP or DMSO treated wild-type MEFs, c-di-GMP or DMSO treated TICAM1 knockout MEFs were analyzed. Gene expression was compared between c-di-GMP treated and DMSO treated samples.

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