Pseudomonas aeruginosa PA14 exposed to gliotoxin versus solvent control
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ABSTRACT: Pseudomonas aeruginosa PA14 was precultured in SCFM2, and the OD600 was adjusted to 0.05 in 8 ml of SCFM2, in 6-well flat bottom cell culture plates. Gliotoxin (20 µM) or MeOH (solvent control) was added at log phase (optical density at 600 nm [OD600] = 0.3), and the cultures were incubated statically for an additional 4 h. 5 ml of each culture (triplicate) was then immediately added to an equal volume of RNA-later.
Project description:Pseudomonas aeruginosa monocultures and co-cultures with Aspergillus fumigatus during growth in SCFM2 were harvested in the stationary phase of growth (triplicates) by aspirating the contents of the petri-dish well into a 15 ml falcon tube (passed over a 40 µm cell strainer) and combining this with an equal volume of RNA-later. Cultures stored in RNAlater were pelleted, resuspended in 1 mL RNA Bee, and RNA was extracted as described previously.
Project description:A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes. 2 Biological replicate experimens conducted 1 month apart. In the first there were 2 dye-swapped duplicates (total 4) of VC+MeOH versus MeOH only. In the second experiment there was one set of dye swapped arrays. Thus 6 arrays were performed including biological replicates, dye swapped replicates and technical duplicates.
Project description:We investigated temperature-dependent regulation of gene expression in E. coli. Liquid cultures of E. coli were grown at 27, 32, 37, 42 degrees Celsius in Tryptone Broth (TB) media in the orbital shaker at 250rpm and harvested by centrifugation at mid-exponential phase (OD600=0.6).
Project description:The trillions of microorganisms in the human gastrointestinal tract are an underexplored aspect of pharmacology. Despite numerous examples of microbial effects on drug efficacy and toxicity, there is often an incomplete understanding of the underlying mechanisms. Here, we dissect the inactivation of the commonly prescribed cardiac glycoside, digoxin, by Eggerthella lenta. Whole genome transcriptional profiling, comparative genomics, and culture-based assays revealed a cytochrome-encoding operon up-regulated by digoxin, absent in non-metabolizing E. lenta strains, and predictive of the efficiency of digoxin inactivation by the human gut microbiome. Digoxin inactivation was further enhanced by microbial interactions and inhibited by arginine. Pharmacokinetic studies using gnotobiotic mice revealed that increasing dietary protein reduces the in vivo metabolism of digoxin by E. lenta, with significant changes to drug concentration in the urine and serum. These results emphasize the importance of viewing pharmacology from the perspective of both our human and microbial genomes. RNA-Seq analysis of Eggerthella lenta cultured with or without digoxin.
Project description:Herbaspirillum seropedicae SmR1 was grown in NFbHPN medium until OD600nm of 0.6, when 5mM phenol or 2,5mM benzoic acid was added to the medium. After 30 minutes of growth, the cells were collected by centrifugation and total RNA was extracted with RiboPureBacteria. Also, a derivative strain from Herbaspirillum seropedicae SmR1 resistant to 1mM phenol (named strain RP) was grown in NFbHPN medium containing 1mM phenol until OD600nm of 0.6, then 5mM phenol was added to the medium. After 30 minutes of growth, the cells were collected by centrifugation and total RNA was extracted with RiboPureBacteria. All samples were depleted for ribossomal RNA e sequenced with Solid plataform.
Project description:Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles. RNA-Seq analysis of the human gut microbiome during consumption of a plant- or animal-based diet.
Project description:Korean ginseng is one of the most valuable medicinal plants worldwide. Yet, our understanding of ginseng proteomics is largely limited due to difficulties in extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone–MeOH/chloroform, Phenol–TCA/acetone, and Phenol–MeOH/chloroform methods. Consequently, the TCA/acetone–MeOH/chloroform method displayed the highest extraction efficiency, thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label–free quantitative proteomics approach. This approach led to the identification of 2,604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis including the methylerythritol 4–phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP–glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ–based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.
Project description:The vertebrate ectoderm gives rise to a variety of cell lineages, including neural, neural crest, placodal and non-neural cell fates. How cell fates are specified at the neural plate border (the region surrounding the neural plate) is not fully understood. We therefore carried out 10x scRNAseq of the chick epiblast to investigate cell fate specification at the neural plate border. Embryos were dissected and pooled according to stage. The tissue was then dissociated and FAC sorted to remove dead cells and remaining doublets before cells were stored in MeOH. Due to the time required to dissect embryos, multiple rounds of collections were carried out, with collections from the same stage pooled prior to 10x sequencing. Libraries were sequenced using an Illumina HiSeq 4000 at the Francis Crick Institute, London. This collection was a follow up to E-MTAB-10408.
Project description:The aim of this study is to evaluate the evolutionary robustness of the quorum sensing inhibitor (QSI) furanone C-30 for the treatment of P. aeruginosa biofilm infections. We repeatedly exposed P. aeruginosa biofilms to furanone C-30 (with or without tobramycin) in the synthetic cystic fibrosis sputum medium (SCFM2) and characterized the genotype and phenotype of the evolved lineages. P. aeruginosa biofilms were grown in SCFM2 for 24 h after which the treatment in fresh SCFM2 was added to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. After each cycle the number of CFU was determined and an aliquot of the culture was stored at -80°C in Microbank vials to allow further tests on the evolved strains. A sample from the treated biofilm was used to prepare a new overnight culture, in order to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. Whole-genome sequencing was performed on the wild type P. aeruginosa PAO1 and on the exposed lineages after 5, 10 and 16 cycles.