Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling in the rpoE deletion mutant strain compared to the wild-type strain of Salmonell typhi in response to ampicillin by miroarray-based profile assay

ABSTRACT: Wild-type and delta-rpoE strains of H:z66 positive S. Typhi were grown respectively in 1 ml of LB broth (pH 7.0) at 37oC with shaking (200 rpm) overnight. 200 ul of each culture was then transfer-incubated in fresh 20 ml of LB broth to log phase (0.5 OD at 600 nm). Ampicillin was added in cultures to a final concentration of 100 mM, and the bacteria were incubated with shaking at 37oC for 30 min. An RNeasy kit (QIAGEN) was used to extract the total RNA according to the manufacturer's instructions. The quantity and quality of the extracted RNA were determined by agarose gel electrophoresis and ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA). The extracted RNA was treated with 1 U of RNase-free DNase I (TaKaRa) at 37oC for 10 min to remove traces of DNA, and then incubated at 85oC for 15 min to inactivate the DNase. Total cDNA were synthesized using 20 ug of RNA (Invitrogen). A genomic cDNA microarray containing 4216 ORFs from the genome of S. Typhi Ty2 was used in this study. The fluorescence labeling of cDNA probe, hybridization, microarray scanning, and data analysis were performed as described previously. Significant differential expression of genes was determined by the value of log2 ratio of Cy3/Cy5 intensity which was larger than 1 or smaller than -1.

ORGANISM(S): Salmonella typhi GIFU10007

SUBMITTER: Hong Du

PROVIDER: E-MTAB-2702 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Infection with Salmonella enterica serovar Typhi in humans causes the systemic, life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. The ensuing disease is characterized by systemic dissemination and colonization of many organs, including the liver, spleen and gallbladder. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment we compared the transcriptome of Salmonella cultures grown in LB or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of phoP as a bile-responsive gene. Repression of phoP expression does not involve PhoPQ sensing of a bile component. Due to its critical role in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease. Salmonella enterica serovar Typhimurium LT2 glass slide microarrays were obtained through the NIAID’s Pathogen Functional Genomics Resource Center, managed and funded by Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Two independent cultures of Salmonella enterica serovar Typhimurium SL1344 were grown overnight in Luria-Bertani (LB) broth with streptomycin (100 μg/mL) at 37oC with shaking (225 RPM). Cells were pelleted by centrifugation and resuspended in sterile Dulbecco’s phosphate-buffered saline (PBS). These suspensions were further diluted 1:50 in PBS and used to inoculate 70 μL of either LB or physiological murine bile (in 0.65-mL tubes) at a 1:100 dilution. Cultures were incubated for 24 hours at 37oC with agitation (225 RPM). Then, samples were diluted in PBS and serial dilutions were plated on LB plates containing 100 μg/mL of streptomycin and incubated overnight at 37oC for bacterial growth and enumeration by colony counting. The remaining cells were used in the subsequent steps, as described below. RNA isolation began with the addition of 2 volumes of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) to the bacterial cultures and incubation at room temperature for approximately 5 minutes. Cells were pelleted by centrifugation and RNA was isolated using the RNeasy Mini Kit (Qiagen) with the on-column DNase digestion, according to the manufacturer’s recommendations. Synthesis of cDNA was performed as described herein. The entire RNA sample (≥3 μg) was mixed with 6 μg of Random Primers (Invitrogen, Burlington, Canada) and 40 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, Burlington, Canada) in a total volume of 18.5 μL and incubated at 70oC for 10 minutes. Samples were incubated on ice for 30 seconds, centrifuged to bring down condensation and incubated with the following components (final concentrations; Invitrogen): First Strand Buffer (1X), dithiothreitol (DTT; 10 mM), dNTPs (dATP, dCTP, dGTP, dTTP; 0.5 mM each) and 100 units of SuperScript II reverse transcriptase in a total volume of 30 μL. The mixture was incubated at 42oC overnight and the reaction was stopped and the RNA degraded through the addition of 0.5 M EDTA and 1 M sodium hydroxide (10 μL each) and incubation at 65oC for 15 minutes. Then, 25 μL of 1 M Tris (pH 7) was added to neutralize the pH of the cDNA solution. cDNA was purified using the MinElute PCR Purification Kit (Qiagen) according to the manufacturer’s recommendations, except that the Qiagen wash and elution buffers were substituted by phosphate buffers, according to the PFGRC microarray protocol. cDNA was then precipitated using ammonium acetate and ethanol and labeled with Cy3 (LB cultures) and Cy5 (bile cultures) using the ULS aRNA Fluorescent Labeling Kit (KREATECH Biotechnology, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Samples were mixed, dried and saved at -80oC until used. Pre-hybridization, hybridization and washing steps were performed essentially as described in the PFGRC protocols, except that the hybridization buffer contained KREAblock blocking buffer (25%; KREATECH Biotechnology). After hybridization, slides were scanned using an Affymetrix 428 Array Scanner from Eurofins MWG Operon (Huntsville, USA).

Project description:Actinosynnema mirum cells were grown in 3 biological replicates and harvested in exponential growth phase either grown in terrific broth (TB) or in glucose-yeast-malt (GYM) medium to analyse the differential gene expression under different grwoth media. A preculture of A. mirum was grown in 20 mL NL148 medium in 100 mL shake flasks for 3 days at 28 °C and 180 rpm (2.5 cm shaking diameter). 1 mL of the preculture was transferred to 60 mL terrific broth (TB) or 60 mL glucose-yeast-malt (GYM) medium in 500 mL shake flasks cultivated for 20 h at 28 °C and 180 rpm (2.5 cm shaking diameter). Pre and main culture were cultivated in triplicates. 495 µL of cultures was transferred to 96 well plates and 5 µL ritonavir in a final concentration of 0.1 mg mL-1 was added. Samples were incubated at 28 °C, 200 rpm for 44 h. Samples were centrifuged at maximum speed for 30 s, the supernatant was removed, and samples were flash frozen in liquid nitrogen. Cells were mechanically homogenized in 1X DNA/RNA Shield™ using Bead Tubes Type B (Macherey-Nagel, Düren, Germany) in a Bead Tube Holder at maximum speed for 20 min. RNA isolation was performed using the Quick-RNA Miniprep Plus (ZYMO research, Freiburg, Germany). An additional in-column DNase I treatment was performed. RNA pellets were eluted in 100 µL DNase/RNase-free water. The purified RNA was quantified with a NanoDrop (Thermo Scientific™, Waltham, USA). RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was stored at -80 °C. Total-RNA with an RNA integrity number (RIN) > 9 was used to prepare cDNA libraries. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. Preparation of cDNA libraries were performed according to the manufacturer’s instructions of TruSeq stranded mRNA Kit (Illumina, San Diego, USA). Subsequently, each cDNA library was sequenced on a HiSeq1500 (2 x 75nt PE rapid v2) Sequencer system (Illumina, San Diego, USA).

Dataset Information

Transcription profiling in the rpoE deletion mutant strain compared to the wild-type strain of Salmonell typhi in response to ampicillin by miroarray-based profile assay

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets