Transcriptomic Profiling of Salmonella enterica Enteritidis and Typhimurium under Chlorine Based Oxidation
Ontology highlight
ABSTRACT:
ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. LT2
SUBMITTER: Kaiping Deng
PROVIDER: E-GEOD-20701 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Investigation of whole genome gene expression level changes in Salmonella enterica serova Enteritidis and Typhimurium under chlorine treatment
2010-03-10 | GSE20701 | GEO
Project description:Mapping of transposon mutant library during growth in Brain Heart Infusion (BHI) broth and BHI broth with 20 ug/ml ampicillin
2012-10-09 | E-MEXP-3501 | biostudies-arrayexpress
Project description:Analysis of the gene expression pattern of a znuA mutant constructed in the O35E background. It's pattern of expression was compared to that of O35E. 3 biological replicates were prepared. Each replicate was grown in BHI broth and RNA extracted with the Ambion Ribopure bacterial kit.
2014-03-16 | E-GEOD-28027 | biostudies-arrayexpress
Project description:Corynebacterium pseudotuberculosis strain 258, isolated from a caprine host in Brazil, was maintained in brain-heart-infusion broth (BHI HiMedia Laboratories Pvt. Ltda, India) at 37C, under rotation. Then, the bacteria was added to 20mL of BHI-Broth with 0.05% of 80% Tween and was maintained at 37C overnight, under rotation. The sample was then diluted 1/100 in 100mL of BHI broth, maintained at 37C, under rotation, and it was monitored until it reached the beginning of the exponential fase (A600 = 0.2). Afterwards, the sample was equally splitted in 4 aliquots of 20mL, centrifuged for 3 minutes under 8000rpm at 37C, and the pellets were re-suspendend in different medias according to the following conditions: acid stress, with HCl (fitting the pH to 5); osmotic stress, with 2M of NaCl; heat shock, where the pellet was re-suspended in pre-heated BHI (50C); and control, where the pellet was re-suspended in BHI under physiologic condition. Then, the samples for the acid stress, osmotic stress and control condition were maintained at 37C for 15 minutes, under rotation, whereas the sample under heat shock condition was maintained at 50C. Next, the samples were serially diluted from 10e-1 to 10e-6, where the samples from 10e-4 to 10e-6 were spread in BHI with agar (1.5%), which was maintained at 37C for 48hs for colony counting (duplicated samples); and the broth was centrifuged at 37C for 3 minutes under 8000rpm and the final pellet was re-suspended in 2mL of RNAlater, following the protocol of the manufacturer. Briefly, the bacteria suspended in RNAlater buffer were subjected to total RNA extraction using the ChargeSwitch total RNA cell kit (Invitrogen, USA) in accordance with the manufacturers recommendations, including the following adaptations: after the addition of the lysis buffer (Invitrogen), the material was transferred to 2mL tubes partially filled with 1mm diameter glass microbeads (Bertin Technologies). The cells were lysed mechanically using a Prescellys 24 homogenizer, set at 6,500 rpm, for 2 cycles (15 seconds per cycle) with an interval of 30 seconds between the cycles. The samples were centrifuged for 1 minute, and the supernatant transferred to fresh 2ml tubes and incubated in a dry bath at 60C for 15 minutes (representing the complete original protocol). DNAse was added to eliminate the residual genomic DNA. The elution of the total RNA from the magnetic beads was performed using 100 uL of milli-Q RNase-free water. The amount of total RNA was assessed using a Qubit 2.0 fluorometer (Invitrogen). In order to construct the library, the SOLiD Total RNA-Seq Kit was used. Briefly, after removing rRNAs, approximately 245ng of RNA was fragmented with RNAIII through incubation in a MJ Research thermocicler at 37C for 10 min and the reaction was interrupted with the addition of 90uL of nuclease-free water. The RNA was, then, concentrated through precipitation with ethanol according to the RiboMinus Concentration Module (Invitrogen - EUA), for subsequent hybridization with adapters and amplification of the cDNA library, which was obtained through reverse transcription using P1 adapters linked to the RNA extremities, according to the protocol SOLiD Total RNA-Seq Kit (Life Technologie, EUA). Next, the cDNA was denatured in polyacrylamide gel electrophoresis (6%) for obtaining fragments in the range from 150 to 250 base pairs for PCR amplification. The PCR amplifications were carried out in a MJ Research thermocycler as follow: the mixture was denatured at 95C for 30 sec, followed by 15 cycles of amplification at 95C for 30 sec, 62C for 30 sec and 72C for 30 sec, with a final extension of 72C for 7 min. Finally, the amplicons were purified using the kit PureLinkTM PCR Micro (Invitrogen - EUA), quantified in Qubit 2.0 Fluorometer-Invitrogen (EUA) and assessed through agarose electrophoresis 2%. The samples were fitted to the concentration of 60pg/mL, which is the suggested concentration for emulsion PCR. After the emulsion PCR, an enrichment reaction was performed for capturing only microbeads with fragments inserted. Next, a workflow analysis (WFA) was performed, where 15,000,000 microbeads were deposited to the slide and have undergone a binding cycle for quantitative and qualitative evaluation. According to this evaluation, 44,000,000 microbeads were deposited for sequencing, based on the manufacturer's description, generating reads of 50 nucleotides in range.
2020-06-20 | E-MTAB-9217 | biostudies-arrayexpress
Project description:M. catarrhalis strain O35E.rpsl was inoculated into the nasopharynx of healthy, male adult chinchillas. 24 hours later the nasopharyngeal tissues were extracted and homogenized. Total RNA was extracted from these tissue samples. Subsequently, M. catarrhalis genome directed primers were utilized to synthesize cDNA from the total RNA sample. As a control, M.catarrhalis strain O35E.rpsl was grown in BHI broth to a Klett density of 200 units and underwent RNA extraction per standard protocols. The genome directed primers mentioned above were utilized to synthesize cDNA. Both cDNA samples were subsequently labelled with either Cy3 or Cy5 and hybridized to a custom Microarrays, Inc. gene chip and scanned after 16 hours. Differential gene expression was measured utilizing the broth grown cells as the baseline and the chinchilla isolated cells as the experimental variable. There are 5 individual sample results included in this series. These represent the data from four individual biological replicates (i.e. 4 different sets of inoculated animals). For each replicate the control samples are simultaneously grown broth samples. Three dye swap experiments were performed.
2012-04-03 | E-GEOD-13559 | biostudies-arrayexpress
Project description:L. monocytogenes EGD-e (Glaser et al., 2001) was grown in Brain Hearth Infusion Broth (Difco) with shaking (200 rpm, New Brunswick C24KC) using 10 ml culture medium in 100 ml conical flasks. An exponentially growing culture was used to inoculate 100 ml of fresh pre-warmed BHI broth in a 500 ml flask. This culture was incubated at 48°C (GFL Type 1083, 60 % shaking speed) and samples for RNA extraction were taken after 3, 10, 20, and 40 min.
2007-10-09 | E-MEXP-1118 | biostudies-arrayexpress
Project description:Two independent aliquots containing approximately 10^7 cells from the transposon mutant of E. faecium E1162 were grown at 37 C for 20 hours in 20 ml of BHI broth. Genomic DNA was isolated from the two replicate cultures and used for the generation of cDNA. The cDNA samples were labeled with Cy3 and Cy5 respectively and hybridized to a microarray that was designed using the E. faecium E1162 genome sequence.
2012-11-02 | E-MEXP-3502 | biostudies-arrayexpress
Project description:In this study, RNA-seq was used to compare the transcriptomes of Listeria monocytogenes 10403S::ÎBCHL Prha-sigH and ÎBCHL Prha. RNA-seq was performed on ÎBCHL Prha-sigH and ÎBCHL Prha RNA samples representing three independent biological replicates at log phase in Brain Heart Infusion (BHI) broth under rhamnose induction. Indexed and purified cDNA libraries (6 libraries including 3 replicates for 2 strains) were loaded together onto an independent flow cell without any other samples; sequencing was carried out by running Hiseq 2500 (single-end, 150-bp per read). Reads alignment was carried out using the Burrows-Wheeler Aligner (BWA). Differential expression of genes in different strains was statistically assessed using the BaySeq method. To identify sigH-dependent promoters, a new method of moving sliding windows of 50 nt along the whole genome was used to compare the normalized RNA-seq coverage (NRC) between the two strains. Using the standard whole gene differential expression analysis, significant upregulation of 5 genes in 4 operons was found in the sigH overexpressing strain. While with the sliding windiow analysis, 2 additional ÏH-dependent promoters were identified. Our results show that three ÏH-dependent transcritption units that encode competence proteins, including the comEABC , comGABCDEFG and coiA. Transcriptome profiles of L. monocytogenes 10403S::ÎBCHL Prha-sigH and ÎBCHL Prha were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500.
2016-02-11 | E-GEOD-73008 | biostudies-arrayexpress
Project description:Background: Inflammatory bowel diseases (IBD) may be caused in part by aberrant immune responses to commensal intestinal microbes including Bacteroides thetaiotaomicron (B.theta). Healthy, germ-free HLA-B27 transgenic (Tg) rats develop chronic colitis when colonized with complex gut commensal bacteria whereas non-transgenic (nTg) rats remain disease-free. However, the role of B.theta, a well-characterized anaerobic commensal bacterium, in causing disease in Tg rats is unknown nor is much known about how microbes respond to host inflammation. Methods: Tg and nTg rats were monoassociated with a human isolate of B.theta. Colonic inflammation was quantified by blinded histological scoring and real-time RT-PCR assays of pro-inflammatory cytokines. Cecal bacterial concentrations were measured by quantitative plating. Whole genome transcriptional profiling of B.theta recovered from ceca was performed using custom GeneChips and data analyzed using dChip, Significance Analysis of Microarrays, and Gene Set Enrichment Analysis (GSEA) software. Results: B.theta monoassociated Tg rats had significantly more colonic inflammation and increased colonic levels of pro-inflammatory cytokine mRNAs compared to nTg controls. Transcriptional profiles of cecal B.theta were significantly different in Tg vs. nTg rats. GSEA revealed that the Gene Ontology molecular function of receptor activity, which is comprised mainly of genes that encode nutrient binding proteins, was significantly enriched with genes upregulated in B.theta from Tg rats. KEGG canonical pathways of ribosome, oxidative phosphorylation, pyrimidine metabolism, purine metabolism, peptidoglycan biosynthesis, and metabolism were significantly enriched with genes downregulated in B.theta from Tg rats. Numbers of viable bacteria/gram cecal contents in Tg vs. nTg rats were not significantly different. Conclusions: B.theta induces mild colitis in HLA-B27 Tg rats, which is associated with changes in the expression of microbial metabolic and nutrient binding pathways, but no difference in concentrations of luminal bacteria. Mechanistic studies of differentially expressed B.theta genes may reveal novel pathways that contribute to IBD. The fully-sequenced human fecal isolate of B.theta (VPI-5482) was grown on Brain-Heart Infusion (BHI) agar and in BHI broth under strict anaerobic conditions using pre-reduced media. Adult germ-free HLA-B27/b2 microglobulin transgenic rats and adult germ-free non-transgenic littermate were monoassociated with B.theta for six weeks in gnotobiotic isolators at the National Gnotobiotic Rodent Resource Center at UNC Chapel Hill. Bacterial RNA was isolated from rat cecal contents and hybridized on Affymetrix human gut microbiota community GeneChip.
2012-01-11 | E-GEOD-34966 | biostudies-arrayexpress
Project description:The foodborne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. While osmotic stress induced changes in expression have been investigated for specific genes, identifying and understanding genome-wide temporal changes in the transcriptome due to salt stress will provide insights into how this pathogen adapts to and survives this stress, leading to development of new intervention strategies. To determine the short-term and long-term responses to salt stress, we exposed exponential phase cells of L. monocytogenes H7858 to 6% NaCl in Brain Heart Infusion (BHI) broth at 7°C and 37°C and extracted RNA after 2.5%, 5%, 10%, and 20% of lag phase and during exponential growth in BHI + 6% NaCl, and evaluated temporal changes in transcript levels with microarrays. The temperature dependent short-term response to salt stress included a significant increase in transcript levels of the alternative sigma factor σB, with maximum expression at 2.5-5% of lag phase at 37°C, and at 20% of lag phase at 7°C. Transcript levels of genes known to be regulated by σB, including inlAB, opuCA, glpFK, and gadBC, were significantly upregulated at the same relative time points as σB As indicated by significantly elevated transcript levels during exponential growth in BHI + 6% NaCl, genes encoding proteins involved in purine and pyrimidine synthesis and amino acid biosynthesis are part of the long-term response to salt stress at 37°C. Transcript levels of virulence genes plcA, mpl, actA, and plcB were also significantly upregulated during exponential growth under salt stress at 37°C. Genes encoding proteins involved in protein degradation and stability and cell membrane modifications are part of the long-term response to salt stress at 7°C. Overall, an initial alteration in the transcriptome occurs, decreasing transcript levels of genes encoding proteins involved in energy conversion and metabolism, while increasing transcript levels of those involved in the stress response controlled by σB, followed by a long-term response, including increased expression of genes encoding compatible solute transporters and general stress proteins, facilitating growth under salt stress. A reference design was used to analyze gene expression differences. Exponential phase cultures at 37C and at 7C were used as a reference for salt stressed samples at each temperature. Salt stressed samples from the same relative time point were compared across temperatures. 4 biological replicates were tested for each comparison.
2011-07-01 | E-GEOD-27521 | biostudies-arrayexpress