Transcriptomic Profiling of Salmonella enterica Enteritidis and Typhimurium under Chlorine Based Oxidation
ABSTRACT: Investigation of whole genome gene expression level changes in Salmonella enterica serova Enteritidis and Typhimurium under chlorine treatment An eighteen chip study using total RNA isolated from three separate cultures of (1) S. Enteritidis in BHI broth (2) S. Typhimurium in BHI broth (3) S. Enteritidis in BHI broth w/ 130 ppm chlorine (4) S. Typhimurium in BHI w/ 130 ppm chlorine (5) S. Enteritidis in BHI broth w/ 390 ppm (6) S. Typhimurium in BHI broth w/ 390 ppm. Each chip measures the expression level of 5,027 ORFs covering the whole genome of S. Enteritidis and S. Typhimurium.
Project description:Investigation of whole genome gene expression level changes in Salmonella enterica serova Enteritidis and Typhimurium under chlorine treatment Overall design: An eighteen chip study using total RNA isolated from three separate cultures of (1) S. Enteritidis in BHI broth (2) S. Typhimurium in BHI broth (3) S. Enteritidis in BHI broth w/ 130 ppm chlorine (4) S. Typhimurium in BHI w/ 130 ppm chlorine (5) S. Enteritidis in BHI broth w/ 390 ppm (6) S. Typhimurium in BHI broth w/ 390 ppm. Each chip measures the expression level of 5,027 ORFs covering the whole genome of S. Enteritidis and S. Typhimurium.
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.
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.
Project description:In this study, RNA-seq was used to compare the transcriptomes of Listeria monocytogenes 10403S::ΔBCHL Prha-sigH and ΔBCHL Prha. RNA-seq was performed on ΔBCHL Prha-sigH and ΔBCHL 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 σH-dependent promoters were identified. Our results show that three σH-dependent transcritption units that encode competence proteins, including the comEABC , comGABCDEFG and coiA. Transcriptome profiles of L. monocytogenes 10403S::ΔBCHL Prha-sigH and ΔBCHL Prha were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500.
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.
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.
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.
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.
Project description:Nucleotide signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to changes in the environment. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP), which is widely distributed among bacteria and is also found in several archaea. This cyclic nucleotide has been shown to involve in several important cellular processes, including maintenance of DNA integrity, cell wall metabolism, stress tolerance, transcription regulation and virulence. However, the mechanisms by which c-di-AMP modulates these physiological changes have remained largely unknown.In the present study, we identified and characterized a c-di-AMP synthase (CdaA) in S. mutans UA159. Furthermore, we investigated the role of CdaA in S. mutans cell physiology and global gene expression by utilizing cdaA gene in-frame deletion mutant. Our findings suggest that CdaA is an important global modulator of optimal growth and environmental adaption in this pathogen. Streptococcus mutans UA159 whole-genome arrays (8 x 15 K) were obtained from Agilent and included 1998 probes for S. mutans transcripts. For microarray analysis, S. mutans UA159 and S. mutans ΔcdaA cells were routinely grown at 37°C anaerobically (90% N2, 5% CO2, 5% H2) in brain heart infusion broth (BHI; Difco, Sparks, MD, USA) to an optical density at 600 nm (OD600) of 0.5. Four RNA samples isolated from four independent cultures of UA159 and cdaA mutant strains were hybridized to the arrays and analyzed.