Project description:Purpose: Examining the transcriptome of Bacteroides thetaiotaomicron VPI-5482 challenged with Bacteroides phage to assess surface molecule expression changes Methods: Bacteroides thetaiotaomicron was grown in BPRM in vitro or Germ-Free mice were monocolonized with Bacteroides thetaiotaomicron and gavaged with ARB25 phage. Fold change was calculated as live phage versus heat-killed phage treated samples with n=3 biological replicates. Once cells reached an optical density corresponding to mid-log phase growth (absorbance between 0.4-0.5), RNA was isolated and rRNA depleted. Samples were multiplexed for sequencing on the Illumina HiSeq platform at the University of Michigan Sequencing Core. Data was analyzed using Arraystar software (DNASTAR, Inc.) using DEseq2 normalization with default parameters. Genes with significant up- or down-regulation were determined by the following criteria: genes with an average fold-change >5-fold and with at least 2/3 biological replicates with a normalized expression level >1% of the overall average, and a p-value < 0.05 (t test with Benjamini-Hochberg correction) Results: Specific capsule expression was increased in wild-type B. thetaiotaomicron during phage infection in vitro and in vivo. Many corresponding in vivo genes were upregulated as well as other surface layer proteins.
Project description:Purpose: Examining the transcriptome of Bacteroides thetaiotaomicron VPI-5482 grown on ribose to look at global changes in regulation in vitro on the defined monosaccharide, ribose as a sole carbon source. Methods: Bacteroides thetaiotaomicron was grown on 5 mg/ml ribose or glucose as a sole carbon source in vitro. Fold change was calculated as ribose over glucose with n=3 biological replicates. Once cells reached an optical density corresponding to mid-log phase growth (absorbance between 0.6-0.8), RNA was isolated and rRNA depleted. Samples were multiplexed for sequencing on the Illumina HiSeq platform at the University of Michigan Sequencing Core. Data was analyzed using Arraystar software (DNASTAR, Inc.) using RPKM normalization with default parameters. Genes with significant up- or down-regulation were determined by the following criteria: genes with an average fold-change >5-fold and with at least 2/3 biological replicates with a normalized expression level >1% of the overall average RPKM expression level in either glucose or ribose, and a p-value < 0.05 (t test with Benjamini-Hochberg correction) Results: We identified 81 genes differentially expressed at a 5-fold cutoff when grown in ribose over the reference glucose condition, many of which are involved in other metabolic processes important for seemingly unrealted nutrients.
Project description:Analysis of the Bacteroides thetaiotaomicron(BT) transcriptome during co-culture with Caco-2 intestinal epithelial cells To identify potential bacterial protein(s) involved in the anti-inflammatory effect of BT in colitis, BT was incubated with Caco-2 human intestinal epithelial cells for 2 hours, and bacterial gene expression was assessed on a Bacteroides thetaiotaomicron VPI-5482 specific microarray. Forty-three BT genes were up-regulated by five-fold or more and of these, twenty genes encoded hypothetical proteins.
Project description:Rag1-/- C57BL/6 or Rag1-/- C57BL/6 injected with the IgA producing hybridoma producing 225.4 anti- Bacteroides thetaiotaomicron (Capsular polysaccharide 4 dependent carbohydrate epitope specific epitope) mice were colonized with B. thetaiotaomicron wildtype strain VPI-5482 for 10 days. Cecal bacteria were harvested and snap frozen and RNA isolated. Keywords: Single time point, experimental and control groups.
Project description:Comparisons of gnotobiotic Rag1-/- mice, with and without subcutaneous 260.8 hybridomas, disclosed that this IgA does not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but does affect bacterial gene expression in ways that are emblematic of a diminished host innate immune response. C57BL/6 wildtype, C57BL/6J Rag1-/- , and C57BL/6J Rag1-/- mice harboring the 260.8 IgA producing hybridoma were colonized for 10 days with Bacteroides thetaiotaomicron VPI-5482.
Project description:The goal of this project was to sequence the transcriptome of wild type Bacteroides thetaiotaomicron VPI-5482 grown in minimal media with either glucose or bovine alpha 1 acid glycoprotein (AAGP) as the sole carbon source. Using these data we could then compare relative gene expression levels under each condition and identify genes specifically upregulated during growth on AAGP. Analysis of the spent media indicated that only the N-glycan component of the AAGP had been used by the cells to support growth.
Project description:We used Affymetrix GeneChips to determine the physiological differences between biofilm and planktonic cells of Bacteroides thetaiotaomicron strain VPI-5482 (ATCC 29148) by comparing gene expression. For this purpose, B. thetaiotaomicron cells were grown in sterile, continuous flow bioreactors fed with tryptone, yeast extract, glucose (TYG) medium. The bioreactors were controlled at a temperature of 37C using a water jacket and a recirculating water heater. After 8 hours post-inoculation, planktonic cells were harvested from the bulk solution in the bioreactor, and after 8 days post-inoculation, the biofilm was scraped from the carbon paper. RNA was harvested from both biofilm and planktonic populations. RNA was extracted by a phenol:chloroform method and purified with a Qiagen RNA Easy mini-kit. Overall growth conditions are summarized above. The experimental conditions were: biofilm (BF), and planktonic (PL).