Comparative genomic hybridization of Bordetella bronchiseptica strains RB50 and 1289
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ABSTRACT: B. bronchiseptica strains RB50 and 1289 strains were grown in SS broth at 37°C with shaking overnight and genomic DNA was isolated from bacterial cultures using a DNA extraction kit (Qiagen, Valencia, CA) and digested with DpnII. For each labeling reaction, 2 ug of digested genomic DNA was randomly primed using Cy-5 and Cy-3 dye-labeled nucleotides, with BioPrime DNA labeling kits (Invitrogen, Carlsbad, CA) and the two differentially labeled reactions to be compared were combined and hybridized to a B. bronchiseptica RB50 specific long-oligonucleotide microarray.
Project description:B. bronchiseptica strains RB50 and 1289 were grown in SS broth, subcultured at a starting OD600 of 0.02 into 50mL of SS broth, grown at 37C for 24 hours while shaking and harvested in log phase (OD600 1.0). Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA), treated with RNase-free DNase I (Invitrogen, Carlsbad, CA) and purified using RNeasy columns (Qiagen, Valencia, CA) according to the manufacturers instructions. RNA was isolated from two independent biological replicates of strains RB50 and 1289. A 2-color hybridization format was used and dye-swap experiments were performed. For each reaction, 5ug of cDNA was fluorescently labeled. The two differentially labeled reactions to be compared were combined and hybridized to a B. bronchiseptica strain RB50 specific long-oligonucleotide microarray.
Project description:comparative genomic hybridization (CGH) analysis was perfromed in order to identify coding regions present in strain T44625 that are either absent or contain a high degree of sequence divergence compared to the RB50 reference strain.
Project description:Bordetella bronchiseptica is a gram-negative respiratory pathogen that causes a diverse spectrum of respiratory disease in a wide-range of hosts. We sought to determine if strains of B. bronchiseptica differed in virulence using the mouse model of infection. Mean lethal doses (LD50) of different B. bronchiseptica strains varied widely in the murine model. B. bronchiseptica strain 253 had a LD50 that was 10-fold lower than the prototypical and fully sequenced B. bronchiseptica strain RB50. Using whole genomic transcriptome analysis covering 100% of B. bronchisetpctica strain RB50ÃÂs predicted open reading frames (ORFs), 253 was identified as lacking expression of adenylate cyclase toxin (ACT). Using whole genomic comparative genomic hybridization analysis and whole genome sequencing, we determined that the cya operon, which is required for ACT production, was absent from the 253 genome.
Project description:Whole genome transcriptome analysis was performed to determine which genes were differentially expressed between strains T44625 and RB50
Project description:Comparison of B. bronchiseptica strains RB50 and 761 grown in either atmospheric concentrations of oxygen and carbon dioxide (normal conditions) or in atmospheric levels of oxygen with the addition of 5% carbon dioxide into a sealed incubator (5% CO2 conditions).
Project description:Bordetella bronchiseptica is a gram-negative respiratory pathogen that causes a diverse spectrum of respiratory disease in a wide-range of hosts. We sought to determine if strains of B. bronchiseptica differed in virulence using the mouse model of infection. Mean lethal doses (LD50) of different B. bronchiseptica strains varied widely in the murine model. B. bronchiseptica strain 253 had a LD50 that was 10-fold lower than the prototypical and fully sequenced B. bronchiseptica strain RB50. Using whole genomic transcriptome analysis covering 100% of B. bronchisetpctica strain RB50ÃÂÃÂs predicted open reading frames (ORFs), 253 was identified as lacking expression of adenylate cyclase toxin (ACT).
Project description:Based on the genomic sequence and using the freely available software ArrayOligoSelector, a long oligonucleotide B. bronchiseptica microarray was designed and assembled. This long-oligonucleotide microarray was subsequently tested and validated by comparing changes in the global expression profiles between B. bronchiseptica RB50 and its Bvg- phase-locked derivative, RB54.
Project description:B. pertussis Tohama I was grown in iron-depleted or iron-replete media and sampled at several time points to assess global gene expression
Project description:Bordetella bronchiseptica RB50 was shifted from iron replete to either iron depleted or iron replete media, and samples were taken post shift for transcriptional profiling
Project description:B. bronchiseptica RB50 was grown in medium either lacking or containing iron (ferrous sulfate). At various time points, samples were taken for gene expression analysis.