Project description:The transcriptional response of Escherichia coli MG1655 to NO released from NOC-5 and NOC-7 under anerobic conditions in contiuous chemostat culture on chemically defined minimal media
Project description:Aspergillus fumigatus was cultured in a chemostat for 12.5 hours, and switched to hypoxia (0.2% oxygen). Samples were collected at the beginning of the experiment, before the switch to hypoxia, and 2, 6, 12 and 24 hours after the switch. RNA was extracted and microarrays performed to compare each time point to the time the experiment was switched. There are 3 biological replicates and 2 technical replicates.
Project description:Peroxynitrite is formed in macrophages by the diffusion-limited reaction of superoxide and nitric oxide. This highly reactive species is capable of causing both oxidative and nitrosative stress in Escherichia coli. Previous studies have focused on the reactions of peroxynitrite with specific proteins or the effects of peroxynitrite on the growth and viability of whole cells. This work shows for the first time the transcriptomic response of E. coli to peroxynitrite, highlighting specific areas targeted by the stress. Upregulation of the cysteine biosynthesis pathway and subsequent identification of an increase in S-nitrosothiol levels suggests S-nitrosylation as a consequence of peroxynitrite exposure. Genes involved in the assembly / repair of iron-sulfur clusters also show enhanced transcription identifying another target of this reactive species. Unexpectedly arginine biosynthesis gene transcription levels were also elevated after treatment with peroxynitrite. Analysis of the negative regulator for these genes, ArgR, showed that the post-translational nitration of tyrosine residues within this protein is responsible for its degradation in vitro. Further upregulation is seen in oxidative stress response genes including katG and ahpCF. Probabilistic modelling of this data identified 5 altered transcription factors in response to peroxynitrite exposure including OxyR and ArgR. Hydrogen peroxide can be present as a contaminant in commercially available peroxynitrite preparations. Transcriptomic analysis of cells treated with hydrogen peroxide also showed an upregulation of oxidative stress response genes; however it did not show increased transcription of many other genes which are upregulated by peroxynitrite suggesting that cellular responses to peroxynitrite and hydrogen peroxide are distinct. Biological experiments (i.e. a comparison of control and plus peroxynitrite cells) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed. Additional biological experiments (i.e. a comparison of control and plus hydrogen peroxide cells) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
Project description:Oxygen transition experiement. Chemostat initially at steady state under oxygen replete conditions (7.5% oxygen input) was perturbed by a reduction in oxygen input (to 1.88% oxygen input). Samples were taken at the 7.5% oxygen and new 1.88% oxygen steady-states and at various points during the transtion between 7.5% and 1.88% oxygen. Type II experiment Biological replicates: At least 3 of each time point. Three independent transition experiements were performed. All samples were analysed with 7.5% oxygen as the reference. gDNA for Cy3 channel from wild-type strain.
Project description:Background: Pyrazinamide (PZA) plays an essential part in the shortened 6-month tuberculosis (TB) treatment course due to its activity against slow-growing, semi-dormant organisms. We tested the paradigm that PZA preferentially targets slow growing cells of Mycobacterium tuberculosis that remain after the initial kill by isoniazid, by observing the response of either slow growing or fast growing bacilli to differing concentrations of PZA. Methods: M. tuberculosis H37Rv was grown in continuous culture at either a constant fast growth rate (Mean Generation Time [MGT] of 23.1 h) or slow growth rate (69.3 h MGT) at a controlled dissolved oxygen tension of 10% and a controlled acidity at pH 6.3 ± 0.1. The cultures were exposed to step-wise increases in the concentration of PZA (25 µg ml-1 to 250 µg ml-1) every 2 MGTs, and bacterial survival was measured. PZA-induced global gene expression was explored for each increase in PZA-concentration, using microarray.
Project description:Staphylococcus aureus can survive in the low redox potential environment of the host cytosol. How low redox potential affects S. aureus USA300 Je2 was investigated by establishing aerobic steady state chemostat cultures and then perturbing the steady state by addition of the reducing agent dithiothreitol. These highly controlled steady state cultures allowed only one parameter (redox potential) changed. Dithiothreitol was used as a non-metabolizable reductant. Before and after perturbation samples were taken for gene expression profiling. Three independent biological replicates were analyzed.
Project description:We used the previously designed oligonucleotide-based microarray (Burgmann et al. Environmental Microbiology 2007, 9: 2742-2755) to detect the transcripts of R. pomeroyi DSS-3 genes when the cells were cultured under steady-state carbon (glucose), nitrogen (ammonium), phosphorus (phosphate), or sulfur (sulfate) limitation. A total of 14 mRNA samples were hybridized to the arrays (three biological replicates from glucose, ammonium, phosphate, or sulfate limitation and one technical replicate each for ammonium or sulfate limitation)
Project description:We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications. Three experiments are gene expression comparisons between three ministat cultures and a single Sixfors sample. The four CGH arrays are individual clones evolved in four sulfate limitation ministats compared to a wt ancestor strain.