Aerobic NO-exposed Chemostat Comparison of Wt & norR mutant Responses

Escherichia coli strains MG1655 and an isogenic norR::Tn5 mutant were grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions.. Cells were grown in defined media containing 8 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.1 h-1. For aerobic growth, the air-flow rate was 1 l/min, and the dissolved oxygen tension was maintained at 40% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode and automated adjustment of stirring rate. Cells were grown as above to steady-state, At steady-state, NOC-5 and NOC-7 was added to the chemostat culture and to the nutrient feed at a final concentration of 10 uM of each unless stated otherwise. Samples were taken after a period of 5 min exposure to NOC for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. Equal quantities of RNA from Wild type and norR::Tn5 cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values Keywords: Mutant Comparison, stress response, nitric oxide, NorR, chemostat, continuous culture Each strain was grown twice in seperate chemostat runs, exposed to NO. Samples were hybridised as WT vs NorR::Tn5 and each hybridisation had a corresponding dye-swap performed