e., glucose) allowing steady state growth of cells (i.e., at steady state the specific growth rate of cells is equal to the dilution rate). At these conditions, transient growth effects and other stress-induced responses are avoided that could mask effects resulting specifically from nutrient limitation. Three dilution rates were chosen based on previous results obtained in our laboratory that suggest that the effect of the Inhibitors,research,lifescience,medical nutrient

limitation and, consequently, the RelA activity, is much lower at higher dilution rates. Thus, the steady state metabolism analyses of the wild-type and ΔrelA mutant cultures were performed at two low (0.05 and 0.1 h−1) and one higher (0.2 h−1) dilution rates. The aim of this study was to analyse the growth rate-dependent behaviour of E. coli cells and observe how the mutation in the relA gene affects the cellular responses to nutrient-limiting conditions. This will provide us further information to evaluate ppGpp-deficient strains as potential hosts for recombinant E. coli bioprocesses. 2. Experimental Section 2.1. Inhibitors,research,lifescience,medical Bacterial Strains and Growth Conditions E. coli K12 W3110 (F-, LAM-, IN[rrnD-rrnE]1, rph-1) and the isogenic Inhibitors,research,lifescience,medical mutant ΔrelA (obtained from M. Cashel [13]) were grown under controlled conditions in a chemostat culture at 37 ºC, pH 7 and dissolved oxygen above 30%. The minimal medium consisted of 5 g·L−1 of glucose, 6 g·L−1 of Na2HPO4, 3 g·L−1 of KH2PO4, 0.5 g·L−1 of NaCl,

1 g·L−1 of NH4Cl, 0.015 g·L−1 of CaCl2, 0.12 g·L−1 of MgSO4•7H2O, 0.34 g·L−1 of thiamine, 2 mL·L−1 of trace-element Inhibitors,research,lifescience,medical solution (described elsewhere [16]) and 2 mL·L−1

of vitamins solution (described elsewhere [16]). The minimal medium was further supplemented with 20 mg·L−1 of L-isoleucine to grow the W3110 strain and 20 mg·L−1 of L-isoleucine and L-valine along with 25 mg·L−1 of kanamycin to grow the ΔrelA mutant strain. Inhibitors,research,lifescience,medical Chemostat cultivations were carried out in a 3 L fermenter (BioFlo 3000, New Brunswick Scientific, USA) with a working volume of 1.5 L. The described minimal medium was continuously fed to the respective E. coli culture, at least for five residence times, at a given dilution rate (0.05, 0.1 and 0.2 h−1), and the working volume was kept constant by withdrawing the culture broth through level control. Steady-state conditions were verified by constant optical density Cilengitide and glucose measurements. The pH of the culture was maintained at 7.0 by adding 2.0 M NaOH and 2.0 M HCl. Dissolved oxygen was maintained above 30% saturation through a cascade mode controlling the agitation speed and airflow. 2.2. Analytical Techniques The biomass concentration was determined by measuring culture absorbance (OD600nm) in a Jenway 6300 spectrophotometer and using a standard calibration curve (OD600nm against cell dry weight (CDW)). In order to determine CDW, 10 mL of broth were filtered using 0.2 µm membrane filters and the filters with cell biomass were dried in the microwave to a constant weight [17].