Analytical methods

Extracellular glucose was measured using an enzymatic assay (GOPOD kit, Megazyme, Bray, Ireland). Extracellular acetate, lactate and pyruvate were measured by high pressure liquid chromatography with a UV absorbance detector (Agilent, Santa Clara, CA, USA). For growing cells, yields of consumed glucose/gcdw were calculated by linear regression, and the specific uptake rate was calculated by multiplying by the calculated growth rate. For all calculations, a constant of 0.41 gcdw/(L×OD₆₀₀) was assumed.

For intracellular metabolite extracts, samples were collected by vacuum filtering 1 ml of culture onto a 0.22 μm Durapore filter (Millipore, Billerica, MA, USA) and immediately placing into 4 ml of cold (−20 °C) 40/40/20 (v/v/v) acetonitrile/methanol/water mixture. Sodium glutarate was added as an internal standard. Cell debris was pelleted by centrifugation at −9 °C and the supernatants were frozen in liquid nitrogen and then lyophilized to dryness. Samples were resuspended in 50 μl of cold 50/50 methanol/water for mass spectrometry analysis. UHPLC-TOF-MS was performed on an Agilent 6550 Q-TOF in negative mode. Absolute quantification of ATP, ADP, AMP and α-ketoglutarate was obtained by comparison to a calibration curve of pure standards (Sigma-Aldrich, St Louis, MO, USA).

For analysis of fatty alcohols, 500 μl of cell culture was extracted with 500 μl of 2:1 (v/v) chloroform/methanol containing 100 mg/l pentadecanol as an internal standard (odd chain lengths are not produced by E. coli). Samples were vortexed for 5 min and centrifuged at room temperature; 100 μl of the chloroform phase was collected and dried overnight. Samples were resuspended in 100 μl hexanes and analyzed by GC-MS. Total abundance was calculated on the basis of the ratio between the sum of the C₁₄, C₁₆ and C₁₈ peaks (including saturated and monounsaturated species) to the C₁₅ peak.

Proteomic samples were prepared as described previously⁴⁰ and analyzed as described in ref. 41.