Acetyl coenzyme A measurement

Bacterial intracellular acetyl-coenzyme A (Ac-CoA) content was determined using the PicoProbe Ac-CoA assay kit (Abcam) according to manufacturer's instructions. Briefly, PA14 and the pvrA mutant were grown for 10 h in the Glu-M9 or FA-M9 medium. 30 ml of the bacterial cultures were collected by centrifugation at 12 000 × g for 5 min. After deproteinization with perchloric acid, the coenzyme A (CoA) Quencher and Quencher remover were added into each sample to correct the background generated by free CoA and succinyl-coenzyme A (succ-CoA). The sample was then diluted with the reaction mix, and the fluorescence was measured using a Versamax Tunable microplate reader (Molecular Devices) at the following settings: λ_ex 535 nm; λ_em 587 nm. The acetyl-CoA standard curve was made in the range of 0–100 pM and the correlation coefficient was 0.990 or higher.