Luciferase-Based Determination of ATP/NAD(H) Pools in a Marine (Environmental) Bacterium

Abstract

In all living organisms, adenosine triphosphate (ATP) and NAD(H) represent universal molecular currencies for energy and redox state, respectively, and are thus widely applicable molecular proxies for an organism’s viability and activity. To this end, corresponding luciferase-based assays in combination with a microplate reader were established with the marine model bacterium <i>Phaeobacter inhibens</i> DSM 17395 (<i>Escherichia coli</i> K12 served as reference). Grey multiwell plates best balanced sensitivity and crosstalk, and optimal incubation times were 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD⁺, and NADH, respectively. Quenching of bacterial cell samples involved Tris-EDTA-DTAB and bicarbonate base-DTAB for ATP and NAD(H) assays, respectively. The ATP and NAD(H) yields determined for <i>P. inhibens</i> DSM 17395 at ¼ OD_max were found to reside well within the range previously reported for <i>E. coli</i> and other bacteria, e.g., 3.28 µmol ATP (g cells_dry)⁻¹. Thus, the here described methods for luciferase-based determination of ATP/NAD(H) pools open a promising approach to investigate energy and redox states in marine (environmental) bacteria.