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

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 Phaeobacter inhibens DSM 17395 (Escherichia coli 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 P. inhibens DSM 17395 at ¼ ODmax were found to reside well within the range previously reported for E. coli and other bacteria, e.g., 3.28 µmol ATP (g cellsdry)−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.在所有活生物体中，三磷酸腺苷（adenosine triphosphate, ATP）与烟酰胺腺嘌呤二核苷酸（NAD(H)）分别是能量与氧化还原状态的通用分子通货，因此二者可作为生物体生存力与活性的通用分子替代标志物。为此，本研究以海洋模式细菌抑藻杆菌（Phaeobacter inhibens DSM 17395）为实验对象，以大肠杆菌K12（Escherichia coli K12）作为参照菌株，建立了结合酶标仪（microplate reader）的配套荧光素酶检测法。灰色多孔板在检测灵敏度与孔间串扰之间实现了最佳平衡；针对ATP与NAD(H)检测的最优孵育时间分别为5分钟与30分钟，三者的检测限分别可达0.042 nM、0.470 nM与0.710 nM，对应检测物为ATP、NAD+与NADH。针对细菌细胞样品的淬灭处理，ATP检测与NAD(H)检测分别采用三(羟甲基)氨基甲烷-乙二胺四乙酸-十二烷基三甲基溴化铵（Tris-EDTA-DTAB）体系与碳酸氢盐缓冲-十二烷基三甲基溴化铵（bicarbonate base-DTAB）体系。在四分之一最大光密度（ODmax）条件下测定的抑藻杆菌P. inhibens DSM 17395的ATP与NAD(H)产出量，均处于此前已报道的大肠杆菌与其他细菌的对应范围内，例如每克干细胞重可生成3.28 μmol ATP。综上，本文所述基于荧光素酶的ATP/NAD(H)库测定方法，为研究海洋（环境）细菌的能量与氧化还原状态提供了极具潜力的技术途径。