Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus

Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A-Dependent NAD(P)H Sulfur Oxidoreductase The hyperthermophilic archaeon Pyrococcus furiosus, uses carbohydrates as a carbon source and produces acetate, CO2 and H2 as end products. When S° is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After one hour cells contain high NADPH- and coenzyme A-dependent S° reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (Mr 100 kDa) and is encoded by PF1186. This was previously assigned to an enzyme that reduces coenzyme A disulfide, which is a side-reaction of NSR. Whole genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to 7-fold within 10 min of S° addition. This primary response to S° also involves the up-regulation (> 16-fold) of a 13 gene cluster encoding a membrane-bound oxidoreductase (MBX). MBX is proposed replace the homologous 14 gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S° addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S°. A secondary response to S° is observed 30 min after S° addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins, termed SipA and SipB. This novel S°-reducing system involving NSR and MBX is so far unique to the heterotrophic Thermococcales, and is in contrast to the cytochrome- and quinone-based S°-reducing system in autotrophic archaea and bacteria. Keywords: time course, kinetic, sulfur metabolism, archaea, Pyrococcus furiosus, hyperthermophile Each kinetic experiment (growth date) containing 5 timepoints (0, 10, 20, 30 and 60 minutes) was performed twice. Each hybridization was performed twice and in case of 30 min 3 times.

《炽热嗜热古菌激烈火球菌（Pyrococcus furiosus）单质硫代谢解析：辅酶A依赖型NAD(P)H硫氧化还原酶的表征》
炽热嗜热古菌激烈火球菌以碳水化合物为碳源，代谢终产物为乙酸、二氧化碳与氢气。当向处于增殖期的培养物中添加单质硫（S°）后，10分钟内氢气生成速率迅速下降，且可检测到硫化氢（H₂S）生成。培养1小时后，胞质内可检测到高活性的NADPH与辅酶A依赖型S°还原活性（0.7 单位/毫克蛋白，85℃）。负责该活性的酶经纯化后达到电泳均一性（比活性达100 单位/毫克蛋白），将其命名为NAD(P)H单质硫氧化还原酶（NAD(P)H elemental sulfur oxidoreductase，NSR）。

NSR为同源二聚体黄素蛋白（分子质量100 kDa），由PF1186基因编码。此前该基因曾被注释为催化还原辅酶A二硫键的酶，而该活性实为NSR的副反应。全基因组DNA微阵列与定量PCR分析显示，添加单质硫后10分钟内，NSR的表达量可上调至初始水平的7倍。该针对单质硫的初级应答还包括一个含13个基因的基因簇的上调（上调幅度超过16倍），该基因簇编码膜结合氧化还原酶（membrane-bound oxidoreductase，MBX）。研究推测MBX可替代同源的14基因簇——后者编码铁氧还蛋白氧化、产氢型膜结合氢酶（membrane-bound hydrogenase，MBH），而MBH在添加单质硫后10分钟内表达量下调超过12倍。尽管尚未证实MBX的具体催化活性，但研究推测其可通过氧化铁氧还蛋白并还原NADP⁺来储备能量，所产生的NADPH可被NSR用于还原单质硫。

添加单质硫30分钟后可观察到次级应答，包括参与氨基酸生物合成与铁代谢的基因上调，以及两种被称为硫诱导蛋白（sulfur-induced proteins，SipA和SipB）的编码基因的上调。这种涉及NSR与MBX的新型单质硫还原系统，目前仅在异养型热球菌目（Thermococcales）中被发现，与自养型古菌和细菌中基于细胞色素与醌的单质硫还原系统截然不同。

关键词：时间进程、动力学、硫代谢、古菌、激烈火球菌、嗜热菌

每项动力学实验（含生长数据）设置5个时间点（0、10、20、30与60分钟），重复进行两次。每次杂交实验重复两次，针对30分钟时间点的实验则重复三次。