Succinate:Quinol Oxidoreductases in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Presence and Function in Metabolism and Electron Transport

The open reading frames sll1625 and sll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp. strain PCC 6803. When the organic acid content in the Δsll1625 and Δsll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Δsll1625 Δsll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.

在蓝细菌集胞藻（Synechocystis sp. strain PCC 6803）中，对与编码琥珀酸脱氢酶（succinate dehydrogenase）和延胡索酸还原酶（fumarate reductase）的铁硫（FeS）亚基的基因具有显著序列相似性的开放阅读框（open reading frames）sll1625和sll0823，分别单独及联合开展了敲除实验。对Δsll1625与Δsll0823单突变株的有机酸含量进行分析后发现，相较于野生型菌株，琥珀酸与延胡索酸的浓度均下降了100倍。针对双突变株Δsll1625 Δsll0823的同类分析显示，其琥珀酸水平仅保留野生型的17%，而延胡索酸水平仅为野生型的1%至2%。在对双突变株细胞内有机酸进行检测前，向其培养基中添加2-氧代戊二酸（2-oxoglutarate）可诱导琥珀酸积累。该结果表明，基因敲除操作阻断了琥珀酸脱氢酶的活性；同时证实，尽管该蓝细菌缺少传统的2-氧代戊二酸脱氢酶，但2-氧代戊二酸仍可在其体内（in vivo）转化为琥珀酸。此外，在黑暗环境且存在氰化钾（KCN）的条件下，突变株类囊体质醌库的还原速率相较于野生型最多慢5倍。进一步的体外（in vitro）实验显示，野生型类囊体膜组分中可检测到琥珀酸脱氢酶活性，但双突变株分离得到的膜组分中完全缺失该活性，单突变株的膜组分中该活性也有所降低；这进一步表明，sll1625和sll0823开放阅读框所编码的正是在蓝细菌类囊体膜中具有活性的琥珀酸脱氢酶复合物亚基。