Specific RubisCO activities of yet uncultured Proteobacteria depending on flanking genes@en

Here we used the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. Hydrothermal vent fluids derived from the interface zone between hot fluids emanating from Drachenschlund and ambient cold seawater at 8°18'S/13°30'W served as initial sample for library construction. Among 1056 screened fosmid clones 12 exhibited RubisCO activity. The metagenomic fragments of all twelve clones resembled genes from Hydrogenovibrio crunogenus. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of 12 genes and 2 intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly-characterized gene and RubisCO activity.本研究采用首个完全基于活性筛选的方法，从宏基因组文库中筛选获得具有核酮糖-1,5-二磷酸羧化酶/加氧酶（RubisCO）活性的黏粒（fosmid）克隆。本研究以位于南纬8°18′、西经13°30′的Drachenschlund热液喷口热流体与周围冷海水交界带采集的热液流体，作为宏基因组文库构建的初始样品。在筛选的1056个黏粒克隆中，共有12个表现出RubisCO活性。所有12个克隆的宏基因组片段均与克鲁诺根氢弧菌（Hydrogenovibrio crunogenus）的基因序列高度相似。选取其中1个克隆进行后续分析，其携带的35.2 kb宏基因组插入片段包含RubisCO基因簇及其侧翼DNA区域。对该宏基因组片段上的12个基因及2个基因间区进行基因敲除实验后发现，RubisCO活性显著受损，该现象归因于编码推定转录调控因子的基因以及被认为对RubisCO激活至关重要的基因发生了缺失。本研究建立的新技术揭示了一类功能尚未明确的基因与RubisCO活性之间的全新关联。