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.

Drachenschlund (Nibelungen hydrothermal vent field), sample type: fluid sample.Fluid sample was collected within the DFG-SPP 1144 priority program “From Mantle to Ocean: Energy-, Material-, and Life-cycles at Spreading Axes"Work was granded by the Graduate School for C1-Chemistry in Resource and Energy Management and the DFG (Project PE1549/5 1 "Linkage between the distribution and biochemical properties of RubisCO and CODH enzymes and abiotic properties in thermally and chemically distinct deep-sea hydrothermal vent systems")Crude extracts used for activity measurements were prepared after autoindiction. All fosmid clones were constructed with pCC1FOS vector and Epi300 as a host. The activity measurements were performed at 25°C in buffer A [100 mM Tris HCl (pH 7.8), 10 mM MgCl2, 1 mM EDTA, 25 mM NaHCO3, and 1 mM DTT] with 0.2 mg unpurified, total protein, and 5 mM ribulose 1.5 bisphosphate (Rubp), whereby Rubp addition is the initiation step. Quantification of RuPB and 3-PGA was done using High Pressure Liquid Chromatography (HPLC) (LaChrom Elite® system, Hitachi) with a Lichrospher® 100 RP 18e column (VWR International GmbH, Darmstadt, Germany), detection at 200 nm, acetonitrile as an eluent, and a flow of 0.6 ml per minute.