Since the majority of all microorganisms are currently not culturable, we used a metagenomic approach to identify genes and enzymes associated with RubisCO expression. The investigated metagenomic DNA fragment originates from the deep-sea hydrothermal vent field Nibelungen at 8°18'S along the Mid-Atlantic Ridge. It is 13,023 bp and resembles genes from Thiomicrospira crunogena. The fragment encodes nine open reading frames (ORFs) which include two types of RubisCO, form I (CbbL/S) and form II (CbbM), two LysR transcriptional regulators (LysR1 and LysR2), two von Willebrand factor type A (CbbO-m and CbbO-1), and two AAA+ ATPases (CbbQ-m and CbbQ-1), expected to function as RubisCO activating enzymes. To date, only one study has ever investigated regulatory mechanisms in metagenome-derived RubisCO gene clusters (Böhnke and Perner 2015). Here, total RubisCO activity was significantly influenced when cbbL and cbbM neighboring genes were knocked out (Böhnke and Perner 2015), but it remained unclear which of the two RubisCOs was primarily affected by these mutations. Our study suggests that CbbQ-m and CbbO-m activate CbbL and that LysR1 and LysR2 proteins promote CbbQ-m/CbbO-m expression. CbbO-1 seems to activate CbbM and CbbM itself appears to contribute to intensifying LysR's binding ability and thus its own transcriptional regulation. CbbM furthermore appears to impair cbbL expression.

Drachenschlund (Nibelungen hydrothermal vent field), sample type: fluid sample cruise.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.