Abstract
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth’s primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. 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 twelve genes and two 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. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.
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Acknowledgements
We thank the captain and crews of the RV Meteor as well as the ROV Kiel6000 (GEOMAR, Kiel) for helping us to obtain deep-sea vent samples. We thank Wenke Bahnsen and Nicole Adam for excellent technical assistance in the laboratory and Wolfgang R Streit for stimulating discussions and suggestions during the work. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (PE1549/5-1) and the DFG priority program 1144 ‘From Mantle to Ocean: Energy-, Material- and Life-cycles at Spreading Axes’.
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Böhnke, S., Perner, M. A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity. ISME J 9, 735–745 (2015). https://doi.org/10.1038/ismej.2014.163
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DOI: https://doi.org/10.1038/ismej.2014.163
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