A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity

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.

Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria

“…Co-expressing rubisco with CbbQ and/or CbbO in Escherichia coli was reported to lead higher rubisco activities in cell extracts and more active purified rubisco, suggesting the proteins' involvement in some form of posttranslational rubisco modification 31 32 . Subsequent similar co-expression experiments did not observe such effects 33 34 35 .…”

Novel hydrogenases from deep-sea hydrothermal vent metagenomes identified by a recently developed activity-based screen

“…The large subunit of the S. denitrificans [NiFe]-hydrogenase was cut out of the vector construct pBBR1MCS-2:: Suden_1435 [31] via double digestion with SacI/SacII (Thermo Fisher Scientific). As a negative control a metagenomic insert (84G4II) of 13 kbp without any hydrogenase genes but a RubisCO gene cluster [32] was used. All amplified (or restricted) [NiFe]-hydrogenase genes as well as the 84G4II insert were end-repaired, ligated into pRS44 (for details see "Construction of (meta)genomic fosmid libraries") and the fosmids transferred into E. coli EPI300 T1R cells via heat-shock transformation [33].…”

“…[21,34,35]). Microbial DNA from fluid samples was isolated from polycarbonate filters (on which the samples were concentrated) and all extracts were amplified via multiple displacement amplification [32,36,37].…”

Functional metagenomic selection of ribulose 1, 5‐bisphosphate carboxylase/oxygenase from uncultivated bacteria

“…However, recent massive deoxyribonucleic acid (DNA) sequencing efforts have shown the abundant presence of diverse and novel RubisCO‐encoding genes in environmental communities of largely uncultivated bacteria and new candidate phyla, the so‐called ‘microbial dark matter’ (Wrighton et al ., 2012; submitted; Campbell et al ., ; Guo et al ., ; Castelle et al ., ; Tebo et al ., ). Although the physiological significance of these novel RubisCO enzymes is unquantified, these organisms likely have evolved significant structural and functional adaptations to allow CO 2 to be metabolized in diverse environments such as in marine surface waters, hydrothermal vents and the terrestrial subsurface (Witte et al ., ; Wrighton et al ., 2012; submitted; Böhnke and Perner, ; Castelle et al ., ). Sequencing, cloning and activity screening of purified recombinant proteins is possible on a small scale (Böhnke and Perner, ).…”

“…Although the physiological significance of these novel RubisCO enzymes is unquantified, these organisms likely have evolved significant structural and functional adaptations to allow CO 2 to be metabolized in diverse environments such as in marine surface waters, hydrothermal vents and the terrestrial subsurface (Witte et al ., ; Wrighton et al ., 2012; submitted; Böhnke and Perner, ; Castelle et al ., ). Sequencing, cloning and activity screening of purified recombinant proteins is possible on a small scale (Böhnke and Perner, ). However, it is not always possible to discern physiological function by sequence‐gazing alone, nor is it practical to produce and individually screen and characterize recombinant proteins from the large number of novel RubisCO‐encoding genes recently observed (Wrighton et al ., 2012; submitted; Campbell et al ., ; Guo et al ., ; Castelle et al ., ; Tebo et al ., ).…”