Böhnke-Brandt, Stefanie; Perner, Mirjam (2021): Specific RubisCO activities of yet uncultured Proteobacteria depending on flanking genes [dataset]. PANGAEA, https://doi.org/10.1594/PANGAEA.937559
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Abstract:
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.
Keyword(s):
Supplement to:
Böhnke, Stefanie; Perner, Mirjam (2015): A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity. The ISME Journal, 9(3), 735-745, https://doi.org/10.1038/ismej.2014.163
Project(s):
Funding:
German Research Foundation (DFG), grant/award no. 5471797: From Mantle to Ocean: Energy-, Material- and Life-cycles at Spreading Axes
German Research Foundation (DFG), grant/award no. 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
Coverage:
Latitude: -8.297167 * Longitude: -13.508833
Date/Time Start: 2009-04-27T10:01:00 * Date/Time End: 2009-04-27T10:01:00
Minimum Elevation: -2886.0 m * Maximum Elevation: -2886.0 m
Event(s):
M78/2_314 * Latitude: -8.297167 * Longitude: -13.508833 * Date/Time: 2009-04-27T10:01:00 * Elevation: -2886.0 m * Campaign: M78/2 (MARSUED5) * Basis: Meteor (1986) * Method/Device: Remote operated vehicle (ROV)
Comment:
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.
Parameter(s):
| # | Name | Short Name | Unit | Principal Investigator | Method/Device | Comment |
|---|---|---|---|---|---|---|
| 1 | Identification | ID | Böhnke-Brandt, Stefanie | fosmid clone designation | ||
| 2 | Insert size | Insert size | bp | Böhnke-Brandt, Stefanie | Sanger Sequencing modified after Sanger et al. (1977); PacBio Sequencing according to Rhoads and Au (2015) | |
| 3 | Deleted open-reading frame | Del orf | Böhnke-Brandt, Stefanie | Sequence editing, BioEdit | ||
| 4 | Insertion position | Insertion pos | aa | Böhnke-Brandt, Stefanie | Insertion position in non-coding region (ncr) is given in nucleotides | |
| 5 | Total length of deleted open-reading frame | Total l of del orf | aa | Böhnke-Brandt, Stefanie | Insertion position in non-coding region (ncr) is given in nucleotides | |
| 6 | Specific ribulose-1,5-bisphosphate carboxylase/oxygenase activity | Spec RubisCO act | nmol/mg/min | Böhnke-Brandt, Stefanie | High Performance Liquid Chromatography (HPLC) | |
| 7 | Specific ribulose-1,5-bisphosphate carboxylase/oxygenase activity, standard deviation | Spec RubisCO act std dev | ± | Böhnke-Brandt, Stefanie | Gaussian propagation of error |
License:
Creative Commons Attribution 4.0 International (CC-BY-4.0)
Status:
Curation Level: Enhanced curation (CurationLevelC)
Size:
393 data points