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Böhnke-Brandt, Stefanie; Perner, Mirjam (2021): Specific RubisCO activity of currently uncultured microorganisms inhabiting hydrothermal deep sea vents. PANGAEA, https://doi.org/10.1594/PANGAEA.931785

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Abstract:
In 2014 we described a new activity-based screen suited to seek active recombinant RubisCOs from the environment - independent of the native host's culturability. Within this study we aimed at seeking novel RubisCO active enzymes from the environment. For this purpose we screened six metagenomic fosmid libraries from hydrothermal vent environments, differing with respect to their (i) geographic origin (north or south Mid Atlantic Ridge) and their (ii) environmental characteristics (basalt vs. ultramafic hosted, temperature, and pH). Overall we screened roughly 12500 metagenomic fosmid clones and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.
Keyword(s):
activity-based screen; autotrophic CO2-fixation; functional metagenomic; HPLC; hydrothermal deep sea vents; specific RubisCO activities
Supplement to:
Böhnke, Stefanie; Perner, Mirjam (2019): Seeking active RubisCOs from the currently uncultured microbial majority colonizing deep-sea hydrothermal vent environments. The ISME Journal, 13(10), 2475-2488, https://doi.org/10.1038/s41396-019-0439-3
Project(s):
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 (RuBisCO)
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:
Median Latitude: -0.844444 * Median Longitude: -19.737040 * South-bound Latitude: -8.300000 * West-bound Longitude: -44.966670 * North-bound Latitude: 14.750000 * East-bound Longitude: -12.366670
Date/Time Start: 2009-02-04T12:37:00 * Date/Time End: 2009-04-27T09:53:00
Minimum ELEVATION: -3047 m a.s.l. * Maximum ELEVATION: -2915 m a.s.l.
Event(s):
M78/2_274 * Latitude: -4.804000 * Longitude: -12.372333 * Date/Time: 2009-04-17T18:08:00 * Elevation: -2980.0 m * Campaign: M78/2 (MARSUED5) * Basis: Meteor (1986) * Method/Device: Remote operated vehicle (ROV)
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)
MSM10/3_300 * Latitude: 14.752500 * Longitude: -44.978000 * Date/Time: 2009-02-04T12:37:00 * Elevation: -3003.3 m * Location: South Atlantic Ocean * Campaign: MSM10/3 * Basis: Maria S. Merian * Method/Device: Remote operated vehicle (ROV)
Comment:
Samples were collected within the DFG-SPP 1144 priority program “From Mantle to Ocean: Energy-, Material-, and Life-cycles at Spreading Axes".
Specific enzyme activity measurements were performed within the scope of 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".
For anoxic activity measurements crude extractes were produced aerobically and allowed to become anoxic by placing them under N2/H2 atmosphere (95%/5%; v/v) at 4°C over night. The assay was then performed in an anaerobic chamber under the same atmosphere using anoxic buffer A (100 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 1 mM EDTA, 25 mM NaHCO3 and 1 mM DTT) and anoxic ribulose-1,5-bisphosphate substrate.
Parameter(s):
#NameShort NameUnitPrincipal InvestigatorMethod/DeviceComment
1Event labelEventBöhnke-Brandt, Stefanie
2Sample code/labelSample labelBöhnke-Brandt, Stefanie
3Sample typeSamp typeBöhnke-Brandt, Stefanie
4IdentificationIDBöhnke-Brandt, Stefaniefosmid clone designation
5SiteSiteBöhnke-Brandt, Stefaniesampling site
6Area/localityAreaBöhnke-Brandt, Stefanie
7DATE/TIMEDate/TimeBöhnke-Brandt, StefanieGeocode
8LATITUDELatitudeBöhnke-Brandt, StefanieGeocode
9LONGITUDELongitudeBöhnke-Brandt, StefanieGeocode
10ELEVATIONElevationm a.s.l.Böhnke-Brandt, StefanieGeocode
11Temperature, technicalT tech°CBöhnke-Brandt, Stefanieheat blockenzyme assay temperature
12TreatmentTreatBöhnke-Brandt, StefaniePresence of oxygen during assay (oxic/anoxic)
13Specific ribulose-1,5-bisphosphate carboxylase/oxygenase activitySpec RubisCO actnmol/mg/minBöhnke-Brandt, StefanieHigh Performance Liquid Chromatography (HPLC)
14Specific ribulose-1,5-bisphosphate carboxylase/oxygenase activity, standard deviationSpec RubisCO act std dev±Böhnke-Brandt, StefanieGaussian propagation of error
15Accession number, geneticsAccess no genBöhnke-Brandt, StefanieSanger Sequencing modified after Sanger et al. (1977); PacBio Sequencing according to Rhoads and Au (2015)nt sequences have been deposited in the NCBI (National Center for Biotechnology Information)
16Accession number, geneticsAccess no genBöhnke-Brandt, StefanieSanger Sequencing modified after Sanger et al. (1977); PacBio Sequencing according to Rhoads and Au (2015)nt sequences have been deposited in the NCBI (National Center for Biotechnology Information)
17Accession number, geneticsAccess no genBöhnke-Brandt, StefanieSanger Sequencing modified after Sanger et al. (1977); PacBio Sequencing according to Rhoads and Au (2015)nt sequences have been deposited in the NCBI (National Center for Biotechnology Information)
Status:
Curation Level: Enhanced curation (CurationLevelC)
Size:
279 data points

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