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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):
activity-based RubisCO screen; autotrophic CO2-fixation; functional metagenomic; HPLC; hydrothermal deep sea vent; Thiomicrospira crunogena
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):
From Mantle to Ocean: Energy-, Material- and Life-cycles at Spreading Axes (DERIDGE)
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:
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):
#NameShort NameUnitPrincipal InvestigatorMethod/DeviceComment
1 IdentificationIDBöhnke-Brandt, Stefaniefosmid clone designation
2 Insert sizeInsert sizebpBöhnke-Brandt, StefanieSanger Sequencing modified after Sanger et al. (1977); PacBio Sequencing according to Rhoads and Au (2015)
3 Deleted open-reading frameDel orfBöhnke-Brandt, StefanieSequence editing, BioEdit
4 Insertion positionInsertion posaaBöhnke-Brandt, StefanieInsertion position in non-coding region (ncr) is given in nucleotides
5 Total length of deleted open-reading frameTotal l of del orfaaBöhnke-Brandt, StefanieInsertion position in non-coding region (ncr) is given in nucleotides
6 Specific ribulose-1,5-bisphosphate carboxylase/oxygenase activitySpec RubisCO actnmol/mg/minBöhnke-Brandt, StefanieHigh Performance Liquid Chromatography (HPLC)
7 Specific ribulose-1,5-bisphosphate carboxylase/oxygenase activity, standard deviationSpec RubisCO act std dev±Böhnke-Brandt, StefanieGaussian propagation of error
License:
Creative Commons Attribution 4.0 International (CC-BY-4.0)
Status:
Curation Level: Enhanced curation (CurationLevelC)
Size:
393 data points

Data

Download dataset as tab-delimited text — use the following character encoding:

1 
ID		2 
Insert size [bp]		3 
Del orf		4 
Insertion pos [aa]		5 
Total l of del orf [aa]		6 
Spec RubisCO act [nmol/mg/min]		7 
Spec RubisCO act std dev [±]
CbbLS2623266.4322.78
CbbM226955.148.96
D71C235195481.408.16
D74C1035195431.9525.07
D74E135195442.5937.72
D77H135195447.4834.23
D77F435195461.224.32
D77D935195467.1936.70
D78G535195482.5220.73
D78E1035195427.1116.42
D80G335195473.465.76
D81E135195456.7624.14
D81G735195484.2444.37
D84G435195461.9138.58
D84G4II1302347.8213.75
D71C2II1302354.698.42
TK21II14244Δorf0857875731.5912.46
TK19II14244Δorf0833375737.172.09
TK14II14244Δorf0856975740.649.02
TK17II14244Δorf0818975739.634.66
TK11II14244Δorf0910926663.3810.56
TK8II14244Δorf099426659.342.82
TK12II14244Δorf094626654.664.49
TK22II14244Δorf1017145924.539.13
TK7II14244Δorf11185314301.3320.92
TK6II14244Δorf1226430830.7311.66
TK24II14244Δorf13414728.295.13
TK23II14244Δorf15229272112.5615.43
TK20II14244Δncr_orf15-168510566.079.02
TK4II14244Δorf1630377763.306.47
TK1136416Δorf0228206470.1511.88
TK1936416Δorf0228206463.7113.41
TK8136416Δorf0352270432.6214.09
TK8636416Δorf04246854490.6927.47
TK8836416Δorf04290854450.2530.18
TK1636416Δorf04499854450.990.10
TK23636416Δncr_orf04-05448450384.382.74
TK5836416Δorf05237385351.5928.43
TK23136416Δncr_orf05-0634200311.2628.01
TK9636416Δorf06613580.4820.12
TK13836416Δorf07139218522.1027.38
TK20736416Δorf07212218437.3839.59
TK16136416Δorf12285308409.3456.32
TK16936416Δorf12220308431.5026.61
TK10936416Δorf12178308485.0427.22
TK3836416Δorf14111116417.8423.33
TK336416Δorf15180272455.1519.18
TK836416Δorf1662777510.6721.37
TK436416Δorf16157777481.596.91
TK10036416Δorf17143177460.121.12
TK4436416Δorf18329408449.921.11
TK11736416Δorf1998233446.658.51
TK1036416Δorf20128425435.0013.28
TK536416Δorf21245431507.8010.37
TK4636416Δorf2218260462.6737.20
TK8936416Δorf235291380.8124.68
TK6836416Δorf2410430405.3410.96
TK18036416Δorf25397520444.249.90
TK6936416Δorf268787449.237.60
TK17936416Δorf2722320458.5217.92
TK4536416Δorf27151320438.4010.12
TK9136416Δorf28147294400.819.27
TK7736416Δorf3021449443.6232.41