Assessing the abundance of genes involved in the denitrification pathways collected in marsh and subtidal unvegetated sediments at the Chandeleur Islands, July 2015 to February 2016
Identification InformationDistribution InformationMetadata Maintenance Information
Metadata:
File identifier:
R4.x262.000-0021-metadata.xml
Language:
eng; USA
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utf8
Hierarchy level:
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dataset
Metadata author:
Responsible party:
Individual name:
Lei Hu
Organisation name:
Dauphin Island Sea Lab (DISL) / University Programs
Position name:
Data Manager
Contact info:
Contact:
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Telephone:
Voice:
2518617521
Facsimile:
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101 Bienville Blvd
City:
Dauphin Island
Administrative area:
Alabama
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36528
Country:
USA
Electronic mail address:
lhu@disl.org
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Role code:
pointOfContact
Date stamp:
2018-08-11T13:38:49+00:00
Metadata standard name:
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
Metadata standard version:
ISO 19115-2:2009(E)
Dataset URI:
https://data.gulfresearchinitiative.org/metadata/R4.x262.000:0021
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Identification info:
Data identification:
Citation:
Citation:
Title:
Assessing the abundance of genes involved in the denitrification pathways collected in marsh and subtidal
unvegetated sediments at the Chandeleur Islands, July 2015 to February 2016
Alternate title:
Date:
Date:
Date:
2017-06-23
Date type:
Date type code:
publication
Abstract:
Selected genes involved in nitrogen cycling were enumerated from sediment collected on the marsh platform as
well as from nearby subtidal unvegetated sediments in the Chandeleur Islands. Data were collected seasonally
from July 2015 to February 2016.
Purpose:
The purpose of this study was to enumerate genes involved in denitrification in marsh and subtidal habitats
that were previously exposed to moderately levels of hydrocarbons.
Status:
Progress code:
completed
Point of contact:
Responsible party:
Individual name:
Patricia Sobecky
Organisation name:
The University of Alabama / Department of Biological Sciences
Position name:
Professor
Contact info:
Contact:
Phone:
Telephone:
Voice:
2053481409
Facsimile:
Address:
Address:
Delivery point:
Box 870344
City:
Tuscaloosa
Administrative area:
Alabama
Postal code:
35487
Country:
USA
Electronic mail address:
psobecky@ua.edu
Role:
Role code:
pointOfContact
Descriptive keywords:
Keywords:
Keyword:
sediments
Keyword:
denitrification
Keyword:
nitrogen cycling
Type:
Keyword type code:
theme
Descriptive keywords:
Keywords:
Keyword:
Chandeleur Islands
Keyword:
Red Fish Point
Keyword:
Gulf of Mexico
Keyword:
Louisiana
Type:
Keyword type code:
place
Language:
eng; USA
Topic category:
Topic category code:
environment
Topic category:
Topic category code:
oceans
Extent:
Extent:
Geographic element:
Geographic bounding box:
West bound longitude:
-88.841188
East bound longitude:
-88.841188
South bound latitude:
29.862799
North bound latitude:
29.862799
Geographic element:
BoundingPolygon:
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gml:MultiPoint:
gml:pointMember:
Point:
gml:pos:
29.862799 -88.841188
Temporal element:
Temporal extent:
Extent:
Time period:
Description:
ground condition
Begin date:
2015-07-09
End date:
2016-02-22
Supplemental Information:
Date: month, day and year, Location: Location on the Map, Habitat: Marsh platform or subtidal unvegetated
sediments, Cores: replicate cores that were collected from each site, 16S (gene copies per gram): 16S
ribosomal (rRNA) in copies per gram sediment, napA (gene copies per gram): napA (nitrate reductase) gene
copies per gram sediment, norB (gene copies per gram): norB (nitric oxide reductase) gene copies per gram
sediment, nirS (gene copies per gram): nirS (nitrite reductase) gene copies per gram sediment, Latitude
Degrees: measured in degrees, Latitude Minutes: measured in minutes, Latitude Seconds: measured in seconds,
Longitude Degrees: measured in degrees, Longitude Minutes: measured in minutes, Longitude Seconds: measured
in seconds|Replicate cores were collected in the field and brought back to the laboratory where they were
set up for measurements of denitrification rates with the isotope pairing technique. At the termination of
the rate measurements the top 10cm of the cores were homogenized and frozen at -80oC for later analysis. DNA
was extracted in triplicate from 1 g sediment from each core with the FastDNATM Spin Kit for Soil (MP
Biomedicals) per the manufacturer’s protocol with the addition of two-minute ice incubations after the
homogenization and 4oC centrifugation steps. The triplicate extractions were pooled and purified using the
Zymoclean Gel DNA Recovery Kit (Zymo Research) and eluted with sterile milliQ water (total volume 50 µl).
DNA concentrations were measured via absorption at 260 nm using a NanoDrop ND-1000. Quantitative PCR (qPCR)
was performed to assess the abundance of three genes in the denitrification pathway: napA (nitrate
reductase), nirS (nitrite reductase), and norB (nitric oxide reductase). Total community abundance was
determined by qPCR of 16S ribosomal RNA (rRNA). Each reaction included Platinum SYBR Green qPCR supermix-UDG
with ROX (12.5 µL) (Invitrogen), 1 µL of forward and reverse primer (5 µM, IDT DNA technology), 0.5 µL MgCl2
(50 mM), and 5 ng of DNA template. The total reaction volume was adjusted to 25 µL with PCR grade water
(ThermoFisher). Following 2 min at 50ºC and activation of Platinum SYBR Green qPCR supermix-UDG with ROX at
95ºC for 10 min, thermal cycles consisted of (napA and nirS) 40 cycles of: 15 s at 95ºC (denaturation),
(norB) 30 cycles 15 s at 95ºC, 1 min at 60ºC elongation step, (16S) 30 cycles 15 s at 95oC, and 30 s at
72oC. Samples were run in duplicate on a 7000 Sequence Detection System (ABI Prism) with the primer
combinations, optimal annealing temperatures, and qPCR conditions detailed below. Primers and thermal
profiles (* denotes modified annealing temperature) used for PCR to generate standards and qPCR
quantification of different functional genes. Target gene: napA (qPCR) Primers: napA-1F; napA-1R Sequence(5’
3’): GTY ATG GAR GAA AAA TTC AA; GAR CCG AAC ATG CCR AC References: Smith et al. (2007) Optimal Annealing
Temp.: 55 Celsius - 60 s Target gene: napA (PCR) Primers: napA V67 F; napA V67 R Sequence(5’ 3’): TAY TTY
YTN HSN AAR ATH ATG TAY GG; DAT NGG RTG CAT YTC NGC CAT RTT References: Flanagan et al. (1999) Optimal
Annealing Temp.: 55 Celsius - 30 s* Target gene: nirS (qPCR and PCR) Primers: nirScd3aF; nirS R3cd
Sequence(5’ 3’): GTS AAC GTS AAG GAR ACS GG; GAS TTC GGR TGS GTC TTG A References: Throback et al. (2004)
Optimal Annealing Temp.: 51 Celsius - 60 s Target gene: norB (qPCR and PCR) Primers: cnorB2F; cnorB6R
Sequence(5’ 3’): GAC AAG NNN TAC TGG TGG T; GAA NCC CCA NAC NCC NGC References: Geets et al. (2007) Optimal
Annealing Temp.: 50 Celsius - 60 s Target gene: 16S (PCR) Primers: 341F; 534R Sequence(5’ 3’): CCT ACG GGA
GGC AGC AG; ATT ACC GCG GCT GCT GGC A References: Bru et al. (2008) Optimal Annealing Temp.: 55 Celsius - 45
s Target gene: (q-PCR) Primers: N/A Sequence(5’ 3’): N/A References: López-Gutiérrez et al. (2004) Optimal
Annealing Temp.: 60 Celsius - 30 s Bru D, Martin-Laurent F, Philippot L. 2008. Quantification of the
Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an
Example. Applied and Environmental Microbiology 74:1660-1663. Flanagan DA, Gregory LG, Carter JP,
Karakas-Sen A, Richardson DJ & Spiro S. 1999. Detection of genes for periplasmic nitrate reductase in
nitrate respiring bacteria and in community DNA. Fems Microbiology Letters 177: 263-270. Geets J, de Cooman
M, Wittebolle L, Heylen K, Vanparys B, De Vos P, Verstraete W, & Boon N. 2007. Real-time PCR assay for the
simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge. Applied Microbology
and Biotechnology 75:211-221. López-Gutiérrez JC, Henry S, Hallet S, Martin-Laurent F, Catroux G & Philippot
L. 2004. Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J
Microbiol Meth 57: 399-407. Smith, C. J., D.B. Nedwell, L.F. Dong, and A.M. Osborn. 2007. Diversity and
abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their
transcripts in estuarine sediments. App. Environ. Microbiol. 73: 3612-3622. Throbäck IN, Enwall K, Jarvis A,
Hallin S. 2004. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of
denitrifying bacteria with DGGE. FEMS Microbiology Ecology 49:401-417.||||
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Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC)
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unknown
Maintenance note:
This ISO metadata record was automatically generated from information provided to GRIIDC for dataset:
R4.x262.000:0021 on 2019-02-17T14:28:27+00:00
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<gco:CharacterString>Date: month, day and year, Location: Location on the Map, Habitat: Marsh platform or subtidal unvegetated sediments, Cores: replicate cores that were collected from each site, 16S (gene copies per gram): 16S ribosomal (rRNA) in copies per gram sediment, napA (gene copies per gram): napA (nitrate reductase) gene copies per gram sediment, norB (gene copies per gram): norB (nitric oxide reductase) gene copies per gram sediment, nirS (gene copies per gram): nirS (nitrite reductase) gene copies per gram sediment, Latitude Degrees: measured in degrees, Latitude Minutes: measured in minutes, Latitude Seconds: measured in seconds, Longitude Degrees: measured in degrees, Longitude Minutes: measured in minutes, Longitude Seconds: measured in seconds|Replicate cores were collected in the field and brought back to the laboratory where they were set up for measurements of denitrification rates with the isotope pairing technique. At the termination of the rate measurements the top 10cm of the cores were homogenized and frozen at -80oC for later analysis. DNA was extracted in triplicate from 1 g sediment from each core with the FastDNATM Spin Kit for Soil (MP Biomedicals) per the manufacturer’s protocol with the addition of two-minute ice incubations after the homogenization and 4oC centrifugation steps. The triplicate extractions were pooled and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research) and eluted with sterile milliQ water (total volume 50 µl). DNA concentrations were measured via absorption at 260 nm using a NanoDrop ND-1000. Quantitative PCR (qPCR) was performed to assess the abundance of three genes in the denitrification pathway: napA (nitrate reductase), nirS (nitrite reductase), and norB (nitric oxide reductase). Total community abundance was determined by qPCR of 16S ribosomal RNA (rRNA). Each reaction included Platinum SYBR Green qPCR supermix-UDG with ROX (12.5 µL) (Invitrogen), 1 µL of forward and reverse primer (5 µM, IDT DNA technology), 0.5 µL MgCl2 (50 mM), and 5 ng of DNA template. The total reaction volume was adjusted to 25 µL with PCR grade water (ThermoFisher). Following 2 min at 50ºC and activation of Platinum SYBR Green qPCR supermix-UDG with ROX at 95ºC for 10 min, thermal cycles consisted of (napA and nirS) 40 cycles of: 15 s at 95ºC (denaturation), (norB) 30 cycles 15 s at 95ºC, 1 min at 60ºC elongation step, (16S) 30 cycles 15 s at 95oC, and 30 s at 72oC. Samples were run in duplicate on a 7000 Sequence Detection System (ABI Prism) with the primer combinations, optimal annealing temperatures, and qPCR conditions detailed below. Primers and thermal profiles (* denotes modified annealing temperature) used for PCR to generate standards and qPCR quantification of different functional genes. Target gene: napA (qPCR) Primers: napA-1F; napA-1R Sequence(5’ 3’): GTY ATG GAR GAA AAA TTC AA; GAR CCG AAC ATG CCR AC References: Smith et al. (2007) Optimal Annealing Temp.: 55 Celsius - 60 s Target gene: napA (PCR) Primers: napA V67 F; napA V67 R Sequence(5’ 3’): TAY TTY YTN HSN AAR ATH ATG TAY GG; DAT NGG RTG CAT YTC NGC CAT RTT References: Flanagan et al. (1999) Optimal Annealing Temp.: 55 Celsius - 30 s* Target gene: nirS (qPCR and PCR) Primers: nirScd3aF; nirS R3cd Sequence(5’ 3’): GTS AAC GTS AAG GAR ACS GG; GAS TTC GGR TGS GTC TTG A References: Throback et al. (2004) Optimal Annealing Temp.: 51 Celsius - 60 s Target gene: norB (qPCR and PCR) Primers: cnorB2F; cnorB6R Sequence(5’ 3’): GAC AAG NNN TAC TGG TGG T; GAA NCC CCA NAC NCC NGC References: Geets et al. (2007) Optimal Annealing Temp.: 50 Celsius - 60 s Target gene: 16S (PCR) Primers: 341F; 534R Sequence(5’ 3’): CCT ACG GGA GGC AGC AG; ATT ACC GCG GCT GCT GGC A References: Bru et al. (2008) Optimal Annealing Temp.: 55 Celsius - 45 s Target gene: (q-PCR) Primers: N/A Sequence(5’ 3’): N/A References: López-Gutiérrez et al. (2004) Optimal Annealing Temp.: 60 Celsius - 30 s Bru D, Martin-Laurent F, Philippot L. 2008. Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example. Applied and Environmental Microbiology 74:1660-1663. Flanagan DA, Gregory LG, Carter JP, Karakas-Sen A, Richardson DJ & Spiro S. 1999. Detection of genes for periplasmic nitrate reductase in nitrate respiring bacteria and in community DNA. Fems Microbiology Letters 177: 263-270. Geets J, de Cooman M, Wittebolle L, Heylen K, Vanparys B, De Vos P, Verstraete W, & Boon N. 2007. Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge. Applied Microbology and Biotechnology 75:211-221. López-Gutiérrez JC, Henry S, Hallet S, Martin-Laurent F, Catroux G & Philippot L. 2004. Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J Microbiol Meth 57: 399-407. Smith, C. J., D.B. Nedwell, L.F. Dong, and A.M. Osborn. 2007. Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments. App. Environ. Microbiol. 73: 3612-3622. Throbäck IN, Enwall K, Jarvis A, Hallin S. 2004. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiology Ecology 49:401-417.||||</gco:CharacterString>
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