Suggested Citation:
Flournoy, Nikaela. 2017. 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. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7154F44
Data Parameters and Units:
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
Methods:
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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