Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification

被引:24
作者
Cannon, Jordan [1 ]
Sanford, Robert A. [2 ]
Connor, Lynn [3 ]
Yang, Wendy H. [1 ,2 ]
Chee-Sanford, Joanne [3 ]
机构
[1] Univ Illinois, Dept Plant Biol, Urbana, IL USA
[2] Univ Illinois, Dept Geol, Urbana, IL USA
[3] USDA ARS, Urbana, IL 61801 USA
基金
美国国家科学基金会;
关键词
nrfA; PCR primers; Nitrite ammonification; DNRA; DISSIMILATORY NITRATE REDUCTION; NITROUS-OXIDE; AMMONIUM DNRA; MICROBIAL COMMUNITY; RAPID METHOD; SOIL; DYNAMICS; CYCLE; DENITRIFICATION; PATHWAYS;
D O I
10.1016/j.mimet.2019.03.020
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Dissimilatory nitrate reduction to ammonium (DNRA) is now known to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme cytochrome c nitrite reductase NrfA associated with respiratory nitrite ammonification, is encoded by the nrfA gene in a broad phylogeny of bacteria. The lack of reliable and comprehensive molecular tools to detect diverse nrfA from environmental samples has hampered efforts to meaningfully characterize the genetic potential for DNRA in environmental systems. In this study, modifications were made to optimize the amplification efficiency of previously-designed PCR primers, targeting the diagnostic region of NrfA between the conserved third- and fourth heme binding domains, and to increase coverage to include detection of environmentally relevant Geobacteraceae-like nrfA. Using an alignment of the primers to > 270 bacterial nrfA genes affiliated with 18 distinct clades, modifications to the primer sequences improved coverage, minimized amplification artifacts, and yielded the predicted product sizes from reference-, soil-, and groundwater DNA. Illumina sequencing of amplicons showed the successful recovery of nrfA gene fragments from environmental DNA based on alignments of the translated sequences. The new primers developed in this study are more efficient in PCR reactions, although gene targets with high GC content affect efficiency. Furthermore, the primers have a broader spectrum of detection and were validated rigorously for use in detecting nrfA from natural environments. These are suitable for conventional PCR, qPCR, and use in PCR access array technologies that allow multiplex gene amplification for downstream high throughput sequencing platforms.
引用
收藏
页码:49 / 59
页数:11
相关论文
共 75 条
[1]   Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries [J].
Aird, Daniel ;
Ross, Michael G. ;
Chen, Wei-Sheng ;
Danielsson, Maxwell ;
Fennell, Timothy ;
Russ, Carsten ;
Jaffe, David B. ;
Nusbaum, Chad ;
Gnirke, Andreas .
GENOME BIOLOGY, 2011, 12 (02)
[2]   The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens [J].
Aklujkar, Muktak ;
Krushkal, Julia ;
DiBartolo, Genevieve ;
Lapidus, Alla ;
Land, Miriam L. ;
Lovley, Derek R. .
BMC MICROBIOLOGY, 2009, 9
[3]   Soil microbial sources of nitrous oxide: recent advances in knowledge, emerging challenges and future direction [J].
Baggs, Elizabeth M. .
CURRENT OPINION IN ENVIRONMENTAL SUSTAINABILITY, 2011, 3 (05) :321-327
[4]  
Ben-Dov E, 2015, CURR ISSUES MOL BIOL, V17, P53
[5]   Dissimilatory nitrate reduction to ammonium (DNRA) seasonally dominates NO3 - reduction pathways in an anthropogenically impacted sub-tropical coastal lagoon [J].
Bernard, Rebecca J. ;
Mortazavi, Behzad ;
Kleinhuizen, Alice A. .
BIOGEOCHEMISTRY, 2015, 125 (01) :47-64
[6]  
Bernhard A, 2016, FRONT MICROBIOL, V7
[7]   Nitrogen Fertilization Has a Stronger Effect on Soil Nitrogen-Fixing Bacterial Communities than Elevated Atmospheric CO2 [J].
Berthrong, Sean T. ;
Yeager, Chris M. ;
Gallegos-Graves, Laverne ;
Steven, Blaire ;
Eichorst, Stephanie A. ;
Jackson, Robert B. ;
Kuske, Cheryl R. .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2014, 80 (10) :3103-3112
[8]  
Bhowmik A, 2017, AIMS MICROBIOL, V3, P826, DOI 10.3934/microbiol.2017.4.826
[9]   Design and evaluation of primers targeting genes encoding NO-forming nitrite reductases: implications for ecological inference of denitrifying communities [J].
Bonilla-Rosso, German ;
Wittorf, Lea ;
Jones, Christopher M. ;
Hallin, Sara .
SCIENTIFIC REPORTS, 2016, 6
[10]   Dissimilatory Nitrate Reduction to Ammonium in the Yellow River Estuary: Rates, Abundance, and Community Diversity [J].
Bu, Cuina ;
Wang, Yu ;
Ge, Chenghao ;
Ahmad, Hafiz Adeel ;
Gao, Baoyu ;
Ni, Shou-Qing .
SCIENTIFIC REPORTS, 2017, 7