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Research Area 3 - SIP-Targeted Metagenomics: Combining DNA Stable-Isotope Probing with High-Throughput Pyrosequencing to Investigate Ammonia-Oxidizing Prokaryotes in Agricultural Ecosystems

Advancements have been made in elucidating the composition of ammonium oxidizing bacteria (AOB) communities in soil, which produce NO2-/NO3-., by taking advantage of variations in gene sequences of 16S ribosomal DNA (rDNA) or the catalytic subunit of NH3 monooxygenase  gene (amoA)( Mintie et al. 2003).   Analysis of these functional genes provides a vigorous, culture-independent method of examining ammonia-oxidizing bacterial diversity and community composition in agroecosystems (Yeager et al. 2005).  Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without cultivation. Stable-isotope-labeled nitrogen (15N) sources are essentially assimilated into the microbial biomass of environmental samples (Neufeld, 2007). Molecular analysis will reveal phylogenetic and functional information about the ammonia-oxidizing prokaryotes in agricultural systems here in the United States (Huntsville, AL) and in China (Nanjing). In addition, bacterial genome diversity has been found to be significantly higher with pyrosequencing analysis as compared to the usual culture-based analysis (Price et al. 2009) and microarray technology (Gilbert et al. 2008).  


These techniques have limitations when exploring the diversity of natural environments.  Contributions of most organisms are missed through cloning because only a select number of clones are chosen for analysis, and microarray technology only gives information about those sequences for which it was designed. This proposed research involving pyrosequencing methods will provide substantial information of both microbial species, such as Nitrosospira and genes responsible for ammonium oxidation and thus increase the knowledge of the diversity and distribution of that group of microorganisms.  We hypothesize that ammonia-oxidizers play an important role in N2 cycling in soil-water systems, and ammonia monooxygenase (amoA) genes may not only be phylogenetically diverse, but also geographically widespread. The REU students selected should have a strong interest in environmental microbiology. They will become knowledgeable of the role the nitrogen cycle plays in the environment, more specifically ammonium oxidizers.  The students will also become skilled in basic molecular techniques such as DNA extraction, polymerase chain reaction (PCR), and more advanced high-throughput techniques such as pyrosequencing.​