Comparing DNA, RNA and protein levels for measuring microbial dynamics in soil microcosms amended with nitrogen fertilizer

Authors: ORELLANA, L - GEORGIA TECH; HATT, J - GEORGIA TECH; IYER, S - OAK RIDGE NATIONAL LABORATORY; CHOUREY, K - OAK RIDGE NATIONAL LABORATORY; HETTICH, R - OAK RIDGE NATIONAL LABORATORY; SPAIN, J - GEORGIA TECH; YANG, W - UNIVERSITY OF ILLINOIS; Chee Sanford, Joanne; SANFORD, R - UNIVERSITY OF ILLINOIS; LOEFFLER, F - UNIVERSITY OF TENNESSEE; KONSTANTINIDIS, K - GEORGIA TECH

Submitted to: Nature Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/14/2019
Publication Date: 11/26/2019
Citation: Orellana, L.H., Hatt, J.K., Iyer, S., Chourey, K., Hettich, R.L., Spain, J.C., Yang, W.H., Chee Sanford, J.C., Sanford, R.A., Loeffler, F.E., Konstantinidis, K.T. 2019. Comparing DNA, RNA and protein levels for measuring microbial dynamics in soil microcosms amended with nitrogen fertilizer. Nature Scientific Reports. 9. Article 17630. https://doi.org/10.1038/s41598-019-53679-0.
DOI: https://doi.org/10.1038/s41598-019-53679-0

Interpretive Summary: Multi-omic techniques are the latest technologies used to characterize microbial communities at the gene (DNA), transcript (RNA) and protein levels. Research to measure in-situ microbial process rates clearly requires multiple approaches that can accurately detect and quantify microbial responses and activities. These approaches are especially difficult in highly complex habitats such as soils. We used metagenomic, transcriptomic, and proteomic methods to follow soil microcosms set up from a field with a long history of agricultural management. The microcosms were amended with ammonium and urea to simulate a fertilization event, and included either 15N-labeled ammonium or urea to specifically monitor the process of nitrification (NH4+'NH2OH'NO2-'NO3-). Nitrification was indicated by an increase in NO3--N after two days and accumulation of N2O after 192 hrs of incubation. A 6-fold increase in ammonia-oxidizing bacteria (AOB) Nitrosomonas and Nitrosospira occurred between 10 and 192 hrs of incubation. Surprisingly, ammonia-oxidizing archaea (AOA) Nitrososphaera and Nitrosopumilus, and novel complete ammonia oxidizer (comammox) nitrifiers showed stable gene expression during incubations but were generally more abundant (DNA level) than their bacterial nitrifier counterparts. A strong relationship was observed between nitrification activity and bacterial ammonia monooxygenase genes (amoA; NH4+'NH2OH) and nitrite oxidoreductase gene (nxrA; NO2-'NO3-) mRNA abundances, revealing that mRNA levels quantitatively reflected measured activity, and were generally more sensitive than monitoring only DNA under our microcosm conditions. Although peptides related to cellular housekeeping proteins from nitrite-oxidizing organisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. The significance of these findings underscored the strengths and limitations of multi-omic approaches for assessing complex microbial communities and provided means to measure nitrification processes in soils. Further, the study provided evidence that novel archaeal ammonia oxidizers abundantly dominate in root zones and respond rapidly to N-fertilizer input, contrasting long-held conventional wisdom that target bacterial (AOB) nitrifiers as the key microbial populations. These results provide the basis for future studies that will more accurately inform predictive models for nitrogen fate in soils.

Technical Abstract: Multi-omic techniques can offer a comprehensive overview of microbial communities at the gene, transcript and protein levels. However, to what extent these levels reflect in-situ process rates is less clear, especially in highly complex habitats such as soils. Here we performed microcosm incubations using soil from a site with a long history of agricultural management. The microcosms were amended with ammonium and urea to simulate a fertilization event, and included either 15N-labeled ammonium or urea as isotopic tracers for nitrification. Nitrification was measured (0.34 µg NO3--N g-1 soil d-1) after two days and accumulation of N2O after 192 hrs of incubation. Nitrification activity (NH4+'NH2OH'NO2-'NO3-) was accompanied by 6-fold increase in 16S rRNA gene relative expression (cDNA/DNA) for ammonia-oxidizing bacteria (AOB) Nitrosomonas and Nitrosospira between 10 and 192 hrs of incubation. In contrast, ammonia-oxidizing archaea (AOA) Nitrososphaera and Nitrosopumilus, and complete ammonia oxidizer (comammox) nitrifiers showed stable gene expression during incubations but were generally more abundant (DNA level) than their Betaproteobacteria AOB counterparts. A strong relationship was observed between nitrification activity and (mostly) betaproteobacterial ammonia monooxygenase (amoA; NH4+'NH2OH) and nitrite oxidoreductase (nxrA; NO2-'NO3-) transcript abundances, revealing that mRNA levels quantitatively reflected measured activity, and were generally more sensitive than the DNA level under our microcosm conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing organisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscored the strengths and limitations of multi-omic approaches for assessing complex microbial communities and provided means to measure nitrification processes in soils.