Comparing DNA, RNA and protein levels for measuring microbial activity in nitrogen-amended soils

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 history of agricultural management. Microcosms, amended with isotopically labelled ammonium and urea to simulate a fertilization event, showed nitrification (up to 4.1 {+/-} 0.87 {micro}g N-NO3- g-1 dry soil d-1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+[->]NH2OH[->]NO2-[->]NO3-) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria (AOB) Nitrosomonas and Nitrosospira. In contrast, ammonia-oxidizing archaea (AOA) 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 between nitrification activity and (mostly) betaproteobacterial ammonia monooxygenase (amoA; NH4+[->]NH2OH) and nitrite oxidoreductase (nxrA; NO2-[->]NO3-) transcript abundances revealed that mRNA levels quantitatively reflected measured activity and were generally more sensitive than the DNA level in the microcosm incubations. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing complex microbial communities and provide the molecular means to assess nitrification processes in soils.\n\nIMPORTANCEEven though the use of omic approaches has expanded our knowledge of the diversity of microbial communities in natural and engineered systems, it is less clear how well the use of whole community DNA-, RNA- or protein-based approaches reflect microbial activities. To this end, we directly compared the different levels of molecular information (i.e., DNA, RNA or proteins) in order to assess which level best correlated with isotope-based measurements of nitrification activity in agricultural soils after fertilization. This work reveals the strengths as well as the associated limitations of metagenomic, metatranscriptomic, and metaproteomic approaches in serving as reliable proxies for examining microbial activities in highly diverse environments like soils.