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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Sustainable Agricultural Systems Laboratory » Research » Publications at this Location » Publication #315668
Title: Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils

Author
item DLOTT, GLADE - University Of Maryland
item Maul, Jude
item Buyer, Jeffrey
item YARWOOD, STEPHANIE - University Of Maryland

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2015
Publication Date: 6/6/2015
Citation: Dlott, G., Maul, J.E., Buyer, J.S., Yarwood, S. 2015. Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils. Journal of Microbiological Methods. doi: 10.1016/j.apsoil.2015.06.001.

Interpretive Summary: DNA encodes the instructions of life whereas RNA encodes the action of life. Plants, bacteria and animals transcribe DNA into RNA to carry out all life activities (via production of enzymes), and the quantity and specific sequence of RNA can be used to gain a snapshot into what activities an organism is engaging in at that particular moment. For example, when a bacterium encounters an energy source, it ramps up activity by first producing ribosomal RNA followed by transcription of the RNAs encoding enzymes to aquire and catabolize the material. Finally, the RNAs for core metabolic pathways are regulated to metabolize the new material into substances useful for the bacterium. Microbial ecologists measure the soil microbial community structure primarily because microbes play critical roles in soil biochemical transformations, crop diease ecology and plant productivity. In many cases, capturing the “activity” of a soil is difficult, both due to the transient nature of the RNA signal and the lability of the RNA molecule itself. When conducting studies of microbial community structure in the environment, microbial genomic DNA is typically used as an indication of which genera are present in a sample. This can lead to misinterpretation of which genera are actually living since there is evidence that extracellular DNA (eDNA) can bind to soil mineral fractions even after the microbe that produced the DNA is dead. To test the question of whether or not genomic DNA extracted from the soil environment is representative of the active community of soil microbes we extracted, both DNA and RNA from the same sample and compared the correspondence between them. We found that the ratio of rRNA to rDNA in our samples was on average 0.24, suggesting significant extracellular DNA preservation. This information will be useful to scientists studying microbial ecology. These scientists may want to reconsider using strictly rDNA for microbial community analysis. In cases where quantitation of active cells is nessessary, researchers may want to employ alternative indirect rDNA and rRNA extraction.

Technical Abstract: We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communities. ‘Activity ratios’ were calculated for bacteria and archaea in soil sampled from a tropical rainforest and temperate agricultural field, and incubated for one year at two levels of moisture availability, with and without carbon additions. Prior to calculating activity ratios, we corrected the relative abundances of OTUs to account for multiple copies of the 16S gene per genome. Although necessary to ensure accurate activity ratios, this correction did not change our interpretation of differences in microbial community composition across treatments. Activity ratios in this study were lower than those previously published (0.0003 – 210, logarithmic mean = 0.24), suggesting significant extracellular DNA preservation. After controlling for the influence of individual incubation jars, significant differences in activity ratios between all members of each phylum were observed. Planctomycetes and Firmicutes had the highest activity ratios, and Crenarchaeota had the lowest activity overall. Our results suggest that greater caution should be taken in interpreting soil microbial community data derived from extracted DNA. Indirect extraction methods may be useful in ensuring that microbes identified from extracellular DNA are not erroneously interpreted as components of an active microbial community.