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Title: 16S rRNA/rRNA Gene Ratios and Cell Activity Staining Reveal Consistent Patterns of Microbial Activity in Plant-Associated Soil

Abstract

ABSTRACT At any given time, only a subset of microbial community members are active in their environment. The others are in a state of dormancy, with strongly reduced metabolic rates. It is of interest to distinguish active and inactive microbial cells and taxa to understand their functional contributions to ecosystem processes and to understand shifts in microbial activity in response to change. Of the methods used to assess microbial activity-dormancy dynamics, 16S rRNA/rRNA gene amplicons (16S ratios) and active cell staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) are two of the most common, yet each method has limitations. Given that in situ activity-dormancy dynamics are proxied only by laboratory methods, further study is needed to assess the level of agreement and potential complementarity of these methods. We conducted two experiments investigating microbial activity in plant-associated soils. First, we treated corn field soil with phytohormones to simulate plant soil stress signaling, and second, we used rhizosphere soil from common bean plants exposed to drought or nutrient enrichment. Overall, the 16S ratio and CTC methods exhibited similar patterns of relative activity across treatments when treatment effects were large, and the instances in which they differed could be attributed to changes in community sizemore » (e.g., cell death or growth). Therefore, regardless of the method used to assess activity, we recommend quantifying community size to inform ecological interpretation. Our results suggest that the 16S ratio and CTC methods report comparable patterns of activity that can be applied to observe ecological dynamics over time, space, or experimental treatment. IMPORTANCE Although the majority of microorganisms in natural ecosystems are dormant, relatively little is known about the dynamics of the active and dormant microbial pools through both space and time. The limited knowledge of microbial activity-dormancy dynamics is in part due to uncertainty in the methods currently used to quantify active taxa. Here, we directly compared two of the most common methods (16S ratios and active cell staining) for estimating microbial activity in plant-associated soil and found that they were largely in agreement in the overarching patterns. Our results suggest that 16S ratios and active cell staining provide complementary information for measuring and interpreting microbial activity-dormancy dynamics in soils. They also support the idea that 16S rRNA/rRNA gene ratios have comparative value and offer a high-throughput, sequencing-based option for understanding relative changes in microbiome activity, as long as this method is coupled with quantification of community size.« less

Authors:
; ; ORCiD logo;
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1504292
Alternate Identifier(s):
OSTI ID: 1609267
Grant/Contract Number:  
SC0018409; FC02-07ER64494
Resource Type:
Published Article
Journal Name:
mSystems
Additional Journal Information:
Journal Name: mSystems Journal Volume: 4 Journal Issue: 2; Journal ID: ISSN 2379-5077
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Microbiology; 16S rRNA; 5-cyano-2,3-ditolyl tetrazolium chloride; beta diversity; community dynamics; dormancy; flow cytometry; microbiome; phantom taxa; resuscitation; rhizosphere

Citation Formats

Bowsher, Alan W., Kearns, Patrick J., Shade, Ashley, and Deveau, ed., Aurélie. 16S rRNA/rRNA Gene Ratios and Cell Activity Staining Reveal Consistent Patterns of Microbial Activity in Plant-Associated Soil. United States: N. p., 2019. Web. doi:10.1128/mSystems.00003-19.
Bowsher, Alan W., Kearns, Patrick J., Shade, Ashley, & Deveau, ed., Aurélie. 16S rRNA/rRNA Gene Ratios and Cell Activity Staining Reveal Consistent Patterns of Microbial Activity in Plant-Associated Soil. United States. doi:10.1128/mSystems.00003-19.
Bowsher, Alan W., Kearns, Patrick J., Shade, Ashley, and Deveau, ed., Aurélie. Tue . "16S rRNA/rRNA Gene Ratios and Cell Activity Staining Reveal Consistent Patterns of Microbial Activity in Plant-Associated Soil". United States. doi:10.1128/mSystems.00003-19.
@article{osti_1504292,
title = {16S rRNA/rRNA Gene Ratios and Cell Activity Staining Reveal Consistent Patterns of Microbial Activity in Plant-Associated Soil},
author = {Bowsher, Alan W. and Kearns, Patrick J. and Shade, Ashley and Deveau, ed., Aurélie},
abstractNote = {ABSTRACT At any given time, only a subset of microbial community members are active in their environment. The others are in a state of dormancy, with strongly reduced metabolic rates. It is of interest to distinguish active and inactive microbial cells and taxa to understand their functional contributions to ecosystem processes and to understand shifts in microbial activity in response to change. Of the methods used to assess microbial activity-dormancy dynamics, 16S rRNA/rRNA gene amplicons (16S ratios) and active cell staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) are two of the most common, yet each method has limitations. Given that in situ activity-dormancy dynamics are proxied only by laboratory methods, further study is needed to assess the level of agreement and potential complementarity of these methods. We conducted two experiments investigating microbial activity in plant-associated soils. First, we treated corn field soil with phytohormones to simulate plant soil stress signaling, and second, we used rhizosphere soil from common bean plants exposed to drought or nutrient enrichment. Overall, the 16S ratio and CTC methods exhibited similar patterns of relative activity across treatments when treatment effects were large, and the instances in which they differed could be attributed to changes in community size (e.g., cell death or growth). Therefore, regardless of the method used to assess activity, we recommend quantifying community size to inform ecological interpretation. Our results suggest that the 16S ratio and CTC methods report comparable patterns of activity that can be applied to observe ecological dynamics over time, space, or experimental treatment. IMPORTANCE Although the majority of microorganisms in natural ecosystems are dormant, relatively little is known about the dynamics of the active and dormant microbial pools through both space and time. The limited knowledge of microbial activity-dormancy dynamics is in part due to uncertainty in the methods currently used to quantify active taxa. Here, we directly compared two of the most common methods (16S ratios and active cell staining) for estimating microbial activity in plant-associated soil and found that they were largely in agreement in the overarching patterns. Our results suggest that 16S ratios and active cell staining provide complementary information for measuring and interpreting microbial activity-dormancy dynamics in soils. They also support the idea that 16S rRNA/rRNA gene ratios have comparative value and offer a high-throughput, sequencing-based option for understanding relative changes in microbiome activity, as long as this method is coupled with quantification of community size.},
doi = {10.1128/mSystems.00003-19},
journal = {mSystems},
number = 2,
volume = 4,
place = {United States},
year = {2019},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
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DOI: 10.1128/mSystems.00003-19

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