Community composition and activity state of estuarine bacterioplankton assessed using differential staining and metagenomic analysis of 16S rDNA and rRNA

Abstract

To better understand how resource availability controls estuarine microbial communities, we compared community composition at 2 sites in the tidal freshwater James River (Virginia, USA) which differed in nutrient concentrations and autochthonous production. In addition, we conducted laboratory microcosm experiments, wherein bacteria from the 2 sites received nutrient, light, and organic carbon amendments. Traditional, microscopy-based methods of staining and enumeration were used to determine the abundance of total, live, and active cells. DNA fingerprinting (terminal restriction fragment length polymorphism, TRFLP) and dual 16S rDNArRNA pyrosequencing were used to assess community composition and taxon-specific activity (from rRNA:rDNA). Enumeration revealed that total, live, and active (CTC+) bacteria were more abundant at the site where autochthonous production was higher, while DNA fingerprinting (TRFLP) and 16S rDNA pyrosequencing indicated high similarity in community composition at the 2 sites. In contrast, pyrosequencing results from rRNA revealed greater variation and suggest that the metabolically active fraction of the community differed between sites. Differences in rDNAand rRNA-based libraries were also apparent in microcosm experiments, where resource amendments resulted in shifts in both the active and overall communities. In both environmental and microcosm samples, dual rDNA-rRNA pyrosequencing yielded higher estimates of the proportion of active cells (32 ± 3%) compared to differential staining and microscopy (13 ± 1%). Though characterization of active taxa was sensitive to the presumed activity threshold (rRNA:rDNA), our findings suggest that variable resource conditions can give rise to unique assemblages of active taxa despite similarities in overall community composition.KEY WORDS: Activity state · Bacteria · Community composition · Estuary · Pyrosequencing · Chesapeake Bay Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 69: [247][248][249][250][251][252][253][254][255][256][257][258][259][260][261] 2013 viral lysis (Thingstad & Lignell 1997). Determining the relative importance of these factors is challenging, particularly in estuaries, which experience large and frequent changes in environmental conditions. In these systems, tidal-driven exchanges with the marine environment and episodic riverine inputs alter salinity, inorganic nutrients, and the quality and quantity of organic matter along the longitudinal axis of the estuary. Mechanisms of bacterial community response to these factors are poorly understood, but can include shifts in bacterial abundance, community composition, and activity state.The analysis of whole-community DNA extracts from environmental samples has been used to characterize variation in estuarine bacterioplankton in response to changes in resources and physicalchemical conditions (Troussellier et al. 2002, Crump et al. 2004, Henriques et al. 2004, Kirchman et al. 2005. However, DNA is present in dormant ...

16S rRNA:rDNA ratios and cell activity staining reveal consistent patterns of soil microbial activity

“…Given the relatively short half-life of ribosomal RNA, the presence of 16S ribosomal transcripts (hereafter “rRNA) is generally assumed to indicate recent metabolic activity, and numerous studies have used rRNA to characterize growing or active communities (reviewed in (13)). In particular, pairing both 16S ribosomal transcripts (rRNA) as well as the 16S ribosomal gene (rDNA) sequencing allows for calculation of 16S rRNA:rDNA ratios, which attempts to normalize rRNA levels by the abundance of that taxon in the community and quantify its relative level of activity (8, 11, 14, 15). Taxa with 16S ratios greater than a specified threshold are considered ‘active’, and most studies report using a threshold of 1.0, which indicates more rRNA reads than rDNA reads for those taxa (6, 8, 16).…”

“…those with low abundance in rDNA sequences) exhibiting disproportionately high activity compared to more abundant taxa; thus phantom taxa arise largely due to sampling stochasticity (18), or alternatively, by in-process transitions from rarity to prevalence (i.e., conditionally rare taxa, (19)). Although these considerations have led researchers to suggest that 16S ratios may be best interpreted as ‘potential microbial activity’, 16S ratios have nevertheless been used to inform microbial activity-dormancy dynamics in a broad range of ecosystems (11, 14, 15, 18).…”

“…Actively respiring cells convert CTC to an insoluble red fluorescent formazan product, which can be visualized by fluorescence microscopy (23). In addition, CTC staining can be coupled with a general DNA stain to compare active and total cell counts in a microbial community, allowing for determination of percent activity (15, 24). As with the 16S ratio method for determining microbial activity, the CTC staining method has several limitations which can affect its interpretation.…”

“…For example, CTC staining can be toxic to some bacterial species (20, 25), and not all actively respiring strains are able to take up the stain efficiently (20, 24), potentially leading CTC staining to underestimate the true proportion of active cells in the community (26). Despite these potential caveats, CTC staining remains a popular method for analyses of microbial activity in a broad range of environmental samples (7, 15, 24).…”

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“…The classification of dormant versus active OTUs is sensitive to the specific cutoff 208 selected (Franklin et al, 2013). Therfore, we estimated dormancy across a range of cutoffs from 0.1 -0.9.…”