Members of the Candidate Phyla Radiation Are Functionally Differentiated by Carbon- and Nitrogen-Cycling Capabilities R
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Danczak et al. Microbiome (2017) 5:112 DOI 10.1186/s40168-017-0331-1 RESEARCH Open Access Members of the Candidate Phyla Radiation are functionally differentiated by carbon- and nitrogen-cycling capabilities R. E. Danczak1, M. D. Johnston2, C. Kenah3, M. Slattery3, K. C. Wrighton1 and M. J. Wilkins1,2* Abstract Background: The Candidate Phyla Radiation (CPR) is a recently described expansion of the tree of life that represents more than 15% of all bacterial diversity and potentially contains over 70 different phyla. Despite this broad phylogenetic variation, these microorganisms appear to feature little functional diversity, with members generally characterized as obligate fermenters. Additionally, much of the data describing CPR phyla has been generated from a limited number of environments, constraining our knowledge of their functional roles and biogeographical distribution. To expand our understanding of subsurface CPR microorganisms, we sampled four separate groundwater wells over 2 years across three Ohio counties. Results: Samples were analyzed using 16S rRNA gene amplicon and shotgun metagenomic sequencing. Amplicon results indicated that CPR members comprised between 2 and 20% of the microbial communities with relative abundances stable through time in Athens and Greene samples but dynamic in Licking groundwater. Shotgun metagenomic analyses generated 71 putative CPR genomes, representing roughly 32 known phyla and 2 putative new phyla, Candidatus Brownbacteria and Candidatus Hugbacteria. While these genomes largely mirrored metabolic characteristics of known CPR members, some features were previously uncharacterized. For instance, nitrite reductase, encoded by nirK,was found in four of our Parcubacteria genomes and multiple CPR genomes from other studies, indicating a potentially undescribed role for these microorganisms in denitrification. Additionally, glycoside hydrolase (GH) family profiles for our 71 genomes and over 2000 other CPR genomes were analyzed to characterize their carbon-processing potential. Although common trends were present throughout the radiation, differences highlighted potential mechanisms that could allow microorganisms across the CPR to occupy various subsurface niches. For example, members of the Microgenomates superphylum appear to potentially degrade a wider range of carbon substrates than other CPR phyla. Conclusions: CPR members are present across a range of environments and often constitute a significant fraction of the microbial population in groundwater systems, particularly. Further sampling of such environments will resolve this portion of the tree of life at finer taxonomic levels, which is essential to solidify functional differences between members that populate this phylogenetically broad region of the tree of life. Keywords: Candidate phyla, Groundwater, Subsurface, Carbon, Nitrogen * Correspondence: [email protected] 1Department of Microbiology, The Ohio State University, Columbus, OH, USA 2School of Earth Sciences, The Ohio State University, Columbus, OH, USA Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Danczak et al. Microbiome (2017) 5:112 Page 2 of 14 Background subsurface locations account for nearly all the phylogen- The Candidate Phyla Radiation (CPR) is a recently etic diversity within the CPR. To better resolve both the described expansion of the tree of life that lacked any biogeography of CPR members and uncover new phylo- sequenced genomes until 2012 [1], with all prior know- genetic diversity within this radiation, we performed ledge derived from various marker gene studies [2, 3]. time-resolved metagenomic sampling on planktonic This absence of genomic information limited inferences biomass from four aquifers across southern Ohio (Fig. 1a). on the phylogenetic diversity and biogeochemical roles From the resulting data, we were able to obtain 71 near- of these organisms. Early work predicted that this radi- complete or incomplete CPR genomes. Furthermore, we ation represents roughly 15% of the bacterial domain [4]. profiled glycoside hydrolases encoded across over 2000 More recently, extensive genomic sampling of this separate CPR genomes and identified a potentially new radiation yielded over 2000 genomes, many of which role for many of these microorganisms in the nitrogen were closed, significantly expanding the phylogenetic cycle via a critical step in denitrification (conversion diversity within the radiation [4–6]. With this increased ofnitritetonitricoxide).Thesedataexpandour metagenomic sampling, CPR membership has grown to current understanding of the phylogeny of the CPR include over 70 phyla including two superphyla (Parcu- and suggest that these microorganisms are cosmopol- bacteria and Microgenomates) [6, 7]. Despite this itan in subsurface environments. Moreover, our re- radiation constituting a large portion of bacterial diver- sults advance knowledge of functional differentiation sity, the absence of cultured representatives means that across the radiation, with new roles in nitrogen and our current understanding is derived exclusively from carbon cycling highlighted here. various omics datasets, with a large fraction derived from a single field site near Rifle, CO. Since then, Methods additional genomes have been reconstructed from other Sample collection subsurface locations [8–10] and habitats [11, 12] around Groundwater samples were collected from the Ohio the globe. Department of Natural Resources (ODNR) Observation Based upon current data, there appear to be a number Well Network used to monitor ground water level fluc- of conserved traits throughout the entire CPR. Firstly, tuations in three different counties (Fig. 1a). These three despite representing a broad phylogenetic diversity, CPR wells are located within separate buried valley aquifers, members are typically characterized by very limited valleys that have been back filled with glacial sands and biosynthetic capabilities (i.e., the inability to produce gravels and some till. The observation wells were amino acids, lipids, some conserved genes, etc.) [4, 5]. sampled on a quarterly basis over a 2-year period from Originally, these organisms were inferred to be obligate July 2014 to July 2016. Additionally, one private drinking fermenters based upon the complete absence of respira- water well, located in a sand and gravel aquifer within a tory genes. Recently, however, CPR metabolic versatility thick till sequence in western Licking County (Fig. 1a), was expanded for members of the Parcubacteria with was sampled once in June 2016. new genomes that encode putative components of the Wells were purged of more than 250 L of water to dissimilatory nitrate reduction to ammonia pathway ensure that aquifer-derived water was being sampled [12, 13] as well as potential hydroxylamine oxidation (dedicated pumps were placed at the screened interval [13]. Metaproteomic analyses also suggested that fermen- for the ODNR wells). Approximately 38 L of post-purge tative CPR likely play a significant role in hydrogen and groundwater was pumped sequentially through a 0.2-μm carbon cycling in subsurface ecosystems [4, 14, 15]. then 0.1-μm Supor PES Membrane Filter (Pall Corpor- Beyond metabolism, small genome size (~ 1 Mb) is ation, NY, USA). The filters were then immediately flash also generally conserved throughout the radiation [4]. frozen and kept on dry ice until they were returned to the Consistent with small genome sizes, high-resolution Ohio State University where they were stored at −80 °C cryo-TEM of the CPR revealed extremely small cell sizes until DNA extraction. During sampling, oxidation- of 0.009 ± 0.002 mm3 [16]. Taken together, phylogenetic, reduction potential (ORP), temperature, and pH were metabolic, and cell biology inferences suggest that measured using a handheld Myron Ultrameter II (Myron members of the CPR may lead a symbiotic or syntrophic L Company, CA, USA). General observational data for lifestyle, dependent upon some partner (or partners) for each of these groundwater wells is provided in Additional necessary metabolites while potentially providing labile file 1: Table S1. fermentation waste products such as acetate in return [4, 5, 9, 11]. Such a co-dependent lifestyle could potentially DNA extraction, sequencing, and processing account for our current lack of cultured CPR members. DNA was extracted from roughly a quarter of each Supor While these conclusions are well supported by existing PES membrane filter by using the PowerSoil DNA Isola- genomic datasets, samples from only a limited range of tion Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA). Danczak et al. Microbiome (2017) 5:112 Page 3 of 14 a b Fig. 1 Map of Ohio and CPR relative abundance through time. a A map of Ohio with studied colors indicated by various colors; colored circles represent the approximate location of the Ohio Department of Natural Resources (ODNR) sampling wells