The Taxonomic and Functional Diversity of Microbes at a Temperate Coastal Site: A ‘Multi-Omic’ Study of Seasonal and Diel Temporal Variation

et al. (2010) The Taxonomic and Functional Diversity of Microbes at a Temperate Coastal Site: A 'Multi-Omic' Study of Seasonal and Diel Temporal Variation. PLoS ONE 5(11): e15545. doi:10.1371/journal.pone.0015545 The Taxonomic and Functional Diversity of Microbes at a Temperate Coastal Site: A 'Multi-Omic' Study of Seasonal and Diel Temporal Variation Jack A Gilbert 0 Dawn Field 0 Paul Swift 0 Simon Thomas 0 Denise Cummings 0 Ben Temperton 0 Karen Weynberg 0 Susan Huse 0 Margaret Hughes 0 Ian Joint 0 Paul J. Somerfield 0 Martin Mu hling 0 Francisco Rodriguez-Valera, Universidad Miguel Hernandez, Spain 0 1 Plymouth Marine Laboratory , Plymouth , United Kingdom , 2 Argonne National Laboratory, Argonne, Illinois, United States of America, 3 Department of Ecology and Evolution, University of Chicago , Chicago , Illinois, United States of America, 4 National Environment Research Council (NERC) Centre for Ecology and Hydrology , Wallingford, Oxfordshire , United Kingdom , 5 Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts, United States of America, 6 School of Biological Sciences, University of Liverpool , Liverpool , United Kingdom , 7 TU Bergakademie Freiberg , IO Z - Interdisciplinary Centre for Ecology , Freiberg , Germany How microbial communities change over time in response to the environment is poorly understood. Previously a six-year time series of 16S rRNA V6 data from the Western English Channel demonstrated robust seasonal structure within the bacterial community, with diversity negatively correlated with day-length. Here we determine whether metagenomes and metatranscriptomes follow similar patterns. We generated 16S rRNA datasets, metagenomes (1.2 GB) and metatranscriptomes (157 MB) for eight additional time points sampled in 2008, representing three seasons (Winter, Spring, Summer) and including day and night samples. This is the first microbial 'multi-omic' study to combine 16S rRNA amplicon sequencing with metagenomic and metatranscriptomic profiling. Five main conclusions can be drawn from analysis of these data: 1) Archaea follow the same seasonal patterns as Bacteria, but show lower relative diversity; 2) Higher 16S rRNA diversity also reflects a higher diversity of transcripts; 3) Diversity is highest in winter and at night; 4) Community-level changes in 16S-based diversity and metagenomic profiles are better explained by seasonal patterns (with samples closest in time being most similar), while metatranscriptomic profiles are better explained by diel patterns and shifts in particular categories (i.e., functional groups) of genes; 5) Changes in key genes occur among seasons and between day and night (i.e., photosynthesis); but these samples contain large numbers of orphan genes without known homologues and it is these unknown gene sets that appear to contribute most towards defining the differences observed between times. Despite the huge diversity of these microbial communities, there are clear signs of predictable patterns and detectable stability over time. Renewed and intensified efforts are required to reveal fundamental deterministic patterns in the most complex microbial communities. Further, the presence of a substantial proportion of orphan sequences underscores the need to determine the gene products of sequences with currently unknown function. - Funding: Funding for this work was provided by a Natural Environmental Research Council (www.nerc.ac.uk) grant, NE/F00138X/1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Jack A. Gilbert is a PLoS ONE Academic Editor. The authors have declared that no competing interests exist. The diversity of bacteria, as revealed by 16S rRNA, is wellknown to be extremely high [110]. Therefore it is expected that the functional (phenotypic) diversity of these organisms will also be vast. This is already evidenced by biogeographic studies [e.g. 11,8] that highlight the huge store of microbial proteins present in marine communities. For example, Rusch and colleagues [11] found approximately 4.4 million unique genetic fragments in a study of 7.7 million sequences. However, to the best of our knowledge there have been no studies that have made direct comparisons of overall diversity at the 16S rRNA, metagenomic and metatranscriptomic levels over time. Here we apply such a multi-omic approach to begin to unravel relationships between genetic and functional diversity in a temperate coastal marine microbial community. Marine bacteria demonstrate seasonal patterns in diversity with, generally, higher diversity during the winter than the summer in pelagic ecosystems [1214]. Numerous environmental factors have been suggested to influence this diversity (e.g. temperature and nutrients: [13,10,15]), yet our characterization of the long-term coastal ocean observatory site, L4, in the Western English Channel (http://www. westernchannelobservatory.org.uk/all_parameters.html) suggests that the robust seasonal pattern in species richness is most closely correlated to day length [14]. It is possible that an ability of organisms to respond to day length could explain the resilience of metabolic circadian oscillators, allowing organisms to respond to changing environmental conditions [16]. Recently, the transcriptional profiles from one pair of night and a day samples of bacterioplankton in the oligotrophic North Pacific Ocean were examined [17], showing that transcriptional activity was correlated to the diel cycle and the estimated diversity of the COG functional categories was higher at night. While previous genomic and metagenomic research has mainly focused on the diel rhythm in photosynthetic microorganisms [e.g. 1820], we now extend this to the whole prokaryotic community, including the non-photosynthetic microorganisms, by comparing night- and day-time samples collected within the same 24 hr period. Specifically, we test two hypotheses about the microbial community found at L4. Firstly, that bacterial and archaeal functional potential (genetic capacity) and functional actuality (transcriptional response) will track diversity (based on the 16S rRNA marker) and show similar seasonal patterns. Secondly, that within short time periods, metatranscriptomes will show more differences than 16S rRNA and metagenomic profiles, reflecting the relationship between expression of particular sets of genes and environmental variation, such as on a day/night cycle. To test these hypotheses, datasets were generated from pelagic water samples taken from the L4 station during the day and night at 3 sampling time points in 2008: January, April and August, representing winter, spring and summer. Samples were collected once during day time and once during night in January and April, while in August four samples, taken at six hourly intervals, were collected o (...truncated)