14Universal SSU rRNA primers allow comprehensive quantitative profiling of 15 natural communities by simultaneously amplifying templates from Bacteria, Archaea, 16 and Eukaryota in a single PCR reaction. Despite the potential to show all rRNA gene 17 relative gene abundances, they are rarely used due to concerns about length bias 18 against 18S amplicons and bioinformatic challenges converting mixed 16S/18S 19 sequences into amplicon sequence variants. We thus developed 16S and 18S rRNA 20 mock communities and a bioinformatic pipeline to validate this three-domain approach. 21To test for length biases, we mixed eukaryotic and prokaryotic mocks before PCR, and 22 found consistent two-fold underestimation of longer 18S sequences due to sequencing 23 but not PCR bias. Using these mocks, we show universal V4-V5 primers (515Y/926R) 24 outperformed eukaryote-specific V4 primers in observed vs. expected abundance 25 correlations and sequences with single mismatches to the primer were strongly 26 underestimated (3-8 fold). A year of monthly time-series data from a protist-enriched 27 1.2-80 µm size fraction yielded an average of 9% 18S, 17% chloroplast 16S, and 74% 28 prokaryote 16S rRNA gene amplicons. These data demonstrate the potential for 29 universal primers to generate quantitative and comprehensive microbiome profiles, 30 although gene copy and genome size variability should be considered -as for any 31 quantitative genetic analysis. 32 33
The ability of ecosystems to adapt to environmental perturbations depends on the duration and intensity of change and the overall biological diversity of the system. While studies have indicated that rare microbial taxa may provide a biological reservoir that supports long-term 40 ecosystem stability, how this dynamic population is influenced by environmental parameters remains unclear. In this study, a microbial mat ecosystem located on San Salvador Island, The Bahamas was used as a model to examine how environmental disturbance affects the activity of rare and abundant archaeal and bacterial communities and how these changes impact potential biogeochemical processes. While this ecosystem undergoes a range of 45 seasonal variation, it experienced a large shift in salinity (230 to 65 g kg -1 ) during 2011-2012 following the landfall of Hurricane Irene on San Salvador Island. High throughput sequencing and analysis of 16S rRNA and rRNA genes from samples before and after the pulse disturbance showed significant changes in the diversity and activity of abundant and rare taxa, suggesting overall functional and compositional sensitivity to environmental 50 change. In both archaeal and bacterial communities, while the majority of taxa showed low activity across conditions, the total number of active taxa and overall activity increased postdisturbance, with significant shifts in activity occurring among abundant and rare taxa across and within phyla. Broadly, following the post-disturbance reduction in salinity, taxa within Halobacteria decreased while those within Crenarchaeota, Thaumarchaeota, Thermoplasmata, 55Cyanobacteria, and Proteobacteria, increased in abundance and activity. Quantitative PCR of genes and transcripts involved in nitrogen and sulfur cycling showed concomitant shifts in biogeochemical cycling potential. Post-disturbance conditions increased the expression of genes involved in N-fixation, nitrification, denitrification, and sulfate reduction. Together, our findings show complex community adaptation to environmental change and help 60 elucidate factors connecting disturbance, biodiversity, and ecosystem function that may enhance ecosystem models.
Viruses are an important top-down control on microbial communities, yet their direct 11 study in natural environments has been hindered by culture limitations [1][2][3] . The advance of 12 sequencing and bioinformatics over the last decade enabled the cultivation independent 13 study of viruses. Many studies focus on assembling new viral genomes 4-6 and studying viral 14 diversity using marker genes amplified from free viruses with POLA being the most impacted and SPOT resembling open ocean conditions. In all of these 33 sites free virus-like particles outnumber bacteria and archaea roughly 10:1 (sup. fig. 1). We 34 examined only the 0.2-1 µm size-fraction, which includes most bacteria, archaea and some 35 picoeukaryotes. Via assembly of metatranscriptomes, we obtained 1455 contigs longer than 5 kb 36 of which 57 (3.9%) were characterized as viral using virSorter and virFinder (see methods). 37Additionally, a cross-assembly of the metatranscriptomic viral contigs with metagenomes of the 38 same samples (N=12) yielded 9 more contigs (mean length 26,563 bp) characterized as viral. 39Most of the contigs represent dsDNA viruses (N= 65) as apparent from their presence in 40 metagenomes, but one appears to be an RNA virus possibly infecting a eukaryotic host. This 41 contig contained an RNA-dependent-RNA-polymerase whose nearest match in NCBI non-42 redundant database was marine Antarctic phytoplankton RNA virus PAL_E4 9 . These 66 viral 43 contigs revealed varied patterns of presence (in metagenomes) and activity (in 44 metatranscriptomes) in the three sites over a year (fig.
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