Characterizing the "fungal shunt": Parasitic fungi on diatoms affect carbon flow and bacterial communities in aquatic microbial food webs.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed "fungal shunt" can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton-bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Constraining the composition and quantity of organic matter used by abundant marine Thaumarchaeota.

Marine Group I (MGI) Thaumarchaeota were originally described as chemoautotrophic nitrifiers, but molecular and isotopic evidence suggests heterotrophic and/or mixotrophic capabilities. Here, we investigated the quantity and composition of organic matter assimilated by individual, uncultured MGI cells from the Pacific Ocean to constrain their potential for mixotrophy and heterotrophy. We observed that most MGI cells did not assimilate carbon from any organic substrate provided (glucose, pyruvate, oxaloacetate, protein, urea, and amino acids). The minority of MGI cells that did assimilate it did so exclusively from nitrogenous substrates (urea, 15% of MGI and amino acids, 36% of MGI), and only as an auxiliary carbon source (<20% of that subset's total cellular carbon was derived from those substrates). At the population level, MGI assimilation of organic carbon comprised just 0.5%-11% of total biomass carbon. We observed extensive assimilation of inorganic carbon and urea- and amino acid-derived nitrogen (equal to that from ammonium), consistent with metagenomic and metatranscriptomic analyses performed here and previously showing a widespread potential for MGI to perform autotrophy and transport and degrade organic nitrogen. Our results constrain the quantity and composition of organic matter used by MGI and suggest they use it primarily to meet nitrogen demands for anabolism and nitrification.

Rates and physicochemical drivers of microbial anabolic activity in deep-sea sediments and implications for deep time.

Sediment microorganisms influence global climate and redox by altering rates of organic carbon burial. However, the activity and ecology of benthic microorganisms are poorly characterized, especially in the deep sea. Here, we conducted nearly 300 stable isotope tracer experiments in sediments from the Pacific and Atlantic oceans (100-4500 m water depth) to determine the rates, spatial distribution, and physicochemical controls on microbial total anabolic activity, nitrogen fixation, and inorganic/organic carbon uptake. Using correlative and manipulative approaches, we find that total activity is limited primarily by organic carbon and/or energy. Activity correlates significantly with distance from shore, sediment depth, C:N ratios, and overlying chlorophyll concentrations and is stimulated by carbon but not nitrogen additions. Consistent with this, nitrogen fixation was undetected despite relatively low concentrations of porewater ammonium and the previous detection of nifH genes. Inorganic carbon uptake accounted for 7%-55% of carbon assimilation per sample (median 21%), suggesting chemoautotrophy is an important and unappreciated source of labile carbon in deep-sea sediments. Community 16S rRNA was dominated by Bacteria (<2% Archaea), primarily Desulfobacterales of the Deltaproteobacteria. Leveraging our findings, we modelled global benthic microbial activity through geologic time and find the potential for significant shifts in total activity with supercontinental cycles.

Genome and epigenome of a novel marine Thaumarchaeota strain suggest viral infection, phosphorothioation DNA modification and multiple restriction systems.

Marine Thaumarchaeota are abundant ammonia-oxidizers but have few representative laboratory-cultured strains. We report the cultivation of Candidatus Nitrosomarinus catalina SPOT01, a novel strain that is less warm-temperature tolerant than other cultivated Thaumarchaeota. Using metagenomic recruitment, strain SPOT01 comprises a major portion of Thaumarchaeota (4-54%) in temperate Pacific waters. Its complete 1.36 Mbp genome possesses several distinguishing features: putative phosphorothioation (PT) DNA modification genes; a region containing probable viral genes; and putative urea utilization genes. The PT modification genes and an adjacent putative restriction enzyme (RE) operon likely form a restriction modification (RM) system for defence from foreign DNA. PacBio sequencing showed >98% methylation at two motifs, and inferred PT guanine modification of 19% of possible TGCA sites. Metagenomic recruitment also reveals the putative virus region and PT modification and RE genes are present in 18-26%, 9-14% and <1.5% of natural populations at 150 m with ≥85% identity to strain SPOT01. The presence of multiple probable RM systems in a highly streamlined genome suggests a surprising importance for defence from foreign DNA for dilute populations that infrequently encounter viruses or other cells. This new strain provides new insights into the ecology, including viral interactions, of this important group of marine microbes.

Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples.

Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated (e.g. Gammaproteobacteria) common marine taxa. We evaluated marine samples and mock communities (containing 11 or 27 marine 16S clones), showing alternative primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA) and 926R (5'-CCGYCAATTYMTTTRAGTTT) yield more accurate estimates of mock community abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, and amplify eukaryotic 18S rRNA. Mock communities amplified with 515F-Y/926R yielded closer observed community composition versus expected (r(2) = 0.95) compared with 515F-Y/806R (r(2) ∼ 0.5). Unexpectedly, biases with 515F-Y/806R against SAR11 in field samples (∼4-10-fold) were stronger than in mock communities (∼2-fold). Correcting a mismatch to Thaumarchaea in the 515F-C increased their apparent abundance in field samples, but not as much as using 926R rather than 806R. With plankton samples rich in eukaryotic DNA (> 1 µm size fraction), 18S sequences averaged ∼17% of all sequences. A single mismatch can strongly bias amplification, but even perfectly matched primers can exhibit preferential amplification. We show that beyond in silico predictions, testing with mock communities and field samples is important in primer selection.

Characterizing Chemoautotrophy and Heterotrophy in Marine Archaea and Bacteria With Single-Cell Multi-isotope NanoSIP.

Characterizing and quantifying in situ metabolisms remains both a central goal and challenge for environmental microbiology. Here, we used a single-cell, multi-isotope approach to investigate the anabolic activity of marine microorganisms, with an emphasis on natural populations of Thaumarchaeota. After incubating coastal Pacific Ocean water with 13C-bicarbonate and 15N-amino acids, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to isotopically screen 1,501 individual cells, and 16S rRNA amplicon sequencing to assess community composition. We established isotopic enrichment thresholds for activity and metabolic classification, and with these determined the percentage of anabolically active cells, the distribution of activity across the whole community, and the metabolic lifestyle-chemoautotrophic or heterotrophic-of each cell. Most cells (>90%) were anabolically active during the incubation, and 4-17% were chemoautotrophic. When we inhibited bacteria with antibiotics, the fraction of chemoautotrophic cells detected via nanoSIMS increased, suggesting archaea dominated chemoautotrophy. With fluorescence in situ hybridization coupled to nanoSIMS (FISH-nanoSIMS), we confirmed that most Thaumarchaeota were living chemoautotrophically, while bacteria were not. FISH-nanoSIMS analysis of cells incubated with dual-labeled (13C,15N-) amino acids revealed that most Thaumarchaeota cells assimilated amino-acid-derived nitrogen but not carbon, while bacteria assimilated both. This indicates that some Thaumarchaeota do not assimilate intact amino acids, suggesting intra-phylum heterogeneity in organic carbon utilization, and potentially their use of amino acids for nitrification. Together, our results demonstrate the utility of multi-isotope nanoSIMS analysis for high-throughput metabolic screening, and shed light on the activity and metabolism of uncultured marine archaea and bacteria.

High-quality genome sequences of uncultured microbes by assembly of read clouds.

Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques. Read-cloud metagenomic sequencing and Athena assembly produced the most comprehensive individual genome drafts with high contiguity (>200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20×) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which were complete (>90% complete, <5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.

Marine archaeal dynamics and interactions with the microbial community over 5 years from surface to seafloor.

Marine archaea are critical contributors to global carbon and nitrogen redox cycles, but their temporal variability and microbial associations across the water column are poorly known. We evaluated seasonal variability of free living (0.2-1 µm size fraction) Thaumarchaea Marine Group I (MGI) and Euryarchaea Marine Group II (MGII) communities and their associations with the microbial community from surface to seafloor (890 m) over 5 years by 16S rRNA V4-V5 gene sequencing. MGI and MGII communities demonstrated distinct compositions at different depths, and seasonality at all depths. Microbial association networks at 150 m, 500 m and 890 m, revealed diverse assemblages of MGI (presumed ammonia oxidizers) and Nitrospina taxa (presumed dominant nitrite oxidizers, completing the nitrification process), suggesting distinct MGI-Nitrospina OTUs are responsible for nitrification at different depths and seasons, and depth- related and seasonal variability in nitrification could be affected by alternating MGI-Nitrospina assemblages. MGII taxa also showed distinct correlations to possibly heterotrophic bacteria, most commonly to members of Marine Group A, Chloroflexi, Marine Group B, and SAR86. Thus, both MGI and MGII likely have dynamic associations with bacteria based on similarities in activity or other interactions that select for distinct microbial assemblages over time. The importance of MGII taxa as members of the heterotrophic community previously reported for photic zone appears to apply throughout the water column.

Seasonal and interannual variability of the marine bacterioplankton community throughout the water column over ten years.

Microbial activities that affect global oceanographic and atmospheric processes happen throughout the water column, yet the long-term ecological dynamics of microbes have been studied largely in the euphotic zone and adjacent seasonally mixed depths. We investigated temporal patterns in the community structure of free-living bacteria, by sampling approximately monthly from 5 m, the deep chlorophyll maximum (∼15-40 m), 150, 500 and 890 m, in San Pedro Channel (maximum depth 900 m, hypoxic below ∼500 m), off the coast of Southern California. Community structure and biodiversity (inverse Simpson index) showed seasonal patterns near the surface and bottom of the water column, but not at intermediate depths. Inverse Simpson's index was highest in the winter in surface waters and in the spring at 890 m, and varied interannually at all depths. Biodiversity appeared to be driven partially by exchange of microbes between depths and was highest when communities were changing slowly over time. Meanwhile, communities from the surface through 500 m varied interannually. After accounting for seasonality, several environmental parameters co-varied with community structure at the surface and 890 m, but not at the intermediate depths. Abundant and seasonally variable groups included, at 890 m, Nitrospina, Flavobacteria and Marine Group A. Seasonality at 890 m is likely driven by variability in sinking particles, which originate in surface waters, pass transiently through the middle water column and accumulate on the seafloor where they alter the chemical environment. Seasonal subeuphotic groups are likely those whose ecology is strongly influenced by these particles. This surface-to-bottom, decade-long, study identifies seasonality and interannual variability not only of overall community structure, but also of numerous taxonomic groups and near-species level operational taxonomic units.

Temporal variability and coherence of euphotic zone bacterial communities over a decade in the Southern California Bight.

Time-series are critical to understanding long-term natural variability in the oceans. Bacterial communities in the euphotic zone were investigated for over a decade at the San Pedro Ocean Time-series station (SPOT) off southern California. Community composition was assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and coupled with measurements of oceanographic parameters for the surface ocean (0-5 m) and deep chlorophyll maximum (DCM, average depth ≈ 30 m). SAR11 and cyanobacterial ecotypes comprised typically more than one-third of the measured community; diversity within both was temporally variable, although a few operational taxonomic units (OTUs) were consistently more abundant. Persistent OTUs, mostly Alphaproteobacteria (SAR11 clade), Actinobacteria and Flavobacteria, tended to be abundant, in contrast to many rarer yet intermittent and ephemeral OTUs. Association networks revealed potential niches for key OTUs from SAR11, cyanobacteria, SAR86 and other common clades on the basis of robust correlations. Resilience was evident by the average communities drifting only slightly as years passed. Average Bray-Curtis similarity between any pair of dates was ≈ 40%, with a slight decrease over the decade and obvious near-surface seasonality; communities 8-10 years apart were slightly more different than those 1-4 years apart with the highest rate of change at 0-5 m between communities <4 years apart. The surface exhibited more pronounced seasonality than the DCM. Inter-depth Bray-Curtis similarities repeatedly decreased as the water column stratified each summer. Environmental factors were better predictors of shifts in community composition than months or elapsed time alone; yet, the best predictor was community composition at the other depth (that is, 0-5 m versus DCM).