Novel integrative elements and genomic plasticity in ocean ecosystems.

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.

Marine viral particles reveal an expansive repertoire of phage-parasitizing mobile elements.

Phage satellites are mobile genetic elements that propagate by parasitizing bacteriophage replication. We report here the discovery of abundant and diverse phage satellites that were packaged as concatemeric repeats within naturally occurring bacteriophage particles in seawater. These same phage-parasitizing mobile elements were found integrated in the genomes of dominant co-occurring bacterioplankton species. Like known phage satellites, many marine phage satellites encoded genes for integration, DNA replication, phage interference, and capsid assembly. Many also contained distinctive gene suites indicative of unique virus hijacking, phage immunity, and mobilization mechanisms. Marine phage satellite sequences were widespread in local and global oceanic virioplankton populations, reflecting their ubiquity, abundance, and temporal persistence in marine planktonic communities worldwide. Their gene content and putative life cycles suggest they may impact host-cell phage immunity and defense, lateral gene transfer, bacteriophage-induced cell mortality and cellular host and virus productivity. Given that marine phage satellites cannot be distinguished from bona fide viral particles via commonly used microscopic techniques, their predicted numbers (∼3.2 × 1026 in the ocean) may influence current estimates of virus densities, production, and virus-induced mortality. In total, the data suggest that marine phage satellites have potential to significantly impact the ecology and evolution of bacteria and their viruses throughout the oceans. We predict that any habitat that harbors bacteriophage will also harbor similar phage satellites, making them a ubiquitous feature of most microbiomes on Earth.

Diversity and origins of bacterial and archaeal viruses on sinking particles reaching the abyssal ocean.

Sinking particles and particle-associated microbes influence global biogeochemistry through particulate matter export from the surface to the deep ocean. Despite ongoing studies of particle-associated microbes, viruses in these habitats remain largely unexplored. Whether, where, and which viruses might contribute to particle production and export remain open to investigation. In this study, we analyzed 857 virus population genomes associated with sinking particles collected over three years in sediment traps moored at 4000 m in the North Pacific Subtropical Gyre. Particle-associated viruses here were linked to cellular hosts through matches to bacterial and archaeal metagenome-assembled genome (MAG)-encoded prophages or CRISPR spacers, identifying novel viruses infecting presumptive deep-sea bacteria such as Colwellia, Moritella, and Shewanella. We also identified lytic viruses whose abundances correlated with particulate carbon flux and/or were exported from the photic to abyssal ocean, including cyanophages. Our data are consistent with some of the predicted outcomes of the viral shuttle hypothesis, and further suggest that viral lysis of both autotrophic and heterotrophic prokaryotes may play a role in carbon export. Our analyses revealed the diversity and origins of prevalent viruses found on deep-sea sinking particles and identified prospective viral groups for future investigation into processes that govern particle export in the open ocean.

Microbial Sources of Exocellular DNA in the Ocean.

Exocellular DNA is operationally defined as the fraction of the total DNA pool that passes through a membrane filter (0.1 µm). It is composed of DNA-containing vesicles, viruses, and free DNA and is ubiquitous in all aquatic systems, although the sources, sinks, and ecological consequences are largely unknown. Using a method that provides separation of these three fractions, we compared open ocean depth profiles of DNA associated with each fraction. Pelagibacter-like DNA dominated the vesicle fractions for all samples examined over a depth range of 75 to 500 m. Viral DNA consisted predominantly of myovirus-like and podovirus-like DNA and contained the highest proportion of unannotated sequences. Euphotic zone free DNA (75 to 125 m) contained primarily bacterial and viral sequences, with bacteria dominating samples from the mesopelagic zone (500 to 1,000 m). A high proportion of mesopelagic zone free DNA sequences appeared to originate from surface waters, including a large amount of DNA contributed by high-light Prochlorococcus ecotypes. Throughout the water column, but especially in the mesopelagic zone, the composition of free DNA sequences was not always reflective of cooccurring microbial communities that inhabit the same sampling depth. These results reveal the composition of free DNA in different regions of the water column (euphotic and mesopelagic zones), with implications for dissolved organic matter cycling and export (by way of sinking particles and/or migratory zooplankton) as a delivery mechanism. IMPORTANCE With advances in metagenomic sequencing, the microbial composition of diverse environmental systems has been investigated, providing new perspectives on potential ecological dynamics and dimensions for experimental investigations. Here, we characterized exocellular free DNA via metagenomics, using a newly developed method that separates free DNA from cells, viruses, and vesicles, and facilitated the independent characterization of each fraction. The fate of this free DNA has both ecological consequences as a nutrient (N and P) source and potential evolutionary consequences as a source of genetic transformation. Here, we document different microbial sources of free DNA at the surface (0 to 200 m) versus depths of 250 to 1,000 m, suggesting that distinct free DNA production mechanisms may be present throughout the oligotrophic water column. Examining microbial processes through the lens of exocellular DNA provides insights into the production of labile dissolved organic matter (i.e., free DNA) at the surface (likely by viral lysis) and processes that influence the fate of sinking, surface-derived organic matter.

A Primer for Microbiome Time-Series Analysis.

Time-series can provide critical insights into the structure and function of microbial communities. The analysis of temporal data warrants statistical considerations, distinct from comparative microbiome studies, to address ecological questions. This primer identifies unique challenges and approaches for analyzing microbiome time-series. In doing so, we focus on (1) identifying compositionally similar samples, (2) inferring putative interactions among populations, and (3) detecting periodic signals. We connect theory, code and data via a series of hands-on modules with a motivating biological question centered on marine microbial ecology. The topics of the modules include characterizing shifts in community structure and activity, identifying expression levels with a diel periodic signal, and identifying putative interactions within a complex community. Modules are presented as self-contained, open-access, interactive tutorials in R and Matlab. Throughout, we highlight statistical considerations for dealing with autocorrelated and compositional data, with an eye to improving the robustness of inferences from microbiome time-series. In doing so, we hope that this primer helps to broaden the use of time-series analytic methods within the microbial ecology research community.

Assembly-free single-molecule sequencing recovers complete virus genomes from natural microbial communities.

Viruses are the most abundant biological entities on Earth and play key roles in host ecology, evolution, and horizontal gene transfer. Despite recent progress in viral metagenomics, the inherent genetic complexity of virus populations still poses technical difficulties for recovering complete virus genomes from natural assemblages. To address these challenges, we developed an assembly-free, single-molecule nanopore sequencing approach, enabling direct recovery of complete virus genome sequences from environmental samples. Our method yielded thousands of full-length, high-quality draft virus genome sequences that were not recovered using standard short-read assembly approaches. Additionally, our analyses discriminated between populations whose genomes had identical direct terminal repeats versus those with circularly permuted repeats at their termini, thus providing new insight into native virus reproduction and genome packaging. Novel DNA sequences were discovered, whose repeat structures, gene contents, and concatemer lengths suggest they are phage-inducible chromosomal islands, which are packaged as concatemers in phage particles, with lengths that match the size ranges of co-occurring phage genomes. Our new virus sequencing strategy can provide previously unavailable information about the genome structures, population biology, and ecology of naturally occurring viruses and viral parasites.

Double-stranded DNA virioplankton dynamics and reproductive strategies in the oligotrophic open ocean water column.

Microbial communities are critical to ecosystem dynamics and biogeochemical cycling in the open oceans. Viruses are essential elements of these communities, influencing the productivity, diversity, and evolution of cellular hosts. To further explore the natural history and ecology of open-ocean viruses, we surveyed the spatiotemporal dynamics of double-stranded DNA (dsDNA) viruses in both virioplankton and bacterioplankton size fractions in the North Pacific Subtropical Gyre, one of the largest biomes on the planet. Assembly and clustering of viral genomes revealed a peak in virioplankton diversity at the base of the euphotic zone, where virus populations and host species richness both reached their maxima. Simultaneous characterization of both extracellular and intracellular viruses suggested depth-specific reproductive strategies. In particular, analyses indicated elevated lytic interactions in the mixed layer, more temporally variable temperate phage interactions at the base of the euphotic zone, and increased lysogeny in the mesopelagic ocean. Furthermore, the depth variability of auxiliary metabolic genes suggested habitat-specific strategies for viral influence on light-energy, nitrogen, and phosphorus acquisition during host infection. Most virus populations were temporally persistent over several years in this environment at the 95% nucleic acid identity level. In total, our analyses revealed variable distributional patterns and diverse reproductive and metabolic strategies of virus populations in the open-ocean water column.

Diel cycling of the cosmopolitan abundant Pelagibacter virus 37-F6: one of the most abundant viruses on earth.

The spatiotemporal dynamics for marine viral populations has only recently been explored. However, nothing is known about temporal activities of the uncultured Pelagibacter virus vSAG 37-F6, which was discovered by single-virus genomics as potentially the most abundant marine virus. Here, we investigate the diel cycling of 37-F6 virus and the putative SAR11 host using coastal and oceanic transcriptomic and viromic time-series data from Osaka Bay and North Pacific Subtropical Gyre. Virus 37-F6 and relatives displayed diel cycling of transcriptional activities synchronized with its putative host. In both virus and host, the lowest transcription rates were observed at 14:00-15:00, coinciding roughly with maximum solar irradiance, while higher transcriptional rates were detected during the night/early morning and afternoon. Diel abundance of free viruses of 37-F6 in seawater roughly mirrored the transcriptional activities of both virus and host. In Osaka Bay, among viral relatives (genus level), virus 37-F6 specifically showed the highest ratio of transcriptional activity to virome abundance, a proxy for viral transcriptional activity relative to free viral particle abundance. This high ratio suggests high infection rate efficiencies in vSAG 37-F6 virus compared to viral relatives. Thus, time-series data revealed temporal transcript activities in one of the most abundant viruses in Earth.

Bacteriophage Distributions and Temporal Variability in the Ocean's Interior.

Bacteriophages are numerically the most abundant DNA-containing entities in the oligotrophic ocean, yet how specific phage populations vary over time and space remains to be fully explored. Here, we conducted a metagenomic time-series survey of double-stranded DNA phages throughout the water column in the North Pacific Subtropical Gyre, encompassing 1.5 years from depths of 25 to 1,000 m. Viral gene sequences were identified in assembled metagenomic samples, yielding an estimated 172,385 different viral gene families. Viral marker gene distributions suggested that lysogeny was more prevalent at mesopelagic depths than in surface waters, consistent with prior prophage induction studies using mitomycin C. A total of 129 ALOHA viral genomes and genome fragments from 20 to 108 kbp were selected for further study, which represented the most abundant phages in the water column. Phage genotypes displayed discrete population structures. Most phages persisted throughout the time-series and displayed a strong depth structure that mirrored the stratified depth distributions of co-occurring bacterial taxa in the water column. Mesopelagic phages were distinct from surface water phages with respect to diversity, gene content, putative life histories, and temporal persistence, reflecting depth-dependent differences in host genomic architectures and phage reproductive strategies. The spatiotemporal distributions of the most abundant open-ocean bacteriophages that we report here provide new insight into viral temporal persistence, life history, and virus-host-environment interactions throughout the open-ocean water column.IMPORTANCE The North Pacific Subtropical Gyre represents one of the largest biomes on the planet, where microbial communities are central mediators of ecosystem dynamics and global biogeochemical cycles. Critical members of these communities are the viruses of marine bacteria, which can alter microbial metabolism and significantly influence their survival and productivity. To better understand these viral assemblages, we conducted genomic analyses of planktonic viruses over a seasonal cycle to ocean depths of 1,000 m. We identified 172,385 different viral gene families and 129 unique virus genotypes in this open-ocean setting. The spatiotemporal distributions of the most abundant open-ocean viruses that we report here provide new insights into viral temporal variability, life history, and virus-host-environment interactions throughout the water column.

Three's a Crowd: Trade-Offs between Attracting Pollinators and Ant Bodyguards with Nectar Rewards in Turnera.

Many plants attract insect pollinators with floral nectar (FN) and ant "bodyguards" with extrafloral nectar (EFN). If nectar production is costly or physiologically linked across glands, investment in one mutualism may trade off with investment in the other. We confirmed that changes in FN and EFN availability alter pollination and ant defense mutualisms in a field population of Turnera ulmifolia. Plants with additional FN tended to produce more seeds, while plants with reduced EFN production experienced less florivory. We then mimicked the consumptive effects of mutualists by removing FN or EFN daily for 50 days in a full factorial design using three Turnera species (T. joelii, T. subulata, and T. ulmifolia) in a glasshouse experiment. For T. ulmifolia and T. subulata, but not T. joelii, removing either nectar reduced production of the other, showing for the first time that EFN and FN production can trade off. In T. subulata, increased investment in FN decreased seed set, suggesting that nectar production can have direct fitness costs. Through the linked expression of EFN and FN, floral visitors may negatively affect biotic defense, and extrafloral nectary visitors may negatively affect pollination.