Space, the final frontier: The spatial component of phytoplankton-bacterial interactions.

Microscale interactions between marine phytoplankton and bacteria shape the microenvironment of individual cells, impacting their physiology and ultimately influencing global-scale biogeochemical processes like carbon and nutrient cycling. In dilute environments such as the ocean water column, metabolic exchange between microorganisms likely requires close proximity between partners. However, the biological strategies to achieve this physical proximity remain an understudied aspect of phytoplankton-bacterial associations. Understanding the mechanisms by which these microorganisms establish and sustain spatial relationships and the extent to which spatial proximity is necessary for interactions to occur, is critical to learning how spatial associations influence the ecology of phytoplankton and bacterial communities. Here, we provide an overview of current knowledge on the role of space in shaping interactions among ocean microorganisms, encompassing behavioural and metabolic evidence. We propose that characterising phytoplankton-bacterial interactions from a spatial perspective can contribute to a mechanistic understanding of the establishment and maintenance of these associations and, consequently, an enhanced ability to predict the impact of microscale processes on ecosystem-wide phenomena.

Internal carbon recycling by heterotrophic prokaryotes compensates for mismatches between phytoplankton production and heterotrophic consumption.

Molecular observational tools are useful for characterizing the composition and genetic endowment of microbial communities but cannot measure fluxes, which are critical for the understanding of ecosystems. To overcome these limitations, we used a mechanistic inference approach to estimate dissolved organic carbon (DOC) production and consumption by phytoplankton operational taxonomic units and heterotrophic prokaryotic amplicon sequence variants and inferred carbon fluxes between members of this microbial community from Western English Channel time-series data. Our analyses focused on phytoplankton spring and summer blooms, as well as bacteria summer blooms. In spring blooms, phytoplankton DOC production exceeds heterotrophic prokaryotic consumption, but in bacterial summer blooms heterotrophic prokaryotes consume three times more DOC than produced by the phytoplankton. This mismatch is compensated by heterotrophic prokaryotic DOC release by death, presumably from viral lysis. In both types of summer blooms, large amounts of the DOC liberated by heterotrophic prokaryotes are reused through internal recycling, with fluxes between different heterotrophic prokaryotes being at the same level as those between phytoplankton and heterotrophic prokaryotes. In context, internal recycling accounts for approximately 75% and 30% of the estimated net primary production (0.16 vs 0.22 and 0.08 vs 0.29 µmol l-1 d-1) in bacteria and phytoplankton summer blooms, respectively, and thus represents a major component of the Western English Channel carbon cycle. We have concluded that internal recycling compensates for mismatches between phytoplankton DOC production and heterotrophic prokaryotic consumption, and we encourage future analyses on aquatic carbon cycles to investigate fluxes between heterotrophic prokaryotes, specifically internal recycling.

Linking the impact of bacteria on phytoplankton growth with microbial community composition and co-occurrence patterns.

The interactions between microalgae and bacteria have recently emerged as key control factors which might contribute to a better understanding on how phytoplankton communities assemble and respond to environmental disturbances. We analyzed partial 16S rRNA and 18S rRNA genes from a total of 42 antibiotic bioassays, where phytoplankton growth was assessed in the presence or absence of an active bacterial community. A significant negative impact of bacteria was observed in 18 bioassays, a significant positive impact was detected in 5 of the cases, and a non-detectable effect occurred in 19 bioassays. Thalasiossira spp., Chlorophytes, Vibrionaceae and Alteromonadales were relatively more abundant in the samples where a positive effect of bacteria was observed compared to those where a negative impact was observed. Phytoplankton diversity was lower when bacteria negatively affect their growth than when the effect was beneficial. The phytoplankton-bacteria co-occurrence subnetwork included many significant Chlorophyta-Alteromonadales and Bacillariophyceae-Alteromonadales positive associations. Phytoplankton-bacteria co-exclusions were not detected in the network, which contrasts with the negative effect of bacteria on phytoplankton growth frequently detected in the bioassays, suggesting strong competitive interactions. Overall, this study adds strong evidence supporting the key role of phytoplankton-bacteria interactions in the microbial communities.

High-resolution phylogenetic analysis reveals long-term microbial dynamics and microdiversity in phytoplankton microbiome.

Phytoplankton-bacteria interactions represent the evolution of complex cross-kingdom networks requiring niche specialization of diverse microbes. Unraveling this co-evolutionary process has proven challenging because microbial partnerships are complex, and their assembly can be dynamic as well as scale- and taxon-dependent. Here, we monitored long-term experimental evolution of phytoplankton-bacteria interactions by reintroducing the intact microbiome into an axenized dinoflagellate Alexandrium tamarense to better understand microbiome assembly dynamics and how microbiome composition could shift and stabilize over 15 months. We examined host functioning by growth rate, photosynthetic capability, cell size, and other physiological signatures and compared it to associated microbial communities determined by 16S rRNA gene sequences. Our results showed that microbiome reconstitution did not restore the intact microbiome, instead a distinct microbial community shift to Roseobacter clade was observed in the re-established cultures. In-depth comparisons of microbial interactions revealed no apparent coupling between host physiology and specific bacterial taxa, indicating that highly represented, abundant taxa might not be essential for host functioning. The emergence of highly divergent Roseobacter clade sequences suggests fine-scale microbial dynamics driven by microdiversity could be potentially linked to host functioning. Collectively, our results indicate that functionally comparable microbiomes can be assembled from markedly different, highly diverse bacterial taxa in changing environments.

Role of Bacterial Community Composition as a Driver of the Small-Sized Phytoplankton Community Structure in a Productive Coastal System.

We present here the first detailed description of the seasonal patterns in bacterial community composition (BCC) in shelf waters off the Ría de Vigo (Spain), based on monthly samplings during 2 years. Moreover, we studied the relationship between bacterial and small-sized eukaryotic community composition to identify potential biotic interactions among components of these two communities. Bacterial operational taxonomic unit (OTU) richness and diversity systematically peaked in autumn-winter, likely related to low resource availability during this period. BCC showed seasonal and vertical patterns, with Rhodobacteraceae and Flavobacteriaceae families dominating in surface waters, and SAR11 clade dominating at the base of the photic zone (30 m depth). BCC variability was significantly explained by environmental variables (e.g., temperature of water, solar radiation, or dissolved organic matter). Interestingly, a strong and significant correlation was found between BCC and small-sized eukaryotic community composition (ECC), which suggests that biotic interactions may play a major role as structuring factors of the microbial plankton in this productive area. In addition, co-occurrence network analyses revealed strong and significant, mostly positive, associations between bacteria and small-sized phytoplankton. Positive associations likely result from mutualistic relationships (e.g., between Dinophyceae and Rhodobacteraceae), while some negative correlations suggest antagonistic interactions (e.g., between Pseudo-nitzchia sp. and SAR11). These results support the key role of biotic interactions as structuring factors of the small-sized eukaryotic community, mostly driven by positive associations between small-sized phytoplankton and bacteria.

Exploring the phycosphere of Emiliania huxleyi: From bloom dynamics to microbiome assembly experiments.

Coccolithophores have global ecological and biogeochemical significance as the most important calcifying marine phytoplankton group. The structure and selection of prokaryotic communities associated with the most abundant coccolithophore and bloom-forming species, Emiliania huxleyi, are still poorly known. In this study, we assessed the diversity of bacterial communities associated with an E. huxleyi bloom in the Celtic Sea (Eastern North Atlantic), exposed axenic E. huxleyi cultures to prokaryotic communities derived from bloom and non-bloom conditions, and followed the dynamics of their microbiome composition over one year. Bloom-associated prokaryotic communities were dominated by SAR11, Marine group II Euryarchaeota and Rhodobacterales and contained substantial proportions of known indicators of phytoplankton bloom demises such as Flavobacteriaceae and Pseudoalteromonadaceae. The taxonomic richness of bacteria derived from natural communities associated with axenic E. huxleyi rapidly shifted and then stabilized over time. The succession of microorganisms recruited from the environment was consistently dependent on the composition of the initial bacterioplankton community. Phycosphere-associated communities derived from the E. huxleyi bloom were highly similar to one another, suggesting deterministic processes, whereas cultures from non-bloom conditions show an effect of stochasticity. Overall, this work sheds new light on the importance of the initial inoculum composition in microbiome recruitment and elucidates the temporal dynamics of its composition and long-term stability.

Emiliania huxleyi-Bacteria Interactions under Increasing CO2 Concentrations.

The interactions established between marine microbes, namely phytoplankton-bacteria, are key to the balance of organic matter export to depth and recycling in the surface ocean. Still, their role in the response of phytoplankton to rising CO2 concentrations is poorly understood. Here, we show that the response of the cosmopolitan Emiliania huxleyi (E. huxleyi) to increasing CO2 is affected by the coexistence with bacteria. Specifically, decreased growth rate of E. huxleyi at enhanced CO2 concentrations was amplified in the bloom phase (potentially also related to nutrient concentrations) and with the coexistence with Idiomarina abyssalis (I. abyssalis) and Brachybacterium sp. In addition, enhanced CO2 concentrations also affected E. huxleyi's cellular content estimates, increasing organic and decreasing inorganic carbon, in the presence of I. abyssalis, but not Brachybacterium sp. At the same time, the bacterial isolates only survived in coexistence with E. huxleyi, but exclusively I. abyssalis at present CO2 concentrations. Bacterial species or group-specific responses to the projected CO2 rise, together with the concomitant effect on E. huxleyi, might impact the balance between the microbial loop and the export of organic matter, with consequences for atmospheric carbon dioxide.

Host genotype structures the microbiome of a globally dispersed marine phytoplankton.

Phytoplankton support complex bacterial microbiomes that rely on phytoplankton-derived extracellular compounds and perform functions necessary for algal growth. Recent work has revealed sophisticated interactions and exchanges of molecules between specific phytoplankton-bacteria pairs, but the role of host genotype in regulating those interactions is unknown. Here, we show how phytoplankton microbiomes are shaped by intraspecific genetic variation in the host using global environmental isolates of the model phytoplankton host Thalassiosira rotula and a laboratory common garden experiment. A set of 81 environmental T. rotula genotypes from three ocean basins and eight genetically distinct populations did not reveal a core microbiome. While no single bacterial phylotype was shared across all genotypes, we found strong genotypic influence of T. rotula, with microbiomes associating more strongly with host genetic population than with environmental factors. The microbiome association with host genetic population persisted across different ocean basins, suggesting that microbiomes may be associated with host populations for decades. To isolate the impact of host genotype on microbiomes, a common garden experiment using eight genotypes from three distinct host populations again found that host genotype influenced microbial community composition, suggesting that a process we describe as genotypic filtering, analogous to environmental filtering, shapes phytoplankton microbiomes. In both the environmental and laboratory studies, microbiome variation between genotypes suggests that other factors influenced microbiome composition but did not swamp the dominant signal of host genetic background. The long-term association of microbiomes with specific host genotypes reveals a possible mechanism explaining the evolution and maintenance of complex phytoplankton-bacteria chemical exchanges.

Tight Adherence (Tad) Pilus Genes Indicate Putative Niche Differentiation in Phytoplankton Bloom Associated Rhodobacterales.

The multiple interactions of phytoplankton and bacterioplankton are central for our understanding of aquatic environments. A prominent example of those is the consistent association of diatoms with Alphaproteobacteria of the order Rhodobacterales. These photoheterotrophic bacteria have traditionally been described as generalists that scavenge dissolved organic matter. Many observations suggest that members of this clade are specialized in colonizing the microenvironment of diatom cells, known as the phycosphere. However, the molecular mechanisms that differentiate Rhodobacterales generalists and phycosphere colonizers are poorly understood. We investigated Rhodobacterales in the North Sea during the 2010-2012 spring blooms using a time series of 38 deeply sequenced metagenomes and 10 metaproteomes collected throughout these events. Rhodobacterales metagenome assembled genomes (MAGs) were recurrently abundant. They exhibited the highest gene enrichment and protein expression of small-molecule transporters, such as monosaccharides, thiamine and polyamine transporters, and anaplerotic pathways, such as ethylmalonyl and propanoyl-CoA metabolic pathways, all suggestive of a generalist lifestyle. Metaproteomes indicated that the species represented by these MAGs were the dominant suppliers of vitamin B12 during the blooms, concomitant with a significant enrichment of genes related to vitamin B12 biosynthesis suggestive of association with diatom phycospheres. A closer examination of putative generalists and colonizers showed that putative generalists had persistently higher relative abundance throughout the blooms and thus produced more than 80% of Rhodobacterales transport proteins, suggesting rapid growth. In contrast, putative phycosphere colonizers exhibited large fluctuation in relative abundance across the different blooms and correlated strongly with particular diatom species that were dominant during the blooms each year. The defining feature of putative phycosphere colonizers is the presence of the tight adherence (tad) gene cluster, which is responsible for the assembly of adhesive pili that presumably enable attachment to diatom hosts. In addition, putative phycosphere colonizers possessed higher prevalence of secondary metabolite biosynthetic gene clusters, particularly homoserine lactones, which can regulate bacterial attachment through quorum sensing. Altogether, these findings suggest that while many members of Rhodobacterales are competitive during diatom blooms, only a subset form close associations with diatoms by colonizing their phycospheres.

The Influence of Genes on the "Killer Plasmid" of Dinoroseobacter shibae on Its Symbiosis With the Dinoflagellate Prorocentrum minimum.

The marine bacterium Dinoroseobacter shibae shows a Jekyll-and-Hyde behavior in co-culture with the dinoflagellate Prorocentrum minimum: In the initial symbiotic phase it provides the essential vitamins B12 (cobalamin) and B1 (thiamine) to the algae. In the later pathogenic phase it kills the dinoflagellate. The killing phenotype is determined by the 191 kb plasmid and can be conjugated into other Roseobacters. From a transposon-library of D. shibae we retrieved 28 mutants whose insertion sites were located on the 191 kb plasmid. We co-cultivated each of them with P. minimum in L1 medium lacking vitamin B12. With 20 mutant strains no algal growth beyond the axenic control lacking B12 occurred. Several of these genes were predicted to encode proteins from the type IV secretion system (T4SS). They are apparently essential for establishing the symbiosis. With five transposon mutant strains, the initial symbiotic phase was intact but the later pathogenic phase was lost in co-culture. In three of them the insertion sites were located in an operon predicted to encode genes for biotin (B7) uptake. Both P. minimum and D. shibae are auxotrophic for biotin. We hypothesize that the bacterium depletes the medium from biotin resulting in apoptosis of the dinoflagellate.

Diatom modulation of select bacteria through use of two unique secondary metabolites.

Unicellular eukaryotic phytoplankton, such as diatoms, rely on microbial communities for survival despite lacking specialized compartments to house microbiomes (e.g., animal gut). Microbial communities have been widely shown to benefit from diatom excretions that accumulate within the microenvironment surrounding phytoplankton cells, known as the phycosphere. However, mechanisms that enable diatoms and other unicellular eukaryotes to nurture specific microbiomes by fostering beneficial bacteria and repelling harmful ones are mostly unknown. We hypothesized that diatom exudates may tune microbial communities and employed an integrated multiomics approach using the ubiquitous diatom Asterionellopsis glacialis to reveal how it modulates its naturally associated bacteria. We show that A. glacialis reprograms its transcriptional and metabolic profiles in response to bacteria to secrete a suite of central metabolites and two unusual secondary metabolites, rosmarinic acid and azelaic acid. While central metabolites are utilized by potential bacterial symbionts and opportunists alike, rosmarinic acid promotes attachment of beneficial bacteria to the diatom and simultaneously suppresses the attachment of opportunists. Similarly, azelaic acid enhances growth of beneficial bacteria while simultaneously inhibiting growth of opportunistic ones. We further show that the bacterial response to azelaic acid is numerically rare but globally distributed in the world's oceans and taxonomically restricted to a handful of bacterial genera. Our results demonstrate the innate ability of an important unicellular eukaryotic group to modulate select bacteria in their microbial consortia, similar to higher eukaryotes, using unique secondary metabolites that regulate bacterial growth and behavior inversely across different bacterial populations.

Ecological drivers of bacterial community assembly in synthetic phycospheres.

In the nutrient-rich region surrounding marine phytoplankton cells, heterotrophic bacterioplankton transform a major fraction of recently fixed carbon through the uptake and catabolism of phytoplankton metabolites. We sought to understand the rules by which marine bacterial communities assemble in these nutrient-enhanced phycospheres, specifically addressing the role of host resources in driving community coalescence. Synthetic systems with varying combinations of known exometabolites of marine phytoplankton were inoculated with seawater bacterial assemblages, and communities were transferred daily to mimic the average duration of natural phycospheres. We found that bacterial community assembly was predictable from linear combinations of the taxa maintained on each individual metabolite in the mixture, weighted for the growth each supported. Deviations from this simple additive resource model were observed but also attributed to resource-based factors via enhanced bacterial growth when host metabolites were available concurrently. The ability of photosynthetic hosts to shape bacterial associates through excreted metabolites represents a mechanism by which microbiomes with beneficial effects on host growth could be recruited. In the surface ocean, resource-based assembly of host-associated communities may underpin the evolution and maintenance of microbial interactions and determine the fate of a substantial portion of Earth's primary production.

Bacterial Communities of Diatoms Display Strong Conservation Across Strains and Time.

Interactions between phytoplankton and bacteria play important roles in shaping the microenvironment surrounding these organisms and in turn influence global biogeochemical cycles. This microenvironment, known as the phycosphere, is presumed to shape the bacterial diversity around phytoplankton and thus stimulate a diverse array of interactions between both groups. Although many studies have attempted to characterize bacterial communities that associate and interact with phytoplankton, bias in bacterial cultivation and consistency and persistence of bacterial communities across phytoplankton isolates likely impede the understanding of these microbial associations. Here, we isolate four strains of the diatom Asterionellopsis glacialis and three strains of the diatom Nitzschia longissima and show through metabarcoding of the bacterial 16S rDNA gene that though each species possesses a unique bacterial community, the bacterial composition across strains from the same species are highly conserved at the genus level. Cultivation of all seven strains in the laboratory for longer than 1 year resulted in only small changes to the bacterial composition, suggesting that despite strong pressures from laboratory culturing conditions associations between these diatoms and their bacterial communities are robust. Specific operational taxonomic units (OTUs) belonging to the Roseobacter-clade appear to be conserved across all strains and time, suggesting their importance to diatoms. In addition, we isolate a range of cultivable bacteria from one of these cultures, A. glacialis strain A3, including several strains of Shimia marina and Nautella sp. that appear closely related to OTUs conserved across all strains and times. Coculturing of A3 with some of its cultivable bacteria as well as other diatom-associated bacteria shows a wide range of responses that include enhancing diatom growth. Cumulatively, these findings suggest that phytoplankton possess unique microbiomes that are consistent across strains and temporal scales.

Co-occurrence Analysis of Microbial Taxa in the Atlantic Ocean Reveals High Connectivity in the Free-Living Bacterioplankton.

We determined the taxonomic composition of the bacterioplankton of the epipelagic zone of the Atlantic Ocean along a latitudinal transect (51°S-47°N) using Illumina sequencing of the V5-V6 region of the 16S rRNA gene and inferred co-occurrence networks. Bacterioplankon community composition was distinct for Longhurstian provinces and water depth. Free-living microbial communities (between 0.22 and 3 µm) were dominated by highly abundant and ubiquitous taxa with streamlined genomes (e.g., SAR11, SAR86, OM1, Prochlorococcus) and could clearly be separated from particle-associated communities which were dominated by Bacteroidetes, Planktomycetes, Verrucomicrobia, and Roseobacters. From a total of 369 different communities we then inferred co-occurrence networks for each size fraction and depth layer of the plankton between bacteria and between bacteria and phototrophic micro-eukaryotes. The inferred networks showed a reduction of edges in the deepest layer of the photic zone. Networks comprised of free-living bacteria had a larger amount of connections per OTU when compared to the particle associated communities throughout the water column. Negative correlations accounted for roughly one third of the total edges in the free-living communities at all depths, while they decreased with depth in the particle associated communities where they amounted for roughly 10% of the total in the last part of the epipelagic zone. Co-occurrence networks of bacteria with phototrophic micro-eukaryotes were not taxon-specific, and dominated by mutual exclusion (~60%). The data show a high degree of specialization to micro-environments in the water column and highlight the importance of interdependencies particularly between free-living bacteria in the upper layers of the epipelagic zone.