Seasonal shifts in Fe-acquisition strategies in Southern Ocean microbial communities revealed by metagenomics and autonomous sampling.

Iron (Fe) governs the cycling of organic carbon in large parts of the Southern Ocean. The strategies of diverse microbes to acquire the different chemical forms of Fe under seasonally changing organic carbon regimes remain, however, poorly understood. Here, we report high-resolution seasonal metagenomic observations from the region off Kerguelen Island (Indian Sector of the Southern Ocean) where natural Fe-fertilization induces consecutive spring and summer phytoplankton blooms. Our data illustrate pronounced, but distinct seasonal patterns in the abundance of genes implicated in the transport of different forms of Fe and organic substrates, of siderophore biosynthesis and carbohydrate-active enzymes. The seasonal dynamics suggest a temporal decoupling in the prokaryotic requirements of Fe and organic carbon during the spring phytoplankton bloom and a concerted access to these resources after the summer bloom. Taxonomic assignments revealed differences in the prokaryotic groups harbouring genes of a given Fe-related category and pronounced seasonal successions were observed. Using MAGs we could decipher the respective Fe- and organic substrate-related genes of individual taxa assigned to abundant groups. The ecological strategies related to Fe-acquisition provide insights on how this element could shape microbial community composition with potential implications on organic matter transformations in the Southern Ocean.

Resource Colimitation Drives Competition Between Phytoplankton and Bacteria in the Southern Ocean.

Across the Southern Ocean, phytoplankton growth is governed by iron and light, while bacterial growth is regulated by iron and labile dissolved organic carbon (LDOC). We use a mechanistic model to examine how competition for iron between phytoplankton and bacteria responds to changes in iron, light, and LDOC. Consistent with experimental evidence, increasing iron and light encourages phytoplankton dominance, while increasing LDOC and decreasing light favors bacterial dominance. Under elevated LDOC, bacteria can outcompete phytoplankton for iron, most easily under lower iron. Simulations reveal that bacteria are major iron consumers and suggest that luxury storage plays a key role in competitive iron uptake. Under seasonal conditions typical of the Southern Ocean, sources of LDOC besides phytoplankton exudation modulate the strength of competitive interactions. Continued investigations on the competitive fitness of bacteria in driving changes in primary production in iron-limited systems will be invaluable in refining these results.

Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler.

The Southern Ocean remains one of the least explored marine environments. The investigation of temporal microbial dynamics has thus far been hampered by the limited access to this remote ocean. We present here high-resolution seasonal observations of the prokaryotic community composition during phytoplankton blooms induced by natural iron fertilization. A total of 18 seawater samples were collected by a moored remote autonomous sampler over 4 months at 5-11 day intervals in offshore surface waters (central Kerguelen Plateau). Illumina sequencing of the 16S rRNA gene revealed that among the most abundant amplicon sequence variants, SAR92 and Aurantivirga were the first bloom responders, Pseudomonadaceae, Nitrincolaceae and Polaribacter had successive peaks during the spring bloom decline, and Amylibacter increased in relative abundance later in the season. SAR11 and SUP05 were abundant prior to and after the blooms. Using network analysis, we identified two groups of diatoms representative of the spring and summer bloom that had opposite correlation patterns with prokaryotic taxa. Our study provides the first seasonal picture of microbial community dynamics in the open Southern Ocean and thereby offers biological insights to the cycling of carbon and iron, and to an important puzzling issue that is the modest nitrate decrease associated to iron fertilization.

Diatoms shape the biogeography of heterotrophic prokaryotes in early spring in the Southern Ocean.

The interplay among microorganisms profoundly impacts biogeochemical cycles in the ocean. Culture-based work has illustrated the diversity of diatom-prokaryote interactions, but the question of whether these associations can affect the spatial distribution of microbial communities is open. Here, we investigated the relationship between assemblages of diatoms and of heterotrophic prokaryotes in surface waters of the Indian sector of the Southern Ocean in early spring. The community composition of diatoms and that of total and active prokaryotes were different among the major ocean zones investigated. We found significant relationships between compositional changes of diatoms and of prokaryotes. In contrast, spatial changes in the prokaryotic community composition were not related to geographic distance and to environmental parameters when the effect of diatoms was accounted for. Diatoms explained 30% of the variance in both the total and the active prokaryotic community composition in early spring in the Southern Ocean. Using co-occurrence analyses, we identified a large number of highly significant correlations between abundant diatom species and prokaryotic taxa. Our results show that key diatom species of the Southern Ocean are each associated with a distinct prokaryotic community, suggesting that diatom assemblages contribute to shaping the habitat type for heterotrophic prokaryotes.

Microbial iron metabolism as revealed by gene expression profiles in contrasted Southern Ocean regimes.

Iron (Fe) is a limiting nutrient in large regions of the ocean, but the strategies of prokaryotes to cope with this micronutrient are poorly known. Using a gene-specific approach from metatranscriptomics data, we investigated seven Fe-related metabolic pathways in microbial communities from high nutrient low chlorophyll and naturally Fe-fertilized waters in the Southern Ocean. We observed major differences in the contribution of prokaryotic groups at different taxonomic levels to transcripts encoding Fe-uptake mechanisms, intracellular Fe storage and replacement and Fe-related pathways in the tricarboxylic acid (TCA) cycle. The composition of the prokaryotic communities contributing to the transcripts of a given Fe-related pathway was overall independent of the in situ Fe supply, indicating that microbial taxa utilize distinct Fe-related metabolic processes. Only a few prokaryotic groups contributed to the transcripts of more than one Fe-uptake mechanism, suggesting limited metabolic versatility. Taxa-specific expression of individual genes varied among prokaryotic groups and was substantially higher for all inspected genes in Fe-limited as compared to naturally fertilized waters, indicating the link between transcriptional state and Fe regime. Different metabolic strategies regarding low Fe concentrations in the Southern Ocean are discussed for two abundant prokaryotic groups, Pelagibacteraceae and Flavobacteriaceae.

Central role for ferritin in the day/night regulation of iron homeostasis in marine phytoplankton.

In large regions of the open ocean, iron is a limiting resource for phytoplankton. The reduction of iron quota and the recycling of internal iron pools are among the diverse strategies that phytoplankton have evolved to allow them to grow under chronically low ambient iron levels. Phytoplankton species also have evolved strategies to cope with sporadic iron supply such as long-term storage of iron in ferritin. In the picophytoplanktonic species Ostreococcus we report evidence from observations both in the field and in laboratory cultures that ferritin and the main iron-binding proteins involved in photosynthesis and nitrate assimilation pathways show opposite diurnal expression patterns, with ferritin being maximally expressed during the night. Biochemical and physiological experiments using a ferritin knock-out line subsequently revealed that this protein plays a central role in the diel regulation of iron uptake and recycling and that this regulation of iron homeostasis is essential for cell survival under iron limitation.

Efficient gene targeting and removal of foreign DNA by homologous recombination in the picoeukaryote Ostreococcus.

With fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant-specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome.

Fronts divide diazotroph communities in the Southern Indian Ocean.

Dinitrogen (N2) fixation represents a key source of reactive nitrogen in marine ecosystems. While the process has been rather well-explored in low latitudes of the Atlantic and Pacific Oceans, other higher latitude regions and particularly the Indian Ocean have been chronically overlooked. Here, we characterize N2 fixation and diazotroph community composition across nutrient and trace metals gradients spanning the multifrontal system separating the oligotrophic waters of the Indian Ocean subtropical gyre from the high nutrient low chlorophyll waters of the Southern Ocean. We found a sharp contrasting distribution of diazotroph groups across the frontal system. Notably, cyanobacterial diazotrophs dominated north of fronts, driving high N2 fixation rates (up to 13.96 nmol N l-1 d-1) with notable peaks near the South African coast. South of the fronts non-cyanobacterial diazotrophs prevailed without significant N2 fixation activity being detected. Our results provide new crucial insights into high latitude diazotrophy in the Indian Ocean, which should contribute to improved climate model parameterization and enhanced constraints on global net primary productivity projections.

Effect of natural iron fertilization on carbon sequestration in the Southern Ocean.

The availability of iron limits primary productivity and the associated uptake of carbon over large areas of the ocean. Iron thus plays an important role in the carbon cycle, and changes in its supply to the surface ocean may have had a significant effect on atmospheric carbon dioxide concentrations over glacial-interglacial cycles. To date, the role of iron in carbon cycling has largely been assessed using short-term iron-addition experiments. It is difficult, however, to reliably assess the magnitude of carbon export to the ocean interior using such methods, and the short observational periods preclude extrapolation of the results to longer timescales. Here we report observations of a phytoplankton bloom induced by natural iron fertilization--an approach that offers the opportunity to overcome some of the limitations of short-term experiments. We found that a large phytoplankton bloom over the Kerguelen plateau in the Southern Ocean was sustained by the supply of iron and major nutrients to surface waters from iron-rich deep water below. The efficiency of fertilization, defined as the ratio of the carbon export to the amount of iron supplied, was at least ten times higher than previous estimates from short-term blooms induced by iron-addition experiments. This result sheds new light on the effect of long-term fertilization by iron and macronutrients on carbon sequestration, suggesting that changes in iron supply from below--as invoked in some palaeoclimatic and future climate change scenarios--may have a more significant effect on atmospheric carbon dioxide concentrations than previously thought.

The interplay between iron limitation, light and carbon in the proteorhodopsin-containing Photobacterium angustum S14.

Iron (Fe) limitation is known to affect heterotrophic bacteria within the respiratory electron transport chain, therefore strongly impacting the overall intracellular energy production. We investigated whether the gene expression pattern of the light-sensitive proton pump, proteorhodopsin (PR), is influenced by varying light, carbon and Fe concentrations in the marine bacterium Photobacterium angustum S14 and whether PR can alleviate the physiological processes associated with Fe starvation. Our results show that the gene expression of PR increases as cells enter the stationary phase, irrespective of Fe-replete or Fe-limiting conditions. This upregulation is coupled to a reduction in cell size, indicating that PR gene regulation is associated with a specific starvation-stress response. We provide experimental evidence that PR gene expression does not result in an increased growth rate, cell abundance, enhanced survival or ATP concentration within the cell in either Fe-replete or Fe-limiting conditions. However, independent of PR gene expression, the presence of light did influence bacterial growth rates and maximum cell abundances under varying Fe regimes. Our observations support previous results indicating that PR phototrophy seems to play an important role within the stationary phase for several members of the Vibrionaceae family, but that the exact role of PR in Fe limitation remains to be further explored.

Hydrothermal vents trigger massive phytoplankton blooms in the Southern Ocean.

Hydrothermal activity is significant in regulating the dynamics of trace elements in the ocean. Biogeochemical models suggest that hydrothermal iron might play an important role in the iron-depleted Southern Ocean by enhancing the biological pump. However, the ability of this mechanism to affect large-scale biogeochemistry and the pathways by which hydrothermal iron reach the surface layer have not been observationally constrained. Here we present the first observational evidence of upwelled hydrothermally influenced deep waters stimulating massive phytoplankton blooms in the Southern Ocean. Captured by profiling floats, two blooms were observed in the vicinity of the Antarctic Circumpolar Current, downstream of active hydrothermal vents along the Southwest Indian Ridge. These hotspots of biological activity are supported by mixing of hydrothermally sourced iron stimulated by flow-topography interactions. Such findings reveal the important role of hydrothermal vents on surface biogeochemistry, potentially fueling local hotspot sinks for atmospheric CO2 by enhancing the biological pump.

Major changes in the composition of a Southern Ocean bacterial community in response to diatom-derived dissolved organic matter.

In the Southern Ocean, natural iron fertilization in the wake of islands leads to annually occurring spring phytoplankton blooms associated with enhanced heterotrophic activity through the release of labile dissolved organic matter (DOM). The aim of this study was to investigate experimentally how diatom-derived DOM affects the composition of Southern Ocean winter water bacterial communities and to identify the most responsive taxa. A bacterial community collected in the naturally iron-fertilized region off Kerguelen Island (KEOPS2 October-November 2011) was grown onboard in continuous cultures, on winter water alone or amended with diatom-derived DOM supplied at identical DOC concentrations. 454 sequencing of 16S amplicons revealed that the two DOM sources sustained strikingly different bacterial communities, with higher relative abundances of Sulfitobacter, Colwellia and Methylophaga operational taxonomic units (OTUs) and lower relative abundances of Polaribacter, Marinobacter, NAC11-7 and SAR11 OTUs in diatom-DOM compared to winter water conditions. Using a modeling approach, we obtained growth rates for phylogenetically diverse taxa varying between 0.12 and 0.49 d-1 under carbon-limited conditions. Our results identify diatom DOM as a key factor shaping Southern Ocean winter water bacterial communities and suggest a role for niche partitioning and microbial interactions in organic matter utilization.

Acclimation of a low iron adapted Ostreococcus strain to iron limitation through cell biomass lowering.

Iron is an essential micronutrient involved in many biological processes and is often limiting for primary production in large regions of the World Ocean. Metagenomic and physiological studies have identified clades or ecotypes of marine phytoplankton that are specialized in iron depleted ecological niches. Although less studied, eukaryotic picophytoplankton does contribute significantly to primary production and carbon transfer to higher trophic levels. In particular, metagenomic studies of the green picoalga Ostreococcus have revealed the occurrence of two main clades distributed along coast-offshore gradients, suggesting niche partitioning in different nutrient regimes. Here, we present a study of the response to iron limitation of four Ostreococcus strains isolated from contrasted environments. Whereas the strains isolated in nutrient-rich waters showed high iron requirements, the oceanic strains could cope with lower iron concentrations. The RCC802 strain, in particular, was able to maintain high growth rate at low iron levels. Together physiological and transcriptomic data indicate that the competitiveness of RCC802 under iron limitation is related to a lowering of iron needs though a reduction of the photosynthetic machinery and of protein content, rather than to cell size reduction. Our results overall suggest that iron is one of the factors driving the differentiation of physiologically specialized Ostreococcus strains in the ocean.

Shifts in bacterial community composition associated with increased carbon cycling in a mosaic of phytoplankton blooms.

Marine microbes have a pivotal role in the marine biogeochemical cycle of carbon, because they regulate the turnover of dissolved organic matter (DOM), one of the largest carbon reservoirs on Earth. Microbial communities and DOM are both highly diverse components of the ocean system, yet the role of microbial diversity for carbon processing remains thus far poorly understood. We report here results from an exploration of a mosaic of phytoplankton blooms induced by large-scale natural iron fertilization in the Southern Ocean. We show that in this unique ecosystem where concentrations of DOM are lowest in the global ocean, a patchwork of blooms is associated with diverse and distinct bacterial communities. By using on-board continuous cultures, we identify preferences in the degradation of DOM of different reactivity for taxa associated with contrasting blooms. We used the spatial and temporal variability provided by this natural laboratory to demonstrate that the magnitude of bacterial production is linked to the extent of compositional changes. Our results suggest that partitioning of the DOM resource could be a mechanism that structures bacterial communities with a positive feedback on carbon cycling. Our study, focused on bacterial carbon processing, highlights the potential role of diversity as a driving force for the cycling of biogeochemical elements.

The transcriptional regulation of the glyoxylate cycle in SAR11 in response to iron fertilization in the Southern Ocean.

The tricarboxylic acid (TCA) cycle is a central metabolic pathway that is present in all aerobic organisms and initiates the respiration of organic material. The glyoxylate cycle is a variation of the TCA cycle, where organic material is recycled for subsequent assimilation into cell material instead of being released as carbon dioxide. Despite the importance for the fate of organic matter, the environmental factors that induce the glyoxylate cycle in microbial communities remain poorly understood. In this study, we assessed the expression of isocitrate lyase, the enzyme that induces the switch to the glyoxylate cycle, of the ubiquitous SAR11 clade in response to natural iron fertilization in the Southern Ocean. The cell-specific transcriptional regulation of the glyoxylate cycle, as determined by the ratio between copy numbers of isocitrate lyase gene transcripts and isocitrate genes, was consistently lower in iron fertilized than in high-nutrient, low chlorophyll waters (by 2.4- to 16.5-fold). SAR11 cell-specific isocitrate lyase gene transcription was negatively correlated to chlorophyll a, and bulk bacterial heterotrophic metabolism. We conclude that the glyoxylate cycle is a metabolic strategy for SAR11 that is highly sensitive to the degree of iron and carbon limitation in the marine environment.

A method for the use of the radiotracer 55Fe for microautoradiography and CARD-FISH of natural bacterial communities.

Heterotrophic bacteria are key players in the biogeochemical cycle of iron (Fe) in the ocean, but the capability of different bacterial groups to access this micronutrient is ignored thus far. The aim of our study was to develop a protocol for the combined application of microautoradiography (MICRO) and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) using the radioisotope 55Fe. Among the different washing solutions tested, Ti-citrate-EDTA was the most efficient for the removal of extracellular 55Fe providing sufficiently low background values. We further demonstrate that the washing of cells with Ti-citrate-EDTA and the fixation with paraformaldehyde or formaldehyde do not induce leakage of intracellular 55Fe. Incubating natural bacterial communities collected from contrasting environments, the NW Mediterranean Sea and the Southern Ocean, with 55Fe revealed that 3-29% of bacterial cells were associated with silver grains. Combining microautoradiography with CARD-FISH, we demonstrate that the contribution of different bacterial groups to total 55Fe-incorporating cells was overall reflected by their relative contribution to abundance. An exception to this pattern was the proportionally higher contribution of Gammaproteobacteria, SAR86 and Alteromonas. Our study demonstrates the feasibility of MICRO-CARD-FISH using the radiotracer 55Fe and provides the first description of marine bacterial assemblages actively incorporating Fe.

Analysis of the global ocean sampling (GOS) project for trends in iron uptake by surface ocean microbes.

Microbial metagenomes are DNA samples of the most abundant, and therefore most successful organisms at the sampling time and location for a given cell size range. The study of microbial communities via their DNA content has revolutionized our understanding of microbial ecology and evolution. Iron availability is a critical resource that limits microbial communities' growth in many oceanic areas. Here, we built a database of 2319 sequences, corresponding to 140 gene families of iron metabolism with a large phylogenetic spread, to explore the microbial strategies of iron acquisition in the ocean's bacterial community. We estimate iron metabolism strategies from metagenome gene content and investigate whether their prevalence varies with dissolved iron concentrations obtained from a biogeochemical model. We show significant quantitative and qualitative variations in iron metabolism pathways, with a higher proportion of iron metabolism genes in low iron environments. We found a striking difference between coastal and open ocean sites regarding Fe(2+) versus Fe(3+) uptake gene prevalence. We also show that non-specific siderophore uptake increases in low iron open ocean environments, suggesting bacteria may acquire iron from natural siderophore-like organic complexes. Despite the lack of knowledge of iron uptake mechanisms in most marine microorganisms, our approach provides insights into how the iron metabolic pathways of microbial communities may vary with seawater iron concentrations.

Determination of iron-porphyrin-like complexes at nanomolar levels in seawater.

A new method for the non-specific determination of iron-porphyrin-like complexes in natural waters has been developed. It is based on the chemiluminescent oxidation of the luminol in the presence of dioxygen (O2) at pH 13. The method has been implemented in a FIA manifold that allowed the direct injection of seawater. The limit of detection is 0.11 nM of equivalent hemin (Fe-protoporphyrin IX). Fe2+, Fe3+, H2O2, siderophore (deferoxamin mesylate), humic acid and phytic acid did not interfere when they were present at the concentrations expected in seawater. Metal free porphyrin and Mg, Cu, Co porphyrin complexes did not induce a significant chemiluminescent signal. Poisoned unfiltered samples could be stored for several weeks before analyses. The new method was successfully applied to the determination of the Fe-porphyrin complexes contained in cultured phytoplankton and in natural samples.