Seasonal patterns in microbial carbon and iron transporter expression in the Southern Ocean.

BACKGROUND: Heterotrophic microbes in the Southern Ocean are challenged by the double constraint of low concentrations of organic carbon (C) and iron (Fe). These essential elements are tightly coupled in cellular processes; however, the prokaryotic requirements of C and Fe under varying environmental settings remain poorly studied. Here, we used a combination of metatranscriptomics and metaproteomics to identify prokaryotic membrane transporters for organic substrates and Fe in naturally iron-fertilized and high-nutrient, low-chlorophyll waters of the Southern Ocean during spring and late summer. RESULTS: Pronounced differences in membrane transporter profiles between seasons were observed at both sites, both at the transcript and protein level. When specific compound classes were considered, the two approaches revealed different patterns. At the transcript level, seasonal patterns were only observed for subsets of genes belonging to each transporter category. At the protein level, membrane transporters of organic compounds were relatively more abundant in spring as compared to summer, while the opposite pattern was observed for Fe transporters. These observations suggest an enhanced requirement for organic C in early spring and for Fe in late summer. Mapping transcripts and proteins to 50 metagenomic-assembled genomes revealed distinct taxon-specific seasonal differences pointing to potentially opportunistic clades, such as Pseudomonadales and Nitrincolaceae, and groups with a more restricted repertoire of expressed transporters, such as Alphaproteobacteria and Flavobacteriaceae. CONCLUSION: The combined investigations of C and Fe membrane transporters suggest seasonal changes in the microbial requirements of these elements under different productivity regimes. The taxon-specific acquisition strategies of different forms of C and Fe illustrate how diverse microbes could shape transcript and protein expression profiles at the community level at different seasons. Our results on the C- and Fe-related metabolic capabilities of microbial taxa provide new insights into their potential role in the cycling of C and Fe under varying nutrient regimes in the Southern Ocean. Video Abstract.

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.

Effects of Phosphorus Limitation on the Bioavailability of DOM Released by Marine Heterotrophic Prokaryotes.

Heterotrophic prokaryotes (HP) contribute largely to dissolved organic matter (DOM) processing in the ocean, but they also release diverse organic substances. The bioavailability of DOM released by HP under varying environmental conditions has not been fully elucidated. In this study, we investigated the bioavailability of DOM released by a single bacterial strain (Sphingopyxis alaskensis) and 2 natural HP communities grown under P-replete and P-limited conditions. The released DOM (HP-DOM) was used as a substrate for natural HP communities at a coastal site in the Northwestern Mediterranean Sea. We followed changes in HP growth, enzymatic activity, diversity, and community composition together with the consumption of HP-DOM fluorescence (FDOM). HP-DOM produced under P-replete and P-limited conditions promoted significant growth in all incubations. No clear differences in HP-DOM lability released under P-repletion and P-limitation were evidenced based on the HP growth, and P-limitation was not demonstrated to decrease HP-DOM lability. However, HP-DOM supported the growth of diverse HP communities, and P-driven differences in HP-DOM quality were selected for different indicator taxa in the degrading communities. The humic-like fluorescence, commonly considered recalcitrant, was consumed during the incubations when this peak was initially dominating the FDOM pool, and this consumption coincided with higher alkaline phosphatase activity. Taken together, our findings emphasize that HP-DOM lability is dependent on both DOM quality, which is shaped by P availability, and the composition of the consumer community.

Functional responses of key marine bacteria to environmental change - toward genetic counselling for coastal waters.

Coastal ecosystems deteriorate globally due to human-induced stress factors, like nutrient loading and pollution. Bacteria are critical to marine ecosystems, e.g., by regulating nutrient cycles, synthesizing vitamins, or degrading pollutants, thereby providing essential ecosystem services ultimately affecting economic activities. Yet, until now bacteria are overlooked both as mediators and indicators of ecosystem health, mainly due to methodological limitations in assessing bacterial ecosystem functions. However, these limitations are largely overcome by the advances in molecular biology and bioinformatics methods for characterizing the genetics that underlie functional traits of key bacterial populations - "key" in providing important ecosystem services, being abundant, or by possessing high metabolic rates. It is therefore timely to analyze and define the functional responses of bacteria to human-induced effects on coastal ecosystem health. We posit that categorizing the responses of key marine bacterial populations to changes in environmental conditions through modern microbial oceanography methods will allow establishing the nascent field of genetic counselling for our coastal waters. This requires systematic field studies of linkages between functional traits of key bacterial populations and their ecosystem functions in coastal seas, complemented with systematic experimental analyses of the responses to different stressors. Research and training in environmental management along with dissemination of results and dialogue with societal actors are equally important to ensure the role of bacteria is understood as fundamentally important for coastal ecosystems. Using the responses of microorganisms as a tool to develop genetic counselling for coastal ecosystems can ultimately allow for integrating bacteria as indicators of environmental change.

SAR11 clade microdiversity and activity during the early spring blooms off Kerguelen Island, Southern Ocean.

The ecology of the SAR11 clade, the most abundant bacterial group in the ocean, has been intensively studied in temperate and tropical regions, but its distribution remains largely unexplored in the Southern Ocean. Through amplicon sequencing of the 16S rRNA gene, we assessed the contribution of the SAR11 clade to bacterial community composition in the naturally iron fertilized region off Kerguelen Island. We investigated the upper 300 m at seven sites located in early spring phytoplankton blooms and at one high-nutrient low-chlorophyll site. Despite pronounced vertical patterns of the bacterioplankton assemblages, the SAR11 clade had high relative abundances at all depths and sites, averaging 40% (±15%) of the total community relative abundance. Micro-autoradiography combined with CARD-FISH further revealed that the clade had an overall stable contribution (45%-60% in surface waters) to bacterial biomass production (determined by 3 H-leucine incorporation) during different early bloom stages. The spatio-temporal partitioning of some of the SAR11 subclades suggests a niche specificity and periodic selection of different subclades in response to the fluctuating extreme conditions of the Southern Ocean. These observations improve our understanding of the ecology of the SAR11 clade and its implications in biogeochemical cycles in the rapidly changing Southern Ocean.

Microbial iron and carbon metabolism as revealed by taxonomy-specific functional diversity in the Southern Ocean.

Marine microbes are major drivers of all elemental cycles. The processing of organic carbon by heterotrophic prokaryotes is tightly coupled to the availability of the trace element iron in large regions of the Southern Ocean. However, the functional diversity in iron and carbon metabolism within diverse communities remains a major unresolved issue. Using novel Southern Ocean meta-omics resources including 133 metagenome-assembled genomes (MAGs), we show a mosaic of taxonomy-specific ecological strategies in naturally iron-fertilized and high nutrient low chlorophyll (HNLC) waters. Taxonomic profiling revealed apparent community shifts across contrasting nutrient regimes. Community-level and genome-resolved metatranscriptomics evidenced a moderate association between taxonomic affiliations and iron and carbon-related functional roles. Diverse ecological strategies emerged when considering the central metabolic pathways of individual MAGs. Closely related lineages appear to adapt to distinct ecological niches, based on their distribution and gene regulation patterns. Our in-depth observations emphasize the complex interplay between the genetic repertoire of individual taxa and their environment and how this shapes prokaryotic responses to iron and organic carbon availability 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.

Dissolved organic matter released by two marine heterotrophic bacterial strains and its bioavailability for natural prokaryotic communities.

Marine heterotrophic prokaryotes (HP) play a key role in organic matter processing in the ocean; however, the view of HP as dissolved organic matter (DOM) sources remains underexplored. In this study, we quantified and optically characterized the DOM produced by two single marine bacterial strains. We then tested the availability of these DOM sources to in situ Mediterranean Sea HP communities. Two bacterial strains were used: Photobacterium angustum (a copiotrophic gammaproteobacterium) and Sphingopyxis alaskensis (an oligotrophic alphaproteobacterium). When cultivated on glucose as the sole carbon source, the two strains released from 7% to 23% of initial glucose as bacterial derived DOM (B-DOM), the quality of which (as enrichment in humic or protein-like substances) differed between strains. B-DOM induced significant growth and carbon consumption of natural HP communities, suggesting that it was partly labile. However, B-DOM consistently promoted lower prokaryotic growth efficiencies than in situ DOM. In addition, B-DOM changed HP exoenzymatic activities, enhancing aminopeptidase activity when degrading P. angustum DOM, and alkaline phosphatase activity when using S. alaskensis DOM, and promoted differences in HP diversity and composition. DOM produced by HP affects in situ prokaryotic metabolism and diversity, thus changing the pathways for DOM cycling (e.g. respiration over biomass production) in the ocean.

Use of organic exudates from two polar diatoms by bacterial isolates from the Arctic Ocean.

Global warming affects primary producers in the Arctic, with potential consequences for the bacterial community composition through the consumption of microalgae-derived dissolved organic matter (DOM). To determine the degree of specificity in the use of an exudate by bacterial taxa, we used simple microalgae-bacteria model systems. We isolated 92 bacterial strains from the sea ice bottom and the water column in spring-summer in the Baffin Bay (Arctic Ocean). The isolates were grouped into 42 species belonging to Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Forty strains were tested for their capacity to grow on the exudate from two Arctic diatoms. Most of the strains tested (78%) were able to grow on the exudate from the pelagic diatom Chaetoceros neogracilis, and 33% were able to use the exudate from the sea ice diatom Fragilariopsis cylindrus. 17.5% of the strains were not able to grow with any exudate, while 27.5% of the strains were able to use both types of exudates. All strains belonging to Flavobacteriia (n = 10) were able to use the DOM provided by C. neogracilis, and this exudate sustained a growth capacity of up to 100 times higher than diluted Marine Broth medium, of two Pseudomonas sp. strains and one Sulfitobacter strain. The variable bioavailability of exudates to bacterial strains highlights the potential role of microalgae in shaping the bacterial community composition. This article is part of the theme issue 'The changing Arctic Ocean: consequences for biological communities, biogeochemical processes and ecosystem functioning'.

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.

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.

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.

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.

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.

Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean.

In the naturally iron-fertilized surface waters of the northern Kerguelen Plateau region, the early spring diatom community composition and contribution to plankton carbon biomass were investigated and compared with the high nutrient, low chlorophyll (HNLC) surrounding waters. The large iron-induced blooms were dominated by small diatom species belonging to the genera Chaetoceros (Hyalochaete) and Thalassiosira, which rapidly responded to the onset of favorable light-conditions in the meander of the Polar Front. In comparison, the iron-limited HNLC area was typically characterized by autotrophic nanoeukaryote-dominated communities and by larger and more heavily silicified diatom species (e.g. Fragilariopsis spp.). Our results support the hypothesis that diatoms are valuable vectors of carbon export to depth in naturally iron-fertilized systems of the Southern Ocean. Furthermore, our results corroborate observations of the exported diatom assemblage from a sediment trap deployed in the iron-fertilized area, whereby the dominant Chaetoceros (Hyalochaete) cells were less efficiently exported than the less abundant, yet heavily silicified, cells of Thalassionema nitzschioides and Fragilariopsis kerguelensis Our observations emphasize the strong influence of species-specific diatom cell properties combined with trophic interactions on matter export efficiency, and illustrate the tight link between the specific composition of phytoplankton communities and the biogeochemical properties characterizing the study area.

Deciphering ocean carbon in a changing world.

Dissolved organic matter (DOM) in the oceans is one of the largest pools of reduced carbon on Earth, comparable in size to the atmospheric CO2 reservoir. A vast number of compounds are present in DOM, and they play important roles in all major element cycles, contribute to the storage of atmospheric CO2 in the ocean, support marine ecosystems, and facilitate interactions between organisms. At the heart of the DOM cycle lie molecular-level relationships between the individual compounds in DOM and the members of the ocean microbiome that produce and consume them. In the past, these connections have eluded clear definition because of the sheer numerical complexity of both DOM molecules and microorganisms. Emerging tools in analytical chemistry, microbiology, and informatics are breaking down the barriers to a fuller appreciation of these connections. Here we highlight questions being addressed using recent methodological and technological developments in those fields and consider how these advances are transforming our understanding of some of the most important reactions of the marine carbon cycle.

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.

Phenotypic plasticity in heterotrophic marine microbial communities in continuous cultures.

Phenotypic plasticity (PP) is the development of alternate phenotypes of a given taxon as an adaptation to environmental conditions. Methodological limitations have restricted the quantification of PP to the measurement of a few traits in single organisms. We used metatranscriptomic libraries to overcome these challenges and estimate PP using the expressed genes of multiple heterotrophic organisms as a proxy for traits in a microbial community. The metatranscriptomes captured the expression response of natural marine bacterial communities grown on differing carbon resource regimes in continuous cultures. We found that taxa with different magnitudes of PP coexisted in the same cultures, and that members of the order Rhodobacterales had the highest levels of PP. In agreement with previous studies, our results suggest that continuous culturing may have specifically selected for taxa featuring a rather high range of PP. On average, PP and abundance changes within a taxon contributed equally to the organism's change in functional gene abundance, implying that both PP and abundance mediated observed differences in community function. However, not all functional changes due to PP were directly reflected in the bulk community functional response: gene expression changes in individual taxa due to PP were partly masked by counterbalanced expression of the same gene in other taxa. This observation demonstrates that PP had a stabilizing effect on a community's functional response to environmental change.