Communal metabolism by Methylococcaceae and Methylophilaceae is driving rapid aerobic methane oxidation in sediments of a shallow seep near Elba, Italy.

The release of abiotic methane from marine seeps into the atmosphere is a major source of this potent greenhouse gas. Methanotrophic microorganisms in methane seeps use methane as carbon and energy source, thus significantly mitigating global methane emissions. Here, we investigated microbial methane oxidation at the sediment-water interface of a shallow marine methane seep. Metagenomics and metaproteomics, combined with 13 C-methane stable isotope probing, demonstrated that various members of the gammaproteobacterial family Methylococcaceae were the key players for methane oxidation, catalysing the first reaction step to methanol. We observed a transfer of carbon to methanol-oxidizing methylotrophs of the betaproteobacterial family Methylophilaceae, suggesting an interaction between methanotrophic and methylotrophic microorganisms that allowed for rapid methane oxidation. From our microcosms, we estimated methane oxidation rates of up to 871 nmol of methane per gram sediment per day. This implies that more than 50% of methane at the seep is removed by microbial oxidation at the sediment-water interface, based on previously reported in situ methane fluxes. The organic carbon produced was further assimilated by different heterotrophic microbes, demonstrating that the methane-oxidizing community supported a complex trophic network. Our results provide valuable eco-physiological insights into this specialized microbial community performing an ecosystem function of global relevance.

Comparative Genomics and Mutational Analysis Reveals a Novel XoxF-Utilizing Methylotroph in the Roseobacter Group Isolated From the Marine Environment.

The Roseobacter group comprises a significant group of marine bacteria which are involved in global carbon and sulfur cycles. Some members are methylotrophs, using one-carbon compounds as a carbon and energy source. It has recently been shown that methylotrophs generally require a rare earth element when using the methanol dehydrogenase enzyme XoxF for growth on methanol. Addition of lanthanum to methanol enrichments of coastal seawater facilitated the isolation of a novel methylotroph in the Roseobacter group: Marinibacterium anthonyi strain La 6. Mutation of xoxF5 revealed the essential nature of this gene during growth on methanol and ethanol. Physiological characterization demonstrated the metabolic versatility of this strain. Genome sequencing revealed that strain La 6 has the largest genome of all Roseobacter group members sequenced to date, at 7.18 Mbp. Multilocus sequence analysis (MLSA) showed that whilst it displays the highest core gene sequence similarity with subgroup 1 of the Roseobacter group, it shares very little of its pangenome, suggesting unique genetic adaptations. This research revealed that the addition of lanthanides to isolation procedures was key to cultivating novel XoxF-utilizing methylotrophs from the marine environment, whilst genome sequencing and MLSA provided insights into their potential genetic adaptations and relationship to the wider community.

Methylamine as a nitrogen source for microorganisms from a coastal marine environment.

Nitrogen is a key limiting resource for biomass production in the marine environment. Methylated amines, released from the degradation of osmolytes, could provide a nitrogen source for marine microbes. Thus far, studies in aquatic habitats on the utilization of methylamine, the simplest methylated amine, have mainly focussed on the fate of the carbon from this compound. Various groups of methylotrophs, microorganisms that can grow on one-carbon compounds, use methylamine as a carbon source. Non-methylotrophic microorganisms may also utilize methylamine as a nitrogen source, but little is known about their diversity, especially in the marine environment. In this proof-of-concept study, stable isotope probing (SIP) was used to identify microorganisms from a coastal environment that assimilate nitrogen from methylamine. SIP experiments using 15 N methylamine combined with metagenomics and metaproteomics facilitated identification of active methylamine-utilizing Alpha- and Gammaproteobacteria. The draft genomes of two methylamine utilizers were obtained and their metabolism with respect to methylamine was examined. Both bacteria identified in these SIP experiments used the γ-glutamyl-methylamide pathway, found in both methylotrophs and non-methylotrophs, to metabolize methylamine. The utilization of 15 N methylamine also led to the release of 15 N ammonium that was used as nitrogen source by other microorganisms not directly using methylamine.

Regrowth in ship's ballast water tanks: Think again!

With the imminent ratification of the International Maritime Organisation's Ballast Water Management Convention, ship owners and operators will have to choose among a myriad of different Ballast Water Treatment Systems (BWTS) and technologies to comply with established discharge standards. However, it has come to our attention that decision-makers seem to be unaware of the problem of regrowth occurring in ballast water tanks after treatment. Furthermore, the information available on the subject in the literature is surprisingly and unfortunately very limited. Herein we summarise previous research findings that suggest that regrowth of bacteria and phytoplankton could occur 18h to 7days and 4 to 20days after treatment, respectively. By highlighting the problem of regrowth, we would like to encourage scientists and engineers to further investigate this issue and to urge ship owners and ship operators to inform themselves on the risks of regrowth associated with the implementation of different BWTS.

Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.

Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.

Dominant oceanic bacteria secure phosphate using a large extracellular buffer.

The ubiquitous SAR11 and Prochlorococcus bacteria manage to maintain a sufficient supply of phosphate in phosphate-poor surface waters of the North Atlantic subtropical gyre. Furthermore, it seems that their phosphate uptake may counter-intuitively be lower in more productive tropical waters, as if their cellular demand for phosphate decreases there. By flow sorting (33)P-phosphate-pulsed (32)P-phosphate-chased cells, we demonstrate that both Prochlorococcus and SAR11 cells exploit an extracellular buffer of labile phosphate up to 5-40 times larger than the amount of phosphate required to replicate their chromosomes. Mathematical modelling is shown to support this conclusion. The fuller the buffer the slower the cellular uptake of phosphate, to the point that in phosphate-replete tropical waters, cells can saturate their buffer and their phosphate uptake becomes marginal. Hence, buffer stocking is a generic, growth-securing adaptation for SAR11 and Prochlorococcus bacteria, which lack internal reserves to reduce their dependency on bioavailable ambient phosphate.

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph.

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined deoxyribonucleic acid-stable isotope probing (DNA-SIP) with metagenomics and metaproteomics to characterize an uncultivated marine methylotroph that actively incorporated carbon from (13) C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which the culture-independent techniques of DNA-SIP and protein-SIP have been used to characterize the metabolism of a naturally occurring Methylophaga-like bacterium in the marine environment (i.e. Methylophaga thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment.

XoxF encoding an alternative methanol dehydrogenase is widespread in coastal marine environments.

The xoxF gene, encoding a pyrroloquinoline quinone-dependent methanol dehydrogenase, is found in all known proteobacterial methylotrophs. In several newly discovered methylotrophs, XoxF is the active methanol dehydrogenase, catalysing the oxidation of methanol to formaldehyde. Apart from that, its potential role in methylotrophy and carbon cycling is unknown. So far, the diversity of xoxF in the environment has received little attention. We designed PCR primer sets targeting clades of the xoxF gene, and used 454 pyrosequencing of PCR amplicons obtained from the DNA of four coastal marine environments for a unique assessment of the diversity of xoxF in these habitats. Phylogenetic analysis of the data obtained revealed a high diversity of xoxF genes from two of the investigated clades, and substantial differences in sequence composition between environments. Sequences were classified as being related to a wide range of both methylotrophs and non-methylotrophs from Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. The most prominent sequences detected were related to the family Rhodobacteraceae, the genus Methylotenera and the OM43 clade of Methylophilales, and are thus related to organisms that employ XoxF for methanol oxidation. Furthermore, our analyses revealed a high degree of so far undescribed sequences, suggesting a high number of unknown bacterial species in these habitats.

Cell-specific CO2 fixation rates of two distinct groups of plastidic protists in the Atlantic Ocean remain unchanged after nutrient addition.

To assess the role of open-ocean ecosystems in global CO2 fixation, we investigated how picophytoplankton, which dominate primary production, responded to episodic increases in nutrient availability. Previous experiments have shown nitrogen alone, or in combination with phosphorus or iron, to be the proximate limiting nutrient(s) for total phytoplankton grown over several days. Much less is known about how nutrient upshift affects picophytoplankton CO2 fixation over the duration of the light period. To address this issue, we performed a series of small volume (8-60 ml) - short term (10-11 h) nutrient addition experiments in different regions of the Atlantic Ocean using NH4 Cl, FeCl3 , K medium, dust and nutrient-rich water from 300 m depth. We found no significant nutrient stimulation of group-specific CO2 fixation rates of two taxonomically and size-distinct groups of plastidic protists. The above was true regardless of the region sampled or nutrient added, suggesting that this is a generic phenomenon. Our findings show that at least in the short term (i.e. daylight period), nutrient availability does not limit CO2 fixation by the smallest plastidic protists, while their taxonomic composition does not determine their response to nutrient addition.

Efficient CO2 fixation by surface Prochlorococcus in the Atlantic Ocean.

Nearly half of the Earth's surface is covered by the ocean populated by the most abundant photosynthetic organisms on the planet--Prochlorococcus cyanobacteria. However, in the oligotrophic open ocean, the majority of their cells in the top half of the photic layer have levels of photosynthetic pigmentation barely detectable by flow cytometry, suggesting low efficiency of CO2 fixation compared with other phytoplankton living in the same waters. To test the latter assumption, CO2 fixation rates of flow cytometrically sorted (14)C-labelled phytoplankton cells were directly compared in surface waters of the open Atlantic Ocean (30°S to 30°N). CO2 fixation rates of Prochlorococcus are at least 1.5-2.0 times higher than CO2 fixation rates of the smallest plastidic protists and Synechococcus cyanobacteria when normalised to photosynthetic pigmentation assessed using cellular red autofluorescence. Therefore, our data indicate that in oligotrophic oceanic surface waters, pigment minimisation allows Prochlorococcus cells to harvest plentiful sunlight more effectively than other phytoplankton.

Elemental composition of natural populations of key microbial groups in Atlantic waters.

Intracellular carbon (C), nitrogen (N) and phosphorus (P) content of marine phytoplankton and bacterioplankton can vary according to cell requirements or physiological acclimation to growth under nutrient limited conditions. Although such variation in macronutrient content is well known for cultured organisms, there is a dearth of data from natural populations that reside under a range of environmental conditions. Here, we compare C, N and P content of Synechococcus, Prochlorococcus, low nucleic acid (LNA) content bacterioplankton and small plastidic protists inhabiting surface waters of the North and South subtropical gyres and the Equatorial Region of the Atlantic Ocean. While intracellular C:N ratios ranged between 3.5 and 6, i.e. below the Redfield ratio of 6.6, all the C:P and N:P ratios were up to 10 times higher than the corresponding Redfield ratio of 106 and 16, respectively, reaching and in some cases exceeding maximum values reported in the literature. Similar C:P or N:P ratios in areas with different concentrations of inorganic phosphorus suggests that this is not just a response to the prevailing environmental conditions but an indication of the extremely low P content of these oceanic microbes.

Comparable light stimulation of organic nutrient uptake by SAR11 and Prochlorococcus in the North Atlantic subtropical gyre.

Subtropical oceanic gyres are the most extensive biomes on Earth where SAR11 and Prochlorococcus bacterioplankton numerically dominate the surface waters depleted in inorganic macronutrients as well as in dissolved organic matter. In such nutrient poor conditions bacterioplankton could become photoheterotrophic, that is, potentially enhance uptake of scarce organic molecules using the available solar radiation to energise appropriate transport systems. Here, we assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions using (33)P-ATP, (3)H-ATP and (35)S-methionine tracers. Light-stimulated uptake of these substrates was assessed in two dominant bacterioplankton groups discriminated by flow cytometric sorting of tracer-labelled cells and identified using catalysed reporter deposition fluorescence in situ hybridisation. One group of cells, encompassing 48% of all bacterioplankton, were identified as members of the SAR11 clade, whereas the other group (24% of all bacterioplankton) was Prochlorococcus. When exposed to light, SAR11 cells took 31% more ATP and 32% more methionine, whereas the Prochlorococcus cells took 33% more ATP and 34% more methionine. Other bacterioplankton did not demonstrate light stimulation. Thus, the SAR11 and Prochlorococcus groups, with distinctly different light-harvesting mechanisms, used light equally to enhance, by approximately one-third, the uptake of different types of organic molecules. Our findings indicate the significance of light-driven uptake of essential organic nutrients by the dominant bacterioplankton groups in the surface waters of one of the less productive, vast regions of the world's oceans-the oligotrophic North Atlantic subtropical gyre.

Mixotrophic basis of Atlantic oligotrophic ecosystems.

Oligotrophic subtropical gyres are the largest oceanic ecosystems, covering >40% of the Earth's surface. Unicellular cyanobacteria and the smallest algae (plastidic protists) dominate CO(2) fixation in these ecosystems, competing for dissolved inorganic nutrients. Here we present direct evidence from the surface mixed layer of the subtropical gyres and adjacent equatorial and temperate regions of the Atlantic Ocean, collected on three Atlantic Meridional Transect cruises on consecutive years, that bacterioplankton are fed on by plastidic and aplastidic protists at comparable rates. Rates of bacterivory were similar in the light and dark. Furthermore, because of their higher abundance, it is the plastidic protists, rather than the aplastidic forms, that control bacterivory in these waters. These findings change our basic understanding of food web function in the open ocean, because plastidic protists should now be considered as the main bacterivores as well as the main CO(2) fixers in the oligotrophic gyres.

Invariable biomass-specific primary production of taxonomically discrete picoeukaryote groups across the Atlantic Ocean.

Oceanic photosynthetic picoeukaryotes (< 3 µm) are responsible for > 40% of total primary production at low latitudes such as the North-Eastern tropical Atlantic. In the world ocean, warmed by climate changes, the expected gradual shift towards smaller primary producers could render the role of photosynthetic picoeukaryotes even more important than they are today. Little is still known, however, about how the taxonomic composition of this highly diverse group affects primary production at the basin scale. Here, we combined flow cytometric cell sorting, NaH¹4CO3 radiotracer incubations and class-specific fluorescence in situ hybridization (FISH) probes to determine cell- and biomass-specific inorganic carbon fixation rates and taxonomic composition of two major photosynthetic picoeukaryote groups on a ∼7500-km-long latitudinal transect across the Atlantic Ocean (Atlantic Meridional Transect, AMT19). We show that even though larger cells have, on average, cell-specific CO2 uptake rates ∼5 times higher than the smaller ones, the average biomass-specific uptake is statistically similar for both groups. On the other hand, even at a high taxonomic level, i.e. class, the contributions to both groups by Prymnesiophyceae, Chrysophyceae and Pelagophyceae are significantly different (P < 0.001 in all cases). We therefore conclude that these group's carbon fixation rates are independent of the taxonomic composition of photosynthetic picoeukaryotes across the Atlantic Ocean. Because the above applies across different oceanic regions the diversity changes seem to be a secondary factor determining primary production.

Comparison of phosphate uptake rates by the smallest plastidic and aplastidic protists in the North Atlantic subtropical gyre.

The smallest phototrophic protists (<3 µm) are important primary producers in oligotrophic subtropical gyres - the Earth's largest ecosystems. In order to elucidate how these protists meet their inorganic nutrient requirements, we compared the phosphate uptake rates of plastidic and aplastidic protists in the phosphate-depleted subtropical and tropical North Atlantic (4-29°N) using a combination of radiotracers and flow cytometric sorting on two Atlantic Meridional Transect cruises. Plastidic protists were divided into two groups according to their size (<2 and 2-3 µm). Both groups of plastidic protists showed higher phosphate uptake rates per cell than the aplastidic protists. Although the phosphate uptake rates of protist cells were on average seven times (P<0.001) higher than those of bacterioplankton, the biomass-specific phosphate uptake rates of protists were one fourth to one twentieth of an average bacterioplankton cell. The unsustainably low biomass-specific phosphate uptake by both plastidic and aplastidic protists suggests the existence of a common alternative means of phosphorus acquisition - predation on phosphorus-rich bacterioplankton cells.