Single-cell measurements and modelling reveal substantial organic carbon acquisition by Prochlorococcus.

Marine phytoplankton are responsible for about half of the photosynthesis on Earth. Many are mixotrophs, combining photosynthesis with heterotrophic assimilation of organic carbon, but the relative contribution of these two lifestyles is unclear. Here single-cell measurements reveal that Prochlorococcus at the base of the photic zone in the Eastern Mediterranean Sea obtain only ~20% of carbon required for growth by photosynthesis. This is supported by laboratory-calibrated calculations based on photo-physiology parameters and compared with in situ growth rates. Agent-based simulations show that mixotrophic cells could grow tens of metres deeper than obligate photo-autotrophs, deepening the nutricline by ~20 m. Time series from the North Atlantic and North Pacific indicate that, during thermal stratification, on average 8-10% of the Prochlorococcus cells live without enough light to sustain obligate photo-autotrophic populations. Together, these results suggest that mixotrophy underpins the ecological success of a large fraction of the global Prochlorococcus population and its collective genetic diversity.

Biofilm enhances the copper (II) adsorption on microplastic surfaces in coastal seawater: Simultaneous evidence from visualization and quantification.

Microplastics (MPs) exposed to the urban coastal seawater could form biofilms, which facilitate the adsorption and transportation of hazardous contaminants. However, influence of biofilms on the metal adsorption of MPs, especially the co-existence of biofilm and metals on MPs, is still less known. In this study, the adsorption of copper (Cu) on biofilm-coated MPs (BMPs) was visually analyzed and quantified. The results of scanning electron microscopy in combination with energy dispersive X-ray showed that biofilm and metals co-occurred on MPs in seawater. The nanoscale secondary ion mass spectrometry images further exhibited that the distribution of Cu, chlorine (Cl) and biofilm on MP surfaces was highly consistent. Moreover, the adsorption of Cu(II) on BMPs was enhanced as quantified by inductively coupled plasma-mass spectrometer. Furthermore, different species on BMPs with and without Cu were identified, and their potential functions of metal or Cl metabolism were predicted based on KEGG pathway database. Overall, for the first time, this study provides visual and quantified evidences for the enhancement of Cu(II) adsorption on BMPs based on co-localization, and it may shed a light on the development of methodologies for investigating the interaction among MPs, biofilms and pollutants in marine environment.

Phytoplankton exudates provide full nutrition to a subset of accompanying heterotrophic bacteria via carbon, nitrogen and phosphorus allocation.

Marine bacteria rely on phytoplankton exudates as carbon sources (DOCp). Yet, it is unclear to what extent phytoplankton exudates also provide nutrients such as phytoplankton-derived N and P (DONp, DOPp). We address these questions by mesocosm exudate addition experiments with spent media from the ubiquitous pico-cyanobacterium Prochlorococcus to bacterial communities in contrasting ecosystems in the Eastern Mediterranean - a coastal and an open-ocean, oligotrophic station with and without on-top additions of inorganic nutrients. Inorganic nutrient addition did not lower the incorporation of exudate DONp, nor did it reduce alkaline phosphatase activity, suggesting that bacterial communities are able to exclusively cover their nitrogen and phosphorus demands with organic forms provided by phytoplankton exudates. Approximately half of the cells in each ecosystem took up detectable amounts of Prochlorococcus-derived C and N, yet based on 16S rRNA sequencing different bacterial genera were responsible for the observed exudate utilization patterns. In the coastal community, several phylotypes of Aureimarina, Psychrosphaera and Glaciecola responded positively to the addition of phytoplankton exudates, whereas phylotypes of Pseudoalteromonas increased and dominated the open-ocean communities. Together, our results strongly indicate that phytoplankton exudates provide coastal and open-ocean bacterial communities with organic carbon, nitrogen and phosphorus, and that phytoplankton exudate serve a full-fledged meal for the accompanying bacterial community in the nutrient-poor eastern Mediterranean.

Structural and Functional Characteristics of Microplastic Associated Biofilms in Response to Temporal Dynamics and Polymer Types.

The colonization of bacterial communities and biofilm formation on microplastics (MPs) have aroused great concern recently. However, the influence of time and polymer types on the structural and functional characteristics of biofilms remains unclear. In this study, three types of MPs (polyethylene, polypropylene, and polystyrene) were exposed for different time periods (10, 20 and 30 days) in seawater using a microcosm experiment. Microscopic spectroscopy and high-throughput gene sequencing techniques were used to reveal the temporal changes of structural and functional characteristics of MPs associated biofilms. The results indicate that the biofilm formation is affected by both the incubation time and the polymer type. In addition, bacterial diversity and community structure in the biofilms show selectivity towards seawater, and tend to shift over time and among different polymer types. Moreover, biofilms are shown to harbor plastic degrading bacteria, leading to the changes of functional groups and surface hydrophobicity, and thereby enhancing the biodegradation of MPs.

Presynaptic activity and protein turnover are correlated at the single-synapse level.

Synaptic transmission relies on the continual exocytosis and recycling of synaptic vesicles. Aged vesicle proteins are prevented from recycling and are eventually degraded. This implies that active synapses would lose vesicles and vesicle-associated proteins over time, unless the supply correlates to activity, to balance the losses. To test this hypothesis, we first model the quantitative relation between presynaptic spike rate and vesicle turnover. The model predicts that the vesicle supply needs to increase with the spike rate. To follow up this prediction, we measure protein turnover in individual synapses of cultured hippocampal neurons by combining nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence microscopy. We find that turnover correlates with activity at the single-synapse level, but not with other parameters such as the abundance of synaptic vesicles or postsynaptic density proteins. We therefore suggest that the supply of newly synthesized proteins to synapses is closely connected to synaptic activity.

Candidatus Sulfurimonas marisnigri sp. nov. and Candidatus Sulfurimonas baltica sp. nov., thiotrophic manganese oxide reducing chemolithoautotrophs of the class Campylobacteria isolated from the pelagic redoxclines of the Black Sea and the Baltic Sea.

Species of the genus Sulfurimonas are reported and isolated from terrestrial habitats and marine sediments and water columns with steep redox gradients. Here we report on the isolation of strains SoZ1 and GD2 from the pelagic redoxcline of the Black Sea and the Baltic Sea, respectively. Both strains are gram-stain-negative and appear as short and slightly curved motile rods. The autecological preferences for growth of strain SoZ1 were 0-25°C (optimum 20°C), pH 6.5-9.0 (optimum pH 7.5-8.0) and salinity 10-40gL-1 (optimum 25gL-1). Preferences for growth of strain GD2 were 0-20°C (optimum 15°C), pH 7.0-8.0 (optimum pH 7.0-7.5) and salinity 5-40gL-1 (optimum 21gL-1). Strain SoZ1 grew chemolithoautotrophically, while strain GD2 also showed heterotrophic growth with short chained fatty acids as carbon source. Both species utilized hydrogen (H2), sulfide (H2S here taken as the sum of H2S, HS- and S2-), elemental sulfur (S0) and thiosulfate (S2O32-) as electron donors and nitrate (NO3-), oxygen (O2) and particulate manganese oxide (MnO2) as electron acceptors. Based on 16S rRNA gene sequence similarity, both strains cluster within the genus Sulfurimonas with Sulfurimonas gotlandica GD1T as the closest cultured relative species with a sequence similarity of 96.74% and 96.41% for strain SoZ1 and strain GD2, respectively. Strains SoZ1 and GD2 share a ribosomal 16S sequence similarity of 99.27% and were demarcated based on average nucleotide identity and average amino acid identity of the whole genome sequence. These calculations have been applied to the whole genus. We propose the names Candidatus Sulfurimonas marisnigri sp. nov. and Candidatus Sulfurimonas baltica sp. nov. for the thiotrophic manganese reducing culture isolates from the Black Sea and Baltic Sea, respectively.

Nitrogen Flow in Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Is Altered by Cyanophage Infection.

Viruses can significantly influence cyanobacteria population dynamics and activity, and through this the biogeochemical cycling of major nutrients. However, surprisingly little attention has been given to understand how viral infections alter the ability of diazotrophic cyanobacteria for atmospheric nitrogen fixation and its release to the environment. This study addressed the importance of cyanophages for net 15N2 assimilation rate, expression of nitrogenase reductase gene (nifH) and changes in nitrogen enrichment (15N/14N) in the diazotrophic cyanobacterium Aphanizomenon flos-aquae during infection by the cyanophage vB_AphaS-CL131. We found that while the growth of A. flos-aquae was inhibited by cyanophage addition (decreased from 0.02 h-1 to 0.002 h-1), there were no significant differences in nitrogen fixation rates (control: 22.7 × 10-7 nmol N heterocyte-1; infected: 23.9 × 10-7 nmol N heterocyte-1) and nifH expression level (control: 0.6-1.6 transcripts heterocyte-1; infected: 0.7-1.1 transcripts heterocyte-1) between the infected and control A. flos-aquae cultures. This implies that cyanophage genome replication and progeny production within the vegetative cells does not interfere with the N2 fixation reactions in the heterocytes of these cyanobacteria. However, higher 15N enrichment at the poles of heterocytes of the infected A. flos-aquae, revealed by NanoSIMS analysis indicates the accumulation of fixed nitrogen in response to cyanophage addition. This suggests reduced nitrogen transport to vegetative cells and the alterations in the flow of fixed nitrogen within the filaments. In addition, we found that cyanophage lysis resulted in a substantial release of ammonium into culture medium. Cyanophage infection seems to substantially redirect N flow from cyanobacterial biomass to the production of N storage compounds and N release.

Prochlorococcus Cells Rely on Microbial Interactions Rather than on Chlorotic Resting Stages To Survive Long-Term Nutrient Starvation.

Many microorganisms produce resting cells with very low metabolic activity that allow them to survive phases of prolonged nutrient or energy stress. In cyanobacteria and some eukaryotic phytoplankton, the production of resting stages is accompanied by a loss of photosynthetic pigments, a process termed chlorosis. Here, we show that a chlorosis-like process occurs under multiple stress conditions in axenic laboratory cultures of Prochlorococcus, the dominant phytoplankton linage in large regions of the oligotrophic ocean and a global key player in ocean biogeochemical cycles. In Prochlorococcus strain MIT9313, chlorotic cells show reduced metabolic activity, measured as C and N uptake by Nanoscale secondary ion mass spectrometry (NanoSIMS). However, unlike many other cyanobacteria, chlorotic Prochlorococcus cells are not viable and do not regrow under axenic conditions when transferred to new media. Nevertheless, cocultures with a heterotrophic bacterium, Alteromonas macleodii HOT1A3, allowed Prochlorococcus to survive nutrient starvation for months. We propose that reliance on co-occurring heterotrophic bacteria, rather than the ability to survive extended starvation as resting cells, underlies the ecological success of ProchlorococcusIMPORTANCE The ability of microorganisms to withstand long periods of nutrient starvation is key to their survival and success under highly fluctuating conditions that are common in nature. Therefore, one would expect this trait to be prevalent among organisms in the nutrient-poor open ocean. Here, we show that this is not the case for Prochlorococcus, a globally abundant and ecologically important marine cyanobacterium. Instead, Prochlorococcus relies on co-occurring heterotrophic bacteria to survive extended phases of nutrient and light starvation. Our results highlight the power of microbial interactions to drive major biogeochemical cycles in the ocean and elsewhere with consequences at the global scale.

Distinctive tasks of different cyanobacteria and associated bacteria in carbon as well as nitrogen fixation and cycling in a late stage Baltic Sea bloom.

Cyanobacteria and associated heterotrophic bacteria hold key roles in carbon as well as nitrogen fixation and cycling in the Baltic Sea due to massive cyanobacterial blooms each summer. The species specific activities of different cyanobacterial species as well as the N- and C-exchange of associated heterotrophic bacteria in these processes, however, are widely unknown. Within one time series experiment we tested the cycling in a natural, late stage cyanobacterial bloom by adding 13C bi-carbonate and 15N2, and performed sampling after 10 min, 30 min, 1 h, 6 h and 24 h in order to determine the fixing species as well as the fate of the fixed carbon and nitrogen in the associations. Uptake of 15N and 13C isotopes by the most abundant cyanobacterial species as well as the most abundant associated heterotrophic bacterial groups was then analysed by NanoSIMS. Overall, the filamentous, heterocystous species Dolichospermum sp., Nodularia sp., and Aphanizomenon sp. revealed no or erratic uptake of carbon and nitrogen, indicating mostly inactive cells. In contrary, non-heterocystous Pseudanabaena sp. dominated the nitrogen and carbon fixation, with uptake rates up to 1.49 ± 0.47 nmol N h-1 l-1 and 2.55 ± 0.91 nmol C h-1 l-1. Associated heterotrophic bacteria dominated the subsequent nitrogen remineralization with uptake rates up to 1.2 ± 1.93 fmol N h-1 cell -1, but were also indicative for fixation of di-nitrogen.

Simultaneous Visualization of Enzymatic Activity in the Cytoplasm and at Polyphosphate Inclusions in Beggiatoa sp. Strain 35Flor Incubated with 18O-Labeled Water.

Here we report on a new nanoscale secondary ion mass spectrometry (nanoSIMS) approach based on enzyme-mediated oxygen isotope exchange, which combines the visualization of general metabolic activity in the cytoplasm with insights into the activity of enzymes related to polyphosphate (polyP) inclusions. The polyP-accumulating strain of the large sulfur bacterium Beggiatoa was used as a model organism. Beggiatoa cultures were grown under oxic and anoxic conditions when exposed to either low- or high-sulfide conditions, which are known to influence polyP metabolism in this strain. Subsequent incubation with 18O-labeled water led to high 18O enrichments above the natural background in the cytoplasm and polyP granules derived from enzymatically mediated oxygen isotope exchange. The relative importance of polyP under the different sulfide regimes became evident by an apparent continued metabolic activity at polyP inclusions under stressfully high sulfide concentrations, in contrast to a decreased general metabolic activity in the cytoplasm. This finding confirms the role of polyP as a critical component in bacterial stress response and maintenance of a survival metabolism.IMPORTANCE Microbial organisms exert a large influence on the environment as they directly affect the turnover of essential elements. This is particularly true for polyphosphate-accumulating large sulfur bacteria, which can either accumulate phosphate as polyphosphate or degrade it and release phosphate into the environment, depending on environmental conditions. This study presents a new approach to simultaneously visualize general metabolic activity and enzymatic activity at polyphosphate granules by incubation with 18O-labeled water as the only stable isotope tracer. For this purpose, the well-studied Beggiatoa sp. strain 35Flor was used as a model organism and was exposed to different stress regimes. General metabolic activity was strongly impaired during high-stress regimes. In contrast, intense intracellular polyP cycling was not restricted to favorable or stressful conditions, highlighting the importance of polyP for general cell physiology, especially during hostile conditions. The nanoSIMS approach adds a new tool to study microorganisms involved in phosphorus cycling in the environment together with the identification of general metabolic activity.

Potential trade-offs between biomineralization and immunity revealed by shell properties and gene expression profiles of two closely related Crassostrea species.

Species of the Ostreidae family are key ecosystem engineers and many of them - including Crassostrea gigas and Crassostreavirginica - are commercially important aquaculture species. Despite similarities in their morphology and ecology, these two species differ in their ability to defend against pathogens, potentially reflecting species-specific differential specialization of hemocytes on immune defense versus biomineralization. To test this hypothesis, we investigated the expression levels of immune- and biomineralization-related genes as well as mineralogical and mechanical properties of the shells and the calcium sequestration ability of the hemocytes of C. gigas and C. virginica The expression of biomineralization-related genes was higher in C. virginica than in C. gigas in multiple tissues including the mantle edge and hemocytes, while the expression of immune genes was higher in the hemocytes of C. gigas Hemocytes of C. virginica contained more calcium (stored intracellularly as calcium carbonate mineral) compared with those of C. gigas Analysis of the adult shells showed that the crystallinity of calcite was higher and the laths of the foliated layer of the shell were thicker in C. virginica than in C. gigas Mechanically, the shells of C. virginica were stiffer, harder and stronger than those of C. gigas Taken together, our results show that the species-specific differences in physiology (such as disease resistance and exoskeleton properties) are reflected at the cellular and molecular levels in the differential specialization of hemocytes on potentially competing functions (immunity and biomineralization) as well as different expression profiles of other tissues involved in biomineralization (such as the mantle edge).

Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission.

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non-recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24-48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.

Differences in the accumulation of phosphorus between vegetative cells and heterocysts in the cyanobacterium Nodularia spumigena.

The cyanobacterium Nodularia spumigena is a species that frequently forms blooms in the Baltic Sea. Accumulation of the vital nutrient phosphorus (P) apparently plays an important role in the ability of this and other cyanobacteria to grow even when dissolved inorganic phosphorus is depleted. However, until now, this has not been studied in N. spumigena at the cellular level. Therefore, in this study, phosphorus incorporation and distribution in cyanobacterial filaments over time was examined by scanning electron microscopy in combination with energy dispersive X-ray analysis (SEM/EDX) and nanoscale secondary ion mass spectrometry (NanoSIMS). Immediately after phosphate addition to a phosphorus-depleted population, the phosphate concentration decreased in the water while intracellular polyphosphate accumulated. Microscopically, phosphorus in form of polyphosphate granules was stored preferentially in vegetative cells, whereas heterocysts remained low in intracellular phosphorus. This information is an essential step towards understanding the phosphorus dynamics of this species and demonstrates that the division of tasks between vegetative cells and heterocysts is not restricted to nitrogen fixation.

Review of combined isotopic and optical nanoscopy.

Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the "history" of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology.

Success of chemolithoautotrophic SUP05 and Sulfurimonas GD17 cells in pelagic Baltic Sea redox zones is facilitated by their lifestyles as K- and r-strategists.

Chemolithoautotrophic sulfur-oxidizing and denitrifying Gamma- (particularly the SUP05 cluster) and Epsilonproteobacteria (predominantly Sulfurimonas subgroup GD17) are assumed to compete for substrates (electron donors and acceptors) in marine pelagic redox gradients. To elucidate their ecological niche separation we performed 34 S0 , 15 NO3- and H13 CO3- stable-isotope incubations with water samples from Baltic Sea suboxic, chemocline and sulfidic zones followed by combined phylogenetic staining and high-resolution secondary ion mass spectrometry of single cells. SUP05 cells were small-sized (0.06-0.09 µm3 ) and most abundant in low-sulfidic to suboxic zones, whereas Sulfurimonas GD17 cells were significantly larger (0.26-0.61 µm3 ) and most abundant at the chemocline and below. Together, SUP05 and GD17 cells accumulated up to 48% of the labelled substrates but calculation of cell volume-specific rates revealed that GD17 cells incorporated labelled substrates significantly faster throughout the redox zone, thereby potentially outcompeting SUP05 especially at high substrate concentrations. Thus, in synopsis with earlier described features of SUP05/GD17 we conclude that their spatially overlapping association in stratified sulfidic zones is facilitated by their different lifestyles: whereas SUP05 cells are streamlined, non-motile K-strategists adapted to low substrate concentrations, GD17 cells are motile r-strategists well adapted to fluctuating substrate and redox conditions.

Superposition of Individual Activities: Urea-Mediated Suppression of Nitrate Uptake in the Dinoflagellate Prorocentrum minimum Revealed at the Population and Single-Cell Levels.

Dinoflagellates readily use diverse inorganic and organic compounds as nitrogen sources, which is advantageous in eutrophied coastal areas exposed to high loads of anthropogenic nutrients, e.g., urea, one of the most abundant organic nitrogen substrates in seawater. Cell-to-cell variability in nutritional physiology can further enhance the diversity of metabolic strategies among dinoflagellates of the same species, but it has not been studied in free-living microalgae. We applied stable isotope tracers, isotope ratio mass spectrometry and nanoscale secondary ion mass spectrometry (NanoSIMS) to investigate the response of cultured nitrate-acclimated dinoflagellates Prorocentrum minimum to a sudden input of urea and the effect of urea on the concurrent nitrate uptake at the population and single-cell levels. We demonstrate that inputs of urea lead to suppression of nitrate uptake by P. minimum, and urea uptake exceeds the concurrent uptake of nitrate. Individual dinoflagellate cells within a population display significant heterogeneity in the rates of nutrient uptake and extent of the urea-mediated inhibition of the nitrate uptake, thus forming several groups characterized by different modes of nutrition. We conclude that urea originating from sporadic sources is rapidly utilized by dinoflagellates and can be used in biosynthesis or stored intracellularly depending on the nutrient status; therefore, sudden urea inputs can represent one of the factors triggering or supporting harmful algal blooms. Significant physiological heterogeneity revealed at the single-cell level is likely to play a role in alleviation of intra-population competition for resources and can affect the dynamics of phytoplankton populations and their maintenance in natural environments.

Innovative methods in soil phosphorus research: A review.

Phosphorus (P) is an indispensable element for all life on Earth and, during the past decade, concerns about the future of its global supply have stimulated much research on soil P and method development. This review provides an overview of advanced state-of-the-art methods currently used in soil P research. These involve bulk and spatially resolved spectroscopic and spectrometric P speciation methods (1 and 2D NMR, IR, Raman, Q-TOF MS/MS, high resolution-MS, NanoSIMS, XRF, XPS, (µ)XAS) as well as methods for assessing soil P reactions (sorption isotherms, quantum-chemical modeling, microbial biomass P, enzymes activity, DGT, 33P isotopic exchange, 18O isotope ratios). Required experimental set-ups and the potentials and limitations of individual methods present a guide for the selection of most suitable methods or combinations.

Allocate carbon for a reason: priorities are reflected in the ¹³C/¹²C ratios of plant lipids synthesized via three independent biosynthetic pathways.

It has long been theorized that carbon allocation, in addition to the carbon source and to kinetic isotopic effects associated with a particular lipid biosynthetic pathway, plays an important role in shaping the carbon isotopic composition ((13)C/(12)C) of lipids (Park and Epstein, 1961). If the latter two factors are properly constrained, valuable information about carbon allocation during lipid biosynthesis can be obtained from carbon isotope measurements. Published work of Chikaraishi et al. (2004) showed that leaf lipids isotopic shifts from bulk leaf tissue Δδ(13)C(bk-lp) (defined as δ(13)C(bulkleaftissue)-δ(13)C(lipid)) are pathway dependent: the acetogenic (ACT) pathway synthesizing fatty lipids has the largest isotopic shift, the mevalonic acid (MVA) pathway synthesizing sterols the lowest and the phytol synthesizing 1-deoxy-D-xylulose 5-phosphate (DXP) pathway gives intermediate values. The differences in Δδ(13)C(bk-lp) between C3 and C4 plants Δδ(13)C(bk-lp,C4-C3) are also pathway-dependent: Δδ(13)C(ACT)(bk-lp,C4-C3) > Δδ(13)C(DXP(bk-lp,C4-C3) > Δδ(13)C(MVA)(bk-lp,C4-C3). These pathway-dependent differences have been interpreted as resulting from kinetic isotopic effect differences of key but unspecified biochemical reactions involved in lipids biosynthesis between C3 and C4 plants. After quantitatively considering isotopic shifts caused by (dark) respiration, export-of-carbon (to sink tissues) and photorespiration, we propose that the pathway-specific differences Δδ(13)C(bk-lp,C4-C3) can be successfully explained by C4-C3 carbon allocation (flux) differences with greatest flux into the ACT pathway and lowest into the MVA pathways (when flux is higher, isotopic shift relative to source is smaller). Highest carbon allocation to the ACT pathway appears to be tied to the most stringent role of water-loss-minimization by leaf waxes (composed mainly of fatty lipids) while the lowest carbon allocation to the MVA pathway can be largely explained by the fact that sterols act as regulatory hormones and membrane fluidity modulators in rather low concentrations.

Correlated optical and isotopic nanoscopy.

The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes ((15)N, (13)C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures.

Chemoautotrophic growth of ammonia-oxidizing Thaumarchaeota enriched from a pelagic redox gradient in the Baltic Sea.

Ammonia-oxidizing archaea (AOA) are an important component of the planktonic community in aquatic habitats, linking nitrogen and carbon cycles through nitrification and carbon fixation. Therefore, measurements of these processes in culture-based experiments can provide insights into their contributions to energy conservation and biomass production by specific AOA. In this study, by enriching AOA from a brackish, oxygen-depleted water-column in the Landsort Deep, central Baltic Sea, we were able to investigate ammonium oxidation, chemoautotrophy, and growth in seawater batch experiments. The highly enriched culture consisted of up to 97% archaea, with maximal archaeal numbers of 2.9 × 10(7) cells mL(-1). Phylogenetic analysis of the 16S rRNA and ammonia monooxygenase subunit A (amoA) gene sequences revealed an affiliation with assemblages from low-salinity and freshwater habitats, with Candidatus Nitrosoarchaeum limnia as the closest relative. Growth correlated significantly with nitrite production, ammonium consumption, and CO2 fixation, which occurred at a ratio of 10 atoms N oxidized per 1 atom C fixed. According to the carbon balance, AOA biomass production can be entirely explained by chemoautotrophy. The cellular carbon content was estimated to be 9 fg C per cell. Single-cell-based (13)C and (15)N labeling experiments and analysis by nano-scale secondary ion mass spectrometry provided further evidence that cellular carbon was derived from bicarbonate and that ammonium was taken up by the cells. Our study therefore revealed that growth by an AOA belonging to the genus Nitrosoarchaeum can be sustained largely by chemoautotrophy.