The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid doublestaining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

Oxygen availability drives changes in microbial diversity and biogeochemical cycling between the aerobic surface layer and the anaerobic core in nitrite-rich anoxic marine zones (AMZs), which constitute huge oxygen-depleted regions in the tropical oceans. The current paradigm is that primary production and nitrification within the oxic surface layer fuel anaerobic processes in the anoxic core of AMZs, where 30-50% of global marine nitrogen loss takes place. Here we demonstrate that oxygenic photosynthesis in the secondary chlorophyll maximum (SCM) releases significant amounts of O 2 to the otherwise anoxic environment. The SCM, commonly found within AMZs, was dominated by the picocyanobacteria Prochlorococcus spp. Free O 2 levels in this layer were, however, undetectable by conventional techniques, reflecting a tight coupling between O 2 production and consumption by aerobic processes under apparent anoxic conditions. Transcriptomic analysis of the microbial community in the seemingly anoxic SCM revealed the enhanced expression of genes for aerobic processes, such as nitrite oxidation. The rates of gross O 2 production and carbon fixation in the SCM were found to be similar to those reported for nitrite oxidation, as well as for anaerobic dissimilatory nitrate reduction and sulfate reduction, suggesting a significant effect of local oxygenic photosynthesis on Pacific AMZ biogeochemical cycling.Prochlorococcus | oxygen minimum zone | secondary chlorophyll maximum | metatranscriptomics | aerobic metabolism

Potentially pathogenic bacteria, such as Escherichia coli and Vibrio cholerae, become non-culturable during stasis. The analysis of such cells has been hampered by difficulties in studying bacterial population heterogeneity. Using in situ detection of protein oxidation in single E. coli cells, and using a density-gradient centrifugation technique to separate culturable and non-culturable cells, we show that the proteins in non-culturable cells show increased and irreversible oxidative damage, which affects various bacterial compartments and proteins. The levels of expression of specific stress regulons are higher in non-culturable cells, confirming increased defects relating to oxidative damage and the occurrence of aberrant, such as by amino-acid misincorporation, proteins. Our data suggest that non-culturable cells are produced due to stochastic deterioration, rather than an adaptive programme, and pinpoint oxidation management as the 'Achilles heel' of these cells.

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Diatoms are one of the major primary producers in the ocean, responsible annually for ~20% of photosynthetically fixed CO2 on Earth. In oceanic models, they are typically represented as large (>20 µm) microphytoplankton. However, many diatoms belong to the nanophytoplankton (2–20 µm) and a few species even overlap with the picoplanktonic size-class (<2 µm). Due to their minute size and difficulty of detection they are poorly characterized. Here we describe a massive spring bloom of the smallest known diatom (Minidiscus) in the northwestern Mediterranean Sea. Analysis of Tara Oceans data, together with literature review, reveal a general oversight of the significance of these small diatoms at the global scale. We further evidence that they can reach the seafloor at high sinking rates, implying the need to revise our classical binary vision of pico- and nanoplanktonic cells fueling the microbial loop, while only microphytoplankton sustain secondary trophic levels and carbon export.

International audienceNitrogen is essential for life but is often a major limiting nutrient for growth in the ocean. Biological dinitrogen fixation is a major source of new nitrogen to surface waters and promotes marine productivity. Yet the fate of diazotroph-derived nitrogen (DDN) in marine ecosystems has been poorly studied, and its transfer to auto- and heterotrophic plankton has not been measured. Here, we use high-resolution nanometer scale secondary ion mass spectrometry (nanoSIMS) coupled with N-15(2) isotopic labelling and flow cytometry cell sorting to examine the DDN transfer to specific groups of natural phytoplankton and bacteria during three diazotroph blooms dominated by the cyanobacterium Trichodesmium spp. in the South West Pacific. During these experiments, 13%+/- 2% to 48%+/- 5% of the fixed N-15(2) was released into the dissolved pool and 6%+/- 1% to 8%+/- 2% of this DDN was transferred to non-diazotrophic plankton after 48 h. The primary beneficiaries of this DDN were diatoms (45%+/- 4% to 61%+/- 38%) and bacteria (22%+/- 27% to 38%+/- 12%), followed by pico-phytoplankton (3%+/- 1% to 21%+/- 14%). The DDN was quickly converted to non-diazotrophic plankton biomass, in particular that of diatoms, which increased in abundance by a factor of 1.4-15 over the course of the three experiments. The single-cell approach we used enabled quantification of the actual transfer of DDN to specific groups of autotrophic and heterotrophic plankton in the surface ocean, revealing a previously unseen level of complexity in the pathways that occur between N-2 fixation and the eventual export of DDN from the photic zone

Apoptosis is a physiologically regulated process of programmed cell death involved in embryonic development and in the maintenance of homeostasis (33, 58). The dysregulation of apoptosis results in disease, e.g., cancer and autoimmune and neurodegenerative disorders (27,75,88). Apoptosis is also the basis for therapies designed to target cancerous cells and limit cytotoxicity that results from drug treatment (42). Thus, the molecular mechanisms and signaling pathways regulating apoptosis are of great significance.The caspases mediating apoptosis include initiator caspases 8 and 9 and effector caspase 3 (25). Apoptosis occurs via two pathways: the extrinsic (death receptor) pathway, initiated by activation of members of the death receptor superfamily (Fas and tumor necrosis factor receptor 1 [TNFR1]), leading to caspase 8 activation (61), and the intrinsic (mitochondrial) pathway, resulting in the mitochondrial release of cytochrome c and caspase 9 activation (80). These two pathways converge upon the activation of caspase 3 (33). Mitochondrial involvement in apoptosis is determined by the balance of antiapoptotic (Bcl-2 and Bcl-X L ) and proapoptotic (Bax, Bad, Bid, and Noxa) Bcl-2 family members (46). Importantly, the extrinsic and intrinsic pathways are linked via the function of the protein Bid (80,81).Apoptosis is regulated by the p38 mitogen-activated protein (MAP) kinase/c-Jun N-terminal kinase (JNK) cellular stress pathways (47, 76). These pathways, members of the mitogenic family of signaling cascades (39), also mediate proliferation and differentiation (20,26,42,62,64,65). Evidence in support of JNK and p38 MAP kinase pathways in regulating apoptosis is derived from studies employing treatments simulating cellular stress. These stresses include growth factor withdrawal (21, 85), the presence of proinflammatory cytokines (8,56,67) and drugs (17,29,57,63), UV radiation (79), and overexpression of constitutively active effectors, e.g., MEKK1 (40), ASK-1 (31, 41), and JNK1 (51). However, despite reports of a role for the JNK and p38 MAP kinase in apoptosis and the demonstration that ASK-1 is upstream of both JNK and p38 MAP kinase pathways (37, 78), whether both pathways are necessary for apoptosis remains unresolved. For example, although both the p38 MAP kinase and JNK pathways are activated upon exposure to UV radiation (34), only the JNK pathway mediates UV-induced apoptosis in primary mouse fibroblasts (79). With other apoptotic treatments, e.g., by overexpression of constitutively active ASK-1 (31, 41), activation of the JNK pathway is the dominant event in mediating apoptosis, although ASK-1 is known to activate both the p38 MAP kinase and JNK pathways (37,78). Thus, despite the demonstrated role of the JNK pathway in apoptosis under robust apoptotic conditions, it is not yet

-1). However, orthosilicic acid concentrations in the incubations under increasing pressure approached those of the atmospheric pressure incubations by the end of the experiment. In contrast, the taxonomic composition of prokaryotic communities was not affected by increasing pressure, but the input of fresh diatom detritus led to an increase in the relative abundance of the Cytophago-Flavobacter cluster and γ-Proteobacteria. These results suggest that hydrostatic pressure affects the function rather than the broad taxonomic structure of prokaryotic communities associated with sinking detrital particles.