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Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of aerobic anoxygenic phototrophic (AAP) bacteria, the pufM gene has been established as the genetic marker for this particular functional group, although available primers are known to have amplification biases. We review here the existing primers for pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from contrasting marine environments to evaluate their performance. By comparing the taxonomic composition of communities retrieved with metagenomics and with different amplicon approaches, we show that the commonly used PCR primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach, as well as the use of other combinations of the existing and newly designed primers, show that these groups are in fact less abundant than previously observed, and that a great proportion of pufM sequences are affiliated to uncultured representatives, particularly in the open ocean. Altogether, the framework developed here becomes a better alternative for future studies based on the pufM gene and, additionally, serves as a reference for primer evaluation of other functional genes.

The symbiotic cyanobacterium UCYN-A is one of the most globally abundant marine dinitrogen (N2)-fixers, but cultures have not been available and its biology and ecology are poorly understood. We used cultivation-independent approaches to investigate how UCYN-A single-cell N2 fixation rates (NFRs) and nifH gene expression vary as a function of depth and photoperiod. Twelve-hour day/night incubations showed that UCYN-A only fixed N2 during the day. Experiments conducted using in situ arrays showed a light-dependence of NFRs by the UCYN-A symbiosis, with the highest rates in surface waters (5–45 m) and lower rates at depth (≥ 75 m). Analysis of NFRs versus in situ light intensity yielded a light saturation parameter (Ik) for UCYN-A of 44 μmol quanta m−2 s−1. This is low compared with other marine diazotrophs, suggesting an ecological advantage for the UCYN-A symbiosis under low-light conditions. In contrast to cell-specific NFRs, nifH gene-specific expression levels did not vary with depth, indicating that light regulates N2 fixation by UCYN-A through processes other than transcription, likely including host–symbiont interactions. These results offer new insights into the physiology of the UCYN-A symbiosis in the subtropical North Pacific Ocean and provide clues to the environmental drivers of its global distributions. En prensa

1.AbstractMesoscale eddies have been shown to support elevated dinitrogen (N2) fixation rates (NFRs) and abundances of N2-fixing microorganisms (diazotrophs), but the mechanisms underlying these observations are not well understood. We explored relationships among NFRs and cyanobacterial diazotroph abundances in eddy pairs of opposite polarity sampled in the North Pacific Subtropical Gyre and compared our observations to seasonal trends from the Hawaii Ocean Time-series (HOT) program. Consistent with previous reports, we found that NFRs were anomalously high for this region (up to 3.7-fold above previous monthly HOT observations) in the centers of the sampled anticyclones, coinciding with elevated abundances of Crocosphaera in the summertime. We then coupled our field-based observations, together with transcriptomic analyses of nutrient stress marker genes and ecological models, to evaluate potential mechanisms controlling diazotroph abundance and activity at the mesoscale. Specifically, we evaluated the role of biological (via estimates of growth and grazing rates) and physical controls on populations of Crocosphaera, Trichodesmium, and diatom symbionts. Our results suggest that increased Crocosphaera abundances associated with summertime anticyclones resulted from the alleviation of phosphate limitation, allowing cells to grow at rates exceeding grazing losses. In contrast, distributions of larger, buoyant taxa (Trichodesmium and diatom symbionts) appeared less affected by eddy-driven biological controls. Instead, they appeared driven by physical dynamics along frontal boundaries that separate cyclonic and anticyclonic eddies. Together, the interplay of eddy-specific changes in bottom-up control, top-down control, and the physical accumulation of cells likely explains the elevated diazotroph abundances and NFRs associated with anticyclones and eddy fronts.

The fine vertical distribution of phytoplankton groups within the deep chlorophyll maximum (DCM) was studied in the NE Atlantic during summer stratification. A simple but unconventional sampling strategy allowed examining the vertical structure with ca. 2 m resolution. The distribution of Prochlorococcus, Synechococcus, chlorophytes, pelagophytes, small prymnesiophytes, coccolithophores, diatoms, and dinoflagellates was investigated with a combination of pigment-markers, flow cytometry and optical and FISH microscopy. All groups presented minimum abundances at the surface and a maximum in the DCM layer. The cell distribution was not vertically symmetrical around the DCM peak and cells tended to accumulate in the upper part of the DCM layer. The more symmetrical distribution of chlorophyll than cells around the DCM peak was due to the increase of pigment per cell with depth. We found a vertical alignment of phytoplankton groups within the DCM layer indicating preferences for different ecological niches in a layer with strong gradients of light and nutrients. Prochlorococcus occupied the shallowest and diatoms the deepest layers. Dinoflagellates, Synechococcus and small prymnesiophytes preferred shallow DCM layers, and coccolithophores, chlorophytes and pelagophytes showed a preference for deep layers. Cell size within groups changed with depth in a pattern related to their mean size: the cell volume of the smallest group increased the most with depth while the cell volume of the largest group decreased the most. The vertical alignment of phytoplankton groups confirms that the DCM is not a homogeneous entity and indicates groups’ preferences for different ecological niches within this layer.

The symbiosis between a marine alga and a N2-fixing cyanobacterium (UCYN-A) is geographically widespread in the oceans and is important in the marine N cycle. UCYN-A is uncultivated, and is an unusual unicellular cyanobacterium because it lacks many metabolic functions, including oxygenic photosynthesis and carbon fixation, which are typical in cyanobacteria. It is now presumed to be an obligate symbiont of haptophytes closely related to Braarudosphaera bigelowii. N2-fìxing cyanobacteria use different strategies to avoid inhibition of N2 fixation by the oxygen evolved in photosynthesis. Most unicellular cyanobacteria temporally separate the two incompatible activities by fixing N2 only at night, but surprisingly UCYN-A appears to fix N2 during the day. The goal of this study was to determine how the unicellular UCYN-A coordinates N2 fixation and general metabolism compared to other marine cyanobacteria. We found that UCYN-A has distinct daily cycles of many genes despite the fact that it lacks two of the three circadian clock genes found in most cyanobacteria. We also found that transcription patterns in UCYN-A are most similar to marine cyanobacteria that are capable of aerobic N2 fixation in the light such as Trichodesmium and heterocyst-forming cyanobacteria, rather than Crocosphaera or Cyanothece species, which are more closely related to unicellular marine cyanobacteria evolutionarily. Our findings suggest that the symbiotic interaction has resulted in a shift of transcriptional regulation to coordinate UCYN-A metabolism with the phototrophic eukaryotic host, thus allowing efficient coupling of N2 fixation (by the cyanobacterium) to the energy obtained from photosynthesis (by the eukaryotic unicellular alga) in the light.ImportanceThe symbiotic N2-fixing cyanobacterium UCYN-A and its eukaryotic algal host, which is closely related to Braarudosphaera bigelowii, have been shown to be globally distributed and important in open ocean N2 fixation. These unique cyanobacteria have reduced metabolic capabilities, even lacking genes for oxygenic photosynthesis and carbon fixation. Cyanobacteria generally use energy from photosynthesis for nitrogen fixation, but require mechanisms for avoiding inactivation of the oxygen-sensitive nitrogenase enzyme by ambient oxygen (O2) or the O2 evolved through photosynthesis. This study shows that the symbiosis between the N2-fixing cyanobacterium UCYN-A and its eukaryotic algal host has led to adaptation of its daily gene expression pattern in order to enable daytime aerobic N2 fixation, which is likely more energetically efficient than fixing N2 at night, as in other unicellular marine cyanobacteria.

14 pages, 6 figures, 1 table, A marine symbiosis has been recently discovered between prymnesiophyte species and the unicellular diazotrophic cyanobacterium UCYN-A. At least two different UCYN-A phylotypes exist, the clade UCYN-A1 in symbiosis with an uncultured small prymnesiophyte and the clade UCYN-A2 in symbiosis with the larger Braarudosphaera bigelowii. We targeted the prymnesiophyte–UCYN-A1 symbiosis by double CARD-FISH (catalyzed reporter deposition-fluorescence in situ hybridization) and analyzed its abundance in surface samples from the MALASPINA circumnavigation expedition. Our use of a specific probe for the prymnesiophyte partner allowed us to verify that this algal species virtually always carried the UCYN-A symbiont, indicating that the association was also obligate for the host. The prymnesiophyte–UCYN-A1 symbiosis was detected in all ocean basins, displaying a patchy distribution with abundances (up to 500 cells ml− 1) that could vary orders of magnitude. Additional vertical profiles taken at the NE Atlantic showed that this symbiosis occupied the upper water column and disappeared towards the Deep Chlorophyll Maximum, where the biomass of the prymnesiophyte assemblage peaked. Moreover, sequences of both prymnesiophyte partners were searched within a large 18S rDNA metabarcoding data set from the Tara-Oceans expedition around the world. This sequence-based analysis supported the patchy distribution of the UCYN-A1 host observed by CARD-FISH and highlighted an unexpected homogeneous distribution (at low relative abundance) of B. bigelowii in the open ocean. Our results demonstrate that partners are always in symbiosis in nature and show contrasted ecological patterns of the two related lineages, Financial support has been provided by the Spanish Ministry of Economy and Competitivity through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077) to CMD, FLAME (CGL2010-16304) to RM, and PANGENOMICS (CGL2011-26848/BOS) to SGA. AMC was recipient of a Spanish FPI grant (BES-2009-027194)

29 pages,8 figures, 9 tables, supporting information https://doi.org/10.1371/journal.pone.0151699, We examine the large-scale distribution patterns of the nano- and microphytoplankton collected from 145 oceanic stations, at 3 m depth, the 20% light level and the depth of the subsurface chlorophyll maximum, during the Malaspina-2010 Expedition (December 2010-July 2011), which covered 15 biogeographical provinces across the Atlantic, Indian and Pacific oceans, between 35°N and 40°S. In general, the water column was stratified, the surface layers were nutrient-poor and the nano- and microplankton (hereafter phytoplankton, for simplicity, although it included also heterotrophic protists) community was dominated by dinoflagellates, other flagellates and coccolithophores, while the contribution of diatoms was only important in zones with shallow nutriclines such as the equatorial upwelling regions. We applied a principal component analysis to the correlation matrix among the abundances (after logarithmic transform) of the 76 most frequent taxa to synthesize the information contained in the phytoplankton data set. The main trends of variability identified consisted of: 1) A contrast between the community composition of the upper and the lower parts of the euphotic zone, expressed respectively by positive or negative scores of the first principal component, which was positively correlated with taxa such as the dinoflagellates Oxytoxum minutum and Scrippsiella spp., and the coccolithophores Discosphaera tubifera and Syracosphaera pulchra (HOL and HET), and negatively correlated with taxa like Ophiaster hydroideus (coccolithophore) and several diatoms, 2) a general abundance gradient between phytoplankton-rich regions with high abundances of dinoflagellate, coccolithophore and ciliate taxa, and phytoplankton-poor regions (second principal component), 3) differences in dominant phytoplankton and ciliate taxa among the Atlantic, the Indian and the Pacific oceans (third principal component) and 4) the occurrence of a diatom-dominated assemblage (the fourth principal component assemblage), including several pennate taxa, Planktoniella sol, Hemiaulus hauckii and Pseudo-nitzschia spp., in the divergence regions. Our findings indicate that consistent assemblages of co-occurring phytoplankton taxa can be identified and that their distribution is best explained by a combination in different degrees of both environmental and historical influences., This work was supported by Consolider-Ingenio 2010, CSD2008-00077 of the former Spanish Ministerio de Ciencia e Innovación (ES) (Now Ministerio de Economía y Competitividad, www.mineco.gob.es); Consejo Superior de Investigaciones Científicas (CSIC) (ES)

Background Published genotype/phenotype data on McArdle disease are limited in sample size. A single national (Spanish) registry of patients with McArdle disease was created with the purpose of analysing their genotypic and phenotypic characteristics. Methods A cross sectional study was conducted, collecting demographic, family history, clinical, genotype and functional capacity data from all patients diagnosed with McArdle disease in the Spanish National Health System up to December 2010. Results 239 cases were recorded (all of Caucasian descent, 102 women; mean±SD age 44±18 years (range 9, 93)); prevalence of ∼1/167 000 people. Two mutant PYGM alleles were identified in 99.6% of cases. Although there was heterogeneity in the severity of symptoms, there were four common diagnostic features: (1) 99.5% of patients reported a history of acute crises of exercise intolerance (accompanied by recurrent myoglobinuria in 50% of cases); (2) in 58% of patients, symptoms started in the first decade of life; (3) 86% of patients repeatedly experienced the ‘second wind’ phenomenon over life; and (4) 99% of patients had a high basal serum level of total creatine kinase (>200 U/l). Clinical presentation of the disease was similar in men and women and worsened with age. Patients who were physically active had higher levels of cardiorespiratory fitness (by 23%, p=0.003) and were more likely to improve their clinical course over a 4 year period compared with inactive patients (OR 225; 95% CI 20.3 to 2496.7). Conclusions The main clinical features of McArdle disease are generally homogeneous and frequently appear during childhood; clinical condition deteriorates with ageing. Active patients have a better clinical outcome and functional capacity.

Acylhomoserine lactone (AHLs)-mediated quorum-sensing (QS) processes seem to be common in the marine environment and among marine pathogenic bacteria, but no data are available on the prevalence of bacteria capable of interfering with QS in the sea, a process that has been generally termed 'quorum quenching' (QQ). One hundred and sixty-six strains isolated from different marine dense microbial communities were screened for their ability to interfere with AHL activity. Twenty-four strains (14.4%) were able to eliminate or significantly reduce N-hexanoyl-l-homoserine lactone activity as detected by the biosensor strain Chromobacterium violaceum CV026, a much higher percentage than that reported for soil isolates, which reinforces the ecological role of QS and QQ in the marine environment. Among these, 15 strains were also able to inhibit N-decanoyl-l-homoserine lactone activity and all of them were confirmed to enzymatically inactivate the AHL signals by HPLC-MS. Active isolates belonged to nine different genera of prevalently or exclusively marine origin, including members of the Alpha- and Gammaproteobacteria (8), Actinobacteria (2), Firmicutes (4) and Bacteroidetes (1). Whether the high frequency and diversity of cultivable bacteria with QQ activity found in near-shore marine isolates reflects their prevalence among pelagic marine bacterial communities deserves further investigation in order to understand the ecological importance of AHL-mediated QS and QQ processes in the marine environment.

Lima-Mendez, Gipsi ... et. al.-- 9 pages, 4 figures, 1 table, supplementary materials www.sciencemag.org/content/348/6237/1262073/suppl/DC1, Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic zone interactome using environmental factors and organismal abundance profiles and found that environmental factors are incomplete predictors of community structure. We found associations across plankton functional types and phylogenetic groups to be nonrandomly distributed on the network and driven by both local and global patterns. We identified interactions among grazers, primary producers, viruses, and (mainly parasitic) symbionts and validated networkgenerated hypotheses using microscopy to confirm symbiotic relationships. We have thus provided a resource to support further research on ocean food webs and integrating biological components into ocean models. © 2015, American Association for the Advancement of Science. All rights reserved, We thank the commitment of the following people and sponsors:Centre National de la Recherche Scientifique (CNRS) (in particular, Groupement de Recherche GDR3280), European Molecular Biology Laboratory (EMBL), Genoscope/CEA, VIB, Stazione Zoologica Anton Dohrn, UNIMIB, Fund for Scientific Research – Flanders (G.L.M., K.F., S.C., and J.R.), Rega Institute (J.R.), KU Leuven(J.R.), The French Ministry of Research, the French Government “Investissements d’Avenir” programmes OCEANOMICS (ANR-11-BTBR-0008), FRANCE GENOMIQUE (ANR-10-INBS-09-08), MEMO LIFE (ANR-10-LABX-54), PSL* Research University (ANR-11-IDEX-0001-02), ANR (projects POSEIDON/ANR-09-BLAN-0348, PHYTBACK/ANR-2010-1709-01, PROMETHEUS/ANR-09-PCSGENM- 217, TARA GIRUS/ANR-09-PCS-GENM-218, European Union FP7 (MicroB3/No.287589, IHMS/HEALTH-F4-2010-261376, ERC Advanced Grant Awards to CB (Diatomite: 294823), Gordon and Betty Moore Foundation grant (3790) to M.B.S., Spanish Ministry of Science and Innovation grant CGL2011-26848/BOS MicroOcean PANGENOMICS to S.G.A., TANIT (CONES 2010-0036) from the Agència de Gestió d´Ajusts Universitaris i Reserca funded to S.G.A., JSPS KAKENHI grant number 26430184 to H.O., FWO, BIO5, Biosphere 2, Agnès b., the Veolia Environment Foundation, Region Bretagne, Lorient Agglomeration, World Courier, Illumina, the EDF Foundation, FRB, the Prince Albert II de Monaco Foundation, Etienne Bourgois, the Tara schooner, and its captain and crew. We are also grateful to the French Ministry of Foreign Affairs for supporting the expedition and to the countries that graciously granted sampling permissions. Tara Oceans would not exist without continuous support from 23 institutes (http://oceans.taraexpeditions.org)

Acylhomoserine lactone (AHLs)-mediated quorum-sensing (QS) processes seem to be common in the marine environment and among marine pathogenic bacteria, but no data are available on the prevalence of bacteria capable of interfering with QS in the sea, a process that has been generally termed 'quorum quenching' (QQ). One hundred and sixty-six strains isolated from different marine dense microbial communities were screened for their ability to interfere with AHL activity. Twenty-four strains (14.4%) were able to eliminate or significantly reduce N-hexanoyl-l-homoserine lactone activity as detected by the biosensor strain Chromobacterium violaceum CV026, a much higher percentage than that reported for soil isolates, which reinforces the ecological role of QS and QQ in the marine environment. Among these, 15 strains were also able to inhibit N-decanoyl-l-homoserine lactone activity and all of them were confirmed to enzymatically inactivate the AHL signals by HPLC-MS. Active isolates belonged to nine different genera of prevalently or exclusively marine origin, including members of the Alpha- and Gammaproteobacteria (8), Actinobacteria (2), Firmicutes (4) and Bacteroidetes (1). Whether the high frequency and diversity of cultivable bacteria with QQ activity found in near-shore marine isolates reflects their prevalence among pelagic marine bacterial communities deserves further investigation in order to understand the ecological importance of AHL-mediated QS and QQ processes in the marine environment.

[Fundamento y objetivo]: Un amplio espectro de enfermedades mitocondriales está producido por mutaciones de POLG y se caracterizan por una alteración en la integridad del genoma mitocondrial. La oftalmoplejía progresiva externa suele ser el marcador clínico en los casos de deleciones múltiples, pero no lo es en aquellas enfermedades que cursan con depleción del ácido desoxirribonucleico mitocondrial (ADNmt). En este trabajo presentamos un paciente con la tríada clínica que define el síndrome de neuropatía sensorial atáxica, disartria y oftalmoplejía, las mutaciones del gen POLG y la presencia inusual de una marcada disminución en el contenido del ADNmt en el músculo esquelético. [Paciente y método]: El paciente presentó un cuadro clínico caracterizado por neuropatía sensorial atáxica, disartria y oftalmoplejía. El diagnóstico se realizó mediante estudios histológicos y análisis molecular del ADNmt y del gen POLG. [Resultados]: La biopsia del nervio sural detectó una pérdida intensa de las fibras nerviosas mielinizadas gruesas. El estudio molecular reveló mutaciones en el gen POLG, así como deleciones múltiples y una marcada depleción del genoma mitocondrial. [Conclusiones]: Los pacientes con síndromes atáxicos de origen mitocondrial presentan fenotipos mitocondriales moleculares diferentes, por lo que se aconseja la búsqueda de mutaciones del gen POLG en todos ellos, independientemente de la anomalía que presenten en el genoma mitocondrial.