High bicarbonate assimilation in the dark by Arctic bacteria

10 páginas, 4 figuras, 1 tabla.Although both autotrophic and heterotrophic microorganisms incorporate CO2 in the dark through different metabolic pathways, this process has usually been disregarded in oxic marine environments. We studied the significance and mediators of dark bicarbonate assimilation in dilution cultures inoculated with winter Arctic seawater. At stationary phase, bicarbonate incorporation rates were high (0.5–2.5 μg C L−1 d−1) and correlated with rates of bacterial heterotrophic production, suggesting that most of the incorporation was due to heterotrophs. Accordingly, very few typically chemoautotrophic bacteria were detected by 16S rRNA gene cloning. The genetic analysis of the biotin carboxylase gene accC putatively involved in archaeal CO2 fixation did not yield any archaeal sequence, but amplified a variety of bacterial carboxylases involved in fatty acids biosynthesis, anaplerotic pathways and leucine catabolism. Gammaproteobacteria dominated the seawater cultures (40–70% of cell counts), followed by Betaproteobacteria and Flavobacteria as shown by catalyzed reporter deposition fluorescence in situ hybridization (CARDFISH). Both Beta- and Gammaproteobacteria were active in leucine and bicarbonate uptake, while Flavobacteria did not take up bicarbonate, as measured by microautoradiography combined with CARDFISH. Within Gammaproteobacteria, Pseudoalteromonas-Colwellia and Oleispira were very active in bicarbonate uptake (ca. 30 and 70% of active cells, respectively), while the group Arctic96B-16 did not take up bicarbonate. Our results suggest that, potentially, the incorporation of CO2 can be relevant for the metabolism of specific Arctic heterotrophic phylotypes, promoting the maintenance of their cell activity and/or longer survival under resource depleted conditions.This work is a contribution to the International Polar Year – Circumpolar Flaw Lead system study (IPY-CFL 2007/2008) lead by D. Barber (University of Manitoba) supported through grants from the Canadian IPY Federal Program Office, the National Sciences and Engineering Research Council, grant BOREAL (CLG2007-28872-E/ANT) from the Spanish Ministry of Science and Innovation to C.P.-A., and grants from the Swedish Research Council to S.B and L.A.S. L.A.S. was supported by a Marie Curie Intraeuropean Fellowship (CHEMOARC PIEF-GA-2008- 221121), E.O.C by the Spanish grant CGL2009-13318- BOS, and P. E. G by a Marie Curie grant (CRENARC MEIF-CT-2007-040247).Peer reviewe

Patterns of Gene Flow Define Species of Thermophilic Archaea

A genomic view of speciation in Archaea shows higher rates of gene flow within coexisting microbial species than between them

Neue linguistische Methoden und arbeitstechnische Verfahren in der Erschliessung der ägyptischen Grammatik

15 páginas, 1 tabla, 6 figuras.Does diversity beget diversity? Diversity includes a diversity of concepts because it is linked to variability in and of life and can be applied to multiple levels. The connections between multiple levels of diversity are poorly understood. Here, we investigated the relationships between genetic, bacterial, and chemical diversity of the endangered Atlanto-Mediterranean sponge Spongia lamella. These levels of diversity are intrinsically related to sponge evolution and could have strong conservation implications. We used microsatellite markers, denaturing gel gradient electrophoresis and quantitative polymerase chain reaction, and high performance liquid chromatography to quantify genetic, bacterial, and chemical diversity of nine sponge populations. We then used correlations to test whether these diversity levels covaried. We found that sponge populations differed significantly in genetic, bacterial, and chemical diversity. We also found a strong geographic pattern of increasing genetic, bacterial, and chemical dissimilarity with increasing geographic distance between populations. However, we failed to detect significant correlations between the three levels of diversity investigated in our study. Our results suggest that diversity fails to beget diversity within a single species and indicates that a diversity of factors regulates a diversity of diversities, which highlights the complex nature of the mechanisms behind diversityResearch funded by grants from the Agence Nationale de la Recherche (ECIMAR), from the Spanish Ministry of Science and Technology SOLID (CTM2010-17755) and Benthomics (CTM2010-22218-C02-01) and the BIOCAPITAL project (MRTN-CT-2004-512301) of the European Union. This is a contribution of the Consolidated Research Group ‘‘Grupo de Ecologı´a Bento´nica,’’ SGR2009-655.Peer reviewe

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists

The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.This work was supported by the projects CGL2012-39627-C03-01 and 02 of the Spanish Ministry of Economy and Competitiveness, which were also co-financed with FEDER support from the European Union. TG group research is funded in part by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund grant (NPRP 5-298-3-086) and a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement no. ERC-2012-StG-310325)

Pleiotropic Roles of a Ribosomal Protein in Dictyostelium discoideum

The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over- or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over- or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect – specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels

Evolution of the TOR Pathway

The TOR kinase is a major regulator of growth in eukaryotes. Many components of the TOR pathway are implicated in cancer and metabolic diseases in humans. Analysis of the evolution of TOR and its pathway may provide fundamental insight into the evolution of growth regulation in eukaryotes and provide a practical framework on which experimental evidence can be compared between species. Here we performed phylogenetic analyses on the components of the TOR pathway and determined their point of invention. We find that the two TOR complexes and a large part of the TOR pathway originated before the Last Eukaryotic Common Ancestor and form a core to which new inputs have been added during animal evolution. In addition, we provide insight into how duplications and sub-functionalization of the S6K, RSK, SGK and PKB kinases shaped the complexity of the TOR pathway. In yeast we identify novel AGC kinases that are orthologous to the S6 kinase. These results demonstrate how a vital signaling pathway can be both highly conserved and flexible in eukaryotes

Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye

Background: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation. Methods: S. cerevisiae W303-1A wt strain and gup1Δ mutant were used as previously described to generate biofilmlike mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. Results: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.Conclusions: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1Δ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.This work was funded by the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and by national funds from FCT I.P. through the strategic funding UID/BIA/04050/2013. Fábio Faria-Oliveira was supported by a PhD scholarship (SFRH/BD/45368/2008) from FCT (Fundação para a Ciência e a Tecnologia). We thank David Caceres and Montserrat MartinezGomariz from the Unidad de Proteómica, Universidad Complutense de Madrid – Parque Científico de Madrid, Spain for excellent technical assistance in the successful implementation of all proteomics procedures including peptide identification, and Joana Tulha from the CBMA, Universidade do Minho, Portugal, for helping with the SDS-PAGE experiments, and the tedious and laborious ECM extraction procedures. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with the dataset identifier PXD001133. We would like to thank the PRIDE team for all the help and support during the submission process.info:eu-repo/semantics/publishedVersio

Effect of Saharan dust inputs on bacterial activity and community composition in Mediterranean lakes and reservoirs

11 páginas, 5 tablas, 6 figuras.We assessed the effects of Saharan dust inputs of particulate matter (PM), total phosphorus (TP), total nitrogen, and water soluble organic carbon (WSOC) on bacterial abundance (BA) in two alpine lakes and two reservoirs in the Mediterranean region. We also experimentally assessed the effects of dust inputs on bacterial activity and community composition and explored the presence of airborne bacteria. We found synchronous BA dynamics at least in one of the study years for each corresponding pair of ecosystems, suggesting an external control. The link between BA dynamics and inputs of PM, WSOC, or TP occurred only in those ecosystems with severe P-limitation and low dissolved organic carbon. The response was most intense in the most P-limited ecosystem. Dust addition had a significant positive effect on bacterial growth and abundance, but not on richness, diversity, or composition of the indigenous bacterial assemblages. We also obtained experimental evidence that some airborne bacteria could develop in oligotrophic waters by observing the growth of gamma-proteobacteria, a group poorly represented in natural aquatic environments.This research was supported by the projects ECOSENSOR (Fundación BBVA, BIOCON04/009) and MICROBIOGEOGRAPHY (080-2007) to IR, REN03-03038 (Ministerio de Ciencia y Tecnologı´a) to RMB, and AERBAC (079-2007) to EOC, FPU (Formacio´n del Profesorado Universitario), and FPI (Formacio´n del Personal Investigador) grants from Spanish Government to E. Ortega-Retuerta, E. Pulido-Villena, and O. Romera.Peer reviewe

Contrasting activity patterns determined by BrdU incorporation in bacterial ribotypes from the Arctic Ocean in winter

7 páginas, 2 tablas, 3 figuras.The winter Arctic Ocean is one of the most unexplored marine environments from a microbiological perspective. Heterotrophic bacteria maintain their activity at a baseline level during the extremely low-energy conditions of the winter, but little is known about the specific phylotypes that have the potential to survive and grow in such harsh environment. In this study, we aimed at identifying actively growing ribotypes in winter Arctic Ocean seawater cultures by experimental incubations with the thymidine analog bromodeoxyuridine (BrdU), followed by immunocapturing, terminal restriction fragment length polymorphism fingerprinting, cloning, and sequencing the 16S rRNA gene. We incubated water collected at different months over the Arctic winter and showed that the actively growing bacterial fraction, taking up BrdU, represented only a subset of the total community. Among the BrdU-labeled bacterial taxa we identified the Flavobacteria Polaribacter, the Alphaproteobacteria SAR11, the Gammaproteobacteria Arctic 96B-16 cluster and, predominately, members of Colwellia spp. Interestingly, Colwellia sequences formed three clusters (93 and 97% pairwise 16S rRNA identity) that contributed in contrasting ways to the active communities in the incubations. Polaribacter, Arctic 96B-16 and one cluster of Colwellia were more abundant in the active community represented by the BrdU-labeled DNA. In contrast, SAR11 and two other Colwellia clusters were underrepresented in the BrdU-labeled community compared to total communities. Despite the limitation of the long incubations needed to label slow growing arctic communities, the BrdU approach revealed the potential for active growth in low-energy conditions in some relevant groups of polar bacteria, includingPolaribacter and Arctic 96B-16. Moreover, under similar incubation conditions, the growth of differentColwellia ribotypes varied, suggesting that related clusters of Colwellia may have distinct metabolic features.Thisworkisacontribution totheInternationalPolarYear–CircumpolarFlawLeadsys- temstudy(IPY-CFL2007/2008)andArcticNet.PierreE.Galand and LauraAlonso-SáezweresupportedbyMarieCurieIntra- EuropeanFellowshipgrantsCRENARCMEIF-CT-2007-040247 and CHEMOARCPIEF-GA-2008-221121,respectively.Connie LovejoywassupportedbytheNaturalSciencesandEngineering ResearchCouncilofCanada(NSERC)SROandDiscoverygrants and ArcticNet.TheworkdoneinEmilioO.Casamayorlabwas supportedbygrantPIRENACGL2009-13318fromtheSpanish MinisteriodeCienciaeInnovación(MICINN)andthemolecu- lar workcarriedoutinUppsalawasfundedbygrantsfromthe SwedishResearchCounciltoStefanBertilsson.Peer reviewe

Inter-annual recurrence of archaeal assemblages in the coastal NW Mediterranean Sea (Blanes Bay Microbial Observatory)

9 páginas, 5 figuras.We report a long-term (i.e., 4.5 consecutive yr) monitoring of surface marine archaeal assemblages of the coastal Mediterranean Sea using quantitative polymerase chain reaction against specific phylogenetic and functional genes, and, for some specific samples, clone libraries of the 16S ribosomal ribonucleic acid gene. Archaea had a marked seasonal periodicity, with recurrent peaks of abundance in December and January and very low occurrence during summer, parallel to temporal changes in community composition. Group II.b Euryarchaeota sequences were mostly present during winter when water was nutrient-enriched, and phytoplankton were abundant. Group II.a sequences were, in turn, more abundant during summer when the water column is stratified, and nutrient concentrations and phytoplankton stocks were lower. Group I Crenarchaeota abundance was highest during winter and significantly correlated with that of archaeal ammonia monooxygenase (amoA) gene copies and nitrite concentrations, suggesting that Group I Crenarchaeota were ammonia oxidizers. The periodicity of archaeal assemblages matched the strong and predictable seasonality of the surface-water conditions in the northwestern Mediterranean Sea, and suggests a low degree of functional redundancy between archaeal groups. The distinct seasonal dynamics for Group II.a and II.b Euryarchaeota, and their close association with major ecosystem processes, indicate that they may play an important but as yet largely unknown role in the ocean.PEG was supported by a European Marie Curie grant (CRENARC MEIFCT- 2007-040247), and CGP by CONSOLIDER-INGENIO 2010 project GRACCIE CSD2007-00067 from the Spanish Ministerio de Ciencia e Innovación (MICINN). Sampling was funded by various projects: European Union project BASICS (EVK3-CT- 2002-00078), and Spanish grants ESTRAMAR (CTM2004-12631/ MAR, MEC), MODIVUS (CTM2005-04795/MAR), and SUMMER (CTM2008-03309/MAR). Lab research was founded by grants CRENARC and MICINN grant CRENYC CGL2006- 12058-BOS to EOC.Peer reviewe