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Este artículo contiene 8 páginas, 4 figuras., The atmosphere is a potential pathway for global-scale and long-range dispersal of viable microorganisms, promoting biological interconnections among the total environment. We aimed to provide relevant baseline information for long-range long-term intercontinental exchange of potentially infectious airborne microorgan-isms of major interest in environmental and health-related disciplines. We used an interannual survey (7-y) with wet depositions fortnightly collected above the boundary layer (free troposphere) at a remote high-elevation LTER (Long-Term-Ecological-Research) site, analyzed by 16S and 18S rRNA genes, and compared to a data-base of 475 well-known pathogens. We applied a conservative approach on close relatives of pathogenic species (>98% identity) standing their theoretical upper limit for atmospheric baseline relative abundances. We iden-tified c. 2–3% of the total airborne microbiota as potential pathogens. Their most frequent environmental origins were soil, aquatic, and anthropogenic sources. Phytopathogens (mostly fungi) were the potential infectious agents most widely present. We uncovered consistent interannual dynamics with taxa foreseeable over time (i.e., predictable seasonal behavior) and under recurrent environmental scenarios (e.g., Saharan dust intrusions), respectively, being highly valuable microbial forensic environmental indicators. Up to 8 bacterial and 21 fungal genera consistently showed temporal abundances and recurrences unevenly distributed. Incidence of allergenic fungi was lower in summer, and significantly higher in spring. Close relatives to Coccidioides posadasii consis-tently showed higher signals (i.e., high specificity and high fidelity) in winter, whereas Cryptococcus neoformans had a significant signal in spring. Along Saharan dust intrusions, the bacterial phytopathogens Acidovorax avenae and Agrobacterium tumefaciens and the fungal phytopathogens Pseudozyma hubeiensis and Peniophora sp. consis-tently showed higher signals. Potential human pathogens showed low proportion, being mostly fungal allergens. Microorganisms related to obligated human, amphibian and fish pathogens were commonly found in winter. More studies in remote field sites above the boundary layer will unveil whether or not a similar trend is found globally.

The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity.

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

Este artículo contiene 13 páginas, 7 figuras., Studies connecting microbiome composition and functional performance in wildlife have received little attention and understanding their connections with wildlife physical condition are sorely needed. We studied the variation in gut microbiota (hard fecal pellets) between allopatric subspecies of the European wild rabbit in wild populations and in captured individuals studied under captivity. We evaluated the infuence of environmental and host-specifc factors. The microbiome of wild rabbit populations reduced its heterogeneity under controlled conditions. None of the host-specifc factors tested correlated with the microbiota composition. We only observed signifcant intra-group dispersion for the age factor. The most diverse microbiomes were rich in Ruminococcaceae potentially holding an enriched functional profle with dominance of cellulases and xylanases, and suggesting higher efciency in the digestion of fber-rich food. Conversely, low diversity gut microbiomes showed dominance of Enterobacteriaceae potentially rich in amylases. We preliminary noticed geographical variations in feld populations with higher dominance of Ruminococcaceae in south-western than in north-eastern Spain. Spatial diferences appeared not to be subspecies driven, since they were lost in captivity, but environmentally driven, although diferences in social structure and behavior may also play a role that deserve further investigations. A marginally signifcant relationship between the Ruminococcaceae/Enterobacteriaceae ratio and potential life expectancy was observed in captive rabbits. We hypothesize that the gut microbiome may determine the efciency of feeding resource exploitation, and can also be a potential proxy for life expectancy, with potential applications for the management of declining wild herbivorous populations. Such hypotheses remain to be explored in the future., Funding was provided by grants INTERACTOMA RTI2018-101205-B-I00 and CGL2013-43197-R from the Spanish Ofce of Science (MICIUN, MINECO) and European funding (ERDF/FEDER) to EOC and RF, respectively, and GECA-2017SGR910, Generalitat de Catalunya.

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis., This research was funded by grant BFU2017-82574-P to JA and AC, and BFU2016-75352-P to JdlC (AEI/FEDER, EU; Ministerio de Industria, Economía y Competitividad, Spain, and ERDF). CC and JF-F were recipient of a PhD fellowship from the Ministerio de Economía y Competitividad (Spain). CZ was recipient of a PhD fellowship from the China Scholarship Council. DV was recipient of a PhD fellowship from UAB.

Este artículo contiene 10 páginas, 5 figuras, 1 tabla, We studied the core bacterial communities of 19 sponge species from Nha Trang Bay (Central Vietnam), with particular emphasis on the contribution of planktonic seawater bacteria to the sponge core microbiomes. To ensure consistent sponge-microbe associations and accurate identification of planktonic bacteria transmitted from seawater, we were very restrictive with the definition of the sponge core microbiomes (present in all the replicates), and with the identification of valid biological 16S rRNA gene sequences (100% sequence identity) that belonged to potentially different bacterial taxa. We found a high overlap (>50% relative abundance) between the sponge species core microbiome and the seawater bacterial core in ca. a half of the studied species, including representatives of both, HMA and LMA sponges. From our restrictive analysis, we point to horizontal transmission as a relevant way of symbiont acquisition in sponges. Some species-specific recognition mechanisms may act in sponges to enrich specific seawater bacteria in their tissues. These mechanisms would allow the maintenance of bacterial communities in a species across geographical ranges. Moreover, besides contrasting preferences in bacteria selection from seawater, divergent physiological traits may also account for the different microbiomes in species of HMA and LMA sponges., This research was partially funded by projects MarSymbiOmics (CTM2013-43287-P) and PopCOmics (CTM2017-88080, MINECO/AEI/FEDER/UE) from the Spanish Government. JC and EOC were supported by the project BRIDGES (CGL2015-69043-P).

Este artículo contiene 6 páginas, 4 figuras, 1 tabla, The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have highoccurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential rolein nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probeSAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequencesin databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabiliza-tion for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditionswere optimized on enrichment cultures of the target species N. devanaterra, as well as the non-targetammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competi-tor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design andapplication of two adjacent helpers. Probe performance was tested in soil samples along a pH gradient,and counting results matched the expected in situ distributions. Probe SAG357 and the CARD-FISH pro-tocol developed in the present study will help to improve the current understanding of the ecology andphysiology of N. devanaterra and its relatives in natural environments., This research was supported by grantsPIRENA-CGL2009-13318 and DARKNESS-CGL2012-32747 from theSpanish Office of Science (MINECO) to EOC, and by a Spanish FPIpre-doctoral scholarship (MINECO-PIRENA) to CRO. JBC and BFacknowledge funding from the Max Planck Society.

Este artículo contiene 11 páginas, 2 tablas, 5 figuras., A rich eukaryotic planktonic community exists in high-mountain lakes despite the diluted, oligotrophic and cold, harsh prevailing conditions. Attempts of an overarching appraisal have been traditionally hampered by observational limitations of small, colorless, and soft eukaryotes. We aimed to uncover the regional eukaryotic biodiversity of a mountain lakes district to obtain general conclusions on diversity patterns, dominance, geographic diversification, and food-web players common to oligotrophic worldwide distributed freshwater systems. An unprecedented survey of 227 high-altitude lakes comprising large environmental gradients was carried out using Illumina massive tag sequencing of the 18S rRNA gene. We observed a large Chrysophyceae dominance in richness, abundance and novelty, and unveiled an unexpected richness in heterotrophic phagotrophs and parasites. In particular, Cercozoa and Chytridiomycota showed diversity features similar to the dominant autotrophic groups. The prominent beta-dispersion shown by parasites suggests highly specific interactions and a relevant role in food webs. Interestingly, the freshwater Pyrenean metacommunity contained more diverse specific populations than its closest marine oligotrophic equivalent, with consistently higher betadiversity. The relevance of unseen groups opens new perspectives for the better understanding of planktonic food webs. Mountain lakes, with remarkable environmental idiosyncrasies, may be suitable environments for the genetic diversification of microscopic eukaryotic life forms., This research was funded by Grants BRIDGES CGL2015–69043-P from the Spanish Office of Science (MINECO) and European funding (ERDF) and DISPERSAL (Ref. 829/2013) from the Spanish National Parks research program (OAPN-MAGRAMA), to EOC, and LACUS CGL2013–45348-P (MINECO-ERDF) to JC. We thank technicians and students who participated in the field sampling expeditions, and specially M Sala and C Palacin for their excellent performance, and X Torras for ArcGIS support

We characterized the rich Archaea microbiome of shallow inland lakes (Monegros Desert, NE Spain) by 16S rRNA gene tag sequencing covering a wide salinity range (0.1-40% w/v) along three years. Up to 990 OTUs (370 OTUs each), followed by Halobacteria (105), Nanohaloarchaeota (62) and Thermoplasmata (19). Overall, the distribution of genetic richness was strongly correlated with environmental niche amplitude, but not with occurrence. We unveiled high occurrence for a very rich Woesearchaeota assemblage, and an unexpected positive correlation of Pacearchaeota abundance with salinity at >15% dissolved salt content. The estimated dynamic behaviour (temporal “turnover” rates of presence/absence data) unveiled Thaumarchaeota and Halobacteria as the most dynamic groups, and Aenigmarchaeota and Thermoplasmata as the most stable. The DPANN Pacearchaeota, Woesearchaeota, and Nanohaloarchaeota showed intermediate rates, suggesting higher resilience to environmental perturbations. A rich and dynamic Archaea microbiome was unveiled including unseen ecological traits for relevant members of the still largely unknown DPANN group, supporting a strong ecological differentiation between Pacearchaeota and Woesearchaeota., Funding was provided by grant BRIDGES, CGL2015-69043-P from the Spanish Office of Science (MINECO) and European funding (ERDF) to EOC and DA. MMS was funded by the Spanish FPI PhD scholarships program (BRIDGES MINECO)

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood-associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI-TOF MS, serotyped and characterized by pulsed-field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (formerly biotype 2) or to any of the fish-related serovars, they all had identical phenotypes, antimicrobial susceptibility profiles and PFGE patterns, which together with their isolation in pure culture from internal organs is strongly indicative of their clinical significance. Moreover, Vv isolates harboured important genetic markers of human virulence potential: they had the clinical variant of the vcg gene, gave the 338 bp DNA amplification product of the pilF gene and resisted the bactericidal activity of human serum. All these results strongly suggest that these Vv isolates should be considered potentially virulent for humans. These results extend the range of fish species affected by V. vulnificus, confirm the threat that this pathogen represents to aquatic animals and highlight the risk that this bacterial pathogen poses to human health.

Este artículo contiene 13 páginas, 5 figuras, 1 tabla., Sponge diversity has been reported to decrease from well-preserved to polluted environments, but whether diversity and intra-species variation of their associated microbiomes also change as function of environmental quality remains unknown. Our study aimed to assess whether microbiome composition and structure are related to the proliferation of some sponges and not others under degraded conditions. We characterized the most frequent sponges and their associated bacteria in two close areas (impacted and well-preserved) of Nha Trang Bay (Indo-Pacific). Sponge assemblages were richer and more diverse in the well-preserved reefs, but more abundant (individuals/m. transect) in the impacted environments, where two species (Clathria reinwardti and Amphimedon paraviridis) dominated. Sponge microbiomes from the polluted zones had, in general, lower bacterial diversity and core size and consequently, higher intra-species dispersion than microbiomes of sponges from the well-preserved environments. Microbial communities reflect the reduction of diversity and richness shown by their host sponges. In this sense, sponges with less complex and more variable microbiomes proliferate under degraded environmental conditions, following the ecological paradigm that negatively correlates community diversity and environmental degradation. Thereby, the diversity and structure of sponge microbiomes might indirectly determine the presence and proliferation of sponge species in certain habitats., This research was partially funded by projects MarSymbiOmics (CTM2013-43287-P) and PopCOmics (CTM2017-88080 and MCIU/AEI/FEDER/UE) from the Spanish MINECO. JC, XT-M, and EC were supported by the project INTERACTOMA (RTI2018-101205-B-I00).

11 páginas, 6 figuras., A natural planktonic bloom of a brown-pigmented photosynthetic green sulfur bacteria (GSB) from the disphotic zone of karstic Lake Banyoles (NE Spain) was studied as a natural enrichment culture from which a nearly complete genome was obtained after metagenomic assembly. We showed in situ a case where horizontal gene transfer (HGT) explained the ecological success of a natural population unveiling ecosystem-specific adaptations. The uncultured brown-pigmented GSB was 99.7% identical in the 16S rRNA gene sequence to its green-pigmented cultured counterpart Chlorobium luteolum DSM 273T. Several differences were detected for ferrous iron acquisition potential, ATP synthesis and gas vesicle formation, although the most striking trait was related to pigment biosynthesis strategy. Chl. luteolum DSM 273T synthesizes bacteriochlorophyll (BChl) c, whereas Chl. luteolum CIII incorporated by HGT a 18-kbp cluster with the genes needed for BChl e and specific carotenoids biosynthesis that provided ecophysiological advantages to successfully colonize the dimly lit waters. We also genomically characterized what we believe to be the first described GSB phage, which based on the metagenomic coverage was likely in an active state of lytic infection. Overall, we observed spread HGT and we unveiled clear evidence for virus-mediated HGT in a natural population of photosynthetic GSB., This research was funded by grant DARKNESS CGL2012- 32747 from the Spanish Office of Science (MINECO) to EOC and by the Global Ocean Sampling Project supported by the Beyster Family Foundation Fund of the San Diego Foundation and the Life Technology Foundation (to JCVI). Work on BChl e biosynthesis and the genomics of GSB in the laboratory of DAB was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-94ER20137.

Lack of the yeast Ptc1 Ser/Thr protein phosphatase results in numerous phenotypic defects. A parallel search for high-copy number suppressors of three of these phenotypes (sensitivity to Calcofluor White, rapamycin and alkaline pH), allowed the isolation of 25 suppressor genes, which could be assigned to three main functional categories: maintenance of cell wall integrity (CWI), vacuolar function and protein sorting, and cell cycle regulation. The characterization of these genetic interactions strengthens the relevant role of Ptc1 in downregulating the Slt2-mediated CWI pathway. We show that under stress conditions activating the CWI pathway the ptc1 mutant displays hyperphosphorylated Cdc28 kinase and that these cells accumulate with duplicated DNA content, indicative of a G2-M arrest. Clb2-associated Cdc28 activity was also reduced in ptc1 cells. These alterations are attenuated by mutation of the MKK1 gene, encoding a MAP kinase kinase upstream Slt2. Therefore, our data show that Ptc1 is required for proper G2-M cell cycle transition after activation of the CWI pathway.

8 páginas, 4 figuras, Although elevational gradients in microbial biodiversity have attracted increasing attention recently, the generality in the patterns and underlying mechanisms are still poorly resolved. Further, previous studies focused mostly on species richness, while left understudied evenness, another important aspect of biodiversity. Here, we studied the elevational patterns in species richness and evenness of stream biofi lm bacteria and diatoms in six mountains in Asia and Europe. We also reviewed published results for elevational richness patterns for soil and stream microbes in a literature analysis. Our results revealed that even within the same ecosystem type (that is, stream) or geographical region, bacteria and diatoms showed contrasting patterns in diversity. Stream microbes, including present stream data, tend to show signifi cantly increasing or decreasing elevational patterns in richness, contrasting the fi ndings for soil microbes that typically showed nonsignifi cant or signifi cantly decreasing patterns. In all six mountains for bacteria and in four mountains for diatoms, species richness and evenness were positively correlated. Th e variation in bacteria and diatom richness and evenness were substantially explained by anthropogenic driven factors, such as total phosphorus (TP). However, diatom richness and evenness were also related to diff erent main drivers as richness was mostly related to pH, while evenness was most explained by TP. Our results highlight the lack of consistent elevational biodiversity patterns of microbes and further indicate that the two facets of biodiversity may respond diff erently to environmental gradients., JW was supported by NSFC grant 41273088, 41571058, 40903031 and CAS oversea visiting scholarship (2011-115). JS and JW were supported by Emil Aaltonen Foundation. JS and JW were supported by 973 Program (2012CB956100). Th e fi eld trips were partly supported by Air and Water Conservation Fund (GEFC12-14, National Geography of Science) to JW, and DISPERSAL 829/2013 from the Spanish National Parks Research Programme OAPNMAGRAMA to EOC.

We carried out a regional survey on the archaea composition from surface waters of > 300 high-altitude Pyrenean lakes (average altitude 2300 m, pH range 4.4-10.1) by 16S rRNA gene tag sequencing. Relative Archaea abundances ranged between 0% and 6.3% of total prokaryotes amplicons in the polymerase chain reaction (PCR) mixture, and we detected 769 operational taxonomic units (OTUs; grouped at 97% identity) that split into 13 different lineages, with altitude and pH having a significant effect on the community composition. Woesearchaeota and Pacearchaeota (formerly Euryarchaeota DHVEG-6 cluster) dominated the data set (83% of total OTUS), showed a high occurrence (presence in c. 75% of the lakes) and had relative abundances significantly and positively correlated with the phylogenetic diversity of bacterial communities. Micrarchaeota-Diapherotrites (formerly Euryarchaeota MEG cluster), Methanomicrobia, Thermoplasmata and ammonia-oxidizing thaumarchaeota (AOA) showed relative abundances between 1% and 3% and occurrences between 14% and 26%. Minor lineages were SM1K20, Aenigmarchaeota (formerly Euryarchaeota DSEG cluster), Methanobacteria, Bathyarchaeota and SCG. Environmental preferences substantially differed among lineages, with Aenigmarchaeota and Methanomicrobia having the largest habitat breadth, and Thermoplasmata, AOA and Micrarchaeota having the smallest. Pacearchaeota and Woesearchaeota had been mostly reported from saline habitats and sediments, but surface waters of oligotrophic alpine lakes are suitable environments for such ecologically spread and genetically diverse archaeal lineages.

High mountain lakes are, in general, highly sensitive systems to external forcing and good sentinels of global environmental changes. For a better understanding of internal lake processes, we examined microbial biodiversity and potential biogeochemical interactions in the oligotrophic deep high-mountain Lake Redon (Pyrenees, 2240m altitude) using shotgun metagenomics. We analyzed the two ends of the range of environmental conditions found in Lake Redon, at 2 and 60 m depths. Bacteria were the most abundant component of the metagenomic reads (> 90%) and the diversity indices of both taxonomic (16S and 18S rRNA) and functional (carbon-, nitrogen-, sulfur-, and phosphorous-cycling) related genes were higher in the bottom dark layer than in the upper compartment. A marked segregation was observed both in biodiversity and in the dominant energy and biomass generating pathways between the extremes. The aerobic respiration was mainly dominated by heterotrophic Burkholderiales at the top and Actinobacteria and Burkholderiales at the lake bottom. The potential for an active nitrogen cycle (nitrogen fixation, nitrification, nitrite oxidation, and nitrate reduction) was mainly found at 60 m, and potential for methanogenesis, anaerobic ammonia oxidation and dissimilatory sulfur pathways were only observed there. Some unexpected and mostly unseen energy and biomass pathways were found relevant for the biogeochemical cycling in lake Redon, i.e., those related to carbon monoxide oxidation and phosphonates processing. We provide a general scheme of the main biogeochemical processes that may operate in the sentinel deep Lake Redon. This framework may help for a better understanding of the whole lake metabolism., This research was funded by Grants INTERACTOMA RTI2018-101205-BI00 to EOC and TRANSFER CGL2016-80124-C2-1-P to JC from the Spanish Office of Science (MICIU/AEI/MINECO) and European funding (ERDF/FEDER).

Este artículo contiene 11 páginas, 6 figuras, 1 tabla., Soil microbial communities (both Bacteria and Archaea) were studied after 16S rRNA genes massive sequencing in two hypersaline and gypsum-rich contrasted sites located in NE Spain. Soilmicrobial communitieswere also locally analysed according to environmental variables, including geological, physico-chemical, biogeochemically, and climatic data. Typical soil characteristics, climate data, and plant composition clearly split the two sites andmajor differences among the microbial communities for the areas were initially expected. Overall, high values of microbial species richness (up to 2300 taxa) and ecological diversity was detected in both sites. High genetic novelty levels were found mostly to environmental sequences, highlighting the high potential for microbiological studies. In contrast to the initial expectations, a substantial overlapping betweenMonegros andGallocantamicrobeswas observed, indicating a high similarity despite of the geographical, botanical and environmental distances between sites, in agreement with both high dispersal and local selection inherent to the microbial world. The potential biogeochemical cycling showed small differences between sites, with presence of photosynthetic green and purple sulfur bacteria, cyanobacteria and aerobic and anaerobic chemolitotrophs. Potential for aerobic methane oxidation and anaerobic methanogenesis was observed in both sites, with predominance of potential nitrification mostly by ammonia-oxidizing archaea, nitrite oxidation and denitrification, and minor contribution for nitrate reduction and nitrate ammonification. The predicted functions based on the taxonomic composition showed high overlapping between the two studied regions, despite their difference in gypsum richness., This research was granted by Spanish projects BRIDGES CGL2015- 69043-P to EOC, and BASIL (PCIN-2014-106) funded by the Ministry of Economy, Industry and Competitiveness (MINECO). MMS holds a FPI predoctoral fellowship from BRIDGES-MINECO.

Root functional diversity of submerged vegetation exerts a major effect on nitrogen (N) cycling in lake sediments. This fact, however, is neglected in current N-balance models because the links between the engineering role of plants and in situ microbial N cycling are poorly understood. We hypothesized that macrophyte species with high root oxygen loss (ROL) capacity promote the highest denitrification because of a higher abundance of ammonia oxidizers and tighter coupling between nitrifiers and denitrifier communities. We sampled five small ultraoligotrophic shallow lakes with abundant macrophyte cover including sediments dominated either by Isoetes spp. (high ROL), mixed communities of natopotamids (low ROL), and unvegetated sandy sediments. At each site, we quantified denitrification (DNT) rates and proxies for the abundance of denitrifiers (nirS and nirK genes), and both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) and the diversity of nirS-harboring bacteria. Vegetated sediments showed significantly higher abundances of N-cycling genes than bare sediments. Plant communities dominated by Isoetes generated sediments with higher redox and NO3− concentrations and significantly higher DNT rates than natopotamids-dominated landscapes. Accordingly, increasing DNT rates were observed along the gradient from low ROL plants-bare sediments-high ROL plants. Significantly higher abundance of the archaeal amoA gene was recorded in sediments colonized by high ROL plants unveiling a key biogeochemical role for AOA in coupling macrophyte landscape and ecosystem denitrification.

Summary Diversity of small protists was studied in sulfidic and anoxic (euxinic) stratified karstic lakes and coastal lagoons by 18S rRNA gene analyses. We hypothesized a major sulfide effect, reducing protist diversity and richness with only a few specialized populations adapted to deal with low-redox conditions and high-sulfide concentrations. However, genetic fingerprinting suggested similar ecological diversity in anoxic and sulfurous than in upper oxygen rich water compartments with specific populations inhabiting euxinic waters. Many of them agreed with genera previously identified by microscopic observations, but also new and unexpected groups were detected. Most of the sequences matched a rich assemblage of Ciliophora (i.e., Coleps, Prorodon, Plagiopyla, Strombidium, Metopus, Vorticella and Caenomorpha, among others) and algae (mainly Cryptomonadales). Unidentified Cercozoa, Fungi, Stramenopiles and Discoba were recurrently found. The lack of GenBank counterparts was higher in deep hypolimnetic waters and appeared differentially allocated in the different taxa, being higher within Discoba and lower in Cryptophyceae. A larger number of populations than expected were specifically detected in the deep sulfurous waters, with unknown ecological interactions and metabolic capabilities.

13 páginas, 6 figuras, 2 tablas, The brine shrimp Artemia (Crustacea) and a diversity of halophilic microorganisms coexist in natural brines, salterns and laboratory cultures; part of such environmental microbial diversity is represented in the gut of Artemia individuals. Bacterial diversity in these environments was assessed by 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) fingerprinting. Eight natural locations in Chile, where A. franciscana or A. persimilis occur, were sampled for analysis of free-living and gutassociated bacteria in water from nature and laboratory cultures. The highest ecological diversity (Shannon’s index, H’) was found in brines, it decreased in the gut of wild and laboratory animals, and in laboratory water. Significant differences in H’ existed between brines and laboratory water, and between brines and gut of wild animals. The greatest similarity of bacterial community composition was between brines and the gut of field animals, suggesting a transient state of the gut microbiota. Sequences retrieved from DGGE patterns (n = 83) exhibited an average of 97.8 % identity with 41 bacterial genera from the phyla Proteobacteria (55.4 % of sequences match), Bacteroidetes (22.9 %), Actinobacteria (16.9 %) and Firmicutes (4.8 %). Environment-exclusive genera distribution was seen in Sphingomonas and Paenibacillus (gut of field animals), Amaricoccus and Ornithinimicrobium (gut of laboratory animals), and Hydrogenophaga (water of laboratory cultures). The reported ecological and physiological capabilities of such bacteria can help to understand Artemia adaptation to natural and laboratory conditions., Mauricio Quiroz acknowledges project FONDEF D09I1256 (Artemia), National Commission for Scientific and Technological Research (CONICYT), Chile, for support to carry out this work at the Centre for Advanced Studies of Blanes (CEAB), Girona, Spain, which is part of his Master thesis in Natural Resources at Universidad de los Lagos, Osorno. Work in Spain was supported by project DARKNESS (CGL2012-32747) from the Spanish Office of Science (MINECO) to EOC.

The phylogenetic composition of freshwater anoxygenic photoheterotrophs (APs) has been poorly investigated as compared with their marine counterparts. In this study, we explored a set of ultraoligotrophic cold high mountain lakes (Central Pyrenees, Spain) by both pufM gene denaturing gradient gel electrophoresis fingerprinting, and cloning and sequencing of selected lakes samples. Different ranges of limnological and physico-chemical values were explored as environmental drivers of APs richness and composition. We did not observe significant relationships between richness/diversity of pufM and any of the limnological characteristics measured or trophic status, but a negative correlation with ammonia concentration. Conductivity, pH and nitrate concentration were significantly related to changes in APs community composition, whereas lake area, altitude, temperature and trophic status did not. Most of the sequences (> 85%) had the pufM sequences of Limnohabitans (Betaproteobacteria) as the closest relative in databases, whereas less abundant clones were more closely related to Rhodobacter, Sulfitobacter and Brevundimonas (Alphaproteobacteria), in agreement with 16S rRNA gene sequences previously found in the area. Congregibacter-like Gammaproteobacteria were not detected. Comparison with available studies in inland waters showed taxonomic partitioning along salinity gradients, and Congregibacter-like sequences restricted to high saline conditions in continental water bodies.

Quantitative environmental distribution of two widely distributed uncultured freshwater Euryarchaeota with unknown functional role was explored by newly designed quantitative PCR primers targeting the 16S rRNA gene of clades Miscellaneous Euryarchaeota Group (MEG, containing the groups pMC2A384 and VALII/Eury4) and Deep-Sea Euryarchaeotal Groups (DSEG, targeting the cluster named VALIII containing the DHVE-3/DSEG, BC07-2A-27/DSEG-3 and DSEG-2 groups), respectively. The summer surface plankton of 28 lakes was analysed, and one additional dimictic deep alpine lake, Lake Redon, was temporally and vertically surveyed covering seasonal limnological variability. A trophic range between 0.2 and 5.2 μg l(-1) Chl a, and pH span from 3.8 to 9.5 was explored at altitudes between 632 and 2590 m above sea level. The primers showed to be highly selective with c. 85% coverage and 100% specificity. Only pH significantly explained the changes observed in gene abundances and environment. In Lake Redon, DSEG bloomed in deep stratified waters both in summer and early spring, and MEG at intermediate depths during the ice-cover period. Overall, MEG and DSEG showed a differential ecological distribution although correlational analyses indicated lack of coupling of both Euryarchaeota with phytoplankton (chlorophyll a). However, an intriguing positive and significant relationship was found between DSEG and putative ammonia oxidizing thaumarchaeota.

Supplementation of nutrients by symbionts enables consumers to thrive on resources that might otherwise be insufficient to meet nutritional demands. Such nutritional subsidies by intracellular symbionts has been well studied; however, supplementation of de novo synthesized nutrients to hosts by extracellular gut symbionts is poorly documented, especially for generalists with relatively undifferentiated intestinal tracts. Although gut symbionts facilitate degradation of resources that would otherwise remain inaccessible to the host, such digestive actions alone cannot make up for dietary insufficiencies of macronutrients such as essential amino acids (EAA). Documenting whether gut symbionts also function as partners for symbiotic EAA supplementation is important because the question of how some detritivores are able to subsist on nutritionally insufficient diets has remained unresolved. To answer this poorly-understood nutritional aspect of symbiont-host interactions, we studied the enchytraeid worm, a bulk soil feeder that thrives in arctic peatlands. In a combined field and laboratory study, we employed stable isotope fingerprinting of amino acids to identify the biosynthetic origins of amino acids to bacteria, fungi and plants in enchytraeids. Enchytraeids collected from arctic peatlands derived more than 80% of their EAA from bacteria. In a controlled feeding study with the enchytraeid Enchytraeus crypticus, EAA derived almost exclusively from gut bacteria when the worms fed on higher fiber diets, whereas most of the enchytraeids’ EAA derived from dietary sources when fed on lower fiber diets. Our gene sequencing results of gut microbiota showed that the worms harbor several taxa in their gut lumen absent from their diets and substrates. Almost all gut taxa are candidates for EAA supplementation because almost all belong to clades capable of biosynthesizing EAA. Our study provides the first evidence of extensive symbiotic supplementation of EAA by microbial gut symbionts, and demonstrate that symbiotic bacteria in the gut lumen appear to function as partners for both symbiotic EAA supplementation as well as for digestion of insoluble plant fibers., The study was financed by the Danish Council for Independent Research, Technology and Production Sciences (09-069881), Carlsbergfondet (2007_01_0301) and the National Science Foundation Grant No. IOB 05–52015 to DO. TL was supported by Juan de la Cierva (JCI- 2009-049330) from the Spanish Office of Science (MINECO) and the DFG‐supported Cluster of Excellence “The Future Ocean”; MV was supported by the Spanish government projects Fundalzoo (CGL2010-14841, MINECO), Invasivefish (427/2011) and Biodiversity Conservation Plan from ENDESA S. A. (6900014499); YW was supported by the DFG - supported INTERDYNAMIK Research Priority Program SPP1266. Bacterial analyses were supported by grant DARKNESS (CGL2012-32747, MINECO) to EOC.

Summary Potassium is the major intracellular cation in most living cells, including yeasts. Although K+ has been demonstrated to be necessary for diverse cellular functions, such as enzyme activation, additional, still uncharacterized cellular targets may exist. We show here that in Saccharomyces cerevisiae short-term potassium deprivation impacts in the mRNA level of over one thousand genes. Lack of potassium drastically alters sulfur metabolism (mainly Met and Cys metabolism), triggers an oxidative stress response and activates the retrograde pathway, possibly due to the ammonium accumulation that occurs through the Trk1 potassium transporter. We also observe a remarkable halt in the expression of genes required for ribosome biogenesis and translation, a decrease in expression of diverse components (cyclins, protein kinases) required for progression through the cell cycle and a blockage in septins assembly. Only specific subsets of these changes were observed in a strain deleted for the TRK1 and TRK2 genes growing in the presence of sufficient potassium (50 mM). Therefore, a shortage of potassium in the environment triggers an acute transcriptional response, which covers different aspects of the cell biology so far unexplored, and whose investigation will likely reveal novel functional roles for this cation.

The genetic diversity of planktonic eukaryotic microorganisms (size range 3-40 µm) inhabiting 11 alpine lakes of the Central Pyrenees (Spain) was analysed by cloning and sequencing of the 18S rRNA gene. The selected lakes covered a wide range of environmental conditions representative of the regional landscape heterogeneity. Overall, we obtained 953 sequences (averaged length 750 bp) that were grouped in 343 representative OTUs (98% identity). The genetic richness was high, and the 18S rRNA gene sequences spread within nine high-rank taxonomic groups and grouped in 26 eukaryal classes. Most of the sequences affiliated with Stramenopiles (> 55% of total sequences, mostly Chrysophyceae), Cryptophyta and Alveolata (15% each). Three groups had relative abundance < 5%, i.e. Opisthokonta (mostly Fungi), Viridiplantae (mostly Chlorophyceae) and Rhizaria (cercomonads). Finally, minor groups were related to Katablepharidophyta, Euglenozoa and Telonemida. The lakes showed a different community structure being pH, and phosphorous and Chl a concentrations the main environmental drivers. The novelty level was high, and a quarter of the retrieved OTUs were notably divergent (< 97% identity) from any previously known sequence, mainly for Rhizaria and Opisthokonta. More than 50% of the sequences affiliated with clusters exclusively formed by uncultured protists. Cryptophyta and Viridiplantae showed the largest number of sequences closely related to cultured counterparts. This work is the first description of the genetic diversity of eukaryotic assemblages in ultraoligotrophic high mountain lakes, and the study unveils alpine environments as an important reservoir of microbial eukaryotic biodiversity.

Alkaline pH stress invokes a potent and fast transcriptional response in Saccharomyces cerevisiae that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and -response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. In the present study we show that the addition of glucose to the medium improves the growth of wild-type cells at high pH, fully abolishes the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr210. Lack of Elm1, one of the three upstream Snf1 kinases (Tos3, Elm1 and Sak1), markedly increases alkali sensitivity, whereas the phenotype of the triple mutant tos3 elm1 sak1 is even more pronounced than that of snf1 cells and is poorly rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of the genes induced in the short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those encoding high-affinity hexose transporters and phosphorylating enzymes. The induction of genes encoding enzymes involved in glycogen, but not trehalose, metabolism is largely dependent of the presence of Snf1. Therefore the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium resulted in an additive effect and allows near-normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.

Aims: Genetic comparison of Lactococcus garvieae isolated from mammals and fish. Methods and Results: One hundred and ninety-seven L. garvieae isolates obtained from trout (n = 153), cow (n = 7) and pigs (n = 37) were genetically characterized by determining their pulsed-field gel electrophoresis (PFGE) profiles after macrorestriction with Bsp120I. Overall, L. garvieae isolates from pigs, cow and trout exhibited distinct PFGE patterns, with a low genetic relationship between them. Isolates from trout generated two pulsotypes [Genetic diversity (GD) 0·01] showing that the fish isolates were more genetically homogenous than the others. The L. garvieae isolates from cows displayed five (GD 0·71) different pulsotypes, while the swine isolates displayed 13 different pulsotypes (GD 0·35). Twenty-one of the 37 swine strains (56·8%) were grouped in a single cluster that included two closely related (93% similarity) pulsotypes. These pulsotypes exhibited a high frequency of isolation from different organs of the animals, and they were also broadly distributed among herds, suggesting a wide distribution across the swine population. This suggests that L. garvieae might be able to colonize different organs of the swine cardio-respiratory system. Conclusions: Results indicate that most L. garvieae isolates from pigs and trout exhibited a distinct genetic background. Significance and Impact of the Study: The present study describes the isolation of L. garvieae from both diseased and healthy pigs for the first time, and the findings suggest that pigs could be a previously unknown reservoir of this pathogen.

Summary Bacterial community composition was analysed in the slush layers of snow-covered Lake Redon (2240 m altitude, Limnological Observatory of the Pyrenees, LOOP, NE Spain) in winter and spring and compared with bacteria from the lake water column, using 16S rRNA gene clone libraries and CARD-FISH counts. The set of biological data was related to changes in bacterial production and to other relevant environmental variables measured in situ. In winter, up to 70% of the 16S rRNA sequences found in the slush were closely related to planktonic bacteria from the water column beneath the ice. Conversely, during spring ablation, 50% of the sequences had > 97% identity with bacteria from the cryosphere (i.e. globally distributed glaciers, snow and ice) and may have originated from remote aerosol deposition. The transition winter to spring was characterized by consistent community changes switching from assemblages dominated by Betaproteobacteria, Verrucomicrobia and Bacteroidetes during snowpack growth to communities essentially dominated by the Bacteroidetes of classes Cytophagia and Sphingobacteria. This strong bacterial composition switch was associated with consistent increases in bacterial abundance and production, and decreasing bacterial diversity.

We studied the carbon dioxide fixation activity in a stratified hypereutrophic karstic lagoon using a combination of fingerprinting techniques targeting bacterial and archaeal 16S rRNA genes, functional gene cloning [the acetyl-CoA carboxylase ( accC )], and isotopic labelling (14C-bicarbonate) coupled to single-cell analyses [microautoradiography combined with catalyzed reported deposition-FISH (MAR-CARD-FISH)]. The microbial planktonic community was dominated by bacteria with maximal abundances of archaea just below the oxic/anoxic transition zone (7% of total cells). In situ incubations with radiolabelled bicarbonate showed maximal photoassimilation activity in the oxic epilimnion, whereas dark CO2 fixation was consistently observed throughout the water column, with a maximum at the oxic/anoxic interface (8.6 mg C m−3 h−1). The contributions of light and dark carbon fixation activities in the whole water column were 69% and 31% of the total C incorporated, respectively. MAR-CARD-FISH incubations corroborated these results and revealed that the highest fraction of bacterial and archaeal cells actively uptaking bicarbonate in the light was found at the surface. The bacterial community was mainly composed of green sulfur bacteria ( Chlorobi ) and members of the Betaproteobacteria and the Bacteroidetes . The archaeal assemblage was composed of phylotypes of the Miscellaneous Crenarchaeotic Group and a few methanogens. Clone libraries of the accC gene showed an absolute dominance of bacterial carboxylases. Our results suggest that the dark carbon fixation activity measured was mainly related to CO2 incorporation by heterotrophs rather than to the activity of true chemoautotrophs.

Despite its widespread distribution and high levels of phylogenetic diversity, microbes are poorly understood creatures. We applied a phylogenetic ecology approach in the Kingdom Euryarchaeota (Archaea) to gain insight into the environmental distribution and evolutionary history of one of the most ubiquitous and largely unknown microbial groups. We compiled 16S rRNA gene sequences from our own sequence libraries and public genetic databases for two of the most widespread mesophilic Euryarchaeota clades, Lake Dagow Sediment (LDS) and Rice Cluster-V (RC-V). The inferred population history indicated that both groups have undergone specific nonrandom evolution within environments, with several noteworthy habitat transition events. Remarkably, the LDS and RC-V groups had enormous levels of genetic diversity when compared with other microbial groups, and proliferation of sequences within each single clade was accompanied by significant ecological differentiation. Additionally, the freshwater Euryarchaeota counterparts unexpectedly showed high phylogenetic diversity, possibly promoted by their environmental adaptability and the heterogeneous nature of freshwater ecosystems. The temporal phylogenetic diversification pattern of these freshwater Euryarchaeota was concentrated both in early times and recently, similarly to other much less diverse but deeply sampled archaeal groups, further stressing that their genetic diversity is a function of environment plasticity. For the vast majority of living beings on Earth (i.e. the uncultured microorganisms), how they differ in the genetic or physiological traits used to exploit the environmental resources is largely unknown. Inferring population history from 16S rRNA gene-based molecular phylogenies under an ecological perspective may shed light on the intriguing relationships between lineage, environment, evolution and diversity in the microbial world.

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes in Saccharomyces cerevisiae , where seven PP2C-encoding genes ( PTC1 to -7 ) with diverse functions can be found. More recently, data on several Candida albicans PP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.

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.

6 páginas, 1 figura, 2 tablas., Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Ciso´ and Lake Vilar, NE Spain) where planktonic photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial biomass. Several extraction, solubilization, and electrophoresis methods were tested to develop an optimal protocol for the best resolution of the SDS-PAGE. Protein composition from different water depths and at different times of the year was visualized within a molecular mass range between 100 and 15 kDa yielding up to 20 different protein bands. Protein banding patterns were reproducible and changed in time and with depth in agreement with changes in photosynthetic bacteria composition. When a taxonomically stable community was followed in time, differences were observed in the intensity but not in the composition of the SDS-PAGE banding pattern. Three environmental variables directly related to the activity of sulfur bacteria (light, oxygen, and sulfide concentrations) had a significant effect on protein banding patterns and explained 33% of the variance. Changes in natural protein profiles of the bacterial blooms agreed with changes in species composition and in the in situ metabolic state of the populations., Work supported by grants VIARC REN2003-08333 and CRENYC CGL2006-12058 from the Spanish Ministerio de Educacio´n y Ciencia (MEC) to EOC. MBO received a I3P postdoctoral fellow from the Spanish Council for Research (CSIC).

13 páginas, 5 figuras, 2 tablas., It has been long debated as to whether marine microorganisms have a ubiquitous distribution or patterns of biogeography, but recently a consensus for the existence of microbial biogeography is emerging. However, the factors controlling the distribution of marine bacteria remain poorly understood. In this study, we combine pyrosequencing and traditional Sanger sequencing of the 16S rRNA gene to describe in detail bacterial communities from the deep Arctic Ocean. We targeted three separate water masses, from three oceanic basins and show that bacteria in the Arctic Ocean have a biogeography. The biogeographical distribution of bacteria was explained by the hydrography of the Arctic Ocean and subsequent circulation of its water masses. Overall, this first taxonomic description of deep Arctic bacteria communities revealed an abundant presence of SAR11 (Alphaproteobacteria), SAR406, SAR202 (Chloroflexi) and SAR324 (Deltaproteobacteria) clusters. Within each cluster, the abundance of specific phylotypes significantly varied among water masses. Water masses probably act as physical barriers limiting the dispersal and controlling the diversity of bacteria in the ocean. Consequently, marine microbial biogeography involves more than geographical distances, as it is also dynamically associated with oceanic processes. Our ocean scale study suggests that it is essential to consider the coupling between microbial and physical oceanography to fully understand the diversity and function of marine microbes., Financial and ship time support from Fisheries and Oceans Canada and the Canadian International Polar Year Program’s Canada’s Three Oceans project and the Nansen and Amundsen Basins Observational System project. PE Galand was supported by a Marie Curie Grant (CRENARC MEIF-CT-2007-040247) and EO Casamayor by the Spanish Grant CGL2006-12058-BOS. C Lovejoy would like to acknowledge the support of the Natural Sciences and Engineering Council, Canada (NSERC) Special Research Opportunity Fund and ArcticNet. Deep Arctic samples were collected by K Scarcella, E Didierjean and M-E´ Garneau. Pyrosequencing was supported by a Keck foundation grant to M Sogin and L Ameral Zettler. This is a contribution to the International Census of Marine Microbes (ICOMM).

12 pages, 3 figures, 2 tables.-- PMID: 19453609 [PubMed].-- Printed version published Jun 2009., Supporting information (Suppl. table S1) available at: http://www3.interscience.wiley.com/journal/122369858/suppinfo, Research reviewed in Caroline Ash's "Microbiology: Aeolian Microbes" (Editor's choice: Highlights of the recent literature, Science 324(5930): 989-991, May 22, 2009, doi: 10.1126/science.324_989d), available OA at: http://www.iac.ethz.ch/people/knuttir/papers/smith09sci.pdf, We have analysed the diversity of the bacteria, which grow after addition of concentrated airborne particles and desert dust in different microcosms combinations with water samples from oligotrophic alpine lakes. We used, on the one hand, airborne bacteria transported by an African dust plume and collected in a high mountain area in the central Pyrenees (Spain). On the other hand, we collected desert dust in Mauritania (c. 3000 km distance, and a few days estimated airborne journey), a known source region for dust storms in West Africa, which originates many of the dust plumes landing on Europe. In all the dust-amended treatments we consistently observed bacterial growth of common phyla usually found in freshwater ecosystems, i.e. Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, and a few Bacteroidetes, but with different composition based on lake water pretreatment and dust type. Overall, we tentatively split the bacterial community in (i) typical freshwater non-airborne bacteria, (ii) cosmopolitan long-distance airborne bacteria, (iii) non-freshwater low-distance airborne bacteria, (iv) non-freshwater long-distance airborne soil bacteria and (v) freshwater non-soil airborne bacteria. We identified viable long-distance airborne bacteria as immigrants in alpine lakes (e.g. Sphingomonas-like) but also viable putative airborne pathogens with the potential to grow in remote alpine areas (Acinetobacter-like and Arthrobacter-like). Generation of atmospheric aerosols and remote dust deposition is a global process, largely enhanced by perturbations linked to the global change, and high mountain lakes are very convenient worldwide model systems for monitoring global-scale bacterial dispersion and pathogens entries in remote pristine environments., This research was carried out in the LTER site Limnological Observatory of the Pyrenees and was initially funded by Grant ECOSENSOR BIOCON04/009 from the Fundación BBVA, and further by projects AERBAC 079-2007 from the Ministerio de Medio Ambiente-Red de Parques Nacionales, and CONSOLIDER grant GRACCIE CSD2007-00004 from the Spanish Ministerio de Ciencia e Innovación (MICINN). We are thankful to the Authorities of AiguesTortes and Estany de St Maurici National Park for sampling facilities in the protected areas and continuous support, to Jose Carlos Brito from Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO, Portugal) for sand collection in Mauritania, and to Carmen Gutiérrez-Provecho and JC Auguet for field and laboratory assistance. A.H. had a PhD research scholarship granted from the BBVA Foundation.

10 páginas, 4 figuras, 1 tabla., The Arctic Ocean plays a critical role in controlling nutrient budgets between the Pacific and Atlantic Ocean. Archaea are key players in the nitrogen cycle and in cycling nutrients, but their community composition has been little studied in the Arctic Ocean. Here, we characterize archaeal assemblages from surface and deep Arctic water masses using massively parallel tag sequencing of the V6 region of the 16S rRNA gene. This approach gave a very high coverage of the natural communities, allowing a precise description of archaeal assemblages. This first taxonomic description of archaeal communities by tag sequencing reported so far shows that it is possible to assign an identity below phylum level to most (95%) of the archaeal V6 tags, and shows that tag sequencing is a powerful tool for resolving the diversity and distribution of specific microbes in the environment. Marine group I Crenarchaeota was overall the most abundant group in the Arctic Ocean and comprised between 27% and 63% of all tags. Group III Euryarchaeota were more abundant in deep-water masses and represented the largest archaeal group in the deep Atlantic layer of the central Arctic Ocean. Coastal surface waters, in turn, harbored more group II Euryarchaeota. Moreover, group II sequences that dominated surface waters were different from the group II sequences detected in deep waters, suggesting functional differences in closely related groups. Our results unveiled for the first time an archaeal community dominated by group III Euryarchaeota and show biogeographical traits for marine Arctic Archaea., P E Galand is supported by a Marie Curie grant (CRENARC MEIF-CT-2007–040247). EO Casamayor was supported by a Spanish grant CGL2006–12058-BOS, and D L Kirchman by NSF OPP ARC-0632233. C Lovejoy would like to acknowledge the support of the Natural Sciences and Engineering Council, Canada (NSERC) Special Research Opportunity Fund. We thank C Pedros-Alio for support during CASES. Deep Arctic samples were collected by K Scarcella and E Didierjean, and we thank the Chief Scientist, Officers and Crew of the CCGS Louis St Laurent and support from Fisheries and Oceans Canada. Tag sequencing was supported by a Keck foundation grant to M Sogin and L Ameral Zettler. This is a contribution to the International Census of Marine Microbes (ICOMM).

Biochemical and molecular genetic studies were performed on an unknown Gram-positive, catalase-negative, coccus-shaped organism isolated from clinical samples from pigs. On the basis of the results of cellular morphological and biochemical tests, the organism was identified as a streptococcal species. 16S rRNA gene sequence comparisons confirmed its identification as a member of the genus Streptococcus, but the organism was distinct from any recognized species of this genus. The closest phylogenetic relative of the unknown organism corresponded to Streptococcus suis NCTC 10234(T) (97.2 % 16S rRNA gene sequence similarity) and this phylogenetic position was confirmed by analysis of rpoB and sodA sequences. DNA-DNA hybridization studies showed that the unidentified organism produced a DNA reassociation value of 36.6 % with respect to S. suis NCTC 10234(T). The novel bacterium was distinguished from S. suis and other Streptococcus species using biochemical tests. On the basis of phenotypic and phylogenetic data, the unidentified organism represents a novel species of the genus Streptococcus, for which the name Streptococcus plurextorum sp. nov. is proposed. The type strain is 1956-02(T) (=CECT 7308(T)=CCUG 52972(T)).

The bacterioneuston (bacteria inhabiting the air–water interface) is poorly characterized and possibly forms a unique community in the aquatic environment. In high mountain lakes, the surface film is subjected to extreme conditions of life, suggesting the development of a specific and adapted bacterioneuston community. We have studied the surface film of a remote high mountain lake in the Pyrenees by cloning the PCR-amplified 16S rRNA gene and comparing with bacteria present in underlying waters (UW), and airborne bacteria from the dust deposited on the top of the snow pack. We did not detect unusual taxa in the neuston but rather very common and widespread bacterial groups. Betaproteobacteria and Actinobacteria accounted for >75% of the community composition. Other minor groups were Gammaproteobacteria (between 8% and 12%), Alphaproteobacteria (between 1% and 5%), and Firmicutes (1%). However, we observed segregated populations in neuston and UW for the different clades within each of the main phylogenetic groups. The soil bacterium Acinetobacter sp. was only detected in the snow–dust sample. Overall, higher similarities were found between bacterioneuston and airborne bacteria than between the former and bacterioplankton. The surface film in high mountain lakes appears as a direct interceptor of airborne bacteria useful for monitoring long-range bacterial dispersion.

Spatial distributions of both total (i.e. 16S rDNA-based fingerprints) and active (i.e. 16S rRNA-based fingerprints) bacterial populations, together with total bacterial activity measured by 3H-leucine incorporation, were studied along a 98 km transect in the NW Mediterranean Sea. Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) fingerprinting was coupled to a clone library, allowing CE-SSCP peaks identification and the monitoring of the spatial variation of bacterial phylotypes. Up to 80% of the community peaks matched those obtained from clone library sequences, accounting for 86.7% of the total fingerprinting area. A good agreement was found between the relative abundance of Prochlorococcus in the CE-SSCP fingerprints and flow cytometry counts (r2=0.66, P 1 both at the surface and at the DCM. These results emphasize the need for combining rDNA- and rRNA-based analyses to better understand the functional role of individual bacterial populations in situ.

We surveyed the archaeal assemblage in a stratified sulfurous lake (Lake Vilar, Banyoles, Spain) over 5 consecutive years to detect potential seasonal and interannual trends in the free-living planktonic Archaea composition. The combination of different primer pairs and nested PCR steps revealed an unexpectedly rich archaeal community. Overall, 140 samples were analyzed, yielding 169 different 16S rRNA gene sequences spread over 14 Crenarchaeota (109 sequences) and six Euryarchaeota phylogenetic clusters. Most of the Crenarchaeota (98% of the total crenarchaeotal sequences) affiliated within the Miscellaneous Crenarchaeota Group (MCG) and were related to both marine and freshwater phylotypes. Euryarchaeota mainly grouped within the Deep Hydrothermal Vent Euryarchaeota (DHVE) cluster (80% of the euryarchaeotal sequences) and the remaining 20% distributed into three less abundant taxa, most of them composed of soil and sediment clones. The largest fraction of phylotypes from the two archaeal kingdoms (79% of the Crenarchaeota and 54% of the Euryarchaeota) was retrieved from the anoxic hypolimnion, indicating that these cold and sulfide-rich waters constitute an unexplored source of archaeal richness. The taxon rank-frequency distribution showed two abundant taxa (MCG and DHVE) that persisted in the water column through seasons, plus several rare ones that were only detected occasionally. Differences in richness distribution and seasonality were observed, but no clear correlations were obtained when multivariate statistical analyses were carried out.

Summary We designed and tested a set of specific primers for specific PCR amplification of the biotin carboxylase subunit gene (accC) of the Acetyl CoA carboxylase (ACCase) enzyme. The primer set yielded a PCR product of c. 460 bp that was suitable for denaturing gradient gel electrophoresis (DGGE) fingerprinting followed by direct sequencing of excised DGGE bands and sequence analysis. Optimization of PCR conditions for selective amplification was carried out with pure cultures of different bacteria and archaea, and laboratory enrichments. Next, fingerprinting comparisons were done in several aerobic and anaerobic freshwater planktonic samples. The DGGE fingerprints showed between 2 and 19 bands in the different samples, and the primer set provided specific amplification in both pure cultures and natural samples. Most of the samples had sequences grouped with bacterial accC, hypothetically related to the anaplerotic fixation of inorganic carbon. Some other samples, however, yielded accC gene sequences that clustered with Crenarchaeota and were related to the 3-hydroxypropionate/4-hydroxybutyrate cycle of autotrophic crenarchaeota. Such samples came from oligotrophic high mountain lakes and the hypolimnia of a sulfide-rich lake, where crenarchaeotal populations had been previously reported by 16S rRNA surveys. This study provided a fast tool to look for presence of accC genes in natural environments as potential marker for studies of carbon dioxide assimilation in the dark. After further refinement for better specificity against archaea, the new and novel primers could be very helpful to establish a target for crenarchaeota with implications for our understanding of archaeal carbon biogeochemistry.

7 páginas, 1 figura, 2 tablas., We have surveyed the first 1 m of 10 oligotrophic high mountain lakes in the Central Pyrenees (Spain) for both abundance and predominant phylotypes richness of the archaeaplankton assemblage, using CARD-FISH and 16S rRNA gene sequencing respectively. Archaea inhabiting the air-water surface microlayer (neuston) ranged between 3% and 37% of total 4,6-diamidino-2-phenylindole (DAPI) counts and were mainly Crenarchaeota of a new freshwater cluster distantly related to the Marine Group 1.1a. Conversely, most of the Archaea from the underlying waters (the remaining first 1 m integrated) were mainly Euryarchaeota of three distantly related branches ranging between 0.4% and 27% of total DAPI counts. Therefore, a consistent qualitative and quantitative spatial segregation was observed for the two main archaeal phyla between neuston and underlying waters at a regional scale. We also observed a consistent pattern along the lakes surveyed between lake area, lake depth and water residence time, and the archaeal enrichment in the neuston: the larger the lake the higher the proportion of archaea in the neuston as compared with abundances from the underlying waters (n = 10 lakes; R2 > 0.80; P < 0.001, in all three cases). This is the first report identifying a widespread non-thermophilic habitat where freshwater planktonic Crenarchaeota can be found naturally enriched. High mountain lakes offer great research opportunities to explore the ecology of one of the most enigmatic and far from being understood group of prokaryotes., This research was supported by projects VIARC REN2003-08333 and CRENYC CGL2006-12058 to E.O.C. from the Spanish Ministerio de Educación y Ciencia (MEC). J.C.A. benefits from a SEUI-MEC postdoctoral fellow and E.O.C. by the Programa Ramón y Cajal from the Spanish MEC and FEDER.

14 pages, 7 figures, 3 tables, Lake Tebenquiche is one of the largest saline water bodies in the Salar de Atacama at 2,500 m above sea level in northeastern Chile. Bacteria inhabiting there have to deal with extreme changes in salinity, temperature and UV dose (i.e., high environmental dissimilarity in the physical landscape). We analyzed the bacterioplankton structure of this lake by 16S rRNA gene analyses along a spatio–temporal survey. The bacterial assemblage within the lake was quite heterogeneous both in space and time. Salinity changed both in space and time ranging between 1 and 30% (w/v), and total abundances of planktonic prokaryotes in the different sampling points within the lake ranged between two and nine times 106 cells mL−1. Community composition changed accordingly to the particular salinity of each point as depicted by genetic fingerprinting analyses (denaturing gradient gel electrophoresis), showing a high level of variation in species composition from place to place (beta-diversity). Three selected sites were analyzed in more detail by clone libraries. We observed a predominance of Bacteroidetes (about one third of the clones) and Gammaproteobacteria (another third) with respect to all the other bacterial groups. The diversity of Bacteroidetes sequences was large and showed a remarkable degree of novelty. Bacteroidetes formed at least four clusters with no cultured relatives in databases and rather distantly related to any known 16S rRNA sequence. Within this phylum, a rich and diverse presence of Salinibacter relatives was found in the saltiest part of the lake. Lake Tebenquiche included several novel microorganisms of environmental importance and appeared as a large unexplored reservoir of unknown bacteria, Sampling and measurements carried out in Chile were funded by grants FONDECYT 1030441 and FONDEF D99I1026. Measurements carried out in Barcelona were funded by grant "ATACAMA-2002" (CICYT, BOS2002-10258-E). Grant "BIOARSENICO" from Fundación BBVA funds current work

Biochemical and molecular genetic studies were performed for five isolates of unknown Gram-positive, catalase-negative, cocci-shaped micro-organisms obtained from clinical samples from pigs. The micro-organisms were tentatively identified as Aerococcus species on the basis of the results from cellular morphological and biochemical tests. 16S rRNA gene sequencing studies confirmed the provisional identification of the isolates as members of the genus Aerococcus, but the micro-organism did not correspond to any recognized species of this genus. The nearest phylogenetic relatives of these unknown cocci isolated from pigs were Aerococcus viridans (95.9 % 16S rRNA gene sequence similarity) and Aerococcus urinaeequi (95.8 %). The unknown bacterium, however, was distinguishable from these two species and from other animal aerococci by using biochemical tests. On the basis of both phenotypic and phylogenetic findings, the isolates represent a novel species of the genus Aerococcus, for which the name Aerococcus suis sp. nov. is proposed. The type strain is 1821/02T (=CECT 7139T=CCUG 52530T).

Tacita Muta fue una náyade a la que Júpiter arrancó la lengua por hablar más de la cuenta. Convertida en divinidad, simbolizaba para los romanos la prerrogativa del silencio femenino. El objetivo del presente artículo es el de, tomando como punto de partida el mito de Tacita, ilustrar cómo el patriarcado romano impuso a las mujeres la obligatoriedad del silencio. Además, se presentan diversos ejemplos de divinidades, personajes mitológicos y mujeres reales que también ejemplificaron esta característica femenina, junto con otros que transgredieron la norma establecida. Todo ello con el objetivo de profundizar en las relaciones de género de época romana. Tacita Muta was a naiad punished by Jupiter who ripped her tongue out for talking excessively. She became a divinity that symbolized the prerogative of the feminine silence. The aim of the present article is to illustrate how the roman patriarchy imposed the duty of silence on women, taking the myth of Tacita as the starting point. We present several examples of deities, mythological characters and real women who also exemplified this female duty, along with others that transgressed the norm. All of this with the aim of getting deeper on gender relations in Roman times.

9 páginas, 4 figuras, 1 tabla, Microbial communities growing under extreme low redox conditions are present in anoxic and sulfide-rich (euxinic) environments such as karstic lakes and experience limitation of electron acceptors. The fine natural chemical gradients and the large diversity of organic and inorganic compounds accumulated in bottom waters are impossible to mimic under laboratory conditions, and only a few groups have been cultured. We investigated the bacterial composition in the oxic-anoxic interface and in the deep waters of three sulfurous lakes from the Lake Banyoles karstic area (NE Spain) through 16S rRNA gene tag sequencing and identified the closest GenBank counterpart. High diversity indices were found in most of the samples with >15 phyla/classes and >45 bacterial orders. A higher proportion of operational taxonomic units (OTUs) of the Bhighest novelty^ was found in the hypolimnia (38 % of total sequences) than in themetalimnia (17%),whereas the percentage of OTUs closer to cultured counterparts (i.e., 97 % identity in the 16S rRNA gene) was 6 to 21 %, respectively. Elusimicrobia, Chloroflexi, Fibrobacteres, and Spirochaetes were the taxa with the highest proportion of novel sequences. Interestingly, tag sequencing results comparison with metagenomics data available from the same dataset, showed a systematic underestimation of sulfuroxidizing Epsilonproteobacteria with the currently available 907R Buniversal^ primer. Overall, despite the limitation of electron acceptors, a highly diverse and novel assemblage was present in dark and euxinic hypolimnetic freshwaters, unveiling a hotspot of microbial diversity with a remarkable gap with cultured counterparts., This research was funded by grants GOS-LAKES CGL2009-08523-E and DARKNESS CGL2012-32747 to EOC and ARCANOX CGL2009-13318-C02-02 to CBM from the Spanish Office of Science (MINECO) and from the financial support by the Beyster Family Fund of the San Diego Foundation and the Life Technologies Foundation to the J. Craig Venter Institute.

Este artículo contiene 13 páginas, 7 figuras, 1 tabla., Members of the archaeal Miscellaneous Crenarchaeotic Group (MCG) are among the most successful microorganisms on the planet. During its evolutionary diversification, this very diverse group has managed to cross the saline–freshwater boundary, one of the most important evolutionary barriers structuring microbial communities. However, the current understanding on the ecological significance of MCG in freshwater habitats is scarce and the evolutionary relationships between freshwater and saline MCG remains poorly known. Here, we carried out molecular phylogenies using publicly available 16S rRNA gene sequences from various geographic locations to investigate the distribution of MCG in freshwater and saline sediments and to evaluate the implications of saline– freshwater transitions during the diversification events. Our approach provided a robust ecological framework in which MCG archaea appeared as a core generalist group in the sediment realm. However, the analysis of the complex intragroup phylogeny of the 21 subgroups currently forming the MCG lineage revealed that distinct evolutionary MCG subgroups have arisen in marine and freshwater sediments suggesting the occurrence of adaptive evolution specific to each habitat. The ancestral state reconstruction analysis indicated that this segregation was mainly due to the occurrence of a few saline–freshwater transition events during the MCG diversification. In addition, a network analysis showed that both saline and freshwater MCG recurrently co-occur with archaea of the class Thermoplasmata in sediment ecosystems, suggesting a potentially relevant trophic connection between the two clades., This research was funded by projects ARCANOX (ref. CGL2009-13318-C02-02) and ARCOS (ref. CGL2012-33033) to CMB, and DARKNESS CGL2012-32747 to EOC from the Spanish Ministry of Economy and Competitiveness (MINECO). J-CA was funded by project METMIC from the Pyrenean Work Community and the Aquitaine region.

14 páginas, 7 figuras, 3 tablas., Stratified sulfurous lakes are appropriate environments for studying the links between composition and functionality in microbial communities and are potentially modern analogs of anoxic conditions prevailing in the ancient ocean. We explored these aspects in the Lake Banyoles karstic area (NE Spain) through metagenomics and in silico reconstruction of carbon, nitrogen and sulfur metabolic pathways that were tightly coupled through a few bacterial groups. The potential for nitrogen fixation and denitrification was detected in both autotrophs and heterotrophs, with a major role for nitrogen and carbon fixations in Chlorobiaceae. Campylobacterales accounted for a large percentage of denitrification genes, while Gallionellales were putatively involved in denitrification, iron oxidation and carbon fixation and may have a major role in the biogeochemistry of the iron cycle. Bacteroidales were also abundant and showed potential for dissimilatory nitrate reduction to ammonium. The very low abundance of genes for nitrification, the minor presence of anammox genes, the high potential for nitrogen fixation and mineralization and the potential for chemotrophic CO2 fixation and CO oxidation all provide potential clues on the anoxic zones functioning. We observed higher gene abundance of ammonia-oxidizing bacteria than ammonia-oxidizing archaea that may have a geochemical and evolutionary link related to the dominance of Fe in these environments. Overall, these results offer a more detailed perspective on the microbial ecology of anoxic environments and may help to develop new geochemical proxies to infer biology and chemistry interactions in ancient ecosystems., This research was funded by Grants GOS-LAKES CGL2009-08523-E and DARKNESS CGL2012-32747 to EOC from the Spanish Office of Science (MINECO), from financial support by the Beyster Family Fund of the San Diego Foundation and the Life Technologies Foundation to the J Craig Venter Institute, and the NASA Astrobiology Institute to CLD.

Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes.