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- Metagenome-assembled genome sequences of three uncultured planktomarina sp. strains from the Northeast Atlantic OceanPublication . Marques, Matilde; Borges, Nuno; Silva, Sandra Godinho; da Rocha, Ulisses Nunes; Lago-Lestón, Asunción; Keller-Costa, Tina; Da Silva Costa, RodrigoWe report three metagenome-assembled genomes (MAGs) of Planktomarina strains from coastal seawater (Portugal) to help illuminate the functions of understudied Rhodobacteraceae bacteria in marine environments. The MAGs encode proteins involved in aerobic anoxygenic photosynthesis and a versatile carbohydrate metabolism, strengthening the role of Planktomarina species in oceanic carbon cycling.
- Genomic insights into aquimarina sp. strain EL33, a bacterial symbiont of the gorgonian coral eunicella labiata.Publication . Keller-Costa, Tina; Silva, Rúben; Lago-Lestón, Asunción; Costa, RodrigoTo address the metabolic potential of symbiotic Aquimarina spp., we report here the genome sequence of Aquimarina sp. strain EL33, a bacterium isolated from the gorgonian coral Eunicella labiata This first-described (to our knowledge) animal-associated Aquimarina genome possesses a sophisticated repertoire of genes involved in drug/antibiotic resistance and biosynthesis.
- Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library constructionPublication . E, Varela-Álvarez; Andreakis, N.; Lago-Lestón, Asunción; Pearson, G. A.; Serrão, Ester; Procaccini, G.; Duarte, C. M.; Marbà, N.A method for isolating high-quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μg genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A260/A280 ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum. The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.
- Metatranscriptomes reveal functional variation in diatom communities from the Antarctic PeninsulaPublication . Pearson, Gareth; Lago-Lestón, Asunción; Canovas, Fernando; Cox, C. J.; Verret, Frédéric; Lasternas, Sebastian; Duarte, Carlos M.; Agusti, Susana; Serrao, Ester A.Functional genomics of diatom-dominated communities from the Antarctic Peninsula was studied using comparative metatranscriptomics. Samples obtained from diatom-rich communities in the Bransfield Strait, the western Weddell Sea and sea ice in the Bellingshausen Sea/Wilkins Ice Shelf yielded more than 500K pyrosequencing reads that were combined to produce a global metatranscriptome assembly. Multi-gene phylogenies recovered three distinct communities, and diatom-assigned contigs further indicated little read-sharing between communities, validating an assembly-based annotation and analysis approach. Although functional analysis recovered a core of abundant shared annotations that were expressed across the three diatom communities, over 40% of annotations (but accounting for <10% of sequences) were community-specific. The two pelagic communities differed in their expression of N-metabolism and acquisition genes, which was almost absent in post-bloom conditions in the Weddell Sea community, while enrichment of transporters for ammonia and urea in Bransfield Strait diatoms suggests a physiological stance towards acquisition of reduced N-sources. The depletion of carbohydrate and energy metabolism pathways in sea ice relative to pelagic communities, together with increased light energy dissipation (via LHCSR proteins), photorespiration, and NO3 uptake and utilization all pointed to irradiance stress and/or inorganic carbon limitation within sea ice. Ice-binding proteins and cold-shock transcription factors were also enriched in sea ice diatoms. Surprisingly, the abundance of gene transcripts for the translational machinery tracked decreasing environmental temperature across only a 4 degrees C range, possibly reflecting constraints on translational efficiency and protein production in cold environments.
- Frayed at the edges: selective pressure and adaptive response to abiotic stressors are mismatched in low diversity edge populationsPublication . Pearson, Gareth; Lago-Lestón, Asunción; Mota, CatarinaTheory predicts that population structure and dynamics affect a population's capacity for adaptation to environmental change. For isolated, small and fragmented populations at the trailing edge of species distributions, loss of genetic diversity through random genetic drift may reduce adaptive potential and fitness levels for complex traits. This has important consequences for understanding population responses to, for example changing climate, but has rarely been tested in natural populations. We measured the intertidal thermal environment and tidal exposure (emersion) times for natural populations of the intertidal seaweed Fucus serratus at the centre (southwest UK) and southern edge (northern Portugal) of its range in the Eastern Atlantic, and for a congener, F. vesiculosus, whose range extends further south to Morocco. Fitness-related traits of individuals at each location were measured in common garden experiments: physiological resilience to desiccation and heat shock (PSII quantum yield), and the molecular phenotype of the heat shock response (quantitative PCR of heat shock protein gene transcripts). The realized thermal environment experienced by F. serratus was similar at the centre and southern edge of its distribution because the maximum shore height (and emersion period) was reduced in southern populations. For F. vesiculosus, thermal maxima were higher and occurred more frequently in the south, although maximum vertical height (emersion time) remained similar to central populations. Edge populations of F. serratus were less resilient to desiccation and heat shock than central populations, and expression of heat shock genes was higher at the same temperature, suggesting greater cellular stress. In contrast, there was no evidence for physiological divergence in heat shock response in F. vesiculosus, and little variation in gene expression. Synthesis. We provide evidence that compared with range-centre populations upper intertidal limits of F. serratus at the southern edge are 'pruned back' by abiotic stressors. Rather than being locally adapted, these small populations are less resilient to abiotic stresses and experience greater cellular stress during heat shock. These results suggest that ongoing climate forcing factors may threaten small, fragmented rear edge populations because of inherently reduced fitness and lower adaptive capacity relative to larger central populations.
- An expressed sequence tag analysis of the intertidal brown seaweeds Fucus serratus (L.) and F. vesiculosus (L.) (Heterokontophyta, Phaeophyceae) in response to abiotic stressorsPublication . Pearson, G. A.; Hoarau, G.; Lago-Lestón, Asunción; Coyer, J. A.; Kube, M.; Reinhardt, Richard; Henckel, K.; Serrão, Ester; Corre, E.; Olsen, J. L.In order to aid gene discovery and uncover genes responding to abiotic stressors in stress-tolerant brown algae of the genus Fucus, expressed sequence tags (ESTs) were studied in two species, Fucus serratus and Fucus vesiculosus. Clustering of over 12,000 ESTs from three libraries for heat shock/recovery and desiccation/rehydration resulted in identification of 2,503, 1,290, and 2,409 unigenes from heat-shocked F. serratus, desiccated F. serratus, and desiccated F. vesiculosus, respectively. Low overall annotation rates (18–31%) were strongly associated with the presence of long 3′ untranslated regions in Fucus transcripts, as shown by analyses of predicted protein-coding sequence in annotated and nonannotated tentative consensus sequences. Posttranslational modification genes were overrepresented in the heat shock/recovery library, including many chaperones, the most abundant of which were a family of small heat shock protein transcripts, Hsp90 and Hsp70 members. Transcripts of LI818-like light-harvesting genes implicated in photoprotection were also expressed during heat shock in high light. The expression of several heat-shock-responsive genes was confirmed by quantitative reverse transcription polymerase chain reaction. However, candidate genes were notably absent from both desiccation/rehydration libraries, while the responses of the two species to desiccation were divergent, perhaps reflecting the species-specific physiological differences in stress tolerance previously established. Desiccation-tolerant F. vesiculosus overexpressed at least 17 ribosomal protein genes and two ubiquitinribosomal protein fusion genes, suggesting that ribosome function and/or biogenesis are important during cycles of rapid desiccation and rehydration in the intertidal zone and possibly indicate parallels with other poikilohydric organisms such as desiccation-tolerant bryophytes.
- Rapid identification of differentially expressed genes by in situ screening of bacteriaPublication . Fonseca, V. G.; Lago-Lestón, Asunción; Laizé, Vincent; Cancela, LeonorThe identification of differentially expressed genes is a key step in the understanding of specific molecular mechanisms. Various methods have been developed to search for differences in expression but most of them are time or money consuming. We present here an alternative technique that connects standard suppression subtractive hybridization with in situ screening of bacteria to isolate and identify differentially expressed transcripts. The in situ differential screening is based on the transfer of bacteria directly from cultures onto nylon membranes with no need of phenol/chloroform extraction, colony lifting, or polymerase chain reaction amplification. This improved method was successfully applied and must be seen as a simple, low-cost, time-saving, and reproducible approach to identify differentially expressed genes.
- Simple and rapid RNA extraction from freeze-dried tissue of brown algae and seagrassesPublication . Pearson, G. A.; Lago-Lestón, Asunción; Serrão, Ester; Bernardo, Marta Sofia Mendes ValenteAn inexpensive and rapid RNA extraction protocol for brown algae and seagrasses is presented, based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride. The protocol avoids the use of toxic chaotropic agents and phenol; high concentrations of dithiothreitol are used to inhibit RNase activity and to prevent oxidative cross-linking of nucleic acids by phenolics. A relatively high throughput of about 100 samples in 24 h can be achieved for, for example, Northern analysis. Yields of 50–200 µg g−1 fresh weight are comparable with those obtained for higher plants using commercially available kits. Tests of the extraction procedure demonstrate that high quality, intact RNA can be obtained from a variety of lyophilized brown algal tissues, even after prolonged storage at room temperature. Lyophilization is therefore suggested as an alternative to freezing tissue at −70°C to −80°C. The RNA obtained was used directly in several downstream applications to detect Fucus plastid-encoded transcripts by RNA-labelling, RT-PCR and Northern analysis.