In situ metagenomics: A platform for rapid sequencing and analysis of metagenomes in less than one day.

We present here a complete system for metagenomic analysis that allows performing the sequencing and analysis of a medium-size metagenome in less than one day. This unprecedented development was possible due to the conjunction of state-of-the-art experimental and computational advances: a portable laboratory suitable for DNA extraction and sequencing with nanopore technology; the powerful metagenomic analysis pipeline SqueezeMeta, capable to provide a complete analysis in a few hours and using scarce computational resources; and tools for the automatic inspection of the results via a graphical user interface, that can be coupled to a web server to allow remote visualization of data (SQMtools and SQMxplore). We have tested the feasibility of our approach in the sequencing of the microbiota associated to volcanic rocks in La Palma, Canary Islands. Also, we did a two-day sampling campaign of marine waters in which the results obtained on the first day guided the experimental design of the second day. We demonstrate that it is possible to generate metagenomic information in less than one day, making it feasible to obtain taxonomic and functional profiles fast and efficiently, even in field conditions. This capacity can be used in the further to perform real-time functional and taxonomic monitoring of microbial communities in remote areas.

Linking the impact of bacteria on phytoplankton growth with microbial community composition and co-occurrence patterns.

The interactions between microalgae and bacteria have recently emerged as key control factors which might contribute to a better understanding on how phytoplankton communities assemble and respond to environmental disturbances. We analyzed partial 16S rRNA and 18S rRNA genes from a total of 42 antibiotic bioassays, where phytoplankton growth was assessed in the presence or absence of an active bacterial community. A significant negative impact of bacteria was observed in 18 bioassays, a significant positive impact was detected in 5 of the cases, and a non-detectable effect occurred in 19 bioassays. Thalasiossira spp., Chlorophytes, Vibrionaceae and Alteromonadales were relatively more abundant in the samples where a positive effect of bacteria was observed compared to those where a negative impact was observed. Phytoplankton diversity was lower when bacteria negatively affect their growth than when the effect was beneficial. The phytoplankton-bacteria co-occurrence subnetwork included many significant Chlorophyta-Alteromonadales and Bacillariophyceae-Alteromonadales positive associations. Phytoplankton-bacteria co-exclusions were not detected in the network, which contrasts with the negative effect of bacteria on phytoplankton growth frequently detected in the bioassays, suggesting strong competitive interactions. Overall, this study adds strong evidence supporting the key role of phytoplankton-bacteria interactions in the microbial communities.

Role of Bacterial Community Composition as a Driver of the Small-Sized Phytoplankton Community Structure in a Productive Coastal System.

We present here the first detailed description of the seasonal patterns in bacterial community composition (BCC) in shelf waters off the Ría de Vigo (Spain), based on monthly samplings during 2 years. Moreover, we studied the relationship between bacterial and small-sized eukaryotic community composition to identify potential biotic interactions among components of these two communities. Bacterial operational taxonomic unit (OTU) richness and diversity systematically peaked in autumn-winter, likely related to low resource availability during this period. BCC showed seasonal and vertical patterns, with Rhodobacteraceae and Flavobacteriaceae families dominating in surface waters, and SAR11 clade dominating at the base of the photic zone (30 m depth). BCC variability was significantly explained by environmental variables (e.g., temperature of water, solar radiation, or dissolved organic matter). Interestingly, a strong and significant correlation was found between BCC and small-sized eukaryotic community composition (ECC), which suggests that biotic interactions may play a major role as structuring factors of the microbial plankton in this productive area. In addition, co-occurrence network analyses revealed strong and significant, mostly positive, associations between bacteria and small-sized phytoplankton. Positive associations likely result from mutualistic relationships (e.g., between Dinophyceae and Rhodobacteraceae), while some negative correlations suggest antagonistic interactions (e.g., between Pseudo-nitzchia sp. and SAR11). These results support the key role of biotic interactions as structuring factors of the small-sized eukaryotic community, mostly driven by positive associations between small-sized phytoplankton and bacteria.

Seasonal Dynamics in Carbon Cycling of Marine Bacterioplankton Are Lifestyle Dependent.

Although free-living (FL) and particle-attached (PA) bacteria are recognized as ecologically distinct compartments of marine microbial food-webs, few, if any, studies have determined their dynamics in abundance, function (production, respiration and substrate utilization) and taxonomy over a yearly cycle. In the Baltic Sea, abundance and production of PA bacteria (defined as the size-fraction >3.0 µm) peaked over 3 months in summer (6 months for FL bacteria), largely coinciding with blooms of Chitinophagales (Bacteroidetes). Pronounced changes in the growth efficiency (range 0.05-0.27) of FL bacteria (defined as the size-fraction <3.0 µm) indicated the magnitude of seasonal variability of ecological settings bacteria experience. Accordingly, 16S rRNA gene analyses of bacterial community composition uncovered distinct correlations between taxa, environmental variables and metabolisms, including Firmicutes associated with elevated hydrolytic enzyme activity in winter and Verrucomicrobia with utilization of algal-derived substrates during summer. Further, our results suggested a substrate-controlled succession in the PA fraction, from Bacteroidetes using polymers to Actinobacteria and Betaproteobacteria using monomers across the spring to autumn phytoplankton bloom transition. Collectively, our findings emphasize pronounced seasonal changes in both the composition of the bacterial community in the PA and FL size-fractions and their contribution to organic matter utilization and carbon cycling. This is important for interpreting microbial ecosystem function-responses to natural and human-induced environmental changes.

Rapid bacterioplankton transcription cascades regulate organic matter utilization during phytoplankton bloom progression in a coastal upwelling system.

Coastal upwelling zones are hotspots of oceanic productivity, driven by phytoplankton photosynthesis. Bacteria, in turn, grow on and are the principal remineralizers of dissolved organic matter (DOM) produced in aquatic ecosystems. However, the molecular processes that key bacterial taxa employ to regulate the turnover of phytoplankton-derived DOM are not well understood. We therefore carried out comparative time-series metatranscriptome analyses of bacterioplankton in the Northwest Iberian upwelling system, using parallel sampling of seawater and mesocosms with in situ-like conditions. The mesocosm experiment uncovered a taxon-specific progression of transcriptional responses from bloom development (characterized by a diverse set of taxa in the orders Cellvibrionales, Rhodobacterales, and Pelagibacterales), over early decay (mainly taxa in the Alteromonadales and Flavobacteriales), to senescence phases (Flavobacteriales and Saprospirales taxa). Pronounced order-specific differences in the transcription of glycoside hydrolases, peptidases, and transporters were found, supporting that functional resource partitioning is dynamically structured by temporal changes in available DOM. In addition, comparative analysis of mesocosm and field samples revealed a high degree of metabolic plasticity in the degradation and uptake of carbohydrates and nitrogen-rich compounds, suggesting these gene systems critically contribute to modulating the stoichiometry of the labile DOM pool. Our findings suggest that cascades of transcriptional responses in gene systems for the utilization of organic matter and nutrients largely shape the fate of organic matter on the time scales typical of upwelling-driven phytoplankton blooms.

Microbial Plankton Community Structure and Function Responses to Vitamin B12 and B1 Amendments in an Upwelling System.

B vitamins are essential cofactors for practically all living organisms on Earth and are produced by a selection of microorganisms. An imbalance between high demand and limited production, in concert with abiotic processes, may explain the low availability of these vitamins in marine systems. Natural microbial communities from surface shelf water in the productive area off northwestern Spain were enclosed in mesocosms in winter, spring, and summer 2016. In order to explore the impact of B-vitamin availability on microbial community composition (16S and 18S rRNA gene sequence analysis) and bacterial function (metatranscriptomics analysis) in different seasons, enrichment experiments were conducted with seawater from the mesocosms. Our findings revealed that significant increases in phytoplankton or prokaryote biomass associated with vitamin B12 and/or B1 amendments were not accompanied by significant changes in community composition, suggesting that most of the microbial taxa benefited from the external B-vitamin supply. Metatranscriptome analysis suggested that many bacteria were potential consumers of vitamins B12 and B1, although the relative abundance of reads related to synthesis was ca. 3.6-fold higher than that related to uptake. Alteromonadales and Oceanospirillales accounted for important portions of vitamin B1 and B12 synthesis gene transcription, despite accounting for only minor portions of the bacterial community. Flavobacteriales appeared to be involved mostly in vitamin B12 and B1 uptake, and Pelagibacterales expressed genes involved in vitamin B1 uptake. Interestingly, the relative expression of vitamin B12 and B1 synthesis genes among bacteria strongly increased upon inorganic nutrient amendment. Collectively, these findings suggest that upwelling events intermittently occurring during spring and summer in productive ecosystems may ensure an adequate production of these cofactors to sustain high levels of phytoplankton growth and biomass. IMPORTANCE B vitamins are essential growth factors for practically all living organisms on Earth that are produced by a selection of microorganisms. An imbalance between high demand and limited production may explain the low concentration of these compounds in marine systems. In order to explore the impact of B-vitamin availability on bacteria and algae in the coastal waters off northwestern Spain, six experiments were conducted with natural surface water enclosed in winter, spring, and summer. Our findings revealed that increases in phytoplankton or bacterial growth associated with B12 and/or B1 amendments were not accompanied by significant changes in community composition, suggesting that most microorganisms benefited from the B-vitamin supply. Our analyses confirmed the role of many bacteria as consumers of vitamins B12 and B1, although the relative abundance of genes related to synthesis was ca. 3.6-fold higher than that related to uptake. Interestingly, prokaryote expression of B12 and B1 synthesis genes strongly increased when inorganic nutrients were added. Collectively, these findings suggest that upwelling of cold and nutrient-rich waters occurring during spring and summer in this coastal area may ensure an adequate production of B vitamins to sustain high levels of algae growth and biomass.

Labile Dissolved Organic Matter Compound Characteristics Select for Divergence in Marine Bacterial Activity and Transcription.

Bacteria play a key role in the planetary carbon cycle partly because they rapidly assimilate labile dissolved organic matter (DOM) in the ocean. However, knowledge of the molecular mechanisms at work when bacterioplankton metabolize distinct components of the DOM pool is still limited. We, therefore, conducted seawater culture enrichment experiments with ecologically relevant DOM, combining both polymer and monomer model compounds for distinct compound classes. This included carbohydrates (polysaccharides vs. monosaccharides), proteins (polypeptides vs. amino acids), and nucleic acids (DNA vs. nucleotides). We noted pronounced changes in bacterial growth, activity, and transcription related to DOM characteristics. Transcriptional responses differed between compound classes, with distinct gene sets ("core genes") distinguishing carbohydrates, proteins, and nucleic acids. Moreover, we found a strong divergence in functional transcription at the level of particular monomers and polymers (i.e., the condensation state), primarily in the carbohydrates and protein compound classes. These specific responses included a variety of cellular and metabolic processes that were mediated by distinct bacterial taxa, suggesting pronounced functional partitioning of organic matter. Collectively, our findings show that two important facets of DOM, compound class and condensation state, shape bacterial gene expression, and ultimately select for distinct bacterial (functional) groups. This emphasizes the interdependency of marine bacteria and labile carbon compounds for regulating the transformation of DOM in surface waters.

High Frequency Multi-Year Variability in Baltic Sea Microbial Plankton Stocks and Activities.

Marine bacterioplankton are essential in global nutrient cycling and organic matter turnover. Time-series analyses, often at monthly sampling frequencies, have established the paramount role of abiotic and biotic variables in structuring bacterioplankton communities and productivities. However, fine-scale seasonal microbial activities, and underlying biological principles, are not fully understood. We report results from four consecutive years of high-frequency time-series sampling in the Baltic Proper. Pronounced temporal dynamics in most investigated microbial variables were observed, including bacterial heterotrophic production, plankton biomass, extracellular enzyme activities, substrate uptake rate constants of glucose, pyruvate, acetate, amino acids, and leucine, as well as nutrient limitation bioassays. Spring blooms consisting of diatoms and dinoflagellates were followed by elevated bacterial heterotrophic production and abundances. During summer, bacterial productivity estimates increased even further, coinciding with an initial cyanobacterial bloom in early July. However, bacterial abundances only increased following a second cyanobacterial bloom, peaking in August. Uptake rate constants for the different measured carbon compounds varied seasonally and inter-annually and were highly correlated to bacterial productivity estimates, temperature, and cyanobacterial abundances. Further, we detected nutrient limitation in response to environmental conditions in a multitude of microbial variables, such as elevated productivities in nutrient bioassays, changes in enzymatic activities, or substrate preferences. Variations among biotic variables often occurred on time scales of days to a few weeks, yet often spanning several sampling occasions. Such dynamics might not have been captured by sampling at monthly intervals, as compared to more predictable transitions in abiotic variables such as temperature or nutrient concentrations. Our study indicates that high resolution analyses of microbial biomass and productivity parameters can help out in the development of biogeochemical and food web models disentangling the microbial black box.

Growth rates of different phylogenetic bacterioplankton groups in a coastal upwelling system.

Microbial degradation of dissolved organic matter (DOM) in planktonic ecosystems is carried out by diverse prokaryotic communities, whose growth rates and patterns of DOM utilization modulate carbon and nutrient biogeochemical cycles at local and global scales. Nine dilution experiments (September 2007 to June 2008) were conducted with surface water from the highly productive coastal upwelling system of the Ría de Vigo (NW Iberian Peninsula) to estimate bacterial growth rates of six relevant marine bacterial groups: Roseobacter, SAR11, Betaproteobacteria,Gammaproteobacteria, SAR86 and Bacteroidetes. Surprisingly, SAR11 dominated over the other bacterial groups in autumn, likely associated to the entry of nutrient-rich, DOC-poor Eastern North Atlantic Central Water (ENACW) into the embayment. Roseobacter and SAR11 showed significantly opposing growth characteristics. SAR11 consistently grows at low rates (range 0.19-0.71 day(-1) ), while Roseobacter has a high growth potential (range 0.70-1.64 day(-1) ). In contrast, Betaproteobacteria, Bacteroidetes, SAR86 and Gammaproteobacteria growth rates widely varied among experiments. Regardless of such temporal variability, mean SAR86 growth rate (range 0.1-1.4 day(-1) ) was significantly lower than that of Gammaproteobacteria (range 0.3-2.1 day(-1) ). Whereas the relative abundance of different bacterial groups showed strong correlations with several environmental variables, group-specific bacterial growth rates did not co-vary with ambient conditions. Our results suggest that different bacterial groups exhibit characteristic growth rates, and, consequently, distinct competitive abilities to succeed under contrasting environmental conditions.

Osteoporosis-related life habits and knowledge about osteoporosis among women in El Salvador: a cross-sectional study.

BACKGROUND: Osteoporosis is a systemic skeletal disorder, characterized by reduced bone mass, deterioration of bone structure, increased bone fragility, and increased fracture risk. It is more frequent to find among women than men at a 4:1 ratio. Evidence suggests that to adopt changes on some life habits can prevent or delay development of osteoporosis. Several osteoporosis-risk factors have been confirmed in the US and western Europe, but in El Salvador there are neither reliable epidemiological statistics about this skeletal disorder nor studies addressing osteoporosis-risk factors in women. The aim of this study was to determinate the extent of osteoporosis knowledge, the levels of both daily calcium intake and weight-bearing physical activity, and the influence of several osteoporosis-risk factors on these variables in three age groups of Salvadorean women. METHODS: In this exploratory cross-sectional study, an osteoporosis knowledge assessment questionnaire including a food frequency and a physical activity record section were used to collect data and it was delivered through a face-to-face interview. A convenience sample (n = 197) comprised of three groups of women aged 25-35 years, 36-49 years, and over 49 years was taken. Among-group comparisons of means were analyzed by two-way ANOVA. To determinate the overall influence of osteoporosis-risk factors, the multivariate analysis was used. RESULTS: Study results indicated that better educated women had more knowledge about osteoporosis than women with a low education level, regardless of age, even though this knowledge was rather fair. Older women got more weight-bearing physical activity at home and less at place of employment than reported by the younger women; however, neither group performed sufficient high-intensity WBPA to improve bone mass. Regardless of age, the most women consumed 60% or less than the Dietary Reference Intake of calcium and depend on household income, lactose intolerance and coffee rather than milk consumption. CONCLUSION: In summary, the majority of women in this study have modest knowledge on osteoporosis. The knowledge base is not linked to preventive health habits, including sufficient calcium intake and performance of weight-bearing physical activities. They are thus at increased risk for low bone mass.