High Nucleic Acid Content Research Articles

Bacterial community structure along the subtidal sandy sediment belt of a high Arctic fjord (Kongsfjorden, Svalbard Islands)

Open fjords are subject to contrasting environmental conditions, owing to meltwater glacial inputs, terrestrial runoff, and marine water mass exchanges, which are exacerbated by anthropogenic and climate perturbations. Following a slope-dependent water circulation, the subtidal sandy sediment belt regulates the convergent transport of nutrients downward the fjord depths, and the effective entrapment of suspended particles and microorganisms. In this study, we aimed at testing how glacial and seawater inputs may influence the bacterial community structure of subtidal sand deposits in the Kongsfjorden. Through total and viable cell counting and an amplicon sequencing approach, we found relevant differences in bacterial community structure along the glacio-marine sampling transect. Viable and high nucleic acid content (HNA) cells represented an important fraction of the total community, generally decreasing toward the glacier front. Besides the predominance of Alpha- and Gammaproteobacteria, Bacteroidetes, Firmicutes and Parcubacteria, the bacterial community structure was likely affected by the glacial activity in the inner fjord, with the occurrence of distinctive phylotypes belonging to Gemmatimonadates, Nitrospirae, Acidobacteria, and Chloroflexi. Overall, our outcomes highlighted that exploring the bacterial community distribution and structure can provide new insights into the active role of sand deposits in coastal cold environments.

Fronts at the Surface Ocean Can Shape Distinct Regions of Microbial Activity and Community Assemblages Down to the Bathypelagic Zone: The Azores Front as a Case Study

Oceanic fronts are widespread features which separate distinct water masses. They are well known to control the distribution of microbial communities in surface waters, although there is scarce information on their role in delimiting critical functions that microbes perform, and on whether their effects can be translated down into the dark ocean. Here we carried out the first study on the variability of hydrolysis of organic matter (extracellular enzymatic activities; EEA) across a permanent front (the Azores Front), coupled with changes in microbial assemblage composition, from the surface down to the bathypelagic zone. The front separated the study area (enclosed into the North Atlantic Subtropical Gyral Province) into two distinct latitudinal sub-regions with sharp differences in the abundance of autotrophic and heterotrophic microbial assemblages, as well as in the extracellular enzymes activities of glucosidases, alkaline phosphatase and leucine aminopeptidase. South of the front there was an abrupt decline in the abundance of picophytoplankton as well as in heterotrophic prokaryotes with high nucleic-acid content, but an increase in the abundance of prokaryotes with high side-scatter, an indication that cells were growing attached to particles. Concomitantly, there was also an increase in the aminopeptidase to glucosidase ratio, a proxy of higher degradation of proteinaceous material relative to carbohydrates. Interestingly, these sharp changes in microbial assemblages and enzymatic activities north and south of the front were translated down to the deep ocean. Our results suggest that permanent fronts, like the Azores Front, can act as ecological boundaries in the ocean (even within a biogeographical province), in terms of microbial community structure and biogeochemical cycling. Oriented studies on oceanic fronts down to the deep ocean will help to understand how the variability of these widely-extended hydrographic futures will impact microbial communities and carbon cycling in a future ocean affected by trends in global warming, de-oxygenation and acidification.

In-situ features of LNA and HNA bacteria in branch ends of drinking water distribution systems

The knowledge of bacteria with low or high nucleic acid (LNA or HNA, respectively) content was relatively limited in drinking water distribution systems (DWDSs). In the present study, LNA and HNA bacterial growth characteristics and effect of water physicochemical properties on these two subgroups were analyzed at the branch ends of a DWDS. The results showed that LNA bacteria were in the majority in tap water (67.2% ± 16.6%) and the specific growth rate (0.469 ± 0.022/d) was lower than that of HNA bacteria (1.116 ± 0.195/d). LNA and HNA bacteria showed different responses to water physicochemical properties. The LNA bacteria dominated the microbial community under relatively high conductivity conditions, and the HNA bacteria were predominant in high turbidity or alkalescence environments. The LNA bacteria growth rate was positively correlated to the assimilable organic carbon concentration. In addition, multivariate ordination illustrated that there were clear variances in water quality among outlets, which could result from different pipeline distances and hydraulic conditions.

Assessing ultraphytoplankton and heterotrophic prokaryote composition by flow cytometry in a Mediterranean lagoon

In the eutrophic Ghar El Melh Lagoon (GML, Tunisia), the distribution of heterotrophic prokaryotes, pico- and nanophytoplankton was studied at five stations in November 2012 at the single cell level, along with environmental factors. Flow cytometry analysis of ultraplankton (<10μm) resolved (i) two heterotrophic prokaryote groups, low and high nucleic acid contents (LNA and HNA, respectively), and (ii) eight to nine ultraphytoplankton groups (cryptophyte-like cells, two nanoeukaryote subgroups, two picoeukaryote subgroups and three Synechococcus-like cells subgroups). Prochlorococcus was not detected. According to redundancy analysis (RDA), a significant difference was found in the distribution of the ultraplankton between stations (F=2.61, p<0.05); maximum proliferations of heterotrophic prokaryotes were observed in the inner parts of the lagoon at stations 3, 4 and 5 affected by urban, agricultural and industrial discharges. Ultraphytoplankton concentrations were the highest near the outlet of the lagoon at stations 1 and 2 influenced by freshwater outflow and oligotrophic Mediterranean water inflow, respectively. At station 1, the large ultraphytoplankton concentration derives from the high abundance of cryptophyte-like cells favoured by the freshwater outflow whereas at station 2, the input of oligotrophic Mediterranean water enhanced the abundance of Synechococcus and picoeukaryotes at the expense of nanoeukaryotes. Two trophic regimes were thus differentiated in GML.

Geographic distribution pattern of low and high nucleic acid content bacteria on a river-catchment scale

Bacteria with low nucleic acid content (LNA) and high nucleic acid content (HNA) are widely distributed in aquatic environments. Most of the current understanding of these two subgroups is derived from studies in marine environments. In comparison, information on the spatial distribution of these two subgroups in freshwater environments is very limited. The present study analysed the biogeographical pattern of those two groups on a large-river scale (i.e. the Songhua River catchment, &gt;1000km). The results showed that the concentrations of LNA and HNA bacteria were distributed over a wide range from 5.45×104 to 4.43×106cellsmL–1, and from 1.35×105 to 4.37×106cellsmL–1 respectively. The two groups have almost equal proportions in the Songhua River, with the average contribution of LNA bacteria reaching 47.0%. In comparison, the abundance of LNA bacteria in the mainstream was significantly higher than in the tributaries. The cytometric expressions (green fluorescence and side scatter) within LNA and HNA were strongly covaried, which implies that these two subgroups are intrinsically linked. Multivariate redundancy analysis indicated that both the abundance and cytometric characteristics of co-occurring LNA and HNA bacteria were regulated differently in the Songhua River. This suggests that LNA and HNA bacteria play different ecological roles in river ecosystems.

Coupled Response of Bacterial Production to a Wind-Induced Fall Phytoplankton Bloom and Sediment Resuspension in the Chukchi Sea Shelf, Western Arctic Ocean

Heterotrophic bacterial abundance and production, dissolved free amino acid (DFAA) and dissolved combined amino acid (DCAA) concentrations, and other microbial parameters were determined for seawater samples collected at a fixed station (maximum water depth, 56 m) deployed on the Chukchi Sea Shelf, in the western Arctic Ocean, during a 16-day period in September 2013. During the investigation period, the sampling station experienced strong winds and a subsequent phytoplankton bloom, which was thought to be triggered by enhanced vertical mixing and upward nutrient fluxes. In this study, we investigated whether bacterial and dissolved amino acid parameters changed in response to these physical and biogeochemical events. Bacterial abundance and production in the upper layer increased with increasing chlorophyll a concentration, despite a concomitant decrease in seawater temperature from 3.2°C to 1.5°C. The percentage of bacteria with high nucleic acid content during the bloom was significantly higher than that during the prebloom period. The ratio of the depth-integrated (0–20 m) bacterial production to primary production differed little between the prebloom and bloom period, with an overall average value of 0.14 ± 0.03 (± standard deviation, n = 8). DFAA and DCAA concentrations varied over a limited range throughout the investigation, indicating that the supply and consumption of labile dissolved amino acids were balanced. These results indicate that there was a tightly coupled, large flow of organic carbon from primary producers to heterotrophic bacteria during the fall bloom. Our data also revealed that bacterial production and abundance were high in the bottom nepheloid (low transmittance) layer during strong wind events, which was associated with sediment resuspension due to turbulence near the seafloor. The impacts of fall wind events, which are predicted to become more prominent with the extension of the ice-free period, on bacterial processes and the dynamics of organic matter in the Chukchi Sea Shelf could have far-reaching influences on biogeochemical cycles and ecosystem dynamics in broader regions of the Arctic Ocean.

Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities.

Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures.

Functional Responses of Bacterioplankton Diversity and Metabolism to Experimental Bottom-Up and Top-Down Forcings.

We conducted an experimental approach using microcosms to simultaneously examine the functional response of natural freshwater bacterial assemblages to the impact of resources (nutrients) and top-down factors (viruses and grazers) on bacterial physiological state and their community structure. Addition of organic and inorganic nutrients led to the proliferation of high nucleic acid content bacterial cells accompanied by high bacterial growth efficiency (considered as proxy of bacterial carbon metabolism) estimates, suggesting that this subgroup represented the most active fraction of bacterial community and had a high capacity to incorporate carbon into its biomass. However, their rapid growth induced the pressure of viral lytic infection which led to their lysis toward the end of the experiment. In microcosms with flagellates plus viruses, and with viruses alone, the selective removal of metabolically active high nucleic acid cells through viral lysis benefitted the less active low nucleic acid content cells, perhaps via the use of lysis products for its growth and survival. Changes in bacterial physiological state in microcosms were reflected in their community structure which was examined using 16S ribosomal RNA (rRNA) gene sequencing by Illumina's Miseq platform. Chao estimator and Shannon diversity index values suggested that bacterial species richness was highest in the presence of both the top-down factors, indicating a tighter control of bacterioplankton dominants within a relatively stable bacterial community. The increase in bacterial metabolism with nutrient addition followed by subsequent lysis of bacterial dominants indicate that both resources and top-down factors work in concert for the sustenance of stable bacterial communities.

Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River.

Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.

Immunocorrective dairy bacterial and yeast drink high in nucleic acids for clinical use in the practice of family doctor

In hepatology nucleic acids (usually preparation nucleinate) are usually administered to patients with chronic hepatitis C, who receive interferon therapy, for stimulation of leucopoiesis. However, nucleic acids have a significantly wider spectrum of biological action. Results of experimental investigations concerning influence of milk bacterialyeast beverage with a considerable content of nucleic acids on different functions of the body have been presented in our research. Materials and methods. Milk bacterial1yeast beverage, containing nucleic acids, was tested. Authors also elaborated technique of koumiss production in dry state (content of DNA – 0,76 mg/g, and RNA – 0,61 mg/g). Mechanism of immunopotentiating action of koumiss from cow’s milk was studied in experiment on animals and birds. Immunopotantiating action of the beverage on human beings was estimated by its influence on the efficacy of preventive immunization against diphtheria. Results. In experiment on piglets it was established that administration of food supplement promoted probable decrease in incidence of gastrointestinal diseases: in experimental group 10% of piglets were ill during observation period, in control group – 70,9%. Average haematological indices, content of erythrocytes and haemoglobin in experimental group of piglets, were higher and constituted 8,14± 0,32 million and 8,16±0,37 g% respectively, and in control group – 7,95±3,4 million and 7,57±0,38 g% respectively (Р 1:1280, constituted only 60%, in experimental – 100%. The obtained data concerning influence of koumiss on synthesis of antitoxin confirm its immunopotentiating action and simultaneously indicate expediency of administration of immunostimulants during vaccination. Conclusions. It has been established that food supplement has a considerable biological activity, in particular, it influences positively immunity indices. In our opinion, it is important that besides high content of ribonucleic acid (1800–2100 mcg/ml), the beverage contains complete set of essential amino acids, glutamine acid (to 6 g/l), vitamins from B group and other useful substances (all components in optimal ratio). Adding of milk bacterial-yeast beverage with high content of nucleic acids to patients’ diet has certain advantages over pharmacological preparations.

Impact of Treated Sewage Effluent on the Microbiology of a Small Brook Using Flow Cytometry as a Diagnostic Tool

Flow cytometry was applied to assess the microbiological impact of treated sewage effluent discharge into a small brook carrying surface runoff water. Increases in dissolved organic carbon and soluble reactive phosphorous were accompanied by increases in counts of intact bacteria by up to eightfold. Effluent ingress furthermore resulted in a pronounced shift of bacterial clusters. Whereas brook water upstream of the discharge point was characterised by a bacterial cluster with low nucleic acid (LNA) content, downstream water showed a shift to bacteria with high nucleic acid (HNA) content. Changes in the LNA/HNA ratio were largely maintained along the course of the brook. Results suggest that the LNA/HNA ratio can under certain conditions serve as an indicator of anthropogenic nutrient impact. Measuring impact on this low trophic level might be more sensitive and straightforward than measuring macroindicators. More evidence will however be required to assess the usefulness of LNA/HNA measurements to assess the ecological nutrient status of natural waters and the impact of nutrient pollution.

Ultra- and microplankton assemblages as indicators of trophic status in a Mediterranean lagoon

The seasonal abundance distribution of heterotrophic prokaryotes, pico- and nanophytoplankton, was investigated in connection with environmental variables and microplankton abundance at five stations in Ghar El Melh Lagoon (northeastern Tunisia). Flow cytometry analysis of ultraplankton resolved (i) five heterotrophic prokaryote groups labelled LNA1, LNA2 (low nucleic acid content), HNA1, HNA2 and HNA3 (high nucleic acid content) and (ii) at least 14 ultraphytoplankton groups assigned to picoeukaryotes, picoprokaryotes, nanoeukaryotes, cryptophyte-like cells and some unknown communities. Redundancy analysis (RDA) revealed (i) autumn-summer outbreaks of heterotrophic prokaryotes dominated by HNA groups and (ii) winter-summer proliferation of ultraphytoplankton dominated by nanophytoplankton groups. Generalized additive models (GAM) highlighted the role of (i) water temperature and orthophosphate concentrations in heterotrophic prokaryote distribution and (ii) water temperature and salinity in ultraphytoplankton abundance variation. Based on Spearman's rank correlation, significant negative correlations were established between ultra- and microplankton communities suggesting that, through grazing pressure, microplankton may be behind the drastic decrease in ultraplankton abundances in spring.

Effects of Oxygen Loss on Carbon Processing and Heterotrophic Prokaryotes from an Estuarine Ecosystem: Results from Stable Isotope Probing and Cytometry Analyses

Many aquatic ecosystems are experiencing a decline in their oxygen (O2) content and this is predicted to continue. Implications of this change on several properties of bacterioplankton (heterotrophic prokaryotes) remain however are poorly known. In this study, oxic samples (∼170 μM O2 = controls) from an oligohaline region of the Scheldt Estuary were purged with N2 to yield low-O2 samples (∼69 μM O2 = treatments); all were amended with 13C-glucose and incubated in dark to examine carbon incorporation and cell size of heterotrophic prokaryotes, and relationships between organic matter (OM) degradation and phosphate (P) availability in waters following O2 loss. Stable isotope (13C) probing of phospholipid fatty acids (PLFA) and flow cytometry were used. In samples that have experienced O2 loss, PLFA biomass became higher, prokaryotic cells had significantly larger size and higher nucleic acid content, but P concentrations was lower, compared to controls. P concentration and OM degradation were positively related in controls, but uncoupled in low-O2 samples. Moreover, the dominant PLFA 16:1ω7c (likely mainly from Gram-negative bacteria) and the nucleic acid content of heterotrophic prokaryotic cells in low-O2 samples explained (62–72 %) differences between controls and low-O2 samples in P amounts. Shortly after incubations began, low-O2 samples had consistently lower bacterial PLFA 13C-enrichments, suggesting involvement of facultatively anaerobic metabolism in carbon incorporation, and supporting the view that this metabolic pathway is widespread among pelagic bacteria in coastal nutrient-rich ecosystems. Estimates based on 13C-enrichment of PLFAs indicated that grazing by protozoa on some bacteria was stronger in low-O2 samples than in controls, suggesting that the grazing pressure on some heterotrophic prokaryotes may increase at the onset of O2 deficiency in nutrient-rich aquatic systems. These findings also suggest that physiological responses of heterotrophic prokaryotes to O2 loss in such ecosystems include increases in cell activity, high carbon incorporation, and possibly phosphorus retention by cells that may contribute to reduce phosphate availability in waters.

Sample dilution and bacterial community composition influence empirical leucine-to-carbon conversion factors in surface waters of the world's oceans.

The transformation of leucine incorporation rates to prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world's oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e., removal of grazers [F treatment]) or filtration combined with dilution (i.e., also relieving resource competition [FD treatment]). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu(-1) and were significantly lower in the FD than in the F samples. Also, changes in bacterial community composition during the incubations, as assessed by automated ribosomal intergenic spacer analysis (ARISA), were more pronounced in the FD than in the F treatments, compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCF determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyll a concentration, picocyanobacterial abundance (mostly Prochlorococcus), and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCF variability in the F treatment, supporting the view that both Prochlorococcus and HNA prokaryotes incorporate leucine in substantial amounts, although this results in relatively low carbon production rates in the oligotrophic ocean.

Cationic Mucic Acid Polymer-Based siRNA Delivery Systems.

Nanoparticle (NP) delivery systems for small interfering RNA (siRNA) that have good systemic circulation and high nucleic acid content are highly desired for translation into clinical use. Here, a family of cationic mucic acid-containing polymers is synthesized and shown to assemble with siRNA to form NPs. A cationic mucic acid polymer (cMAP) containing alternating mucic acid and charged monomers is synthesized. When combined with siRNA, cMAP forms NPs that require steric stabilization by poly(ethylene glycol) (PEG) that is attached to the NP surface via a 5-nitrophenylboronic acid linkage (5-nitrophenylboronic acid-PEGm (5-nPBA-PEGm)) to diols on mucic acid in the cMAP in order to inhibit aggregation in biological fluids. As an alternative, cMAP is covalently conjugated with PEG via two methods. First, a copolymer is prepared with alternating cMAP-PEG units that can form loops of PEG on the surface of the formulated siRNA-containing NPs. Second, an mPEG-cMAP-PEGm triblock polymer is synthesized that could lead to a PEG brush configuration on the surface of the formulated siRNA-containing NPs. The copolymer and triblock polymer are able to form stable siRNA-containing NPs without and with the addition of 5-nPBA-PEGm. Five formulations, (i) cMAP with 5-nPBA-PEGm, (ii) cMAP-PEG copolymer both (a) with and (b) without 5-nPBA-PEGm, and (iii) mPEG-cMAP-PEGm triblock polymer both (a) with and (b) without 5-nPBA-PEGm, are used to produce NPs in the 30-40 nm size range, and their circulation times are evaluated in mice using tail vein injections. The mPEG-cMAP-PEGm triblock polymer provides the siRNA-containing NP with the longest circulation time (5-10% of the formulation remains in circulation at 60 min postdosing), even when a portion of the excess cationic components used in the formulation is filtered away prior to injection. A NP formulation using the mPEG-cMAP-PEGm triblock polymer that is free of excess components could contain as much as ca. 30 wt % siRNA.

More, smaller bacteria in response to ocean's warming?

Heterotrophic bacteria play a major role in organic matter cycling in the ocean. Although the high abundances and relatively fast growth rates of coastal surface bacterioplankton make them suitable sentinels of global change, past analyses have largely overlooked this functional group. Here, time series analysis of a decade of monthly observations in temperate Atlantic coastal waters revealed strong seasonal patterns in the abundance, size and biomass of the ubiquitous flow-cytometric groups of low (LNA) and high nucleic acid (HNA) content bacteria. Over this relatively short period, we also found that bacterioplankton cells were significantly smaller, a trend that is consistent with the hypothesized temperature-driven decrease in body size. Although decadal cell shrinking was observed for both groups, it was only LNA cells that were strongly coherent, with ecological theories linking temperature, abundance and individual size on both the seasonal and interannual scale. We explain this finding because, relative to their HNA counterparts, marine LNA bacteria are less diverse, dominated by members of the SAR11 clade. Temperature manipulation experiments in 2012 confirmed a direct effect of warming on bacterial size. Concurrent with rising temperatures in spring, significant decadal trends of increasing standing stocks (3% per year) accompanied by decreasing mean cell size (−1% per year) suggest a major shift in community structure, with a larger contribution of LNA bacteria to total biomass. The increasing prevalence of these typically oligotrophic taxa may severely impact marine food webs and carbon fluxes by an overall decrease in the efficiency of the biological pump.

Heterotrophic prokaryote distribution along a 2300 km transect in the North Pacific subtropical gyre during a strong La Niña conditions: relationship between distribution and hydrological conditions

Abstract. The spatial distribution of heterotrophic prokaryotes was investigated during the Tokyo–Palau cruise in the western part of the North Pacific subtropical gyre (NPSG) along a north–south transect between 33.60 and 13.25° N. The cruise was conducted in three different hydrological areas identified as the Kuroshio region, the subtropical gyre area and the transition zone. Two eddies were crossed along the transect: one cold-core cyclonic eddy and one warm-core anticyclonic eddy and distributions of the heterotrophic prokaryotes were recorded. By using analytical flow cytometry and a nucleic acid staining protocol, heterotrophic prokaryotes were discriminated into three subgroups depending on their nucleic acid content (low, high and very high nucleic acid contents labelled LNA, HNA and VHNA, respectively). Statistical analyses performed on the data set showed that LNA, mainly associated with low temperature and low salinity, were dominant in all the hydrological regions. In contrast, HNA distribution seemed to be associated with temperature, salinity, Chl a and silicic acid. A latitudinal increase in the HNA / LNA ratio was observed along the north–south transect and was related to higher phosphate and nitrate concentrations. However, the opposite relationship observed for the VHNA / HNA ratio suggested that the link between nucleic acid content and oligotrophic conditions is not linear, underlying the complexity of the biodiversity in the VHNA, HNA and LNA subgroups. In the Kuroshio Current, it is suggested that the high concentration of heterotrophic prokaryotes observed at station 4 was linked to the path of the cold cyclonic eddy core. In contrast, it is thought that low concentrations of heterotrophic prokaryotes in the warm core of the anticyclonic gyre (Sta. 9) are related to the low nutrient concentrations measured in the seawater column. Our results showed that the high variability between the various heterotrophic prokaryote cluster abundances depend both on the mesoscale structures and the oligotrophic gradient.

Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

Viral metagenomic approaches are increasingly being used for viral discovery. Various strategies are applied to enrich viral sequences, but there is often a lack of knowledge about their effective influence on the viral discovery sensitivity. We evaluate some convenient and widely used approaches for RNA virus discovery in clinical samples in order to reveal their sensitivity and potential bias introduced by the enrichment or amplifications steps. An RNA virus was artificially spiked at a fixed titer in serum and lung tissue, respectively, low and high nucleic acid content matrices. For serum, a simple DNase treatment on the RNA extract gave the maximum gain in proportion of viral sequences (83×), and a subsequent ribosomal RNA removal nearly doubled once more the proportion of viral sequences. For lung tissue, a ribosomal RNA depletion step on the RNA extract had the biggest gain in proportion of viral sequences (32×). We show also that direct sequencing of cDNA is recommended above an extra random PCR amplification step, and a that the virion enrichment strategy (filtration and nuclease treatment) has a beneficial effect for sequencing-based virus discovery. Our findings provide sample-dependent guidelines for targeted virus discovery strategies.

Microbial metabolism of transparent exopolymer particles during the summer months along a eutrophic estuary system.

This study explores the role of transparent exopolymer particles (TEP) as an additional carbon source for heterotrophic microbial activity in the eutrophic Qishon estuary. From the coastal station and upstream the estuary; TEP concentrations, β-glucosidase activity, bacterial production and abundance have gradually increased. TEP were often found as bio-aggregates, scaffolding algae, detritus matter and bacteria that likely formed “hotspots” for enhance microbial activity. To further demonstrate the link between TEP and heterotrophic bacterial activity, confined incubations with ambient and polysaccharide-enriched estuary water were carried out. Following polysaccharide addition, elevated (~50%) β-glucosidase activity rates were observed, leading to TEP hydrolysis. This newly formed bioavailable carbon resulted in significantly higher growth rates, with up to a 5-fold increase in heterotrophic bacterial biomass, comprising mostly high nucleic acid content bacteria. Taking together the findings from this research, we conclude that even in highly eutrophic environments heterotrophic bacteria may still be carbon limited. Further, TEP as a polysaccharide matrix can act as a metabolic surrogate, adding fresh bioavailable carbon through tight associations with bacteria in eutrophic ecosystems such as the Qishon estuary.

Using coagulation to restrict microbial re-growth in tap water by phosphate limitation in water treatment

Extensive microbial re-growth in a drinking water distribution system can deteriorate water quality. The limiting factor for microbial re-growth in a tap water produced by a conventional drinking water treatment plant in China was identified by determining the microbial re-growth potential (MRP) by adding different nutrients to stimulate growth of a natural microbial consortium as inoculum and flow-cytometric enumeration. No obvious change of MRP was found in tap water after addition of carbon, whereas, a 1- to 2-fold increase of MRP was observed after addition of phosphate (P). This clearly demonstrated that microbial re-growth in this tap water was limited by P. Most of the re-grown microbial flora (>85%) consisted of high nucleic acid content cells.A subsequent investigation of the MRP in the actual water treatment plant demonstrated that coagulation was the crucial step for decreasing MRP and producing P-limited water. Therefore, a comparison concerning the control of MRP by three different coagulants was conducted. It showed that all the three coagulants efficiently reduced the MRP and shifted the limitation regime from C to P, but the required dose was different. The study shows that it is feasible to restrict microbial re-growth by P limitation using coagulation in water treatment.