Catalyzed Reporter Deposition Research Articles

Bacterial community succession in response to dissolved organic matter released from live jellyfish

Jellyfish blooms have increased worldwide, and the outbreaks of jellyfish population not only affect the food web structures via voracious predation but also play an important role in the dynamics of nutrients and oxygen in planktonic food webs. However, it remains unclear whether specific carbon compounds released through jellyfish metabolic processes have the potential to shape bacterial community composition. Therefore, in this study, we aimed to investigate the compositional succession of the bacterioplankton community in response to the dissolved organic matter (DOM) released by the live Scyphomedusae Cyanea lamarckii and Chrysaora hysoscella collected from Helgoland Roads of the North Sea. The bacterial community was significantly stimulated by the DOM released form live jellyfish and different dominant phylotypes were observed for these two Scyphomedusae species. Furthermore, the bacterial community structures in the different DOM sources, jellyfish-incubated media, Kabeltonne seawater, and artificial seawater (DOM-free) were significantly different, as revealed by automated ribosomal intergenic spacer analysis fingerprints. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) revealed a rapid species-specific shift in bacterial community composition. Gammaproteobacteria dominated the community instead of the Bacteroidetes community for C. lamarckii, whereas Gammaproteobacteria and Bacteroidetes dominated the community for C. hysoscella. The significant differences in the bacterial community composition and succession indicate that the components of the DOM released by jellyfish might differ with jellyfish species.

Rapid and Sensitive Quantification of Anammox Bacteria by Flow Cytometric Analysis Based on Catalyzed Reporter Deposition Fluorescence In Situ Hybridization.

The quantification of anammox bacteria is crucial to manipulation and management of anammox biosystems. In this study, we proposed a protocol specifically optimized for quantification of anammox bacteria abundance in anammox sludge samples using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and flow cytometry (FCM) in combination (Flow-CARD-FISH). We optimized the pretreatment procedures for FCM-compatibility, as well as the permeabilization, hybridization and staining protocols of the CARD-FISH. The developed method was compared with other methods for specific bacteria quantification (standard FISH, 16S rRNA sequencing and quantitative polymerase chain reaction). Anammox sludge samples could be disaggregated effectively by sonication (specific energy of 90 kJ·L-1 with MLVSS of 3-5 g·L-1) with the mixed ionic and nonionic dispersants Triton X-100 (5%) and sodium pyrophosphate (10 mM). Lysozyme treatment for permeabilizing bacterial cell walls and H2O2 incubation for completely quenching endogenous peroxidase of anammox sludges were essential to fluorescence enhancement and false positive signals control, respectively. Horseradish peroxidase molecules labeling at 20 °C for 12 h and the fluorescent tyramide labeling at 25 °C for 30 min with a fluorescent substrate concentration of 1:50 maintained the balance between increasing the signal and preventing nonspecific binding. Flow-CARD-FISH results showed that anammox bacteria absolute abundance in two different sludge samples were (2.31 ± 0.01) × 107 and (1.20 ± 0.06) × 107 cells·mL-1, respectively, with the relative abundances of 36.7 ± 4.1% and 26.5 ± 3.7%, respectively, comparable with those of qPCR and 16S rRNA sequencing analysis. The enhanced fluorescence signals induced by CARD-FISH combined with the high quantitative fluorescence sensitivity of FCM provide a rapid and sensitive method that yields accurate quantification results that will be valuable in future studies of microbial community determination.

Modulation of microbial growth and enzymatic activities in the marine environment due to exposure to organic contaminants of emerging concern and hydrocarbons

Organic pollutants are continuously being introduced in seawater with uncharacterized impacts on the engines of the marine biogeochemical cycles, the microorganisms. The effects on marine microbial communities were assessed for perfluoroalkyl substances, organophosphate esters flame retardants and plasticizers, polycyclic aromatic hydrocarbons, and n-alkanes. Dose-response experiments were performed at three stations and at three depths in the NW Mediterranean with contrasted nutrient and pollutant concentrations. In these experiments, the microbial growth rates, the abundances of the main bacterial groups, measured by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH), and extracellular enzymatic activities, were quantified. Increasing concentrations of organic pollutants (OPs) promoted different responses in the communities that were compound, organism and nutrient availability (trophic status). The largest differences between OP treatments and controls in the growth rates of both heterotrophic and phototrophic microbial groups were observed in seawater from the deep chlorophyll maxima. Furthermore, there was a compound specific stimulation of different extracellular enzymatic activities after the exposure to OPs. Our results revealed that marine microbial communities reacted not only to hydrocarbons, known to be used as a carbon source, but also to low concentrations of organic pollutants of emerging concern in a complex manner, reflecting the variability of various environmental variables. Multiple linear regressions suggested that organic pollutants modulated the bacterial growth and extracellular enzymatic activities, but this modulation was of lower magnitude than the observed pronounced response of the microbial community to nutrient availability.

Biofilm diversity, structure and matrix seasonality in a full-scale cooling tower

Biofilms commonly colonise cooling water systems, causing equipment damage and interference with the operational requirements of the systems. In this study, next-generation sequencing (NGS), catalysed reporter deposition fluorescence in situ hybridisation (CARD-FISH), lectin staining and microscopy were used to evaluate temporal dynamics in the diversity and structure of biofilms collected seasonally over one year from an open full-scale cooling tower. Water samples were analysed to evaluate the contribution of the suspended microorganisms to the biofilm composition and structure. Alphaproteobacteria dominated the biofilm communities along with Beta- and Gammaproteobacteria. The phototrophic components were mainly cyanobacteria, diatoms and green algae. Bacterial biodiversity decreased from winter to autumn, concurrently with an increase in cyanobacterial and microalgal richness. Differences in structure, spatial organisation and glycoconjugates were observed among assemblages during the year. Overall, microbial variation appeared to be mostly affected by irradiance and water temperature rather than the source of the communities. Variations in biofilms over seasons should be evaluated to develop specific control strategies.

Experimental evidence of the quantitative relationship between the prokaryote ingestion rate and the food vacuole content in mixotrophic phytoflagellates.

The verification that many phytoflagellates ingest prokaryotes has changed the view of the microbial loop in aquatic ecosystems. Still, progress is limited because the phagotrophic activity is difficult to quantify in natural assemblages. Linking the food vacuole content in protist with the ingestion rate of prokaryotes would provide a crucial step forward. In this study, using the catalysed reporter deposition - fluorescence in situ hybridization protocol (CARD-FISH), which allows the visualization of labelled prokaryotes inside protists without relying on incubation procedures, we experimentally relate the food vacuole content of prokaryotes (Vc ) to the population-averaged ingestion rates (Ir ) estimated using bacteria-size fluorescent microspheres. The two variables relate according to the equation Ir = 7.52 Vc 0.9 , which indicates a prokaryote half-life of about 6 min in the protist vacuole. Five mixotrophic flagellate species from natural and culture populations were evaluated seven times during 24 h; they provided a broad range of average vacuole content (0.01 to 2.02 prokaryote protist-1 ) and ingestion rates (0.18 to 23 prokaryote protist-1 h-1 ). Consequently, the relationship found can be applied to quantify the mixotrophy activity in a large variety of field and experimental studies.

Microbial communities in the nepheloid layers and hypoxic zones of the Canary Current upwelling system.

Eastern boundary upwelling systems (EBUSs) are among the most productive marine environments in the world. The Canary Current upwelling system off the coast of Mauritania and Morocco is the second most productive of the four EBUS, where nutrient‐rich waters fuel perennial phytoplankton blooms, evident by high chlorophyll a concentrations off Cape Blanc, Mauritania. High primary production leads to eutrophic waters in the surface layers, whereas sinking phytoplankton debris and horizontally dispersed particles form nepheloid layers (NLs) and hypoxic waters at depth. We used Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD‐FISH) in combination with fatty acid (measured as methyl ester; FAME) profiles to investigate the bacterial and archaeal community composition along transects from neritic to pelagic waters within the “giant Cape Blanc filament” in two consecutive years (2010 and 2011), and to evaluate the usage of FAME data for microbial community studies. We also report the first fatty acid profile of Pelagibacterales strain HTCC7211 which was used as a reference profile for the SAR11 clade. Unexpectedly, the reference profile contained low concentrations of long chain fatty acids 18:1 cis11, 18:1 cis11 11methyl, and 19:0 cyclo11–12 fatty acids, the main compounds in other Alphaproteobacteria. Members of the free‐living SAR11 clade were found at increased relative abundance in the hypoxic waters in both years. In contrast, the depth profiles of Gammaproteobacteria (including Alteromonas and Pseudoalteromonas), Bacteroidetes, Roseobacter, and Synechococcus showed high abundances of these groups in layers where particle abundance was high, suggesting that particle attachment or association is an important mechanisms of dispersal for these groups. Collectively, our results highlight the influence of NLs, horizontal particle transport, and low oxygen on the structure and dispersal of microbial communities in upwelling systems.

Enhanced molecular visualization of root colonization and growth promotion by Bacillus subtilis EA-CB0575 in different growth systems

Bacillus subtilis EA-CB0575 is a plant growth-promoting bacterium (PGPB) associated with banana and tomato crops. Root colonization is an important trait for PGPB microorganisms and potentiates the bacterial effect related to the mechanisms of plant growth promotion. Therefore, detection of bacterial colonization of roots in different culture systems is important in the study of plant–microorganism interactions. In this study, fluorescent in situ hybridization (FISH) and catalyzed reporter deposition–FISH (CARD–FISH) were evaluated to determine the colonization ability of B. subtilis EA-CB0575 on banana and tomato roots planted on solid and liquid Murashige and Skoog medium (MS(S) and MS(L), respectively) and in soil for tomato plants. Results showed B. subtilis colonization 0–30 days post inoculation for banana and tomato plants in different culture systems with differential distribution of bacterial cells along tomato and banana roots. FISH and CARD–FISH methodologies were both successful in detecting B. subtilis colonies, but CARD–FISH proved to be superior due to its enhanced fluorescence signal. The presence of bacteria correlated with the promotion of plant growth in both plant species, providing clues to relate rhizospheric colonization with improvement in plant growth. FISH and CARD–FISH analysis results suggested the presence of native microbiota on the roots of in vitro banana plants, but not on those of tomato plants.

Dalangtan Playa (Qaidam Basin, NW China): Its microbial life and physicochemical characteristics and their astrobiological implications.

Dalangtan Playa is the second largest salt playa in the Qaidam Basin, north-western China. The hyper saline deposition, extremely arid climate and high UV radiation make Dalangtan a Mars analogue both for geomorphology and life preservation. To better understand microbial life at Dalangtan, both culture-dependent and culture-independent methods were examined and simultaneously, environment conditions and the evaporitic mineral assemblages were investigated. Ten and thirteen subsurface samples were collected along a 595-cm deep profile (P1) and a 685-cm deep profile (P2) respectively, and seven samples were gathered from surface sediments. These samples are composed of salt minerals, minor silicate mineral fragments and clays. The total bacterial cell numbers are (1.54±0.49) ×105 g−1 for P1 and (3.22±0.95) ×105 g−1 for P2 as indicated by the CAtalyzed Reporter Deposition- Fluorescent in situ Hybridization (CARD-FISH). 76.6% and 75.7% of the bacteria belong to Firmicutes phylum respectively from P1 and P2. In total, 47 bacteria and 6 fungi were isolated from 22 subsurface samples. In contrast, only 3 bacteria and 1 fungus were isolated from 3 surface samples. The isolated bacteria show high homology (≥97%) with members of the Firmicutes phylum (47 strains, 8 genera) and the Actinobacteria phylum (3 strains, 2 genera), which agrees with the result of CARD-FISH. Isolated fungi showed ≥98% ITS1 homology with members of the phylum Ascomycota. Moisture content and TOC values may control the sediments colonization. Given the deliquescence of salts, evaporites may provide refuge for microbial life, which merits further investigation. Halotolerant and spore-forming microorganisms are the dominant microbial groups capable of surviving under extreme conditions. Our results offer brand-new information on microbial biomass in Dalangtan Playa and shed light on understanding the potential microbial life in the dried playa or paleo-lakes on Mars.

Visualisation of the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans in co-cultures with micro-algae by CARD-FISH

Some studies have described the isolation and 16S rRNA gene sequence-based identification of hydrocarbon-degrading bacteria living associated with marine eukaryotic phytoplankton, and thus far the direct visual observation of these bacteria on micro-algal cell surfaces (‘phycosphere’) has not yet been reported. Here, we developed two new 16S rRNA-targeted oligonucleotide probes, PCY223 and ALGAR209, to respectively detect and enumerate the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). To enhance the hybridization specificity with the ALGAR209 probe, a competitor probe was developed. These probes were tested and optimized using pure cultures, and then used in enrichment experiments with laboratory cultures of micro-algae exposed to phenanthrene, and with coastal water enriched with crude oil. Microscopic analysis revealed these bacteria are found in culture with the micro-algal cells, some of which were found attached to algal cells, and whose abundance increased after phenanthrene or crude oil enrichment. These new probes are a valuable tool for identifying and studying the ecology of P. algicola and A. aromaticivorans in laboratory and field samples of micro-algae, as well as opening new fields of research that could harness their ability to enhance the bioremediation of contaminated sites.

Development of a 16S rRNA-targeted fluorescence in situ hybridization probe for quantification of the ammonia-oxidizer Nitrosotalea devanaterra and its relatives

The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have high occurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential role in nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probe SAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequences in databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabilization for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditions were optimized on enrichment cultures of the target species N. devanaterra, as well as the non-target ammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competitor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design and application 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 protocol developed in the present study will help to improve the current understanding of the ecology and physiology of N. devanaterra and its relatives in natural environments.

Cryptophyta as major bacterivores in freshwater summer plankton

Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator–prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Římov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

Abundance and diversity of aerobic heterotrophic microorganisms and their interaction with cyanobacteria in the oxic layer of an intertidal hypersaline cyanobacterial mat.

Aerobic heterotrophic microorganisms (AH) play a significant role in carbon cycling in cyanobacterial mats; however, little is known about their abundance, diversity and interaction with cyanobacteria. Using catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), bacterial counts in the mat's oxic layer reached a mean of 2.23±0.4×1010 cells g-1. Cultivation of AH yielded strains belonging to Actinobacteria, Bacteroidetes, Firmicutes, Gammaproteobacteria and Haloarchaea. 16S rRNA bacterial sequences retrieved from the mat's oxic layer were related to Bacteroidetes, Chloroflexi and Proteobacteria, whereas archaeal sequences belonged to Crenarchaeota and Haloarchaea. Monocultures of cyanobacteria from the same mat were associated with different AH, although Bacteroidetes were found in most cultures. CARD-FISH showed that Bacteroidetes- and Chloroflexi-related bacteria were closely associated with filaments of Microcoleus chthonoplastes. The growth of an axenic culture of M. chthonoplastes PCC7420 was stimulated on the addition of a filtrate obtained from a non-axenic Microcoleus culture and containing only AH and released substances. In contrast, a similar filtrate from a non-axenic Cyanothece-related culture killed Cyanothece PCC 7418. We conclude that a diverse community of AH exist in close association with cyanobacteria in microbial mats and the interactions between AH and cyanobacteria are species-specific and involve the release of substances.

High dark inorganic carbon fixation rates by specific microbial groups in the Atlantic off the Galician coast (NW Iberian margin).

Bulk dark dissolved inorganic carbon (DIC) fixation rates were determined and compared to microbial heterotrophic production in subsurface, meso- and bathypelagic Atlantic waters off the Galician coast (NW Iberian margin). DIC fixation rates were slightly higher than heterotrophic production throughout the water column, however, more prominently in the bathypelagic waters. Microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH) allowed us to identify several microbial groups involved in dark DIC uptake. The contribution of SAR406 (Marinimicrobia), SAR324 (Deltaproteobacteria) and Alteromonas (Gammaproteobacteria) to the dark DIC fixation was significantly higher than that of SAR202 (Chloroflexi) and Thaumarchaeota, in agreement with their contribution to microbial abundance. Q-PCR on the gene encoding for the ammonia monooxygenase subunit A (amoA) from the putatively high versus low ammonia concentration ecotypes revealed their depth-stratified distribution pattern. Taken together, our results indicate that chemoautotrophy is widespread among microbes in the dark ocean, particularly in bathypelagic waters. This chemolithoautotrophic biomass production in the dark ocean, depleted in bio-available organic matter, might play a substantial role in sustaining the dark ocean's food web.

Microbial life on a sand grain: from bulk sediment to single grains

Globally, marine surface sediments constitute a habitat for estimated 1.7 × 1028 prokaryotes. For benthic microbial community analysis, usually, several grams of sediment are processed. In this study, we made the step from bulk sediments to single sand grains to address the microbial community directly in its micro-habitat: the individual bacterial diversity on 17 sand grains was analyzed by 16S ribosomal RNA gene sequencing and visualized on sand grains using catalyzed reporter deposition fluorescence in situ hybridization. In all, 104–105 cells were present on grains from 202 to 635 μm diameter. Colonization was patchy, with exposed areas largely devoid of any epi-growth (mean cell–cell distance 4.5±5.9 μm) and protected areas more densely populated (0.5±0.7 μm). Mean cell–cell distances were 100-fold shorter compared with the water column. In general, growth occurred in monolayers. Each sand grain harbors a highly diverse bacterial community as shown by several thousand species-level operational taxonomic units (OTU)0.97. Only 4–8 single grains are needed to cover 50% of OTU0.97 richness found in bulk sediment. Although bacterial communities differed between sand grains, a core community accounting for >50% of all cells was present on each sand grain. The communities between sediment grains are more similar than between soil macroaggregates.

Using Click-Chemistry for Visualizing in Situ Changes of Translational Activity in Planktonic Marine Bacteria.

A major challenge in microbial ecology is linking diversity and function to determine which microbes are actively contributing to processes occurring in situ. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising technique for detecting and quantifying translationally active bacteria in the environment. This technique consists of incubating a bacterial sample with an analog of methionine and using click-chemistry to identify the cells that have incorporated the substrate. Here, we established an optimized protocol for the visualization of protein-synthesizing cells in oligotrophic waters that can be coupled with taxonomic identification using Catalyzed Reporter Deposition Fluorescent in Situ Hybridization. We also evaluated the use of this technique to track shifts in translational activity by comparing it with leucine incorporation, and used it to monitor temporal changes in both cultures and natural samples. Finally, we determined the optimal concentration and incubation time for substrate incorporation during BONCAT incubations at an oligotrophic site. Our results demonstrate that BONCAT is a fast and powerful semi-quantitative approach to explore the physiological status of marine bacteria.

Major role of nitrite-oxidizing bacteria in dark ocean carbon fixation.

Carbon fixation by chemoautotrophic microorganisms in the dark ocean has a major impact on global carbon cycling and ecological relationships in the ocean's interior, but the relevant taxa and energy sources remain enigmatic. We show evidence that nitrite-oxidizing bacteria affiliated with the Nitrospinae phylum are important in dark ocean chemoautotrophy. Single-cell genomics and community metagenomics revealed that Nitrospinae are the most abundant and globally distributed nitrite-oxidizing bacteria in the ocean. Metaproteomics and metatranscriptomics analyses suggest that nitrite oxidation is the main pathway of energy production in Nitrospinae. Microautoradiography, linked with catalyzed reporter deposition fluorescence in situ hybridization, indicated that Nitrospinae fix 15 to 45% of inorganic carbon in the mesopelagic western North Atlantic. Nitrite oxidation may have a greater impact on the carbon cycle than previously assumed.

The Identification of Cable Bacteria Attached to the Anode of a Benthic Microbial Fuel Cell: Evidence of Long Distance Extracellular Electron Transport to Electrodes.

Multicellular, filamentous, sulfur-oxidizing bacteria, known as cable bacteria, were discovered attached to fibers of a carbon brush electrode serving as an anode of a benthic microbial fuel cell (BMFC). The BMFC had been operated in a temperate estuarine environment for over a year before collecting anode samples for scanning electron microscopy and phylogenetic analyses. Individual filaments were attached by single terminus cells with networks of pilus-like nano-filaments radiating out from these cells, across the anode fiber surface, and between adjacent attachment locations. Current harvesting by the BMFC poised the anode at potentials of ~170–250 mV vs. SHE, and these surface potentials appear to have allowed the cable bacteria to use the anode as an electron acceptor in a completely anaerobic environment. A combination of catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) and 16S rRNA gene sequence analysis confirmed the phylogeny of the cable bacteria and showed that filaments often occurred in bundles and in close association with members of the genera Desulfuromonas. However, the Desulfobulbaceae Operational Taxonomic Units (OTUs) from the 16S sequencing did not cluster closely with other putative cable bacteria sequences suggesting that the taxonomic delineation of cable bacteria is far from complete.

Unravelling the core microbiome of biofilms in cooling tower systems

In this study, next generation sequencing and catalyzed reporter deposition fluorescence in situ hybridization, combined with confocal microscopy, were used to provide insights into the biodiversity and structure of biofilms collected from four full-scale European cooling systems. Water samples were also analyzed to evaluate the impact of suspended microbes on biofilm formation. A common core microbiome, containing members of the families Sphingomonadaceae, Comamonadaceae and Hyphomicrobiaceae, was found in all four biofilms, despite the water of each coming from different sources (river and groundwater). This suggests that selection of the pioneer community was influenced by abiotic factors (temperature, pH) and tolerances to biocides. Members of the Sphingomonadaceae were assumed to play a key role in initial biofilm formation. Subsequent biofilm development was driven primarily by light availability, since biofilms were dominated by phototrophs in the two studied ‘open’ systems. Their interactions with other microbial populations then shaped the structure of the mature biofilm communities analyzed.

Composition of Total and Cell-Proliferating Bacterioplankton Community in Early Summer in the North Sea - Roseobacters Are the Most Active Component.

Heterotrophic bacterioplankton communities play an important role in organic matter processing in the oceans worldwide. In order to investigate the significance of distinct phylogenetic bacterial groups it is not only important to assess their quantitative abundance but also their growth dynamics in relation to the entire bacterioplankton. Therefore bacterial abundance, biomass production and the composition of the entire and cell-proliferating bacterioplankton community were assessed in North Sea surface waters between the German Bight and 58°N in early summer by applying catalyzed reporter deposition (CARD-FISH) and bromodeoxyuridine fluorescence in situ hybridization (BrdU-FISH). Bacteroidetes and the Roseobacter group dominated the cell-proliferating fraction with 10–55 and 8–31% of total BrdU-positive cells, respectively. While Bacteroidetes also showed high abundances in the total bacterial fraction, roseobacters constituted only 1–9% of all cells. Despite abundances of up to 55% of total bacterial cells, the SAR11 clade constituted <6% of BrdU-positive cells. Gammaproteobacteria accounted for 2–16% of the total and 2–13% of the cell-proliferating cells. Within the two most active groups, BrdU-positive cells made up 28% of Bacteroidetes as an overall mean and 36% of roseobacters. Estimated mean growth rates of Bacteroidetes and the Roseobacter group were 1.2 and 1.5 day-1, respectively, and much higher than bulk growth rates of the bacterioplankton whereas those of the SAR11 clade and Gammaproteobacteria were 0.04 and 0.21 day-1, respectively, and much lower than bulk growth rates. Only numbers of total and cell-proliferating roseobacters but not those of Bacteroidetes and the other groups were significantly correlated to chlorophyll fluorescence and bacterioplankton biomass production. The Roseobacter group, besides Bacteroidetes, appeared to be a major player in processing phytoplankton derived organic matter despite its low partitioning in the total bacterioplankton community.

Sensitive immunosensor for respiratory syncytial virus based on dual signal amplification of gold nanopaticle layer-modified plates and catalyzed reporter deposition

It is still a challenge to identify and control the outbreak of infectious diseases at their earliest stages because of the lack of ultrasensitive, rapid, and selective diagnostic technologies. We have developed and describe here a highly sensitive immuno-biosensor for the fast and unambiguous detection of respiratory syncytial virus (RSV) by accompanying catalyzed reporter deposition with gold nanoparticle layer-modified plate which can significantly amplify the binding density of capture antibody. The captured RSV pulls down the horseradish peroxidase (HRP)-labeled detection antibody through sandwiched immunoreactions on the plate, where the HRP drives the deposition and accumulation of biotinalyated-tyramide molecules in the presence of hydrogen peroxide, in turn increasing the load of streptavidin-HRP by the biotin-streptavidin interaction. As a result, the developed immunosensor displays high sensitivity for the detection of RSV with a dynamic range from 0.05pg/mL to 30pg/mL and the lowest detectable concentration of 0.01pg/mL. Intra-assay and interassay coefficients of variation are 2.63% and 5.52%, respectively. Furthermore, it can be adapted to the detection of RSV spiked in complex samples, showing good accordance with the conventional ELISA method.