Composition of bacterioplankton in the North Atlantic subtropical gyre

Subtropical oceanic gyres are the most extensive biomes on Earth where SAR11 and Prochlorococcus bacterioplankton numerically dominate the surface waters depleted in inorganic macronutrients as well as in dissolved organic matter. In such nutrient poor conditions bacterioplankton could become photoheterotrophic. We assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions. The experimental work was performed on board the Royal Research Ship James Cook (cruise no. JC53, October-November 2010) as part of the Atlantic Meridional Transect programme, and on board the Royal Research Ship Discovery (cruise no. D369, August-September 2011). At each station, samples were collected from 20m depth with a sampling rosette of 20-l Niskin bottles mounted on aconductivity-temperature-depth profiler. Samples were collected in 1 l thermos flasks (washed with10% v/v HCl) in the dark and processed immediately. Depth of 20m was chosen because it represents the mixed layer and it was the shallowest depth unaffected by the ship's movement, including thrusting, that could artificially affect microbial metabolism in nutrient-depleted stratified surfacewaters. Molecular identification of flow-sorted cells CARD-FISH was performed on flow-sorted cells to identify the groups for which uptake rates were measured. High nucleic acid-containing bacteria, based on SYBR Green DNA staining, that had virtually undetectable chlorophyll autofluorescence, were phylogenetically affiliated with Prochlorococcus,in agreement with our previously reported results (Zubkov et al., 2007; doi:10.1111/j.1462-2920.2007.01324.x).

Anaerobic methanotrophic archaea-2 and Desulfosarcina/Desulfococcus group in sediments at different stations

The anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). In sediment samples from Hydrate Ridge, the Isis Mud Volcano and the Gulf of Mexico, DSS cells accounted for 3-6% of all DAPI-stained single cells. Out of these, 8-17% were labelled with probe SEEP1a-1441. This translated into relative abundances of single SEEP-SRB1a cells of 0.3% to 0.7%. Contrastingly, in a sediment sample from the Gullfaks oil field, DSS cells accounted for 18% and SEEP-SRB1a for 9% of all single cells. This sediment sample also featured an unusually high abundance of single ANME-2 cells and only very few ANME-2/DSS aggregates in comparison with other AOM habitats. Considering also the nature of the sample, it is likely that the high number of single ANME-2 and SEEP-SRB1a cells were an artifact of sample preparation. Here, harsher sonication was required to remove the microorganisms from coarse sand prior to CARD-FISH analysis.

Microbial community, biomarker concentrations and their isotopic signature in sediment of Gullfaks and Tommeliten methane seeps in the Northern North Sea

Gullfaks is one of the four major Norwegian oil and gas fields, located in the northeastern edge of the North Sea Plateau. Tommeliten lies in the greater Ekofisk area in the central North Sea. During the cruises HE 208 and AL 267 several seep locations of the North Sea were visited. At the Heincke seep at Gullfaks, sediments were sampled in May 2004 (HE 208) using a video-guided multiple corer system (MUC; Octopus, Kiel). The samples were recovered from an area densely covered with bacterial mats where gas ebullition was observed. The coarse sands limited MUC penetration depth to maximal 30 centimeters and the highly permeable sands did not allow for a high-resolution, vertical subsampling because of pore water loss. The gas flare mapping and videographic observation at Tommeliten indicated an area of gas emission with a few small patches of bacterial mats with diameters <50 cm from most of which a single stream of gas bubbles emerged. The patches were spaced apart by 10-100 m. Sampling of sediments covered by bacterial mats was only possible with 3 small push cores (3.8 cm diameter) mounted to ROV Cherokee. These cores were sampled in 3 cm intervals. Lipid biomarker extraction from 10 -17 g wet sediment was carried out as described in detail elsewhere (Elvert et al., 2003; doi:10.1080/01490450303894). Briefly, defined concentrations of cholestane, nonadecanol and nonadecanolic acid with known delta 13C-values were added to the sediments prior to extraction as internal standards for the hydrocarbon, alcohol and fatty acid fraction, respectively. Total lipid extracts were obtained from the sediment by ultrasonification with organic solvents of decreasing polarity. Esterified fatty acids (FAs) were cleaved from the glycerol head group by saponification with methanolic KOH solution. From this mixture, the neutral fraction was extracted with hexane. After subsequent acidification, FAs were extracted with hexane. For analysis, FAs were methylated using BF3 in methanol yielding fatty acid methyl esters (FAMES). The fixation for total cell counts and CARD-FISH were performed on-board directly after sampling. For both methods, sediments were fixed in formaldehyde solution. After two hours, aliquots for CARD-FISH staining were washed with 1* PBS (10mmol/l sodium phosphate solution, 130mmol/l NaCl, adjusted to a pH of 7.2) and finally stored in a 1:1 PBS:ethanol solution at -20°C until further processing. Samples for total cell counts were stored in formalin at 4°C until analysis. For sandy samples, the total cell count/CARD-FISH protocol was optimized to separate sand particles from the cells. Cells were dislodged from sediment grains and brought into solution with the supernatant by sonicating each sample onice for 2 minutes at 50W. This procedure was repeated four times and supernatants were combined. The sediment samples were brought to a final dilution of 1:2000 to 1:4000 and filtered onto 0.2µm GTTP filters (Millipore, Eschbonn, Germany).

(Table T1) Total procaryotes, and living bacteria and archaea in black shales of ODP Leg 207 sites

Subseafloor sediments harbor over half of all prokaryotic cells on Earth (Whitman et al., 1998). This immense number is calculated from numerous microscopic acridine orange direct counts (AODCs) conducted on sediment cores drilled during the Ocean Drilling Program (ODP) (Parkes et al., 1994, doi:10.1038/371410a0, 2000, doi:10.1007/PL00010971). Because these counts cannot differentiate between living and inactive or even dead cells (Kepner and Pratt, 1994; Morita, 1997), the population size of living microorganisms has recently been enumerated for ODP Leg 201 sediment samples from the equatorial Pacific and the Peru margin using ribosomal ribonucleic acid targeting catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) (Schippers et al., 2005, doi:10.1038/nature03302). A large fraction of the subseafloor prokaryotes were alive, even in very old (16 Ma) and deep (>400 m) sediments. In this study, black shale samples from the Demerara Rise (Erbacher, Mosher, Malone, et al., 2004, doi:10.2973/odp.proc.ir.207.2004) were analyzed using AODC and CARD-FISH to find out if black shales also harbor microorganisms.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Investigations of artificial sediments from two deep-sea in situ recolonization experiments deployed for one year at the central HAUSGARTEN station IV during ARK-XIX/3c

Commercial exploitation and abrupt changes of the natural conditions may have severe impacts on the Arctic deep-sea ecosystem. The present recolonisation experiment mimicked a situation after a catastrophic disturbance (e.g. by turbidites caused by destabilized continental slopes after methane hydrate decomposition) and investigated if the recolonisation of a deep-sea habitat by meiobenthic organisms is fostered by variations innutrition and/or sediment structure. Two "Sediment Tray Free Vehicles" were deployed for one year in summer 2003 at 2500 m water depth in the Arctic deep-sea in the eastern Fram Strait. The recolonisation trays were filled with different artificial and natural sediment types (glass beads, sand, sediment mixture, pure deep-sea sediment) and were enriched with various types of food (algae, yeast, fish). After one year, meiobenthos abundances and various sediment related environmental parameters were investigated. Foraminifera were generally the most successful group: they dominated all treatments and accounted for about 87% of the total meiobenthos. Colonizing meiobenthos specimens were generally smaller compared to those in the surrounding deep-sea sediment, suggesting an active recolonisation by juveniles. Although experimental treatments with fine-grained, algae-enriched sediment showed abundances closest to natural conditions, the results suggest that food availability was the main determining factor for a successful recolonisation by meiobenthos and the structure of recolonised sediments was shown to have a subordinate influence.

Relative abundance of prokaryotes in sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

CARD-FISH was performed as previously described in Ruff et al., (2013; doi:10.1371/journal.pone.0072627) with the following modifications. 4-6 µl of 25-fold diluted sediment were used for filtration. Archaeal cell walls were permeabilized with 0.1M HCl for 2 min to detect ANME-3 cells, or Proteinase K solution (15 µg ml-1 (Merck, Darmstadt, Germany) in 0.05 M EDTA (pH 8), 0.1 M Tris-HCl (pH 8), 0.5 M NaCl) for 2-4 min at room temperature for all other archaea. Bacterial cell walls were permeabilized with lysozyme solution (1000kU/ml) for 60 min at 37°. Cells were stained with DAPI (1µg/ml), embedded in mounting medium and counted in 40-60 independent microscopic fields using an Axiophot II epifluorescence microscope (Carl Zeiss, Jena, Germany).

Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization of a dominant group of human gut bacteria related to Roseburia spp. or Eubacterium rectale

Peer reviewed

Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization of a dominant group of human gut bacteria related to Roseburia spp. or Eubacterium rectale

Peer reviewed

Quantification of microbial communities in subsurface marine sediments of the Black Sea and off Namibia

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 10(9) to 10(10) cells/mL at the sediment surface to 10(7)-10(9) cells/mL below one meter depth. Based on CARD FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

Anaerobic oxidation of methane at a marine methane seep in a forearc sediment basin off Sumatra, Indian Ocean

A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by C-13-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO2 was confirmed in a C-13-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 x 10(9) cells cm(-3) sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (delta-Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible.

Purification, biochemical characterization and localization at single cell resolution of Matrix Gla Protein (MGP) and Bone Gla Protein (BGP) in the teleost fish Argyrosomus regius

Tese de dout. em Química, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2002

RNA-FISH Technique: Probe Cocktails vs Single Probes

RNA-Fluorescence In Situ Hybridization (RNA-FISH) enables to analyze and visualize the microorganisms of interest within microbial communities in their natural environments by fluorescent labelling of specific RNA sequences. Poor accessibility or low content of the RNA target region can cause false positives/negatives due to low fluorescence of the cells. To reduce the chances of this occurring, probe cocktails – i.e. mixture of several probes that hybridize to different regions of the target RNA- has been proposed as an alternative to single probes use for increasing the Fluorescence Intensities (FI). However, is this really a good solution? The key finding of this work was that the use of probe cocktails is not always a good solution for maximizing the FI as at high concentrations the single probe EUK516-6 FAM yielded higher FI than the probe cocktails.