Investigation of cell fate decision in Schizosaccharomyces pombe by ncRNA annotation and statistical analyses

Studies in the fission yeast Schizosaccharomyces pombe (S. pombe) have done much to inform the view of heterochromatin and its control by the RNA interference (RNAi) machinery. Using cDNA synthesised from poly(A)-enriched RNA samples, numerous novel ncRNA loci were discovered, and the 50 and 30 ends of many other genes were refined in previous studies. Although some of these transcripts may encode novel proteins the function of the majority is yet to be determined. The authors have used strand-specific deep sequencing of RNA, irrespective of poly(A) status, to reveal a highly structured antisense programme that modulates gene expression to dictate cell fate decisions during sexual differentiation. They show that an extensive and elaborate array of ncRNA production accompanies sexual differentiation in the fission yeast S. pombe. Experimental manipulation suggests that these transcripts specifically regulate the function of the target genes.

Microbial community composition in fresh water at different stations

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea. Cells were concentrated from water samples (1 to 100 ml) on white polycarbonate filters (diameter, 47 mm; pore size, 0.2 mm; type GTTP 4700 [Millipore, Eschborn, Germany]) by applying a vacuum of <25 kPa. They were subsequently fixed by covering the filter with 3 ml of a freshly prepared, phosphate-buffered saline (pH 7.2)-4% paraformaldehyde (Sigma, Deisenhofen, Germany) solution for 30 min at room temperature. Airdried filters are ready for hybridization and can be stored at 220°C or room temperature for several months without showing apparent changes. Probes BET42a, GAM42a, and PLA886 were used with competitor oligonucleotides as described previously amongst others in Manz et al., (1992; doi:10.1016/S0723-2020(11)80121-9). The filters were transferred to a vial containing 50 ml of prewarmed (48°C) washing solution (70 mM NaCl, 20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 0.01% sodium dodecyl sulfate) and incubated freely floating without shaking at 48°C for 15 min. The filter sections were dried on Whatman 3M paper (Whatman Ltd., Maidstone, United Kingdom) and covered with 50 ml of DAPI solution (1 mg/ml in distilled water filtered through at 0.2-mm filter) for 5 min at room temperature in the dark. For each sample and probe, more than 500 cells were enumerated; for the DAPI examination, more than 1,500 cells were counted per sample. All probe-specific cell counts are presented as the percentage of cells visualized by DAPI. The mean abundances and standard deviations were calculated from the counts of 10 to 20 randomly chosen fields on each filter section. All counts were corrected by subtracting the counts obtained with the negative control NON338. Mean and standard deviation were calculated from the counts of 10 to 20 randomly chosen fields on each filter section.

Chemical analysis and Microbial community composition of surface water samples from the Southern Lagoon of Venice

The Lagoon of Venice is a large water basin that exchanges water with the Northern Adriatic Sea through three large inlets. We examined two adjacent sites within the Southern Basin and at the Chioggia inlet in autumn 2007 and summer 2008. A pilot study in June 2007 on a surface water sample from Chioggia with a rather high salinity of 36.9 PSU had revealed a conspicuous bloom of CF319a-positive cells likely affiliated with the Cytophaga /Flavobacteria cluster of Bacteroidetes. These flavobacterial abundances were one to two orders of magnitude higher than in other marine surface waters. DAPI-stained cells were identified as bacteria with the general bacterial probe mixture EUB338 I-III. CARD-FISH counts with group-specific probes confirmed the dominance of Bacteroidetes (CF319a), Alphaproteobacteria (ALF968), and Gammaproteobacteria (GAM42a). CARD-FISH showed thatBetaproteobacteria and Planctomycetes were minor components of the bacterioplankton in the Lagoon of Venice.

Chloroflexus spp. and Sulfurihydrogenibium spp. in microbial mats of the Nakabusa hot spring, Japan

Microbial mats develop in a wide range of aquatic habitats, such as geothermal hot springs, hypersaline ponds, marine cold seeps or hydrothermal vents. The Nakabusa hot spring is located in the Nagano Prefecture, Japan (36.3875N, 137.75E), dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65°C. The microbial community of such mats was analyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Microbial mat samples were kept in sterile plastic tubes (for molecular analysis) or glass bottles completely filled with hot spring water to avoid oxidation. Samples were transferred to the laboratory on ice and used for physiological experiments within 8h. Quantification of cell biovolumes was carried out based on images of mat sections hybridized with Sulfurihydrogenibium- and Chloroflexi-specific probes, and stained with DAPI. In situ hybridizations (CARD-FISH) of thin matsections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfurihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume).

Verrucomicrobia in situ abundance and water chemistry in a humic lake during the year 2000

Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in various terrestrial and aquatic habitats. They are key players in soils, but little is known about their role in aquatic systems. Thus, we applied newly designed 16S rRNA-targeted probe set for the identification of Verrucomicrobia and of clades within this phylum to a study concerning the seasonal abundance of Verrucomicrobia in waters of the humic lake Große Fuchskuhle (Germany) by catalyzed reporter deposition fluorescence in situ hybridization. The Lake Große Fuchskuhle is located in the large Mecklenburg-Brandenburg lake district near Berlin (53°10'N, 13°02'E). The lake was artificially divided into four basins (northwest, northeast, southwest, and southeast). We chose the two most contrasting basins, the acidotrophic humic southwestern (SW) basin with a high influx of allochthonous dissolved organic carbon (DOC) and the more mesotrophic northeastern (NE) basin, to study abundance and seasonality of Verrucomicrobia. Lake water was collected from depths of 0.5 m (oxic) and 4.5 m (seasonally anoxic) approximately trimonthly in 2000 (March, June, September and December). The lake hosted diverse Verrucomicrobia clades in all seasons. Either Spartobacteria (up to 19%) or Opitutus spp. (up to 7%) dominated the communities, whereas Prosthecobacter spp. were omnipresent in low numbers (<1%). Verrucomicrobial abundance and community composition varied between the seasons, and between more and less humic basins, but were rather stable in oxic and seasonally anoxic waters.

NOR5/OM60 clade of the Yangtze River estuary

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes "Congregibacter litoralis" strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. Most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil. The abundance of members of the NOR5/OM60 clade in various marine sites was determined by fluorescence in situ hybridization using a newly designed and optimized probe set. Relative abundances in coastal marine waters off the Yangtze estuary were up to 3% of the total 4',6-diamidino-2-phenylindole (DAPI) counts. A small cruise was undertaken from 2006-09-06 to 2006-09-08 in the Yangtze River estuary. Samples were taken from surface water, and immediately fixed with 1% paraformaldehyde (PFA) for 1 h, filtered onto polycarbonate filters (Millipore, 47 mm in diameter, 0.2 µm pore size) and stored frozen at -20 °C.

Microbial community composition and bacterioplankton at time series station Helgoland Roads, North Sea

A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria, most notably during massive marine algae blooms. Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades, most prominently members of the Flavobacteriia, Gammaproteobacteria and the alphaproteobacterial Roseobacter clade. This study explores such successions during spring phytoplankton blooms in the southern North Sea (German Bight) for four consecutive years. The surface water samples were taken at Helgoland Island about 40 km offshore in the southeastern North Sea in the German Bight at the station 'Kabeltonne' (54° 11.3' N, 7° 54.0' E) between the main island and the minor island, Düne (German for 'dune') using small research vessels (http://www.awi.de/en/expedition/ships/more-ships.html). Water depths at this site fluctuate from 6 to 10 m over the tidal cycle. Samples were processed as described previously (Teeling et al., 2012; doi:10.7554/eLife.11888.001) in the laboratory of the Biological Station Helgoland within less than two hours after sampling. Assessment of absolute cell numbers and bacterioplankton community composition was carried out as described previously (Thiele et al., 2011; doi:10.1016/B978-0-444-53199-5.00056-7). To obtain total cell numbers, DNA of formaldehyde fixed cells filtered on 0.2 mm pore sized filters was stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescently labeled cells were subsequently counted on filter sections using an epifluores-cence microscope. Likewise, bacterioplankton community composition was assessed by catalyzedreporter deposition fluorescence in situ hybridization (CARD-FISH) of formaldehyde fixed cells on 0.2 mm pore sized filters.

Microbial community composition in sediments of the Hydrate Ridge (Cascadian Margin) collected during RV SONNE cruises SO143 (1999) and SO148-1 (2000)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH). Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6×10**10cells/cm**3). Sediment samples were obtained during RV SONNE cruises SO143-2 and SO148-1 at the crest of southern Hydrate Ridge at the Cascadia convergent margin off the coast of Oregon. Sediment cores of 20 - 40 cm length were obtained using a video-guided multiple corer from gas hydrate bearing sediments and from reference sites not enriched in methane in the surface sediments. Samples for total cell counts were obtained from 1 cm core slices, fixed with 2% formaldehyde and stored cold (4°C) and the quantification of aggregates was done via epifluorescence microscopy after staining the sediments with Acridine Orange Direct Counts (AODC) according to the method of Meyer- Reil (1983, doi:10.1007/BF00395813). Total cell counts were defined as the sum of single cells plus the aggregated cells in the syntrophic consortia. DAPI staining was used to measure ANME2/DSS aggregate sizes via epifluorescence microscopy of FISH-treated samples. For FISH, subsamples of sediment cores were sliced into 1 cm intervals and fixed for 2-3 h with 3% formaldehyde (final concentration), washed twice with 1×PBS (10 mM sodium phosphate; 130 mM NaCl), and finally stored in 1×PBS/EtOH (1:1) at -20°C.

Hydrochemical and microbiological properties of groundwater from pristine aquifer sampled at the Lower Rhine Embayment

The hydrochemistry and the microbial diversity of a pristine aquifer system near Garzweiler, Germany next to the open-pit lignite mine Garzweiler 1, were characterized. Hydrogeochemical and isotopic data indicate a recent activity of sulfate-reducing bacteria in the Tertiary marine sands. The community structure in the aquifer was studied by fluorescence in situ hybridization (FISH). Up to 7.3 x 10**5 cells/ml were detected by DAPIstaining. Bacteria (identified by the probe EUB338) were dominant, representing 51.9% of the total cell number (DAPI). Another 25.7% of total cell were affiliated with the domain Archaea as identified by the probe ARCH915. Within the domain Bacteria, the beta-Proteobacteria were most abundant (21.0% of total cell counts). Using genusspecific probes for sulfate-reducing bacteria (SRB), 2.5% of the total cells were identified as members of the genus Desulfotomaculum. This reflects the predominant role these microorganisms have been found to play in sulfatereducing zones of aquifers at other sites. Previously, all SRB cultured from this site were from the spore-forming genera Desulfotomaculum and Desulfosporosinus. Samples were taken after pumping for >= 40 min and after parameters such as temperature, pH, redox potential, oxygen and conductivity of the groundwater had remained stable for >= 15 min due to recharge of aquifer water. Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)- stained cells were performed as described in Snaidr et al., (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800-1000 DAPI stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations and oligonucleotide probes used please see further details.

Microbial community in the sediment of the arctic Smeerenburgfjorden

Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations are given in further details. FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides delta-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the gamma-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts), showing no clear zonation along the vertical profile. Gram-positive bacteria and the beta-proteobacteria were near the detection limit in all sediments.

Community structure of sulfate reducing bacteria in the sediment of the arctic Smeerenburgfjorden

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. Samples stored in PBS-ethanol were diluted and treated by mild sonication. A 10-ml aliquot of a 1:40 dilution was filtered onto a 0.2-mm-pore-size type GTTP polycarbonate filter (Millipore, Eschborn, Germany). Hybridization and microscopic counting of hybridized and 49,69-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Details of probes and formamide concentrations which were used are listed in futher details.. Means were calculated by using 10 to 20 randomly chosen fields for each filter section, which corresponded to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals observed with probe NON338. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected.

Microbial community composition of sandy intertidal sediments of Sylt-Rømø Basin, Wadden Sea

Molecular biological methods were used to investigate the microbial diversity and community structure in intertidal sandy sediments near the island of Sylt (Wadden Sea) at a site which was characterized for transport and mineralization rates in de Beer et al., (2005, hdl:10013/epic.21375). The sampling was performed during low tide in the middle of the flat, approximately 40 m in the offshore direction from the high water line on October 6, 1999, March 7, 2000, and July 5, 2000. Two parallel cores were collected from each season for molecular analyses. Within 2 h after sampling the sediment cores were sub-sampled and fixed in formaldehyde for FISH analysis. The cells were hybridized, stained with 4',6'-diamidino-2-phenylindole (DAPI) and microscopically counted as described previously [55]. Details of probes and formamide concentrations which were used are shown in further details. Counts are reported as means calculated from 10-15 randomly chosen microscopic fields corresponding to 700-1000 DAPI-stained cells. Values were corrected for the signals counted with the probe NON338. Fluorescence in situ hybridization (FISH)with group-specific rRNA-targeted oligonucleotide probes were used to characterize the microbial community structure over depth (0-12 cm) and seasons (March, July, October). We found high abundances of bacteria with total cell numbers up to 3×109 cells ml-1 and a clear seasonal variation, with higher values in July and October versus March. The microbial community was dominated by members of the Planctomycetes, the Cytophaga/Flavobacterium group, Gammaproteobacteria, and bacteria of the Desulfosarcina/Desulfococcus group. The high abundance (1.5×10**7 - 1.8×10**8 cells/ml accounting for 3-19% of all cells) of presumably aerobic heterotrophic polymer-degrading planctomycetes is in line with the high permeability, deep oxygen penetration, and the high rates of aerobic mineralization of algal biomass measured in the sandy sediments by de Beer et al., (2005, hdl:10013/epic.21375). The high and stable abundance of members of the Desulfosarcina/Desulfococcus group, both over depth and season, suggests that these bacteria may play a more important role than previously assumed based on low sulfate reduction rates in parallel cores de Beer et al., (2005).