Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Research Articles

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 10(1) cells ml(-1) in the large saline lake Neusiedler See to 5.6 × 10(4) cells ml(-1) in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.

Distribution and in situ abundance of sulfate‐reducing bacteria in diverse marine hydrocarbon seep sediments

Marine gas and hydrocarbon seeps are hot spots of sulfate reduction which is fuelled by methane, other short-chain alkanes or a complex mixture of hydrocarbons. In this study, we investigated the global distribution and abundance of sulfate-reducing bacteria (SRB) in eight gas and hydrocarbon seeps by catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH). The majority of Deltaproteobacteria were assigned to specific SRB groups, i.e. 83 ± 14% at gas seeps and 61 ± 35% at hydrocarbon seeps, indicating that the probe set used was sufficient for classification of marine SRB. Statistical analysis showed that SRB abundance and distribution were significantly influenced by habitat type and sediment depth. Members of the Desulfosarcina/Desulfococcus (DSS) clade strongly dominated all sites. Our data indicated the presence of many diverse and highly specialized DSS species of low abundance rather than a single abundant subgroup. In addition, SEEP-SRB2, an uncultured deep-branching deltaproteobacterial group, was ubiquitously found in high abundances at all sites. SEEP-SRB2 members occurred either in a novel association with methanotrophic archaea in shell-type ANME-2/SEEP-SRB2 consortia, in association with ANME-1 archaea in Black Sea microbial mats or as single cells. Two other uncultured groups, SEEP-SRB3 and SEEP-SRB4, were preferentially detected in surface sediments from mud volcanoes.

Field distribution and activity of chlorinated solvents degrading bacteria by combining CARD-FISH and real time PCR

Nowadays several advanced molecular techniques are applied for quantifying bacteria involved in contaminant degradation processes. However, despite the fact that significant efforts have been taken to make these tools more reliable and specific, their application for the analysis of field samples is hardly ever applied. In this study, a combination of three methods (CARD-FISH, qPCR and RT-qPCR) was successfully applied to evaluate the distribution and the activity of known chlorinated solvent dechlorinating bacteria in a contaminated site where no remedial actions have been undertaken. CAtalysed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) specifically provided the cell densities of known dechlorinating bacteria and was found to be more sensitive than quantitative PCR (qPCR) for the quantification of 'Dehalococcoides' cell numbers in the aquifer. Among the screened dechlorinators, 'Dehalococcoides' spp. were mainly found and nearly homogenously distributed in the aquifers at concentrations ranging from 8.1×10(5)±1.2×10(5) to 2.5×10(7)±5.6×10(6)cells per liter of groundwater (with a relative abundance out of the total Bacteria of 0.7-15%). Further, the dechlorination potentialities of 'Dehalococcoides' species living in the aquifer were evaluated by analyzing the abundance and the expression of 16S rRNA genes and reductive dehalogenase (RDase) encoding functional genes by qPCR and Reverse Transcription qPCR (RT-qPCR). 'Dehalococcoides'tceA gene, known to be associated to strains capable of reducing chlorinated solvents beyond cis-DCE, was found and expressed in the field. Overall, this study proved the existence of a well-established dechlorinating microbial community able to use contaminants as substrates for their metabolic activity and indicated the occurrence of reductive dechlorination at the site.

Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells.The gold-FISH protocol was developed and optimised using pure cultures and environmental samples, such as rice roots and marine sediments. The combination of epifluorescence and scanning electron microscopy provides a promising tool for investigating microorganisms at levels of high resolution. Correlative characterisation of physicochemical properties by EDS will allow for the analysis of microbe-surface interactions.

High abundance of novel environmental chlamydiae in a Tyrrhenian coastal lake (Lago di Paola, Italy)

For a long time the bacterial phylum of Chlamydiae exclusively consisted of one family of obligate intracellular bacteria, the Chlamydiaceae, which encompassed causative agents of severe diseases. In the 1990s, environmental chlamydiae were discovered as symbionts of free-living amoebae and other eukaryotic hosts. During a sampling campaign in September 2008, while monitoring Planctomycetes, we retrieved 20 almost full-length 16S rRNA gene sequences affiliated with Chlamydiales from a lake at the Tyrrhenian coast of central Italy (Lago di Paola, Latium). Two main clusters were identified. The nine sequences within the tight cluster I shared ∼98% identity, just like the six sequences of cluster II. The 16S rRNA sequence identity between the two novel groups was with 88% higher than with all known families of the order Chlamydiales. Four types of less frequent chlamydial 16S rRNA sequences were also detected. Two oligonucleotide probes were designed, and optimized. Chl282 targets the cluster I and almost all other Chlamydiales, while Chl282bis targets the cluster II and few other sequences. By catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH), we identified in the Lago di Paola picoplankton abundant tiny cells with dot-shaped morphology and, interestingly, rarely also protists with intracellular pleomorphic chlamydiae. Abundances of the novel chlamydial clusters were up to 5 × 10(4) cells per millilitre. The two clusters were also detected in similar numbers during a second sampling in October 2010. This confirmed the relevance of the two newly described clusters of chlamydiae in Lago di Paola, not only enlarging the knowledge on the biodiversity of environmental chlamydiae in aquatic habitats, but also raising sanitary issues that should be addressed in the future.

Vertical shifts in the microbial community structure of organic-rich Namibian shelf sediments

This study investigates the diversity and abundance of bacteria in organic-rich Namibian shelf sediments from two sampling stations, using the 16S rRNA library approach and Catalyzed Reporter Deposition Fluorescent in situ Hybridization (CARD-FISH). Six clone libraries were constructed. Clone libraries were dominated by Delta-proteobacteria (up to 48%) and Gamma-proteobacteria (up to 98%). Bacteroidetes were dominant in the clone library of the top 6 cm (up to 17%), while actinobacteria dominated at a depth of 10 to 12 cm (up to 34%). Sequences that were related to bacteria with hydrolytic and fermenting abilities include members from the Gamma-proteobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria. Cloned sequences within the Delta-proteobacteria affiliates to sulfate reducing bacteria, including Desulfarculaceae, Desulfobacteraceae, Desulfobulbaceae,and Desulfuromonadales and were detected throughout the sediment. The two sampling stations differed in microbial diversity with a higher diversity prevailing at the station with higher metabolic rates for organic matter decomposition. At both sampling stations a shift in microbial community composition with depth was observed and is explained by gradients in organic substrate availability within the sediment, which affects the life strategies adopted by bacteria, resulting in niche diversification and ultimately affects bacterial community composition and structure throughout the sediment depth. Key words: Namibia, upwelling, microbial, diversity, abundance.

Archaea of the Miscellaneous Crenarchaeotal Group are abundant, diverse and widespread in marine sediments

Members of the highly diverse Miscellaneous Crenarchaeotal Group (MCG) are globally distributed in various marine and continental habitats. In this study, we applied a polyphasic approach (rRNA slot blot hybridization, quantitative PCR (qPCR) and catalyzed reporter deposition FISH) using newly developed probes and primers for the in situ detection and quantification of MCG crenarchaeota in diverse types of marine sediments and microbial mats. In general, abundance of MCG (cocci, 0.4 μm) relative to other archaea was highest (12-100%) in anoxic, low-energy environments characterized by deeper sulfate depletion and lower microbial respiration rates (P=0.06 for slot blot and P=0.05 for qPCR). When studied in high depth resolution in the White Oak River estuary and Hydrate Ridge methane seeps, changes in MCG abundance relative to total archaea and MCG phylogenetic composition did not correlate with changes in sulfate reduction or methane oxidation with depth. In addition, MCG abundance did not vary significantly (P>0.1) between seep sites (with high rates of methanotrophy) and non-seep sites (with low rates of methanotrophy). This suggests that MCG are likely not methanotrophs. MCG crenarchaeota are highly diverse and contain 17 subgroups, with a range of intragroup similarity of 82 to 94%. This high diversity and widespread distribution in subsurface sediments indicates that this group is globally important in sedimentary processes.

Development of a new CARD-FISH protocol for quantification of Legionella pneumophila and its application in two hospital cooling towers

Open cooling towers are frequent sources of infections with Legionella pneumophila. The gold standard for the detection of Leg. pneumophila is based on cultivation lasting up to 10 days and detecting only culturable cells. Alternative fluorescence in situ hybridization (FISH) protocols have been proposed, but they result in faint fluorescence signals and lack specificity because of cross-hybridization with other Legionella species. Our aim was thus to develop a new FISH protocol for rapid and specific detection of Leg. pneumophila in water samples. A novel catalysed reporter deposition FISH (CARD-FISH) protocol for the detection of Leg. pneumophila was developed, which significantly enhanced signal intensity as well as specificity of the probe through the use of a novel competitor probe. The developed protocol was compared with the culture method for monitoring the seasonal development of culturable and nonculturable Leg. pneumophila in two hospital cooling tower systems. Seasonal fluctuations of Leg. pneumophila concentrations detected via CARD-FISH were related to the development of the total bacterial community in both cooling towers, with temperature and biocide as the main factors controlling this development. Our results clearly showed that the majority of the Leg. pneumophila cells were in a nonculturable state. Thus, detection of Leg. pneumophila with culture methods may underestimate the total numbers of Leg. pneumophila present. Rapid, sensitive and specific detection and quantification of Leg. pneumophila in water systems is prerequisite for reliable risk estimation. The new protocol significantly improves current methodology and can be used to monitor and screen for Leg. pneumophila concentrations in cooling towers or other water systems.

Quantification of Tinto River Sediment Microbial Communities: Importance of Sulfate-Reducing Bacteria and Their Role in Attenuating Acid Mine Drainage

Tinto River (Huelva, Spain) is a natural acidic rock drainage (ARD) environment produced by the bio-oxidation of metallic sulfides from the Iberian Pyritic Belt. This study quantified the abundance of diverse microbial populations inhabiting ARD-related sediments from two physicochemically contrasting sampling sites (SN and JL dams). Depth profiles of total cell numbers differed greatly between the two sites yet were consistent in decreasing sharply at greater depths. Although catalyzed reporter deposition fluorescence in situ hybridization with domain-specific probes showed that Bacteria (>98%) dominated over Archaea (<2%) at both sites, important differences were detected at the class and genus levels, reflecting differences in pH, redox potential, and heavy metal concentrations. At SN, where the pH and redox potential are similar to that of the water column (pH 2.5 and +400 mV), the most abundant organisms were identified as iron-reducing bacteria: Acidithiobacillus spp. and Acidiphilium spp., probably related to the higher iron solubility at low pH. At the JL dam, characterized by a banded sediment with higher pH (4.2 to 6.2), more reducing redox potential (-210 mV to 50 mV), and a lower solubility of iron, members of sulfate-reducing genera Syntrophobacter, Desulfosporosinus, and Desulfurella were dominant. The latter was quantified with a newly designed CARD-FISH probe. In layers where sulfate-reducing bacteria were abundant, pH was higher and redox potential and levels of dissolved metals and iron were lower. These results suggest that the attenuation of ARD characteristics is biologically driven by sulfate reducers and the consequent precipitation of metals and iron as sulfides.

Distribution of putative denitrifying methane oxidizing bacteria in sediment of a freshwater lake, Lake Biwa

Methane oxidation coupled to denitrification is mediated by ‘Candidatus Methylomirabilis oxyfera’, which belongs to the candidate phylum NC10. The distribution of putative denitrifying methane-oxidizing bacteria related to “M. oxyfera” was investigated in a freshwater lake, Lake Biwa, Japan. In the surface layer of the sediment from a profundal site, a phylotype closely related to “M. oxyfera” was most frequently detected among NC10 bacteria in PCR analysis of the 16S rRNA gene. In the sediment, sequences related to “M. oxyfera” were also detected in a pmoA gene library. The presence of NC10 bacteria was also confirmed by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR indicated that the abundance of the “M. oxyfera”-related phylotype was higher in the upper layers of the profundal sediment. The horizontal distribution of the putative methanotrophs in lake sediment was also analyzed by DGGE, which revealed that their occurrence was restricted to deep water areas. These results agreed with those in a previous study of another freshwater lake, and suggested that the upper layer of the profundal sediments is the main habitat for denitrifying methanotrophs.

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 109 to 1010 cells/mL at the sediment surface to 107–109 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.

Reliability of CARD-FISH Procedure for Enumeration of Archaea in Deep-Sea Surficial Sediments

The enumeration of Archaea in deep-sea sediment samples is still limited, although different methodological procedures have been applied. Among these, catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH) technique is a promising tool for estimation of archaeal abundance in deep-sea sediment samples. Comparing different permeabilisation treatments, the best results obtained both on archaeal pure cultures and on natural assemblages were with hydrochloric acid (0.1M) and proteinase K (0.004U/ml) treatments. The application of CARD-FISH on deep-sea sediments revealed that Archaea reach up to 41% of total prokaryotic cells. Specific probes for planktonic Archaea showed that marine Crenarchaea dominated archaeal seafloor communities. No clear bathymetric trends were observed for archaeal abundances and the morphology of continental margin (slope vs. canyon) seems not to have a direct influence on archaeal relative abundances. The site-specific sediment habitat-both abiotic environmental setting and sedimentary organic matter quality-explain up to 65% of variance of archaeal, crenarchaeal and euryarchaeal relative abundance, suggesting a wide ecophysiological adaptation to deep-sea benthic ecosystems. The findings demonstrate that Archaea are an important component of benthic microbial assemblages so far neglected, and hence they lay the groundwork for more focused research on their ecological importance in the functioning of deep-sea benthic ecosystems.

Microbial diversity and composition of the sediment in the drinking water reservoir Saidenbach (Saxonia, Germany)

Sediments contain a huge number and diversity of microorganisms that are important for the flux of material and are pivotal to all major biogeochemical cycles. Sediments of reservoirs are affected by a wide spectrum of allochthous and autochthonous influences providing versatile environments along the flow of water within the reservoir. Here we report on the microbial diversity in sediments of the mesotrophic drinking water reservoir Saidenbach, Germany, featuring a pronounced longitudinal gradient in sediment composition in the reservoir system. Three sampling sites were selected along the gradient, and the microbial communities in two sediment depths were characterized using catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and a bar-coded pyrosequencing approach. Multivariate statistic was used to reveal relationships between sequence diversity and the environmental conditions. The microbial communities were tremendously diverse with a Shannon index of diversity ( H′) ranging from 6.7 to 7.1. 18,986 sequences could be classified into 37 phyla including candidate divisions, but the full extent of genetic diversity was not captured. While CARD-FISH gave an overview about the community composition, more detailed information was gained by pyrosequencing. Bacteria were more abundant than Archaea. The dominating phylum in all samples was Proteobacteria, especially Betaproteobacteria and Deltaproteobacteria. Furthermore, sequences of Bacteroidetes, Verrucomicrobia, Acidobacteria, Chlorobi, Nitrospira, Spirochaetes, Gammaproteobacteria, Alphaproteobacteria, Chloroflexi, and Gemmatimonadetes were found. The site ammonium concentration, water content and organic matter content revealed to be strongest environmental predictors explaining the observed significant differences in the community composition between sampling sites.

Microbial community composition of anoxic marine sediments in the Bay of Cádiz (Spain).

The composition of the microbial community inhabiting the anoxic coastal sediments of the Bay of Cádiz (southern Spain) was investigated using a molecular approach consisting of PCR cloning and denaturing gradient gel electrophoresis (DGGE), based on 16S rRNA sequences. The total cell count was 1-5 × 10⁸ cells/g sediment and, as determined by catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH), the proportion of Bacteria to Archaea was about 70:30. The analysis of 16S-rRNA gene sequences revealed a wide spectrum of microorganisms, which could be grouped into 111 operational taxonomic units (OTUs). Many of the OTUs showed high phylogenetic similarity to microorganisms living in marine sediments of diverse geographic origin. The phylogenetic groups that were predominantly detected were Firmicutes, Deltaproteobacteria, and Gammaproteobacteria, accounting for 23, 15, and 14% of the clones, respectively. Diversity in the domain Archaea was significantly lower than in the domain Bacteria. The majority of the archaeal OTUs belonged to the Crenarchaeota phylum. Since most of the sequences could not be identified precisely at the genus/species level, the functional roles of the microorganisms in the ecosystem could not be inferred. However, seven OTUs affiliated with the Delta- and Epsilonproteobacteria were identified down to the genus level, with all of the identified genera known to occur in sulfate-rich marine environments.

Assimilation of acetate by the putative atmospheric methane oxidizers belonging to the USCα clade

Forest soils are a major biological sink for atmospheric methane, yet the identity and physiology of the microorganisms responsible for this process remain unclear. Although members of the upland soil cluster α (USCα) are assumed to represent methanotrophic bacteria adapted to the oxidation of the trace level of methane in the atmosphere and to be an important sink of this greenhouse gas, so far they have resisted isolation. In particular, the question of whether the atmospheric methane oxidizers are able to obtain all their energy and carbon solely from atmospheric methane still waits to be answered. In this study, we performed stable-isotope probing (SIP) of RNA and DNA to investigate the assimilation of (13) C-methane and (13) C-acetate by USCα in an acidic forest soil. RNA-SIP showed that pmoA mRNA of USCα was not labelled by (13) C of supplemented (13) C methane, although catalysed reporter deposition - fluorescence in situ hybridization (CARD-FISH) targeting pmoA mRNA of USCα detected its expression in the incubated soil. In contrast, incorporation of (13) C-acetate into USCαpmoA mRNA was observed. USCαpmoA genes were not labelled, indicating that they had not grown during the incubation. Our results indicate that the contribution of alternative carbon sources, such as acetate, to the metabolism of the putative atmospheric methane oxidizers in upland forest soils might be substantial.

Planctomycetes diversity associated with macroalgae

Planctomycetes associated with 12 macroalgae from the north coast of Portugal were isolated, using an improved method. A total of 138 isolates were found to comprise 10 operational taxonomic units (OTUs), with 65% of the strains being closely related to the species Rhodopirellula baltica. The other strains are probably new species or genera related to Rhodopirellula, Blastopirellula and Planctomyces. Some of the OTUs isolated are unique and have never been found before in previous studies. Catalyzed reporter deposition-FISH confirmed the presence of Planctomycetes on macroalgal surfaces. This study provides the first report of the cultured diversity of Planctomycetes on the epiphytic macroalgae community and presents clear evidence of their nutritional and intimate relationship.

Temporal variability and phylogenetic characterization of planktonic anammox bacteria in the coastal upwelling ecosystem off central Chile

The phylogenetic affiliation and temporal variability in the abundance of planktonic anammox bacteria were studied at a time-series station above the continental shelf off central Chile (∼36°S; bottom depth 93 m), a wind-driven, seasonal upwelling area, between August 2006 and April 2008. The study was carried out by cloning and sequencing the 16S rRNA gene and by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Our results showed the presence of a single anammox bacteria-like ribotype during both upwelling and non-upwelling seasons, which was phylogenetically associated with a recently described oxygen-minimum-zone subcluster within the Candidatus Scalindua clade. Moreover, clear differences were observed in the temporal and vertical distribution of anammox cells. During the upwelling season (austral spring–summer), relatively high abundances (∼5500 cells mL −1) and large cells (0.8 μm 3–75.7 fg C cell −1) were found below 20 m depth. In contrast, during the non-upwelling season (austral fall–winter), lower abundances (∼600 cells mL −1) and smaller cells (0.1 μm 3–22.8 fg C cell −1) were found, predominantly associated with the bottom layer. Overall, our results indicate that the abundance and vertical distribution of anammox planktonic assemblages are related to the occurrence of seasonal, wind-driven, coastal upwelling, which in turn appears to offer favorable conditions for the development of these microorganisms. The dominance of a unique anammox bacteria-like ribotype could be related to the high environmental variability observed in the system, which prevents the establishment of other anammox lineages.

Temporal variability of coastal Planctomycetes clades at Kabeltonne station, North Sea.

Members of the bacterial phylum Planctomycetes are reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution of Planctomycetes in surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Generally Planctomycetes are more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed that Planctomycetes abundance was correlated to the Centrales diatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ~90% of the Planctomycetes reside in the >3-μm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, named Planctomyces-related group A, uncultured Planctomycetes group B, and Pirellula-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. Uncultured Planctomycetes group B was most abundant in autumn samples, while Planctomyces-related group A was present in high numbers only during late autumn and winter. The levels of Pirellula-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of the Planctomycetes is correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.

Phylogenetic characterisation of picoplanktonic populations with high and low nucleic acid content in the North Atlantic Ocean

In flow cytometric analyses of marine prokaryotic picoplankton often two populations with distinct differences in their apparent nucleic acid content are discernable, one with a high and one with a low nucleic acid content (HNA and LNA, respectively). In this study we determined the phylogenetic composition of flow cytometrically sorted HNA and LNA populations, collected at six stations along a transect across three oceanic provinces from Iceland to the Azores. Catalysed reporter deposition fluorescence in situ hybridisation (CARD-FISH) analysis of sorted cells revealed distinct differences in phylogenetic composition between the LNA and HNA populations with only little overlap. At all stations the LNA population was dominated by the alphaproteobacterial clade SAR11 (45–74%). Also, Betaproteobacteria were always present at 2–4%. While the LNA composition was rather stable, the HNA populations were composed of distinct phylogenetic clades in the different oceanic provinces of Arctic and Tropics. For example Cyanobacteria dominated the North Atlantic Gyre HNA population (29–44%) with Prochlorococcus as the major clade (34–44%), but were low in Arctic and Polar waters (1% and 5%, respectively). In contrast, Bacteroidetes accounted for the majority of HNA cells in the Polar and Arctic province (26% and 32%, respectively), but were low in the Gyre region (3–10%). The DNA content of the HNA population was about 3.5 times higher than that of the LNA populations. This reflects differences in the genome sizes of closely related cultured representatives of HNA clades (3–6 Mbp) and LNA clades (1.3–1.5 Mbp).

Temporal attachment dynamics by distinct bacterial taxa during a dinoflagellate bloom

Limited quantitative information exists on the physical interaction between specific taxa of heterotrophic bacteria and phytoplankton in pelagic aquatic environments. Using catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we quantified bacterial attach- ment to the cells of the dinoflagellate Lingulodinium polyedrum in 39 surface samples collected during a natural bloom in summer 2005 off the coast of La Jolla, California, USA. Using a ribosomal RNA based tunable array with Luminex® bead technology, we also quantified the relative abun- dances of 11 particle-associated bacterial taxa during this time, including 8 members of the Bac- teroidetes division. Bacterial colonization of dinoflagellate cells was generally low (mean <2 bacteria alga -1 ) but increased during the days preceding bloom decline events. This indicates that physical associations, and thus potentially physiological interactions among bacteria and dinoflagellates, changed over the course of the algal bloom cycle. The 11 detected bacterial taxa exhibited diverse patterns of colonization over time, suggesting that they mediated different types of interactions with the dinoflagellates. Some bacterial types were only detected during the early bloom phase, others peaked in abundance during peaks in algal numbers, and still others peaked following bloom decline events. Our data linking the temporal succession of different bacterial colonizers to algal bloom dynamics exemplify the idea that microscale, species-specific interactions between bacteria and pro- tists can result in large-scale ecosystem level changes that can impact phytoplankton community structure in the coastal ocean.