The roles of individual bacterioplankton species in the re-mineralization of algal biomass are poorly understood. Evidence from molecular data had indicated that a spring diatom bloom in the German Bight of the North Sea in 2009 was followed by a rapid succession of uncultivated bacterioplankton species, including members of the genera Ulvibacter, Formosa, Polaribacter (class Flavobacteria) and Reinekea (class Gammaproteobacteria). We isolated strains from the same site during the diatom bloom in spring 2010 using dilution cultivation in an artificial seawater medium with micromolar substrate and nutrient concentrations. Flow cytometry demonstrated growth from single cells to densities of 10(4) -10(6) ?cells?ml(-1) and a culturability of 35%. Novel Formosa, Polaribacter and Reinekea strains were isolated and had 16S rRNA gene sequence identities of >?99.8% with bacterioplankton in spring or summer 2009. Genomes of selected isolates were draft sequenced and used for read recruitment of metagenomes from bacterioplankton in 2009. Metagenome reads covered 93% of a Formosa clade B, 91% of a Reinekea and 74% of a Formosa clade A genome, applying a ??94.5% nucleotide identity threshold. These novel strains represent abundant bacterioplankton species thriving on coastal phytoplankton blooms in the North Sea.
The globally abundant, uncultured unicellular cyanobacterium UCYN-A was recently discovered living in association with a eukaryotic cell closely related to a prymnesiophyte. Here, we established a double CAtalysed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) approach to identify both partners and provided quantitative information on their distribution and abundance across distinct water masses along a transect in the North Atlantic Ocean. The N2 fixation activity coincided with the detection of UCYN-A cells and was only observed in oligotrophic (0.067 N O 3 - ??M and 0.04 P O 4 3 - ??M) and warm (>?18°C) surface waters. Parallel 16S ribosomal RNA gene analyses among unicellular diazotrophs indicated that only UCYN-A cells were present. UCYN-A cells were associated with an algal partner or non-associated using the double CARD-FISH approach. We demonstrated that UCYN-A cells living in association with Haptophyta were the dominant form (87.0?±?6.1%), whereas non-associated UCYN-A cells represented only a minor fraction (5.2?±?3.9%). Interestingly, UCYN-A cells were also detected living in association with unknown single-celled eukaryotes in small amounts (7.8?±?5.2%), presumably Alveolata. The proposed ecological niche of UCYN-A as an oligotrophic, mesophilic and obligate symbiotic nitrogen-fixing microorganism is evident for the North Atlantic Ocean.
Marine and limnic particles are hotspots of organic matter mineralization significantly affecting biogeochemical element cycling. Fluorescence in-situ hybridization and pyrosequencing of 16S rRNA genes were combined to investigate bacterial diversity and community composition on limnic and coastal marine particles >?5 and >?10??m respectively. Limnic particles were more abundant (average: 1?×?10(7) ?l(-1) ), smaller in size (average areas: 471 versus 2050??m(2) ) and more densely colonized (average densities: 7.3 versus 3.6 cells 100??m(-2) ) than marine ones. Limnic particle-associated (PA) bacteria harboured Alphaproteobacteria and Betaproteobacteria, and unlike previously suggested sizeable populations of Gammaproteobacteria, Actinobacteria and Bacteroidetes. Marine particles were colonized by Planctomycetes and Betaproteobacteria additionally to Alphaproteobacteria, Bacteroidetes and Gammaproteobacteria. Large differences in individual particle colonization could be detected. High-throughput sequencing revealed a significant overlap of PA and free-living (FL) bacteria highlighting an underestimated connectivity between both fractions. PA bacteria were in 14/21 cases more diverse than FL bacteria, reflecting a high heterogeneity in the particle microenvironment. We propose that a ratio of Chao 1 indices of PA/FL?1 indicates the presence of rather homogeneously colonized particles. The identification of different bacterial families enriched on either limnic or marine particles demonstrates that, despite the seemingly similar ecological niches, PA communities of both environments differ substantially.
The iron fertilization experiment LOHAFEX was conducted in a cold-core eddy in the Southern Atlantic Ocean during austral summer. Within a few days after fertilization, a phytoplankton bloom developed dominated by nano- and picoplankton groups. Unlike previously reported for other iron fertilization experiments, a diatom bloom was prevented by iron and silicate co-limitation. We used 18S rRNA gene tag pyrosequencing to investigate the diversity of these morphologically similar cell types within the nano- and picoplankton and microscopically enumerated dominant clades after catalyzed reported deposition fluorescence in situ hybridization (CARD-FISH) with specific oligonucleotide probes. In addition to Phaeocystis, members of Syndiniales group II, clade 10-11, and the Micromonas clades ABC and E made up a major fraction of the tag sequences of the nano- and picoplankton community within the fertilized patch. However, the same clades were also dominant before the bloom and outside the fertilized patch. Furthermore, only little changes in diversity could be observed over the course of the experiment. These results were corroborated by CARD-FISH analysis which confirmed the presence of a stable nano- and picoplankton community dominated by Phaeocystis and Micromonas during the entire course of the experiment. Interestingly, although Syndiniales dominated the tag sequences, they could hardly be detected by CARD-FISH, possibly due to the intracellular parasitic life style of this clade. The remarkable stability of the nano- and picoplankton community points to a tight coupling of the different trophic levels within the microbial food web during LOHAFEX.
Nitrogen (N?) fixation is a globally important process often mediated by diazotrophic cyanobacteria in the open ocean. In 2010, seawater was collected near Cape Verde to identify and measure N? and carbon (C) fixation by unicellular diazotrophic cyanobacteria. The nifH gene abundance (10?-10? nifH L?¹) and nifH gene transcript abundance (10²-10? cDNA nifHL?¹) for two unicellular groups, UCYN-A and UCYN-B, were detected. UCYN-A was also identified and quantified (10?-10?cells L?¹) by new probes (UCYN-A732 and UCYN-A159) using Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) assays. The UCYN-A were observed as free cells or attached to a larger unidentified eukaryotic cell. A Halogen In Situ Hybridization-Secondary Ion Mass Spectrometry (HISH-SIMS) assay using the UCYN-A732 probe was applied on samples previously incubated with ¹³C-bicarbonate and ¹?N?. Free UCYN-A cells were enriched in both ¹³C and ¹?N and estimated C and N? fixation rates for UCYN-A were lower compared to co-occurring unicellular cyanobacteria cells similar in size (3.1-5.6 ?m) and pigmentation to diazotroph Crocosphaera watsonii. Here, we identify and quantify two common co-occurring unicellular groups and measure their cellular activities by nanoSIMS.
Aerobic gammaproteobacteria affiliated to the OM60/NOR5 clade are widespread in saline environments and of ecological importance in several marine ecosystems, especially the euphotic zone of coastal areas. Within this group a close relationship between aerobic anoxygenic photoheterotrophs and non-phototrophic members has been found.
The degradation of diatoms is mainly catalyzed by Bacteroidetes and this process is of global relevance for the carbon cycle. In this study, a combination of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and fluorescent lectin binding analysis (FLBA) was used to identify and map glycoconjugates involved in the specific interactions of Bacteroidetes and diatoms, as well as detritus, at the coastal marine site Helgoland Roads (German Bight, North Sea). The study probed both the presence of lectin-specific extracellular polymeric substances (EPS) of Bacteroidetes for cell attachment and that of glycoconjugates on diatoms with respect to binding sites for Bacteroidetes. Members of the clades Polaribacter and Ulvibacter were shown to form microcolonies within aggregates for which FLBA indicated the presence of galactose containing slime. Polaribacter spp. was shown to bind specifically to the setae of the abundant diatom Chaetoceros spp., and the setae were stained with fucose-specific lectins. In contrast, Ulvibacter spp. attached to diatoms of the genus Asterionella which bound, among others, the mannose-specific lectin PSA. The newly developed CARD-FISH/FLBA protocol was limited to the glycoconjugates that persisted after the initial CARD-FISH procedure. The differential attachment of bacteroidetal clades to diatoms and their discrete staining by FLBA provided evidence for the essential role that formation and recognition of glycoconjugates play in the interaction of bacteria with phytoplankton.
Bacteroidetes are widespread in marine systems where they play a crucial role in organic matter degradation. Whole genome analysis of several strains has revealed a broad glycolytic and proteolytic potential. In this study, we used a targeted metagenomic approach to investigate the degradation capabilities of distinct Bacteroidetes clades from two contrasting regions of the North Atlantic Ocean, the Polar Biome (BPLR) and the North Atlantic Subtropical (NAST). We present here the analysis of 76 Bacteroidetes fosmids, of which 28 encode the 16S rRNA gene as phylogenetic marker, and their comparison to complete Bacteroidetes genomes. Almost all of the 16S rRNA harbouring fosmids belonged to clades that we previously identified in BPLR and NAST. The majority of sequenced fosmids could be assigned to Bacteroidetes affiliated with the class Flavobacteria. We also present novel genomic information on the classes Cytophagia and Sphingobacteria, suggesting a capability of the latter for attachment to algal surfaces. In our fosmid set we identified a larger potential for polysaccharide degradation and cell surface attachment in the phytoplankton-rich BPLR. Particularly, two flavobacterial fosmids, one affiliated with the genus Polaribacter, showed a whole armoury of enzymes that likely function in degradation of sulfated polysaccharides known to be major constituents of phytoplankton cell walls. Genes involved in protein and peptidoglycan degradation, although present in both fosmid sets, seemed to have a slight preponderance in NAST. This study provides support for the hypothesis of a distinct specialization among marine Bacteroidetes for the degradation of certain types of polymers.
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-?m 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.
Vibrio species are ubiquitously distributed in marine waters all over the world. High genome plasticity due to frequent mutation, recombination, and lateral gene transfer enables Vibrio to adapt rapidly to environmental changes. The genus Vibrio comprises several human pathogens, which commonly cause outbreaks of severe diarrhea in tropical regions. In recent years, pathogenic Vibrio emerged also in coastal European waters. Little is known about factors driving the proliferation of Vibrio spp. in temperate waters such as the North Sea. In this study a quantification of Vibrio in the North Sea and their response to biotic and abiotic parameters were assessed. Between January and December 2009, Vibrio at Helgoland Roads (North Sea, Germany) were quantified using fluorescence in situ hybridization. Vibrio numbers up to 3.4 × 10(4) cells × mL(-1) (2.2% of total microbial counts) were determined in summer, but their abundance was significantly lower in winter (5 × 10(2) cells × mL(-1)). Correlations between Vibrio and nutrients (SiO(2), PO(4) (3-), DIN), Secchi depth, temperature, salinity, and chlorophyll a were calculated using Spearman rank analysis. Multiple stepwise regression analysis was carried out to analyze the additive influence of multiple factors on Vibrio. Based on these calculations, we found that high water temperature and low salinity best explained the increase of Vibrio cell numbers. Other environmental parameters, especially nutrients and chlorophyll a, also had an influence. All variables were shown to be subject to the overall seasonal dynamics at Helgoland Roads. Multiple regression models could represent an efficient and reliable tool to predict Vibrio abundances in response to the climate change in European waters.
Fluorescence in situ hybridization (FISH) of genes and mRNA is most often based on polynucleotide probes. However, so far there was no published framework for the rational design of polynucleotide probes. The well-established concepts for oligonucleotide probe design cannot be transferred to polynucleotides. Due to the high allele diversity of genes, a single probe is not sufficient to detect all alleles of a gene. Therefore, the main objective of this study was to develop a concept and software (PolyPro) for rational design of polynucleotide probe mixes to target particular genes. PolyPro consists of three modules: a GenBank Taxonomy Extractor (GTE), a Polynucleotide Probe Designer (PPD) and a Hybridization Parameters Calculator (HPC). The new concept proposes the construction of defined polynucleotide mixes to target the habitat specific sequence diversity of a particular gene. The concept and the software are intended as a first step towards a more frequent application of polynucleotides for in situ identification of mRNA and genes in environmental microbiology.
Microbial community structure in the subtropical north-east Atlantic Ocean was compared between 2 years and variation attributed to environmental variables. Surface seawater communities were analysed by flow cytometry and fluorescence in situ hybridisation. Probes specific to Alphaproteobacteria, Cyanobacteria, Gammaproteobacteria and Bacteroidetes identified 67-100% of cells. Due to natural variation in the study region due to the occurrence of major currents and islands, data could not be pooled but were instead divided between distinct water masses. Community structure did not differ greatly around the Cape Verde Islands between sampling periods but varied substantially in the open ocean, suggesting different environmental perturbations favour specific bacterial groups. Wind speed varied significantly between years, with moderate to strong breeze in winter 2008 and gales in winter 2006 (8.9?±?0.2 ms(-1) and 16.0?±?0.4 ms(-1), respectively). Enhanced wind-driven turbulence was associated with domination by the SAR11 clade of Alphaproteobacteria, which were present at 2.4-fold in the abundance of Prochlorococcus (41.8?±?1.6% cells, compared to 17.7?±?7.1%). Conversely, the calmer conditions of 2008 seemed to favour Prochlorococcus (40.0?±?1.2% cells). Prochlorococcus high-light adapted clade HLI were only numerous during wind-driven turbulence, whereas oligotrophic-adapted clade HLII dominated under calm conditions. Bacteroidetes were most prominent in turbulent conditions (9.5?±?1.3% cells as opposed to 4.7?±?0.3%), as were Synechococcus. In 2008, a considerable dust deposition event occurred in the region, which may have led to the substantial Gammaproteobacteria population (22.5?±?4.0% cells compared to 4.6?±?0.6% in 2006). Wind-driven turbulence may have a significant impact on microbial community structure in the surface ocean. Therefore, community change following dust storm events may be linked to associated wind in addition to dust-derived nutrients.
A dimorphic life cycle has been described for the planctomycete Rhodopirellula baltica SH1(T), with juvenile motile, free-swimming cells and adult sessile, attached-living cells. However, attachment as a response to environmental factors was not investigated. We studied the response of R. baltica to nitrogen limitation. In batch cultures, ammonium limitation coincided with a dominance of free-swimming cells and a low number of aggregates. Flow cytometry revealed a quantitative shift with increasing ammonium availability, from single cells towards attached cells in large aggregates. During growth of R. baltica on glucose and ammonium in chemostats, an ammonium addition caused a macroscopic change of the growth behaviour, from homogeneous growth in the liquid phase to a biofilm on the borosilicate glass wall of the chemostat vessel. Thus, an ammonium limitation-a carbon to nitrogen supply ratio of 30:1-sustained free-living growth without aggregate formation. A sudden increase in ammonium supply induced sessile growth of R. baltica. These observations reveal a response of Rhodopirellula baltica cells to ammonium: they abandon the free-swimming life, attach to particles and form biofilms.
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 (3-6Mbp) and LNA clades (1.3-1.5Mbp).
Next-generation sequencing (NGS) technologies have enabled the application of broad-scale sequencing in microbial biodiversity and metagenome studies. Biodiversity is usually targeted by classifying 16S ribosomal RNA genes, while metagenomic approaches target metabolic genes. However, both approaches remain isolated, as long as the taxonomic and functional information cannot be interrelated. Techniques like self-organizing maps (SOMs) have been applied to cluster metagenomes into taxon-specific bins in order to link biodiversity with functions, but have not been applied to broad-scale NGS-based metagenomics yet. Here, we provide a novel implementation, demonstrate its potential and practicability, and provide a web-based service for public usage. Evaluation with published data sets mimicking varyingly complex habitats resulted into classification specificities and sensitivities of close to 100% to above 90% from phylum to genus level for assemblies exceeding 8?kb for low and medium complexity data. When applied to five real-world metagenomes of medium complexity from direct pyrosequencing of marine subsurface waters, classifications of assemblies above 2.5?kb were in good agreement with fluorescence in situ hybridizations, indicating that biodiversity was mostly retained within the metagenomes, and confirming high classification specificities. This was validated by two protein-based classifications (PBCs) methods. SOMs were able to retrieve the relevant taxa down to the genus level, while surpassing PBCs in resolution. In order to make the approach accessible to a broad audience, we implemented a feature-rich web-based SOM application named TaxSOM, which is freely available at http://www.megx.net/toolbox/taxsom. TaxSOM can classify reads or assemblies exceeding 2.5?kb with high accuracy and thus assists in linking biodiversity and functions in metagenome studies, which is a precondition to study microbial ecology in a holistic fashion.
Our knowledge concerning the metabolic potentials of as yet to be cultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes. Therefore, the aim of the present study was to develop geneFISH - a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA-targeted catalysed reporter deposition - fluorescence in situ hybridization and in situ gene detection. To test the protocol, it was applied to seawater samples from the Benguela upwelling system. For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amoA clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. To determine the hybridization parameters, the T(m) of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements. It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amoA gene and were therefore likely to be catalysing the oxidation of ammonia.
The Lagoon of Venice is a large water basin that exchanges water with the Northern Adriatic Sea through three large inlets. In this study, the 16S rRNA approach was used to investigate the bacterial diversity and community composition within the southern basin of the Lagoon of Venice and at one inlet in October 2007 and June 2008. Comparative sequence analysis of 645 mostly partial 16S rRNA gene sequences indicated high diversity and dominance of Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes at the lagoon as well as at the inlet station, therefore pointing to significant mixing. Many of these sequences were close to the 16S rRNA of marine, often coastal, bacterioplankton, such as the Roseobacter clade, the family Vibrionaceae, and class Flavobacteria. Sequences of Actinobacteria were indicators of a freshwater input. The composition of the bacterioplankton was quantified by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) with a set of rRNA-targeted oligonucleotide probes. CARD-FISH counts corroborated the dominance of members of the phyla Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. When assessed by a probe set for the quantification of selected clades within Alphaproteobacteria and Gammaproteobacteria, bacterioplankton composition differed between October 2007 and June 2008, and also between the inlet and the lagoon. In particular, members of the readily culturable copiotrophic gammaproteobacterial genera Vibrio, Alteromonas and Pseudoalteromonas were enriched in the southern basin of the Lagoon of Venice. Interestingly, the alphaproteobacterial SAR11 clade and related clusters were also present in high abundances at the inlet and within the lagoon, which was indicative of inflow of water from the open sea.
Members of the class Flavobacteria in the phylum Bacteroidetes are among the most abundant picoplankton in coastal and polar oceans. Their diversity is high in marine waters. However, quantitative information about distribution patterns of flavobacterial clades is scarce. We analyzed the diversity and clade-specific abundances of individual Flavobacteria in different oceanic provinces in the North Atlantic Ocean. Samples were taken along the 30 degrees W meridian between the East Greenland current and the North Atlantic subtropical gyre. Comparative sequence analysis of 16S ribosomal RNA (rRNA) gene libraries revealed high diversity and significant spatial variability within the class Flavobacteria. Published and newly designed oligonucleotide probes were used to enumerate eleven flavobacterial clades by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). We found that different provinces harbor distinct flavobacterial communities. Clade DE2 accounted for a substantial fraction of total Flavobacteria only in the Polar Biome (BPLR), whereas the VISION clades VIS1 and VIS4 significantly increased in the Arctic (ARCT) province. Members of the genus Polaribacter were the most abundant clade in all the water masses analyzed, with highest absolute numbers in BPLR and ARCT. We improved the CARD-FISH protocol to quantify the rare clades VIS2, VIS3, VIS5 and VIS6, which were present in abundances below 0.5%. They all showed pronounced regional distribution patterns. Microscopic analysis proved a specific enrichment of Flavobacteria in the phycosphere of nanophytoplankton of BPLR and ARCT. Our results suggest that different marine flavobacterial clades have distinct niches and different life strategies.
Members of the prokaryotic picoplankton are the main drivers of the biogeochemical cycles over large areas of the worlds oceans. In order to ascertain changes in picoplankton composition in the euphotic and twilight zones at an ocean basin scale we determined the distribution of 11 marine bacterial and archaeal phyla in three different water layers along a transect across the Atlantic Ocean from South Africa (32.9 degrees S) to the UK (46.4 degrees N) during boreal spring. Depth profiles down to 500 m at 65 stations were analysed by catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and automated epifluorescence microscopy. There was no obvious overall difference in microbial community composition between the surface water layer and the deep chlorophyll maximum (DCM) layer. There were, however, significant differences between the two photic water layers and the mesopelagic zone. SAR11 (35 +/- 9%) and Prochlorococcus (12 +/- 8%) together dominated the surface waters, whereas SAR11 and Crenarchaeota of the marine group I formed equal proportions of the picoplankton community below the DCM (both approximately 15%). However, due to their small cell sizes Crenarchaeota contributed distinctly less to total microbial biomass than SAR11 in this mesopelagic water layer. Bacteria from the uncultured Chloroflexi-related clade SAR202 occurred preferentially below the DCM (4-6%). Distinct latitudinal distribution patterns were found both in the photic zone and in the mesopelagic waters: in the photic zone, SAR11 was more abundant in the Northern Atlantic Ocean (up to 45%) than in the Southern Atlantic gyre (approximately 25%), the biomass of Prochlorococcus peaked in the tropical Atlantic Ocean, and Bacteroidetes and Gammaproteobacteria bloomed in the nutrient-rich northern temperate waters and in the Benguela upwelling. In mesopelagic waters, higher proportions of SAR202 were present in both central gyre regions, whereas Crenarchaeota were clearly more abundant in the upwelling regions and in higher latitudes. Other phylogenetic groups such as the Planctomycetes, marine group II Euryarchaeota and the uncultured clades SAR406, SAR324 and SAR86 rarely exceeded more than 5% of relative abundance.
Coastal waters support approximately 90 per cent of global fisheries and are therefore an important food reserve for our planet. Eutrophication of these waters, due to human activity, leads to severe oxygen depletion and the episodic occurrence of hydrogen sulphide-toxic to multi-cellular life-with disastrous consequences for coastal ecosytems. Here we show that an area of approximately 7,000 km(2) of African shelf, covered by sulphidic water, was detoxified by blooming bacteria that oxidized the biologically harmful sulphide to environmentally harmless colloidal sulphur and sulphate. Combined chemical analyses, stoichiometric modelling, isotopic incubations, comparative 16S ribosomal RNA, functional gene sequence analyses and fluorescence in situ hybridization indicate that the detoxification proceeded by chemolithotrophic oxidation of sulphide with nitrate and was mainly catalysed by two discrete populations of gamma- and epsilon-proteobacteria. Chemolithotrophic bacteria, accounting for approximately 20 per cent of the bacterioplankton in sulphidic waters, created a buffer zone between the toxic sulphidic subsurface waters and the oxic surface waters, where fish and other nekton live. This is the first time that large-scale detoxification of sulphidic waters by chemolithotrophs has been observed in an open-ocean system. The data suggest that sulphide can be completely consumed by bacteria in the subsurface waters and, thus, can be overlooked by remote sensing or monitoring of shallow coastal waters. Consequently, sulphidic bottom waters on continental shelves may be more common than previously believed, and could therefore have an important but as yet neglected effect on benthic communities.
There is accumulating evidence that in some marine environments aerobic bacteriochlorophyll a-producing bacteria represent a significant part of the microbial population. The interaction of photosynthesis and carbon metabolism in these interesting bacteria is still largely unknown and requires further investigation in order to estimate their contribution to the marine carbon cycle.
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. More than 500 16S rRNA sequences affiliated with this clade were retrieved from public databases. By comparative sequence analysis, 13 subclades could be identified, some of which are currently restricted to discrete habitat types. Almost all sequences in the largest subclade NOR5-1 and related subclade NOR5-4 originated from marine surface water samples. Overall, 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 Namibia and the Yangtze estuary were up to 3% of the total 4,6-diamidino-2-phenylindole (DAPI) counts, and in the German Bight off Helgoland the abundance was even up to 7%. In an open-ocean North Atlantic transect, between Iceland and the Azores, the NOR5/OM60 group was much less abundant (0.1-0.5%). Interestingly, the surface layer of North Sea intertidal sediments was very rich in NOR5/OM60, with absolute numbers >10(8) cells cm(-3) (or 4% of the total DAPI). An analysis of the frequencies of NOR5/OM60 16S rRNA genes in the Global Ocean Survey datasets provided further support for a marine cosmopolitan occurrence of NOR5/OM60, and a clear preference for coastal marine waters.
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.
Microbial communities play a key role in the marine carbon cycle, processing much of phytoplankton-derived organic matter. The composition of these communities varies by depth, season, and location in the ocean; the functional consequences of these compositional variations for the carbon cycle are only beginning to be explored. We measured the abilities of microbial communities in the large-particle fraction (retained by a 10-?m pore-size cartridge filter) to enzymatically hydrolyze high molecular weight substrates, and therefore initiate carbon remineralization in four distinct oceanic provinces: the boreal polar (BPLR), the Arctic oceanic (ARCT), the North Atlantic drift (NADR), and the North Atlantic subtropical (NAST) provinces. Since we expected the large-particle fraction to include phytoplankton cells, we measured the hydrolysis of polysaccharide substrates (laminarin, fucoidan, xylan, and chondroitin sulfate) expected to be associated with phytoplankton. Hydrolysis rates and patterns clustered into two groups, the BPLR/ARCT and the NADR/NAST. All four substrates were hydrolyzed by the BPLR/ARCT communities; hydrolysis rates of individual substrate varied by factors of ca. 1-4. In contrast, chondroitin was not hydrolyzed in the NADR/NAST, and hydrolytic activity was dominated by laminarinase. Fluorescence in situ hybridization of the large-particle fraction post-incubation showed a substantial contribution (15-26%) of CF319a-positive cells (Bacteroidetes) to total DAPI-stainable cells. Concurrent studies of microbial community composition and of fosmids from these same stations also demonstrated similarities between BPLR and ARCT stations, which were distinct from the NADR/NAST stations. Together, these data support a picture of compositionally as well as functionally distinct communities across these oceanic provinces.
Magnetotactic bacteria (MTB), which orient along the earths magnetic field using magnetosomes, are ubiquitous and abundant in marine and freshwater environments. Previous phylogenetic analysis of diverse MTB has been limited to few cultured species and the most abundant and conspicuous members of natural populations, which were assigned to various lineages of the Proteobacteria, the Nitrospirae phylum as well as the candidate division OP3. However, their known phylogenetic diversity still not matches the large morphological and ultrastructural variability of uncultured MTB found in environmental communities. Here, we used analysis of 16S rRNA gene clone libraries in combination with microsorting and whole-genome amplification to systematically address the entire diversity of uncultured MTB from two different habitats. This approach revealed extensive and novel diversity of MTB within the freshwater and marine sediment samples. In total, single-cell analysis identified eight different phylotypes, which were only partly represented in the clone libraries, and which could be unambiguously assigned to their respective morphotypes. Identified MTB belonged to the Alphaproteobacteria (seven species) and the Nitrospirae phylum (two species). End-sequencing of a small insert library created from WGA-derived DNA of a novel conspicuous magnetotactic vibrio identified genes with highest similarity to two cultivated MTB as well as to other phylogenetic groups. In conclusion, the combination of metagenomic cloning and single cell sorting represents a powerful approach to recover maximum bacterial diversity including low-abundant magnetotactic phylotypes from environmental samples and also provides access to genomic analysis of uncultivated MTB.
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, that is, potentially enhance uptake of scarce organic molecules using the available solar radiation to energise appropriate transport systems. Here, we assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions using (33)P-ATP, (3)H-ATP and (35)S-methionine tracers. Light-stimulated uptake of these substrates was assessed in two dominant bacterioplankton groups discriminated by flow cytometric sorting of tracer-labelled cells and identified using catalysed reporter deposition fluorescence in situ hybridisation. One group of cells, encompassing 48% of all bacterioplankton, were identified as members of the SAR11 clade, whereas the other group (24% of all bacterioplankton) was Prochlorococcus. When exposed to light, SAR11 cells took 31% more ATP and 32% more methionine, whereas the Prochlorococcus cells took 33% more ATP and 34% more methionine. Other bacterioplankton did not demonstrate light stimulation. Thus, the SAR11 and Prochlorococcus groups, with distinctly different light-harvesting mechanisms, used light equally to enhance, by approximately one-third, the uptake of different types of organic molecules. Our findings indicate the significance of light-driven uptake of essential organic nutrients by the dominant bacterioplankton groups in the surface waters of one of the less productive, vast regions of the worlds oceans-the oligotrophic North Atlantic subtropical gyre.
Iron fertilization experiments in high-nutrient, low-chlorophyll areas are known to induce phytoplankton blooms. However, little is known about the response of the microbial community upon iron fertilization. As part of the LOHAFEX experiment in the southern Atlantic Ocean, Bacteria and Archaea were monitored within and outside an induced bloom, dominated by Phaeocystis-like nanoplankton, during the 38 days of the experiment. The microbial production increased 1.6-fold (thymidine uptake) and 2.1-fold (leucine uptake), while total cell numbers increased only slightly over the course of the experiment. 454 tag pyrosequencing of partial 16S rRNA genes and catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) showed that the composition and abundance of the bacterial and archaeal community in the iron-fertilized water body were remarkably constant without development of typical bloom-related succession patterns. Members of groups usually found in phytoplankton blooms, such as Roseobacter and Gammaproteobacteria, showed no response or only a minor response to the bloom. However, sequence numbers and total cell numbers of the SAR11 and SAR86 clades increased slightly but significantly toward the end of the experiment. It seems that although microbial productivity was enhanced within the fertilized area, a succession-like response of the microbial community upon the algal bloom was averted by highly effective grazing. Only small-celled members like the SAR11 and SAR86 clades could possibly escape the grazing pressure, explaining a net increase of those clades in numbers.
Flow cytometric sorting, based on cellular optical properties and macromolecule content, has been successfully employed to taxonomically affiliate bacterioplankton. However, this approach has not been much used for eukaryotic plankton. To redress this imbalance, we identified a conspicuous group of red autofluorescent picoplankton in surface waters of the South Atlantic Ocean. Using catalysed reporter deposition fluorescence in situ hybridization, virtually, all cells sorted from that group were affiliated with the Mamiellales clade II (84 ± 4%, division Chlorophyta) with a size of 1.6 ± 0.03 ?m. Based on electron microscopy, the Mamiellales clade II-sorted cells have a simple morphology with apparently no scales, flagella or surface features. Their latitudinal distribution resembled the distribution of Synechococcus with very low concentrations in the surface waters of the Southern subtropical gyre (0.6-1.6 × 10(3) cells mL(-1)) and increased concentrations in the Southern temperate waters 8.3 × 10(3) cells mL(-1). Identification of the flow cytometric group as Mamiellales clade II allowed us to characterize the morphology of these enigmatic uncultured picoplanktonic cells by electron microscopy and to determine their apparent preference for temperate rather than subtropical oceanic photic waters.
Phytoplankton blooms characterize temperate ocean margin zones in spring. We investigated the bacterioplankton response to a diatom bloom in the North Sea and observed a dynamic succession of populations at genus-level resolution. Taxonomically distinct expressions of carbohydrate-active enzymes (transporters; in particular, TonB-dependent transporters) and phosphate acquisition strategies were found, indicating that distinct populations of Bacteroidetes, Gammaproteobacteria, and Alphaproteobacteria are specialized for successive decomposition of algal-derived organic matter. Our results suggest that algal substrate availability provided a series of ecological niches in which specialized populations could bloom. This reveals how planktonic species, despite their seemingly homogeneous habitat, can evade extinction by direct competition.
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