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.

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-μm 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.

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ⩽25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ⩾75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.

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.

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.

In this report, we describe the selective cloning of large DNA fragments from magnetotactic metagenomes from various aquatic habitats. This was achieved by a two-step magnetic enrichment which allowed the mass collection of environmental magnetotactic bacteria (MTB) virtually free of nonmagnetic contaminants. Four fosmid libraries were constructed and screened by end sequencing and hybridization analysis using heterologous magnetosome gene probes. A total of 14 fosmids were fully sequenced. We identified and characterized two fosmids, most likely originating from two different alphaproteobacterial strains of MTB that contain several putative operons with homology to the magnetosome island (MAI) of cultivated MTB. This is the first evidence that uncultivated MTB exhibit similar yet differing organizations of the MAI, which may account for the diversity in biomineralization and magnetotaxis observed in MTB from various environments.

Sulfate-reducing bacteria (SRB) belonging to the metabolically versatile Desulfobacteriaceae are abundant in marine sediments and contribute to the global carbon cycle by complete oxidation of organic compounds. Desulfobacterium autotrophicum HRM2 is the first member of this ecophysiologically important group with a now available genome sequence. With 5.6 megabasepairs (Mbp) the genome of Db. autotrophicum HRM2 is about 2 Mbp larger than the sequenced genomes of other sulfate reducers (SRB). A high number of genome plasticity elements (> 100 transposon-related genes), several regions of GC discontinuity and a high number of repetitive elements (132 paralogous genes Mbp−1) point to a different genome evolution when comparing with Desulfovibrio spp. The metabolic versatility of Db. autotrophicum HRM2 is reflected in the presence of genes for the degradation of a variety of organic compounds including long-chain fatty acids and for the Wood–Ljungdahl pathway, which enables the organism to completely oxidize acetyl-CoA to CO2 but also to grow chemolithoautotrophically. The presence of more than 250 proteins of the sensory/regulatory protein families should enable Db. autotrophicum HRM2 to efficiently adapt to changing environmental conditions. Genes encoding periplasmic or cytoplasmic hydrogenases and formate dehydrogenases have been detected as well as genes for the transmembrane TpII-c3, Hme and Rnf complexes. Genes for subunits A, B, C and D as well as for the proposed novel subunits L and F of the heterodisulfide reductases are present. This enzyme is involved in energy conservation in methanoarchaea and it is speculated that it exhibits a similar function in the process of dissimilatory sulfate reduction in Db. autotrophicum HRM2.

Recent studies have shown that the anaerobic oxidation of ammonium by anammox bacteria plays an important role in catalyzing the loss of nitrogen from marine oxygen minimum zones (OMZ). However, in situ oxygen concentrations of up to 25 μM and ammonium concentrations close to or below the detection limit in the layer of anammox activity are hard to reconcile with the current knowledge of the physiology of anammox bacteria. We therefore investigated samples from the Namibian OMZ by comparative 16S rRNA gene analysis and fluorescence in situ hybridization. Our results showed that “Candidatus Scalindua” spp., the typical marine anammox bacteria, colonized microscopic particles that were likely the remains of either macroscopic marine snow particles or resuspended particles. These particles were slightly but significantly (P < 0.01) enriched in Gammaproteobacteria (11.8% ± 5.0%) compared to the free-water phase (8.1% ± 1.8%). No preference for the attachment to particles could be observed for members of the Alphaproteobacteria and Bacteroidetes, which were abundant (12 to 17%) in both habitats. The alphaproteobacterial SAR11 clade, the Euryarchaeota, and group I Crenarchaeota, were all significantly depleted in particles compared to their presence in the free-water phase (16.5% ± 3.5% versus 2.6% ± 1.7%, 2.7% ± 1.9% versus <1%, and 14.9% ± 4.6% versus 2.2% ± 1.8%, respectively, all P < 0.001). Sequence analysis of the crenarchaeotal 16S rRNA genes showed a 99% sequence identity to the nitrifying “Nitrosopumilus maritimus.” Even though we could not observe conspicuous consortium-like structures of anammox bacteria with particle-enriched bacterioplankton groups, we hypothesize that members of Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes play a critical role in extending the anammox reaction to nutrient-depleted suboxic water layers in the Namibian upwelling system by creating anoxic, nutrient-enriched microniches.

Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical importance, little is known about their genetic repertoire because of the lack of pure cultures. Here, we present a unique approach to access the genome of single filaments of Beggiatoa by combining whole genome amplification, pyrosequencing, and optical genome mapping. Sequence assemblies were incomplete and yielded average contig sizes of approximately 1 kb. Pathways for sulfur oxidation, nitrate and oxygen respiration, and CO2 fixation confirm the chemolithoautotrophic physiology of Beggiatoa. In addition, Beggiatoa potentially utilize inorganic sulfur compounds and dimethyl sulfoxide as electron acceptors. We propose a mechanism of vacuolar nitrate accumulation that is linked to proton translocation by vacuolar-type ATPases. Comparative genomics indicates substantial horizontal gene transfer of storage, metabolic, and gliding capabilities between Beggiatoa and cyanobacteria. These capabilities enable Beggiatoa to overcome non-overlapping availabilities of electron donors and acceptors while gliding between oxic and sulfidic zones. The first look into the genome of these filamentous sulfur-oxidizing bacteria substantially deepens the understanding of their evolution and their contribution to sulfur and nitrogen cycling in marine sediments.

Author Summary

In 1888 Winogradsky proposed the concept of chemolithotrophy—growth using inorganic compounds as an energy source—after studying the sulfur bacterium Beggiatoa. These filamentous bacteria and related organisms inhabit the surface of marine and freshwater sediments, where they oxidize hydrogen sulfide using either oxygen or nitrate. In particular, conspicuously large marine representatives accumulate nitrate in vacuoles to survive anoxia, a unique feature among prokaryotes. Since nitrate-storing Beggiatoa are not available in pure culture, we amplified and sequenced the genomic DNA of single multicellular filaments. We comprehensively tested the incomplete sequence assemblies for foreign DNA. We show that the Beggiatoa genome encodes the pathways of chemolithoautotrophy but also appears to support the use of alternative electron donors and acceptors. We propose that vacuolar-type ATPases generate an electrochemical gradient to drive nitrate transport over the vacuole membrane, a mechanism similar to eukaryotic solute accumulation. Intriguingly, we found evidence for substantial gene exchange between Beggiatoa and cyanobacteria. In both phyla, hemagglutinins are possibly involved in filament formation. The breadth of storage and metabolic capabilities encoded in its genome enables Beggiatoa to act as a “rechargeable battery,” which glides between oxic and sulfidic zones to overcome non-overlapping availabilities of electron donors and acceptors.

Examining the genome of single filaments of Beggiatoa deepens our understanding of their contribution to sulfur and nitrogen cycling in marine sediments.

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56% ± 8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94% ± 2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in 13C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of 13C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the 13C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the 13C-labeled compounds. Similar results were obtained with the 13C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the 13C analysis of the captured SSU rRNA.

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5′-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.

The dissimilatory reduction of sulfate is an ancient metabolic process central to today's biogeochemical cycling of sulfur and carbon in marine sediments. Until now its polyphyletic distribution was most parsimoniously explained by multiple horizontal transfers of single genes rather than by a not-yet-identified “metabolic island.” Here we provide evidence that the horizontal transfer of a gene cluster may indeed be responsible for the patchy distribution of sulfate-reducing prokaryotes (SRP) in the phylogenetic tree. We isolated three DNA fragments (32 to 41 kb) from uncultured, closely related SRP from DNA directly extracted from two distinct marine sediments. Fosmid ws39f7, and partially also fosmids ws7f8 and hr42c9, harbored a core set of essential genes for the dissimilatory reduction of sulfate, including enzymes for the reduction of sulfur intermediates and synthesis of the prosthetic group of the dissimilatory sulfite reductase. Genome comparisons suggest that encoded membrane proteins universally present among SRP are critical for electron transfer to cytoplasmic enzymes. In addition, novel, conserved hypothetical proteins that are likely involved in dissimilatory sulfate reduction were identified. Based on comparative genomics and previously published experimental evidence, a more comprehensive model of dissimilatory sulfate reduction is presented. The observed clustering of genes involved in dissimilatory sulfate reduction has not been previously found. These findings strongly support the hypothesis that genes responsible for dissimilatory sulfate reduction were concomitantly transferred in a single event among prokaryotes. The acquisition of an optimized gene set would enormously facilitate a successful implementation of a novel pathway.

Major biogeochemical processes in the water columns of lakes and oceans are related to the activities of heterotrophic microbes, e.g., the mineralization of organic carbon from photosynthesis and allochthonous influx or its transport to the higher trophic levels. During the last 15 years, cultivation-independent molecular techniques have substantially contributed to our understanding of the diversity of the microbial communities in different aquatic systems. In parallel, the complexity of aquatic habitats at a microscale has inspired research on the ecophysiological properties of uncultured microorganisms that thrive in a continuum of dissolved to particulate organic matter. One possibility to link these two aspects is to adopt a “Gleasonian” perspective, i.e., to study aquatic microbial assemblages in situ at the population level rather than looking at microbial community structure, diversity, or function as a whole. This review compiles current knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters. Specifically, we focus on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells. An overview is given of the potential and limitations of methodological approaches, and factors that might control the population sizes of different microbes in pelagic habitats are discussed.

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 μM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.

Background

Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments.

Results

The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool.

Conclusion

Using this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page .

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, ∼300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the γ-Proteobacteria (Gamma 1 and 2 symbionts), two members of the δ-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the δ-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the β-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of β-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the β-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.

A comprehensive assessment of bacterial diversity and community composition in arctic and antarctic pack ice was conducted through cultivation and cultivation-independent molecular techniques. We sequenced 16S rRNA genes from 115 and 87 pure cultures of bacteria isolated from arctic and antarctic pack ice, respectively. Most of the 33 arctic phylotypes were >97% identical to previously described antarctic species or to our own antarctic isolates. At both poles, the α- and γ-proteobacteria and the Cytophaga-Flavobacterium group were the dominant taxonomic bacterial groups identified by cultivation as well as by molecular methods. The analysis of 16S rRNA gene clone libraries from multiple arctic and antarctic pack ice samples revealed a high incidence of closely overlapping 16S rRNA gene clone and isolate sequences. Simultaneous analysis of environmental samples with fluorescence in situ hybridization (FISH) showed that ∼95% of 4′,6′-diamidino-2-phenylindole (DAPI)-stained cells hybridized with the general bacterial probe EUB338. More than 90% of those were further assignable. Approximately 50 and 36% were identified as γ-proteobacteria in arctic and antarctic samples,respectively. Approximately 25% were identified as α-proteobacteria, and 25% were identified as belonging to the Cytophaga-Flavobacterium group. For the quantification of specific members of the sea ice community, new oligonucleotide probes were developed which target the genera Octadecabacter, Glaciecola, Psychrobacter, Marinobacter, Shewanella, and Polaribacter. High FISH detection rates of these groups as well as high viable counts corroborated the overlap of clone and isolate sequences. A terrestrial influence on the arctic pack ice community was suggested by the presence of limnic phylotypes.

Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.

We studied the growth of six culturable bacterial lineages from coastal North Sea picoplankton in environmental samples under different incubation conditions. The grazing pressure of heterotrophic nanoflagellates (HNF) was reduced either by double prefiltration through 0.8-μm-pore-size filters or by 10-fold dilutions with 0.2-μm (pore-size) prefiltered seawater. We hypothesized that those γ-proteobacterial genera that are rapidly enriched would also be most strongly affected by HNF regrowth. In the absence of HNF, the mean protein content per bacterial cell increased in both treatments compared to environmental samples, whereas the opposite trend was found in incubations of unaltered seawater. Significant responses to the experimental manipulations were observed in Alteromonas, Pseudoalteromonas, and Vibrio populations. No treatment-specific effects could be detected for members of the Roseobacter group, the Cytophaga latercula-C. marinoflava lineage, or the NOR5 clade. Statistical analysis confirmed a transient increase in the proportions of Alteromonas, Pseudoalteromonas, and Vibrio cells at reduced HNF densities only, followed by an overproportional decline during the phase of HNF regrowth. Cells from these genera were significantly larger than the community average in the dilution treatments, and changes in their relative abundances were negatively correlated with HNF densities. Our findings suggest that bacteria affiliated with frequently isolated genera such as Alteromonas, Pseudoalteromonas, and Vibrio might be rare in coastal North Sea picoplankton because their rapid growth response to changing environmental conditions is counterbalanced by a higher grazing mortality.

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455T. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml−1) followed by achromopeptidase (60 U ml−1) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.