Thermal Selection of Microbial Communities and Preservation of Microbial Function in Guaymas Basin Hydrothermal Sediments.

The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, hydrothermal activity, and organic matter accumulation on the seafloor due to high sedimentation rates. In the hydrothermal sediments of Guaymas Basin, microbial community compositions and coexistence patterns change across steep gradients of temperature, potential carbon sources, and electron acceptors. Nonmetric multidimensional scaling and guanine-cytosine percentage analyses reveal that the bacterial and archaeal communities adjust compositionally to their local temperature regime. Functional inference using PICRUSt shows that microbial communities consistently maintain their predicted biogeochemical functions in different sediments. Phylogenetic profiling shows that microbial communities retain distinct sulfate-reducing, methane-oxidizing, or heterotrophic lineages within specific temperature windows. The preservation of similar biogeochemical functions across microbial lineages with different temperature adaptations stabilizes the hydrothermal microbial community in a highly dynamic environment. IMPORTANCE Hydrothermal vent sites have been widely studied to investigate novel bacteria and archaea that are adapted to these extreme environments. However, community-level analyses of hydrothermal microbial ecosystems look beyond the presence and activity of particular types of microbes and examine to what extent the entire community of bacteria and archaea is adapted to hydrothermal conditions; these include elevated temperatures, hydrothermally generated carbon sources, and inorganic electron donors and acceptors that are characteristic for hydrothermal environments. In our case study of bacterial and archaeal communities in hydrothermal sediments of Guaymas Basin, we found that sequence-inferred microbial function was maintained in differently structured bacterial and archaeal communities across different samples and thermal regimes. The resulting preservation of biogeochemical functions across thermal gradients is an important factor in explaining the consistency of the microbial core community in the dynamic sedimentary environment of Guaymas Basin.

Interactions between temperature and energy supply drive microbial communities in hydrothermal sediment.

Temperature and bioavailable energy control the distribution of life on Earth, and interact with each other due to the dependency of biological energy requirements on temperature. Here we analyze how temperature-energy interactions structure sediment microbial communities in two hydrothermally active areas of Guaymas Basin. Sites from one area experience advective input of thermogenically produced electron donors by seepage from deeper layers, whereas sites from the other area are diffusion-dominated and electron donor-depleted. In both locations, Archaea dominate at temperatures >45 °C and Bacteria at temperatures <10 °C. Yet, at the phylum level and below, there are clear differences. Hot seep sites have high proportions of typical hydrothermal vent and hot spring taxa. By contrast, high-temperature sites without seepage harbor mainly novel taxa belonging to phyla that are widespread in cold subseafloor sediment. Our results suggest that in hydrothermal sediments temperature determines domain-level dominance, whereas temperature-energy interactions structure microbial communities at the phylum-level and below.

Filamentous Giant Beggiatoaceae from the Guaymas Basin Are Capable of both Denitrification and Dissimilatory Nitrate Reduction to Ammonium.

Filamentous large sulfur-oxidizing bacteria (FLSB) of the family Beggiatoaceae are globally distributed aquatic bacteria that can control geochemical fluxes from the sediment to the water column through their metabolic activity. FLSB mats from hydrothermal sediments of Guaymas Basin, Mexico, typically have a "fried-egg" appearance, with orange filaments dominating near the center and wider white filaments at the periphery, likely reflecting areas of higher and lower sulfide fluxes, respectively. These FLSB store large quantities of intracellular nitrate that they use to oxidize sulfide. By applying a combination of 15N-labeling techniques and genome sequence analysis, we demonstrate that the white FLSB filaments were capable of reducing their intracellular nitrate stores to both nitrogen gas and ammonium by denitrification and dissimilatory nitrate reduction to ammonium (DNRA), respectively. On the other hand, our combined results show that the orange filaments were primarily capable of DNRA. Microsensor profiles through a laboratory-incubated white FLSB mat revealed a 2- to 3-mm vertical separation between the oxic and sulfidic zones. Denitrification was most intense just below the oxic zone, as shown by the production of nitrous oxide following exposure to acetylene, which blocks nitrous oxide reduction to nitrogen gas. Below this zone, a local pH maximum coincided with sulfide oxidation, consistent with nitrate reduction by DNRA. The balance between internally and externally available electron acceptors (nitrate) and electron donors (reduced sulfur) likely controlled the end product of nitrate reduction both between orange and white FLSB mats and between different spatial and geochemical niches within the white FLSB mat.IMPORTANCE Whether large sulfur bacteria of the family Beggiatoaceae reduce NO3- to N2 via denitrification or to NH4+ via DNRA has been debated in the literature for more than 25 years. We resolve this debate by showing that certain members of the Beggiatoaceae use both metabolic pathways. This is important for the ecological role of these bacteria, as N2 production removes bioavailable nitrogen from the ecosystem, whereas NH4+ production retains it. For this reason, the topic of environmental controls on the competition for NO3- between N2-producing and NH4+-producing bacteria is of great scientific interest. Recent experiments on the competition between these two types of microorganisms have demonstrated that the balance between electron donor and electron acceptor availability strongly influences the end product of NO3- reduction. Our results suggest that this is also the case at the even more fundamental level of enzyme system regulation within a single organism.

Visualizing Evolutionary Relationships of Multidomain Proteins: An Example from Receiver (REC) Domains of Sensor Histidine Kinases in the Candidatus Maribeggiatoa str. Orange Guaymas Draft Genome.

For multidomain proteins, evolutionary changes may occur at the domain as well as the whole-protein level. An example is presented here, with suggestions for how such complicated relationships might be visualized. Earlier analysis of the Candidatus Maribeggiatoa str. Orange Guaymas (BOGUAY; Gammaproteobacteria) single-filament draft genome found evidence of gene exchange with the phylogenetically distant Cyanobacteria, particularly for sensory and signal transduction proteins. Because these are modular proteins, known to undergo frequent duplication, domain swapping, and horizontal gene transfer, a single domain was chosen for analysis. Recognition (REC) domains are short (~125 amino acids) and well conserved, simplifying sequence alignments and phylogenetic calculations. Over 100 of these were identified in the BOGUAY genome and found to have a wide range of inferred phylogenetic relationships. Two sets were chosen here for detailed study. One set of four BOGUAY ORFs has closest relatives among other Beggiatoaceae and Cyanobacteria. A second set of four has REC domains with more mixed affiliations, including other Beggiatoaceae, several sulfate-reducing Deltaproteobacteria and Firmicutes, magnetotactic Nitrospirae, one Shewanella and one Ferrimonas strain (both Gammaproteobacteria), and numerous Vibrio vulnificus and V. navarrensis strains (also Gammaproteobacteria). For an overview of the possible origins of the whole proteins and the surrounding genomic regions, color-coded BLASTP results were produced and displayed against cartoons showing protein domain structure of predicted genes. This is suggested as a visualization method for investigation of possible horizontally transferred regions, giving more detail than scans of DNA composition and codon usage but much faster than carrying out full phylogenetic analyses for multiple proteins. As expected, most of the predicted sensor histidine kinases investigated have two or more segments with distinct BLASTP affiliations. For the first set of BOGUAY ORFs, the flanking regions were also examined, and the results suggest they are embedded in genomic stretches with complex histories. An automated method of creating such visualizations could be generally useful; a wish list for its features is given.

Distinct Bacterial Communities in Surficial Seafloor Sediments Following the 2010 Deepwater Horizon Blowout.

A major fraction of the petroleum hydrocarbons discharged during the 2010 Macondo oil spill became associated with and sank to the seafloor as marine snow flocs. This sedimentation pulse induced the development of distinct bacterial communities. Between May 2010 and July 2011, full-length 16S rRNA gene clone libraries demonstrated bacterial community succession in oil-polluted sediment samples near the wellhead area. Libraries from early May 2010, before the sedimentation event, served as the baseline control. Freshly deposited oil-derived marine snow was collected on the surface of sediment cores in September 2010, and was characterized by abundantly detected members of the marine Roseobacter cluster within the Alphaproteobacteria. Samples collected in mid-October 2010 closest to the wellhead contained members of the sulfate-reducing, anaerobic bacterial families Desulfobacteraceae and Desulfobulbaceae within the Deltaproteobacteria, suggesting that the oil-derived sedimentation pulse triggered bacterial oxygen consumption and created patchy anaerobic microniches that favored sulfate-reducing bacteria. Phylotypes of the polycyclic aromatic hydrocarbon-degrading genus Cycloclasticus, previously found both in surface oil slicks and the deep hydrocarbon plume, were also found in oil-derived marine snow flocs sedimenting on the seafloor in September 2010, and in surficial sediments collected in October and November 2010, but not in any of the control samples. Due to the relative recalcitrance and stability of polycyclic aromatic compounds, Cycloclasticus represents the most persistent microbial marker of seafloor hydrocarbon deposition that we could identify in this dataset. The bacterial imprint of the DWH oil spill had diminished in late November 2010, when the bacterial communities in oil-impacted sediment samples collected near the Macondo wellhead began to resemble their pre-spill counterparts and spatial controls. Samples collected in summer of 2011 did not show a consistent bacterial community signature, suggesting that the bacterial community was no longer shaped by the DWH fallout of oil-derived marine snow, but instead by location-specific and seasonal factors.

Phylogeography, Salinity Adaptations and Metabolic Potential of the Candidate Division KB1 Bacteria Based on a Partial Single Cell Genome.

Deep-sea hypersaline anoxic basins and other hypersaline environments contain abundant and diverse microbial life that has adapted to these extreme conditions. The bacterial Candidate Division KB1 represents one of several uncultured groups that have been consistently observed in hypersaline microbial diversity studies. Here we report the phylogeography of KB1, its phylogenetic relationships to Candidate Division OP1 Bacteria, and its potential metabolic and osmotic stress adaptations based on a partial single cell amplified genome of KB1 from Orca Basin, the largest hypersaline seafloor brine basin in the Gulf of Mexico. Our results are consistent with the hypothesis - previously developed based on (14)C incorporation experiments with mixed-species enrichments from Mediterranean seafloor brines - that KB1 has adapted its proteins to elevated intracellular salinity, but at the same time KB1 apparently imports glycine betaine; this compatible solute is potentially not limited to osmoregulation but could also serve as a carbon and energy source.

The Guaymas Basin Hiking Guide to Hydrothermal Mounds, Chimneys, and Microbial Mats: Complex Seafloor Expressions of Subsurface Hydrothermal Circulation.

The hydrothermal mats, mounds, and chimneys of the southern Guaymas Basin are the surface expression of complex subsurface hydrothermal circulation patterns. In this overview, we document the most frequently visited features of this hydrothermal area with photographs, temperature measurements, and selected geochemical data; many of these distinct habitats await characterization of their microbial communities and activities. Microprofiler deployments on microbial mats and hydrothermal sediments show their steep geochemical and thermal gradients at millimeter-scale vertical resolution. Mapping these hydrothermal features and sampling locations within the southern Guaymas Basin suggest linkages to underlying shallow sills and heat flow gradients. Recognizing the inherent spatial limitations of much current Guaymas Basin sampling calls for comprehensive surveys of the wider spreading region.

Microbial Communities in Methane- and Short Chain Alkane-Rich Hydrothermal Sediments of Guaymas Basin.

The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed "Mat Mound") were characterized by porewater geochemistry of methane, C2-C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates.

Abundant Intergenic TAACTGA Direct Repeats and Putative Alternate RNA Polymerase ß' Subunits in Marine Beggiatoaceae Genomes: Possible Regulatory Roles and Origins.

The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. "Maribeggiatoa" filament from the Guaymas Basin (Gulf of California, Mexico) seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. "Maribeggiatoa" Orange Guaymas (BOGUAY) genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein), and Csr (carbon storage regulator) families. No pattern was evident in the predicted functions of the open reading frames (ORFs) downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in translational regulation is suggested. More speculatively, the putative alternate RNA polymerase subunit could be a negative transcriptional regulator.

Sulfide oxidation, nitrate respiration, carbon acquisition, and electron transport pathways suggested by the draft genome of a single orange Guaymas Basin Beggiatoa (Cand. Maribeggiatoa) sp. filament.

A near-complete draft genome has been obtained for a single vacuolated orange Beggiatoa (Cand. Maribeggiatoa) filament from a Guaymas Basin seafloor microbial mat, the third relatively complete sequence for the Beggiatoaceae. Possible pathways for sulfide oxidation; nitrate respiration; inorganic carbon fixation by both Type II RuBisCO and the reductive tricarboxylic acid cycle; acetate and possibly formate uptake; and energy-generating electron transport via both oxidative phosphorylation and the Rnf complex are discussed here. A role in nitrite reduction is suggested for an abundant orange cytochrome produced by the Guaymas strain; this has a possible homolog in Beggiatoa (Cand. Isobeggiatoa) sp. PS, isolated from marine harbor sediment, but not Beggiatoa alba B18LD, isolated from a freshwater rice field ditch. Inferred phylogenies for the Calvin-Benson-Bassham (CBB) cycle and the reductive (rTCA) and oxidative (TCA) tricarboxylic acid cycles suggest that genes encoding succinate dehydrogenase and enzymes for carboxylation and/or decarboxylation steps (including RuBisCO) may have been introduced to (or exported from) one or more of the three genomes by horizontal transfer, sometimes by different routes. Sequences from the two marine strains are generally more similar to each other than to sequences from the freshwater strain, except in the case of RuBisCO: only the Guaymas strain encodes a Type II enzyme, which (where studied) discriminates less against oxygen than do Type I RuBisCOs. Genes subject to horizontal transfer may represent key steps for adaptation to factors such as oxygen and carbon dioxide concentration, organic carbon availability, and environmental variability.

Mobile elements in a single-filament orange Guaymas Basin Beggiatoa ("Candidatus Maribeggiatoa") sp. draft genome: evidence for genetic exchange with cyanobacteria.

The draft genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament collected from a microbial mat at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) shows evidence of extensive genetic exchange with cyanobacteria, in particular for sensory and signal transduction genes. A putative homing endonuclease gene and group I intron within the 23S rRNA gene; several group II catalytic introns; GyrB and DnaE inteins, also encoding homing endonucleases; multiple copies of sequences similar to the fdxN excision elements XisH and XisI (required for heterocyst differentiation in some cyanobacteria); and multiple sequences related to an open reading frame (ORF) (00024_0693) of unknown function all have close non-Beggiatoaceae matches with cyanobacterial sequences. Sequences similar to the uncharacterized ORF and Xis elements are found in other Beggiatoaceae genomes, a variety of cyanobacteria, and a few phylogenetically dispersed pleiomorphic or filamentous bacteria. We speculate that elements shared among filamentous bacterial species may have been exchanged in microbial mats and that some of them may be involved in cell differentiation.

Depth-related differences in organic substrate utilization by major microbial groups in intertidal marine sediment.

Stable isotope probing of magnetic-bead-captured rRNA (Mag-SIP) indicated clear differences in in situ organic substrate utilization by major microbial groups between the more oxidized (0 to 2 cm) and sulfate-reducing (2 to 5 cm) horizons of marine intertidal sediment. We also showed that cyanobacteria and diatoms may survive by glucose utilization under dark anoxic conditions.

Why orange Guaymas Basin Beggiatoa spp. are orange: single-filament-genome-enabled identification of an abundant octaheme cytochrome with hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities.

Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (µLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.

Quantitative PCR methods for RNA and DNA in marine sediments: maximizing yield while overcoming inhibition.

For accurate quantification of DNA and RNA from environmental samples, yield loss during nucleic acid purification has to be minimized. Quantitative PCR (qPCR) and reverse transcription (RT)-qPCR require a trade-off between maximizing yield and removing inhibitors. We compared DNA and RNA yield and suitability for quantitative SYBR Green PCR and RT-PCR using the UltraClean and PowerSoil extraction kits and a bead-beating protocol with phenol/chloroform extraction steps. Purification methods included silica-column-based procedures from the MoBio kits, RNeasy MinElute, WizardPlus miniprep columns, and an acrylamide gel extraction. DNA and RNA purification with WizardPlus and RNeasy, respectively, led to significant losses of nucleic acids and archaeal 16S rRNA or 16S rRNA gene, as measured with RiboGreen or PicoGreen, and RT-qPCR or qPCR. Extraction and purification of DNA with the MoBio DNA UltraClean and DNA PowerSoil kits also decreased the yields slightly, relative to gel purification, in all sediments, except those from the deep sea in the Gulf of Mexico. Organic matter in humic-rich sediments may bind to these silica columns, reducing their nucleic acid-loading capacity. Purification with gel extraction cleans up organic-rich sediment samples sufficiently for quantitative analysis while avoiding the yield loss associated with commonly used silica columns.

Linking microbial community function to phylogeny of sulfate-reducing Deltaproteobacteria in marine sediments by combining stable isotope probing with magnetic-bead capture hybridization of 16S rRNA.

We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine (13)C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [(13)C]glucose, [(13)C]propionate, or [(13)C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that (13)C labeling can be much lower, to the extent that delta(13)C ratios can be studied even at their natural abundances.

Diversity, relative abundance and metabolic potential of bacterial endosymbionts in three Bathymodiolus mussel species from cold seeps in the Gulf of Mexico.

Cold seeps in the Gulf of Mexico are often dominated by mussels of the genus Bathymodiolus that harbour symbiotic bacteria in their gills. In this study, we analysed symbiont diversity, abundance and metabolic potential in three mussel species from the northern Gulf of Mexico: Bathymodiolus heckerae from the West Florida Escarpment, Bathymodiolus brooksi from Atwater Valley and Alaminos Canyon, and 'Bathymodiolus' childressi, which co-occurs with B. brooksi in Alaminos Canyon. Comparative 16S rRNA sequence analysis confirmed a single methanotroph-related symbiont in 'B.' childressi and a dual symbiosis with a methanotroph- and thiotroph-related symbiont in B. brooksi. A previously unknown diversity of four co-occurring symbionts was discovered in B. heckerae: a methanotroph, two phylogenetically distinct thiotrophs and a methylotroph-related phylotype not previously described from any marine invertebrate symbiosis. A gene characteristic of methane-oxidzing bacteria, pmoA, was identified in all three mussel species confirming the methanotrophic potential of their symbionts. Stable isotope analyses of lipids and whole tissue also confirmed the importance of methanotrophy in the carbon nutrition of all of the mussels. Analyses of absolute and relative symbiont abundance in B. heckerae and B. brooksi using fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization indicated a clear dominance of methanotrophic over thiotrophic symbionts in their gill tissues. A site-dependent variability in total symbiont abundance was observed in B. brooksi, with specimens from Alaminos Canyon harbouring much lower densities than those from Atwater Valley. This shows that symbiont abundance is not species-specific but can vary considerably between populations.

Improved 16S rRNA-targeted probe set for analysis of sulfate-reducing bacteria by fluorescence in situ hybridization.

An updated dataset of in silico specificities for 54 previously published 16S rRNA-targeted oligonucleotides was assembled to provide guidance for reliable fluorescence in situ hybridization (FISH) analysis of sulfate-reducing bacteria. Additionally, six new FISH probes were developed for major deltaproteobacterial taxa, including a probe trio targeting most Deltaproteobacteria and Gemmatimonadetes.

Comparison of rRNA and polar-lipid-derived fatty acid biomarkers for assessment of 13C-substrate incorporation by microorganisms in marine sediments.

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.

Single-stranded conformational polymorphism for separation of mixed rRNAS (rRNA-SSCP): a new method for profiling microbial communities.

We show that non-denaturing gel electrophoresis, or single-stranded conformational polymorphism (SSCP), can be used to separate mixtures of full-length rRNAs. Individual bands can then be excised for identification by RT-PCR and sequencing. This has the advantage over profiling methods such as DGGE and T-RFLP that no PCR amplification is involved prior to sequencing; thus, extraction biases aside, it should yield a quantitative picture of community composition in terms of ribosome content. To simplify banding patterns, RNA subsamples (e.g. bacterial 16S rRNA) can first be isolated by magnetic bead capture hybridization. Alternatively, oligonucleotide-directed ribonuclease H (RNase H) digestion can be used to identify bands of interest by running digested samples in parallel to undigested ones. We illustrate the use of this technique to identify a potentially predominant species in a hypersaline microbial mat. We anticipate that rRNA-SSCP will be useful for community profiling; for clone library construction by directed cloning of individual rRNAs; and for following incorporation of radiolabeled substrates at the species level, by gel autoradiography, without advance information or guesswork about which species might be active and abundant.