Bacteria of the class Dehalococcoidia (DEH) (phylum Chloroflexi) are widely distributed in the marine subsurface and are especially prevalent in deep marine sediments. Nevertheless, little is known about the specific distributions of DEH subgroups at different sites and depths. This study therefore specifically examined the distributions of DEH through depths of various marine sediment cores by quantitative PCR and pyrosequencing using newly designed DEH 16S rRNA gene targeting primers. Quantification of DEH showed populations may establish in shallow sediments (i.e. upper centimetres), although as low relative proportions of total Bacteria, yet often became more prevalent in deeper sediments. Pyrosequencing revealed pronounced diversity co-exists within single biogeochemical zones, and that clear and sometimes abrupt shifts in relative proportions of DEH subgroups occur with depth. These shifts indicate varying metabolic properties exist among DEH subgroups. The distributional changes in DEH subgroups with depth may be related to a combination of biogeochemical factors including the availability of electron acceptors such as sulfate, the composition of organic matter and depositional regimes. Collectively, the results suggest DEH exhibit wider metabolic and genomic diversity than previously recognized, and this contributes to their widespread occurrence in the marine subsurface.
There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined "yield" to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.
Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated DEH-J10 obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160?cm below sea floor. A 1.44?Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO2 or oxidation of organics completely to CO2. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.The ISME Journal advance online publication, 22 August 2013; doi:10.1038/ismej.2013.143.
Half of the microbial cells in the Earths oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.
The Guaymas Basin (Gulf of California) hydrothermal vent area is known as a dynamic and hydrothermally vented sedimentary system, where the advection and production of a variety of different metabolic substrates support a high microbial diversity and activity in the seafloor. The main objective of our study was to explore the role of temperature and other environmental factors on community diversity, such as the presence of microbial mats and seafloor bathymetry within one hydrothermally vented field of 200 × 250 m dimension. In this field, temperature increased strongly with sediment depth reaching the known limit of life within a few decimeters. Potential sulfate reduction rate as a key community activity parameter was strongly affected by in situ temperature and sediment depth, declining from high rates of 1-5 ?mol ml(-1) d(-1) at the surface to the detection limit below 5 cm sediment depth, despite the presence of sulfate and hydrocarbons. Automated Ribosomal Intergenic Spacer Analysis yielded a high-resolution fingerprint of the dominant members of the bacterial community. Our analyses showed strong temperature and sediment depth effects on bacterial cell abundance and Operational Taxonomic Units (OTUs) number, both declining by more than one order of magnitude below the top 5 cm of the sediment surface. Another fraction of the variation in diversity and community structure was explained by differences in the local bathymetry and spatial position within the vent field. Nevertheless, more than 80% of all detected OTUs were shared among the different temperature realms and sediment depths, after being classified as cold (T < 10°C), medium (10°C ? T < 40°C) or hot (T ? 40°C) temperature conditions, with significant OTU overlap with the richer surface communities. Overall, this indicates a high connectivity of benthic bacterial habitats in this dynamic and heterogeneous marine ecosystem influenced by strong hydrothermalism.
Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on ?(13)C signatures of CH(4), dissolved inorganic carbon and porewater concentration profiles of CH(4) and sulfate. Cool, warm and hot in-situ temperature regimes (15-20?°C, 30-35?°C and 70-95?°C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.
Uncultured ANaerobic MEthanotrophic (ANME) archaea are often assumed to be obligate methanotrophs that are incapable of net methanogenesis, and are therefore used as proxies for anaerobic methane oxidation in many environments in spite of uncertainty regarding their metabolic capabilities. Anaerobic methane oxidation regulates methane emissions in marine sediments and appears to occur through a reversal of a methane-producing metabolism. We tested the assumption that ANME are obligate methanotrophs by detecting and quantifying gene transcription of ANME-1 across zones of methane oxidation versus methane production in sediments from the White Oak River estuary, North Carolina. ANME-1 consistently transcribe 16S rRNA and mRNA of methyl coenzyme M reductase (mcrA), the key gene for methanogenesis, up to 45 cm into methanogenic sediments. CARD-FISH shows that ANME-1 exist as single rod-shaped cells or pairs of cells. Integrating normalized depth distributions of 16S rDNA and rRNA (measured with qPCR and RT-qPCR respectively) shows that 26-77% of the rDNA (a proxy for ANME-1 cell numbers), and 18-76% of the rRNA (a proxy for ANME-1 activity) occurs within methane-producing sediments. These results, along with a re-assessment of the published Iiterature, change the perspective to ANME-1 as methanogens that are also capable of methane oxidation.
Subsurface fluids from deep-sea hydrocarbon seeps undergo methane- and sulfur-cycling microbial transformations near the sediment surface. Hydrocarbon seep habitats are naturally patchy, with a mosaic of active seep sediments and non-seep sediments. Microbial community shifts and changing activity patterns on small spatial scales from seep to non-seep sediment remain to be examined in a comprehensive habitat study.
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.
A study was conducted evaluating the effect of long-term Cu deficiency, with or without high Mn, on growth, gene expression and Cu status of beef cattle. Twenty-one Angus calves were born to cows receiving one of the following treatments: (1) 10 mg supplemental Cu/kg DM (+Cu); (2) no supplemental Cu and 2 mg Mo/kg DM ( - Cu); (3) - Cu diet plus 500 mg supplemental Mn/kg DM ( - Cu+Mn). Calves were weaned at approximately 183 d of age and individually fed throughout the growing and finishing phases. Plasma Cu was lower (P < 0.01) in - Cu calves compared with +Cu calves while high dietary Mn further depressed (P < 0.01) plasma Cu in - Cu+Mn calves v. - Cu calves. Liver Cu concentrations in +Cu calves were greater (P < 0.01) than in - Cu calves, with no differences between - Cu and - Cu+Mn calves. The daily body-weight gain of +Cu calves was greater (P < 0.01) than - Cu calves during the period from birth to weaning, but did not differ during the growing phase. - Cu+Mn calves gained less (P < 0.05) than - Cu calves during the growing phase. DM intake was lower (P < 0.01) in - Cu+Mn calves v. - Cu calves, and did not differ among +Cu and - Cu calves. The relative gene expression of cytochrome c oxidase in the liver was lower (P < 0.05) in - Cu calves compared with +Cu or - Cu+Mn calves. In conclusion, feeding a Cu - deficient diet in combination with high Mn negatively affected the growth and Cu status of beef cattle.
Members of the highly diverse Miscellaneous Crenarchaeotal Group (MCG) are globally distributed in various marine and continental habitats. In this study, we applied a polyphasic approach (rRNA slot blot hybridization, quantitative PCR (qPCR) and catalyzed reporter deposition FISH) using newly developed probes and primers for the in situ detection and quantification of MCG crenarchaeota in diverse types of marine sediments and microbial mats. In general, abundance of MCG (cocci, 0.4 ?m) relative to other archaea was highest (12-100%) in anoxic, low-energy environments characterized by deeper sulfate depletion and lower microbial respiration rates (P=0.06 for slot blot and P=0.05 for qPCR). When studied in high depth resolution in the White Oak River estuary and Hydrate Ridge methane seeps, changes in MCG abundance relative to total archaea and MCG phylogenetic composition did not correlate with changes in sulfate reduction or methane oxidation with depth. In addition, MCG abundance did not vary significantly (P>0.1) between seep sites (with high rates of methanotrophy) and non-seep sites (with low rates of methanotrophy). This suggests that MCG are likely not methanotrophs. MCG crenarchaeota are highly diverse and contain 17 subgroups, with a range of intragroup similarity of 82 to 94%. This high diversity and widespread distribution in subsurface sediments indicates that this group is globally important in sedimentary processes.
Disclosure of HIV serostatus by HIV-infected individuals is considered a prevention strategy, under the assumption that disclosure will prompt risk reduction practices among sex partners. We examined patients self-reports regarding disclosure messages they found relevant as part of prevention with positives (PwP) interventions in clinical settings. We conducted 52 in-depth interviews with patients participating in 13 PwP interventions. We found that the opportunity to reflect about living with HIV, explore fears of stigma and rejection, develop communication skills and strategies to disclose, and explore a sense of responsibility influenced patients intention to disclose and their disclosure practices. PwP interventions need to include a combination of messages about disclosure strategies, stigma, and communication, as well as helping patients frame disclosure as a process that includes situations and interactions to consider post-disclosure. PwP disclosure counseling can help influence a shift in patients risk towards safer sex practices.
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