Microbial Diversity and Function in Shallow Subsurface Sediment and Oceanic Lithosphere of the Atlantis Massif.

The marine lithospheric subsurface is one of the largest biospheres on Earth; however, little is known about the identity and ecological function of microorganisms found in low abundance in this habitat, though these organisms impact global-scale biogeochemical cycling. Here, we describe the diversity and metabolic potential of sediment and endolithic (within rock) microbial communities found in ultrasmall amounts (101 to 104 cells cm-3) in the subsurface of the Atlantis Massif, an oceanic core complex on the Mid-Atlantic Ridge that was sampled on International Ocean Discovery Program (IODP) Expedition 357. This study used fluorescence-activated cell sorting (FACS) to enable the first amplicon, metagenomic, and single-cell genomic study of the shallow (<20 m below seafloor) subsurface of an actively serpentinizing marine system. The shallow subsurface biosphere of the Atlantis Massif was found to be distinct from communities observed in the nearby Lost City alkaline hydrothermal fluids and chimneys, yet similar to other low-temperature, aerobic subsurface settings. Genes associated with autotrophy were rare, although heterotrophy and aerobic carbon monoxide and formate cycling metabolisms were identified. Overall, this study reveals that the shallow subsurface of an oceanic core complex hosts a biosphere that is not fueled by active serpentinization reactions and by-products. IMPORTANCE The subsurface rock beneath the ocean is one of the largest biospheres on Earth, and microorganisms within influence global-scale nutrient cycles. This biosphere is difficult to study, in part due to the low concentrations of microorganisms that inhabit the vast volume of the marine lithosphere. In spite of the global significance of this biosphere, little is currently known about the microbial ecology of such rock-associated microorganisms. This study describes the identity and genomic potential of microorganisms in the subsurface rock and sediment at the Atlantis Massif, an underwater mountain near the Mid-Atlantic Ridge. To enable our analyses, fluorescence-activated cell sorting (FACS) was used as a means to concentrate cells from low biomass environmental samples for genomic analyses. We found distinct rock-associated microorganisms and found that the capacity for microorganisms to utilize organic carbon was the most prevalent form of carbon cycling. We additionally identified a potential role for carbon monoxide metabolism in the subsurface.

Record of archaeal activity at the serpentinite-hosted Lost City Hydrothermal Field.

Samples of young, outer surfaces of brucite-carbonate deposits from the ultramafic-hosted Lost City hydrothermal field were analyzed for DNA and lipid biomarker distributions and for carbon and hydrogen stable isotope compositions of the lipids. Methane-cycling archaeal communities, notably the Lost City Methanosarcinales (LCMS) phylotype, are specifically addressed. Lost City is unlike all other hydrothermal systems known to date and is characterized by metal- and CO2 -poor, high pH fluids with high H2 and CH4 contents resulting from serpentinization processes at depth. The archaeal fraction of the microbial community varies widely within the Lost City chimneys, from 1-81% and covaries with concentrations of hydrogen within the fluids. Archaeal lipids include isoprenoid glycerol di- and tetraethers and C25 and C30 isoprenoid hydrocarbons (pentamethylicosane derivatives - PMIs - and squalenoids). In particular, unsaturated PMIs and squalenoids, attributed to the LCMS archaea, were identified for the first time in the carbonate deposits at Lost City and probably record processes exclusively occurring at the surface of the chimneys. The carbon isotope compositions of PMIs and squalenoids are remarkably heterogeneous across samples and show highly (13) C-enriched signatures reaching δ(13) C values of up to +24.6‰. Unlike other environments in which similar structural and isotopic lipid heterogeneity has been observed and attributed to diversity in the archaeal assemblage, the lipids here appear to be synthesized solely by the LCMS. Some of the variations in lipid isotope signatures may, in part, be due to unusual isotopic fractionation during biosynthesis under extreme conditions. However, we argue that the diversity in archaeal abundances, lipid structure and carbon isotope composition rather reflects the ability of the LCMS archaeal biofilms to adapt to chemical gradients in the hydrothermal chimneys and possibly to perform either methanotrophy or methanogenesis using dissolved inorganic carbon, methane or formate as a function of the prevailing environmental conditions.

Sources of organic nitrogen at the serpentinite-hosted Lost City hydrothermal field.

The reaction of ultramafic rocks with water during serpentinization at moderate temperatures results in alkaline fluids with high concentrations of reduced chemical compounds such as hydrogen and methane. Such environments provide unique habitats for microbial communities capable of utilizing these reduced compounds in present-day and, possibly, early Earth environments. However, these systems present challenges to microbial communities as well, particularly due to high fluid pH and possibly the availability of essential nutrients such as nitrogen. Here we investigate the source and cycling of organic nitrogen at an oceanic serpentinizing environment, the Lost City hydrothermal field (30°N, Mid-Atlantic Ridge). Total hydrolizable amino acid (THAA) concentrations in the fluids range from 736 to 2300 nm and constitute a large fraction of the dissolved organic carbon (2.5-15.1%). The amino acid distributions, and the relative concentrations of these compounds across the hydrothermal field, indicate they most likely derived from chemolithoautotrophic production. Previous studies have identified the presence of numerous nitrogen fixation genes in the fluids and the chimneys. Organic nitrogen in actively venting chimneys has δ(15) N values as low as 0.1‰ which is compatible with biological nitrogen fixation. Total hydrolizable amino acids in the chimneys are enriched in (13) C by 2-7‰ compared to bulk organic matter. The distribution and absolute δ(13) C(THAA) values are compatible with a chemolithoautotrophic source, an attribution also supported by molar organic C/N ratios in most active chimneys (4.1-5.5) which are similar to those expected for microbial communities. In total, these data indicate nitrogen is readily available to microbial communities at Lost City.

Microbial diversity of Loki's Castle black smokers at the Arctic Mid-Ocean Ridge.

Hydrothermal vent systems harbor rich microbial communities ranging from aerobic mesophiles to anaerobic hyperthermophiles. Among these, members of the archaeal domain are prevalent in microbial communities in the most extreme environments, partly because of their temperature-resistant and robust membrane lipids. In this study, we use geochemical and molecular microbiological methods to investigate the microbial diversity in black smoker chimneys from the newly discovered Loki's Castle hydrothermal vent field on the Arctic Mid-Ocean Ridge (AMOR) with vent fluid temperatures of 310-320 °C and pH of 5.5. Archaeal glycerol dialkyl glycerol tetraether lipids (GDGTs) and H-shaped GDGTs with 0-4 cyclopentane moieties were dominant in all sulfide samples and are most likely derived from both (hyper)thermophilic Euryarchaeota and Crenarchaeota. Crenarchaeol has been detected in low abundances in samples derived from the chimney exterior indicating the presence of Thaumarchaeota at lower ambient temperatures. Aquificales and members of the Epsilonproteobacteria were the dominant bacterial groups detected. Our observations based on the analysis of 16S rRNA genes and biomarker lipid analysis provide insight into microbial communities thriving within the porous sulfide structures of active and inactive deep-sea hydrothermal vents. Microbial cycling of sulfur, hydrogen, and methane by archaea in the chimney interior and bacteria in the chimney exterior may be the prevailing biogeochemical processes in this system.

Molecular characterization of the bacteria adherent to human colorectal mucosa.

AIMS: To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. METHODS AND RESULTS: Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient - MSC 96%, InterQuartile Range - IQR 3.0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2.8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3.3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. CONCLUSIONS: Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. SIGNIFICANCE AND IMPACT OF THE STUDY: This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.

An off-axis hydrothermal vent field near the Mid-Atlantic Ridge at 30 degrees N.

Evidence is growing that hydrothermal venting occurs not only along mid-ocean ridges but also on old regions of the oceanic crust away from spreading centres. Here we report the discovery of an extensive hydrothermal field at 30 degrees N near the eastern intersection of the Mid-Atlantic Ridge and the Atlantis fracture zone. The vent field--named 'Lost City'--is distinctly different from all other known sea-floor hydrothermal fields in that it is located on 1.5-Myr-old crust, nearly 15 km from the spreading axis, and may be driven by the heat of exothermic serpentinization reactions between sea water and mantle rocks. It is located on a dome-like massif and is dominated by steep-sided carbonate chimneys, rather than the sulphide structures typical of 'black smoker' hydrothermal fields. We found that vent fluids are relatively cool (40-75 degrees C) and alkaline (pH 9.0-9.8), supporting dense microbial communities that include anaerobic thermophiles. Because the geological characteristics of the Atlantis massif are similar to numerous areas of old crust along the Mid-Atlantic, Indian and Arctic ridges, these results indicate that a much larger portion of the oceanic crust may support hydrothermal activity and microbial life than previously thought.

Immunostaining for prostate cancer androgen receptor in paraffin identifies a subset of men with a poor prognosis.

BACKGROUND: Knowledge of androgen receptor (AR) content could help predict hormone response and disease course in prostate cancer. However, determination of AR by biochemical assay is difficult. An immunohistochemical assay (ICA) would solve most difficulties and be especially useful if it could be performed on paraffinized tissue. EXPERIMENTAL DESIGN: AR was studied in paraffin sections from 90 men for whom endocrine response, survival (except one case), and/or biochemical AR was known. After Ag retrieval in a microwave oven, a polyclonal anti-AR Ab was used with the peroxidase antiperoxidase method. Results were semiquantified using a Histoscore (Hscore) and were correlated with biochemistry, endocrine response, and survival. RESULTS: Only 15 patients were AR-negative. AR-ICA did not correlate with biochemistry, Gleason score, stage, or ethnicity but did correlate with endocrine response and survival. The average Histoscore was significantly lower in patients with progressive disease (p < 0.05). In a Cox's regression analysis of survival (mean follow-up = 30 months) AR-ICA was a significant predictor (p = 0.015). Risk of death was 2.5 times greater for a patient with a negative assay compared with one with a positive result. CONCLUSIONS: Our data indicate that AR status by ICA may be a useful predictor of survival and endocrine response in prostate cancer. Further studies are needed to confirm these results because the assay could impact significantly on management.