Bacterial genome replication at subzero temperatures in permafrost

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with ¹³C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to −20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize ¹³C-labeled DNA when supplemented with ¹³C-acetate at temperatures of 0 to −20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of ¹³C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.     

Functional Metagenomic Investigations of Microbial Communities in a Shallow-Sea Hydrothermal System

Little is known about the functional capability of microbial communities in shallow-sea hydrothermal systems (water depth of <200 m). This study analyzed two high-throughput pyrosequencing metagenomic datasets from the vent and the surface water in the shallow-sea hydrothermal system offshore NE Taiwan. This system exhibited distinct geochemical parameters. Metagenomic data revealed that the vent and the surface water were predominated by Epsilonproteobacteria (Nautiliales-like organisms) and Gammaproteobacteria ( Thiomicrospira -like organisms), respectively. A significant difference in microbial carbon fixation and sulfur metabolism was found between the vent and the surface water. The chemoautotrophic microorganisms in the vent and in the surface water might possess the reverse tricarboxylic acid cycle and the Calvin−Bassham−Benson cycle for carbon fixation in response to carbon dioxide highly enriched in the environment, which is possibly fueled by geochemical energy with sulfur and hydrogen. Comparative analyses of metagenomes showed that the shallow-sea metagenomes contained some genes similar to those present in other extreme environments. This study may serve as a basis for deeply understanding the genetic network and functional capability of the microbial members of shallow-sea hydrothermal systems.     

Influence of Seasonal and Geochemical Changes on the Geomicrobiology of an Iron Carbonate Mineral Water Spring

Fuschna Spring in the Swiss Alps (Engadin region) is a bicarbonate iron(II)-rich, pH-neutral mineral water spring that is dominated visually by dark green microbial mats at the side of the flow channel and orange iron(III) (oxyhydr)oxides in the flow channel. Gradients of O₂, dissolved iron(II), and bicarbonate establish in the water. Our goals were to identify the dominating biogeochemical processes and to determine to which extent changing geochemical conditions along the flow path and seasonal changes influence mineral identity, crystallinity, and microbial diversity. Geochemical analysis showed microoxic water at the spring outlet which became fully oxygenated within 2.3 m downstream. X-ray diffraction and Mössbauer spectroscopy revealed calcite (CaCO₃) and ferrihydrite [Fe(OH)₃] to be the dominant minerals which increased in crystallinity with increasing distance from the spring outlet. Denaturing gradient gel electrophoresis banding pattern cluster analysis revealed that the microbial community composition shifted mainly with seasons and to a lesser extent along the flow path. 16S rRNA gene sequence analysis showed that microbial communities differ between the flow channel and the flanking microbial mat. Microbial community analysis in combination with most-probable-number analyses and quantitative PCR (qPCR) showed that the mat was dominated by cyanobacteria and the channel was dominated by microaerophilic Fe(II) oxidizers (1.97 × 10⁷ ± 4.36 × 10⁶ 16S rRNA gene copies g⁻¹ using Gallionella-specific qPCR primers), while high numbers of Fe(III) reducers (10⁹ cells/g) were identified in both the mat and the flow channel. Phototrophic and nitrate-reducing Fe(II) oxidizers were present as well, although in lower numbers (10³ to 10⁴ cells/g). In summary, our data suggest that mainly seasonal changes caused microbial community shifts, while geochemical gradients along the flow path influenced mineral crystallinity.     

Culture-Dependent and -Independent Characterization of Microbial Communities Associated with a Shallow Submarine Hydrothermal System Occurring within a Coral Reef off Taketomi Island, Japan

Microbial communities in a shallow submarine hydrothermal system near Taketomi Island, Japan, were investigated using cultivation-based and molecular techniques. The main hydrothermal activity occurred in a craterlike basin (depth, ~23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52°C) contained 175 μM H₂S and gas bubbles mainly composed of CH₄ (69%) and N₂ (29%). A liquid serial dilution cultivation technique targeting a variety of metabolism types quantified each population in the vent fluid and in a white microbial mat located near the vent. The most abundant microorganisms cultivated from both the fluid and the mat were autotrophic sulfur oxidizers, including mesophilic Thiomicrospira spp. and thermophilic Sulfurivirga caldicuralii. Methane oxidizers were the second most abundant organisms in the fluid; one novel type I methanotroph exhibited optimum growth at 37°C, and another novel type I methanotroph exhibited optimum growth at 45°C. The number of hydrogen oxidizers cultivated only from the mat was less than the number of sulfur and methane oxidizers, although a novel mesophilic hydrogen-oxidizing member of the Epsilonproteobacteria was isolated. Various mesophilic to hyperthermophilic heterotrophs, including sulfate-reducing Desulfovibrio spp., iron-reducing Deferribacter sp., and sulfur-reducing Thermococcus spp., were also cultivated. Culture-independent 16S rRNA gene clone analysis of the vent fluid and mat revealed highly diverse archaeal communities. In the bacterial community, S. caldicuralii was identified as the predominant phylotype in the fluid (clonal frequency, 25%). Both bacterial clone libraries indicated that there were bacterial communities involved in sulfur, hydrogen, and methane oxidation and sulfate reduction. Our results indicate that there are unique microbial communities that are sustained by active chemosynthetic primary production rather than by photosynthetic production in a shallow hydrothermal system where sunlight is abundant.     

Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (≈24 μg of chlorophyll a liter⁻¹). At this time bacterial abundance abruptly decreased from 2.8 × 10⁶ to 0.75 × 10⁶ ml⁻¹, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

Microbial diversity and activity in hypersaline high Arctic spring channels

Lost Hammer (LH) spring is a unique hypersaline, subzero, perennial high Arctic spring arising through thick permafrost. In the present study, the microbial and geochemical characteristics of the LH outflow channels, which remain unfrozen at ≥−18°C and are more aerobic/less reducing than the spring source were examined and compared to the previously characterized spring source environment. LH channel sediments contained greater microbial biomass (~100-fold) and greater microbial diversity reflected by the 16S rRNA clone libraries. Phylotypes related to methanogenesis, methanotrophy, sulfur reduction and oxidation were detected in the bacterial clone libraries while the archaeal community was dominated by phylotypes most closely related to THE ammonia-oxidizing Thaumarchaeota. The cumulative percent recovery of ¹⁴C-acetate mineralization in channel sediment microcosms exceeded ~30% and ~10% at 5 and −5°C, respectively, but sharply decreased at −10°C (≤1%). Most bacterial isolates (Marinobacter, Planococcus, and Nesterenkonia spp.) were psychrotrophic, halotolerant, and capable of growth at −5°C. Overall, the hypersaline, subzero LH spring channel has higher microbial diversity and activity than the source, and supports a variety of niches reflecting the more dynamic and heterogeneous channel environment.     

Rapid Methane Oxidation in a Landfill Cover Soil

Methane oxidation rates observed in a topsoil covering a retired landfill are the highest reported (45 g m⁻² day⁻¹) for any environment. This microbial community had the capacity to rapidly oxidize CH₄ at concentrations ranging from <1 ppm (microliters per liter) (first-order rate constant [k] = −0.54 h⁻¹) to >10⁴ ppm (k = −2.37 h⁻¹). The physiological characteristics of a methanotroph isolated from the soil (characteristics determined in aqueous medium) and the natural population, however, were similar to those of other natural populations and cultures: the Q₁₀ and optimum temperature were 1.9 and 31°C, respectively, the apparent half-saturation constant was 2.5 to 9.3 μM, and 19 to 69% of oxidized CH₄ was assimilated into biomass. The CH₄ oxidation rate of this soil under waterlogged (41% [wt/vol] H₂O) conditions, 6.1 mg liter⁻¹ day⁻¹, was near rates reported for lake sediment and much lower than the rate of 116 mg liter⁻¹ day⁻¹ in the same soil under moist (11% H₂O) conditions. Since there are no large physiological differences between this microbial community and other CH₄ oxidizers, we attribute the high CH₄ oxidation rate in moist soil to enhanced CH₄ transport to the microorganisms; gas-phase molecular diffusion is 10⁴-fold faster than aqueous diffusion. These high CH₄ oxidation rates in moist soil have implications that are important in global climate change. Soil CH₄ oxidation could become a negative feedback to atmospheric CH₄ increases (and warming) in areas that are presently waterlogged but are projected to undergo a reduction in summer soil moisture.     

Assessing the Microbial Community and Functional Genes in a Vertical Soil Profile with Long-Term Arsenic Contamination

Arsenic (As) contamination in soil and groundwater has become a serious problem to public health. To examine how microbial communities and functional genes respond to long-term arsenic contamination in vertical soil profile, soil samples were collected from the surface to the depth of 4 m (with an interval of 1 m) after 16-year arsenic downward infiltration. Integrating BioLog and functional gene microarray (GeoChip 3.0) technologies, we showed that microbial metabolic potential and diversity substantially decreased, and community structure was markedly distinct along the depth. Variations in microbial community functional genes, including genes responsible for As resistance, carbon and nitrogen cycling, phosphorus utilization and cytochrome c oxidases were detected. In particular, changes in community structures and activities were correlated with the biogeochemical features along the vertical soil profile when using the rbcL and nifH genes as biomarkers, evident for a gradual transition from aerobic to anaerobic lifestyles. The C/N showed marginally significant correlations with arsenic resistance (p = 0.069) and carbon cycling genes (p = 0.073), and significant correlation with nitrogen fixation genes (p = 0.024). The combination of C/N, NO₃ ⁻ and P showed the highest correlation (r = 0.779, p = 0.062) with the microbial community structure. Contradict to our hypotheses, a long-term arsenic downward infiltration was not the primary factor, while the spatial isolation and nutrient availability were the key forces in shaping the community structure. This study provides new insights about the heterogeneity of microbial community metabolic potential and future biodiversity preservation for arsenic bioremediation management.     

Association of Microbial Community Composition and Activity with Lead, Chromium, and Hydrocarbon Contamination

Microbial community composition and activity were characterized in soil contaminated with lead (Pb), chromium (Cr), and hydrocarbons. Contaminant levels were very heterogeneous and ranged from 50 to 16,700 mg of total petroleum hydrocarbons (TPH) kg of soil⁻¹, 3 to 3,300 mg of total Cr kg of soil⁻¹, and 1 to 17,100 mg of Pb kg of soil⁻¹. Microbial community compositions were estimated from the patterns of phospholipid fatty acids (PLFA); these were considerably different among the 14 soil samples. Statistical analyses suggested that the variation in PLFA was more correlated with soil hydrocarbons than with the levels of Cr and Pb. The metal sensitivity of the microbial community was determined by extracting bacteria from soil and measuring [³H]leucine incorporation as a function of metal concentration. Six soil samples collected in the spring of 1999 had IC₅₀ values (the heavy metal concentrations giving 50% reduction of microbial activity) of approximately 2.5 mM for CrO₄²⁻ and 0.01 mM for Pb²⁺. Much higher levels of Pb were required to inhibit [¹⁴C]glucose mineralization directly in soils. In microcosm experiments with these samples, microbial biomass and the ratio of microbial biomass to soil organic C were not correlated with the concentrations of hydrocarbons and heavy metals. However, microbial C respiration in samples with a higher level of hydrocarbons differed from the other soils no matter whether complex organic C (alfalfa) was added or not. The ratios of microbial C respiration to microbial biomass differed significantly among the soil samples (P < 0.05) and were relatively high in soils contaminated with hydrocarbons or heavy metals. Our results suggest that the soil microbial community was predominantly affected by hydrocarbons.     

CO2 Uptake and Fixation by a Thermoacidophilic Microbial Community Attached to Precipitated Sulfur in a Geothermal Spring

Carbon fixation at temperatures above 73°C, the upper limit for photosynthesis, is carried out by chemosynthetic thermophiles. Yellowstone National Park (YNP), Wyoming possesses many thermal features that, while too hot for photosynthesis, presumably support chemosynthetic-based carbon fixation. To our knowledge, in situ rates of chemosynthetic reactions at these high temperatures in YNP or other high-temperature terrestrial geothermal springs have not yet been reported. A microbial community attached to precipitated elemental sulfur (S^o floc) at the source of Dragon Spring (73°C, pH 3.1) in Norris Geyser Basin, YNP, exhibited a maximum rate of CO₂ uptake of 21.3 ± 11.9 μg of C 10⁷ cells⁻¹ h⁻¹. When extrapolated over the estimated total quantity of S^o floc at the spring''s source, the S^o floc-associated microbial community accounted for the uptake of 121 mg of C h⁻¹ at this site. On a per-cell basis, the rate was higher than that calculated for a photosynthetic mat microbial community dominated by Synechococcus spp. in alkaline springs at comparable temperatures. A portion of the carbon taken up as CO₂ by the S^o floc-associated biomass was recovered in the cellular nucleic acid pool, demonstrating that uptake was coupled to fixation. The most abundant sequences in a 16S rRNA clone library of the S^o floc-associated community were related to chemolithoautotrophic Hydrogenobaculum strains previously isolated from springs in the Norris Geyser Basin. These microorganisms likely contributed to the uptake and fixation of CO₂ in this geothermal habitat.The upper temperature limit for primary production via photosynthesis is ∼73°C (7, 8, 11). At this temperature, photosynthesis is restricted to cyanobacteria of the genus Synechococcus, which generally inhabit alkaline environments (11). In acidic environments (pH < 4.0), the upper temperature limit for photosynthetic-based primary production is ∼56°C. Under these conditions, phototrophic activity is restricted to the unicellular eukaryotic red algae Cyanidium, Galdieria, and Cyanidioschyzon, collectively referred to as “cyanidia” (6, 12, 31, 48). Primary production above this temperature in acidic environments occurs through chemoautotrophy, a metabolism restricted to prokaryotes.Yellowstone National Park (YNP), WY, possesses numerous high-temperature (73 to 93°C) geothermal environments that are thought to support communities of microorganisms through chemoautotrophic-based primary production. Evidence for chemosynthesis in these environments is based on the recovery of 16S rRNA gene sequences that are affiliated with cultivated representatives of the phyla Aquificae and Crenarchaeota, many of which are capable of CO₂ fixation via the oxidation of hydrogen (H₂) and/or sulfide (HS⁻) (15, 17, 21, 24, 26, 28, 41, 46). Surprisingly, CO₂ fixation has yet to be demonstrated in situ in YNP hot spring environments (acidic or alkaline) where temperatures exceed the limits of photosynthesis and where primary production is thought to be driven by chemoautotrophic metabolism (14, 15, 28, 29).Dragon Spring, an acid-sulfate-chloride (ASC) spring located in the Norris Geyser Basin of YNP, is a likely habitat for chemoautotrophic primary production. The pH of the water is ∼3.1, and the temperature of the water at the source fluctuates from 65 to 78°C, which is well above the upper temperature limit for photosynthesis under acidic conditions. Potential electron donors for chemolithoautotrophic growth in the source water include hydrogen (H₂) and sulfide (S²⁻) at concentrations of 13 nM and 65 μM, respectively (15). In addition, submerged substrata at the spring''s source are blanketed by precipitates of elemental sulfur (S°), hereafter referred to as S^o floc (23). Inventories of bacterial and archaeal 16S rRNA genes recovered from S^o floc collected from the source of Dragon Spring indicate the presence of Crenarchaeota and Aquificae (4, 15). The latter are related to chemolithoautotrophic Hydrogenobaculum spp., representatives of which have recently been isolated from the spring (15). In the present study, we demonstrate uptake and fixation of CO₂ at a temperature of 73°C by a Hydrogenobaculum-dominated microbial community associated with S^o floc collected from the source of Dragon Spring. This is the first direct evidence of CO₂ uptake in situ by a thermoacidophilic microbial community at a temperature that precludes photosynthesis in terrestrial geothermal springs.     

Bacterial Activity at −2 to −20°C in Arctic Wintertime Sea Ice

Arctic wintertime sea-ice cores, characterized by a temperature gradient of −2 to −20°C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4′,6′-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O₂-based respiration), the abundances of total, particle-associated (>3-μm), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (−2 to −20°C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (−20°C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to −20°C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.     

Immune Mediated Shaping of Microflora Community Composition Depends on Barrier Site

Barrier surfaces, such as the intestinal lining and the skin, are colonized by a diverse community of commensal microorganisms. Although commensal microorganisms clearly impact the host immune system, whether the immune system also shapes the commensal community is poorly understood. We used 16S rDNA deep sequencing to test whether mice with specific immune defects have an altered commensal microflora. Initially, skin swabs were obtained from wild-type and Langerhans Cell (LC) deficient mice. Despite the intimate contacts that LC make with the upper epidermis, no significant differences were observed in microbial community composition. Similarly, the skin of MyD88/TRIF^−/−, Rag1^−/− and heterozygous littermate controls showed no alteration in their commensal communities. Next we examined mouth swabs and feces. We did not find a difference in the MyD88/TRIF^−/− mice. However, we did observe a significant shift in the microbial composition in the feces and mouths of Rag1^−/− mice. Thus, we conclude that the adaptive immune system modulates the microbial composition at mucosal surfaces in the steady-state but LC, adaptive immunity, and MyD88-dependent innate responses do not affect the skin microbiome revealing a major distinction between barrier sites.     

Community Analysis of Ammonia and Nitrite Oxidizers during Start-Up of Nitritation Reactors

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30°C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25°C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day⁻¹ compared to 0.2 day⁻¹ for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.     

Microbial Community Structure and Arsenic Biogeochemistry in an Acid Vapor-Formed Spring in Tengchong Geothermal Area,China

Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73–0.86). Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation), Sulfolobus (sulfur and iron oxidation), Metallosphaera and Acidicaldus (iron oxidation). Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41–95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs.     

Assembly-Driven Community Genomics of a Hypersaline Microbial Ecosystem

Microbial populations inhabiting a natural hypersaline lake ecosystem in Lake Tyrrell, Victoria, Australia, have been characterized using deep metagenomic sampling, iterative de novo assembly, and multidimensional phylogenetic binning. Composite genomes representing habitat-specific microbial populations were reconstructed for eleven different archaea and one bacterium, comprising between 0.6 and 14.1% of the planktonic community. Eight of the eleven archaeal genomes were from microbial species without previously cultured representatives. These new genomes provide habitat-specific reference sequences enabling detailed, lineage-specific compartmentalization of predicted functional capabilities and cellular properties associated with both dominant and less abundant community members, including organisms previously known only by their 16S rRNA sequences. Together, these data provide a comprehensive, culture-independent genomic blueprint for ecosystem-wide analysis of protein functions, population structure, and lifestyles of co-existing, co-evolving microbial groups within the same natural habitat. The “assembly-driven” community genomic approach demonstrated in this study advances our ability to push beyond single gene investigations, and promotes genome-scale reconstructions as a tangible goal in the quest to define the metabolic, ecological, and evolutionary dynamics that underpin environmental microbial diversity.     

Genotypic and Phenotypic Responses of a Riverine Microbial Community to Polycyclic Aromatic Hydrocarbon Contamination

The phenotypic and genotypic adaptation of a freshwater sedimentary microbial community to elevated (22 to 217 μg g [dry weight] of sediment⁻¹) levels of polycyclic aromatic hydrocarbons (PAHs) was determined by using an integrated biomolecular approach. Central to the approach was the use of phospholipid fatty acid (PLFA) profiles to characterize the microbial community structure and nucleic acid analysis to quantify the frequency of degradative genes. The study site was the Little Scioto River, a highly impacted, channelized riverine system located in central Ohio. This study site is a unique lotic system, with all sampling stations having similar flow and sediment characteristics both upstream and downstream from the source of contamination. These characteristics allowed for the specific analysis of PAH impact on the microbial community. PAH concentrations in impacted sediments ranged from 22 to 217 μg g (dry weight) of sediment⁻¹, while PAH concentrations in ambient sediments ranged from below detection levels to 1.5 μg g (dry weight) of sediment⁻¹. Total microbial biomass measured by phospholipid phosphate (PLP) analysis ranged from 95 to 345 nmol of PLP g (dry weight) of sediment⁻¹. Nucleic acid analysis showed the presence of PAH-degradative genes at all sites, although observed frequencies were typically higher at contaminated sites. Principal component analysis of PLFA profiles indicated that moderate to high PAH concentrations altered microbial community structure and that seasonal changes were comparable in magnitude to the effects of PAH pollution. These data indicate that this community responded to PAH contamination at both the phenotypic and the genotypic level.     

Effect of Soil Ammonium Concentration on N2O Release and on the Community Structure of Ammonia Oxidizers and Denitrifiers

The effect of ammonium addition (6.5, 58, and 395 μg of NH₄⁺-N g [dry weight] of soil⁻¹) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N₂O release rates and a shift toward a higher contribution of nitrification to N₂O release occurred after incubation for 5 days at 4°C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected amoA sequences which probably belonged to Nitrosospira 16S rRNA clusters 3 and 4. Additional clones clustered with Nitrosospira sp. strains Ka3 and Ka4 and within an amoA cluster from unknown species. A Nitrosomonas-like amoA gene was detected in only one clone. In agreement with the amoA results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of nirK genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned nirK genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil nirK genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change.